The Ministry of Health, Labour and
Welfare Ministerial Notification No. 285
Pursuant to Paragraph 1, Article 41 of the Pharmaceutical Affairs Law (Law No.
145, 1960), the Japanese Pharmacopoeia (hereinafter referred to as "new Phar-
macopoeia"), which has been established as follows*, shall be applied on April 1,
2006, and the Ministry of Health, Labour and Welfare Ministerial Notification No.
Ill (Matter of Establishing the Japanese Pharmacopoeia; hereinafter referred to as
"previous Pharmacopoeia"), issued in 2001, shall be abolished on March 31, 2006.
However, in the case of drugs which are listed in the new Pharmacopoeia (limited to
those listed in the previous Pharmacopoeia) and drugs which have been approved as
of April 1, 2006 as prescribed under Paragraph 1, Article 14 of the same law [includ-
ing drugs the Minister of Health, Labour and Welfare specifies (the Ministry of
Health and Welfare Ministerial Notification No. 104, 1994) as those exempted from
marketing approval pursuant to Paragraph 1, Article 14 of the Pharmaceutical
Affairs Law (hereinafter referred to as "drugs exempted from approval")], the Name
and Standards established in the previous Pharmacopoeia (limited to part of the
Name and Standards for the drugs concerned) may be accepted to conform to the
Name and Standards established in the new Pharmacopoeia before and on September
30, 2007. In the case of drugs which are listed in the new Pharmacopoeia (excluding
those listed in the previous Pharmacopoeia) and drugs which have been approved as
of April 1, 2006 as prescribed under Paragraph 1, Article 14 of the same law (includ-
ing those exempted from approval), they may be accepted as those being not listed in
the new Pharmacopoeia before and on September 30, 2007.
Jiro Kawasaki
The Minister of Health, Labour and Welfare
March 31, 2006
Referring the next title to this book: "The Japanese Pharmacopoeia".
(The text referred to by the term "as follows" are omitted here. All of them are made
available for public exhibition at the Evaluation and Licensing Division, Pharmaceu-
tical and Food Safety Bureau, Ministry of Health, Labour and Welfare, at each
Regional Bureau of Health and Welfare, and at each Prefectural Office in Japan).
*The term "as follows" here indicates the contents of the Japanese Pharmacopoeia Fifteenth Edition
from General Notices to Ultraviolet-visible Reference Spectra (pp. 1 - 1654).
CONTENTS
Preface i
The Japanese Pharmacopoeia, Fifteenth
Edition 1
General Notices 1
General Rules for Crude Drugs 5
General Rules for Preparations 7
General Tests, Processes and Apparatus 17
1 . Chemical Methods
1.01 Alcohol Number Determination 17
1.02 Ammonium Limit Test 19
1.03 Chloride Limit Test 20
1.04 Flame Coloration Test 20
1.05 Mineral Oil Test 20
1 .06 Oxygen Flask Combustion Method 20
1.07 Heavy Metals Limit Test 21
1.08 Nitrogen Determination (Semimicro-
Kjeldahl Method) 22
1.09 Qualitative Tests 23
1.10 Iron Limit Test 28
1.11 Arsenic Limit Test 29
1.12 Methanol Test 30
1.13 Fats and Fatty Oils Test 31
1.14 Sulfate Limit Test 33
1.15 Readily Carbonizable Substances Test 33
2. Physical Methods
Chromatographys
2.01 Liquid Chromatography 33
2.02 Gas Chromatography 35
2.03 Thin-layer Chromatography 37
Spectroscopic Methods
2.21 Nuclear Magnetic Resonance
Spectroscopy 38
2.22 Fluorometry 40
2.23 Atomic Absorption
Spectrophotometry 40
2.24 Ultraviolet-visible Spectrophotometry 41
2.25 Infrared Spectrophotometry 43
Other Physical Methods
2.41 Loss on Drying Test 44
2.42 Congealing Point Determination 44
2.43 Loss on Ignition Test 45
2.44 Residue on Ignition Test 45
2.45 Refractive Index Determination 46
2.46 Residual Solvents Test 46
2.47 Osmolarity Determination 46
2.48 Water Determination (Karl Fischer
Method) 47
2.49 Optical Rotation Determination 50
2.50 Endpoint Detection Methods in
Titrimetry 51
2.51 Conductivity Measurement 53
2.52 Thermal Analysis 54
2.53 Viscosity Determination 56
2.54 pH Determination 59
2.55 Vitamin A Assay 60
2.56 Determination of Specific Gravity and
Density 61
2.57 Boiling Point and Distilling Range
Test 63
2.58 X-Ray Powder Diffraction Method 64
2.59 Test for Total Organic Carbon 65
2.60 Melting Point Determination 66
3. Powder Property Determinations
3.01 Determination of Bulk and Tapped
Densities 68
3.02 Specific Surface Area by Gas
Adsorption 69
3.03 Powder Particle Density
Determination 71
3.04 Particle Size Determination 72
4. Biological Tests/Biochemical Tests/
Microbial Tests
4.01 Bacterial Endotoxins Test 77
4.02 Microbial Assay for Antibiotics 81
4.03 Digestion Test 84
4.04 Pyrogen Test 87
4.05 Microbial Limit Test 88
4.06 Sterility Test 93
5. Tests for Crude Drugs
5.01 Crude Drugs Test 97
5.02 Microbial Limit Test for Crude Drugs.... 100
6. Tests for Preparations
6.01 Test for Metal Particles in Ophthalmic
Ointments 106
6.02 Uniformity of Dosage Units 106
6.03 Particle Size Distribution Test for
Preparations 109
6.04 Test for Acid-neutralizing Capacity of
Gastrointestinal Medicines 109
6.05 Test for Extractable Volume of
Parenteral Preparations 110
6.06 Foreign Insoluble Matter Test for
Injections 110
6.07 Insoluble Particulate Matter Test for
Injections Ill
6.08 Insoluble Particulate Matter Test for
Ophthalmic Solutions 113
6.09 Disintegration Test 114
6.10 Dissolution Test 116
Contents
JP XV
7. Tests for Containers and Packing Materials
7.01 Test for Glass Containers for Injections.. 120
7.02 Test Methods for Plastic Containers 121
7.03 Test for Rubber Closure for Aqueous
Infusions 127
8. Other Methods
8.01 Sterilization and Aseptic Manipulation,
and Reverse Osmosis-Ultrafiltration.... 128
9. Reference Standards; Standard Solutions;
Reagents, Test Solutions; Measuring
Instruments, Appliances, Thermometers,
etc.
Reference Standards
9.01 Reference Standards 129
Standard Solutions
9.21 Standard Solutions for Volumetric
Analysis 132
9.22 Standard Solutions 143
9.23 Matching Fluids for Color 145
Reagents, Test Solutions, etc.
9.41 Reagents, Test Solutions 146
9.42 Solid Supports/Column Packing for
Chromatography 259
9.43 Filter Paper, Filters for Filtration,
Test Papers, Crucibles, etc 261
9.44 Reference Particles, etc 261
Measuring Instruments and Appliances,
Thermometers, etc.
9.61 Optical Filters for Wavelength and
Transmission Rate Calibration 262
9.62 Measuring Instruments, Appliances 262
9.63 Thermometers 263
Official Monographs 265
Crude Drugs 1251
Infrared Reference Spectra 1373-1518
Ultraviolet-visible Reference Spectra 1519-1654
General Information
1. Amino Acid Analysis 1655
2. Aristolochic Acid 1662
3. Basic Requirements for Viral Safety of
Biotechnological/Biological Products
listed in Japanese Pharmacopoeia 1663
4. Capillary Electrophoresis 1675
5. Decision of Limit for Bacterial
Endotoxins 1680
6 . Disinfection and Sterilization Methods 1 680
7. Guideline for Residual Solvents, Residual
Solvents Test, and Models for the Test in
Monographs 1682
8. International Harmonization
Implemented in the Japanese
Pharmacopoeia Fifteenth Edition 1684
9. Isoelectric Focusing 1708
10. Laser Diffraction Measurement of
Particle Size 1710
11. Media Fill Test 1712
12. Microbial Attributes of Nonsterile
Pharmaceutical Products 1715
13. Microbiological Evaluation of Processing
Areas for Sterile Pharmaceutical
Products 1717
14. Mycoplasma Testing for Cell Substrates
used for the Production of
Biotechnological/Biological Products 1721
15. Peptide Mapping 1724
16. pH Test for Gastrointestinal Medicine 1727
17. Plastic Containers for Pharmaceutical
Products 1728
18. Powder Flow 1729
19. Preservatives-Effectiveness Tests 1732
20. Qualification of Animals as Origin of
Animal-derived Medicinal Products
provided in the General Notices of
Japanese Pharmacopoeia and Other
Standards 1734
21. Quality Control of Water for
Pharmaceutical Use 1736
22. Rapid Identification of Microorganisms
Based on Molecular Biological Method... 1740
23. SDS-Polyacrylamide Gel Electrophoresis.... 1742
24. Solid and Particle Densities 1746
25. Sterility Assurance for Terminally
Sterilized Pharmaceutical Products 1748
26. Tablet Friability Test 1751
27. Terminal Sterilization and Sterilization
Indicators 1752
28. Test for Trace Amounts of Aluminum in
Trans Parenteral Nutrition (TPN)
Solutions 1754
29. Total Protein Assay 1756
30. Validation of Analytical Procedures 1760
Appendix
Atomic Weight Table (2004) 1763
Standard Atomic Weights 2004 1764
Index 1765
Index in Latin name 1777
Index in Japanese 1779
PREFACE
The 14th Edition of the Japanese Pharmacopoeia
(JP) was promulgated by Ministerial Notification No.
Ill of the Ministry of Health, Labour and Welfare
(MHLW) on March 30, 2001. To keep pace with
progress in medical and pharmaceutical sciences, in
November 2001, the Committee on JP established the
basic principles for the preparation of the JP 15th
Edition, setting out the characteristics and roles of the
JP, the definite measures for the revision, the date of
the revision, and the organization of the JP expert
committees.
At the above Committee, the five basic principles of
JP, which we refer to as the "Five Pillars of JP" were
established as follows.:
1) Making the JP more substantial by including all
drugs which are important from the viewpoint of
health care and medical treatment;
2) Making prompt partial revision as necessary and
facilitating smooth administrative operation;
3) Promoting international harmonization;
4) Ensuring transparency regarding the revision
and dissemination to the public of the JP;
5) Promoting the introduction of new analytical
technology and appropriate modifications to existing
science and technology, and promoting the improve-
ment of reference standards.
It was agreed that the Committee on JP should make
efforts, on the basis of the Five Pillars of JP, to ensure
that the JP is used effectively in health care and medical
treatment, by seeking the understanding and coopera-
tion for interested parties.
The JP should provide an official standard, being
required to assure the quality of medicines in Japan in
response to the progress of science and technology and
medical demands at the time. It should define the
standards for specifications, as well as the methods of
testing to assure the overall quality of every drug in
principle, and it should have a role in clarifying the
criteria for quality assurance of drugs that are recog-
nized to be essential for public health and medical
treatment.
The JP has been prepared with the aid of the
knowledge and experience of many professionals in the
pharmaceutical field. Therefore, JP should have the
characteristics of an official standard, which might be
widely used by all parties concerned. It should provide
information and understanding about the quality of
drugs to the public, and it should be conducive to
smooth and effective regulatory control of the quality
of drugs, as well as promoting and maintaining inter-
national consistency and harmonization of technical
requirements.
It was also agreed that JP articles should cover drugs
which are important from the viewpoint of health care
and medical treatment based on demand, frequency of
use and clinical results. It should ensure prompt phar-
macopeial listing after the introduction of drugs to the
market.
A definite rule for selection of articles and a clarifica-
tion of criteria for selection were also established. The
rule was described by the Pharmaceutical Affairs and
Food Sanitation Council (PAFSC) in an article entitled
" What the future Japanese Pharmacopoeia should
be" on December 2002. The target date for the publica-
tion of JP 15th Edition (the Japanese edition) was set
as April 2006.
At the first stage, JP Expert Committees were or-
ganized into the following twelve Panels and two sub-
committees: Panel on the Principles of Revisions;
Panel on the Selection of Articles; Panel on Nomencla-
ture ; Panel on Pharmaceutical Excipients; First Panel
on Chemical Drugs, Second Panel on Chemical Drugs;
Panel on Biologicals; Panel on Biological Tests; Panel
on Physico-Chemical Methods; Panel on Physical
Methods; Panel on Preparations; Panel on Crude
Drugs; First Sub-committee on the Principles of
Revisions; First Sub-committee on Crude Drugs.
In November 2001, the JP Expert Committees were
reorganized into the following eleven Panels in accor-
dance with the recommendation of the PAFSC: Panel
on the Principles of Revisions; Panel on Nomencla-
ture; Panel on Pharmaceutical Excipients; Panel on
Physico-Chemical Methods; Panel on Medicinal
Chemicals; Panel on Biologicals; Panel on Biological
Tests; Panel on Antibiotics; Panel on Crude Drugs;
Sub-committee on the Principles of Revisions; Panel
on International Harmonization (Pharmacopoeial
Discussion Group (PDG) related Panel). Thereafter,
the Panel on Pharmaceutical Water, Panel on Refer-
ence Standards, and three working groups under the
Panel on Medicinal Chemicals were established to
expedite discussion of revision drafts of Monographs.
In the Committee on JP, Mitsuru Uchiyama took
the role of chairman from January 2001 to December
2002, Tadao Terao from January to June 2003, and
Takao Hayakawa from July 2003 to March 2006.
i
Preface
JP XV
In addition to the regular revision every five years in
line with the basic principles for the preparation of the
JP, it was agreed that partial revision should be done as
necessary to take account of recent progress of science
and in the interests of international harmonization.
In accordance with the above principles, the expert
committees initiated deliberations on selection of
articles, and revisions for General Notices, General
Rules for Preparations, General Tests, Monographs
and so on.
Draft revisions covering subjects, the addition to
General Notices of a sentence "In principle, unless
otherwise specified, animals used as a source of materi-
als for preparing pharmaceutical preparations must be
healthy" and the deletion of a monograph "Phenace-
tin" were examined by the Committee on JP in
November 2001, followed by the PAFSC in December
2001, and then submitted to the MHLW.
The revision was promulgated on March 29, 2002 by
Ministerial Notification No. 151 of the MHLW.
For draft revisions covering subjects in General
Notices, General Rules for Preparations, General
Rules for Crude Drugs, General Tests and Mono-
graphs, deliberations were continued between June
2000 and February 2002, prepared for a supplement to
the JP 14, examined by the Committee on JP in
September 2002, followed by the PAFSC in December
2002, and then submitted to the Minister of MHLW.
The supplement was named "Supplement I to the JP
14th Edition" and promulgated on December 27, 2002
by Ministerial Notification No. 395 of MHLW.
The numbers of meetings in the above process to
prepare the supplement drafts were as follows: Panel
on the Principles of Revisions (6 times); Panel on the
Selection of Articles (2); Panel on Nomenclature (11);
Panel on Pharmaceutical Excipients (10); First Com-
mittee on Chemical Drugs (10); Second Committee on
Chemical Drugs (16); Committee on Biologicals (8);
Committee on Biological Methods (9); Committee on
Physico-Chemical Methods (8); Committee on Physi-
cal Methods (8); Committee on Pharmaceutical
Preparations (5); Committee on Crude Drugs (6); First
Sub-committee on the Principles of Revisions (27);
First Sub-committee on Crude Drugs (7). The numbers
of meetings of newly reorganized Panels were as
follows, Panel on Nomenclature (2), Panel on Physico-
Chemical Methods (1), Panel on Crude Drugs (3), and
Sub-panel on the Principles of Revisions (1).
In consequence of the above revision, the JP 14th
Edition with Supplement I carries 881 articles in Part I,
including 31 articles newly added and 8 articles deleted;
and 481 articles in Part II, including 15 articles newly
added and 3 articles deleted. It should be noted that in
the preparation of the drafts for the Supplement I,
generous cooperation was given by the Technical
Committee of the Pharmaceutical Manufacturer's
Association of Osaka (OPMA) and of Tokyo (PMAT),
the Tokyo Crude Drugs Association (TCDA), the
Japan Pharmaceutical Excipients Council (JPEC), the
Japan Kampo Medicine Manufacturers' Association
(JKMA), the Japan Antibiotics Research Association
(JARA), the Japan Flavor and Fragrance Materials
Association (JFFMA), the Japan Medical Plants
Federation (JMPF), the Japan Pharmaceutical
Manufacturers Association (JPMA), the Japanese
Society of Hospital Pharmacists (JSHP), the Japan
Pharmaceutical Association (JPA), and the Japan
Oilseeds Processors Association (JOPA).
Draft revisions covering subjects in General Rules
for Preparations, General Tests and Monographs, for
which discussions were completed between March 2002
and December 2003, were prepared for a supplement to
the JP 14. They were examined by the Committee on
JP in September 2004, followed by the PAFSC in
December 2004, and then submitted to the Minister of
Health, Labour and Welfare.
The supplement was named "Supplement II to the
JP 14th Edition" and promulgated on December 28,
2004 by Ministerial Notification No.461 of MHLW.
The numbers of meetings in the expert committees to
prepare the supplement drafts were as follows: Panel
on the Principles of Revisions (9); Panel on Nomen-
clatuere (10); Panel on Pharmaceutical Excipients (11);
Panel on Physico-Chemical Methods (30); Panel on
Medicinal Chemicals (containing the WG) (24); Panel
on Biologicals (11); Panel on Biological Tests (10);
Panel on Antibiotics (19); Committee on Crude Drugs
(19); Sub-panel on the Principles of Revisions (12);
Panel on International Harmonization (PDG related
Panel) (9); Panel on Pharmaceutical Water (2); Panel
on Reference Standards (3).
In consequence of this revision, the JP 14th Edition
including the Supplement I and II carries 907 articles in
Part I, including 27 articles newly added and 1 article
deleted; and 484 articles in Part II, including 12 articles
newly added and 9 articles deleted. It should be noted
that in the preparation of the drafts for the Supplement
II, generous cooperation was given by the Technical
Committee of OPMA and PMAT, TCDA , JPEC,
JKMA, JARA, JFFMA, JMPF, JPMA , JSHP, JPA,
and JOPA.
Draft revisions covering subjects, the addition to
General Notices of a sentence "The items in General
Test, Monographs (excipients) or General Information
in JP, which reflect text signed-off by all three phar-
macopoeias, JP, EP and USP (referred to as "the
signed-off item"), are stated at the front", the addition
of a test to General Tests "Test for Extractable Volume
JPXV
Preface
in
of Parenteral Preparations" and the revisions in
General Notices for Preparations and Monographs due
to the addition of the test, were examined by the
Committee on JP in June 2005, followed by the
PAFSC, and then submitted to the Minister MHLW.
The revision was promulgated on July 21, 2005 by
Ministerial Notification No. 344 of MHLW.
Draft revisions covering subjects in General Notices,
General Rules for Crude Drugs, General Rules for
Preparations, General Tests and Monographs, for
which discussions were finished between January 2004
and August 2005, were decided as a supplement to the
JP 15. They were examined by the Committee on JP in
October 2005, followed by the PAFSC in December
2005, and then submitted to the Minister of MHLW.
The numbers of meetings in the expert committees to
prepare the supplement drafts were as follows: Panel
on the Principles of Revisions (2); Panel on Nomencla-
ture (2); Panel on Pharmaceutical Excipients (3); Panel
on Physico-Chemical Methods (6); Panel on Medicinal
Chemicals (including the working groups) (17); Panel
on Biologicals (3); Panel on Biological Tests (2); Panel
on Antibiotics (6); Panel on Crude Drugs (6); Panel on
International Harmonization (PDG) (2); Panel on
Pharmaceutical Water (2); Panel on Reference Stan-
dards (2).
In April 2004, the Pharmaceuticals and Medical
Devices Agency (PMDA) was established, and a part of
the organization for JP preparation was moved from
MHLW to PMDA. Since July 2004, the numbers of
discussions in the expert committees to prepare the
supplement drafts were as follows: Panel on the Princi-
ples of Revisions (6); Panel on International Harmoni-
zation (PDG related)(3); Panel on Pharmaceutical
Water (7); Panel on Reference Standards (4); Panel on
Physico-Chemical Methods (6); Panel on Drug
Formulation (7); Panel on Physical Methods (9); Panel
on Medicinal Chemicals (containing the WG) (32);
Panel on Biologicals (6); Panel on Antibiotics (9);
Panel on Biological Tests (6); Panel on Crude Drugs
(12); Panel on Nomenclature (8); Panel on Pharmaceu-
tical Excipients (7). It should be noted that in the
preparation of the drafts for the supplement, generous
cooperation was given by OPMA and PMAT, TCDA,
JPEC, JKMA, JARA, JFMA, JMPF, JPMA, ISHP,
JPA, and JOPA.
In consequence of this revision, the JP 15th Edition
carries 1483 articles, owing to the addition of 102
articles and the deletion of 8 articles.
The principles of description and the salient points
of the revision in this volume are as follows:
1. The JP 15th Edition comprises the following i-
tems, in order: Notification of MHLW; Contents;
Preface; General Notices; General Rules for Crude
Drugs; General Rules for Preparations; General Tests,
Processes and Apparatus; Official Monographs; then
followed by Infrared Reference Spectra; Ultraviolet-
visual Reference Spectra; General Information; Table
of Atomic Mass as an appendix; and a Cumulative
Index.
2. The articles in General Rules for Preparations,
Official Monographs, Infrared Reference Spectra Offi-
cial and Ultraviolet-visible Reference Spectra are
respectively placed in alphabetical order.
3. The following items in each monograph are put
in the order shown below, except that unnecessary
items are omitted depending on the nature of the drug:
(1
(2
(3
(4
(5
(6
(7
(8
(9
(10
(11
(12
(13
(i4;
(15
(16
(17
(18
(19
(20;
(21
(22;
(23
English title
Commonly used name(s)
Latin title (only for crude drugs)
Title in Japanese
Structural formula or empirical formula
Molecular formula and molecular mass
Chemical name
Origin
Limits of the content of the ingredient(s) and/or
the unit of potency
Labeling requirements
Method of preparation
Description/Description of crude drugs
Identification tests
Specific physical and/or chemical values
Purity tests
Loss on drying, loss on ignition, and/or water
Residue on ignition, total ash, and/or acid-
insoluble ash
Tests being required for pharmaceutical prepara-
tions and other special tests
Isomer ratio
Assay or the content of the ingredient(s)
Containers and storage
Expiration date
Others
4. In each monograph, the following physical and
chemical values representing the properties and quality
of the drug are given in the order indicated below,
except that unnecessary items are omitted depending
on the nature of the drug:
(1) Alcohol number
(2) Absorbance
(3) Congealing point
(4) Refractive index
(5) Osmolarity
(6) Optical rotation
(7) Viscosity
IV
Preface
JP XV
(8)
pH
(9)
Specific gravity
(10)
Boiling point
(11)
Melting point
(12)
Acid value
(13)
Saponification value
(14)
Ester value
(15)
Hydroxyl value
(16)
Iodine value
5. Identification tests comprise the following items,
which are generally put in the order given below:
(1) Coloration reactions
(2) Precipitation reactions
(3) Decomposition reactions
(4) Derivatives
(5) Visible, ultraviolet or infrared spectra
(6) Special reactions
(7) Cations
(8) Anions
6. Purity tests comprise the following items, which
are generally put in the order given below, except that
unnecessary items are omitted depending on the nature
of the drug:
(1)
Color
(2)
Odor
(3)
Clarity and/or color of solution
(4)
Acidity or alkalinity
(5)
Acidity
(6)
Alkalinity
(7)
Chloride
(8)
Sulfate
(9)
Sulfite
(10)
Nitrate
(11)
Nitrite
(12)
Carbonate
(13)
Bromide
(14)
Iodide
(15)
Soluble halide
(16)
Thiocyanide
(17)
Selenium
(18)
Cationic salts
(19)
Ammonium
(20)
Heavy metals
(21)
Iron
(22)
Manganese
(23)
Chromium
(24)
Bismuth
(25)
Tin
(26)
Aluminum
(27)
Zinc
(28)
Cadmium
(29)
Mercury
(30)
Copper
(31)
Lead
(32)
Silver
(33)
Alkaline earth metals
(34)
Arsenic
(35)
Foreign matter
(36)
Related substances
(37)
Residual solvent
(38)
Other mixtures
(39)
Readily carbonizable substances
7. In accordance with the deletion of the prescrip-
tion on composition of JP by the Pharmaceutical
Affairs Law, the composition of Official Monographs
was prescribed in General Notices.
8. The following items in General Notices were
revised:
(1) The criteria for conformity of drugs by Descrip-
tion tests in monograph was defined by para-
graph 5.
(2) The atomic mass table used for JP was changed
to the Standard Atomic Weights 2004 by para-
graph 8.
(3) A provision "the temperature used for the tests
or in storage is described, in principle, in specific
figures" was added by paragraph 15.
(4) The containers of well -closed, tight and her-
metic were defined by paragraphs 38, 39 and 40,
respectively.
(5) Renumbering for paragraph, and other rearran-
gements were done.
9. The following items of the General Rules for
Preparations were revised:
(1) General Notices for Preparations
(2) Extracts
(3) Capsules
(4) Transdermal Systems (newly added)
(5) Tablets
(6) Injections
(7) Plasters and Pressure Sensitive Adhesives Tapes
(8) Arrangements for others
10. The provision of the criteria for conformity of
crude drugs was revised by paragraphs 4 and 5 of
General Rules for Crude Drugs.
11. In the General Tests, Processes and Apparatus,
"Content Uniformity Tests" and "Mass Variation
Tests" were combined and renamed as "Uniformity of
Dosage Units" .
12. The following items of the General Tests,
Processes and Apparatus were revised:
(1) Ammonium Limit Test
(2) Arsenic Limit Test
(3) Boiling Point and Distilling Range Test
JPXV
Preface
(4
(5
(6
(7
(8
(9
(io;
(ii
(12
(13
(14
Crude Drugs Test
Disintegration Test
Dissolution Test
Insoluble Particulate Matter Test for Injections
Melting Point Determination
Microbial Assay for Antibiotics
Microbial Limit Test for Crude Drugs
Powder Particle Size Determination
Residue on Ignition Test
Specific Surface Area Determination
Test Methods for Plastic Containers
13. The following items of the General Tests,
Processes and Apparatus were deleted:
(1) Absorbance Ratio Method
(2) Methoxyl Assay
(3) Paper Chromatography
(4) Volatile Contaminations in Ethanol
14. The following Reference Standards were newly
added:
Cilostazol
Ethyl Icosapentate
Ginsenoside Rbj
Ginsenoside Rgj
Gonadorelin Acetate
Human Menopausal Gonadotrophin
Interleukin-2
Isofiurane
Limaprost
Low-molecular Mass Heparin
Melting Point Standard (Acetanilide)
Melting Point Standard (Acetophenetidin)
Melting Point Standard (Caffeine)
Melting Point Standard (Sulfanilamide)
Melting Point Standard (Sulfapyridine)
Melting Point Standard (Vanillin)
Methylergometrine Maleate
Oxytocin
Phytonadione
Pravastatin 1 , 1 ,3 ,3-Tetramethylbutylammonium
Ritodrine Hydrochloride
Roxatidine Acetate Hydrochloride
Vasopressin
Warfarin Potassium
15. The following Reference Standards were delet-
ed.
Benzylpenicilline Sodium
Cefaloridin
Cefamandole Lithium
Cefetamet Pivoxil Hydrochloride
Cefoselis Sulfate
Cefoxitin
Cefradine
Posterior Pituitary
Secretin
Ticarcillin Sodium
16. English and Latin titles of drugs were based, in
principle, on the International Nonproprietary Names
for Pharmaceutical Substances, and the chemical
names were based on the Rules of the International
Union of Pure and Applied Chemistry (IUPAC).
17. Molecular formulas of organic compounds
begin with C and then H, followed by other involved
elements in the alphabetical order of the symbols of the
elements.
18. Structural formulas of drugs represent, as far
as possible, steric configurations.
19. The test procedures in monographs were
written in full, except within the same monograph and
in the monographs for preparations having a cor-
responding monograph of their principal material sub-
stances.
20. The following articles were newly added to
Official Monographs:
Alacepril
Alacepril Tablets
Amphotericin B for Injection
Amphotericin B Syrup
Amphotericin B Tablets
Arbekacin Sulfate Injection
L-Arginine
L-Aspartic Acid
Atenolol
Benincasa Seed
Betahistine Mesilate Tablets
Betamethasone Tablets
Betamethasone Valerate and Gentamicin Sulfate
Cream
Betamethasone Valerate and Gentamicin Sulfate
Ointment
Bezafibrate
Bezafibrate Sustained Release Tablets
Bucillamine
Cefaclor Capsules
Cefaclor Compound Granules
Cefaclor Fine Granules
Cefcapene Pivoxil Hydrochloride Fine Granules
Cefcapene Pivoxil Hydrochloride Tablets
Cefdinir Capsules
Cefdinir Fine Granules
Cefditoren Pivoxil Fine Granules
Cefditoren Pivoxil Tablets
Cefotiam Hydrochloride for Injection
Cefozopran Hydrochloride for Injection
VI
Preface
JP XV
Cefteram Pivoxil Fine Granules
Celmoleukin (Genetical Recombination)
Chenodeoxycholic Acid
Cilastatin Sodium
Cilostazol
Cilostazol Tablets
Clarithromycin Tablets
Clindamycin Hydrochloride Capsules
Croscarmellose Sodium
Daiokanzoto Extract
Dolichos Seed
Doxifluridine
Doxifluridine Capsules
Eleutherococcus Senticosus Rhizome
Ethyl Icosapentate
Etidronate Disodium
Etidronate Disodium Tablets
Etodolac
Faropenem Sodium for Syrup
Faropenem Sodium Tablets
Fosfomycin Sodium for Injection
Gonadorelin Acetate
Haloperidol Tablets
Hochuekkito Extract
Human Menopausal Gonadotrophin
Idarubicin Hydrochloride for Injection
Imipenem and Cilastatin for Injection
Isofiurane
Kakkonto Extract
Kamishoyosan Extract
Limaprost Alfadex
Lisinopril Hydrate
Lisinopril Tablets
Mefloquine Hydrochloride
Metformin Hydrochloride
Metformin Hydrochloride Tablets
Metoprolol Tartrate
Metoprolol Tartrate Tablets
Metronidazole Tablets
Nelumbo Seed
Nicergoline
Nicergoline Powder
Nicergoline Tablets
Nitrendipine
Nitrendipine Tablets
Parnaparin Sodium
Piperacillin Sodium for Injection
Polygonatum Rhizome
Pravastatin Sodium
Propranolol Hydrochloride Tablets
Pullulan
Rifampicin Capsules
Ritodrine Hydrochloride
Ritodrine Hydrochloride Tablets
Roxatidine Acetate Hydrochloride
Roxatidine Acetate Hydrochloride Extended-release
Capsules
Ryokeijutsukanto Extract
Saccharin
Saireito Extract
Sulpiride Capsules
Sulpiride Tablets
Tamsulosin Hydrochloride
Taurine
Teceleukin (Genetical Recombination)
Teceleukin for Injection (Genetical Recombination)
Timolol Maleate
Trimetazidine Hydrochloride Tablets
Urapidil
Vancomycin Hydrochloride for Injection
Verapamil Hydrochloride Tablets
Voglibose
Voglibose Tablets
Zaltoprofen
Zaltoprofen Tablets
21. The following monographs were revised:
Achyranthes Root
Aclarubicin Hydrochloride
Acrinol Hydrate
Actinomycin D
Akebia Stem
Alisma Rhizome
Powdered Alisma Rhizome
Alprostadil Alfadex
Amikacin Sulfate
Amomum Seed
Powder Amomum Seed
Amoxicillin Hydrate
Amphotericin B
Ampicillin Hydrate
Anhydrous Ampicillin
Ampicillin Sodium
Arbekacin Sulfate
L-Arginine Hydrochloride
Asiasarum Root
Aspoxicillin Hydrate
Astragalus Root
Astromicin Sulfate
Atractylodes Lancea Rhizome
Powdered Atractylodes Lancea Rhizome
Aztreonam
Bacitracin
Bekanamycin Sulfate
Benidipine Hydrochloride Tablets
Benzyl Alcohol
Benzylpenicillin Potassium
Berberine Tannate
JPXV
Preface
vn
Betahistine Mesilate
Betamethasone
Bethanechol Chloride
Bleomycin Hydrochloride
Bleomycin Sulfate
Bromazepam
Bupleurum Root
Burdock Fruit
Butyl Parahydroxybenzoate
Calcium Gluconate Hydrate
Calcium Para-aminosalicylate Granules
Calcium Para-aminosalicylate Hydrate
L-Carbocisteine
Carumonam Sodium
Cefaclor
Cefadroxil
Cefalotin Sodium
Cefatrizine Propylene Glycolate
Cefazolin Sodium
Cefazolin Sodium Hydrate
Cefcapene Pivoxil Hydrochloride Hydrate
Cefdinir
Cefditoren Pivoxil
Cefepime Dihydrochloride Hydrate
Cefixime
Cefmenoxime Hydrochloride
Cefmetazole Sodium
Cefminox Sodium Hydrate
Cefotetan
Cefotiam Hexetil Hydrochloride
Cefotiam Hydrochloride
Cefozopran Hydrochloride
Cefpiramide Sodium
Cefpodoxime Proxetil
Cefroxadine Hydrate
Cefsulodin Sodium
Ceftazidime Hydrate
Ceftibuten Hydrate
Ceftizoxime Sodium
Ceftriaxone Sodium Hydrate
Cefuroxime Axetil
Microcrystalline Cellulose
Powdered Cellulose
Chloramphenicol
Chloramphenicol Palmitate
Chlorpheniramine Maleate
Chlorpheniramine Maleate Injection
Chlorpheniramine Maleate Powder
Chlorpheniramine Maleate Tablets
^-Chlorpheniramine Maleate
Chlorpromazine Hydrochloride Tablets
Ciclacillin
Cinnamon Bark
Powdered Cinnamon Bark
Anhydrous Citric Acid
Citric Acid Hydrate
Citrus Unshiu Peel
Clindamycin Hydrochloride
Clindamycin Phosphate
Clove
Cloxacillin Sodium Hydrate
Colistin Sodium Methanesulfonate
Colistin Sulfate
Coptis Rhizome
Powdered Coptis Rhizome
Corn Starch
Cornus Fruit
Cortisone Acetate
Cyanamide
Cyanocobalamin
Cyanocobalamin Injection
Cycloserine
Daunorubicin Hydrochloride
Demethylchlortetracycline Hydrochloride
Dexamethasone
Dibekacin Sulfate
Dicloxacillin Sodium Hydrate
Dimercaprol
Dimercaprol Injection
Doxorubicin Hydrochloride
Doxycycline Hydrochloride Hydrate
Enfiurane
Enviomycin Sulfate
Epirubicin Hydrochloride
Erythromycin
Erythromycin Ethylsuccinate
Erythromycin Lactobionate
Erythromycin Stearate
Ethanol
Anhydrous Ethanol
Ethanol for Disinfection
Ethyl Parahydroxybenzoate
Etilefrine Hydrochloride
Famotidine Powder
Famotidine Tablets
Faropenem Sodium Hydrate
Powdered Fennel
Flomoxef Sodium
Flopropione Capsules
Foeniculated Ammonia Spirit
Fosfomycin Sodium
Fradiomycin Sulfate
Gardenia Fruit
Gentamicin Sulfate
Ginger
Powdered Ginger
Ginseng
Powdered Ginseng
Vlll
Preface
JP XV
Glycyrrhiza
Powdered Glycyrrhiza
Griseofulvin
Haloperidol
Hemp Fruit
Idarubicin Hydrochloride
Imipenem Hydrate
Insulin
Insulin Human (Genetical Recombination)
Insulin Injection
Insulin Zinc Injection (Aqueous Suspension)
Insulin Zinc Protamine Injection (Aqueous Suspen-
sion)
Isophane Insulin Injection (Aqueous Suspension)
Amorphous Insulin Zinc Injection (Aqueous Sus-
pension)
Crystalline Insulin Zinc Injection (Aqueous Suspen-
sion)
Isepamicin Sulfate
Japanese Angelica Root
Powdered Japanese Angelica Root
Josamycin
Josamycin Propionate
Jujube
Jujube Seed
Kanamycin Monosulfate
Kanamycin Sulfate
Ketoprofen
Kitasamycin
Kitasamycin Acetate
Kitasamycin Tartrate
Lactose Hydrate
Anhydrous Lactose
Latamoxef Sodium
Lenampicillin Hydrochloride
Lincomycin Hydrochloride Hydrate
Loquat Leaf
Meropenem Hydrate
Methylcellulose
Methylergometrine Maleate
Methylergometrine Maleate Tablets
Methyl Parahydroxybenzoate
Metronidazole
Micronomicin Sulfate
Midecamycin
Midecamycin Acetate
Minocycline Hydrochloride
Mitomycin C
Moutan Bark
Powdered Moutan Bark
Mupirocin Calcium Hydrate
Nalidixic Acid
Netilmicin Sulfate
Nicotinamide
Nystatin
Ophiopogon Tuber
Oxytetracycline Hydrochloride
Oxytocin
Oxytocin Injection
Panipenem
Powdered Peach Kernel
Peony Root
Powdered Peony Root
Peplomycin Sulfate
Perilla Herb
Phellodendron Bark
Powdered Phellodendron Bark
Phenethicillin Potassium
Phytonadione
Pimaricin
Pinellia Tuber
Pirarubicin
Pivmecillinam Hydrochloride
Plantago Seed
Platycodon Root
Powdered Platycodon Root
Polygala Root
Powdered Polygala Root
Polymixin B Sulfate
Poria Sclerotium
Powdered Poria Sclerotium
Potassium Clavulanate
Potato Starch
Povidone
Processed Aconite Root
Powdered Processed Aconite Root
Propranolol Hydrochloride
Propyl Parahydroxybenzoate
Pueraria Root
Pyrrolnitrin
Red Ginseng
Rehmannia Root
Rhubarb
Powdered Rhubarb
Ribostamycin Sulfate
Rifampicin
Rokitamycin
Roxithromycin
Saccharin Sodium Hydrate
Saffiower
Santonin
Schisandra Fruit
Scutellaria Root
Powdered Scutellaria Root
Senna Leaf
Powdered Senna Leaf
Siccanin
Sisomicin Sulfate
JP XV
Preface
IX
Sodium Citrate Hydrate
Sodium Fusidate
Spectinomycin Hydrochloride Hydrate
Spiramycin Acetate
Streptomycin Sulfate
Sulbenicillin Sodium
Sulpiride
Swertia Herb
Talampicillin Hydrochloride
Teicoplanin
Tetracycline Hydrochloride
Tobramycin
Tranexamic Acid Capsules
Tranexamic Acid Tablets
Trichomycin
Trichosanthes Root
Trimetazidine Hydrochloride
Uncaria Hook
Vancomycin Hydrochloride
Vasopressin Injection
Warfarin Potassium
Water
Water for Injection
Purified Water
Sterile Purified Water
Wine
Wheat Starch
Zinostatin Stimalamer
22. The following monographs were revised in
Japanese titles:
Acebutolol Hydrochloride
Acetylcholine Chloride for Injection
Acetylspiramycin
Aclarubicin Hydrochloride
Acrinol Hydrate
Adrenaline
Adrenaline Injection
Adrenaline Solution
Alimemazine Tartrate
Alprenolol Hydrochloride
Aluminum Potassium Sulfate Hydrate
Amantadine Hydrochloride
Ambenonium Chloride
Amikacin Sulfate
Aminophylline Hydrate
Amitriptyline Hydrochloride
Amitriptyline Hydrochloride Tablets
Amoxicillin Hydrate
Ampicillin Hydrate
Arbekacin Sulfate
L-Arginine Hydrochloride
L-Arginine Hydrochloride Injection
Arotinolol Hydrochloride
Aspoxicillin Hydrate
Astromicin Sulfate
Atropine Sulfate Hydrate
Atropine Sulfate Injection
Bacampicillin Hydrochloride
Bamethan Sulfate
Beclometasone Dipropionate
Bekanamycin Sulfate
Benidipine Hydrochloride
Benidipine Hydrochloride Tablets
Benserazide Hydrochloride
Benzalkonium Chloride
Benzalkonium Chloride Concentrated Solution 50
Benzalkonium Chloride Solution
Benzethonium Chloride
Benzethonium Chloride Solution
Benzylpenicillin Benzathine Hydrate
Berberine Chloride Hydrate
Betahistine Mesilate
Betamethasone Dipropionate
Betamethasone Sodium Phosphate
Betamethasone Valerate
Bethanechol Chloride
Biperiden Hydrochloride
Bleomycin Hydrochloride
Bleomycin Sulfate
Bromhexine Hydrochloride
Bromocriptine Mesilate
Bromovalerylurea
Bucumolol Hydrochloride
Bufetolol Hydrochloride
Bufexamac Cream
Bunazosin Hydrochloride
Bupranolol Hydrochloride
Butropium Bromide
Caffeine Hydrate
Calcium Chloride Hydrate
Calcium Gluconate Hydrate
Calcium Lactate Hydrate
Calcium Para-aminosalicylate Hydrate
Camostat Mesilate
Carbazochrome Sodium Sulfonate Hydrate
Carbidopa Hydrate
Carteolol Hydrochloride
Cefcapene Pivoxil Hydrochloride Hydrate
Cefepime Dihydrochloride for Injection
Cefepime Dihydrochloride Hydrate
Cefmenoxime Hydrochloride
Cefminox Sodium Hydrate
Cefotiam Hexetil Hydrochloride
Cefotiam Hydrochloride
Cefozopran Hydrochloride
Cefpirome Sulfate
Cefpodoxime Proxetil
Preface
JP XV
Cefroxadine Hydrate
Ceftazidime Hydrate
Cefteram Pivoxil
Ceftibuten Hydrate
Ceftriaxone Sodium Hydrate
Cefuroxime Axetil
Cellacefate
Cetraxate Hydrochloride
Chloramphenicol Palmitate
Chloramphenicol Sodium Succinate
Chlorhexidine Gluconate Solution
Chlorhexidine Hydrochloride
Chlormadinone Acetate
Chlorphenesin Carbamate
Chlorpheniramine Maleate
Chlorpheniramine Maleate Injection
Chlorpheniramine Maleate Powder
Chlorpheniramine Maleate Tablets
^-Chlorpheniramine Maleate
Chlorpromazine Hydrochloride
Chlorpromazine Hydrochloride Injection
Chlorpromazine Hydrochloride Tablets
Citric Acid Hydrate
Clemastine Fumarate
Clindamycin Hydrochloride
Clindamycin Phosphate
Clocapramine Hydrochloride Hydrate
Clofedanol Hydrochloride
Clomifene Citrate
Clomifene Citrate Tablets
Clomipramine Hydrochloride
Clonidine Hydrochloride
Cloperastine Hydrochloride
Cloxacillin Sodium Hydrate
Cocaine Hydrochloride
Codeine Phosphate Hydrate
1 % Codeine Phosphate Powder
10% Codeine Phosphate Powder
Codeine Phosphate Tablets
Colistin Sulfate
Cortisone Acetate
Croconazole Hydrochloride
Cyclopentolate Hydrochloride
Cyclophosphamide Hydrate
Cyproheptadine Hydrochloride Hydrate
Dantrolene Sodium Hydrate
Daunorubicin Hydrochloride
Deferoxamine Mesilate
Demethylchlortetracycline Hydrochloride
Dextromethorphan Hydrobromide Hydrate
Dibasic Calcium Phosphate Hydrate
Dibasic Sodium Phosphate Hydrate
Dibekacin Sulfate
Dibucaine Hydrochloride
Dicloxacillin Sodium Hydrate
Diethylcarbamazine Citrate
Diethylcarbamazine Citrate Tablets
Difenidol Hydrochloride
Dihydrocodeine Phosphate
1% Dihydrocodeine Phosphate Powder
10% Dihydrocodeine Phosphate Powder
Dihydroergotamine Mesilate
Dihydroergotoxine Mesilate
Dilazep Hydrochloride Hydrate
Diltiazem Hydrochloride
Dimemorfan Phosphate
Diphenhydramine and Bromovalerylurea Powder
Diphenhydramine Hydrochloride
Disodium Edetate Hydrate
Distigmine Bromide
Distigmine Bromide Tablets
Dobutamine Hydrochloride
Dopamine Hydrochloride
Dopamine Hydrochloride Injection
Doxapram Hydrochloride Hydrate
Doxorubicin Hydrochloride
Doxycycline Hydrochloride Hydrate
Ecothiopate Iodide
Edrophonium Chloride
Edrophonium Chloride Injection
Enoxacin Hydrate
Enviomycin Sulfate
Eperisone Hydrochloride
Ephedrine Hydrochloride
Ephedrine Hydrochloride Injection
10% Ephedrine Hydrochloride Powder
Ephedrine Hydrochloride Tablets
Epirubicin Hydrochloride
Ergometrine Maleate
Ergometrine Maleate Injection
Ergometrine Maleate Tablets
Ergotamine Tartrate
Erythromycin Ethylsuccinate
Erythromycin Lactobionate
Erythromycin Stearate
Estradiol Benzoate
Estradiol Benzoate Injection
Estradiol Benzoate Injection (Aqueous Suspension)
Ethambutol Hydrochloride
Ethyl L-Cysteine Hydrochloride
Ethylmorphine Hydrochloride Hydrate
Etilefrine Hydrochloride
Etilefrine Hydrochloride Tablets
Faropenem Sodium Hydrate
Fentanyl Citrate
Ferrous Sulfate Hydrate
Flavoxate Hydrochloride
Fluphenazine Enanthate
JPXV
Preface
XI
Flurazepam Hydrochloride
Formoterol Fumarate Hydrate
Fosfomycin Calcium Hydrate
Fradiomycin Sulfate
Fursultiamine Hydrochloride
Gabexate Mesilate
Gentamicin Sulfate
Guanabenz Acetate
Guanethidine Sulfate
Homatropine Hydrobromide
Homochlorcyclizine Hydrochloride
Human Chorionic Gonadotrophin
Human Chorionic Gonadotrophin for Injection
Hydralazine Hydrochloride
Hydralazine Hydrochloride for Injection
Hydralazine Hydrochloride Powder
Hydralazine Hydrochloride Tablets
Hydrocortisone Acetate
Hydrocortisone Butyrate
Hydrocortisone Sodium Phosphate
Hydrocortisone Sodium Succinate
Hydrocortisone Succinate
Hydrocotarnine Hydrochloride Hydrate
Hydroxocobalamin Acetate
Hypromellose Phthalate
Hydroxyzine Hydrochloride
Hydroxyzine Pamoate
Idarubicin Hydrochloride
Ifenprodil Tartrate
Imipenem Hydrate
Imipramine Hydrochloride
Imipramine Hydrochloride Tablets
Indenolol Hydrochloride
Ipratropium Bromide Hydrate
Isepamicin Sulfate
/-Isoprenaline Hydrochloride
Josamycin Propionate
Kainic Acid Hydrate
Kanamycin Monosulfate
Kanamycin Sulfate
Ketamine Hydrochloride
Ketotifen Fumarate
Kitasamycin Acetate
Kitasamycin Tartrate
Lactose Hydrate
Lenampicillin Hydrochloride
Levallorphan Tartrate
Levallorphan Tartrate Injection
Levomepromazine Maleate
Levothyroxine Sodium Hydrate
Lincomycin Hydrochloride Hydrate
Loxoprofen Sodium Hydrate
L-Lysine Hydrochloride
Lysozyme Hydrochloride
Magnesium Sulfate Hydrate
Maltose Hydrate
Maprotiline Hydrochloride
Meclofenoxate Hydrochloride
Mepenzolate Bromide
Mepivacaine Hydrochloride
Mepivacaine Hydrochloride Injection
Mercaptopurine Hydrate
Meropenem Hydrate
Metenolone Acetate
Metenolone Enanthate
Metenolone Enanthate Injection
Methamphetamine Hydrochloride
Methylbenactyzium Bromide
Methyldopa Hydrate
G?/-Methylephedrine Hydrochloride
10% cW-Methylephedrine Hydrochloride Powder
Methylergometrine Maleate
Methylergometrine Maleate Tablets
Methylprednisolone Succinate
Methylrosanilinium Chloride
Mexiletine Hydrochloride
Miconazole Nitrate
Micronomicin Sulfate
Midecamycin Acetate
Minocycline Hydrochloride
Monobasic Calcium Phosphate Hydrate
Morphine Hydrochloride Hydrate
Morphine Hydrochloride Injection
Morphine Hydrochloride Tablets
Mupirocin Calcium Hydrate
Naloxone Hydrochloride
Naphazoline Hydrochloride
Naphazoline Nitrate
Neostigmine Methylsulfate
Neostigmine Methylsulfate Injection
Netilmicin Sulfate
Nicardipine Hydrochloride
Nicardipine Hydrochloride Injection
Noradrenaline
Noradrenaline Injection
Nortriptyline Hydrochloride
Noscapine Hydrochloride Hydrate
Opium Alkaloids Hydrochlorides
Opium Alkaloids Hydrochlorides Injection
Orciprenaline Sulfate
Oxapium Iodide
Oxprenolol Hydrochloride
Oxybuprocaine Hydrochloride
Oxycodone Hydrochloride Hydrate
Oxytetracycline Hydrochloride
Pancuronium Bromide
Papaverine Hydrochloride
Papaverine Hydrochloride Injection
Xll
Preface
JP XV
Penbutolol Sulfate
Pentoxyverine Citrate
Peplomycin Sulfate
Perphenazine Maleate
Perphenazine Maleate Tablets
Pethidine Hydrochloride
Pethidine Hydrochloride Injection
Phenylephrine Hydrochloride
Pilocarpine Hydrochloride
Pipemidic Acid Hydrate
Piperazine Adipate
Piperazine Phosphate Hydrate
Piperazine Phosphate Tablets
Pirenzepine Hydrochloride Hydrate
Pivmecillinam Hydrochloride
Polymixin B Sulfate
Prednisolone Acetate
Prednisolone Sodium Succinate for Injection
Prednisolone Succinate
Procainamide Hydrochloride
Procainamide Hydrochloride Injection
Procainamide Hydrochloride Tablets
Procaine Hydrochloride
Procaine Hydrochloride Injection
Procarbazine Hydrochloride
Procaterol Hydrochloride Hydrate
Prochlorperazine Maleate
Prochlorperazine Maleate Tablets
Promethazine Hydrochloride
Propantheline Bromide
Propranolol Hydrochloride
Protamine Sulfate
Protamine Sulfate Injection
Protirelin Tartrate Hydrate
Pyrantel Pamoate
Pyridostigmine Bromide
Pyridoxine Hydrochloride
Pyridoxine Hydrochloride Injection
Quinidine Sulfate Hydrate
Quinine Ethyl Carbonate
Quinine Hydrochloride Hydrate
Quinine Sulfate Hydrate
Ranitidine Hydrochloride
Retinol Acetate
Retinol Palmitate
Riboflavin Butyrate
Riboflavin Sodium Phosphate
Riboflavin Sodium Phosphate Injection
Ribostamycin Sulfate
Saccharin Sodium Hydrate
Salbutamol Sulfate
Scopolamine Butylbromide
Scopolamine Hydrobromide Hydrate
Sisomicin Sulfate
Sodium Acetate Hydrate
Sodium Carbonate Hydrate
Sodium Citrate Hydrate
Sodium Picosulfate Hydrate
Sodium Prasterone Sulfate Hydrate
Sodium Thiosulfate Hydrate
Sorbitan Sesquioleate
Spectinomycin Hydrochloride Hydrate
Streptomycin Sulfate
Sucralfate Hydrate
Sulfamonomethoxine Hydrate
Sulpyrine Hydrate
Sultamicillin Tosilate Hydrate
Suxamethonium Chloride for Injection
Suxamethonium Chloride Hydrate
Suxamethonium Chloride Injection
Talampicillin Hydrochloride
Terbutaline Sulfate
Testosterone Enanthate
Testosterone Enanthate Injection
Testosterone Propionate
Testosterone Propionate Injection
Tetracaine Hydrochloride
Tetracycline Hydrochloride
Thiamine Chloride Hydrochloride
Thiamine Chloride Hydrochloride Injection
Thiamine Chloride Hydrochloride Powder
Thiamine Nitrate
Thioridazine Hydrochloride
Tiaramide Hydrochloride
Tiaramide Hydrochloride Tablets
Ticlopidine Hydrochloride
Timepidium Bromide Hydrate
Tipepidine Hibenzate
Tipepidine Hibenzate Tablets
Tizanidine Hydrochloride
Tocopherol Acetate
Tocopherol Calcium Succinate
Tocopherol Nicotinate
Todralazine Hydrochloride Hydrate
Tolperisone Hydrochloride
Trihexyphenidyl Hydrochloride
Trihexyphenidyl Hydrochloride Tablets
Trimebutine Maleate
Trimetazidine Hydrochloride
Trimetoquinol Hydrochloride Hydrate
Tubocurarine Chloride Hydrochloride Hydrate
Tubocurarine Chloride Hydrochloride Injection
Tulobuterol Hydrochloride
Vancomycin Hydrochloride
Verapamil Hydrochloride
Vinblastine Sulfate
Vinblastine Sulfate for Injection
Vincristine Sulfate
JPXV
Preface xiii
Zinc Sulfate Hydrate
23. The following three monographs in the JP 14th
Edition were replaced by a new monograph
"Hypromellose", and were deleted due to this revi-
sion:
Hydroxypropylmethylcellulose 2208
Hydroxypropylmethylcellulose 2906
Hydroxypropylmethylcellulose 29 1
24. The following monographs were deleted:
Cefaloridine
Cefamandole Sodium
Cefetamet Pivoxil Hydrochloride
Cefoselis Sulfate
Cefoxitin Sodium
Cefradine
Secretin
Ticarcillin Sodium
Those who were engaged in the preparation of the JP
15th Edition are as follows:
Norio Aimi
Mitsuo Aoki
Kiichi Aonuki
Nobuo Aoyagi**
Yoshichika Arakawa
Keiko Arimoto
Kazuhide Ashizawa
Shinichiro Aso
Yukio Aso
Makoto Emura
Hiroyuki Fuchino
Shigeyuki Fujikura
Kunihiro Fujita
Hiroshi Fujiwara
Goro Funamoto
Akihiro Furukawa
Yoshiharu Geki
Yukihiro Goda
Morio Hamashima
Ruri Hanajiri
Kouji Hasegawa
Ryuichi Hasegawa
Rika Hatano
Takao Hayakawa*
Masahiro Hayashi
Kenji Higuchi
Katsufuto Hiramatsu
Fusayoshi Hirayama
Yukio Hiyama
Kunimoto Hotta
Takanori Ichikawa
Nobukazu Igoshi
Toshio Imanari
Kenichi Inui
Mumio Ishibashi
Shigeru Itai
Mitsuo Ito
Yuji Ito
Takashi Itoh
Shozo Iwagami
Masao Izaki
Kenichi Izutsu
Akemi Kai
Kazuaki Kakehi
Shozo Kamiya
Nahoko Kaniwa
Motoko Kanke
Yoshiaki Kato
Mitsunori Katoh
Noriko Katori
Nobuo Kawahara
Toru Kawanishi
Toshiaki Kawanishi
Nana Kawasaki
Toshisuke Kawasaki
Yoshiaki Kawashima
Keiji Kijima
Fumiyuki Kiuchi
Takao Kiyohara
Takashi Kobayashi
Toyohiko Kobayashi
Masayoshi Kohase
Shigeo Kojima
Hiroyasu Kokubo
Eiichi Kokue
Katsuko Komatsu
Akio Komura
Toshifumi Konda
Seizo Kondo
Hideki Kumakura
Takao Kunisada
Mitsuo Kurashige
Takeshi Kurata
Masaaki Kurihara
Haruo Kuriyama
Fumiyo Kusu
Kumiko Kusuyama
Masako Maeda
Tamio Maitani
Midori Makita
Keiichi Maruyama
Toshio Masaoka
Toshihiko Matsubara
Yoshihisa Matsuda
Norio Matsuki
Shigeru Matsuki
Hayashi Matsukura
Eiichi Mikami
Satoshi Minobe
Katsutoshi Mise
Hiroto Miyamoto
Naoki Miyata
Michinao Mizugaki
Taiichi Mizuta
Kaoru Morikawa
Osamu Morita
Takashi Morita
Toshimi Murai
Shigeru Muraki
Masashi Muroi
Hiroaki Nagashige
Emi Nakajima
Hiroshi Nakamura
Takatoshi Nakamura
Tatsuya Nakano
Hiroyuki Nakazawa
Masaaki Naotsuka
Masao Nasu
Shingo Niimi
Koji Nishijima
Motohiro Nishijima
Tatsumi Nishiyama
Takashi Nomoto
Hiroyasu Ogata
Yoshiyuki Ogawa
Masaru Ohno
Yasuo Ohno
Yoshiro Ohtani
Minoru Okada
Satoshi Okada**
Kimiya Okazaki
Tsuneo Okubo
Haruhiro Okuda
Masakazu Ootani
Masami Otsuka
Tadashi Ouchi
Kazuhiko Sagara
Eiji Sakai
Tomoaki Sakamoto
Hideki Sasaki
Tsuguo Sasaki
Motoyoshi Satake
Akihiro Sato
Kyoko Sato
Michiko Sekiguchi
Setsuko Sekita
Yasuo Shimada
Kesamitsu Shimizu
Kyoko Shimura
Fumitoshi Shincho
Osamu Shirota
Kouichi Shudo
Hisashi Sonobe
Shoko Sueyoshi
Shinji Sugaya
Hisakazu Sunada
Hideyo Suzuki
Senji Suzuki
Yukio Tabuchi
Yoshikazu Takahashi
Tadahiro Takeda
Yasushi Takeda**
Toyoshige Tanabe
Haruo Tanaka
Toshihiro Tanaka
Kenichi Tanamoto
Tsuyoshi Tanimoto
Susumu Terabayashi
Tadao Terao*
Reiko Teraoka
Hiroshi Terashima
Masafumi Teshigawara
Kunikazu Teshima
Hiroshi Tokunaga
Kiyoshi Tomioka
Motowo Tomita
Tatsuru Tomizawa
Hideya Tsuge
Yosuke Tsuji
Nobuchika Tsumagari
Eriko Uchida
Mitsuru Uchiyama*
Yoshimasa Uehara
Kazuichi Umemoto
XIV
Preface
JP XV
Takashi Unno
Haruo Watanabe
Morimasa Yagisawa
Takehiko Yajima
Teruhide Yamaguchi
Keiichi Yamamoto
Keiji Yamamoto
Tosuke Yamamoto
Kenichi Yamazaki
Takeshi Yamazaki
Masato Yasuhara
Hikaru Yoden
Chikako Yomota
Hitoo Yoshida
Kazumasa Yoshikawa
Sumie Yoshioka
*: Chairman, the Committee on JP
**: Acting Chairman, the Committee on JP
GENERAL NOTICES
1. The official name of this pharmacopoeia is
H+Ka&IE B ^H/hj^J , and may be abbreviated as B
M+S, BM15, JP XV or JP 15.
2. The English name of this pharmacopoeia is The
Japanese Pharmacopoeia, Fifteenth Edition.
3. Among drugs, the Japanese Pharmacopoeia
Drugs (the JP Drugs) are those specified in the
monographs. The title names and the commonly used
names adopted in the monographs should be used as
official names. The distinction of the preparations
name of Fine Granules and Powders follows according
to the definition in the section of "Powders" of
General Rules for Preparations. In the drug mono-
graphs, in addition to English name, chemical names
or Latin names can be mentioned in the titles, as
appropriate.
4. "Crude Drugs and related drugs" are placed
together in the posterior part of the Official Mono-
graphs. These include: Crude Drugs being applied the
requirements of the General Rules for Crude Drugs, or
Powders, Extracts, Tinctures, Syrups, Spirits, Lini-
ments or Suppositories containing Crude Drugs as the
active ingredient, combination preparations containing
Crude Drugs as the principal active ingredient.
5. Drugs are to be tested according to the provi-
sions given in the pertinent monographs, General
Notices, General Rules for Crude Drugs, General Rules
for Preparations, and General Tests for their confor-
mity to the Japanese Pharmacopoeia. However, the
items of "Description" and "Storage" under Contain-
ers and storage in the monographs on preparations are
given for information, and should not be taken as in-
dicating standards for conformity.
6. In principle, unless otherwise specified, animals
used for preparing the JP Drugs or their source
materials must be healthy.
7. In this English version, the JP Drugs described
in the monographs begin with a capital letter.
8. The molecular formulas or constitution
formulas in parentheses ( ) after the name of drugs
or chemicals designate chemically pure substances.
Atomic masses adopted in the Japanese Phar-
macopoeia conform to the table of "Standard Atomic
Weights 2004". Molecular masses are indicated to two
decimal places rounded from three decimals.
9. The following abbreviations are used for the
principal units.
meter
m
centimeter
cm
millimeter
mm
micrometer
/urn
nanometer
nm
kilogram
kg
gram
g
milligram
mg
microgram
m
nanogram
ng
picogram
Pg
Celsius degree
°C
square centimeter
cm 2
liter
L
milliliter
mL
microliter
luL
megahertz
MHz
per centimeter
cm -1
newton
N
kilopascal
kPa
pascal
Pa
mole per liter
mol/L
millipascal second
mPa-s
square millimeter second
mmVs
lux
lx
mass per cent
%
mass parts per million
ppm
mass parts per billion
ppb
volume per cent
vol%
volume parts per million
vol ppm
mass per volume per cent
w/v%
hydrogen ion concentration
pH
endotoxin unit
EU
Note: "ppm" used in the Nuclear Magnetic Resonance
Spectroscopy indicates the chemical shift, and
"w/v%" is used in the formula or composition of
preparations.
10. The unit used for expressing the potency of
drug is recognized as the quantity of drug. Usually it is
expressed by a definite quantity of a definite standard
substance which shows a definite biological activity,
and differs according to each drug. The units are
determined, in principle, by comparison with each
reference standard by means of biological methods.
The term "Unit" used for the JP articles indicates the
unit defined in the Japanese Pharmacopoeia.
1
General Notices
JP XV
11. The statement "Being specified separately." in
the monographs means that the tests are to be specified
when the drugs are granted approval based on the
Pharmaceutical Affairs Law.
12. When an assurance that a product is of the JP
Drug quality is obtained consistently from data derived
from the manufacturing process validation studies,
and from the records of appropriate manufacturing
process control and of the test results of the quality
control, some of the test items in the monograph being
performed for the release of a product may be omitted
as occasion demands.
13. The test methods specified in the Japanese
Pharmacopoeia can be replaced by alternative methods
which give better accuracy and precision. However,
where a difference in test results is suspected, only the
result obtained by the procedure given in the
Pharmacopoeia is effective for the final judgment.
14. The details of the biological test methods may
be changed insofar as they do not affect the essential
qualities of the test.
15. The temperature for the tests or storage is
described, in principle, in specific figures. However, the
following expressions may be used instead.
Standard temperature, ordinary temperature, room
temperature, and lukewarm are defined as 20°C, 15 -
25°C, 1-30°C, and 30 - 40°C, respectively. A cold
place, unless otherwise specified, shall be a place
having a temperature of 1 - 15°C.
The temperature of cold water, lukewarm water,
warm water, and hot water are defined as not exceeding
10°C, 30 - 40°C, 60 - 70°C, and about 100°C, respec-
tively.
The term "heated solvent" or "hot solvent" means
a solvent heated almost to the boiling point of the
solvent, and the term "warmed solvent" or "warm
solvent" usually means a solvent heated to a tempera-
ture between 60°C and 70°C. The term "heat on or in a
water bath" indicates, unless otherwise specified,
heating with a boiling water bath or a steam bath at
about 100°C.
Cold extraction and warm extraction are usually
performed at temperatures of 15-25°C and 35-
45 °C, respectively.
16. To measure the number of drops, a dropping
device which delivers 20 drops of Purified Water
weighing 0.90 - 1.10 g at 20°C shall be used.
17. The term "in vacuum" indicates, unless
otherwise specified, a pressure not exceeding 2.0 kPa.
18. The acidity or alkalinity of a solution, unless
otherwise specified, is determined by blue or red litmus
papers. To indicate these properties more precisely, pH
values are used.
19. The terms in Table 1 are used to express the
degree of cutting of Crude Drugs or fineness of powder
Drugs.
Table 1
Sieve No.
4
6.5
8.6
18
50
100
200
Nominal
Designation
of sieve
4750 /mi
2800 /mi
2000 /mi
850 /jm
300 /jm
150 /jm
75 /mi
Names of
the drugs
which pass
through the
respective
sieves
Coarse
cutting
Moderate-
ly
fine
cutting
Fine
cutting
Coarse
powder
Moderate-
ly
fine
powder
Fine
powder
Very
fine
powder
20. Unless otherwise specified, the water to be used
in the tests of drugs shall be Purified Water.
21. As for wording "solution of a solute", where
the name of the solvent is not stated, the term "solu-
tion" indicates a solution in water.
22. For solution an expression such as "(1 in 3)",
"(1 in 10)", or "(1 in 100)" means that 1 g of a solid is
dissolved in, or 1 mL of a liquid is diluted with the
solvent to make the total volume of 3 mL, 10 mL or
100 mL, respectively. For the liquid mixture an
expression such as "(10:1)" or "(5:3:1)" means that
the respective numbers of parts, by volume, of the
designated liquids are to be mixed.
23. The term "weigh accurately" means to weigh
down to the degree of 0.1 mg, 0.01 mg or 0.001 mg by
taking into account the purpose of the test and using a
relevant weighing device. The term "weigh exactly"
means to weigh to the given decimal places.
24. A value of "n" figures in a test of a JP Drug
shall be obtained by rounding off a value of "n+ 1"
figures.
25. Unless otherwise specified, all tests of the drugs
shall be performed at the ordinary temperature and
observations of the results shall follow immediately
after the operations. However, the judgment for a test
which is affected by temperature should be based on the
conditions at the standard temperature.
26. The terms "immediately'V'at once" used in
the test of a JP Drug mean that the procedure is to be
performed within 30 seconds after the preceding
procedure.
27. In the section under the heading Description,
the term "white" is used to indicate white or practical-
ly white, and "colorless" is colorless or practically
colorless. Unless otherwise specified, the test of color is
carried out by placing 1 g of a solid drug on a sheet of
white paper or in a watch glass placed on white paper.
A liquid drug is put into a colorless test tube of 15-mm
internal diameter and is observed in front of a white
JPXV
General Notices
background through a layer of 30 mm. For the test of
clarity of liquid drugs the same procedure is applied
with either a black or white background. For the
observation of fluorescence of a liquid drug, only a
black background shall be used.
28. In the section under the heading Description,
the term "odorless" is used to indicate odorless or
practically odorless. Unless otherwise specified, the test
of odor shall be carried out by placing 1 g of a solid
drug or 1 mL of a liquid drug in a beaker.
29. In the section under the heading Description,
solubilities are expressed by the terms in Table 2.
Unless otherwise specified, solubility means the degree
of dissolution of a JP Drug, previously powdered in
the case of a solid drug, within 30 minutes in a solvent
at 20 + 5°C, by vigorous shaking for 30 seconds each
time at 5-minute intervals.
Table 2
Descriptive term
Volume of solvent required
for dissolving
1 g or 1 mL of solute
Very soluble
Freely soluble
Soluble
Sparingly soluble
Slightly soluble
Very slightly soluble
Less than 1 mL
From 1 mL to less than 10
mL
From 10 mL to less than 30
mL
From 30 mL to less than 100
mL
From 100 mL to less than
1000 mL
From 1000 mL to less than
10000 mL
Practically insoluble, or insolu- 10000 mL and over
ble
30. In the test of a drug, the term "dissolve" or
"miscible" indicates that it dissolves in, or mixes in
arbitrary proportion with the solvent to form a clear
solution or mixture. Insoluble materials other than the
drug including fibers should not be detected or practi-
cally invisible, if any.
31. Identification is the test necessary to identify
the active ingredient(s) of the drug based upon its
specific property.
32. Purity is the test to detect impurities/con-
taminants in drugs, and it, as well as other require-
ments in each monograph, specifies the purity of the
drug usually by limiting the kind/nature and quantity
of the impurities/contaminants. The impurities/
contaminants subject to the purity test are those sup-
posed to generate/contaminate during the manufactur-
ing process or storage, including hazardous agents such
as heavy metals, arsenic, etc. If any foreign substances
are used or supposed to be added, it is necessary to
perform tests to detect or limit the presence of such
substances.
33. The term "constant mass" in drying or igni-
tion, unless otherwise specified, means that the mass
difference after an additional 1 hour of drying or igni-
tion is not more than 0.10% of the preceding mass of
the dried substance or ignited residue. For crude drugs,
the difference is not more than 0.25%. However, when
the difference does not exceed 0.5 mg in a chemical
balance, 0.05 mg in a semi-microbalance, or 0.005 mg
in a microbalance, the difference is considered as
negligible and constant mass has been attained.
34. Assay is the test to determine the composition,
the content of the active ingredients, and the potency
unit of medicine by physical, chemical or biological
procedures.
35. In stating the appropriate quantities to be
taken for assay, the use of the word "about" indicates
a quantity within 10% of the specified mass. The word
"dry" in respect of the sample indicates drying under
the same conditions, as described in Loss on drying in
the monograph.
36. For the content of an ingredient determined by
Assay in the monographs, if it is expressed simply as
"not less than a certain percentage" without indicating
its upper limit, 101.0% is understood as the upper
limit.
37. The container is the device which holds drugs.
The stopper or cap, etc., is considered as part of the
container. The containers have no physical and chemi-
cal reactivity affecting the specified description and
quality of the contents.
38. A well-closed container protects the contents
from extraneous solids and from loss of the drug under
ordinary or customary conditions of handling, ship-
ment, and storage.
Where a well-closed container is specified, it may be
replaced by a tight container.
39. A tight container protects the contents from
extraneous solids or liquids, from loss of the contents,
and from efflorescence, deliquescence, or evaporation
under ordinary or customary conditions of handling,
shipment, and storage.
Where a tight container is specified, it may be
replaced by a hermetic container.
40. A hermetic container is impervious to air or
any other gas under ordinary or customary conditions
of handling, shipment, and storage.
41. The term "light-resistant" means that it can
prevent transmittance of light affecting in the specified
properties and quality of the contents and protect the
contained medicament from the light under ordinary
or customary conditions of handling, shipment, and
storage.
4 General Notices JP XV
42. For the JP Drugs, the contents or potency in
terms of units of the active ingredient(s), or the speci-
fied expiration date in the monographs have to be
shown on the immediate container or wrapping of
them.
43. The origin, numerical value or physical proper-
ties of the JP Drugs, being stipulated by the special
labeling requirements in the monographs, have to be
shown on the immediate container or wrapping of
them.
44. The harmonized General Tests and Mono-
graphs among the Japanese Pharmacopoeia, the
European Pharmacopoeia and the United States
Pharmacopeia are preceded by the statement as such.
The parts of the text, being not harmonized, are
surrounded by the symbols (* ♦).
GENERAL RULES
FOR CRUDE DRUGS
1. Crude drugs in the monographs include medici-
nal parts obtained from plants or animals, cell inclu-
sions and secretes separated from the origins, their
extracts, and minerals. General Rules for Crude Drugs
and Crude Drugs Test are applicable to the following:
Acacia, Achyranthes Root, Agar, Akebia Stem,
Alisma Rhizome, Aloe, Alpinia Offlcinarum Rhizome,
Amomum Seed, Anemarrhena Rhizome, Angelica Da-
hurica Root, Apricot Kernel, Areca, Artemisia Capil-
laris Flower, Asiasarum Root, Asparagus Tuber, As-
tragalus Root, Atractylodes Lancea Rhizome, Atrac-
tylodes Rhizome, Bear Bile, Bearberry Leaf, Belladon-
na Root, Benincasa Seed, Benzoin, Bitter Cardamon,
Bitter Orange Peel, Bupleurum Root, Burdock Fruit,
Calumba, Capsicum, Cardamon, Cassia Seed, Catalpa
Fruit, Chrysanthemum Flower, Cimicifuga Rhizome,
Cinnamon Bark, Citrus Unshiu Peel, Clematis Root,
Clove, Cnidium Monnieri Fruit, Cnidium Rhizome,
Coix Seed, Condurango, Coptis Rhizome, Cornus
Fruit, Corydalis Tuber, Cyperus Rhizome, Digenea,
Dioscorea Rhizome, Dolichos Seed, Eleutherococcus
Senticosus Rhizome, Ephedra Herb, Epimedium Herb,
Eucommia Bark, Evodia Fruit, Fennel, Forsythia
Fruit, Fritillaria Bulb, Gambir, Gardenia Fruit,
Gastrodia Tuber, Gentian, Geranium Herb, Ginger,
Ginseng, Glehnia Root, Glycyrrhiza, Gypsum, Hemp
Fruit, Honey, Houttuynia Herb, Immature Orange,
Imperata Rhizome, Ipecac, Japanese Angelica Root,
Japanese Gentian, Japanese Valerian, Jujube, Jujube
Seed, Lindera Root, Lithospermum Root, Longgu,
Lonicera Leaf and Stem, Loquat Leaf, Lycium Bark,
Lycium Fruit, Magnolia Bark, Magnolia Flower, Mal-
lotus Bark, Mentha Herb, Moutan Bark, Mulberry
Bark, Nelumbo Seed, Notopterygium Rhizome,
Nuphar Rhizome, Nux Vomica, Ophiopogon Tuber,
Oriental Bezoar, Oyster Shell, Panax Japonicus
Rhizome, Peach Kernel, Peony Root, Perilla Herb,
Pharbitis Seed, Phellodendron Bark, Picrasma Wood,
Pinellia Tuber, Plantago Herb, Plantago Seed,
Platycodon Root, Polygala Root, Polygonatum Rhi-
zome, Polygonum Root, Polyporus Sclerotium, Poria
Sclerotium, Powdered Acacia, Powdered Agar, Pow-
dered Alisma Rhizome, Powdered Aloe, Powdered
Amomum Seed, Powdered Atractylodes Lancea Rhi-
zome, Powdered Atractylodes Rhizome, Powdered
Calumba, Powdered Capsicum, Powdered Cinnamon
Bark, Powdered Clove, Powdered Cnidium Rhizome,
Powdered Coix Seed, Powdered Coptis Rhizome,
Powdered Cyperus Rhizome, Powdered Dioscorea
Rhizome, Powdered Fennel, Powdered Gambir,
Powdered Gardenia Fruit, Powdered Gentian, Pow-
dered Geranium Herb, Powdered Ginger, Powdered
Ginseng, Powdered Glycyrrhiza, Powdered Ipecac,
Powdered Japanese Angelica Root, Powdered
Japanese Gentian, Powdered Japanese Valerian, Pow-
dered Magnolia Bark, Powdered Moutan Bark, Pow-
dered Oyster Shell, Powdered Panax Japonicus Rhi-
zome, Powdered Peach Kernel, Powdered Peony
Root, Powdered Phellodendron Bark, Powdered
Picrasma Wood, Powdered Platycodon Root,
Powdered Polygala Root, Powdered Polypourus
Sclerotium, Powdered Poria Sclerotium, Powdered
Processed Aconite Root, Powdered Rhubarb, Pow-
dered Rose Fruit, Powdered Scutellaria Root, Pow-
dered Senega, Powdered Senna Leaf, Powdered Smi-
lax Rhizome, Powdered Sophora Root, Powdered
Sweet Hydrangea Leaf, Powdered Swertia Herb, Pow-
dered Tragacanth, Powdered Zanthoxylum Fruit,
Processed Aconite Root, Processed Ginger, Prunella
Spike, Pueraria Root, Red Ginseng, Rehmannia Root,
Rhubarb, Rice Starch, Rose Fruit, Rosin, Safflower,
Saffron, Saposhnikovia Root, Sappan Wood, Saus-
surea Root, Schisandra Fruit, Schizonepeta Spike,
Scopolia Rhizome, Scutellaria Root, Senega, Senna
Leaf, Sinomenium Stem, Smilax Rhizome, Sophora
Root, Sweet Hydrangea Leaf, Swertia Herb, Termeric,
Toad Venom, Tragacanth, Tribulus Fruit, Trichosan-
thes Root, Uncaria Hook, Zanthoxylum Fruit, Zedoa-
ry.
2. Crude drugs are usually used in the forms of
whole crude drugs, cut crude drugs or powdered crude
drugs.
Whole crude drugs are the medicinal parts or their
ingredients prepared by drying and /or simple proc-
esses, as specified in the monographs.
Cut crude drugs are small pieces or small blocks pre-
pared by cutting or crushing of the whole crude drugs,
and also coarse, medium or fine cutting of the crude
drugs in whole, and, unless otherwise specified, are
required to conform to the specifications of the whole
General Rules for Crude Drugs
JP XV
crude drugs used as original materials.
Powdered crude drugs are coarse, medium, fine or
very fine powder prepared from the whole crude drugs
or the cut crude drugs; usually powdered crude drugs
as fine powder are specified in the monographs.
3. Unless otherwise specified, crude drugs are used
in dried form. The drying is usually carried out at a
temperature not exceeding 60°C.
4. The origin of crude drugs is to serve as the criter-
ia. Such statements as 'other species of the same genus'
and 'allied plants' or 'allied animals' appearing in the
origin of crude drugs usually indicate plants or animals
which may be used as materials for crude drugs con-
taining the same effective constituents.
5. Description in each monograph for crude drugs
usually covers the crude drug derived from its typical
original plant or animal and includes statements of
characteristic properties of the crude drug. As for the
color, odor and solubility, apply correspondingly to
the prescription of the General Notices, except the
odor which is to serve as the criteria. The taste and
aspects obtained by microscopic observation are to
serve as the criteria.
6. Powdered crude drugs, otherwise specified, may
be mixed with diluents so as to attain proper content
and potency.
7. Powdered crude drugs do not contain fragments
of tissues, cells, cell inclusions or other foreign matter
alien to the original crude drugs or cut crude drugs.
8. Crude drugs are as free as possible from con-
taminants and other impurities due to molds, insects
and other animals and from other foreign matters, and
are required to be kept in a clean and hygienic state.
9. Crude drugs are preserved under protection
from moisture and insect damage, unless otherwise
specified. In order to avoid insect damage, suitable
fumigants may be used to preserve crude drugs,
provided that the fumigants are so readily volatilized as
to be harmless at the usual dosage of the crude drugs,
and such fumigants that may affect the therapeutic
efficacy of the crude drugs or interfere with the testing
are precluded.
10. Crude drugs are preserved in well-closed con-
tainers unless otherwise specified.
GENERAL RULES
FOR PREPARATIONS
1. General Notices for Preparations
(1) General notices for preparations present gener-
al rules and definitions for pharmaceutical dosage
forms.
(2) Pharmaceutical excipients are substance(s)
other than drug substance(s) contained in preparations
which are used to increase the utility of the prepara-
tion, to enable manufacturing of drug products easy,
to keep product's integrity, to improve the appearance
of a formulation and so forth. For these purposes, suit-
able excipients such as diluents, stabilizers, preserva-
tives, buffering agents, corrigents, suspending agents,
emulsifiers, aromatics, solubilizers, coloring agents,
and viscous agents may be added. The excipients used,
however, must be non-toxic, harmless and pharmaco-
logically inactive in the amount administered and must
not interfere with the therapeutic efficacy or the quality
test of the preparations.
(3) Vegetable oils used for pharmaceutical prepara-
tions usually indicate the edible oils listed in the Phar-
macopoeia. When starch is called for, any kind of
starch incorporated in the Pharmacopoeia may be
used, unless otherwise specified.
Moreover, ethanol specified in vol% is prepared by
adding purified water or water for injection to ethanol
at the specified vol%.
(4) To pharmaceutical preparations, functions
which control the releasing rate of drug substance(s),
leading to the modified absorption or transfer into the
body may be added for the purpose of controlling the
onset and duration of therapeutic effects and/or
decreasing adverse or side effects. However, modified
release preparations must meet the corresponding
requirements of dissolution test etc. which specify the
releasing rate, unless otherwise specified. In addition,
the functional modification of releasing rate must be
displayed on the pack insert and direct container or
package of the preparation, unless otherwise specified.
(5) Immediate-release and modified-release prepa-
rations exist in oral dosage forms which show different
release characteristics, respectively. Immediate-release
dosage forms are preparations showing a release of
drug substance(s), which is not intentionally modified
and generally dependent on the intrinsic physicochemi-
cal properties of the drug substance. Modified-release
dosage forms are preparations showing a release of
drug substance(s) which is suitably modified by a
specific formulation design and/or manufacturing
method. Modified-release dosage forms include en-
teric-coated and extended release preparations. Enteric
coated preparations are designed to release the majori-
ty drug substance(s) in small intestines rather than in
stomachs in order to prevent the degradation or
decomposition of drug substance(s) in stomach or to
decrease the irritant effect of drug substance(s) on
stomachs. Enteric coated preparations are generally
prepared by applying enteric films to preparations.
Extended release preparations are designed to control
the releasing rate and time of drug substance(s) and the
release sites in gastrointestinal tracts in order to
decrease the dosing times and/or to reduce adverse or
side effects. Extended release preparations are general-
ly prepared using suitable agents that prolong the drug
release. Capsules, tablets, powders, granules, and pills
of oral dosage forms can be coated with appropriate
coating agents, such as sugar, sugar alcohol, or high-
molecular-mass materials to enable the ingestion easy
or to prevent degradation of drug substance(s).
(6) When a high level of sterility assurance is
maintained consistently, based on the records derived
from validation studies of the manufacturing process
and the in-process controls, the sterility test usually
required for the release of the product may be omitted
(Parametric release).
(7) Unless otherwise specified, preserve phar-
maceutical preparations at room temperature.
2. Aerosols
(1) Aerosols are preparations for use by expelling a
solution or suspension of drug substance(s) under a
pressure of liquefied or compressed gas filled in a
common or different container.
Aerosols are used for topical application, space
spray, inhalation, oral administration, etc. Modes of
expelling are available in vapor, powder, foam and
paste, depending on the purpose of use.
(2) Hermetic containers are used for preservation.
Aromatic Waters / General Rules for Preparations
JP XV
3. Aromatic Waters
(1) Aromatic Waters are clear saturated solutions
of essential oils or other volatile substances in water.
(2) Unless otherwise specified, Aromatic Waters
may be usually prepared by the following process.
Shake thoroughly 2 mL of an essential oil or 2 g of a
volatile substance with 1000 mL of lukewarm purified
water for 15 minutes, set the mixture aside for 12 hours
or longer, filter through moistened filter paper, and
add purified water to make 1000 mL. Alternatively,
incorporate thoroughly 2 mL of an essential oil or 2 g
of volatile substances with sufficient refined siliceous
earth or pulped filter-paper, add 1000 mL of purified
water, agitate thoroughly for 10 minutes, and then
filter the mixture. To obtain a clear filtrate, repeat the
filtration, and add sufficient water through the filter
paper to make 1000 mL.
(3) Aromatic Waters have odor and taste derived
from the drug substance(s) and excipients used.
(4) Tight containers are used for preservation.
4. Capsules
(1) Capsules are preparations in which liquefied,
suspended, semi-solid, powdered or granulated drugs
or preparations are enclosed in capsules or wrapped
with capsule bases. There are two kinds of capsules,
which are:
(i) Hard capsules (ii) Soft capsules
(2) Capsules are usually prepared by the following
methods.
(i) Hard capsules: Drug substance(s) or uniform
mixtures of drug substance(s) with diluents and other
suitable excipients, or granules or preparations pre-
pared by a suitable method, are filled as they are or
prepared lightly formed and into hard capsules. Exten-
ded-release or enteric-coated capsules can be prepared
by filling extended-release or enteric-coated prepara-
tions into capsules or by changing the components of
capsule shells or coating the capsule with suitable
coating agents.
(ii) Soft capsules: Drug substance(s) or mixtures of
drug substance(s) with suitable diluents, etc. are en-
closed by a suitable capsule such as gelatin plasticized
by addition of glycerin, sorbitol, etc., and molded in a
suitable shape. If necessary, coloring agents, preserva-
tives, etc. may be added to capsule agents. By changing
the components of capsule shells or applying suitable
coating agents to capsules, extended-release or enteric-
coated capsules can be prepared.
(3) Unless otherwise specified, Capsules meet the
requirements of the Dissolution Test <6.w> or the
Disintegration Test <6.09>.
(4) Unless otherwise specified, Capsules meet the
requirements of the Uniformity of Dosage Units <6.02>.
(5) Well-closed or tight containers are used for
preservation.
5. Cataplasms/Gel Patches
(1) Cataplasms/Gel Patches are generally pasty
preparations containing the mixture of drug substan-
ce^) and water or those prepared by spreading the
mixture on cloth, which are intended for external use.
(2) Unless otherwise specified, Cataplasms/Gel
Patches are usually prepared by mixing drug substan-
ce^) with glycerin, water, or other suitable liquid
materials, or with high-molecular-mass materials(s)
which are soluble in water or absorbent of water until
homogeneity is attained.
(3) Pasty cataplasms which have separated out one
or more of their components during storage are
rehomogenized before use unless the substances have
deteriorated.
(4) Tight containers are used for preservation.
6. Elixirs
(1) Elixirs are usually clear, sweetened, and aro-
matic liquid preparations, containing ethanol, intend-
ed for oral use.
(2) Elixirs are usually prepared by dissolving drugs
or their extractives in ethanol and purified water,
adding aromatic agents and sucrose, other sugars or
sweetening agents, and clarifying by filtration or other
procedures.
(3) Tight containers are used for preservation.
7. Extracts
(1) Extracts are prepared by evaporating the
extractives of crude drugs. There are two kinds of
Extracts which are:
(i) viscous extracts (ii) dry extracts
(2) Unless other-wise specified, Extracts are pre-
pared as follows.
(i) Crude drugs pulverized in suitable sizes, are
usually extracted for a certain period of time with
suitable solvents by cold extraction or warm extrac-
tion, or by percolation as directed in (2) under
Tinctures.
JPXV
General Rules for Preparations / Fluidextracts
The extractive is filtered, and the filtrate is concen-
trated or dried in a suitable method to produce a millet
jelly-like consistency in the case of a viscous extract,
and to make crushable solid masses, granules or
powder in the case of a dry extract.
Extracts for which the content of the drug substan-
ce^) is specified are prepared by assaying the drug
substance(s) in a sample portion and adjusting, if
necessary, with suitable diluents to the specified
strength.
(ii) Weigh crude drugs pulverized in suitable sizes
according to the prescription and heat after adding
10 - 20 times water to them. After liquid-solid separa-
tion by centrifuge etc., the filtrate is concentrated or
dried in a suitable method to produce a millet jelly-like
consistency in the case of a viscous extract, and to
make crushable solid masses, granules or powder in the
case of a dry extract.
(3) Extracts have odor and taste derived from the
crude drugs used.
(4) Unless otherwise specified, Extracts meet the
requirements of the Heavy Metals Limit Test <.07>
when the test solution and the control solution are pre-
pared as follows.
Test solution: Ignite 0.3 g of Extracts to ash, warm
with 3 mL of the dilute hydrochloric acid, and filter.
Wash the residue with two 5 mL portions of water.
Neutralize the combined filtrate and washings (indica-
tor: a drop of phenolphthalein TS) by adding ammonia
TS until the color of the solution changes to pale red,
filter, if necessary, and add 2 mL of the dilute acetic
acid and water to make 50 mL.
Control solution: Proceed with 3 mL of dilute
hydrochloric acid in the same manner as directed in the
preparation of the test solution, and add 3.0 mL of
Standard Lead Solution and water to make 50 mL.
(5) Tight containers are used for preservation.
8. Fluidextracts
(1) Fluidextracts are liquid percolates of crude
drugs, usually prepared so that each mL contains solu-
ble constituents from 1 g of the crude drugs.
(2) Fluidextracts are usually prepared by the perco-
lation process. Mix well 1000 g of coarse powder or fine
cutting of the crude drugs with the first solvent to
moisten it, close the container, and allow it to stand for
about 2 hours at room temperature. Transfer the
content to a suitable percolator, stuff it as tightly as
possible, open the lower opening of the percolator, and
slowly pour the second solvent to cover the crude
drugs. Close the lower opening when the solvent begins
to drop, and allow the mixture to stand for 2 to 3 days
at room temperature. Open the lower opening, and
allow the percolate to run out at the rate of 0.5 to 1.0
mL per minute.
Set aside the first 850 mL of the percolate as the first
percolate. Add the second solvent to the percolator,
then drip the percolate, and use it as the second perco-
late.
The time of maceration and the flow rate during
percolation may be varied depending on the kind and
the amount of the crude drugs used. The flow rate is
usually regulated as follows, depending on the amount
of the crude drugs used.
Mass of crude drug
Volume of solution running
per minute
Not more than 1000 g
Not more than 3000 g
Not more than 10,000 g
0.5- 1.0 mL
1.0- 2.0 mL
2.0- 4.0 mL
Concentrate the second percolate, taking care not to
lose the volatile substances of the crude drug, mix with
the first percolate, and use it as (A). To (A) add the sec-
ond solvent to make 1000 mL, and allow the mixture to
stand for 2 days. Decant the supernatant liquid or filter
the liquid to obtain a clear solution.
Fluidextracts for which the content of the drug
substance(s) is specified are obtained by adjusting the
content of the drug substance(s) with a sufficient
amount of the second solvent, as required on the basis
of the result of the assay made with a portion of (A).
Use the specified solvent only in cases where there is
no distinction between the first and the second solvent.
(3) Fluidextracts have odor and taste derived from
the crude drugs used.
(4) Unless otherwise specified, Fluidextracts meet
the requirements of the Heavy Metals Limit Test <?.07>
when the test solution and the control solution are pre-
pared as follows.
Test solution: Ignite 1.0 g of Fluidextracts to ash,
warm with 3 mL of dilute hydrochloric acid, filter, and
wash the residue with two 5 mL portions of water.
Neutralize the combined filtrate and washings by
adding ammonia TS, filter, if necessary, and add 2 mL
of dilute acetic acid and water to make 50 mL.
Control solution: Proceed with 3 mL of dilute
hydrochloric acid as directed in the preparation of the
test solution, and add 3.0 mL of Standard Lead Solu-
tion and water to make 50 mL.
(5) Tight containers are used for preservation.
10
Granules / General Rules for Preparations
JP XV
9. Granules
(1) Granules are prepared in a form of granules
using drug substance(s) or a mixture of drug substan-
ce^) and excipients.
(2) Granules are made, usually, from drug substan-
ce^) or a uniform mixture of drug substance(s) with
diluents, binders, disintegrators or other suitable
excipients. The granules are prepared by a suitable
method so that the finished granules are preferably
equal in size. Extended-release or enteric coated gran-
ules can also be prepared by a suitable method.
(3) When the Particle Size Distribution Test <6.03>
is performed with granules, all the granules pass
through a No. 10 (1700 fim) sieve, not more than 5% of
total granules remain on a No. 12 (1400 nm) sieve, and
not more than 15% of total granules pass through a
No. 42 (355 /urn) sieve.
(4) Unless otherwise specified, Granules comply
with the Dissolution Test <6.io> or the Disintegration
Test <6.09>, provided that this provision does not apply
to granules not more than 5% of which remain on a
No. 30 (500 fim) sieve when shaken with a No. 30 sieve
as directed under Particle Size Distribution Test <6.03>.
(5) Unless otherwise specified, Granules for single-
dose use meet the requirements of the Uniformity of
Dosage Units.
(6) Well-closed or tight containers are used for
preservation.
10. Infusions and Decoctions
(1) Infusions and Decoctions are liquid prepara-
tions usually obtained by macerating crude drugs in
purified water.
(2) Infusions and Decoctions are usually prepared
by the following method. Cut crude drugs as directed
below, and transfer 50 g to an infusion or decoction
apparatus.
Leaves, flowers, and whole plants: Coarse cutting
Woods, stems, barks, roots, and rhizomes: Medium
cutting
Seeds and fruits: Fine cutting
Infusions: Damp an amount of crude drugs with 50
mL of purified water for about 15 minutes, pour 900
mL of hot purified water, and heat for 5 minutes with
several shakings. Filter through cloth after cooling.
Decoctions: Heat, with several stirrings, an amount
of crude drugs with 950 mL of purified water for 30
minutes, and filter through cloth while warm.
Sufficient purified water is further added to the
filtrate through the residue to make 1000 mL of an
infusion or decoction.
Prepare Infusions or Decoctions before use.
(3) Infusions and Decoctions have odor and taste
derived from the crude drugs used.
(4) Tight containers are used for preservation.
11. Injections
(1) Injections are solutions, suspensions or emul-
sions of drugs or other preparations that contain drugs
to be dissolved or suspended before use. They are
sterile preparations to be administered directly into the
skin or the body through the skin, or mucous mem-
brane.
(2) Unless otherwise specified, Injections are pre-
pared by dissolving, suspending or emulsifying drug
substance(s) in a prescribed volume of the solvent, or
by distributing drug substance(s) in hermetic contain-
ers for Injections. Every care should be taken to
prevent contamination. The entire process of preparing
Injections from the preparation of drug solution to the
sterilization should be completed as rapidly as possible
by taking into consideration the composition of Injec-
tion and the storage condition. The concentration of
Injections expressed as % indicates w/v%.
Water for injection prepared by Reverse Osmosis-
Ultrafiltration shall be sterilized by heating before use.
This provision does not apply to Injections and
attached solvent, if they are sterilized by heating in the
process of manufacture.
Drugs to be dissolved or suspended before use and
designated in the title as "for injection" may be ac-
companied by a suitable solvent.
(3) Solvents used in the preparation of Injections
or attached to Injections must be harmless in the
amounts usually administered and must not interfere
with the therapeutic efficacy or with quality testing.
The solvents are classified into the following two
major groups. They should meet the following require-
ments.
(i) Aqueous vehicles: As the solvent of aqueous in-
jections, water for injection is usually used. Unless
otherwise specified, isotonic sodium chloride solution,
Ringer's solution, or other suitable aqueous solutions
may be used instead. Unless otherwise specified, these
aqueous vehicles other than those exclusively for in-
tracutaneous, subcutaneous or intramuscular adminis-
tration meet the requirements of the Bacterial En-
dotoxins Test <3.0i>.
When the Bacterial Endotoxins Test <<.0/>is not ap-
plicable to aqueous vehicles, the Pyrogen Test <3.04>
may be used.
JP XV
General Rules for Preparations / Lemonades
11
(ii) Non-aqueous vehicles: Vegetable oils are
usually used as solvents for nonaqueous injections.
These oils, unless otherwise specified, are clear at 10°C
and have no odor or taste suggesting rancidity. The
acid value is not more than 0.56, iodine value is
between 79 and 137, and the saponification value falls
in the range between 185 and 200. They meet the
requirements of the Mineral Oil Test <.05>.
Several suitable organic solvents other than the
vegetable oils may be used as nonaqueous vehicles.
(4) The usual size of particles observed in suspen-
sions for injection is not larger than 150 ym, and that
of particles in emulsions for injection is not larger than
7 fim. As a rule, suspensions for injection are not to be
injected into the vessels or spinal cord, and emulsions
for injection, not into the spinal cord.
(5) Unless otherwise specified, any coloring agent
must not be added solely for the purpose of coloring
the preparations.
(6) Unless otherwise specified, sodium chloride or
other suitable excipients may be added to aqueous
injections to render them isotonic with blood or other
body fluids. Acids or alkalis may be added to them to
adjust the pH.
(7) Unless otherwise specified, sufficient amounts
of suitable preservatives to prevent the growth of
microorganisms are added to Injections filled in multi-
ple dose containers.
(8) Unless otherwise specified, Injections other
than those used exclusively for intracutaneous, sub-
cutaneous or intramuscular administration meet the
requirements of the Bacterial Endotoxins Test <3.0i>.
When the Bacterial Endotoxins Test <8.0i>is not ap-
plicable to Injections, the Pyrogen Test <3.04>may be
used.
(9) Unless otherwise specified, Injections and sol-
vents attached to Injections meet the requirements of
the Sterility Test <?.06>
(10) Usual containers of Injections are colorless
and meet the requirements of the Glass Containers for
Injections <.oi>. Where specified in individual mono-
graphs, these containers may be replaced by colored
containers meeting the requirements of the Glass Con-
tainers for Injections <7.0/>or by plastic containers for
aqueous injections meeting the requirements of the
Test Methods for Plastic Containers <7.02>.
(11) Unless otherwise specified, rubber stoppers
used for glass containers of 100 mL or more of aque-
ous infusions meet the requirements of the Rubber
Closures for Aqueous Infusions <.03>.
(12) Unless otherwise specified, Injections meet the
requirements of the Foreign Insoluble Matter Test for
Injections <6.06>.
(13) Unless otherwise specified, Injections meet the
requirements of the Insoluble Particulate Matter Test
for Injections <6.07>.
(14) Unless otherwise specified, the actual volume
of an injection contained in a single-dose container
meets the requirements of Test for Extractable Volume
of Parenteral Preparations <6.05>.
(15) Unless otherwise specified, Injections to be
dissolved or suspended before use meet the require-
ments of the Uniformity of Dosage Units <6.02>.
(16) Unless otherwise specified, the written, print-
ed, or graphic matter in the package, the container, or
the wrapper must include the following information:
(i) Names of employed vehicles and added substan-
ce^), unless the vehicle is water for injection, or sodi-
um chloride solution in concentrations not exceeding
0.9 w/v%, or unless the vehicle contains acids or alka-
lies in order to adjust the pH of the injections.
(ii) In the case that dissolving vehicles are attached
to the preparations, the presence of the vehicles and
their names, quantities, compositions or ratios of the
vehicles on the outer containers or outer wrappers.
(iii) Names and quantities of added stabilizers,
preservatives, and diluents. In the case where nitrogen
or carbon dioxide is enclosed in the container to replace
the inside air, the statement of this replacement is not
necessary.
(17) For ampules or other containers of 2 mL or
less, the designations "injection", "for injection" and
"aqueous suspension for injection" may be replaced
by "inj.", "for inj." and "aq. susp. for inj.", respec-
tively.
For ampules or other containers of more than 2 mL
and not exceeding 10 mL, made of glass or similar
materials, the designations "injection", "for injec-
tion" and "aqueous suspension for injection" may be
replaced by "inj.", "for inj." and "aq. susp. for
inj.", respectively, when information is printed di-
rectly on the surface of ampules or containers.
(18) Hermetic containers are used for preservation.
Plastic containers for aqueous injections may be used
when specified in an individual monograph.
12. Lemonades
(1) Lemonades are sweet, sour, and usually clear
liquid preparations intended for oral use.
(2) Unless otherwise specified, Lemonades are
usually prepared by dissolving hydrochloric acid, citric
acid, L-tartaric acid, or lactic acid in simple syrup and
purified water, and filtering if necessary.
Prepare Lemonades before use.
12 Liniments / General Rules for Preparations
(3) Tight containers are used for preservation.
13. Liniments
(1) Liniments are usually liquid or semisolid prepa-
rations intended for external application to the skin by
inunction.
(2) Unless otherwise specified, Liniments are
usually prepared by adding drugs to water, ethanol,
fatty oils, glycerin, soap, emulsifying agents, suspend-
ing agents, other suitable excipients or their mixtures,
and kneading the mixture until homogeneity is at-
tained.
(3) Liniments which have separated out one or
more of their components during storage are re-
homogenized before use unless the substances have
deteriorated.
(4) Tight containers are used for preservation.
JP XV
16. Ointments
(1) Ointments are usually homogeneous, semisolid
preparations for external application, of such con-
sistency that they may be applied to the skin by inunc-
tion.
(2) Unless otherwise specified, Ointments are usu-
ally prepared by kneading and mixing homogeneously
drug substance(s) with fats, fatty oils, lanolin, petrola-
tum, paraffin, waxes, resins, plastics, glycols, higher
alcohols, glycerin, water, emulsifying agents, suspend-
ing agents, or other suitable excipients, or with above
excipients emulsified in a suitable way as bases.
Prepare before use in the case of Ointments which
are apt to deteriorate.
Ointments which are prepared with emulsified bases
may be described as Cream.
(3) Ointments are free from rancid odor.
(4) Tight containers are used for preservation.
14. Liquids and Solutions
(1) Liquids and Solutions are liquid preparations
intended for oral or external use. They are not identical
with any other preparations under General Rules for
Preparations.
(2) Liquids and Solutions are usually prepared
directly with drug substance(s) or by dissolving drug
substance(s) in a solvent.
(3) Tight containers are used for preservation.
15. Lotions
(1) Lotions are external preparations applied to the
skin by inunction, which are usually prepared by
dissolving drug substance(s) in an aqueous vehicle or
emulsifying or dispersing them homogeneously.
(2) Unless otherwise specified, Lotions are usually
prepared by adding drug substance(s) with solvents,
emulsifying agents, suspending agents, etc. to an aque-
ous vehicle and mixing to complete uniformity by a
suitable method.
Prepare before use in the case of Lotions which are
apt to deteriorate.
(3) Lotions which have separated out one or more
of their components during storage are rehomogenized
before use unless the substances have deteriorated.
(4) Tight containers are used for preservation.
17. Ophthalmic Ointments
(1) Ophthalmic Ointments are aseptic ointments
intended for the application to the conjunctiva.
(2) Ophthalmic Ointments are usually prepared by
the following method. Solution of drug substance(s) or
finely powdered drug substance(s) are thoroughly mix-
ed with petrolatum or other suitable materials as a
base, and are distributed into collapsible tubes or other
tight containers. Sufficient care should be taken to
prevent any kinds of contamination, and to proceed as
fast as possible in the manufacturing of products.
(3) The particle size of drug substance(s) in
Ophthalmic Ointments is usually not larger than 75
/urn.
(4) Unless otherwise specified, Ophthalmic Oint-
ments meet the requirements of the Sterility Test
<3.06>, and unless otherwise specified, carry out the test
by the Membrane filtration method.
(5) Unless otherwise specified, Ophthalmic Oint-
ments meet the requirements of the Test of Metal
Particles in Ophthalmic Ointments <6.0i>.
The requirement is met if a total of not more than 50
metal particles, each measuring 50,um or more in any
dimension, is found in the 10 samples, and if not more
than one sample is found to contain more than 8 such
particles. If Ophthalmic Ointments fail the foregoing
test, repeat the test on 20 additional samples of
Ophthalmic Ointments. The requirement is met if a
total of not more than 150 metal particles, each meas-
uring 50 [im or more in any dimension, is found in the
30 samples, and if not more than three samples are
JP XV
General Rules for Preparations / Plasters and Pressure Sensitive
13
found to contain more than 8 such particles each.
(6) Tight containers are used for preservation.
18. Ophthalmic Solutions
(1) Ophthalmic Solutions are aseptic preparations
intended for application to the conjunctiva. They are
solutions or suspensions of the drug substance(s), or
preparations which contain drug substance(s) to be
dissolved or suspended before use.
(2) Unless otherwise specified, Ophthalmic Solu-
tions are prepared either by dissolving or suspending
drug substance(s) in a prescribed volume of a solvent,
or by placing drug substance(s) in tight containers.
Every caution is required to avoid contamination in
preparing Ophthalmic Solutions. The entire process of
preparing Ophthalmic Solutions should be completed
as rapidly as possible. The concentration of
Ophthalmic Solutions expressed as % of a drug sub-
stance indicates w/v%.
Preparations to be dissolved or suspended before use
and designated as "for ophthalmic solutions" may be
accompanied by a suitable solvent.
(3) Solvents used in the preparation of Ophthalmic
Solutions or attached to Ophthalmic Solutions must be
harmless in the amounts usually administered and must
not interfere with therapeutic efficacy, or with testing.
Solvents for Ophthalmic Solutions are classified into
the following two major groups. They should meet the
following requirements.
(i) Aqueous vehicles: The usual vehicle for aqueous
ophthalmic solutions is purified water or suitable
aqueous solutions. Solvents constituted to Ophthalmic
Solutions are sterilized, purified water or suitable steri-
lized aqueous solutions.
(ii) Non-aqueous vehicles: The vehicles for non-
aqueous ophthalmic solutions are usually vegetable
oils. Also, suitable organic solvents may be used as
non-aqueous solvents for some preparations.
(4) The usual particle size observed in suspensions
for Ophthalmic Solutions is not larger than 75 iim.
(5) Unless otherwise specified, no coloring agent
may be added solely for the purpose of coloring the
preparations.
(6) Unless otherwise specified, sodium chloride or
other suitable excipients may be added to aqueous
preparations to render them isotonic with lachrymal
liquid. Acids or alkalies or other suitable excipients,
may be added to aqueous preparations to adjust the
pH.
(7) Unless otherwise specified, Ophthalmic Solu-
tions and solvents attached to Ophthalmic Solutions
meet the requirements of the Sterility Test <&.06>.
(8) Ophthalmic Solutions prepared as aqueous
solution and aqueous vehicles attached to Ophthalmic
Solutions to be prepared before use should be clear and
free from foreign insoluble matter when inspected with
the unaided eye at a position of luminous intensity of
3000 to 5000 luxes under an incandescent electric bulb.
The containers of Ophthalmic Solutions should have a
transparency which does not interfere with the test for
foreign matter.
(9) Unless otherwise specified, Ophthalmic Solu-
tions meet the Insoluble Particulate Matter Test for
Ophthalmic Solutions <6.08>. The limit of the particu-
lates is not more than 1 particle per mL equal to or
greater than 300 ,um.
(10) Tight containers are used for preservation.
19. Pills
(1) Pills are spherical masses.
(2) Pills are usually prepared by mixing drug sub-
stance^) uniformly with diluents, binders, disintegra-
tors or other suitable excipients, and rolling into spher-
ical form by a suitable method.
(3) Unless otherwise specified, Pills comply with
the Dissolution Test <6.io>ov the Disintegration Test
<6.09>.
(4) Well-closed or tight containers are used for
preservation.
20. Plasters and Pressure Sensitive
Adhesive Tapes
(1) Plasters and Pressure Sensitive Adhesive Tapes
are usually used as topical drugs of external use by
spreading or sealing a mixture of drug substance(s),
bases and excipients on a cloth or on/in a plastic film,
and adhering to the skin in order to deliver the drug
substance(s) to the disease sites located to the skin or
nearby skin.
(2) Unless otherwise specified, Plasters and Pres-
sure Sensitive Adhesive Tapes are usually prepared by
mixing bases such as water soluble or insoluble, natural
or artificial high-molecular-mass compound, or their
mixture uniformly with drug substance(s) and knead-
ing or sealing on a cloth or film into a suitable shape.
Unless otherwise specified, Plasters and Pressure
Sensitive Adhesive Tapes prepared from fats, fatty
oils, salts of fatty acids, waxes, resins, plastics, purified
lanolin, rubber, or a mixture of the above substances,
or prepared by mixing the drug substance(s) with the
14
Powders / General Rules for Preparations
JP XV
above bases uniformly and as a solid at the ordinary
temperature, may be described as plasters.
(3) Well-closed containers are used for preserva-
tion.
21. Powders
(1) Powders are preparations in powdered or finely
granulated form.
(2) Powders are usually prepared by uniformly
mixing drug substance(s) with or without diluents, bin-
ders, disintegrators or other suitable excipients by a
suitable method to produce a pulverized or finely
granulated form.
(3) When the Particle Size Distribution Test <6.03>
is performed with Powders, all the powders pass
through a No. 18 (850 fim) sieve and not more than 5%
of total powders remain on a No. 30 (500 ^/m) sieve.
Powders with not more than 10% of total passing
through a No. 200 (75 fim) sieve may be described as
Fine Granules.
(4) Unless otherwise specified, Powders for single-
dose use meet the requirements of the Uniformity of
Dosage Units <6.02>.
(5) Well-closed or tight containers are used for
preservation.
22. Spirits
(1) Spirits are usually alcoholic or hydro-alcoholic
solutions of volatile drug substance(s).
(2) Unless otherwise specified, Spirits are usually
prepared by dissolving drug substance(s) in ethanol or
in a mixture of ethanol and water.
(3) Tight containers are used for preservation,
remoting from fire.
23. Suppositories
(1) Suppositories are solid preparations intended
for insertion into the rectal or vaginal cavity. Supposi-
tories are usually prepared by molding bases into a
suitable shape.
Suppositories melt or soften at body temperature or
dissolve slowly in the secretions.
(2) Unless otherwise specified, Suppositories are
usually prepared by mixing drug substance(s) with fat-
type bases, watermiscible bases or other suitable
materials, and, if necessary, with emulsifying agents,
suspending agents, etc. into a homogeneous mass, and
molding it into a suitable shape or coating it with a suit-
able coating agent, or prepared as a liquid form -fill-
seal.
(3) Rectal suppositories are usually conical or spin-
dleshaped, and Vaginal suppositories are globular or
oval.
(4) Unless otherwise specified, Suppositories meet
the requirements of the Uniformity of Dosage Units
<6.02>
(5) Well-closed or tight containers are used for
preservation.
24. Suspensions and Emulsions
(1) Suspensions and Emulsions are usually liquid
preparations of finely divided drug substance(s) sus-
pended or emulsified uniformly in liquid vehicles,
respectively.
(2) Suspensions and Emulsions are usually pre-
pared by the following method.
Suspensions: Suspensions are prepared by adding
suspending agents or other suitable excipients and
purified water or oil to drug substance(s), and suspend-
ing to complete uniformity by a suitable method.
Emulsions: Emulsions are prepared by adding
emulsifying agents and purified water to drug substan-
ce^), and emulsifying to complete uniformity by a suit-
able method.
If necessary, preservatives, stabilizers, etc., may be
added.
Prepare before use in the case of Suspensions or
Emulsions which are apt to deteriorate.
(3) Mix uniformly before use, if necessary.
(4) Tight containers are used for preservation.
25. Syrups
(1) Syrups are oral liquid preparations. Syrups are
solutions of sucrose, or viscous liquids or suspensions
of drug substance(s) containing sucrose, other sugars
or sweetening agents.
Syrups include the preparations which are dissolved
or suspended before use depending on the properties of
the drug substance(s).
(2) Unless otherwise specified, Syrups are usually
prepared by dissolving, mixing, suspending or emul-
sifying drug substance(s) in solutions of sucrose, other
sugars or sweetening agents, or in simple syrup. If
necessary, the mixtures are boiled and filtered while
hot.
(3) Unless otherwise specified, Syrups which are
JP XV
General Rules for Preparations / Transdermal Systems
15
dissolved or suspended before use and are for single-
dose use (divided dosage forms) meet the requirements
of the Uniformity of Dosage Units <6.02>.
(4) Tight containers are used for preservation.
26. Tablets
(1) Tablets are prepared by compressing drug sub-
stance^) directly, or by forming or molding drug sub-
stance^) dampened with a solvent into a desired shape
and size. Sugar- and film-coated tablets can be pre-
pared by coating core tablets using suitable coating
agents containing sugars, sugar alcohols and related
substances and by coating with thin films using suitable
film coating agents, respectively. Enteric coated and
extended release tablets can be prepared by suitable
methods.
(2) Tablets are usually prepared by the following
procedures:
(i) Drug substance(s) are first rendered granular in
a suitable method with or without uniform admixture
with a diluent, binder, disintegrator, and other suitable
excipients. The resultant granules are provided with
additives such as a lubricant, and compressed into a
desired shape and size.
(ii) Tablets may also be prepared either by direct
compression of drug substance(s) with or without a
diluent, binder, disintegrator, and other suitable
excipients, or by compression after drug substance(s)
with or without suitable excipients have been added to
previously prepared inactive granules.
(iii) Tablets may also be prepared by drying the
admixture by a suitable method after forming or
molding drug substance(s), uniformly mixed with a
diluent, binder and other suitable excipients and
dampened with a solvent, into a desired shape and size.
(iv) Multilayer tablets can be prepared by com-
pressing different layers of particles or granules in
composition. Press-coated tablets can be prepared by
covering inner core tablets with different layers in
composition by a suitable method.
(3) Unless otherwise specified, Tablets meet the
requirements of the Dissolution Test <6.w> or the
Disintegration Test <6.09>.
(4) Unless otherwise specified, Tablets meet the re-
quirements of the Uniformity of Dosage Units <6.02>.
The requirements for coated tablets are provided in
each monograph.
(5) Well-closed or tight containers are used for
preservation.
27. Tinctures
(1) Tinctures are liquid preparations, and usually
prepared by extracting crude drug substance(s) with
ethanol or with a mixture of ethanol and purified
water.
(2) Unless otherwise specified, Tinctures are
usually prepared from coarse powder or fine cuttings
of crude drug substance(s) either by maceration or by
percolation as described below.
Maceration: Place crude drugs in a suitable contain-
er, and add about three-fourths of the total volume of
a solvent to be used. Stopper, and allow the container
to stand at ordinary temperature with occasional stir-
ring for about 5 days or until the soluble constituents
have satisfactorily dissolved. Filter the liquid through
cloth. Wash the residue with several portions of the
solvent, and press. Combine the filtrate and washings,
and add sufficient solvent to make up the volume.
Allow the mixture to stand for about 2 days, and ob-
tain a clear liquid by decantation or filtration.
Percolation: Pour the solvent in small portions on
crude drugs placed in a container, and mix well to
moisten the crude drugs. Stopper the container, and
allow it to stand for about 2 hours at room tempera-
ture. Pack the contents as tightly as possible in a suita-
ble percolator, open the lower opening, and slowly
pour sufficient solvent to cover the crude drugs. When
the percolate begins to drip, close the opening, and
allow the mixture to stand for 2 to 3 days at room
temperature. Open the opening, and allow the perco-
late to drip at a rate of 1 to 3 mL per minute. Add an
appropriate quantity of the solvent, and continue to
percolate until the desired volume has passed. Mix
thoroughly, allow standing for 2 days, and obtain a
clear liquid by decantation or filtration. The time of
standing and the flow rate may be varied depending on
the kind and amount of crude drugs to be percolated.
Tinctures prepared by either of the above methods
for which the content of the drug substance is specified
are prepared by assaying the drug substance using a
portion of the sample and adjusting, if necessary, with
the percolate or with the solvent to the specified
content.
(3) Tight containers are used for preservation,
remoting from fire.
28. Transdermal Systems
(1) Transdermal Systems are preparations applied
to the skin that are designed to deliver drug substan-
ce^) through the skin to the systemic blood circulation.
16 Troches / General Rules for Preparations JP XV
Transdermal Systems include semisolid mixtures of
drug substance(s) and excipients which are used by
spreading a suitable amount of the mixture on the
backing layer.
(2) Unless otherwise specified, Transdermal Sys-
tems are usually prepared by spreading the mixtures of
emulsified or suspended drug substance(s) and soluble
or insoluble high molecular weight of natural or syn-
thetic bases or their mixtures on the liner or backing
sheet. If necessary, adhesives agents, solvents or skin
permeation enhancers etc., may be added. The trans-
dermal systems are also prepared by filling the mixture
of drug substance(s) and bases or excipients in a reser-
voir made of a backing layer and a membrane which
controls the release of drug substance(s).
(3) Transdermal Systems meet the requirements of
release tests specified.
(4) Well-closed or tight containers are used for
preservation.
29. Troches
(1) Troches are usually preparations of suitable
shape to dissolve or disintegrate slowly in the mouth,
and are intended for application to the mouth or the
throat.
(2) Troches are usually prepared by the following
procedures:
(i) Drug substance(s) are first rendered granular by
a suitable method with or without uniform admixing
with a diluent, binder, and other suitable excipients.
The resultant granules are provided with additives such
as a lubricant, and compressed into a desired shape and
size.
(ii) Troches may also be prepared either by direct
compression of drug substance(s) with or without a
diluent, binder or other suitable excipients, or by
compression of drug substance(s) with or without suit-
able excipients after they have been uniformly mixed
with previously prepared inactive granules.
(iii) Troches are also prepared by mixing drug sub-
stance^) with a diluent such as sucrose, binder,
moistening agent, other suitable excipients, etc., to
make a homogeneous paste, spreading the paste,
stamping out or cutting into a suitable shape and
drying.
(3) Unless otherwise specified, Troches meet the re-
quirements of the Uniformity of Dosage Units <6.02>.
(4) Well-closed or tight containers are used for
preservation.
GENERAL TESTS, PROCESSES
AND APPARATUS
General Tests, Processes and Apparatus includes common
methods for tests, useful test methods for quality recognition
of drugs and other articles related to them. Unless otherwise
specified, the procedures for acid-neutralizing capacity deter-
mination of gastrointestinal medicines, alcohol number de-
termination, ammonium determination, arsenic determina-
tion, atomic absorption spectrophotometry, test for bacterial
endotoxins, boiling point determination, distilling range de-
termination, chloride determination, conductivity measure-
ment, congealing point determination, determination of bulk
and tapped densities, digestion test, disintegration test, disso-
lution test, endpoint detection in titrimetry, test of extracta-
ble volume for injection, flame coloration, fluorometry,
foreign insoluble matter test for injections, gas chro-
matography, heavy metals determination, test for glass
containers for injections, infrared spectrophotometry,
insoluble particulate matter test for injections, insoluble
particulate matter test for ophthalmic solutions, iron deter-
mination, liquid chromatography, loss on drying determina-
tion, loss on ignition determination, melting point determina-
tion, test for metal particles in ophthalmic ointments,
methanol determination, microbial assay for antibiotics, test
for microbial limit, test for microbial limit for crude drugs,
mineral oil determination, nitrogen determination, nuclear
magnetic resonance spectroscopy, optical rotation determi-
nation, osmolarity determination, oxygen flask combustion
method, particle size distribution test for preparations, pH
determination, test for plastic containers, powder particle
density determination, powder particle size determination,
test for pyrogen, qualitative test, test for readily carbonizable
substances, refractive index determination, residual solvents
test, residue on ignition determination, test for rubber
closure for aqueous infusions, specific gravity and density
determination, specific surface area determination, test for
sterility, sulfate determination, thermal analysis, thin-layer
chromatography, test for total organic carbon, ultraviolet-
visible spectrophotometry, uniformity of dosage units (test
for content uniformity, mass variation test), viscosity
determination, vitamin A assay, water determination, and
X-ray powder diffraction are performed as directed in the
corresponding articles under the General Tests, Processes
and Apparatus. The tests for melting point of fats, congeal-
ing point of fatty acids, specific gravity, acid value, saponifi-
cation value, ester value, hydroxyl value, unsaponifiable mat-
ter and iodine value of fats and fatty oils are performed as
directed in the corresponding items under Fats and Fatty Oils
Test, and sampling, preparation of sample for analysis,
microscopic examination, purity test, loss on drying, total
ash, acid-insoluble ash, extract content, and essential oil con-
tent of crude drugs are performed as directed in the corre-
sponding items under Crude Drugs Test.
The number of each test method is a category number
given individually. The number in blackets (< >) appeared
in monograph indicates the number corresponding to the
general test method.
1. Chemical Methods
1.01 Alcohol Number
Determination
Alcohol Number Determination represents the number of
milliliters of ethanol at 15°C obtained from 10 mL of tin-
cture or other preparations containing ethanol by the follow-
ing procedures.
Method 1 Distilling method
This is a method to determine the Alcohol Number by
reading the number of milliliters of ethanol distillate at 15°C
obtained from 10 mL of a sample measured at 15°C by the
following procedures.
(1) Apparatus
Use hard glass apparatus as illustrated in Fig. 1.01-1.
Ground glass may be used for the joints.
(2) Reagent
Alkaline phenolphthalein solution: To 1 g of phenolphtha-
lein add 7 mL of sodium hydroxide TS and water to make 100
mL.
(3) Procedure
Transfer 10 mL of the sample preparation, accurately
measured at 15 ± 2°C, to the distilling flask A, add 5 mL of
water and boiling chips. Distil ethanol carefully into the
glass-stoppered, volumetric cylinder D.
By reference to Table 1.01-1, a suitable volume of distillate
(mL) should be collected, according to the content of ethanol
in the sample preparation.
Prevent bumping during distillation by rendering the
sample strongly acidic with phosphoric acid or sulfuric acid,
or by adding a small amount of paraffin, beeswax or silicone
resin before starting the distillation.
When the samples contain the following substances, carry
out pretreatment as follows before distillation.
(i) Glycerin: Add sufficient water to the sample so that
the residue in the distilling flask, after distillation, contains at
least 50% of water.
(ii) Iodine: Decolorize the sample with zinc powder.
(iii) Volatile substances: Preparations containing ap-
preciable proportions of essential oil, chloroform, diethyl
ether or camphor require treatment as follows. Mix 10 mL of
the sample, accurately measured, with 10 mL of saturated
sodium chloride solution in a separator, add 10 mL of
petroleum benzin, and shake. Collect the separated aqueous
layer. The petroleum benzin layer was extracted with two 5
mL portions of saturated sodium chloride solution. Combine
the aqueous layers, and distill. According to the ethanol
content in the sample, collect a volume of distillate 2 to 3 mL
17
18
Alcohol Number Determination / General Tests
JP XV
Table 1.01-1
Ethanol content in
Distillate
the sample (vol%)
to be collected (mL)
more than 80
13
80-70
12
70-60
11
60-50
10
50-40
9
40-30
8
less than 30
7
ltie figures are in mm.
A; Distilling flask (50-mL)
B: Delivery tube
C: Condenser
D: Glass-stoppered volumetric cylinder
(25 mL, graduated in 0,1 mL)
Fig. 1.01-1
more than that shown in the above Table.
(iv) Other substances: Render preparations containing
free ammonia slightly acidic with dilute sulfuric acid. If
volatile acids are present, render the preparation slightly
alkaline with sodium hydroxide TS, and if the preparations
contain soap along with volatile substances, decompose the
soap with an excess of dilute sulfuric acid before the extrac-
tion with petroleum benzin in the treatment described in (iii).
To the distillate add 4 to 6 g of potassium carbonate and 1
to 2 drops of alkaline phenolphthalein solution, and shake
vigorously. If the aqueous layer shows no white turbidity,
agitate the distillate with additional potassium carbonate.
After allowing to stand in water at 15 ± 2°C for 30 minutes,
read the volume of the upper reddish ethanol layer in mL,
and regard it as the Alcohol Number. If there is no clear
boundary surface between these two layers, shake vigorously
after addition of a few drops of water, then observe in the
same manner.
Method 2 Gas chromatography
This is a method to determine the alcohol number by deter-
mining ethanol (C 2 H 5 OH) content (vol%) from a sample
measured at 15°C by the following procedures.
(1) Reagent
Ethanol for alcohol number: Ethanol (99.5) with deter-
mined ethanol (C 2 H 5 OH) content. The relation between
specific gravity &\\ of ethanol and content of ethanol
(C 2 H 5 OH) is 0.797 : 99.46 vol%, 0.796 : 99.66 vol%, and
0.795 : 99.86 vol%.
(2) Preparation of sample solution and standard solution
Sample solution: Measure accurately a volume of sample
at 15 ± 2°C equivalent to about 5 mL of ethanol (C 2 H 5 OH),
and add water to make exactly 50 mL. Measure accurately
25 mL of this solution, add exactly 10 mL of the internal
standard solution, and add water to make 100 mL.
Standard solution: Measure accurately 5 mL of ethanol for
alcohol number at the same temperature as the sample, and
add water to make exactly 50 mL. Measure accurately 25 mL
of this solution, add exactly 10 mL of the internal standard
solution, and add water to make 100 mL.
(3) Procedure
Place 25 mL each of the sample solution and the standard
solution in a 100-mL, narrow-mouthed, cylindrical glass
bottle sealed tightly with a rubber closure and aluminum
band, immerse the bottle up to the neck in water, allowed to
stand at room temperature for more than 1 hour in a room
with little change in temperature, shake gently so as not to
splash the solution on the closure, and allow to stand for 30
minutes. Perform the test with 1 mL each of the gas in the
bottle with a syringe according to the Gas Chromatography
<2.02> under the following conditions, and calculate the
ratios, Q T and Q s , of the peak height of ethanol to that of the
internal standard.
Alcohol number
Qs
5(mL)
a volume (mL) of sample
ethanol (C 2 H 5 OH) content (vol%) of
ethanol for alcohol number
9.406
Internal standard solution — A solution of acetonitrile (3 in
50).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass tube about 3 mm in inside diameter and
about 1.5 m in length, packed with 150- to 180-^m porous
ethylvinylbenzene-divinylbenzene copolymer (mean pore
size: 0.0075 /um, 500 - 600 m 2 /g) for gas chromatography.
Column temperature: A constant temperature between
105°C and 115°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethanol is 5 to 10 minutes.
JPXV
General Tests / Ammonium Limit Test
19
Selection of column: Proceed with 1 mL of the gas ob-
tained from the standard solution in the bottle under the
above operating conditions, and calculate the resolution. Use
a column giving elution of ethanol and the internal standard
in this order with the resolution between these peaks being
not less than 2.0.
1.02 Ammonium Limit Test
Ammonium Limit Test is a limit test for ammonium con-
tained in drugs.
In each monograph, the permissible limit for ammonium
(as NH 4 + ) is described in terms of percentage (%) in paren-
theses.
Apparatus
Use a distilling apparatus for ammonium limit test as illus-
trated in Fig. 1.02-1. For the distillation under reduced pres-
sure, use the apparatus shown in Fig. 1.02-2. Either appara-
tus are composed of hard glass, and ground-glass joints may
be used. All rubber parts used in the apparatus should be
boiled for 10 to 30 minutes in sodium hydroxide TS and for
30 to 60 minutes in water, and finally washed thoroughly with
water before use.
Procedure
(1) Preparation of test solution and control
solution — Unless otherwise specified, test solutions and con-
trol solution are prepared as directed in the following.
Place an amount of the sample, directed in the mono-
graph, in the distilling flask A. Add 140 mL of water and 2 g
of magnesium oxide, and connect the distillation apparatus.
To the receiver (measuring cylinder) F add 20 mL of boric
acid solution (1 in 200) as an absorbing solution, and im-
merse the lower end of the condenser. Adjust the heating to
[riboiil SlHml.)
A : Distilling flask
B : Spray trap
C : Small hole
D : Condenser
E : Trap
F : Measuring cylinder
G : Stop cock
H and J : Rubber stoppers
K : Rubber tubing
The figures are in mm.
Fig. 1.02-1 Distilling apparatus for ammonium limit test
The figures are in mm
L: Vacuum distillation flask
(200-mL)
M: Receiver (a 200-mL flask)
N: Water bath
O: Thermometer
P: Funnel
Q: Cooling water
R: Glass cock
S: Rubber tube with screw cock
T: Glass tube [or anli-bumpin#
Fig. 1.02-2 Vacuum distilling apparatus for ammonium
limit test
give a rate of 5 to 7 mL per minute of distillate, and distill un-
til the distillate measures 60 mL. Remove the receiver from
the lower end of the condenser, rinsing the end part with a
small quantity of water, add sufficient water to make 100 mL
and designate it as the test solution.
For the distillation under reduced pressure, take the
amount of sample specified in the monograph to the vacuum
distillation flask L, add 70 mL of water and 1 g of magnesium
oxide, and connect to the apparatus (Fig. 1.02-2). To the
receiver M add 20 mL of a solution of boric acid (1 in 200) as
absorbing liquid, put the end of the branch tube of the distil-
lation flask L in the absorbing liquid, and keep at 60°C using
a water bath or alternative equipment. Adjust the reduced
pressure to get the distillate at a rate of 1 to 2 mL per minute,
and continue the distillation until to get 30 mL of the distil-
late. Cool the receiver M with running water during the distil-
lation. Get off the end of the branch tube from surface of the
absorbing liquid, rinse in the end with a small amount of
water, then add water to the liquid to make 100 mL, and per-
form the test using this solution as the test solution.
Place a volume of Standard Ammonium Solution, directed
in the monograph, in the distilling flask A or the vacuum dis-
tillation flask L, proceed as for the preparation of the test so-
lution, and designate it as the control solution.
(2) Test of the test solution and the control
solution — Unless otherwise specified, proceed as directed in
the following.
Place 30 mL each of the test solution and the control solu-
tion in Nessler tubes, add 6.0 mL of phenol-sodium pentac-
yanonitrosylferrate (III) TS to each solution, and mix. Then
add 4 mL of sodium hypochlorite-sodium hydroxide TS and
water to make 50 mL, mix, and allow to stand for 60
20
Chloride Limit Test / General Tests
JP XV
minutes. Compare the color of both solutions against a white
background by viewing downward or transversely: the color
developed in the test solution is not more intense than that of
the control solution.
1.03 Chloride Limit Test
Chloride Limit Test is a limit test for chloride contained in
drugs.
In each monograph, the permissible limit for chloride (as
CI) is described in terms of percentage (%) in parentheses.
Procedure
Unless otherwise specified, transfer the quantity of the
sample, directed in the monograph, to a Nessler tube, and
dissolve it in a proper volume of water to make 40 mL. Add 6
mL of dilute nitric acid and water to make 50 mL, and use
this solution as the test solution. Transfer the volume of 0.01
mol/L hydrochloric acid VS, directed in the monograph, to
another Nessler tube, add 6 mL of dilute nitric acid and water
to make 50 mL, and use this solution as the control solution.
When the test solution is not clear, filter both solutions by
using the same procedure.
Add 1 mL of silver nitrate TS to the test solution and to the
control solution, mix well, and allow to stand for 5 minutes
protecting from direct sunlight. Compare the opalescence
developed in both solutions against a black background by
viewing downward or transversely.
The opalescence developed in the test solution is not more
than that of the control solution.
1.04 Flame Coloration Test
Flame Coloration Test is a method to detect an element, by
means of the property that the element changes the colorless
flame of a Bunsen burner to its characteristic color.
(1) Salt of metal — The platinum wire used for this test is
about 0.8 mm in diameter, and the end part of it is straight.
In the case of a solid sample, make the sample into a gruel by
adding a small quantity of hydrochloric acid, apply a little of
the gruel to the 5-mm end of the platinum wire, and test by
putting the end part in a colorless flame, keeping the plati-
num wire horizontal. In the case of a liquid sample, immerse
the end of the platinum wire into the sample to about 5 mm in
length, remove from the sample gently, and perform the test
in the same manner as for the solid sample.
(2) Halide — Cut a copper net, 0.25 mm in opening and
0.174 mm in wire diameter, into a strip 1.5 cm in width and 5
cm in length, and wind in round one end of a copper wire.
Heat the copper net strongly in the colorless flame of Bunsen
burner until the flame no longer shows a green or blue color,
and then cool it. Repeat this procedure several times, and
coat the net completely with cupric oxide. After cooling, un-
less otherwise specified, apply about 1 mg of the sample to
the copper net, ignite, and burn it. Repeat this procedure
three times, and then test by putting the copper net in the
colorless flame.
The description, "Flame coloration persists", in a mono-
graph, indicates that the reaction persists for 4 seconds.
1.05 Mineral Oil Test
Mineral Oil Test is a method to test mineral oil in nonaque-
ous solvents for injections and for eye drops.
Procedure
Pour 10 mL of the sample into a 100-mL flask, and add 15
mL of sodium hydroxide solution (1 in 6) and 30 mL of
ethanol (95). Put a short-stemmed, small funnel on the neck
of the flask, and heat on a water bath to make clear, with fre-
quent shaking. Then transfer the solution to a shallow por-
celain dish, evaporate the ethanol on a water bath, add 100
mL of water to the residue, and heat on a water bath: no tur-
bidity is produced in the solution.
1.06 Oxygen Flask Combustion
Method
Oxygen Flask Combustion Method is a method for the
identification or the determination of halogens or sulfur
produced by combusting organic compounds, which contain
chlorine, bromine, iodine, fluorine or sulfur, in a flask filled
with oxygen.
Apparatus
Use the apparatus shown in Fig. 1.06-1.
Preparation of test solution and blank solution
Unless otherwise specified, prepare them by the following
method.
(1) Preparation of sample
(i) For solid samples: Place the quantity of the sample
specified in the monograph on the center of the filter illustrat-
ed in the figure, weigh accurately, wrap the sample carefully
along the dotted line without scattering, and place the parcel
in a platinum basket or cylinder B, leaving its fuse-strip on
the outside.
(ii) For liquid samples: Roll a suitable amount of absor-
bent cotton with filter paper, 50 mm in length and 5 mm in
width, so that the end part of the paper is left to a length of
about 20 mm as a fuse-strip, and place the parcel in a plati-
num basket or cylinder B. Place the sample in a suitable glass
tube, weigh accurately, and moisten the cotton with the
quantity of the sample specified in the monograph, bringing
the edge of the sample in contact with the cotton.
(2) Method of combustion
Place the absorbing liquid specified in the monograph in
flask A, fill it with oxygen, moisten the ground part of the
stopper C with water, then ignite the fuse-strip, immediatey
transfer it to the flask, and keep the flask airtight until the
combustion is completed. Shake the flask occasionally until
the white smoke in A vanishes completely, allow to stand for
15 to 30 minutes, and designate the resulting solution as the
test solution. Prepare the blank solution in the same manner,
without sample.
Procedure of determination
Unless otherwise specified in the monograph, perform the
test as follows.
(1) Chlorine and bromine
Apply a small amount of water to the upper part of A, pull
JPXV
General Tests / Heavy Metals Limit Test
21
Filter paper
fuse strip -
Tlie figures are in mm.
-30-
— line to be folded
A: Colorless, thick-walled (about 2mm) , SOO-mL hard glass
flask, the upper part of which is made like a saucer. A flask
made of quartz should be used for the determination of
fluorine.
B: Platinum basket or cylinder made of platinum woven gauge.
(It is hung at the end of the stopper C with platinum wire) .
C: Ground stopper made of hard glass, A stopper made of
quartz should be used for the determination of fluorine.
Fig. 1.06-1
out C carefully, and transfer the test solution to a beaker.
Wash C, B and the inner side of A with 15 mL of 2-propanol,
and combine the washings with the test solution. To this solu-
tion add 1 drop of bromophenol blue TS, add dilute nitric
acid dropwise until a yellow color develops, then add 25 mL
of 2-propanol, and titrate <2.50> with 0.005 mol/L silver ni-
trate VS according to the potentiometric titration under the
Electrometric titration. Perform the test with the blank solu-
tion in the same manner, and make any necessary correction.
Each mL of 0.005 mol/L silver nitrate VS
= 0.1773 mg of CI
Each mL of 0.005 mol/L silver nitrate VS
= 0.3995 mg of Br
(2) Iodine
Apply a small amount of water to the upper part of A, pull
out C carefully, add 2 drops of hydrazine hydrate to the test
solution, put C on A, and decolorize the solution by vigorous
shaking. Transfer the content of A to a beaker, wash C, B
and the inner side of A with 25 mL of 2-propanol, and trans-
fer the washings to the above beaker. To this solution add 1
drop of bromophenol blue TS, then add dilute nitric acid
dropwise until a yellow color develops, and titrate <2.50> with
0.005 mol/L silver nitrate VS according to the Potentiomet-
ric tiration under Electrometric Titration. Perform the test
with the blank solution in the same manner, and make any
necessary correction.
Each mL of 0.005 mol/L silver nitrate VS
= 0.6345 mg of I
(3) Fluorine
Apply a small amount of water to the upper part of A, pull
out C carefully, transfer the test solution and the blank solu-
tion to 50 mL volumetric flasks separately, wash C, B and the
inner side of A with water, add the washings and water to
make 50 mL, and use these solutions as the test solution and
the correction solution. Pipet the test solution (KmL) equiva-
lent to about 30 ng of fluorine, KmL of the correction solu-
tion and 5 mL of standard fluorine solution, transfer to
50-mL volumetric flasks separately, add 30 mL of a mixture
of alizarin complexone TS, acetic acid-potassium acetate
buffer solution, pH 4.3 and cerium (III) nitrate TS (1:1:1),
add water to make 50 mL, and allow to stand for 1 hour. Per-
form the test with these solutions as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using a blank pre-
pared with 5 mL of water in the same manner. Determine the
absorbances, A T , A c and A s , of the subsequent solutions of
the test solution, the correction solution and the standard so-
lution at 600 nm.
Amount (mg) of fluorine (F) in the test solution
= amount (mg) of fluorine in 5 mL of
the standard solution X
50
V
Standard Fluorine Solution: Dry sodium fluoride (standard
reagent) in a platinum crucible between 500°C and 550°C for
1 hour, cool it in a desiccator (silica gel), weigh accuraly
about 66.3 mg of it, and dissolve in water to make exactly 500
mL. Pipet 10 mL of this solution, and dilute with sufficient
water to make exactly 100 mL.
(4) Sulfur
Apply a small amount of water to the upper part of A, pull
out C carefully, and wash C, B and the inner side of A with
15 mL of methanol. To this solution add 40 mL of methanol,
then add exactly 25 mL of 0.005 mol/L barium perchlorate
VS, allow to stand for 10 minutes, add 0.15 mL of arsenazo
TH TS with a measuring pipet, and titrate <2.50> with 0.005
mol/L sulfuric acid VS. Perfrom the test with the blank solu-
tion in the same manner.
Each mL of 0.005 mol/L barium perchlorate VS
= 0.1604 mg of S
1.07 Heavy Metals Limit Test
Heavy Metals Limit Test is a limit test of the quantity of
heavy metals contained as impurities in drugs. The heavy
metals are the metallic inclusions that are darkened with sodi-
um sulfide TS in acidic solution, as their quantity is expressed
in terms of the quantity of lead (Pb).
In each monograph, the permissible limit for heavy metals
(as Pb) is described in terms of ppm in parentheses.
Preparation of test solutions and control solutions
Unless otherwise specified, test solutions and control solu-
tions are prepared as directed in the following:
(1) Method 1
Place an amount of the sample, directed in the mono-
graph, in a Nessler tube. Dissolve in water to make 40 mL.
22
Nitrogen Determination (Semimicro-Kjeldahl Method) / General Tests
JP XV
Add 2 mL of dilute acetic acid and water to make 50 mL, and
designate it as the test solution.
The control solution is prepared by placing the volume of
Standard Lead Solution directed in the monograph in a Ness-
ler tube, and adding 2 mL of dilute acetic acid and water to
make 50 mL.
(2) Method 2
Place an amount of the sample, directed in the mono-
graph, in a quartz or porcelain crucible, cover loosely with a
lid, and carbonize by gentle ignition. After cooling, add 2 mL
of nitric acid and 5 drops of sulfuric acid, heat cautiously un-
til white fumes are no longer evolved, and incinerate by igni-
tion between 500°C and 600°C. Cool, add 2 mL of
hydrochloric acid, evaporate to dryness on a water bath,
moisten the residue with 3 drops of hydrochloric acid, add 10
mL of hot water, and warm for 2 minutes. Then add 1 drop
of phenolphthalein TS, add ammonia TS dropwise until the
solution develops a pale red color, add 2 mL of dilute acetic
acid, filter if necessary, and wash with 10 mL of water.
Transfer the filtrate and washings to a Nessler tube, and add
water to make 50 mL. Designate it as the test solution.
The control solution is prepared as follows: Evaporate a
mixture of 2 mL of nitric acid, 5 drops of sulfuric acid and 2
mL of hydrochloric acid on a water bath, further evaporate
to dryness on a sand bath, and moisten the residue with 3
drops of hydrochloric acid. Hereinafter, proceed as directed
in the test solution, then add the volume of Standard Lead
Solution directed in the monograph and water to make 50
mL.
(3) Method 3
Place an amount of the sample, directed in the mono-
graph, in a quartz or porcelain crucible, heat cautiously,
gently at first, and then increase the heat until incineration is
completed. After cooling, add 1 mL of aqua regia, evaporate
to dryness on a water bath, moisten the residue with 3 drops
of hydrochloric acid, add 10 mL of hot water, and warm for
2 minutes. Add 1 drop of phenolphthalein TS, add ammonia
TS dropwise until the solution develops a pale red color, add
2 mL of dilute acetic acid, filter if necessary, wash with 10
mL of water, transfer the filtrate and washings to a Nessler
tube, and add water to make 50 mL. Designate it as the test
solution.
The control solution is prepared as follows: Evaporate 1
mL of aqua regia to dryness on a water bath. Hereinafter,
proceed as directed for the test soluttion, and add the volume
of Standard Lead Solution directed in the monograph and
water to make 50 mL.
(4) Method 4
Place an amount of the sample, directed in the mono-
graph, in a platinum or porcelain crucible, mix with 10 mL of
a solution of magnesium nitrate hexahydrate in ethanol (95)
(1 in 10), fire the ethanol to burn, and carbonize by gradual
heating. Cool, add 1 mL of sulfuric acid, heat carefully, and
incinerate by ignition between 500°C and 600°C. If a car-
bonized substance remains, moisten with a small amount of
sulfuric acid, and incinerate by ignition. Cool, dissolve the
residue in 3 mL of hydrochloric acid, evaporate on a water
bath to dryness, wet the residue with 3 drops of hydrochloric
acid, add 10 mL of water, and dissolve by warming. Add 1
drop of phenolphthalein TS, add ammonia TS dropwise until
a pale red color develops, then add 2 mL of dilute acetic acid,
filter if necessary, wash with 10 mL of water, transfer the
filtrate and the washing to a Nessler tube, add water to make
50 mL, and use this solution as the test solution.
The control solution is prepared as follows: Take 10 mL of
a solution of magnesium nitrate hexahydrate in ethanol (95)
(1 in 10), and fire the ethanol to burn. Cool, add 1 mL of
sulfuric acid, heat carefully, and ignite between 500°C and
600°C. Cool, and add 3 mL of hydrochloric acid. Here-
inafter, proceed as directed in the test solution, then add the
volume of Standard Lead Solution directed in the mono-
graph and water to make 50 mL.
Procedure
Add 1 drop of sodium sulfide TS to each of the test solu-
tion and the control solution, mix thoroughly, and allow to
stand for 5 minutes. Then compare the colors of both solu-
tions by viewing the tubes downward or transversely against a
white background. The test solution has no more color than
the control solution.
1.08 Nitrogen Determination
(Semimicro-Kjeldahl Method)
Nitrogen Determination is a method to determine ammo-
nia in ammonium sulfate obtained by decomposition of or-
ganic substances containing nitrogen with sulfuric acid.
Apparatus
Use the apparatus illustrated in Fig. 1.08-1. It is thorough-
ly constructed of hard glass, and ground glass surfaces may
be used for joints. All rubber parts used in the apparatus
should be boiled for 10 to 30 minutes in sodium hydroxide TS
and for 30 to 60 minutes in water, and finally washed
thoroughly with water before use.
Procedure
Unless otherwise specified, proceed by the following
method. Weigh accurately or pipet a quantity of the sample
corresponding to 2 to 3 mg of nitrogen (N: 14.01), and place
in the Kjeldahl flask A. Add 1 g of a powdered mixture of 10
g of potassium sulfate and 1 g of cupper (II) sulfate pentahy-
drate. Wash down any adhering sample from the neck of the
flask with a small quantity of water. Add 7 mL of sulfuric
acid, allowing it to flow down the inside wall of the flask.
Then, while shaking the flask, add cautiously 1 mL of
hydrogen peroxide (30) drop by drop along the inside wall of
the flask. Heat the flask gradually, then heat so strong that
the vapor of sulfuric acid is condensed at the neck of the
flask, until the solution changes through a blue and clear to a
vivid green and clear, and the inside wall of the flask is free
from a carbonaceous material. If necessary, add a small
quantity of hydrogen peroxide (30) after cooling, and heat
again. After cooling, add cautiously 20 mL of water, cool the
solution, and connect the flask to the distillation apparatus
(Fig. 1.08-1) washed beforehand by passing steam through it.
To the absorption flask K add 15 mL of boric acid solution (1
in 25), 3 drops of bromocresol green-methyl red TS and
sufficient water to immerse the lower end of the condenser
tube J. Add 30 mL of sodium hydroxide solution (2 in 5)
through the funnel F, rinse cautiously the funnel with 10 mL
of water, immediately close the clamp attached to the rubber
tubing G, then begin the distillation with steam, and continue
until the distillate measures 80 to 100 mL. Remove the ab-
sorption flask from the lower end of the condenser tube J,
JPXV
General Tests / Qualitative Tests
23
I"he figures are in mm.
A: Kjeldahl flask
B: Steam generator, containing water, to which 2 to 3 drops of
sulfuric acid and fragments of boiling tips for preventing
bumping have been added
C: Spray trap
D: Water supply funnel
F: Steam tube
F: Funnel for addition of alkali solution to flask A
G: Rubber tubing with a clamp
H: A small hole having a diameter approximately equial to that
of the delivery tube
J: Condenser, the lower end of which is beveled
K: Absorption flask
Fig. 1.08-1
rinsing the end part with a small quantity of water, and titrate
<2.50> the distillate with 0.005 mol/L sulfuric acid VS until
the color of the solution changes from green through pale
grayish blue to pale grayish red-purple. Perform a blank de-
termination in the same manner, and make any necessary
correction.
Each mL of 0.005 mol/L sulfuric acid VS
= 0.1401 mg of N
1.09 Qualitative Tests
Qualitative Tests are applied to the identification of drugs
and are done generally with quantities of 2 to 5 mL of the test
solution.
Acetate
(1) When warmed with diluted sulfric acid (1 in 2),
acetates evolve the odor of acetic acid.
(2) When an acetate is warmed with sulfuric acid and a
small quantity of ethanol (95), the odor of ethyl acetate is
evolved.
(3) Neutral solutions of acetates produce a red-brown
color with iron (III) chloride TS, and a red-brown precipitate
when boiled. The precipitate dissolves and the color of the so-
lution changes to yellow upon addition of hydrochloric acid.
Aluminum salt
(1) Solutions of aluminum salts, when treated with am-
monium chloride TS and ammonia TS, yield a gelatinous,
white precipitate which does not dissolve in an excess of am-
monia TS.
(2) Solutions of aluminum salts, when treated with sodi-
um hydroxide TS, yield a gelatinous, white precipitate which
dissolves in an excess of the reagent.
(3) Solutions of aluminum salts, when treated with sodi-
um sulfide TS, yield a gelatinous, white precipitate which dis-
solves in an excess of the reagent.
(4) Add ammonia TS to solutions of aluminum salts until
a gelatinous, white precipitate is produced. The color of the
precipitate changes to red upon addition of 5 drops of aliza-
rin red S TS.
Ammonium salt
When heated with an excess of sodium hydroxide TS, am-
monium salts evolve the odor of ammonia. This gas changes
moistened red litmus paper to blue.
Antimony salt, primary
(1) When primary antimony salts are dissolved in a slight
excess of hydrochloric acid for the test and then diluted with
water, a white turbidity is produced. The mixture produces
an orange precipitate upon addition of 1 to 2 drops of sodium
sulfide TS. When the precipitate is separated, and sodium sul-
fide TS is added to one portion of the precipitate and sodium
hydroxide TS is added to another portion, it dissolves in
either of these reagents.
(2) Add water to acidic solutions of primary antimony
salts in hydrochloric acid until a small quantity of precipitate
is produced, and then add sodium thiosulfate TS: the
precipitate dissolves. A red precipitate is reproduced when
the solution is heated.
Aromatic amines, primary
Acidic solutions of primary aromatic amines, when cooled
in ice, mixed with 3 drops of sodium nitrite TS under agita-
tion, allowed to stand for 2 minutes, mixed well with 1 mL of
ammonium amidosulfate TS, allowed to stand for 1 minute,
and then mixed with 1 mL of AT.JV-diethyWV'-l-
naphtylethylenediamine oxalate TS, exhibit a red-purple
color.
Arsenate
(1) Neutral solutions of arsenates produce no precipitate
with 1 to 2 drops of sodium sulfide TS, but produce a yellow
precipitate with hydrochloric acid subsequently added. The
separated precipitate dissolves in ammonium carbonate TS.
(2) Neutral solutions of arsenates produce a dark red-
brown precipitate with silver nitrate TS. When dilute nitric
acid is added to one portion of the suspension, and ammonia
TS is add to another portion, the precipitate dissolves in
either of these reagents.
(3) Neutral or ammonia alkaline solutions of arsenates
produce with magnesia TS a white, crystalline precipitate,
which dissolves by addition of dilute hydrochloric acid.
Arsenite
(1) Acidic solutions of arsenites in hydrochloric acid
24
Qualitative Tests / General Tests
JP XV
produce a yellow precipitate with 1 to 2 drops of sodium sul-
fide TS. When hydrochloric acid is added to one portion of
the separated precipitate, it does not dissolve. When ammo-
nium carbonate TS is added to another portion, the
precipitate dissolves.
(2) Slightly alkaline solutions of arsenites produce a yel-
lowish white precipitate with silver nitrate TS. When ammo-
nia TS is added to one portion of the suspension, and dilute
nitric acid is added to another portion, the precipitate dis-
solves in either of these reagents.
(3) Slightly alkaline solutions of arsenites produce a
green precipitate with copper (II) sulfate TS. When the sepa-
rated precipitate is boiled with sodium hydroxide TS, it
changes to red-brown.
Barium salt
(1) When the Flame Coloration Test (1) <1.04> is applied
to barium salts, a persistent yellow-green color develops.
(2) Solutions of barium salts produce with dilute sulfuric
acid a white precipitate, which does not dissolve upon addi-
tion of dilute nitric acid.
(3) Acidic solutions of barium salts in acetic acid produce
a yellow precipitate with potassium chromate TS. The
precipitate dissolves by addition of dilute nitric acid.
Benzoate
(1) Concentrated solutions of benzoates produce a white,
crysralline precipitate with dilute hydrochloric acid. The
separated precipitate, washed with cold water and dried,
melts between 120°C and 124°C <2.60>.
(2) Neutral solutions of benzoates produce a pale yellow-
red precipitate upon dropwise addition of iron (III) chloride
TS. The precipitate changes to white on subsequent addition
of dilute hydrochloric acid.
Bicarbonate
(1) Bicarbonates effervesce upon addition of dilute
hydrochloric acid, generating a gas, which produces a white
precipitate immediately, when passed into calcium hydroxide
TS (common with carbonates).
(2) Solutions of bicarbonates produce no precipitate with
magnesium sulfate TS, but produce a white precipitate when
boiled subsequently.
(3) A cold solution of bicarbonates remains unchanged
or exhibits only a slightly red color upon addition of 1 drop
of phenolphthalein TS (discrimination from carbonates).
Bismuth salt
(1) Bismuth salts, dissolved in a slight excess of
hydrochloric acid, yield a white turbidity upon dilution with
water. A dark brown precipitate is produced with 1 to 2 drops
of sodium sulfide TS subsequently added.
(2) Acidic solutions of bismuth salts in hydrochloric acid
exhibit a yellow color upon addition of thiourea TS.
(3) Solution of bismuth salts in dilute nitric acid or in di-
lute sulfuric acid yield with potassium iodide TS a black
precipitate, which dissolves in an excess of the reagent to give
an orange-colored solution.
Borate
(1) When ignite a mixture of a borate with sulfuric acid
and methanol, it burns with a green flame.
(2) Turmeric paper, when moistened with acidic solutions
of borates in hydrochloric acid and dried by warming, ex-
hibits a red color, which changes to blue with ammonia TS
added dropwise.
Bromate
(1) Acidic solutions of bromates in nitric acid yield with 2
to 3 drops of silver nitrate TS a white, crystalline precipitate,
which dissolves upon heating. When 1 drop of sodium nitrite
TS is added to this solution, a pale yellow precipitate is
produced.
(2) Acidic solutions of bromates in nitric acid exhibit a
yellow to red-brown color upon addition of 5 to 6 drops of
sodium nitrite TS. When 1 mL of chloroform is added to the
mixture and shaken, the chloroform layer exhibits a yellow to
red-brown color.
Bromide
(1) Solutions of bromides yield a pale yellow precipitate
with silver nitrate TS. Upon addition of dilute nitric acid to a
portion of the separated precipitate, it dose not dissolve.
When ammonia solution (28) is added to another portion and
shaken, the separated solution yields a white turbidity upon
acidifying with dilute nitric acid.
(2) Solutions of bromides exhibit a yellow-brown color
with chlorine TS. The mixture is separated into 2 portions.
When one portion is shaken with chloroform, the chloroform
layer exhibits a yellow-brown to red-brown color. When
phenol is added to the other portion, a white precipitate is
produced.
Calcium salt
(1) When the Flame Coloration Test (1) <1.04> is applied
to calcium salts, a yellow-red color develops.
(2) Solutions of calcium salts yield a white precipitate
with ammonium carbonate TS.
(3) Solutions of calcium salts yield a white precipitate
with ammonium oxalate TS. The separated precipitate does
not dissolve in dilute acetic acid, but dissolves in dilute
hydrochloric acid.
(4) Neutral solutions of calcium salts produce no
precipitate, when mixed with 10 drops of potassium chro-
mate TS and heated (discrimination from strontium salts).
Carbonate
(1) Carbonates effervesce upon addition of dilute
hydrochloric acid, generating a gas, which produces a white
precipitate immediately, when passed into calcium hydroxide
TS (common with bicarbonates).
(2) Solutions of carbonates yield with magnesium sulfate
TS a white precipitate, which dissolves by addition of dilute
acetic acid.
(3) Cold solutions of carbonates exhibit a red color with 1
drop of phenolphthalein TS (discrimination from bicar-
bonates).
Ceric salt
(1) When a cerous salt is mixed with 2.5 times its mass of
lead (IV) oxide, nitric acid is added and the solution is boiled,
it exhibits a yellow color.
(2) Solutions of cerous salts yield a yellow to red-brown
precipitate upon addition of hydrogen peroxide TS and
ammonia TS.
Chlorate
(1) Solutions of chlorates yield no precipitate with silver
nitrate TS. When 2 to 3 drops of sodium nitrite TS and dilute
nitric acid are added to the mixture, a white precipitate is
produced gradually, which dissolves by addition of ammonia
JPXV
General Tests / Qualitative Tests
25
TS.
(2) When indigocarmine TS is added dropwise to neutral
solutions of chlorates until a pale blue color appears, and the
mixture is acidified with dilute sulfuric acid, the blue color
vanishes promptly upon subsequent dropwise addition of so-
dium hydrogensulfite TS.
Chloride
(1) Solution of chlorides evolve an odor of chlorine,
when mixed with sulfuric acid and potassium permanganate,
and heated. The gas evolved turns moistened potassium
iodide starch paper blue.
(2) Solutions of chlorides yield a white precipitate with
silver nitrate TS. When dilute nitric acid is added to a portion
of the separated precipitate, it does not dissolve. When an ex-
cess of ammonia TS is added to another portion, the
precipitate dissolves.
Chromate
(1) Solutions of chromates exhibit a yellow color.
(2) Solutions of chromates produce a yellow precipitate
with lead (II) acetate TS. When acetic acid (31) is added to a
portion of the suspension, the precipitate does not dissolve.
When dilute nitric acid is added to another portion, the
precipitate dissolves.
(3) When acidic solutions of chromates in sulfuric acid
are mixed with an equal volume of ethyl acetate and 1 to 2
drops of hydrogen peroxide TS, shaken immediately and al-
lowed to stand, the ethyl acetate layer exhibits a blue color.
Citrate
(1) When 20 mL of a mixture of pyridine and acetic anhy-
dride (3:1) is added to 1 or 2 drops of a solution of citrate,
and the solution is allowed to stand for 2 to 3 minutes, a red-
brown color develops.
(2) Neutral solutions of citrates, when mixed with an
equal volume of dilute sulfuric acid and two-thirds volume of
potassium permanganate TS, heated until the color of per-
manganate is discharged, and then treated dropwise with bro-
mine TS to one-tenth of total volume, yield a white
precipitate.
(3) Neutral solutions of citrates, when boiled with an ex-
cess of calcium chloride TS, yield a white crystalline
precipitate. When sodium hydroxide TS is added to a portion
of the separated precipitate, it does not dissolve. When dilute
hydrochloric acid is added to another portion, the precipitate
dissolves.
Cupric salt
(1) When a well polished iron plate is immersed in acidic
solutions of cupric salts in hydrochloric acid, a red metallic
film appears on its surface.
(2) Solutions of cupric salts produce a pale blue
precipitate with a small quantity of ammonia TS. The
precipitate dissolves in an excess of the reagent, yielding a
deep blue-colored solution.
(3) Solutions of cupric salts yield a red-brown precipitate
with potassium hexacyanoferrate (II) TS. When dilute nitric
acid is added to a portion of the suspension, the precipitate
does not dissolve. When ammonia TS is added to another
portion, the precipitate dissolves, yielding a deep blue-
colored solution.
(4) Solutions of cupric salts produce a black precipitate
with sodium sulfide TS. When dilute hydrochloric acid, dilute
sulfuric acid or sodium hydroxide TS is added to a portion of
the separated precipitate, it does not dissolve. When hot di-
lute nitric acid is added to another portion, the precipitate
dissolves.
Cyanide
(1) Solutions of cyanides yield a white precipitate with an
excess of silver nitrate TS. When dilute nitric acid is added to
a portion of the separated precipitate, it does not dissolve.
When ammonia TS is added to another portion, the
precipitate dissolves.
(2) Solutions of cyanides yield a blue precipitate, when
mixed by shaking with 2 to 3 drops of iron (II) sulfate TS, 2
to 3 drops of dilute iron (III) chloride TS and 1 mL of sodium
hydroxide TS, and then acidified with dilute sulfuric acid.
Dichromate
(1) Solutions of dichromates exhibit a yellow-red color.
(2) Solutions of dichromates produce a yellow precipitate
with lead (II) acetate TS. When acetic acid (31) is added to
one portion of the suspension, the precipitate dose not dis-
solve. When dilute nitric acid is added to another portion, the
precipitate dissolves.
(3) When acidic solutions of dichromates in sulfuric acid
are mixed with an equal volume of ethyl acetate and with 1 to
2 drops of hydrogen peroxide TS, shaken immediately and al-
lowed to stand, the ethyl acetate layer exhibits a blue color.
Ferric salt
(1) Slightly acidic solutions of ferric salts yield with
potassium hexacyanoferrate (II) TS a blue precipitate, which
does not dissolve in dilute hydrochloric acid subsequently
added.
(2) Solutions of ferric salts yield with sodium hydroxide
TS a gelatinous, red-brown precipitate, which changes to
black upon addition of sodium sulfide TS. The separated
precipitate dissolves in dilute hydrochloric acid, yielding a
white turbidity.
(3) Slightly acidic solutions of ferric salts exhibit a purple
color with 5-sulfosalicylic acid TS.
Ferricyanide
(1) Solutions of ferricyanides exhibit a yellow color.
(2) Solutions of ferricyanides yield with iron (II) sulfate
TS a blue precipitate, which does not dissolve in dilute
hydrochloric acid subsequently added.
Ferrocyanide
(1) Solutions of ferrocyanides yield with iron (III) chlo-
ride TS a blue precipitate, which does not dissolve in dilute
hydrochloric acid subsequently added.
(2) Solutions of ferrocyanides yield with copper (II) sul-
fate TS a red-brown precipitate, which does not dissolve in
dilute hydrochloric acid subsequently added.
Ferrous salt
(1) Slightly acidic solutions of ferrous salts yield with
potassium hexacyanoferrate (III) TS a blue precipitate, which
does not dissolve in dilute hydrochloric acid subsequently
added.
(2) Solutions of ferrous salts yield with sodium hydroxide
TS a greenish gray, gelatinous precipitate, which changes to
black with sodium sulfide TS. The separated precipitate dis-
solves in dilute hydrochloric acid.
(3) Neutral or slightly acidic solutions of ferrous salts ex-
hibit an intense red color upon dropwise addition of a solu-
tion of 1,10-phenanthroline monohydrate in ethanol (95) (1
26
Qualitative Tests / General Tests
JP XV
in 50).
Fluoride
(1) When solutions of fluorides are heated with chromic
acid-sulfuric acid TS, the inside of the test tube is not
moistened uniformly.
(2) Neutral or slightly acidic solutions of fluorides exhibit
a blue-purple color after standing with 1.5 mL of a mixture
of alizarin complexone TS, acetic acid-potassium acetate
buffer solution, pH 4.3, and cerium (III) nitrate TS (1:1:1).
Glycerophosphate
(1) Solutions of glycerophosphates remain unaffected by
addition of calcium chloride TS, but yield a precipitate when
boiled.
(2) Solutions of glycerophosphates yield no precipitate
with hexaammonium heptamolybdate TS in the cold, but
yield a yellow precipitate when boiled for a long time.
(3) When glycerophosphates are mixed with an equal
mass of powdered potassium hydrogen sulfate and heated
gently over a free flame, the pungent odor of acrolein is
evolved.
Iodide
(1) Solutions of iodides yield a yellow precipitate with sil-
ver nitrate TS. When dilute nitric acid is added to one portion
of the suspension, and ammonia solution (28) to another por-
tion, the precipitates do not dissolve in either of these rea-
gents.
(2) Acidic solutions of iodides exhibit a yellow-brown
color with 1 to 2 drops of sodium nitrite TS and then yield a
black-purple precipitate. The solutions exhibit a deep blue
color with starch TS subsequently added.
Lactate
Acidic solutions of lactates in sulfuric acid, when heated
with potassium permanganate TS, evolve the odor of acetal-
dehyde.
Lead salt
(1) Solutions of lead salts yield a white precipitate with
dilute sulfuric acid. When dilute nitric acid is added to a por-
tion of the separated precipitate, it does not dissolve. When
sodium hydroxide TS is added to another portion and
warmed, or when ammonium acetate TS is added to another
portion, the precipitate dissolves.
(2) Solutions of lead salts yield with sodium hydroxide
TS a white precipitate, which dissolves in an excess of sodium
hydroxide TS, and yields a black precipitate upon subsequent
addition of sodium sulfide TS.
(3) Acidic solutions of lead salts in dilute acetic acid yield
with potassium chromate TS a yellow precipitate, which does
not dissolve in ammonia TS but dissolves in sodium
hydroxide TS subsequently added.
Lithium salt
(1) When the Flame Coloratuion Test (1) <1.04> is applied
to lithium salts, a persistent red color develops.
(2) Solutions of lithium salts yield with disodium
hydrogenphosphate TS a white precipitate, which dissolves
upon subsequent addition of dilute hydrochloric acid.
(3) Solutions of lithium salts yield no precipitate with di-
lute sulfuric acid (discrimination from strontium salts).
Magnesium salt
(1) Solutions of magnesium salts yield upon warming
with ammonium carbonate TS a white precipitate, which dis-
solves in ammonium chloride TS. A white, crystalline
precipitate is reproduced by subsequent addition of disodium
hydrogenphosphate TS.
(2) Solutions of magnesium salts yield with sodium
hydroxide TS a white, gelatinous precipitate. When iodine TS
is added to one portion of the suspension, the precipitate de-
velops a dark-brown color. When excess sodium hydroxide
TS is added to another portion, the precipitate does not dis-
solve.
Manganese salt
(1) Solutions of manganese salts yield a white precipitate
with ammonia TS. When silver nitrate TS is added to a por-
tion of the suspension, the precipitate changes to black.
When another portion is allowed to stand, the upper part of
the precipitate exhibits a brownish color.
(2) Acidic solutions of manganese salts in dilute nitric
acid exhibit a purple-red color with a small quantity of pow-
dered bismuth sodium trioxide.
Mercuric salt
(1) A copper plate is immersed in solutions of mercuric
salts, allowed to stand, taken out, and then washed with
water. The plate becomes bright and silvery white in appear-
ance, when rubbed with paper or cloth (common with mer-
curous salts).
(2) Solutions of mercuric salts yield with a small quantity
of sodium sulfide TS a black precipitate, which dissolves in
an excess of the reagent. The black precipitate is reproduced
by subsequent addition of ammonium chloride TS.
(3) When potassium iodide TS is added dropwise to neu-
tral solutions of mercuric salts, a red precipitate is produced.
The precipitate dissolves in an excess of the reagent.
(4) Acidic solutions of mercuric salts in hydrochloric acid
yield with a small quantity of tin (II) chloride TS a white
precipitate, which changes to grayish black upon addition of
an excess of the reagent.
Mercurous salt
(1) A copper plate is immersed in solutions of mercurous
salts, allowed to stand, taken out, and then washed with
water. The plate becomes bright and silvery white in appear-
ance, when rubbed with paper or cloth (common with mer-
curic salts).
(2) Mercurous salts or their solutions exhibit a black
color with sodium hydroxide TS.
(3) Solutions of mercurous salts yield a white precipitate
with dilute hydrochloric acid. The separated precipitate
changes to black upon addition of ammonia TS.
(4) Solutions of mercurous salts yield with potassium
iodide TS a yellow precipitate, which changes to green, when
allowed to stand, and changes again to black upon subse-
quent addition of an excess of the reagent.
Nitrate
(1) When a solution of nitrates is mixed with an equal
volume of sulfuric acid, the mixture is cooled, and iron (II)
sulfate TS is superimposed, a dark brown ring is produced at
the junction of the two liquids.
(2) Solutions of nitrates exhibit a blue color with
diphenylamine TS.
(3) When potassium permanganate TS is added to acidic
solutions of nitrates in sulfuric acid, the red-purple color of
the reagent does not fade (discrimination from nitrites).
JPXV
General Tests / Qualitative Tests
27
Nitrite
(1) Solutions of nitrites, when acidified with dilute sulfur-
ic acid, evolve a yellow-brown gas with a characteristic odor.
The solutions exhibit a dark brown color upon addition of a
small quantity of iron (II) sulfate crystals.
(2) Solutions of nitrites, when 2 to 3 drops of potassium
iodide TS and dilute sulfuric acid are added dropwise, exhibit
a yellow-brown color, and then yield a black-purple
precipitate. When the mixture is shaken with 2 mL of chlo-
roform, the chloroform layer exhibits a purple color.
(3) Solutions of nitrites, when mixed with thiourea TS
and acidified with dilute sulfuric acid, and iron (III) chloride
TS is added dropwise, exhibit a dark red color. When the
mixture is shaken with 2 mL of diethyl ether, the diethyl ether
layer exhibits a red color.
Oxalate
(1) When potassium permanganate TS is added dropwise
to warm acidic solutions of oxalates in sulfuric acid, the rea-
gent is decolorized.
(2) Solutions of oxalates yield a white precipitate with
calcium chloride TS. The separated precipitate does not dis-
solve in dilute acetic acid but dissolves upon subsequent addi-
tion of dilute hydrochloric acid.
Permanganate
(1) Solutions of permanganates exhibit a red-purple
color.
(2) When an excess of hydrogen peroxide TS is added to
acidic solutions of permanganates in sulfuric acid, the solu-
tions effervesce and decolorize permanganates.
(3) Acidic solutions of permanganates in sulfuric acid are
decolorized, when an excess of oxalic acid TS is added and
heated.
Peroxide
(1) Solutions of peroxides are mixed with an equal
volume of ethyl acetate and 1 to 2 drops of potassium dichro-
mate TS, and then acidified with dilute sulfuric acid. When
the mixture is shaken immediately and allowed to stand, the
ethyl acetate layer exhibits a blue color.
(2) Acidic solutions of peroxides in sulfuric acid decolo-
rize dropwise added potassium permanganate TS, and effer-
vesce to evolve a gas.
Phosphate (Orthophosphate)
(1) Neutral solutions of phosphates yield with silver ni-
trate TS a yellow precipitate, which dissolves upon addition
of dilute nitric acid or ammonia TS.
(2) Neutral solutions or acidic solutions in dilute nitric
acid of phosphates yield a yellow precipitate with hexaammo-
nium heptamolybdate TS on warming. The precipitate dis-
solves upon subsequent addition of sodium hydroxide TS or
ammonia TS.
(3) Neutral or ammonia-alkaline solutions of phosphates
yield with magnesia TS a white, crystalline precipitate, which
dissolves upon subsequent addition of dilute hydrochloric
acid.
Potassium salt
(1) When the Flame Coloration Test (1) <1.04> is applied
to potassium salts, a pale purple color develops. When it
gives a yellow color, a red-purple color can be seen through
cobalt glass.
(2) Neutral solutions of potassium salts yield a white,
crystalline precipitate with sodium hydrogen tartrate TS. The
formation of the precipitate is accelerated by rubbing the in-
side wall of the test tube with a glass rod. The separated
precipitate dissolves upon addition of any of ammonia TS,
sodium hydroxide TS or sodium carbonate TS.
(3) Acidic solutions of potassium salts in acetic acid (31)
yield a yellow precipitate with sodium hexanitrocobaltate
(III) TS.
(4) Potassium salts do not evolve the odor of ammonia,
when an excess of sodium hydroxide TS is added and warmed
(discrimination from ammonium salts).
Salicylate
(1) Salicylates evolve the odor of phenol, when an excess
of soda-lime is added and heated.
(2) Concentrated solutions of salicylates yield a white,
crystalline precipitate with dilute hydrochloric acid. The
separated precipitate, washed well with cold water and dried,
melts <2.60> at about 159°C.
(3) Neutral solutions of salicylates exhibit with 5 to 6
drops of dilute iron (III) chloride TS a red color, which
changes to purple and then fades when dilute hydrochloric
acid is added dropwise.
Silver salt
(1) Solutions of silver salts yield a white precipitate with
dilute hydrochloric acid. When dilute nitric acid is added sub-
sequently to a portion of the suspension, the precipitate does
not dissolve. When an excess of ammonia TS is added to
another portion, the precipitate dissolves.
(2) Solutions of silver salts yield with potassium chro-
mate TS a red precipitate, which dissolves upon addition of
dilute nitric acid.
(3) Solutions of silver salts yield a brownish gray
precipitate with ammonia TS added dropwise. When ammo-
nia TS is added dropwise until the precipitate dissolves, then
1 to 2 drops of formaldehyde solution are added and
warmed, a mirror of metallic silver is deposited on the inside
wall of the container.
Sodium salt
(1) When the Flame Coloration Test (1) <1.04> is applied
to sodium salts, a yellow color develops.
(2) Concentrated, neutral or slightly alkaline solutions of
sodium salts yield a white, crystalline precipitate with potassi-
um hexahydroxoantimonate (V) TS. The formation of the
precipitate is accelerated by rubbing the inside wall of the test
tube with a glass rod.
Stannic salt
(1) When the outside bottom of a test tube containing
water is moistened with acidic solutions of stannic salts in
hydrochloric acid and is placed in a nonluminous flame of a
Bunsen burner, a blue flame mantle is seen around the bot-
tom of the test tube (common with stannous salts).
(2) When granular zinc is immersed in acidic solutions of
stannic salts in hydrochloric acid, a spongy, gray substance is
deposited on the surface of the granules (common with stan-
nous salts).
(3) Add iron powder to acidic solutions of stannic salts in
hydrochloric acid, allow to stand, and then filter. When io-
dine-starch TS is added dropwise to the filtrate, the color of
the test solution disappears.
(4) Acidic solutions of stannic salts in hydrochloric acid,
to which ammonia TS is added dropwise until a small quanti-
28
Iron Limit Test / General Tests
JP XV
ty of precipitate is produced, yield a pale yellow precipitate
with 2 to 3 drops of sodium sulfide TS. The separated
precipitate dissolves upon addition of sodium sulfide TS and
pale yellow precipitate is reproduced by subsequent addition
of hydrochloric acid.
Stannous salt
(1) When the outside bottom of a test tube containing
water is moistened with acidic solutions of stannous salts in
hydrochloric acid and is placed in a nonluminous flame of a
Bunsen burner, a blue flame mantle is seen around the bot-
tom of the test tube (common with stannic salts).
(2) When granular zinc is immersed in acidic solutions of
stannous salts in hydrochloric acid, a spongy, gray substance
is deposited on the surface of the granules (common with
stannic salts).
(3) When iodine-starch TS is added dropwise to solutions
of stannous salts, the color of the test solution disappears.
(4) Acidic solutions of stannous salts in hydrochloric
acid, to which ammonia TS is added dropwise until a small
quantity of precipitate is produced, yield a dark brown
precipitate with 2 to 3 drops of sodium sulfide TS. When so-
dium sulfide TS is added to a portion of the separated
precipitate, it does not dissolve. When ammonium polysul-
fide TS is added to another portion, the precipitate dissolves.
Sulfate
(1) Solutions of sulfates yield with barium chloride TS a
white precipitate, which does not dissolve upon addition of
dilute nitric acid.
(2) Neutral solutions of sulfates yield with lead (II)
acetate TS a white precipitate, which dissolves upon subse-
quent addition of ammonium acetate TS.
(3) When an equal volume of dilute hydrochloric acid is
added, solutions of sulfates yield no white turbidity (discrimi-
nation from thiosulfates), and do not evolve the odor of sul-
fur dioxide (discrimination from sulfites).
Sulfide
Most kinds of sulfides evolve the odor of hydrogen sulfide
with dilute hydrochloric acid. This gas blackens lead (II)
acetate paper moistened with water.
Sulfite and Bisulfite
(1) When iodine TS is added dropwise to acidic solutions
of sulfites or bisulfites in acetic acid (31), the color of the
reagent fades.
(2) When an equal volume of dilute hydrochloric acid is
added, solutions of sulfites or bisulfites evolve the odor of
sulfur dioxide but yield no turbidity (discrimination from
thiosulfates). The solutions yield immediately with 1 drop of
sodium sulfide TS a white turbidity, which changes gradually
to a pale yellow precipitate.
Tartrate
(1) Neutral tartrate solutions yield a white precipitate
with silver nitrate TS. When nitric acid is added to a portion
of the separated precipitate, it dissolves. When ammonia TS
is added to another portion and warmed, the precipitate dis-
solves and metallic silver is deposited gradually on the inside
wall of the test tube, forming a mirror.
(2) Solutions of tartrates exhibit a red-purple to purple
color, when 2 drops of acetic acid (31), 1 drop of iron (II) sul-
fate TS, 2 to 3 drops of hydrogen peroxide TS and an excess
of sodium hydroxide TS are added.
(3) When a solution, prepared by mixing 2 to 3 drops of a
solution of resorcinol (1 in 50) and 2 to 3 drops of a solution
of potassium bromide (1 in 10) with 5 mL of sulfuric acid, is
added to 2 to 3 drops of solutions of tartrates, and then heat-
ed for 5 to 10 minutes on a water bath, a deep blue color is
produced. The solution exhibits a red to red-orange color
when poured to 3 mL of water after cooling.
Thiocyanate
(1) Solutions of thiocyanates yield a white precipitate
with an excess of silver nitrate TS. When dilute nitric acid is
added to a portion of the suspension, the precipitate does not
dissolve. When ammonia solution (28) is added to another
portion, the precipitate dissolves.
(2) Solutions of thiocyanates produce with iron (III)
chloride TS a red color, which is not decolored by addition of
hydrochloric acid.
Thiosulfate
(1) When iodine TS is added dropwise to acidic solutions
of thiosulfates in acetic acid (31), the color of the reagent
fades.
(2) When an equal volume of dilute hydrochloric acid is
added, solutions of thiosulfates evolve the odor of sulfur di-
oxide, and yield gradually a white turbidity, which changes to
yellow on standing.
(3) Solutions of thiosulfates yield with an excess of silver
nitrate TS a white precipitate, which changes to black on
standing.
Zinc salt
(1) Neutral to alkaline solutions of zinc salts yield a
whitish precipitate with ammonium sulfide TS or sodium sul-
fide TS. The separated precipitate does not dissolve in dilute
acetic acid but dissolves upon subsequent addition of dilute
hydrochloric acid.
(2) Solutions of zinc salts yield a white precipitate with
potassium hexacyanoferrate (II) TS. When dilute hydro-
chloric acid is added to a portion of the suspension, the
precipitate does not dissolve. When sodium hydroxide TS is
added to another portion, the precipitate dissolves.
(3) Neutral to weakly acidic solutions of zinc salts yield a
white precipitate, when 1 or 2 drops of pyridine and 1 mL of
potassium thiocyanate TS are added.
1.10 Iron Limit Test
Iron Limit Test is a limit test for iron contained in drugs.
The limit is expressed in term of iron (Fe).
In each monograph, the permissible limit for iron (as Fe) is
described in terms of ppm in parentheses.
Preparation of test solutions and control solutions
Unless otherwise specified, test solutions and control solu-
tions are prepared as follows:
(1) Method 1
Weigh the amount of sample specified in indivisual mono-
graph, add 30 mL of acetic acid-sodium acetate buffer solu-
tion for iron limit test, pH 4.5, dissolve by warming if neces-
sary, and designate this solution as the test solution.
Prepare the control solution as follows: To the amount of
Standard Iron Solution specified in individual monograph
add 30 mL of acetic acid-sodium acetate buffer solution for
JPXV
General Tests / Arsenic Limit Test
29
iron limit test, pH 4.5.
(2) Method 2
Weigh the amount of sample specified in individual mono-
graph, add 10 mL of dilute hydrochloric acid, and dissolve
by warming if necessary. Dissolve 0.5 g of L-tartaric acid,
and add one drop of phenolphthalein TS. Add ammonia TS
dropwise untill the solution develops a pale red color. Add 20
mL of acetic acid-sodium acetate buffer solution for iron
limit test, pH 4.5, and designate this solution as the test solu-
tion.
Prepare the control solution as follows: To the amount of
Standard Iron Solution specified in individual monograph
add 10 mL of dilute hydrochloric acid, and proceed as direct-
ed for the test solution.
(3) Method 3
Place the amount of sample specified in individual mono-
graph in a crucible, moisten with a small amount of sulfuric
acid, heat cautiously and gently at first, and then incinerate
by ignition. After cooling, add 1 mL of diluted hydrochloric
acid (2 in 3) and 0.5 mL of diluted nitric acid (1 in 3),
evaporate on a water bath to dryness, and to the residue add
0.5 mL of diluted hydrochloric acid (2 in 3) and 10 mL of
water. After dissolving by warming, add 30 mL of acetic
acid-sodium acetate buffer solution for iron limit test, pH
4.5, and designate this solution as the test solution.
Prepare the control solution as follows: Transfer the
amount of Standard Iron Solution specified in indivisual
monograph to a crucible, and add 1 mL of diluted
hydrochloric acid (2 in 3) and 0.5 mL of diluted nitric acid (1
in 3), evaporate on a water bath to dryness, and proceed as
directed for the test solution.
In this procedure, use a quartz or porcelain crucible, which
is immersed in boiling dilute hydrochloric acid for 1 hour and
washed throughly with water and dried.
Procedure
Unless otherwise specified, proceed as follows:
(1) Method A
Transfer the test solution and the control solution to
separate Nessler tubes, to each add 2 mL of a solution of l-
ascorbic acid (1 in 100), mix well, and allow to stand for 30
minutes. Add 1 mL of a solution of a, a'-dipyridyl in ethanol
(95) (1 in 200), add water to make 50 mL, and allow to stand
for 30 minutes. Then compare the colors developed in both
solutions against a white background. The test solution has
no more color than the control solution.
(2) Method B
Dissolve 0.2 g of L-ascorbic acid in the test solution and the
control solution, and allow to stand for 30 minutes. Add 1
mL of a solution of a, a'-dipyridyl in ethanol (95) (1 in 200),
and allow to stand for 30 minutes. Then add 2 mL of a solu-
tion of 2,4,6-trinitrophenol (3 in 1000) and 20 mL of 1,2-
dichloroethane, shake vigorously, collect the 1,2-
dichloroethane layer, and filter through a pledget of absor-
bent cotton in a funnel on which 5 g of anhydrous sodium
sulfate is placed if necessary. Then compare the colors deve-
loped in both solutions against a white background. The test
solution has no more color than the control solution.
1.11 Arsenic Limit Test
Arsenic Limit Test is a limit test for arsenic contained in
drugs. The limit is expressed in terms of arsenic (III) trioxide
(As 2 3 ).
In each monograph, the permissible limit for arsenic (as
As 2 3 ) is described in terms of ppm in parentheses.
Apparatus
Use the apparatus illustrated in Fig. 1.11-1.
Place glass wool F in the exit tube B up to about 30 mm in
height, moisten the glass wool uniformly with a mixture of an
equal volume of lead (II) acetate TS and water, and apply
gentle suction to the lower end to remove the excess of the
mixture. Insert the tube vertically into the center of the rub-
ber stopper H, and attach the tube to the generator bottle A
so that the small perforation E in the lower end of B extends
slightly below. At the upper end of B, attach the rubber stop-
per J to hold the tube C vertically. Make the lower end to the
exit tube of C level with that of the rubber stopper J.
Preparation of the test solution
Unless otherwise specified, proceed as directed in the
following.
(1) Method 1
Weigh the amount of the sample directed in the mono-
graph, add 5 mL of water, dissolve by heating if necessary,
and designate the solution as the test solution.
(2) Method 2
Weigh the amount of the sample directed in the mono-
graph, add 5 mL of water, and add 1 mL of sulfuric acid
except in the cases that the samples are inorganic acids. Add
10 mL of sulfurous acid solution, transfer to a small beaker,
and evaporate the mixture on a water bath until it is free from
sulfurous acid and is reduced to about 2 mL in volume.
Dilute with water to make 5 mL, and designate it as the test
solution.
(3) Method 3
Weigh the amount of the sample directed in the mono-
graph, and place it in a crucible of platinum, quartz or
porcelain. Add 10 mL of a solution of magnesium nitrate
hexahydrate in ethanol (95) (1 in 50), ignite the ethanol, and
heat gradually to incinerate. If carbonized material still
remains by this procedure, moisten with a small quantity of
nitric acid, and ignite again to incinerate. After cooling, add
3 mL of hydrochloric acid, heat on a water bath to dissolve
the residue, and designate it as the test solution.
(4) Method 4
Weigh the amount of the sample directed in the mono-
graph, and place it in a crucible of platinum, quartz or por-
celain. Add 10 mL of a solution of magnesium nitrate
hexahydrate in ethanol (95) (1 in 10), burn the ethanol, heat
gradually, and ignite to incinerate. If carbonized material still
remains by this procedure, moisten with a small quantity of
nitric acid, and ignite again to incinerate in the same manner.
After cooling, add 3 mL of hydrochloric acid, heat on a
water bath to dissolve the residue, and designate it as the test
solution.
(5) Method 5
Weigh the amount of the sample directed in the mono-
graph, add 10 mL of A^TV-dimethylformamide, dissolve by
heating if necessary, and designate the solution as the test
solution.
Test solutions
Absorbing solution for hydrogen arsenide: Dissolve 0.50 g
30 Methanol Test / General Tests
90
5.G — ■
(ID)
,\
B —
F
II
/
A—
^
-45-
D —
G^
Lfl O ITS
■-I Wl QA
i
i.i hi
-90-
Al
!()[))
5.6
ill i.i
5
Markof_
5mL
^
i s
10 1
llic figures are in mm. (ID) (ID)
A: Generator bottle (capacity up to the shoulder: approxi-
mately 70 ml)
B: Exit tube
C: Glass tube (inside diameter: 5.6 mm, the tip of the part
to be inserted in the absorber tube D is drawn out to 1
mm in diameter)
D: Absorber tube (inside diameter: 10 mm)
E: Small perforation
F: Glass wool (about 0.2 g)
G: Mark of 5 mi-
ll and J: Rubber stoppers
E: Mark of 40 mi-
Fig. 1.11-1 Arsenic limit test apparatus
JP XV
of silver A^A^-diethyldithiocarbamate in pyridine to make 100
mL. Preserve this solution in a glass-stoppered bottle protect-
ed from light, in a cold place.
Standard Arsenic Stock Solution: Weigh accurately 0.100
g of finely powdered arsenic (III) trioxide dried at 105°C for 4
hours, and add 5 mL of sodium hydroxide solution (1 in 5) to
dissolve. Add dilute sulfuric acid to neutralize, add further 10
mL of dilute sulfuric acid, and add freshly boiled and cooled
water to make exactly 1000 mL.
Standard Arsenic Solution: Pipet 10 mL of Standard
Arsenic Stock Solution, add 10 mL of dilute sulfuric acid,
and add freshly boiled and cooled water to make exactly 1000
mL. Each mL of the solution contains 1 fig of arsenic (III)
trioxide (As 2 3 ). Prepare Standard Arsenic Solution just
before use and preserve in a glass-stoppered bottle.
Procedure
Unless otherwise specified, proceed using apparatus shown
in Fig. 1.11-1. Carry out the preparation of the standard
color at the same time.
Place the test solution in the generator bottle A and, if
necessary, wash down the solution in the bottle with a small
quantity of water. Add 1 drop of methyl orange TS, and after
neutralizing with ammonia TS, ammonia solution (28) or
dilute hydrochloric acid, add 5 mL of diluted hydrochloric
acid (1 in 2) and 5 mL of potassium iodide TS, and allow to
stand for 2 to 3 minutes. Add 5 mL of acidic tin (II) chloride
TS, and allow to stand for 10 minutes. Then add water to
make 40 mL, add 2 g of zinc for arsenic analysis, and im-
mediately connect the rubber stopper H fitted with B and C
with the generator bottle A. Transfer 5 mL of the absorbing
solution for hydrogen arsenide to the absorber tube D, insert
the tip of C to the bottom of the absorber tube D, then
immerse the generator bottle A up to the shoulder in water
maintained at 25 °C, and allow to stand for 1 hour. Discon-
nect the absorber tube, add pyridine to make 5 mL, if neces-
sary, and observe the color of the absorbing solution: the
color produced is not more intense than the standard color.
Preparation of standard color: Measure accurately 2 mL
of Standard Arsenic Solution in the generator bottle A. Add
5 mL of diluted hydrochloric acid (1 in 2) and 5 mL of
potassium iodide TS, and allow to stand for 2 to 3 minutes.
Add 5 mL of acidic tin (II) chloride TS, allow to stand at
room temperature for 10 minutes, and then proceed as direct-
ed above. The color produced corresponds to 2,Mg of arsenic
(III) trioxide (As 2 3 ) and is used as the standard.
Note: Apparatus, reagents and test solutions used in the
test should contain little or no arsenic. If necessary, perform
a blank determination.
1.12 Methanol Test
Methanol Test is a method to determine methanol adhering
in ethanol.
Reagents
(1) Standard Methanol Solution — To 1.0 g of methanol,
accurately measured, add water to make exactly 1000 mL. To
5 mL of this solution, exactly measured, add 2.5 mL of
methanol-free ethanol and water to make exactly 50 mL.
(2) Solution A — To 75 mL of phosphoric acid add water
to make 500 mL, then dissove 15 g of potassium perman-
JPXV
ganate in this solution.
(3) Solution B — Add sulfuric acid carefully to an equal
volume of water, cool, and dissolve 25 g of oxalic acid dihy-
drate in 500 mL of this dilute sulfuric acid.
Procedure
Pipet 1 mL of the sample, and add water to make exactly
20 mL. Use this solution as the sample solution. Transfer 5
mL each of the sample solution and the Standard Methanol
Solution, accurately measured, to test tubes, add 2 mL of So-
lution A to each solution, and allow to stand for 15 minutes.
Decolorize these solutions by adding 2 mL of Solution B, and
mix with 5 mL of fuchsin-sulfurous acid TS. Allow to stand
for 30 minutes at ordinary temperature. The sample solution
has no more color than the Standard Methanol Solution.
1.13 Fats and Fatty Oils Test
Fats and Fatty Oils Test is a method applied to fats, fatty
oils, waxes, fatty acids, higher alcohols, and related sub-
stances.
Preparation of test sample
For a solid sample, melt with care, and, if necessary, filter
the melted sample with a dry filter paper by warming. For a
turbid liquid sample, heat at about 50°C. If it is still turbid,
filter it with a dry filter paper while warm. In either case, mix
the sample to make it homogeneous.
Melting point
Proceed by the method described in Method 2 of Melting
Point Determination <2.60>.
Congealing point of fatty acids
(1) Preparation of fatty acids — Dissolve 25 g of potassi-
um hydroxide in 100 g of glycerin. Transfer 75 g of this solu-
tion into a 1-L beaker, and heat at 150°C. Add 50 g of the
sample to this solution, and heat at a temperature not higher
than 150°C for 15 minutes under frequent stirring to saponi-
fy completely. Cool the solution to 100°C, dissolve by addi-
tion of 500 mL of hot water, and add slowly 50 mL of diluted
sulfuric acid (1 in 4). Heat the solution under frequent stir-
ring until the clear layer of fatty acid is separated distinctly.
Separate the fatty acid layer, and wash the fatty acid with hot
water until the washing shows no acidity to methyl orange
TS. Transfer the fatty acid layer to a small beaker, and heat
on a water bath until the fatty acid becomes clear owing to
the separation of water. Filter the warm solution, and com-
plete the evaporation of water by carefully heating the filtered
solution to 130°C.
(2) Measurement of congealing point — Proceed by the
method described in Congealing Point Determination <2.42>.
Specific gravity
(1) Liquid sample at ordinary temperature
Proceed by the method described in Determination of
Specific Gravity and Density <2.56>.
(2) Solid sample at ordinary temperature
Unless otherwise specified, fill a pycnometer with water at
20°C. Weigh accurately the pycnometer, and, after discard-
ing the water and drying, weigh accurately the empty py-
cnometer. Then, fill the pycnometer with the melted sample
to about three-fourths of the depth, and allow to stand at a
temperature a little higher than the melting temperature of
General Tests / Fats and Fatty Oils Test 31
Table 1.13-1
Acid value
Amount (g) of sample
Less than 5
20
5 to 15
10
15 to 30
5
30 to 100
2.5
More than 100
1.0
the sample for 1 hour to drive off the air in the sample. After
keeping at the specified temperature, weigh accurately the py-
cnometer. Fill up the pycnometer with water over the sample
at 20°C, and weigh accurately again.
The other procedure is the same as described in Method 1
of Determination of Specific Gravity and Density <2.56>.
W,-W
{W 1 -W)-(W,-W l )
W : Mass (g) of the empty pycnometer.
Wy. Mass (g) of the pycnometer filled with the sample.
W 2 : Mass (g) of the pycnometer filled with water.
W 3 : Mass (g) of the pycnometer filled with the sample and
water.
Acid value
The acid value is the number of milligrams of potassium
hydroxide (KOH) required to neutralize the free acids in 1 g
of sample.
Procedure: Unless otherwise specified, weigh accurately
the amount of sample shown in Table 1.13-1, according to
the expected acid value of the sample, in a glass-stoppered,
250-mL flask, add 100 mL of a mixture of diethyl ether and
ethanol (95) (1:1 or 2:1) as the solvent, and dissolve the sam-
ple by warming, if necessary. Then, add a few drops of
phenolphthalein TS, and titrate <2.50> with 0.1 mol/L potas-
sium hydroxide-ethanol VS until the solution develops a light
red color which persists for 30 seconds. If the sample solu-
tions is turbid at lower temperature, titration should be done
while warm. To the solvent used add phenolphthalein TS as
an indicator, and add 0.1 mol/L potassium hydroxide-
ethanol VS before use, until the solvent remains light red for
30 seconds.
Acid value =
consumed volume (mL) of 0.1 mol/L
potassium hydroxide-ethanol VS
amount (g) of sample
X5.611
Saponification value
The saponification value is the number of milligrams of
potassium hydroxide (KOH) required to saponify the esters
and to neutralize the free acids in 1 g of the sample.
Procedure: Unless otherwise specified, weigh accurately 1
to 2 g of the sample, transfer to a 200-mL flask, and add ex-
actly 25 mL of 0.5 mol/L potassium hydroxide-ethanol VS.
Attach a short reflux condenser or an air condenser 750 mm
in length and 6 mm in diameter to the neck of the flask, and
heat gently in a water bath for 1 hour with frequent shaking.
Cool the solution, add 1 mL of phenolphthalein TS, and ti-
trate <2.50> immediately the excess potassium hydroxide with
0.5 mol/L hydrochloric aicd VS. If the sample solution is tur-
bid at lower temperature, titration should be done while
warm. Perform a blank determination.
32 Fats and Fatty Oils Test / General Tests
JP XV
Saponification value =
(a-fr)x28.05
amount (g) of sample
a: Volume (mL) of 0.5 mol/L hydrochloric acid VS con-
sumed in the blank determination.
b: Volume (mL) of 0.5 mol/L hydrochloric acid VS con-
sumed for titration of the sample.
Ester value
The ester value is the number of milligrams of potassium
hydroxide (KOH) required to saponify the esters in 1 g of
sample.
Procedure: Unless otherwise specified, designate the differ-
ence between the saponification value and the acid value de-
termined as the ester value.
Hydroxyl value
The hydroxyl value is the number of milligrams of potassi-
um hydroxide (KOH) required to neutralize acetic acid com-
bined with hydroxyl groups, when 1 g of the sample is
acetylated by the following procedure.
Procedure: Place about 1 g of the sample, weighed ac-
curately, in a 200-mL round-bottom flask (shown in Fig.
1.13-1), and add exactly 5 mL of pyridine-acetic anhydride
TS. Place a small funnel on the neck of the flask, and heat by
immersing the flask up to 1 cm from the bottom in an oil bath
between 95°C and 100°C. Put a thick, round paper with a
round hole on the joint of the neck of the flask to protect the
neck from the heat of the oil bath. After heating for 1 hour,
take the flask from the oil bath, and cool by standing. Add 1
mL of water to the flask, and shake to decompose acetic an-
hydride. Heat the flask in the oil bath for 10 minutes again.
After cooling, wash the funnel and neck with 5 mL of neu-
tralized ethanol down into the flask, and titrate <2.50> with
0.5 mol/L potassium hydroxide-ethanol VS (indicator: 1 mL
of phenolphthalein TS). Perform a blank determination.
Hydroxyl value =
(a-fr)x28.05
amount (g) of sample
+ acid value
a: Volume (mL) of 0.5 mol/L potassium hydroxide-
ethanol VS consumed in the blank determination.
b: Volume (mL) of 0.5 mol/L potassium hydroxide-
ethanol VS consumed for titration of the sample.
The figures are in mm.
Fig. 1.13-1 Hydroxyl value determination flask
Unsaponifiable matter
Unsaponifiable matter is calculated as the difference be-
tween the amount of materials, which are unsaponifiable by
the procedure described below, soluble in diethyl ether and
insoluble in water, and the amount of fatty acids expressed in
terms of the amount of oleic acid. Its limit is expressed as a
percentage in the monograph.
Procedure: Transfer about 5 g of the sample, accurately
weighed, to a 250-mL flask. Add 50 mL of potassium
hydroxide-ethanol TS, attach a reflux condenser to the flask,
boil gently on a water bath for 1 hour with frequent shaking,
and then transfer to the first separator. Wash the flask with
100 mL of warm water, and transfer the washing to the sepa-
rator. Further, add 50 mL of water to the separator, and cool
to room temperature. Wash the flask with 100 mL of diethyl
ether, add the washing to the separator, extract by vigorous
shaking for 1 minute, and allow to stand until both layers are
separated clearly. Transfer the water layer to the second sepa-
rator, add 50 mL of diethyl ether, shake, and allow to stand
in the same manner. Transfer the water layer in the second
separator to the third separator, add 50 mL of diethyl ether,
and extract by shaking again in the same manner. Combine
the diethyl ether extracts in the second and third separators
into the first separator, wash each separator with a small
amount of diethyl ether, and combine the washings into the
first separator. Wash the combined extracts in the first sepa-
rator with 30 mL portions of water successively, until the
washing does not develop a light red color with 2 drops of
phenolpohthalein TS. Add a small amount of anhydrous so-
dium sulfate to the diethyl ether extracts, and allow to stand
for 1 hour. Filter the diethyl ether extracts with dry filter
paper, and collect the filtrates into a tared flask. Wash well
the first separator with diethyl ether, and add the washing to
the flask through the above filter paper. After evaporation of
the filtrate and washing almost to dryness on a water bath,
add 3 mL of acetone, and evaporate again to dryness on a
water bath. Complete the drying between 70°C and 80°C un-
der reduced pressure (about 2.67 kPa) for 30 minutes, allow
to stand for cooling in a desiccator (reduced pressure, silica
gel) for 30 minutes, and then weigh. After weighing, add 2
mL of diethyl ether and 10 mL of neutralized ethanol, and
dissolve the residue by shaking well. Add a few drops of
phenolphthalein TS, and titrate <2.50> the remaining fatty
acids in the residue with 0.1 mol/L potassium hydroxide-
ethanol VS until the solution develops a light red color which
persists for 30 seconds.
Unsaponifiable matter (%) --
a -(bx 0.0282)
amount (g) of sample
xlOO
a: Amount (g) of the extracts.
b: Volume (mL) of 0.1 mol/L potassium hydroxide-
ethanol VS consumed for titration.
Iodine value
The iodine value, when measured under the following con-
ditions, is the number of grams of iodine (I), representing the
corresponding amount of halogen, which combines with 100
g of sample.
Procedure: Unless otherwise specified, weigh accurately
the amount of sample shown in Table 1.13-2, according to
the expected iodine value of the sample, in a small glass con-
tainer. In a 500-mL glass-stoppered flask place the container
containing the sample, add 20 mL of cyclohexane to dissolve
JPXV
General Tests / Liquid Chromatography
33
the sample, then add exactly 25 mL of Wijs' TS, and mix
well. Stopper the flask, and allow to stand, protecting against
light, between 20°C and 30°C for 30 minutes (when the
expected iodine value is more than 100, for 1 hour) with oc-
casional shaking. Add 20 mL of potassium iodide solution
(1 in 10) and 100 mL of water, and shake. Then, titrate <2.50>
the liberated iodine with 0.1 mol/L sodium thiosulfate VS
(indicator: 1 mL of starch TS). Perform a blank determina-
tion.
Iodine value =
(a- b) x 1.269
amount (g) of sample
a: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
sumed in the blank determination.
b: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
sumed for titration of the sample.
Before use, wash the Nessler tubes thoroughly with sulfuric
acid for readily carbonizable substances. Unless otherwise
specified, proceed as follows. When the sample is solid, place
5 mL of sulfuric acid for readily carbonizable substances in a
Nessler tube, to which add a quantity of the finely powdered
sample, little by little, as directed in the monograph, and dis-
solve it completely by stirring with a glass rod. When the
sample is liquid, transfer a volume of the sample, as directed
in the monograph, to a Nessler tube, add 5 mL of sulfuric
acid for readily carbonizable substances, and mix by shaking.
If the temperature of the content of the tube rises, cool the
content; maintain it at the standard temperature, if the reac-
tion may be affected by the temperature. Allow to stand for
15 minutes, and compare the color of the liquid with that of
the matching fluid in the Nessler tube specified in the mono-
graph, by viewing transversely against a white background.
Table 1.13-2
Iodine value
Amount (g) of sample
Less than 30
30 to 50
50 to 100
More than 100
1.0
0.6
0.3
0.2
1.14 Sulfate Limit Test
Sulfate Limit Test is a limit test for sulfate contained in
drugs.
In each monograph, the permissible limit for sulfate (as
S0 4 ) is described in terms of percentage (%) in parentheses.
Procedure
Unless otherwise specified, transfer the quantity of the
sample, directed in the monograph, to a Nessler tube, dis-
solve it in sufficient water, and add water to make 40 mL.
Add 1 mL of dilute hydrochloric acid and water to make 50
mL, and use this solution as the test solution. Transfer the
volume of 0.005 mol/L sulfuric acid VS, directed in the
monograph, to another Nessler tube, add 1 mL of dilute
hydrochloric acid and water to make 50 mL, and use this so-
lution as the control solution. When the test solution is not
clear, filter both solutions according to the same procedure.
Add 2 mL of barium chloride TS to the test solution and to
the control solution, mix well, and allow to stand for 10
minutes. Compare the white turbidity produced in both solu-
tions against a black background by viewing downward or
transversely.
The turbidity produced in the test solution is not thicker
than that of the control solution.
1.15 Readily Carbonizable
Substances Test
Readily Carbonizable Substances Test is a method to exa-
mine the minute impurities contained in drugs, which are rea-
dily colored by addition of sulfuric acid.
Procedure
2. Physical Methods
Chromatography
2.01 Liquid Chromatography
Liquid Chromatography is a method to develop a mixture
injected into a column prepared with a suitable stationary
phase by passing a liquid as a mobile phase through the
column, in order to separate the mixture into its components
by making use of the difference of retention capacity against
the stationary phase, and to determine the components. This
method can be applied to a liquid or soluble sample, and is
used for identification, purity test, and quantitative determi-
nation.
A mixture injected into the column is distributed between
the mobile phase and the stationary phase with a characteris-
tic ratio (k) for each component.
, _ amount of compound in the stationary phase
amount of compound in the mobile phase
The ratio k represents the mass distribution ratio (or the
capacity factor) k' in liquid chromatography.
Since the relation given below exists among the ratio (k),
the time for which the mobile phase is passed through the
column (t : time measured from the time of injection of a
compound with k = to the time of elution at the peak maxi-
mum), and the retention time (t K : time measured from the
time of injection of a compound to be determined to the time
of elution at the peak maximum), the retention time for a
compound on a column has a characteristic value under fixed
chromatographic conditions.
t R = (l+k)t
Apparatus
Basically, the apparatus required for the liquid chromato-
graphic procedure consists of a pumping system for the
mobile phase, a sample injection port, a column, a detector
and a recorder. A mobile phase component regulator, a ther-
mostat for the column, a pumping system for reaction rea-
gents and a chemical reaction chamber are also used, if neces-
sary. The pumping system serves to deliver the mobile phase
34 Liquid Chromatography / General Tests
JP XV
and the reagents into the column and connecting tube at a
constant flow rate. The sample injection port is used to
deliver a quantity of the sample to the apparatus with high
reproducibility. The column is a tube with a smooth interior,
made of inert metal, etc., in which a packing material for
liquid chromatography is uniformly packed. A column with a
stationary phase chemically bound on the inside wall instead
of the column packed with the packing material may be used.
The detector is used to detect a property of the samples which
is different from that of the mobile phase, and may be an
ultraviolet or visible spectrophotometer, fluorometric detec-
tor, differential refractometer, electrochemical detector,
chemiluminescence detector, electric conductivity detector,
mass spectrophotometer, etc. The output signal is usually
proportional to the concentration of samples at amounts of
less than a few fig. The recorder is used to record the output
signals of the detector. As required, a data processor may be
used as the recorder to record or output the chromatogram,
retention times or amounts of the components. The mobile
phase component regulator is used to vary the ratio of the
mobile phase components in a stepwise or gradient fashion.
Procedure
Fix the detector, column and mobile phase to the appara-
tus, and adjust the flow rate and the column temperature to
the values described in the operating conditions specified in
the individual monograph. Inject a volume of the sample so-
lution or the standard solution specified in the individual
monograph with the sample injector into the column through
the sample injection port. The separated components are de-
tected by the detector, and recorded by the recorder as a
chromatogram. If the components to be analyzed have no
readily detectable physical properties such as absorbance or
fluorescence, the detection is achieved by changing the com-
ponents to suitable derivatives. Usually, the derivatization is
performed as a pre- or post-column labeling.
Identification and purity test
Identification of a component of a sample is performed by
confirming agreement of the retention time of the sample
with that of an authentic specimen, or by confirming that the
peak shape of the sample is unchanged after mixing the sam-
ple with an authentic specimen.
In general, the purity of the sample is determined by com-
paring the sample solution with a standard solution which is
prepared by diluting the sample solution to a concentration
corresponding to the specified limit amount of the impurity,
or by the peak area percentage method. Unless otherwise spe-
cified, if a sample is separated into isomers in the chromato-
gram, the isomer ratio is calculated by using the peak area
percentage method.
The peak area percentage method is a method to calculate
the proportion of the components from the ratio of the peak
area of each component to the sum of the peak areas of every
peak recorded in the chromatogram. In order to obtain ac-
curate results in evaluating the proportion of the compo-
nents, it is necessary to correct the area of each component
based on the relative sensitivity to the principal component.
Assay
(1) Internal standard method — In the internal standard
method, choose a stable compound as an internal standard
which shows a retention time close to that of the compound
to be assayed, and whose peak is well separated from all
other peaks in the chromatogram. Prepare several kinds of
standard solutions containing a fixed amount of the internal
standard and several graded amounts of the authentic speci-
men specified in the individual monograph. Based on the
chromatogram obtained by injection of a fixed volume of in-
dividual standard solutions, calculate the ratio of peak area
or peak height of the authentic specimen to that of the inter-
nal standard, and prepare a calibration curve by plotting
these ratios on the ordinate against the amount of the authen-
tic specimen or the ratio of the amount of the authentic speci-
men to that of the internal standard on the abscissa. The
calibration curve is usually obtained as a straight line passing
through the origin. Then, prepare a sample solution contain-
ing the internal standard in the same amount as in the stan-
dard solutions used for the preparation of the calibration
curve according to the method specified in the individual
monograph, perform the liquid chromatography under the
same operating conditions as for the preparation of the
calibration curve, calculate the ratio of the peak area or peak
height of the objective compound to that of the internal stan-
dard, and read the amount of the compound from the
calibration curve.
In an individual monograph, generally one of the standard
solutions with a concentration within the linear range of the
calibration curve and a sample solution with a concentration
close to that of the standard solution are prepared, and the
chromatography is performed with these solutions under fix-
ed conditions to determine the amount of the objective com-
pound. Generally, the relative standard deviation (variation
coefficient) is calculated to confirm the reproducibility of the
ratios of the peak area or peak height of the objective com-
pound to those of the internal standard, which are obtained
by repeating the injection of a fixed volume of the standard
solution.
(2) Absolute calibration curve method — Prepare stan-
dard solutions with several graded amounts of the authentic
specimen, and inject accurately a fixed volume of these stan-
dard solutions. With the chromatogram obtained, prepare a
calibration curve by plotting the peak areas or peak heights
on the ordinate against the amount of the authentic specimen
on the abscissa. The calibration curve is generally obtained as
a straight line passing through the origin. Then, prepare a
sample solution according to the method specified in the in-
dividual monograph, perform the liquid chromatography un-
der the same conditions as for the preparation of the calibra-
tion curve, measure the peak area or peak height of the objec-
tive compound, and read the amount of the compound from
the calibration curve.
In an individual monograph, generally one of the standard
solutions with a concentration within the linear range of the
calibration curve and a sample solution with a concentration
close to that of the standard solution are prepared, and the
chromatography is performed with these solutions under a
fixed condition to obtain the amount of the component. In
this method, all procedures, such as the injection procedure,
must be carried out under a strictly constant condition.
Generally, the relative standard deviation (variation
coefficient) is calculated to confirm the reproducibility of the
peak areas of peak heights of the objective compound which
are obtained by repeating the injection of a fixed volume of
the standard solution.
JPXV
General Tests / Gas Chromatography 35
Method for peak measuring
Generally, the following methods are used.
(1) Peak height measuring method
(i) Peak height method: Measure the distance between
the maximum of the peak and the intersecting point of a per-
pendicular line from the maximum of the peak to the
horizontal axis of recording paper with a tangent linking the
baselines on both sides of the peak.
(ii) Automatic peak height method: Measure the signals
from the detector as the peak height using a data processing
system.
(2) Peak area measuring method
(i) Width at half-height method: Multiply the peak width
at the half-height by the peak height.
(ii) Automatic integration method: Measure the signals
from the detector as the peak area using a data processing
system.
Terminology
Reproducibility of test: Reproducibility of test is used as
a method to ensure that the results obtained by a given proce-
dure truly meet the requirements of the test described in the
individual monograph. It is given as the relative standard
deviation (5 R (%)).
Symmetry factor: Symmetry factor shows the degree of
symmetry of a peak in the chromatogram, and is denned as S
in the following equation.
WVo5 h
2/
Wo.o5h: Width of the peak at one-twentieth of the peak
height,
/: Distance between the perpendicular from the peak maxi-
mum and the leading edge of the peak at one-twentieth
of the peak height,
where Wo.osh and /have the same unit.
Relative standard deviation: Generally, it is given as S R
(%) defined by the following equation.
S R( o/o)=^X
X>,-*) 2
n-\
x x : Measured value
X: Mean of measured values
n: Number of repeated measurements
Complete separation of peak: Complete separation of
the peak means that the resolution between two peaks is not
less than 1.5.
Separation factor: Separation factor shows the relation
between the retention times of peaks in the chromatogram,
and is defined as a in the following equation.
^ri> ^R2 : Retention times of two compounds used for the
resolution measurement (t Rl < t R2 ).
t : Time of passage of the mobile phase through the
column (time measured from the time of injection of a
compound with k = to the time of elution at the peak
maximum).
The separation factor (a) is a characteristic of the ther-
modynamic difference in partition of two compounds. It is
basically the ratio of their partition equilibrium coefficients
or of their mass-distribution ratios, and is obtained from the
chromatogram as the ratio of the retention times of the two
compounds.
Resolution: Resolution shows the relation between the
retention time and the peak width of peaks in the chromato-
gram, and is defined as R s in the following equation.
R*=lASx
tR2 t Ri
W , 5bl +W gjh2 )
t Rl , t R2 : Retention times of two compounds used for meas-
urement of the resolution (t Rl < t R2 ),
Wo.shi. W .5h2- Peak widths at half peak height,
where t Rl , t R2 , W .5hi and W 05h2 have the same unit.
Number of theoretical plates: Number of theoretical
plates is generally defined in terms of the following equation
to indicate the extent of the band broadening of a compound
in the column.
N=5.54x
W , 5h 2
t R : Retention time of compound,
Wo. 5h : Width of the peak at half peak height,
where t R and W , 5 b have the same unit.
Note: Avoid the use of authentic specimens, internal stan-
dards, reagents or solvents containing substances that may
interfere with the determination.
Among the operating conditions specified in the individual
monograph, inside diameter and length of the column, parti-
cle size of the column packing material, column temperature,
composition ratio of the mobile phase, composition of buffer
solutions in the mobile phase, pH of the mobile phase, con-
centration of ion pair-forming agents in the mobile phase,
ionic strength of the mobile phase, numbers of condition
changes, timing of such changes, gradient program, composi-
tion and flow rate of derivative-producing reagents, reaction
time and temperature of reaction chamber and flow rate of
mobile phase may be modified within limits which allow the
required elution order, resolution, symmetry factor, and rela-
tive standard deviation to be obtained.
2.02 Gas Chromatography
Gas Chromatography is a method to develop a mixture in-
jected into a column prepared with a suitable stationary
phase by passing a gas (carrier gas) as a mobile phase through
the column, in order to separate the mixture into its compo-
nents by making use of the difference of retention capacity
against the stationary phase, and to determine the compo-
nents. This method can be applied to a gaseous or vaporiza-
ble sample, and is used for identification, purity test, and
quantitative determination.
A mixture injected into the column is distributed between
the mobile phase and the stationary phase with a characteris-
tic ratio (k) for each component.
k =
amount of compound in the stationary phase
amount of compound in the mobile phase
36 Gas Chromatography / General Tests
JP XV
Since the relation given below exists among the ratio (k),
the time for which the mobile phase is passed through the
column (t : time measured from the time of injection of a
compound with k = to the time of elution at the peak maxi-
mum), and the retention time (t R : time measured from the
time of injection of a compound to be determined to the time
of elution at the peak maximum), the retention time for a
compound on a column has a characteristic value under fixed
chromatographic conditions.
t R = (l+k) t
Apparatus
Basically, the apparatus required for the gas chromato-
graphic procedure consists of a carrier gas-introducing port
and flow regulator, a sample injection port, a column, a
column oven, a detector and a recorder. Gas introducing port
and flow regulator for a combustion gas, a burning support-
ing gas and an accessory gas and sample injection port for
headspace are also used, if necessary. The carrier gas-in-
troducing port and flow regulator serves to deliver the carrier
gas into the column at a constant flow rate, and usually con-
sist of a pressure regulation valve, a flow rate regulation valve
and a pressure gauge. The sample injection port is used to
deliver a quantity of the sample to the flow line of carrier gas
with high reproducibility. There are sample injection ports
for packed column and for capillary column. There are both
divided injection mode and non-divided injection mode to
sample injection port for capillary column. The columns are
usually classified as packed column or capillary column. The
packed column is a tube made of inert metal, glass or synthet-
ic resin, in which a packing material for gas chromatography
is uniformly packed. The packed column with not more than
1 mm in inside diameter is also called a packed capillary
column (micro packed column). A capillary column is a tube
made of inert metal, glass, quartz or synthetic resin, whose
inside wall is bound chemically with stationary phase for gas
chromatography. The column oven has the setting capacity
for a column with required length and the temperature regu-
lation system for keeping the constant column temperature.
The detector is used to detect a component separated on the
column, and may be an alkaline thermal ionization detector,
a flame photometry detector, mass spectrophotometer,
hydrogen flame-ionization detector, an electron capture de-
tector, a thermal conductivity detector, etc. The recorder is
used to record the output signals of the detector.
Procedure
Unless otherwise specified, proceed by the following
method. Fix the detector, column and carrier gas to the ap-
paratus, and adjust the flow rate and the column temperature
to the values described in the operating conditions specified in
the individual monograph. Inject a volume of the sample so-
lution or the standard solution specified in the individual
monograph with the sample injector into the column system
through the sample injection port. The separated compo-
nents are detected by the detector, and recorded by the
recorder as a chromatogram.
Identification and purity test
Identification of a component of a sample is performed by
confirming agreement of the retention time of the sample
with that of an authentic specimen, or by confirming that the
peak shape of the sample is unchanged after mixing the sam-
ple with an authentic specimen.
In general, the purity of the sample is determined by com-
paring the sample solution with a standard solution which is
prepared by diluting the sample solution to a concentration
corresponding to the specified limit amount of the impurity,
or by the peak area percentage method. Unless otherwise spe-
cified, if a sample is separated into isomers in the chromato-
gram, the isomer ratio is calculated by using the peak area
percentage method.
The peak area percentage method is a method to calculate
the proportion of the components from the ratio of the peak
area of each component to the sum of the peak areas of every
peak recorded in the chromatogram. In order to obtain ac-
curate results in evaluating the proportion of the compo-
nents, it is necessary to correct the area of each component
based on its relative sensitivity to the principal component.
Assay
In general, perform the assay by using the internal stan-
dard method. The absolute calibration curve method is used
when a suitable internal standard is not available. Perform
the assay by using the standard addition method when the
effect of the component other than the compound to be
assayed on the quantitative determination is not negligible
against a result of the determination.
(1) Internal standard method — In the internal standard
method, choose a stable compound as an internal standard
which shows a retention time close to that of the compound
to be assayed, and whose peak is well separated from all
other peaks in the chromatogram. Prepare several kinds of
standard solutions containing a fixed amount of the internal
standard and several graded amounts of the authentic speci-
men specified in the individual monograph. Based on the
chromatogram obtained by injection of a fixed volume of
individual standard solutions, calculate the ratio of peak area
or peak height of the authentic specimen to that of the inter-
nal standard, and prepare a calibration curve by plotting
these ratios on the ordinate against the amount of the authen-
tic specimen or the ratio of the amount of the authentic speci-
men to that of the internal standard on the abscissa. The
calibration curve is usually obtained as a straight line passing
through the origin. Then, prepare a sample solution contain-
ing the internal standard in the same amount as in the stan-
dard solutions used for the preparation of the calibration
curve according to the method specified in the individual
monograph, perform the gas chromatography under the
same operating conditions as for the preparation of the
calibration curve, calculate the ratio of the peak area or peak
height of the objective compound to that of the internal stan-
dard, and read the amount of the compound from the
calibration curve.
In an individual monograph, generally one of the standard
solutions with a concentration within the linear range of the
calibration curve and a sample solution with a concentration
close to that of the standard solution are prepared, and the
chromatography is performed with these solutions under fix-
ed conditions to determine the amount of the objective com-
pound. Generally, the relative standard deviation (variation
coefficient) is calculated to confirm the reproducibility of the
ratios of the peak area or peak height of the objective com-
pound to those of the internal standard, which are obtained
by repeating the injection of a fixed volume of the standard
solution.
JPXV
General Tests / Thin-layer Chromatography
37
(2) Absolute calibration curve method — Prepare stan-
dard solutions with several graded amounts of the authentic
specimen, and inject accurately a fixed volume of these stan-
dard solutions. With the chromatogram obtained, prepare a
calibration curve by plotting the peak areas or peak heights
on the ordinate against the amount of the authentic specimen
on the abscissa. The calibration curve is generally obtained as
a straight line passing through the origin. Then, prepare a
sample solution according to the method specified in the
individual monograph, perform the liquid chromatography
under the same conditions as for the preparation of the
calibration curve, measure the peak area or peak height of
the objective compound, and read the amount of the com-
pound from the calibration curve.
In an individual monograph, generally one of the standard
solutions with a concentration within the linear range of the
calibration curve and a sample solution with a concentration
close to that of the standard solution are prepared, and the
chromatography is performed with these solutions under a
fixed condition to obtain the amount of the component. In
this method, all procedures, such as the injection procedure,
must be carried out under a strictly constant condition.
Generally, the relative standard deviation (variation
coefficient) is calculated to confirm the reproducibility of the
peak areas or peak heights of the objective compound, which
are obtained by repeating the injection of a fixed volume of
the standard solution.
(3) Standard addition method — Pipet a fixed volume of
more than 4 sample solutions, add exactly the standard solu-
tion so that stepwise increasing amounts of the object com-
pound are contained in the solutions except 1 sample solu-
tion, diluted exactly each solution with and without standard
solution to a definite volume, and use each solution as the
sample solution. Based on the chromatogram obtained by ex-
act injection of a fixed volume of individual sample solutions,
measure the peak area of individual sample solutions. Calcu-
late the concentration of standard objective compound added
into each sample solution, plot the amounts (concentration)
of added standard object compound on the abscissa and the
peak area on the ordinate on the graph, extend the calibra-
tion curve obtained by linking the plots, and determine the
amount of object compound to be assayed from the distance
between the origin and the intersecting point of the calibra-
tion curve with the abscissa. Generally, the relative standard
deviation (variation coefficient) is calculated to confirm the
reproducibility of the peak areas of the objective compound,
which are obtained by repeating the injection of a fixed
volume of the standard solution. This method is available
only in the case that the calibration curve is a straight line,
and passes through the origin when the absolute calibration
curve method is employed. In this method, all procedures
must be carried out under a strictly constant condition.
Method for peak measuring
Generally, the following methods are used.
(1) Peak height measuring method
(i) Peak height method: Measure the distance between
the maximum of the peak and the intersecting point of a per-
pendicular line from the maximum of the peak to the
horizontal axis of recording paper with a tangent linking the
baselines on either side of the peak.
(ii) Automatic peak height method: Measure the signals
from the detector as the peak height using a data processing
system.
(2) Peak area measuring method
(i) Width at half-height method: Multiply the peak width
at the half-height by the peak height.
(ii) Automatic integration method: Measure the signals
from the detector as the peak area using a data processing
system.
Terminology
The definition of terms described under Liquid Chro-
matography <2.01> shall apply in Gas Chromatography
<2.02>.
Note: Avoid the use of authentic specimens, internal stan-
dards, reagents or solvents containing substances that may
interfere with the determination.
Among the operating conditions specified in the individual
monograph, inside diameter and length of the column, parti-
cle size of the column packing material, concentration of the
stationary phase, column temperature, and flow rate of carri-
er gas may be modified within limits which allow the required
elution order, resolution, symmetry factor and relative stan-
dard deviation to be obtained. The sample injection port and
the operating conditions for headspace may be also modified
within limits which allow the accuracy and precision more
than those of a prescribed method to be obtained.
2.03 Thin-layer Chromatography
Thin-layer Chromatography is a method to separate each
ingredient by developing a mixture in a mobile phase, using a
thin-layer made of a suitable stationary phase, and is applied
for identification, purity test, etc. of substances.
Preparation of thin-layer plate
Generally, proceed by the following method.
A smooth and uniformly thick glass plate having a size of
50 mm x 200 mm or 200 mm x 200 mm is used for preparing
a thin-layer plate. Using a suitable apparatus, apply a water
suspension of powdered solid substance for the stationary
phase, directed in the monograph, on one side of the glass
plate to make a uniform layer of 0.2 to 0.3 mm in thickness.
After air-drying, dry further by heating at a fixed tempera-
ture between 105 C C and 120 C C for 30 to 60 minutes. A suita-
ble plastic plate may be used instead of the glass plate.
Preserve the dried plate with protection from moisture.
Procedure
Unless otherwise specified, proceed by the following
method.
Designate a line about 20 mm distant from the bottom of
the thin-layer plate as the starting line, spot 2 to 6 mm in di-
ameter the directed volumes of the sample solution or the
standard solution in the monograph using micropipets at
points on this line, separated by more than 10 mm, and air-
dry. Unless otherwise specified, attach the filter paper along
with the inside wall of the container, and wet the filter paper
with the developingt solvent. In the container, the developing
solvent is placed up to about 10 mm in height from the bot-
tom beforehand, seal the container closely, and allow it to
stand for 1 hour at ordinary temperature. Place the plate in
the container, avoiding contact with the inside wall, and seal
the container. Develop it at ordinary temperature.
When the solvent front has ascended from the starting line
38
Nuclear Magnetic Resonance Spectroscopy / General Tests
JP XV
to the distance directed in the monograph, remove the plate
from the container. Immediately put a mark at the solvent
front. After air-drying, observe the location, color, etc., of
each spot by the method specified in the monograph. Calcu-
late the Ri value by using the following equation:
Rf
distance from the starting line to the center of the spot
distance from the starting line to the solvent front
Spectroscopic Methods
2.21 Nuclear Magnetic Resonance
Spectroscopy
Nuclear Magnetic Resonance (NMR) Spectroscopy is
based on the phenomenon that specific radio frequency radia-
tion is absorbed by magnetic nuclei in a sample placed in a
magnetic field; target nuclei are 'H, 13 C, 15 N, 19 F, 31 P, etc.
These nuclei have intrinsic spin angular momentum, of which
the magnitude is given by / (/+ l)/h/2n, where / is the spin
quantum number and is integral or half-integral (/= 1/2 for
'H and 13 C). When the magnetic nuclei are placed in a mag-
netic field, they are oriented in 21+ 1 possible orientations
corresponding to 21+ 1 equally spaced energy levels (two
energy levels for 'H and 13 C). The transition between two suc-
cessive quantized energy levels corresponding to adjacent
orientations can be induced by electromagnetic radiation
with a suitable frequency. The precise relation between the
field strength and the resonant frequency v is given by
H
where H is the strength of the applied external magnetic field
and y is the gyromagnetic ratio, a constant characterizing a
particular isotope. The absorption of radiation (NMR signal)
can occur only when the irradiating radio frequency satisfies
the resonance condition. Since the absorption coefficient (the
transition probability) does not depend on the environment
in which the nuclei are located, the intensity is basically
proportional to the number of nuclei. The excess spins shift-
ed to the higher energy levels by the transition process return
to the thermal equilibrium state at various rates determined
by a characteristic time constant (known as the relaxation
time).
A nucleus is shielded from the applied magnetic field by the
electrons belonging to its own atom and to the molecule.
Therefore nuclei in different environments are shielded to
different extents and resonate at different frequencies. The
difference in resonance frequencies is defined as chemical
shift (<5), which is independent of the strength of the magnetic
field, and is given by
S=^^+S R
v R
where,
v s : The resonance frequency of the observed signal,
v R : The resonance frequency of the reference signal,
v R : The chemical shift of the reference signal (in the case of
the value not being 0).
probe
r \ — \
it
n M
5 I
spectrum
Analog to digital
convener
Fig. 2.21-1 FT-NMR spectrometer
The chemical shifts are normally expressed in ppm, a
dimensionless unit, by assuming the chemical shift of the
reference compound as ppm. When the chemical shift of
the reference compound is not assumed to be ppm, chemi-
cal shifts of samples are corrected accordingly.
In addition to the shielding due to electrons, the nucleus is
subjected to effects due to the spin orientations of other mag-
netic nuclei through chemical bonds, resulting in an
additional splitting of the signal. The spacing between two
adjacent components of the signal is known as the spin-spin
coupling constant (J). Coupling constants are measured in
hertz and are independent of the strength of the external
magnetic field. The increased number of interacting nuclei
will make the multiplet pattern more complex.
From the NMR spectrum the following four parameters
can be obtained: chemical shift, spin-spin coupling constant,
resonance intensity (intensities of 'H are proportional to the
number of nuclei and those of 13 C and others are susceptible
to the nuclear Overhauser effect (NOE) and relaxation) and
relaxation time. These parameters are useful for structural
determination, identification and quantitative analysis of
molecules. Spin decoupling, NOE, and two-dimensional
NMR techniques are also available for structural analysis.
Spectrometer
There are two types of spectrometers.
(1) Fourier transform NMR (FT-NMR) spectrometers
(Fig. 2.21-1)
Target nuclei are simultaneously excited in all frequency
range of the nuclei by means of an intense radio frequency
pulse. The FID (free induction decay) after the pulse is
detected, which is a time domain signal, is converted to a
frequency domain spectrum by Fourier transformation.
Number of data points suitable for the spectral range, flip an-
gle, acquisition time, delay time and number of scans should
be set appropriately.
Recently FT-NMR is commonly used because of its high
sensitivity and various advanced applications.
(2) Continuous wave NMR (CW-NMR) spectrometers
(Fig. 2.21-2)
In the case of the CW method, a spectrum is obtained by
sweeping the radio frequency or magnetic field continuously
over the frequency range of the nuclei being observed.
Measurement
Prior to measurements, the sensitivity and resolution of the
JPXV
General Tests / Nuclear Magnetic Resonance Spectroscopy
39
^^A
probe
rs^
t- sweep gcuci'alor
spectrum
Fig. 2.21-2 CW-NMR spectrometer
instrument must be adjusted to the optimum levels using a
standard sample (ethylbenzene, 1,2-dichlorobenzene or
acetaldehyde) dissolved in an appropriate NMR solvent.
(1) The sample dissolved in a suitable solvent is trans-
ferred into an NMR tube. The reference compound can be
added directly to the sample solution (internal reference), or
a sealed capillary tube containing the reference compound
can be inserted into the NMR tube (external reference). The
sample solutions should be completely homogeneous. In par-
ticular, solid contaminants should be removed in order to ob-
tain good spectra. Various deuterated NMR solvents are
commonly used for NMR measurement and the following
points should be considered in selecting an appropriate
solvent: (i) The solvent signals do not overlap with the sample
signals, (ii) The sample must be soluble in the solvent select-
ed, (hi) The solvent does not react with the sample. Further-
more, it should be noted that chemical shifts can depend
upon the solvent employed, sample concentration and deu-
terium ion concentration, and that viscous solutions usually
give rather broad, poorly resolved spectra.
(2) For the reference standards use the reagents for
nuclear magnetic resonance spectroscopy. For 'H and 13 C
spectra, tetramethylsilane (TMS) is usually used as the
reference compound for samples dissolved in organic
solvents. For samples dissolved in deuterium oxide, sodium
2,2-dimethyl-2-silapentane-5-sulfonate (DSS) or sodium 3-
(trimethylsilyl)propionate (TSP) is used. For other nuclei,
nitromethane, trichlorofluoromethane and phosphoric acid
are used as reference compounds for 15 N, 19 F and 31 P, respec-
tively. Furthermore, chemical shifts of residual protons in
deuterated solvents and 13 C in the solvent instead of a refer-
ence compound can be used for 'H and 13 C NMR.
Record of apparatus and measurement conditions
Type of instrument, frequency, solvent, temperature, sam-
ple concentration, reference compound, experimental tech-
nique, etc. should be recorded to allow appropriate compari-
son of spectra, because NMR spectra depend on the measure-
ment conditions.
Identification
The sample solution is prepared and tested by the method
directed in each monograph. Usually in the case of 'H NMR,
the sample is identified by the following method.
(1) Identification by the use of chemical shift, multiplicity
and relative intensity
When chemical shifts, multiplicities and relative intensities
of signals are defined, the sample can be identified as being
the same substance when all chemical shifts, multiplicities
and relative intensities are the same as those prescribed.
(2) Identification by the use of a Reference Standard
Measurement conditions should be the same as those used
in the case of the Reference Standard. When the spectra of a
sample and the Reference Standard exhibit the same
multiplicities and relative intensities of signal at the same
chemical shifts, the sample can be identified as being the same
substance as the Reference Standard.
Experimental techniques of ] H and 13 C NMR spectroscopy
NMR spectroscopy includes one-, two- and multi-dimen-
sional techniques, which are used for various purposes.
Spin decoupling, and NOE are available in one-dimen-
sional 'H spectroscopy. Spin decoupling can assign coupling
correlations. As NOE can observe correlations among
spatially proximate protons, the configuration and the
conformation can be analyzed.
Broadband decoupling, INEPT and DEPT are usually
applied in one-dimensional ,3 C spectroscopy. The broadband
decoupling technique simplifies a spectrum and achieves en-
hancement of sensitivity. INEPT (insensitive nuclei enhanced
by polarization transfer) and DEPT (distortionless enhance-
ment of polarization transfer) enhance the sensitivity of 13 C
by means of polarization transfer from directly bonded *H
with a large magnetic moment. They can be applied to identi-
fy primary, secondary, tertiary or quarternary carbon.
Two-dimensional spectroscopy can observe all correlation
peaks between nuclei through spin-spin coupling or NOE in a
single experiment, and there are many techniques for
homonuclear and heteronuclear measurements. Representa-
tive techniques are described below.
COSY (2D correlation spectroscopy), HOHAHA
(homonuclear Hartmann-Hahn spectroscopy) or TOCSY
(total correlation spectroscopy): Correlation between protons
through scalar spin-spin coupling is obtained and
intramolecular connectivities of hydrogen atoms are
revealed.
NOESY (2D nuclear Overhauser enhancement and
exchange spectroscopy): NOE is measured by a two-dimen-
sional technique. Approximate distances between spatially
proximate hydrogen atoms are obtained to analyze the three-
dimensional structure.
INADEQUATE (incredible natural abundance double
quantum transfer experiment): Although this technique is in-
sensitive because it involves double quantum transfer by
13 C- 13 C scalar coupling in a sample with natural isotopic
abundance, the connectivities of all neighboring 13 C nuclei
can be obtained to analyze the carbon skeleton.
HMQC (heteronuclear multiple quantum coherence): This
technique observes correlations between 'H and 13 C with
direct spin-spin coupling using 'H detection and reveals
intramolecular chemical bonds between hydrogen and carbon
atoms.
HMBC (heteronuclear multiple bond connectivity): This
technique observes correlations between 'H and 13 C with long
range spin-spin coupling using >H detection and reveals in-
tramolecular connectivities of hydrogen and carbon atoms.
There are many other techniques such as DQF-COSY
(double quantum filtered COSY) and HSQC (heteronuclear
single quantum coherence). Furthermore, multidimensional
NMR techniques are used to analyze macromolecules.
40
Fluorometry / General Tests
JP XV
2.22 Fluorometry
Fluorometry is a method to measure the intensity of
fluorescence emitted from a solution of fluorescent substance
irradiated with an exciting light in a certain wavelength
range. This method is also applied to the phosphorescent sub-
stances.
Fluorescence intensity Fin a dilute solution is proportional
to the concentration c in mol per liter of the solution and the
pathlength / of light through the solution in centimeter.
F=kI 4>Ecl
k: Constant
I : Intensity of exciting light
4>: Quantum yield of fluorescence or phosphorescence
number of quanta emitted as
fluorescence or phosphorescence
number of quanta absorbed
e: Molar extinction coefficient of the substance at the exci-
tation wavelength
Apparatus
Spectrofluorometer is usually used. Generally, a xenon
lamp, laser, an alkaline halide lamp, etc. which provide sta-
ble exciting light are used as the light source. Usually, a non-
fluorescent quarz cell (1 cm x 1 cm) with four transparent
sides is used as the container for sample solution.
Procedure
Excitation spectrum is obtained by measuring fluorescence
intensities of sample solution with varying excitation
wavelengths at a fixed emission wavelength (in the vicinity of
the fluorescence maximum) and drawing a curve showing the
relationship between the excitation wavelength and the
fluorescence intensity. Fluorescence spectrum is obtained by
measuring fluorescence intensities of sample solution with
varying emission wavelengths at a fixed excitation wavelength
(in the vicinity of the excitation maximum) and drawing the
same curve as described for the excitation spectrum. If neces-
sary, the spectra are corrected with regard to the optical
characteristics of the apparatus.
The fluorescence intensity is usually measured at the excita-
tion and the emission wavelengths in the vicinity of excitation
and emission maxima of the fluorescent substance. The
fluorescence intensity is expressed as a value relative to that
of a standard solution, because it is readily affected even by a
slight change in the condition for the measurement.
Unless otherwise specified, the instrument is operated as
follows with standard, sample, and reference solutions pre-
pared as directed in the monograph: Fix the excitation and
fluorescence wavelength scales at the designated positions,
adjust the dark current to zero, put the quarz cell containing
the standard solution in the light path, and adjust the instru-
ment so that the standard solution shows the fluorescence in-
tensity of 60% to 80% of full scale. Then perform the meas-
urements with the cells containing the sample solution and
the control solution, and read the fluorescence intensity as %
under the same condition. Set the width of the wavelength
properly unless otherwise specified.
Note: The fluorescence intensity is readily affected by the
concentration, temperature and pH of the solution, and na-
ture and purity of solvents or reagents used.
2.23 Atomic Absorption
Spectrophotometry
Atomic Absorption Spectrophotometry is a method to de-
termine the amount or the concentration of an element in a
sample specimen being examined, by utilizing the phenomen-
on that atoms being in the ground state absorb the light of
specific wavelength, characteristic of the respective atom,
when the light passes through an atomic vapor layer of the
element to be determined.
Apparatus
Usually, the apparatus consists of a light source, a sample
atomizer, a spectroscope, a photometer and a recording sys-
tem. Some are equipped with a background compensation
system. As a light source, usually a hollow cathode lamp
specified for each element is used and sometimes a discharge
lamp is also used. There are three types of sample atomizer:
the flame type, the electrothermal type, and the cold-vapor
type. The first one is composed of a burner and a gas-flow
regulator, the second one is composed of an electric furnace
and a power source, and the third one is composed of a
mercury generator and an absorption cell. The third one is
further classified into two subtypes, which differ in the
atomizing method for mercury containing-compounds: one
utilizes chemical reduction-vaporization and the other utilizes
a thermal reduction-vaporization method.
For the selection of an appropriate analytical wavelength
in a spectroscope, a grating for light diffraction or an inter-
ference filter can be used. A recording system is composed of
a display and a recording device. A background compensa-
tion system is employed for the correction of atmospheric
effects on the measuring system. Several principles can be
utilized for background compensation, using continuous
spectrum sources, the Zeeman splitted spectrum, the non-
resonance spectrum, or self-inversion phenomena.
Another special options such as a hydride generator and a
heating cell, can also be used for analyzing such as selenium.
As a hydride generator, a batch method and/or a continuous
flow method can be applied. While as a heating cell, there are
two kinds of cell: one for heating by flame and the other for
heating by electric furnace.
Procedure
Unless otherwise specified, proceed by any of the following
methods.
(1) Flame type — Fit the specific light source to the lamp
housing and switch on the instrument. After lighting the
lamp and selecting the analytical wavelength specified in the
monograph, set an appropriate electric current and slit-
width. Next, a mixture of a combustible gas and a supporting
gas is ignited and the gas flow rate and/or pressure should be
adjusted to optimum conditions. The zero adjustment of the
detecting system must be done through nebulizing the blank
solvent into the flame. After setting up the measuring system,
the sample solution prepared by the specified procedure is
introduced into the flame and the light absorption at the
characteristic wavelength of the element to be determined is
measured.
JPXV
General Tests / Ultraviolet-visible Spectrophotometry
41
(2) Electrothermal type — Fit the specific light source to
the lamp housing and switch on the instrument. After light-
ing the lamp and selecting the analytical wavelength specified
in the monograph, set an appropriate electric current and slit-
width. Further, set an electric furnace to the appropriate tem-
perature, electric current, and heating program, as directed
separately in the monograph. When a suitable amount of
sample is injected into the heated furnace with an appropriate
stream of inert gas, the sample is dried and ashed, simultane-
ously with atomization of the metallic compound included in
the specimen. The atomic absorption specified is observed
and the intensity of absorption is measured. Details of the
sample preparation method are provided separately in the
monograph.
(3) Cold vapor type — Fit the mercury lamp to the lamp
housing and switch on the instrument. After lighting the
lamp and selecting the analytical wavelength specified in the
monograph, set an appropriate electric current and a slit-
width. In the chemical atomization-vaporization method, a
mercury containing compound in the sample solution, pre-
pared by the specified procedure, is chemically reduced to
metallic mercury by adding a proper reducing reagent to the
closed vessel and the generated mercury is vaporized and in-
troduced into the absorption cell with a flow of inert gas. In
the thermal atomization-vaporization method, the sample
specimen on a quartz dish is heated electrically and the gener-
ated atomic mercury is vaporized and introduced into the
absorption cell with a flow of inert gas. Thus, in both
methods, the generated atomic mercury is carried into the
absorption cell as cold vapor and the intensity of the charac-
teristic atomic absorption of mercury is measured.
Determination
Usually, proceed by any of the following methods. In the
determination, the possibility of interference for various
reasons and the background effect must be considered and
avoided if possible.
(1) Calibration curve method — Prepare standard solu-
tions at more than 3 concentration levels, measure the specific
absorption due to these standard solutions, and prepare the
calibration curve of the atomic absorption against the con-
centration. Then measure the atomic absorption due to the
sample specimen, in which the concentration of the element
to be determined should be adjusted to be within the concen-
tration range of the standard solutions, and determine the
amount or the concentration of the element to be examined
using the calibration curve.
(2) Standard addition method — To equal volumes of
more than 3 sample solutions, prepared as directed in the
monograph, add a measured quantity of the standard solu-
tions to produce a series of solutions containing increasing
amounts of the element to be examined, and further add a
solvent to make up a constant volume. Measure the atomic
absorption for the respective solutions, and plot the obtained
values on a graph with the added amount or the concentra-
tion on the abscissa and the absorbance on the ordinate.
Extrapolate the linear plot obtained by linking the data
points, and determine the amount or the concentration of the
element to be examined from the distance between the origin
and the point where the plot intersects with the abscissa. This
method is available only when the calibration curve obtained
by Method (1) is confirmed to be linear and to pass through
the origin.
(3) Internal standard method — Prepare a series of stan-
dard solutions of the element to be determined, each contain-
ing a definite amount of the internal standard element direct-
ed in the monograph. For these standard solutions, measure
the atomic absorption due to the standard element and the
internal standard element separately at the respective
wavelengths under the same operating conditions, and obtain
the ratio of absorbance by the standard element to that by the
internal standard element. Prepare a calibration curve for the
element to be determined, with the amount or the concentra-
tion of the standard element on the abscissa and the above-
mentioned ratio of the absorbance on the ordinate. Then pre-
pare sample solutions, adding the same amount of the inter-
nal standard element as contained in the standard solutions.
Measure the ratio of the absorbance due to the element to be
determined to that due to the internal standard element under
the same conditions as employed for preparing the calibra-
tion curve, and determine the amount or the concentration of
the element being examined by using the calibration curve.
Note: Reagents, test solutions, and gases used in this test
should not interfere in any process of the measurement.
2.24 Ultraviolet-visible
Spectrophotometry
Ultraviolet-visible Spectrophotometry is a method to meas-
ure the degree of absorption of light between the wavelengths
of 200 nm and 800 nm by substances for the tests of their
identity and purity, and for assay. When an atomic absorp-
tion spectrophotometer is used for these purposes, proceed as
directed under Atomic Absorption Spectrophotometry <2.23
>. When monochromatic light passes through a substance in
the solution, the ratio of transmitted light intensity / to inci-
dent light intensity I is called transmittance t; transmittance
expressed in the percentage is called percent transmission T,
and common logarithm of the reciprocal of transmittance is
called absorbance A.
t =
T=
x 100=100?
yl=log-
The absorbance A is proportional to the concentration c of
a substance in the solution and the length / of the layer of the
solution through which light passes.
A = kcl (k: constant)
The absorbance, calculated on the basis that / is 1 cm and c
is 1 mol/L, is called molar absorption coefficient e. The mo-
lar absorption coefficient at the wavelength of maximum ab-
sorption is expressed as e max .
When a light beam passes through a substance in the solu-
tion, the absorbance by the sample differs depending on the
wavelengh of the light. So, an absorption spectrum is ob-
tained by determining the absorbances of a light beam at
various wavelengths and by graphically plotting the relation
between absorbance and wavelength. From the absorption
spectrum, it is possible to determine the wavelength of maxi-
mum absorption A max and that of minimum absorption A min .
The absorption spectrum of a substance in the solution is
characteristic, depending on its chemical structure. There-
fore, it is possible to identify a substance by comparing the
42
Ultraviolet-visible Spectrophotometry / General Tests
JP XV
spectrum of a sample within the specified wavelength range
with the Reference Spectrum or the spectrum of Reference
Standard, by determing the wavelengths of maximum ab-
sorption, or by measuring the ratio of absorbances at two
specified wavelengths. For the purpose of assay, the absor-
bance by a sample solution with a certain concentration is
measured at the wavelength of the maximum absorption A max
and compared it with the absorbance of a standard solution
with a certain concentration.
Apparatus and adjustment
A spectrophotometer or a photoelectric photometer is used
for the measurement of absorbance.
After adjusting the spectrophotometer or photoelectric
photometer based on the operation manual of the apparatus,
it should be confirmed that the wavelength and the transmis-
sion rate meet the specifications of the tests described below.
The calibration of wavelength should be carried out as
follows. Using an optical filter for wavelength calibration,
measure the transmission rate in the vicinity of the standard
wavelength value shown in the test results form, under the
test conditions given in the test results form attached to each
of the filters. When performing a test to determine the
wavelength which shows minimal transmission rate, the
difference between the measured wavelength and the standard
wavelength value should be within ± 0.5 nm. When the
measurement is repeated three times, each value obtained
should be within the mean ± 0.2 nm. It is also possible to
carry out the test using a low-pressure mercury lamp at bright
line wavelengths of 253.65 nm, 365.02 nm, 435.84 nm and
546.07 nm, or a deuterium discharge lamp at bright line
wavelengths of 486.00 nm and 656.10 nm. In the case of these
tests, the difference between the measured wavelength and the
wavelength of the bright line should be within ± 0.3 nm.
When the measurement is repeated three times, each value
obtained should be within the mean ± 0.2 nm.
The calibration of transmission rate or absorbance should
be carried out as follows. Using an optical filter for transmis-
sion rate calibration, determine the transmission rate at the
standard wavelength value under the test conditions given in
the test results form attached to each of the filters. The differ-
ence between the measured transmission rate and the stan-
dard transmission rate value should be within the range of
from 1% larger of the upper limit to 1% smaller of the lower
limit for the relative accuracy shown in the test results form.
When the measurement is repeated three times, each absor-
bance obtained (or calculated from the transmission rate)
should be within the mean ± 0.002 when the absorbance is
not more than 0.500, and within the mean ± 0.004 when the
absorbance is more than 0.500. In addition, it will be desira-
ble to confirm the linearity of transmission rate at the same
wavelength using several optical filters for calibration of
transmission rate with different transmission rates.
Procedure
After adjusting the apparatus as directed in the Apparatus
and adjustment, select and set the light source, detector,
mode of measurement, measuring wavelength or wavelength
range, spectrum width and scanning speed.
Subsequently, allow the apparatus to stand for a certain
time to confirm its stability. Then, usually adjust the appara-
tus so that the transmittance is 0% at measuring wavelength
or over measuring wavelength range after shutting the sample
side of light path. Then open the shutter and adjust the trans-
mittance to 100% (the absorbance is zero). Adjusting the
transmittance to 100% is usually done by putting cells con-
taining the control solution in both light paths. For the con-
trol solution, unless otherwise specified, blank solvent is
used.
Then perform the measurement with the cell containing the
sample solution, and read the absorbance at measuring
wavelength, or measure the spectrum over measuring
wavelength range. Unless otherwise specified, a cell with a
path length of 1 cm, made of quartz for ultraviolet range and
of quartz or glass for visible range, is used. Special considera-
tion is needed with the absorption of solvents in the ultravio-
let range; use a solvent which does not disturb accurate meas-
urement.
Specific absorbance
In the Japanese Pharmacopoeia, the absorbance, calculat-
ed on the basis that /is 1 cm and c (concentration of a
medicament) is 1 w/v%, is called specific absorbance, and is
expressed as E 1 ^.
-*-■ 1cm
CX/
/ : Length of the layer of the solution (cm)
A : Absorbance value
c: Concentration of the sample in the solution (w/v%)
The description of, for example, "E 1 ^ (241 nm): 500 -
530 (after drying, 2 mg, methanol, 200 mL)" in the mono-
graph, indicates that observed E 1 ^ value is between 500 and
530, when the test is performed in the following manner: The
sample is dried under the conditions specified in the Test for
Loss on Drying, and about 2 mg of the sample is weighed ac-
curately with a microbalance, and dissolved in methanol to
make exactly 200 mL, then the absorbance of the solution is
measured as directed in the Procedure at a wavelength of 241
nm using a cell with a path length of 1 cm.
Identification
Prepare the sample solution as directed in the monograph,
and test as directed in the Procedure. Usually, the test is per-
formed by a single method or in a combination of a few
methods in the following methods using the absorbance or
absorption spectrum obtained from the sample solution.
Subtle differences in the absorption spectrum arising from
differences in the apparatus used may be neglected.
(1) Identification using Reference Spectrum
When the absorption spectrum obtained from the sample
solution exhibits similar intensities of absorption at the same
wavelengths as those of the Reference Spectrum, the identity
of the sample and the reference may be confirmed. In this
case, the range of the wavelength to be compared is the range
shown on the Reference Spectrum.
Reference spectrum: Reference spectra are specified under
the Ultraviolet-visual Reference Spectra, which are used as
the reference for the test of identification specified in the
monograph.
(2) Identification using Reference Standard
When the absorption spectrum obtained from the sample
solution exhibits similar intensities of absorption at the same
wavelengths as those of the spectrum obtained from the
Reference Standard, the identity of the sample and the refer-
ence may be confirmed. In this case, the range of the
wavelength to be compared is the range shown on the Refer-
JPXV
General Tests / Infrared Spectrophotometry
43
ence Spectrum. When the relevant Reference Spectrum is not
available, the range is that specified in the monograph.
(3) Identification using absorption wavelength
When maximum absorption wavelengths of the spectrum
obtained from the sample solution match the wavelengths
specified in the monograph, the identity of the substance may
be confirmed. In this case, the range of the wavelength to be
compared is the range shown on the Reference Spectrum.
(4) Identification using the ratio of the absorbances ob-
tained at two or more wavelengths
When the ratios of absorbances at the specified
wavelengths in the spectrum obtained from the sample solu-
tion meet the specifications in the monograph, the identity of
the substance may be confirmed.
Assay
Prepare the control solution, the sample solution and the
standard solution as directed in the monograph, measure the
absorbances of the sample solution and the standard solution
according to the method described in the Procedure, and de-
termine the amount of the substance to be assayed in the
sample by comparing the absorbances.
2.25 Infrared Spectrophotometry
Infrared Spectrophotometry is a method of measurement
of the extent, at various wave numbers, of absorption of in-
frared radiation when it passes through a layer of a sub-
stance. In the graphic representation of infrared spectra, the
plot usually shows units of wave numbers as the abscissa and
units of transmittance or absorbance as the ordinate. Wave
number and transmittance or absorbance at each absorption
maximum may be read graphically on an absorption spec-
trum and/or obtained by a data-processor. Since the wave
number and the respective intensity of an absorption maxi-
mum depend on the chemical structure of a substance, this
measurement can be used to identify or determine a sub-
stance.
Instrument and adjustment
Several models of dispersive infrared spectrophotometers
or Fourier-transform infrared spectrophotometers are availa-
ble.
The instruments, adjusted according to the instruction
manual of each individual instrument, should comply with
the following test for resolving power, transmittance repro-
ducibility and wave number reproducibility. When the spec-
trum of a polystyrene film about 0.04 mm thick is recorded,
the depth of the trough from the maximum absorption at
about 2850 cm -1 to the minimum at about 2870 cm -1 should
be not less than 18% transmittance and that from the maxi-
mum at about 1583 cm -1 to the minimum at about 1589
cm -1 should be not less than 12% transmittance.
The wave number (cm -1 ) scale is usually calibrated by the
use of several characteristic absorption wave numbers (cm -1 )
of a polystyrene film shown below. The number in paren-
theses indicates the permissible range.
3060.0 (±1.5)
1601.2 (±1.0)
1028.3 (±1.0)
2849.5 (±1.5)
1583.0 (±1.0)
1942.9 (±1.5)
1154.5 (±1.0)
When the dispersive infrared spectrophotometer is used,
the permissible range of the absorption wave numbers at
1601.2 cm -1 and at 1028.3 cm -1 should be both within ±2.0
cm -1 .
As the repeatability of transmittance and wave number,
the difference of transmittance should be within 0.5% when
the spectrum of a polystyrene film is measured twice at sever-
al wave numbers from 3000 to 1000 cm -1 , and the difference
of wave number should be within 5 cm -1 at about 3000 cm -1
and within 1 cm -1 at about 1000 cm -1 .
Preparation of samples and measurement
Unless otherwise specified, when it is directed to perform
the test "after drying the sample", use a sample dried under
the conditions specified in the monograph. Prepare the speci-
men for the measurement according to one of the following
procedures so that the transmittance of most of the absorp-
tion bands is in the range of 5% to 80% . Single crystals of so-
dium chloride, potassium bromide, etc. are available for the
optical plate. Generally, the reference cell or material is
placed in the reference beam for double-beam instruments,
while for single-beam instruments, it is placed in the same op-
tical path in place of the specimen and measured separately
under the same operating conditions. The composition and
preparation of the reference depend on the sample prepara-
tion methods, and sometimes the background absorption of
the atmosphere can be utilized.
Unless otherwise specified in the monograph, the spectrum
is usually recorded between 4000 cm -1 and 400 cm -1 . The
spectrum should be scanned using the same instrumental con-
ditions as were used to ensure compliance with the require-
ments for the resolving power and for the precision of wave
number scale and of wave numbers.
(1) Potassium bromide disk or potassium chloride disk
method — Powder 1 to 2 mg of a solid sample in an agate
mortar, triturate rapidly with 0.10 to 0.20 g of potassium
bromide or potassium chloride for infrared spectrophotomet-
ry with precautions against moisture absorption, and com-
press the mixture with a press in a suitable die (disk-forming
container) to make the sample disk. If necessary to obtain a
transparent disk, press the mixture under vacuum in a die
with pressure applied to the die of 50 to 100 kN per cm 2 for 5
to 8 minutes. Prepare a potassium bromide reference disk or
a potassium chloride reference disk in the same manner as the
sample disk.
(2) Solution method — Place the sample solution prepared
by the method directed in each monograph in a fixed cell for
liquid, and usually measure the spectrum against the refer-
ence solvent used for preparing the sample solution. The sol-
vent used in this method should not show any interaction or
chemical reaction with the specimen to be examined and
should not damage the optical plate. The thickness of the fix-
ed cell is usually 0.1 mm or 0.5 mm.
(3) Paste method — Powder 5 to 10 mg of a solid speci-
men in an agate mortar, and, unless otherwise specified,
triturate the specimen with 1 to 2 drops of liquid paraffin to
give a homogeneous paste. After spreading the paste to make
a thin film in the center of an optical plate, place the plate
upon another optical plate with precautions against intrusion
of air, bubbles in the film, and examine its absorption spec-
trum.
(4) Liquid film method — Examine 1 to 2 drops of a liquid
specimen as a thin film held between two optical plates. When
the absorption intensity is not sufficient, place spacers of
44
Loss on Drying Test / General Tests
JP XV
aluminum foil, etc., between the two optical plates to make a
thicker liquid film.
(5) Film method — Examine a thin film just as it is or a
prepared thin film as directed in each monograph.
(6) Gas sampling method — Put a sample gas in a gas cell
previously evacuated under the pressure directed in the
monograph, and examine its absorption spectrum. The path
length of the gas cell is usually 5 cm or 10 cm, but, if necessa-
ry, may exceed 1 m.
(7) ^477? method — Place a specimen in close contact with
an attenuated total reflectance (ATR) prism, and examine its
reflectance spectrum.
(8) Diffuse reflectance method — Powder 1 to 3 mg of a
solid specimen into a fine powder of not more than about 50
/um particle size in an agate mortar, and triturate rapidly with
0.05 to 0.10 g of potassium bromide or potassium chloride
for infrared spectrophotometry with precautions against
moisture absorption. Place the mixture in a sample cup, and
examine its reflectance spectrum.
Identification
When the spectrum of a specimen and the Reference Spec-
trum of the substance expected to be found or the spectrum
of the Reference Standard exhibit similar intensities of ab-
sorption at the same wave numbers, the specimen can be
identified as being the substance expected to be found. Fur-
thermore, when several specific absorption wave numbers are
specified in the monograph, the identification of a specimen
with the substance expected to be found can be confirmed by
the appearance of absorption bands at the specified wave
numbers.
(1) Identification by the use of a Reference Standard
When the spectra of a specimen and the Reference Stan-
dard exhibit similar intensities of absorption at the same
wave numbers, the specimen can be identified as being the
same substance as the Reference Standard. When a sample
treatment method for a solid specimen is indicated in the
monograph in the case of nonconformity of the spectrum
with that of the Reference Standard, treat the specimen being
examined and the Reference Standard in the same manner as
directed in the monograph, then repeat the measurement.
(2) Identification by the use of a Reference Spectrum
When the spectra of a specimen and the Reference Spec-
trum exhibit similar intensities of absorption at the same
wave numbers, the specimen can be identified as being the
same substance associated with the Reference Spectrum.
When a sample treatment method for a solid specimen is indi-
cated in the monograph in the case of nonconformity of the
spectrum with the Reference Spectrum, treat the specimen
being examined as directed in the monograph, then repeat the
measurement.
(3) Identification by the use of absorption wave number
When several specific absorption wave numbers of the sub-
stance being examined are specified in the monograph, a
specimen can be identified as being the same substance as the
expected substance by confirmation of clear appearance of
the absorption bands at all the specified wave numbers.
Reference spectra
Infrared Reference Spectra, in the range between 4000
cm ' and 400 cm -1 , are shown at the end of this book for the
monographs requiring the identification test by Infrared
Spectrophotometry <2.25>, except for monographs in which
"Identification by absorption wave number" is specified.
Other Physical Methods
2.41 Loss on Drying Test
Loss on Drying Test is a method to measure the loss in
mass of the sample, when dried under the conditions specified
in each monograph. This method is applied to determine the
amount of water, all or a part of water of crystallization, or
volatile matter in the sample, which is removed during the
drying.
The description, for example, "not more than 1.0% (1 g,
105°C, 4 hours)" in a monograph, indicates that the loss in
mass is not more than 10 mg per 1 g of the substance in the
test in which about 1 g of the substance is accurately weighed
and dried at 105 °C for 4 hours, and "not more than 0.5% (1
g, in vacuum, phosphorus (V) oxide, 4 hours)," indicates
that the loss in mass is not more than 5 mg per 1 g of the sub-
stance in the test in which about 1 g of the substance is ac-
curately weighed, transferred into a desiccator (phosphorus
(V) oxide), and dried in vacuum for 4 hours.
Procedure
Weigh accurately a weighing bottle that has been dried for
30 minutes according to the method specified in the mono-
graph. Take the sample within the range of ± 10% of the
amount directed in the monograph, transfer into the weigh-
ing bottle, and, unless otherwise specified, spread the sample
so that the layer is not thicker than 5 mm, then weigh it ac-
curately. Place the loaded bottle in a drying chamber, and
dry under the conditions specified in the monograph. When
the size of the sample is large, convert it to small particles
having a size not larger than 2 mm in diameter by quick
crushing, and use the crushed sample for the test. After
drying, remove from the drying chamber, and reweigh ac-
curately. When the sample is dried by heating, the tempera-
ture is within the range of ±2°C of that directed in the
monograph, and, after drying the bottle, the sample is al-
lowed to cool in a desiccator (silica gel) before weighing.
If the sample melts at a temperature lower than that speci-
fied in the monograph, expose the sample for 1 to 2 hours to a
temperature between 5°C and 10°C below the melting tem-
perature, dry under the conditions specified in the mono-
graph. Use a desiccant specified in the monograph, and renew
frequently.
2.42 Congealing Point
Determination
The congealing point is the temperature measured by the
following method.
Apparatus
Use the apparatus illustrated in Fig. 2.42-1.
Procedure
Transfer the sample into sample container B up to the
marked line C. When the sample is solid, melt the sample by
heating to a temperature not higher than 20 °C above the
expected congealing point, and transfer to B. Fill the glass or
JPXV
General Tests / Residue on Ignition Test
45
*-D
The figures are in mm.
A: Cylinder made of glass (the tube is painted with silicone oil
on both sides of the wall to prevent clouding),
B: Sample container (a hard glass test tube, which is painted
wild silicone oil to prevent clouding, except a! the region of
the wall in contact with the sample; insert it into cylinder A,
and fix with cork stopper).
C: A marked line.
D: Bath made of glass or plastics.
E: Stirring rod made ol glass or stainless steel (3 mm in diam-
eter, the lower end part of it is bent to make a loop, about
18 mm in diameter).
F: Thermometer with an immersion line.
G: Thermometer with an immersion line or a total immersion
thermometer.
II: Immersion line
Fig. 2.42-1
plastic bath D with water at a temperature about 5°C below
the expected congealing point. When the sample is liquid at
room temperature, fill bath D with water at a temperature be-
tween 10°C and 15°C lower than the expected congealing
point.
Insert the sample container B containing the sample into
cylinder A. Adjust the immersion line H of thermometer F to
the same level of the meniscus of the sample. After cooling
the sample to about 5°C above the expected congealing
point, move vertically the stirrer E at the rate of about 60 to
80 strokes per minute, and observe the thermometer readings
at 30-second intervals. The temperature falls gradually. Dis-
continue stirring, when an appreciable amount of crystals has
formed and the temperature is constant or has begun to rise.
Usually, read the maximum temperature (reading of F), that
is constant for a while after a rise of temperature. If no rise of
temperature occurs, read the temperature that is constant for
a while. The average of not less than four consecutive read-
ings that lie within a range of 0.2°C constitutes the congeal-
ing point.
Note: If a state of super cooling is anticipated, rub the in-
ner wall of bath B or put a small fragment of the solid sample
into bath B for promoting the congealment, when the tem-
perature approaches near the expected congealing point.
2.43 Loss on Ignition Test
Loss on Ignition Test is a method to measure the loss in
mass when the sample is ignited under the conditions speci-
fied in each monograph. This method is usually applied to in-
organic drugs which lose a part of the components or impuri-
ties during ignition.
The description, for example, "40.0-52.0% (1 g, 450 -
550°C, 3 hours)" in a monograph, indicates that the loss in
mass is 400 to 520 mg per g of the substance in the test in
which about 1 g of the substance is weighed accurately and ig-
nited between 450°C and 550°C for 3 hours.
Procedure
Previously ignite a crucible or a dish of platinum, quartz or
porcelain to constant mass, at the temperature directed in the
monograph, and weigh accurately after cooling.
Take the sample within the range of ± 10% of the amount
directed in the monograph, transfer into the above ignited
container, and weigh it accurately. Ignite under the condi-
tions directed in the monograph, and, after cooling, reweigh
accurately. Use a desiccator (silica gel) for the cooling.
2.44 Residue on Ignition Test
This test is harmonized with the Sulphated Ash Test of the
European Pharmacopoeia and the Residue on Igntion Test of
the U. S. Pharmacopeia. The parts of the text that are not
harmonized are marked with symbols (* ♦).
*The Residue on Ignition Test is a method to measure the
amount of residual substance not volatilized from a sample
when the sample is ignited in the presence of sulfuric acid ac-
cording to the procedure described below. This test is usually
used for determining the content of inorganic impurities in an
organic substance.
The description, for example, "not more than 0.1% (1
g)", in a monograph, indicates that the mass of the residue is
not more than 1 mg per 1 g of the substance in the test in
which about 1 g of the substance is weighed accurately and ig-
nited by the procedure described below, and "after drying"
indicates that the sample is tested after being dried under the
conditions specified in the test for Loss on drying. »
Procedure
Ignite a suitable crucible (for example, silica, platinum,
quartz or porcelain) at 600±50°C for 30 minutes, cool the
crucible in a desiccator (silica gel or other suitable desiccant)
and weigh it accurately.
Take the amount of test sample specified in the individual
monograph in the crucible and weigh the crucible accurately.
Moisten the sample with a small amount (usually 1 mL) of
sulfuric acid, then heat gently at a temperature as low as
practicable until the sample is thoroughly charred. After
cooling, moisten the residue with a small amount (usually 1
mL) of sulfuric acid, heat gently until white fumes are no Ion-
46
Refractive Index Determination / General Tests
JP XV
ger evolved, and ignite at 600 ± 50°C until the residue is com-
pletely incinerated. Ensure that flames are not produced at
any time during the procedure. Cool the crucible in a desicca-
tor (silica gel or other suitable desiccant), weigh accurately
and calculate the percentage of residue.
Unless otherwise specified, if the amount of the residue so
obtained exceeds the limit specified in the individual mono-
graph, repeat the moistening with sulfuric acid, heating and
ignition as before, using a 30-minute ignition period, until
two consecutive weighings of the residue do not differ by
more than 0.5 mg or until the percentage of residue complies
with the limit in the individual monograph.
2.45 Refractive Index
Determination
Refractive Index Determination is a method to measure the
ratio of the velocity of light in air to that in the sample.
Generally, when light proceeds from one medium into
another, the direction is changed at the boundary surface.
This phenomenon is called refraction. When light passes
from the first isotropic medium into the second, the ratio of
the sine of the angle of incidence, i, to that of the angle of
refraction, r, is constant with regard to these two media and
has no relation to the angle of incidence. This ratio is called
the refractive index of the second medium with respect to the
first, or the relative refractive index, n.
The refractive index obtained when the first medium is a
vacuum is called the absolute refractive index, N, of the
second medium.
In isotropic substances, the refractive index is a charac-
teristic constant at a definite wavelength, temperature, and
pressure. Therefore, this measurement is applied to purity
test of substances, or to determination of the composition of
homogeneous mixtures of two substances.
The measurement is usually carried out at 20°C, and the D
line of the sodium spectrum is used for irradiation. This value
is expressed as ri^.
Procedure
For the measurement of refractive index, usually the Abbe
refractometer is used at a temperature in the range of ± 0.2
°C of that directed in the monograph. Use of the Abbe
refractometer permits direct reading of n D under incandes-
cent light, with a measurable range from 1.3 to 1.7, and an
attainable precision of 0.0002.
2.46 Residual Solvents Test
Residual Solvents Test is a test to determine the amounts of
residual organic solvents in pharmaceuticals by using the gas
chromatography to monitor adherence to the limits which are
advised for the safety of patients by "Guideline for Residual
Solvents: ICH Harmonized Tripartite Guideline".
Unless otherwise specified, the limit of the residual solvents
is described in ppm in the individual monograph, and unless
otherwise specified, the limit should be not more than the
limit advised in the Guideline.
Apparatus, Procedure, and Test Method
Prepare the sample solution and the standard solution as
directed in the relevant monograph, and perform the test as
directed under Gas Chromatography <2.02>.
In monographs, the quantity for the test of sample and
reference standard (reference substances), the method for
preparation of the sample and standard solutions, the injec-
tion amount of the sample and standard solutions for the gas
chromatography, the operating conditions for the head-space
apparatus and the gas chromatography, the system suitabili-
ty, the calculation formula, and other items concerning the
test are specified.
2.47 Osmolarity Determination
Osmolarity Determination is a method for measuring the
osmotic concentration of the sample solution from the extent
of the freezing-point depression.
When a solution and a pure solvent are separated by a
semipermeable membrane, through which the solvent can
pass freely, but the solute cannot, a part of the solvent passes
into the solution compartment through the membrane. The
pressure difference produced between the two compartments
concomitantly with the solvent migration through the mem-
brane, is defined as the osmotic pressure II (Pa). The osmotic
pressure is a physical quantity depending on the total of the
molecular species present, including neutral molecules and
ions, and does not depend on the kind of solute. A solution
property, such as osmotic pressure, freezing-point depres-
sion, boiling-point elevation etc., which depends not on the
kind of solute, but on the total number of all molecular spe-
cies, is called a colligative property of a solution.
The osmotic pressure of a polymer solution can be meas-
ured directly as the hydrostatic pressure difference between
two compartments separated by a semipermeable membrane,
such as a cellulose membrane. However, this is not applicable
to a solution containing low molecular species, which can
pass through a semipermeable membrane. Though the os-
motic pressure of such a solution cannot be measured direct-
ly, the direction and extent of solvent migration through bio-
logical membranes can be predicted from the total number of
all molecular species present when the solution is placed un-
der physiological conditions. Other colligative properties of a
solution such as freezing-point depression, boiling-point ele-
vation, vapor-pressure depression, etc. can be directly ob-
tained by observing changes of temperature and/or pressure,
etc. These solution properties depend on the total number of
ionic and neutral species in the solution in the same way as
the osmotic pressure, and the molecular particle concentra-
tion is defined as the osmotic concentration. The osmotic
concentration can be defined in two ways, one being mass-
based concentration (osmolality, mol/kg) and the other,
volume-based concentration (osmolarity, mol/L). In prac-
tice, the latter is more convenient.
Unless otherwise specified, the freezing-point depression
method is used for measuring the osmotic concentration. The
method is based on the linear dependency of the freezing-
point depression A T (°C) upon the osmolality in (mol/kg), as
expressed in the following equation,
JPXV
General Tests / Water Determination (Karl Fischer Method)
47
AT = Km
In this equation, K is the molal freezing-point depression
constant, and it is known to be 1.86°C kg/mol for water.
Since the constant K is defined on the basis of molarity, the
molar osmotic concentration can be obtained from the above
equation. In the dilute osmotic concentration range, os-
molality m (mol/kg) can be assumed to be numerically equal
to osmolarity c (mol/L). Thus, the conventional osmolarity
(mol/L) and the unit of osmole (Osm) are adopted in this test
method. One Osm means that the Avogadro number (6.022
x 10 23 /mol) of species is contained in 1 L of solution. Usually
the osmotic concentration is expressed as the submultiple mil-
liosmole (mOsm, mosmol/L) in the Pharmacopoeia.
Apparatus
Usually, the osmotic concentration of a solution can be ob-
tained by measuring the extent of the freezing-point depres-
sion. The apparatus (osmometer) is composed of a sample
cell for a fixed volume of sample solution and a cell holder, a
cooling unit and bath with a temperature regulator, and a
thermistor for detecting temperature.
Procedure
A fixed volume of the test solution is introduced into the
sample cell, as indicated for the individual apparatus.
The apparatus must first be calibrated by the two-point
calibration method by using osmolal standard solutions. For
the calibration, select two different standard solutions just
covering the expected osmolar concentration of a sample so-
lution. Other than the indicated osmolal standard solutions
in the Table below, water can also be used as a standard solu-
tion (0 mOsm) for measuring low osmolar sample solutions
(0 - 100 mOsm). Next, after washing the sample cell and the
thermistor as indicated for the individual apparatus, measure
the degree of the freezing-point depression caused by a sam-
ple solution. Using the above-mentioned relation of osmolar
concentration m and A T, the osmolarity of a sample solution
can be obtained, and it is assumed to be numerically equal to
the osmolarity.
In the case of higher osmolar solutions over 1000 mOsm,
dilute the sample by adding distilled water and prepare n
times diluted sample solution (1 in ri). Measure the osmolari-
ty of the diluted solution, as described above. In this case, it
is necessary to state that the calculated osmolarity for the
sample (see below) is an apparent osmolarity obtained by the
dilution method. When the dilution method is applied, the di-
lution number should be selected so that the expected os-
molarity is nearly equal to that of physiological saline solu-
tion.
In the case of solid samples, such as freeze-dried medi-
cines, prepare a sample solution by dissolving the solid using
the indicated solution for dissolution.
Suitability of the apparatus
After the calibration of the apparatus, a suitability test
must be done by repeating the measurement of osmolarity for
one of the standard solutions not less than 6 times. In per-
forming the test, it is advisable that the osmolarity of a sam-
ple solution and the selected standard solution are similar to
each other. In this test, the repeatability of measured values
and the deviation of the average from the indicated value
should be less than 2.0% and 3.0%, respectively. When the
requirement is not met, calibrate the apparatus again by the
two-point calibration method, and repeat the test.
Preparation of the osmolar standard solutions
Weigh exactly an amount indicated in Table 2.47-1 of sodi-
um chloride (standard reagent), previously dried between 500
°C and 650°C for 40 to 50 minutes and allowed to cool in a
desiccator (silica gel). Dissolve the weighed sodium chloride
in exactly 100 g of water to make the corresponding osmolar
standard solution.
Table 2.47-1
Standard solution for osmometer
Amount of
calibration (milliosmoles)
sodium chloride (g)
100
0.309
200
0.626
300
0.946
400
1.270
500
1.593
700
2.238
1000
3.223
Osmolar ratio
In this test method the osmolar ratio is defined as the ratio
of osmolarity of a sample solution to that of the isotonic so-
dium chloride solution. The ratio can be used as a measure of
isotonicity of sample solution. Since the osmolarity of the
isotonic sodium chloride solution (NaCl 0.900 g/100 mL) c s
(mOsm) is assumed to be constant (286 mOsm), the osmolar
ratio of a sample solution, of which the osmolarity is c T
(mOsm), can be calculated by means of the following equa-
tion,
Osmolar ratio =
Cs
c s : 286 mOsm
When the measurement is done by the dilution method, be-
cause the sample has an osmolarity over 1000 mOsm, the ap-
parent osmolarity of the sample solution c T can be calculated
as n ■ c' T = c T , in which n is the dilution number and c' T is the
measured osmolarity for the diluted solution. In this calcula-
tion, a linear relation between osmolarity and solute concen-
tration is assumed. Thus when the dilution measurement is
performed, the dilution number must be stated as (1 in ri).
2.48 Water Determination
(Karl Fischer Method)
Water Determination is a method to determine water con-
tent in sample materials, utilizing the fact that water reacts
with iodine and sulfur dioxide quantitatively in the presence
of a lower alcohol such as methanol, and an organic base
such as pyridine. The reaction proceeds in the manner shown
in the following equation:
I 2 + S0 2 + 3C 5 H 5 N + CH 3 OH + H 2
-» 2(C 5 H 5 N + H)I- +(C 5 H 5 N + H)-OS0 2 OCH 3
In this measurement there are two methods different in io-
dine-providing principle: one is the volumetric titration
method and the other, the coulometric titration method. In
the former, iodine is previously dissolved in a reagent for
water determination, and water content is determined by
48
Water Determination (Karl Fischer Method) / General Tests
JP XV
measuring the amount of iodine consumed as a result of reac-
tion with water. In the latter, iodine is produced by electroly-
sis of Karl Fisher reagent containing iodide ion. Based on the
quantitative reaction of the generated iodine with water, the
water content in a sample specimen can be determined by
measuring the quantity of electricity which is required for the
production of iodine during the titration.
21-
I 2 + 2e-
1. Volumetric titration
Apparatus
Generally, the apparatus consists of automatic burettes, a
titration flask, a stirrer, and equipment for amperometric
titration at constant voltage or potentiometric titration at
constant current.
The Karl Fischer TS is extremely hygroscopic, so the ap-
paratus should be designed to be protected from atmospheric
moisture. Desiccants such as silica gel or calcium chloride for
water determination are used for moisture protection.
Reagents
(1) Chloroform for water determination — To 1000 mL of
chloroform add 30 g of synthetic zeolite for drying, stopper
tightly, allow to stand for about 8 hours with occasional gen-
tle shaking, then allow to stand for about 16 hours, and col-
lect the clear layer of chloroform. Preserve the chloroform,
protecting it from moisture. The water content of this chlo-
roform should not be more than 0.1 mg per mL.
(2) Methanol for water determination — To 1000 mL of
methanol add 30 g of synthetic zeolite for drying, stopper
tightly, allow to stand for about 8 hours with occasional gen-
tle shaking, then allow to stand for about 16 hours, and col-
lect the clear layer of methanol. Preserve the methanol, pro-
tecting it from moisture. The water content of this methanol
should not be more than 0.1 mg per mL.
(3) Propylene carbonate for water determination — To
1000 mL of propylene carbonate add 30 g of synthetic zeolite
for drying, stopper tightly, allow to stand for about 8 hours
with occasional gentle shaking, then allow to stand for about
16 hours, and collect the clear propylene carbonate layer.
Preserve this protecting from moisture. The water content
should not be more than 0.3 mg per mL.
(4) Diethylene glycol monoethyl ether for water deter-
mination — To 1000 mL of diethylene glycol monoethyl ether
add 30 g of synthetic zeolite for drying, stopper tightly, allow
to stand for about 8 hours with occasional gentle shaking,
then allow to stand for about 16 hours, and collect the clear
layer of diethylene glycol monoethyl ether. Preserve the
diethylene glycol monoethyl ether, protecting it from
moisture. The water content of this diethylene glycol
monoethyl ether should not be more than 0.3 mg per mL.
(5) Pyridine for water determination — Add potassium
hydroxide or barium oxide to pyridine, stopper tightly, and
allow to stand for several days. Distill and preserve the puri-
fied and dried pyridine, protecting it from moisture. The
water content of this pyridine should not be more than 1 mg
per mL.
(6) Imidazole for water determination — Use imidazole
for thin-layer chromatography, of which the water content
should not be more than 1 mg per g.
(7) 2-Methylaminopyridine for water determination —
Distill and preserve 2-methylaminopyridine, protecting it
from moisture. The water content of this 2-methylaminopyri-
dine should not be more than 1 mg per mL.
Preparation of test solutions and standard solutions
(1) Karl Fischer TS for water determination
Prepare according to the following method (i), (ii) or (iii).
(i) Preparation 1
Dissolve 63 g of iodine in 100 mL of pyridine for water de-
termination, cool the solution in ice bath, and pass dried sul-
fur dioxide gas through this solution until the mass increase
of the solution reaches 32 g. Then make up to 500 mL by
adding chloroform for water determination or methanol for
water determination, and allow to stand for more than 24
hours before use.
(ii) Preparation 2
Dissolve 102 g of imidazole for water determination in 350
mL of diethylene glycol monoethyl ether for water determi-
nation, cool the solution in ice bath, and pass dried sulfur di-
oxide gas through this solution until the mass increase of the
solution reaches 64 g, keeping the temperature between 25 °C
and 30°C. Then dissolve 50 g of iodine in this solution, and
allow to stand for more than 24 hours before use.
(iii) Preparation 3
Pass dried sulfur dioxide gas through 220 mL of propylene
carbonate until the mass increase of the solvent reaches 32 g.
To this solution, cooled in ice bath, add 180 mL of propylene
carbonate or diethylene glycol monoethyl ether for water de-
termination, in which 81 g of 2-methylaminopyridine for
water determination is dissolved. Then dissolve 36 g of iodine
in this solution, and allow to stand for more than 24 hours
before use.
The Karl Fischer TS, prepared by any one of the methods
described above, must be standardized before every use, be-
cause its activity for water determination changes with the
lapse of time. Further preserve the TS in a cold place, pro-
tecting it from light and moisture.
Standardization — According to the procedure described
below, take a suitable quantity of methanol for water deter-
mination in a dried titration flask, and titrate the solvent with
a Karl Fischer TS to make the content of the flask anhydrous.
Then, add quickly about 30 mg of water weighed accurately
to the solution in the flask, and titrate the water dissolved in
the solvent with a Karl Fischer TS to the end point, under
vigorous stirring. Calculate the water equivalence factor,
/(mg/mL), corresponding to the amount of water (H 2 0) in
mg per 1 mL of the test solution by using the following equa-
tion:
/(mg/mL) =
Amount of water (H 2 0) (mg)
Volume of Karl Fischer TS consumed
for titration of water (H 2 0) (mL)
(2) Standard water-methanol solution
Preparation — Take 500 mL of methanol for water deter-
mination in a dried 1000-mL volumetric flask, add 2.0 mL of
water, and adjust with the methanol to make 1000 mL.
Perform the standardization of this solution, followed by
the procedure for Karl Fischer TS. Preserve it in a cool place,
protecting it from light and moisture.
Standardization — According to the procedure described
below, take a suitable quantity of methanol for water deter-
mination in a dried titration flask, and titrate the water con-
taminated with Karl Fischer TS to make the content of the
flask anhydrous. Then, add exactly 10 mL of Karl Fischer TS
to this solution in the flask, and titrate it with the prepared
JP XV
General Tests / Water Determination (Karl Fischer Method)
49
standard water-methanol solution to the end point. Calculate
the water concentration in the standard water-methanol solu-
tion, /'(mg/mL), by using the following equation:
/'(mg/mL) =
/(mg/mL) x 10 (mL)
Volume of the standard water-methanol
solution consumed for titration (mL)
Procedure
As a rule, the titration of water with a Karl Fischer TS
should be performed at the same temperature as that at which
the standardization was done, with protection from
moisture. The apparatus is equipped with a variable resistor
in the circuit, and this resistor is manipulated so as to main-
tain a constant voltage (mV) between two platinum electrodes
immersed in the solution to be titrated. The variable current
(/uA) can be measured (Amperometric titration at constant
voltage). During titration with Karl Fischer TS, the current in
the circuit varies noticeably, but returns to the original value
within several seconds. At the end of a titration, the current
stops changing and persists for a certain time (usually, longer
than 30 seconds). When this electric state has been attained, it
is designated as the end point of titration.
Otherwise, the manipulation of the resistor serves to pass a
definite current between two platinum electrodes. The varia-
ble potential (mV) can be measured (Potentiometric titration
at constant current). With the progress of titration of water
with a Karl Fischer TS, the value indicated by the potentiom-
eter in the circuit decreases suddenly from a polarization state
of several hundreds (mV) to the non-polarization state, but it
returns to the original value within several seconds. At the
end of titration, the non-polarization state persists for a cer-
tain time (usually, longer than 30 seconds). When this electric
state has been attained, it is designated as the end point of
titration.
In the case of back titration, when the amperometric titra-
tion method is used at constant voltage, the needle of
microammeter is out of scale during excessive presence of
Karl Fischer TS, and it returns rapidly to the original position
when the titration system has reached the end point. In the
case of the potentiometric titration method at constant cur-
rent in the back titration mode, the needle of the mil-
livoltmeter is at the original position during excessive
presence of Karl Fischer TS. Finally a definite voltage is indi-
cated when the titration system has reached the end point.
Unless otherwise specified, the titration of water with Karl
Fischer TS can be performed by either one of the following
methods. Usually, the end point of the titration can be ob-
served more clearly in the back titration method, compared
with the direct titration method.
(1) Direct titration
Unless otherwise specified, proceed by the following
method. Take a suitable quantity of methanol for water de-
termination in a dried titration flask, and titrate the water
contaminated with Karl Fischer TS to make the content of
the flask anhydrous. Take a quantity of sample specimen
containing 5 to 30 mg of water, transfer it quickly into the
titration flask, dissolve by stirring, and titrate the solution to
be examined with Karl Fischer TS to the end point under
vigorous stirring.
In the case of an insoluble sample specimen, powder the
sample quickly, weigh a suitable amount of the sample con-
taining 5 to 30 mg of water, and transfer it quickly into the
titration vessel, stir the mixture for 5-30 minutes, protecting
it from moisture, and perform a titration under vigorous stir-
ring. Alternatively, in the case of a sample specimen which is
insoluble in the solvent for water determination or which in-
terfere with the Karl Fisher reaction, water in the sample can
be removed by heating under a stream of nitrogen gas, and
introduced into the titration vessel by using a water evapora-
tion technique.
Though the titration procedure should be performed under
atmospheric conditions at low humidity, if the effect of at-
mospheric moisture cannot be avoided, for instance, if a long
time is required for extraction and titration of water, a blank
test must be done under the same conditions as used for the
sample test, and the data must be corrected, accordingly.
Water (H 2 0) %
Volume of Karl Fischer
TS consumed for x/(mg/mL)
titration (mL)
Amount of sample (mg)
x 100
(2) Back titration
Unless otherwise specified, proceed by the following
method. Take a suitable quantity of methanol for water de-
termination in the dried titration vessel, and titrate the water
contaminated with Karl Fischer TS to make the content of
the flask anhydrous. Take a suitable quantity of sample speci-
men having 5-30 mg of water, transfer the sample quickly
into the titration vessel, dissolve it in the solution by stirring,
add an excessive and definite volume of Karl Fischer TS, and
then titrate the solution with the standard water-methanol so-
lution to the end point under vigorous stirring.
In the case of an insoluble sample specimen, powder the
sample quickly, weigh a suitable amount accurately, transfer
it quickly into the titration vessel, and add an excessive and
definite volume of Karl Fischer TS. After stirring for 5-30
minutes, with protection from moisture, perform the titra-
tion under vigorous stirring.
Water (H 2 0) %
Volume of Karl
Fischer TS
added (mL)
x/(mg/mL)
Volume of the standard water-
methanol solution consumed
. for titration (mL)
x/' (mg/mL)
Amount of sample (mg)
X100
2. Coulometric titration
Apparatus
Usually, the apparatus is comprised of a titration flask
equipped with an electrolytic cell for iodine production, a
stirrer, and a potentiometric titration system at constant cur-
rent. The iodine production system is composed of an anode
and a cathode, separated by a diaphragm. The anode is im-
mersed in the anolyte solution for water determination and
the cathode is immersed in the catholyte solution for water
determination. Both electrodes are usually made of platinum-
mesh.
Because both the anolyte and the catholyte solutions for
water determination are strongly hygroscopic, the titration
system should be protected from atmospheric moisture. For
this purpose, silica gel or calcium chloride for water determi-
nation can be used.
50
Optical Rotation Determination / General Tests
JP XV
Preparation of anolyte and catholyte solutions for water
determination
Electrolytic solutions shall consist of an anolyte solution
and a catholyte solution, the preparations of which are
described below.
Preparation — Any of methods (1), (2), and (3) can be used
for the preparation of the electrolytes for coulometric titra-
tion.
(1) Preparation 1
Anolyte for water determination — Dissolve 102 g of imida-
zole for water determination in 900 mL of methanol for
water determination, cool the solution in ice bath, and pass
dried sulfur dioxide gas through the solution, which is kept
below 30°C. When the mass increase of the solution has
reached 64 g, the gas flow is stopped and 12 g of iodine is dis-
solved by stirring. Then drop a suitable amount of water into
the solution until the color of liquid is changed from brown
to yellow, and add methanol for water determination to
make up 1000 mL.
Catholyte for water determination — Dissolve 24 g of
diethanolamine hydrochloride in 100 mL of methanol for
water determination.
(2) Preparation 2
Anolyte for water determination — Dissolve 40 g of 1,3-
di(4-pyridyl)propane and 30 g of diethanolamine in about
200 mL of methanol for water determination, and pass dried
sulfur dioxide gas through the solution. When the mass in-
crease of the solution has reached 25 g, the gas flow is
stopped. Add 50 mL of propylene carbonate, and dissolve 6 g
of iodine in the solution. Then make up the solution to 500
mL by addition of methanol for water determination and
drop in a suitable amount of water until the color of liquid is
changed from brown to yellow.
Catholyte for water determination — Dissolve 30 g of cho-
line hydrochloride into methanol for water determination
and adjust the volume to 100 mL by adding the methanol.
(3) Preparation 3
Anolyte for water determination — Dissolve 100 g of
diethanolamine in 900 mL of methanol for water determina-
tion or a mixture of methanol and chloroform for water de-
termination^ : 1), and pass dried sulfur dioxide gas through
the solution. When the mass increase of the solution has
reached 64 g, the gas flow is stopped. Dissolve 20 g of iodine
in the solution, and drop in a suitable amount of water until
the color of liquid is changed from brown to yellow.
Catholyte for water determination — Dissolve 25 g of lithi-
um chloride in 1000 mL of a mixture of methanol for water
determination and nitromethane (4 : 1).
Procedure
Take a suitable volume of an anolyte for water determina-
tion in the titration vessel, immerse in this solution a pair of
platinum electrodes for potentiometric titration at constant
current. Then immerse the iodine production system filled
with a catholyte for water determination in the anolyte solu-
tion. Switch on the electrolytic system and make the content
of the titration vessel anhydrous. Next take an accurately
weighed amount of a sample specimen containing 0.2 - 5 mg
of water, add it quickly to the vessel, dissolve by stirring, and
perform the titration to the end point under vigorous stirring.
When a sample specimen cannot be dissolved in the ano-
lyte, powder it quickly, and add an accurately weighed
amount of the sample estimated to contain 0.2 - 5 mg of
water to the vessel. After stirring the mixture for 5-30
minutes, with protection from atmospheric moisture, per-
form the titration under vigorous stirring. Alternatively, in
the case of an insoluble solid or a sample containing a com-
ponent which interferes with the Karl Fisher reaction, water
in the sample can be removed by heating, and carried by a
nitrogen gas flow into the titration vessel, by using a water
evaporation technique.
Determine the quantity of electricity (C) [ = electric current
(A) x time (s)] required for the production of iodine during
the titration, and calculate the water content (%) in the sam-
ple specimen by use of the following equation.
Though the titration procedure should be performed under
atmospheric conditions at low humidity, if the effect of at-
mospheric moisture cannot be avoided, for instance, if a long
time is required for extraction and titration of water, a blank
test must be done under the same conditions as used for the
sample test, and the data must be corrected, accordingly.
Water (H 2 0) % =
Quantity of electricity required
for iodine production (C)
10.72 (C/mg)
x Amount of sample (mg)
X100
10.72: quantity of electricity corresponding to 1 mg of
water (C/mg)
2.49 Optical Rotation
Determination
Optical Rotation Determination is a method for the meas-
urement of the angular rotation of the sample using a
polarimeter.
Generally, the vibrations of light take place on planes per-
pendicular to the direction of the beam. In the case of ordina-
ry light, the directions of the planes are unrestricted. In the
case of plane polarized light, commonly designated as pola-
rized light, however, the vibrations take place on only one
plane that includes the direction of the beam (plane of polari-
zation). Some drugs in the solid state or in solution have the
property of rotating the plane of the polarized light either to
the right or to the left. This property is referred to as optical
activity or optical rotation, and is inherently related to the
chemical constitution of the substance.
The extent of the rotation, expressed in degrees of rotation
of the angle of the plane of polarized light caused by the opti-
cally active substance or its solution, is measured with a
polarimeter. This value is proportional to the length of the
polarimeter tube, and is related to the concentration of the
solution, the temperature and the wavelength. The character
of the rotation is indicated by placing a plus sign ( + ) for that
which rotates the plane of the polarized light to the right,
when facing the direction of the beam, referred to as dex-
trorotatory, or a minus sign ( — ) for that which rotates the
plane to the left, referred to as levorotatory, before the num-
ber indicating the degrees of rotation, as like as +20°, mean-
ing 20° to the right, or -20°, meaning 20° to the left.
The angular rotation a' x is that which is measured with
specific monochromatic light of x (described in terms of the
wavelength or the name) at a temperature of t°C Usually the
measurement is performed at 20°C, with a polarimeter tube
JPXV
General Tests / Endpoint Detection Methods in Titrimetry
51
of 100 mm in length, and with the D line of sodium as the
light source.
The specific rotation is represented by the following equa-
tion:
i«ii=
100,
Ic
t: The temperature of measurement.
x: The wavelength or the name of the specific monochro-
matic light of the spectrum used (in the case of the D
line, described as D).
a: The angle, in degrees, of rotation of the plane of the
polarized light.
/: The thickness of the layer of sample solution, i.e., the
length of the polarimeter tube (mm).
c: For the purpose of the Japanese Pharmacopoeia, the
number of grams of a drug present in 1 mL of the solu-
tion. When an intact liquid drug is used for determina-
tion, not in solution, c represents the density. However,
unless otherwise specified, the specific gravity is used in-
stead of the density.
The description, for example, "[a]p°: -33.0- —36.0° (af-
ter drying, 1 g, water, 20 mL, 100 mm)," in a monograph,
indicates that the [o\d is between - 33.0° and - 36.0° in the
determination in which the substance is dried under the con-
ditions described in the test for Loss on Drying, and about 1 g
of the substance is accurately weighed, and dissolved by add-
ing water to make exactly 20 mL, then the solution is meas-
ured with a polarimeter tube 100 mm in length.
2.50 Endpoint Detection Methods
in Titrimetry
Titrimetry is a method or a procedure for volumetric anal-
ysis, which is usually classified into acid-base titration (neu-
tralization titration or pH titration), precipitation titration,
complexation titration, oxidation-reduction titration, etc.,
according to the kind of reaction or the nature of the
phenomenon occurring between the titrate and the titrant
(standard solution for volumetric analysis). Furthermore,
titration performed in a nonaqueous solvent is generally
called nonaqueous titration, which is frequently used for
volumetric analysis of weak acids, weak bases, and their
salts. The endpoint in titrimetry can be detected by color
changes of indicators and/or by changes of electrical signals
such as electrical potential or electrical current.
The indicator method is one of the endpoint detection
methods in titrimetry. In this method the color of an indica-
tor dye, dissolved in the titrate, changes dramatically in the
vicinity of the equivalence point due to its physico-chemical
character, and this property is used for visual endpoint detec-
tion. Selection of an indicator and specification of the color
change induced in the respective titration system, should be
described in the individual monograph. An appropriate indi-
cator should change color clearly, in response to a slight
change in physico-chemical properties of the titrate, such as
pH, etc., in the vicinity of the equivalence point.
Regarding the electrical endpoint detection methods, there
are an electrical potential method and an electrical current
method, which are called potentiometric and amperometric
titration methods, respectively. They are generically named
electrometric titration. In the potentiometric titration
method, the endpoint of a titration is usually determined to
be the point at which the differential potential change
becomes maximum or minimum as a function of the quantity
of titrant added. In the amperometric titration method, un-
less otherwise specified, a bi-amperometric titration method
is used, and the endpoint is determined by following the
change of microcurrent during the course of a titration. Fur-
thermore, the quantity of electricity (electrical current x time)
is often used as another electrochemical signal to follow a
chemical reaction, as described in Coulometric Titration un-
der Water Determination <2.48>.
The composition of a titration system, such as amount of
specimen, solvent, standard solution for volumetric analysis,
endpoint detection method, equivalent amount of substance
to be examined (mg)/standard solution (mL), should be spe-
cified in the individual monograph. Standardization of the
standard solution and titration of a specimen are recom-
mended to be done at the same temperature. When there is a
marked difference in the temperatures at which the former
and the latter are performed, it is necessary to make an ap-
propriate correction for the volume change of the standard
solution due to the temperature difference.
Indicator Method
Weigh an amount of a specimen in a flask or a suitable ves-
sel as directed in the monograph or in "Standard Solutions
for Volumetric Analysis" , and add a specified quantity of
solvent to dissolve the specimen. After adding a defined indi-
cator to the solution to prepare the titrate, titrate by adding a
standard solution for volumetric analysis by using a buret. In
the vicinity of the endpoint, observe the color change induced
by the cautious addition of 0.1 mL or less of the titrant. Cal-
culate the quantity of titrant added from the readings on the
scale of the buret used for the titration at the starting point
and at the endpoint at which the specified color change ap-
pears, as directed in the individual monograph or in the
"Standard Solutions for Volumetric Analysis" . Although
addition of the volumetric standard solution by buret is
usually done manually, an automatic buret can also be used.
Unless otherwise specified, perform a blank determination
according to the following method, and make any necessary
correction.
Measure a specified quantity of solvent, as directed in the
monograph or in the "Standard Solutions for Volumetric
Analysis" , and titrate as directed. The required quantity of
the standard solution added to reach a specified color change,
is assumed to be the blank quantity for the titration system.
However, when the blank quantity is too small to evaluate ac-
curately, the quantity can be assumed to be zero.
Electrical Endpoint Detection Methods
1. Potentiometric titration
(1) Apparatus
The apparatus consists of a beaker to contain the speci-
men, a buret for adding a standard solution, an indicator
electrode and a reference electrode, a potentiometer for
measuring potential difference between the electrodes or an
adequate pH meter, a recorder, and a stirrer for gentle stir-
ring of the solution to be examined. Separately, an automatic
titration apparatus assembled from suitable units and/or
parts, including a data processing system, can also be used.
In this titration method, unless otherwise specified, indica-
52
Endpoint Detection Methods in Titrimetry / General Tests
JP XV
tor electrodes designated in Table 2.50-1 are used according
to the kind of titration. As a reference electrode, usually a sil-
ver-silver chloride electrode is used. Besides the single indica-
tor electrodes as seen in Table 2.50-1, a combined reference
electrode and indicator electrode can also be used.
Table 2.50-1 Kind of Titration and Indicator Electrode
Kind of titration
Indicator electrode
Acid-base titration
Glass electrode
(Neutralization titra-
tion, pH titration)
Precipitation titration
Silver electrode. A silver-
(Titration of halogen
silver chloride electrode
ion by silver nitrate)
is used as a reference
electrode, which is con-
nected with the titrate
by a salt bridge of satu-
rated potassium nitrate
solution.
Oxidation-reduction titra-
Platinum electrode
tion
(Diazo titration, etc.)
Complexation titration
Mercury-mercury chloride
(Chelometric titration)
(II) electrode
Nonaqueous titration
Glass electrode
(Perchloric acid titra-
tion, Tetramethylam-
monium hydroxide
titration)
When the potentiometric titration is carried out by the pH
measurement method, the pH meter should be adjusted ac-
cording to the pH Determination <2.54>.
(2) Procedure
Weigh a defined amount of a specimen in a beaker, and
add an indicated quantity of solvent to dissolve the specimen,
as directed in the monograph. After the potential difference E
(mV) or the pH value of the solvent to be used for titration
has reached a stable value, immerse both reference and indi-
cator electrodes, which have previously been washed with the
solvent being used, in the solution to be examined, and titrate
with a standard solution for volumetric analysis with gentle
stirring of the solution. During the titration, the tip of the
buret should be dipped into the solution, to be examined. The
endpoint of titration is determined by following the variation
of the potential difference between two electrodes as a func-
tion of the quantity of titrant added. In the vicinity of the en-
dpoint, the amounts of a titrant added should be 0.1 mL or
less for adequate titrimetry. Plot the obtained potential
values along the ordinate and the quantity of a titrant added
V (mL) along the abscissa to draw a titration curve, and ob-
tain the endpoint from the maximum or the minimum value
of AE/A V or from the value of electromotive force or pH
corresponding to the equivalence point.
Unless otherwise specified, the decision of the endpoint in
this method is usually made by either of the following
methods.
(i) Drawing method
Usually, draw two parallel tangent lines with a slope of
about 45° to the obtained titration curve. Next, draw a 3rd
parallel line at the same distance from the previously drawn
two parallel lines, and decide the intersection point of this
line with the titration curve. Further, from the intersection
point, draw a vertical line to the abscissa, and read the quan-
tity of titrant added as the endpoint of the titration.
Separately, the endpoint of the titration can also be
otained from the maximum or the minimum of the differen-
tial titration curve (AE/AV vs. V).
(ii) Automatic detection method
In the case of potentiometric titration using an automatic
titiration system, the endpoint can be determined by follow-
ing the respective instrumental indications. The endpoint is
decided either by following the variation of the differential
potential change or the absolute potential difference as a
function of the quantity of titrant added: in the former case
the quantity given by the maximum or the minimum of the
differential values, and in the latter the quantity given by the
indicator reaching the endpoint potential previously set for
the individual titration system, are assumed to be the en-
dpoint volumes, respectively.
2. Amperometric titration
(1) Apparatus
The apparatus consists of a beaker for holding a specimen,
a buret for adding a standard solution for volumetric analy-
sis, two small platinum plates or wires of the same shape as
the indicator electrode, a device to load direct current
microvoltage between two electrodes, a microammeter to
measure the indicator current between the two electrodes, a
recorder, and a stirrer which can gently stir the solution in a
beaker. Separately, an automatic titration apparatus assem-
bled from suitable units and/or parts, including a data
processing system, can also be used.
(2) Procedure
Weigh a defined amount of a specimen in a beaker, and
add an indicated quantity of solvent to dissolve the specimen,
as directed in the individual monograph. Next, after washing
the two indicator electrodes with water, immerse both elec-
trodes in the solution to be examined, apply a constant vol-
tage suitable for measurement across two electrodes by using
an appropriate device, and titrate the solution with a stan-
dard solution for volumetric analysis. During the titration,
the tip of the buret should be dipped into the solution to be
examined. The endpoint of titration is determined by follow-
ing the changes of microcurrent between the two electrodes as
a function of the quantity of titrant added. In the vicinity of
the endpoint, the amounts of the titrant added should be 0.1
mL or less for adequate titrimetry. Plot the obtained current
values along the ordinate and the quantity of the titrant ad-
ded F(mL) along the abscissa to draw a titration curve, and
usually take the inflection point of the titration curve (the
point of intersection given by the extrapolation of two
straight lines before and after the inflection) as the endpoint
in amperometric titration.
The blank test in this titration is usually performed as fol-
lows: Take a volume of the solvent specified in the individual
monograph or in the "Standard Solution for Volumetric
Analysis", and use this as the sample solution. Determine the
amount of the volumetric standard solution needed for giving
the endpoint, and use this volume as the blank. If this volume
is too small to determine accurately, the blank may be consi-
dered as (mL).
Unless otherwise specified, the endpoint in this titration is
decided by either of the following methods.
(i) Drawing method
Usually, extrapolate the two straight lines before and after
the inflection, and obtain the inflection point of the titration
JPXV
General Tests / Conductivity Measurement
53
curve. Next, read the quantity of titrant added at the inflec-
tion point, and assume this point to be the endpoint.
(ii) Automatic detection method
In the case of amperometric titration using an automatic
titration system, the endpoint can be determined by following
the instrumental indications. The endpoint is decided by fol-
lowing the variation of the indicator current during the
course of a titration, and the quantity of titrant added is as-
sumed to be that at which the current has reached the en-
dpoint current set previously for the individual titration sys-
tem.
When atmospheric carbon dioxide or oxygen is expected to
influence the titration, a beaker with a lid should be used, and
the procedure should be carried out in a stream of an inert
gas, such as nitrogen gas. Further, when a specimen is expect-
ed to be influenced by light, use a light-resistant container to
avoid exposure of the specimen to direct sunlight.
2.51 Conductivity Measurement
Conductivity Measurement is a method for the measuring
the flowability of electric current in an aqueous solution. The
measurement is made with a conductivity meter or a resistiv-
ity meter, and is used, for example, in the purity tests in
monographs. The method is applied to evaluate the test item
"Conductivity (Electrical Conductivity)" specified in the
monographs. Further it is also used for monitoring the quali-
ty of water in the preparation of highly purified water.
However, when applying this method for monitoring the
quality of water, the details of measurement should be speci-
fied by the user, based on the principles described here.
Conductivity of a solution «:(S-m _1 ) is defined as the
reciprocal of resistivity p(Q-m), which is an indicator of the
strength of ionic conductivity for a fluid conductor. Resistiv-
ity is defined as the product of electrical resistance per unit
length and cross-sectional area of a conductor. When resistiv-
ity is p, cross-section area A (m 2 ), and length / (m), resistance
R (Q) can be expressed by the following equation.
R=p(l/A)
Thus, conductivity k is expressed as follows,
K=\/p = {\/R)(l/A)
If l/A is known, the conductivity k can be obtained by
measuring resistance R or conductance G (= R 1 ).
In the International System (SI), the unit of conductivity is
the Siemens per meter (S-m~'). In practice, conductivity of a
solution is generally expressed by ^S-cm -1 , and resistivity by
Q-cm.
Unless otherwise specified, the reference temperature for
the expression of conductivity or resistivity is 20 °C.
Apparatus
A conductivity meter or a resistivity meter is composed of
an indicator part (operating panel, display, recording unit)
and a detector part, the latter of which includes a conductivi-
ty cell. In the conductivity cell a pair of platinum electrodes is
embedded. The cell is immersed in a solution, and the
resistance or the resistivity of the liquid column between the
electrodes is measured. Alternating current is supplied to this
apparatus to avoid the effects of electrode polarization. Fur-
ther, a temperature compensation system is generally con-
tained in the apparatus.
Conductivity measurement is generally performed by using
an immersion-type cell. A pair of platinum electrodes, the
surfaces of which are coated with platinum black, is fixed in
parallel. Both electrodes are generally protected by a glass
tube to prevent physical shocks.
When the surface area of the electrode is A (cm 2 ), and the
separation distance of the two electrodes is / (cm), the cell
constant C (cm -1 ) is given by the following equation.
C = a-(l/A)
a is a dimensionless numerical coefficient, and it is character-
istic of the cell design.
In addition to the immersion-type cell, there are flow-
through-type and insert-in-pipe-type cells. These cells are set
or inserted in an appropriate position in the flow system for
monitoring the quality of water continuously or intermittent-
ly, during the preparation of highly purified water.
Standard Solution of Potassium Chloride
After pulverizing an appropriate amount of potassium
chloride for conductivity measurement, dry it at 500 - 600°C
for 4 hours. Take an indicated amount of the dried potassi-
um chloride, as shown in Table 2.51-1, dissolve it in distilled
or purified water (conductivity less than 2//S-cm~ 1 ), previ-
ously boiled and cooled, and adjust to make 1000.0 g, for
preparation of the standard solutions. The conductivity and
the resistivity of the respective standard solutions at 20°C are
indicated in Table 2.51-1. These standard solutions should be
kept in tightly closed polyethylene or hard glass bottles.
Table 2.51-1. Conductivity and Resistivity of the Standard
Solutions of Potassium Chloride at 20°C
Concentration
(g/lOOO.Og)
Conductivity K
CuS-cm" 1 )
Resistivity p
(0 • cm)
0.7455
0.0746
0.0149
1330
133.0
26.6
752
7519
37594
When measurement at 20°C can not be done, the indicated
value of conductivity for the respective standard solution
(Table 2.51-1), can be corrected by using the equation below.
However, the equation is valid only within the range of 20 ±
5°C.
/c T = K 20 [l+0. 021(77- 20)]
T: Measuring temperature specified in the monograph
Kj\ Calculated conductivity of the KC1 standard solution
at T°C
K 2 o'- Conductivity of the KC1 standard solution at 20°C
Operating Procedure
(1) Cell Constant
An appropriate conductivity cell should be chosen accord-
ing to the expected conductivity of the sample solution. The
higher the expected conductivity, the larger the cell constant
required for the conductivity cell, so that the electrical
resistance is within the measuring range of the apparatus
being used. Conductivity cells with a cell constant of the ord-
er of 0.1 cm -1 , 1 cm -1 , or 10 cm -1 , are generally used.
For determination or confirmation of the cell constant, an
54
Thermal Analysis / General Tests
JP XV
appropriate KC1 standard solution should be chosen and pre-
pared, taking account of the expected conductivity of the
sample solution to be measured. Rinse the cell several times
with distilled water. Next, after rinsing the cell 2-3 times
with the standard solution used for the cell constant determi-
nation, immerse the cell in the standard solution contained in
a measuring vessel. After confirming that the temperature of
the standard solution is maintained at 20 ± 0.1 °C or at the
temperature specified in the monograph, measure the
resistance R KC \ or the conductance G K ci of the standard solu-
tion, and calculate the cell constant C (cm -1 ) by use of the
following equation.
C = R*
or C = K KCl /G K
Rkci- Measured resistance (Mfi)
G K ci: Measured conductance (/xS)
/c KC1 : Conductivity of the standard solution being used
(juS-cm- 1 )
The measured cell constant should be consistent with the
given value within 5%. If it is not consistent, coat the elec-
trodes with platinum black again, or replace the cell with a
new one.
(2) Suitability Test for the Apparatus
Using an appropriate KC1 standard solution according to
the expected conductivity of the sample solution, perform the
suitability test for the apparatus. Rinse the conductivity cell
several times with distilled water, and rinse again 2-3 times
with the selected standard solution. Fill the standard solution
in the measuring vessel. After confirming that the tempera-
ture of the measuring system is maintained at 20 ± 0.1 °C,
measure the conductivity of the standard solution. When this
measuring procedure is repeated several times, the average
conductivity should be consistent with an indicated value in
Table 1 within 5%. Further, the relative standard deviation
should be less than 2%.
(3) Measurement
After confirmation of the suitability of the apparatus, per-
form the conductivity measurement for the sample solution.
Unless otherwise specified, the preparation method for sam-
ple solution should be as specified in the respective mono-
graph. Rinse the conductivity cell several times with distilled
water, and rinse again 2-3 times with sample solution. Im-
merse the cell in the sample solution placed in a measuring
vessel. If necessary, agitate gently the sample solution. After
confirming that the temperature of the sample solution is
maintained at 20 ± 0.1 °C or at the temperature specified in
the monograph, measure the resistance R T (MO) or conduc-
tance G T (,«S) of the sample solution, and calculate the con-
ductivity k-y by using the following equation.
k t = CGj or k t = C/R T
Items such as the sample preparation method, the necessity
of blank correction, the calculation method, the specification
value, and the measuring temperature should be described in
the monograph, if necessary.
2.52 Thermal Analysis
"Thermal Analysis" is a generic term for a variety of tech-
niques to measure the physical properties of a substance as a
function of temperature and/or time.
Among the physical properties, phase transitions such as
solid/liquid phase transition (melting, freezing) and crystal
polymorphism or thermal behavior such as heat evolution or
absorption accompanying thermal degradation or chemical
reaction can be detected by the techniques of differential ther-
mal analysis (DTA) or differential scanning calorimetry
(DSC). DTA is a method for detecting the thermal behavior
of a specimen in terms of the temperature change, while DSC
employs the heat quantity (enthalpy) change. There is also a
method, thermogravimetry (TG), in which the mass change
of a specimen caused by dehydration, adsorption, elimina-
tion or oxidation etc., is detected as a function of tempera-
ture and/or time.
Among the above three different methods, TG can be used
as an alternative method for "Loss on Drying <2.41>" or
"Water Determination <2.48>". However, it must be con-
firmed beforehand that no volatile component except for
water is included in the test specimen when TG is used as an
alternative method for "Water Determination".
Method 1 Differential Thermal Analysis (DTA) or Differen-
tial Scanning Calorimetry (DSC)
Apparatus Apparatus for DTA or DSC is usually com-
posed of a heating furnace, a temperature-controller, a detec-
tor, a device for controlling the atmosphere, and an indicator
/recorder.
Differential Thermal Analysis (DTA) In a DTA appara-
tus, a sample specimen and an inert reference material placed
in the heating furnace are heated or cooled at a constant rate,
and the temperature difference evolved between the sample
and reference material is detected continuously by a device
such as a thermocouple and recorded as a function of time
and/or temperature. As an inert reference material, a-Alumi-
na for thermal analysis is usually adopted.
Differential Scanning Calorimetry (DSC) Two kinds of
DSC apparatus, based upon different principles are available
as shown below.
1. Input compensation-type differential scanning calorimet-
ry (Input compensation DSC)
A sample specimen and the reference material in twin fur-
naces are programmed to be heated or cooled at a constant
rate, and the temperature difference between the sample and
the reference, which is detected by a device such as a plati-
num resistance thermometer, is kept at null by controlling the
heating unit with a compensation feed-back circuit. The
instrument is designed to measure and record continuously
the balance of thermal energy applied to each furnace as a
function of temperature and/or time.
2. Heat flux-type differential scanning calorimetry (Heat
flux DSC)
A sample specimen and the reference material in twin fur-
naces are programmed to be heated or cooled at a constant
rate, and the temperature difference between the sample and
the reference is detected as a difference of heat flux and
recorded as a function of temperature and/or time. In heat
flux DSC, thermal conductors are adopted so that the heat
flux between the sample and the heat reservoir is proportional
to the temperature difference between them.
In usual DSC analysis, a-Alumina is used as a reference
material, both in Input compensation DSC and in Heat flux
DSC. But in some cases, an empty sample container can also
be used without any reference material.
JPXV
General Tests / Thermal Analysis
55
Procedure
A sample specimen and the reference material are put in
sample pans, and the furnace is heated or cooled under a con-
trolled temperature program. As the temperature changes,
the temperature difference (DTA) or heat quantity change
(DSC) that develops between the specimen and the reference
is detected and recorded continuously. Apparatus equipped
with a data-processor is operated according to the instruction
manual provided with the instrument.
A preliminary experiment is needed to determine the ap-
propriate temperature range of measurement, within which a
predicted physical change such as melting or polymorphic
phase transition will occur, and to confirm that unpredicted
thermal changes are not induced in a specimen in that tem-
perature range. In this preliminary test, a wide temperature
range (room temperature-the temperature at which degrada-
tion begins) can be scanned at a rapid heating rate (10 - 20°C
/min). Thereafter, tests by DSC or DTA should be performed
at a low heating rate, usually 2°C/min, in the chosen temper-
ature range. However, when a clear heat change cannot be
observed, such as in a case of glass-transition, the heating
rate may be changed to a higher or a lower rate, as appropri-
ate for the kind of physical change being observed. By
analyzing the measured DTA-curve or DSC-curve, a quantity
of heat change and/or a specific temperature (ignition, peak
or end temperature) that accompanies a physical change,
such as melting or polymorphic phase transition, can be ob-
tained.
Calibration of the apparatus
1. Temperature calibration for DTA and DSC
Temperature calibration for DTA and/or DSC apparatus
can be performed by using reference substances having an in-
trinsic thermal property, such as melting point of pure metals
or organic substances, or phase transition point of crystalline
inorganic salts or oxides. Melting points of Indium for ther-
mal analysis and/or Tin for thermal analysis are usually em-
ployed for calibration.
2. Heat-quantity calibration for DSC
For accurate estimation of a quantity of heat change (en-
thalpic change) of a sample specimen, caused by a certain
physical change accompanying a temperature change, it is
necessary to calibrate the apparatus by using appropriate
reference substances. As indicated in the section of Tempera-
ture calibration, heat-quantity calibration for DSC apparatus
can be performed by using appropriate reference substances
having a known definite enthalpic change caused by such
physical changes as melting of pure metals and/or organic
substances, or phase transition of crystalline inorganic salts.
Melting points of Indium for thermal analysis and/or Tin for
thermal analysis are usually employed for calibration.
Notes on operating conditions
When DTA or DSC measurements are made, the following
items must be recorded: sample size, discrimination of open-
or closed-type sample container, heating or cooling rate,
measuring temperature range, and kind and flow rate of at-
mospheric gas.
Method 2 Thermogravimetry (TG)
Apparatus The construction of a TG apparatus is fun-
damentally similar to that of DTA or DSC apparatus.
However, the detector for TG is a balance, called a ther-
mobalance, which can be classified to hanging-, RobervaFs-,
and horizontal-type balances. The TG apparatus is designed
to detect small mass changes of a specimen, placed at a fixed
position on a thermobalance, caused by temperature change
of the furnace under a controlled temperature program. Mass
change with time and/or temperature is recorded continuous-
ly.
Procedure
A specimen is put in a sample container, which is placed at
a fixed position of the thermobalance, then the heating fur-
nace is run under a controlled temperature program. During
this temperature change of the furnace, the mass change of a
specimen with time and/or temperature is recorded continu-
ously. Apparatus equipped with a data-processor is operated
according to the instruction manual provided with the instru-
ment.
When TG is used as an alternative method for "Loss on
Drying" or "Water Determination", the measurement starts
at room temperature and ends at a temperature above which
no further mass change due to drying and/or vaporization of
water can be observed. The standard heating rate is usually
5°C/min, and a linear heating program is recommended.
However heating conditions (rate and time span) can be
changed as necessary, depending on the kind of specimen and
the extent of the measuring temperature range. Further, in
TG measurement, dry air or dry nitrogen is usually passed
through the heating furnace to ensure rapid elimination of
evolved water or other volatile components and to avoid the
occurrence of any chemical reaction, such as oxidation. By
analyzing the TG curve plotted against time and/or tempera-
ture, absolute mass change and/or relative mass change with
respect to the initial quantity(%) is obtained.
When the mass change caused by oxidation or degradation
of a specimen is measured, a specific temperature range has
to be determined beforehand so that stable baselines can be
obtained before and after a targetted chemical reaction. Sub-
sequent operating procedures are the same as described
above.
Calibration of the apparatus
1. Temperature calibration
The Curie temperature of a ferromagnetic substance such
as pure Nickel can be used for temperature calibration for
TG, based on the occurrence of an apparent mass change at
the Curie point. In the case of a TG apparatus capable of
simultaneously conducting DSC and DTA, the same refer-
ence substances as those for DTA and DSC can be adopted.
2. Scale calibration and confirmation
The scale calibration for TG must be done by using refer-
ence masses for chemical balances and/or semimicrobalances
in the appropriate range. This is called a primary scale
calibration, and is performed under ordinary temperature
and pressure when the apparatus is set up initially and period-
ically, thereafter.
In usual measurement by TG, scale calibration or confir-
mation is done by using Calcium Oxalate Monohydrate
Reference Standard to take account of such effects as
buoyancy and convection due to atmospheric gas flow in the
real measurement state. This is called secondary scale calibra-
tion, and is performed under the standard operation condi-
tions stated below by using the above-mentioned Reference
Standard, with a certified water content. When the difference
of water content between the measured value and the certified
one for the Reference Standard is less than 0.3%, normal
56 Viscosity Determination / General Tests
JP XV
operation of the apparatus is confirmed. However, when the
difference is more than 0.3%, scale calibration for TG must
be done, based on the certified water content of the Reference
Standard.
The standard operation conditions are as follows,
Amount of Calcium Oxalate Monohydrate Reference
Standard: 0.01 g
Heating rate: 5°C/min
Temperature range: from room temperature to 250°C
Atmospheric gas: dried Nitrogen or dried Air
Flow rate of atmospheric gas,
hanging- or Roberval's-type balance: 40 mL/min
horizontal-type balance: 100 mL/min
Notes on operating conditions
In TG measurement, the following operation conditions
must be recorded: sample size, heating rate, temperature
range, kind and flow rate of atmospheric gas, etc.
2.53 Viscosity Determination
Viscosity Determination is a method to determine the vis-
cosity of liquid samples using a viscometer.
When a liquid moves in a definite direction, and the liquid
velocity has a gradient with respect to the direction rectangu-
lar to that of flow, a force of internal friction is generated
along both sides of a hypothetical plane parallel to the move-
ment. This flow property of a liquid is expressed in terms of
viscosity. The internal friction per unit area on the parallel
plane is called slip stress or shear stress, and the velocity
gradient with respect to the direction rectangular to that of
flow is called slip velocity or shear velocity. A liquid of which
the slip velocity is proportional to its slip stress is called a
Newtonian liquid. The proportionality constant, n, is a
characteristic of a liquid at a certain temperature and is called
viscosity. The viscosity is expressed in the unit of Pascal se-
cond (Pas), and usually milli-Pascal second (mPa-s).
A liquid whose slip velocity is not proportional to its slip
stress is called a non-Newtonian liquid. Since the viscosity for
a sample of a non-Newtonian liquid changes with its slip
velocity, the viscosity measured at a certain slip velocity is
called an apparent viscosity. In that case, the value of slip
stress divided by the corresponding slip velocity is called an
apparent viscosity. Thus, the relationship between apparent
viscosity and slip velocity will permit characterization of the
flow properties of a given non-Newtonian liquid.
The value of the viscosity, n, divided by the density, p, at
the same temperature is defined as a kinematic viscosity, v,
which is expressed in the unit of meters squared per second
(m 2 /s), and usually millimeters squared per second (mm 2 /s).
The viscosity of a liquid is determined either by the follow-
ing Method I or Method II.
Method I Viscosity measurement by capillary tube viscome-
ter
For measuring the viscosity of a Newtonian liquid, a capil-
lary tube viscometer is usually used, in which the downflow-
ing time of a liquid, t(s), required for a definite volume of the
liquid to flow through a capillary tube is measured and the
kinematic viscosity, v, is calculated according to the follow-
ing equation.
v = Kt
Further, the viscosity, n, is calculated from the next equa-
tion:
n = vp = Ktp
where p (g/mL) is the density of the liquid measured at the
same temperature, t ( C C).
The parameter K (mm 2 /s 2 ) represents the viscometer con-
stant and is previously determined by using the Standard Liq-
uids for Calibrating Viscometers with known kinematic vis-
cosity. In the case of a liquid having a similar viscosity to
water, water itself can be used as a reference standard liquid
for the calibration. The kinematic viscosity of water is
1.0038 mm 2 /s at 20 °C. In the cases of liquids having a slight-
ly higher viscosity than water, the Standard Liquids
for Calibrating Viscometers should be used for the calibra-
tion.
The intrinsic viscosity, [n] (dL/g), of a polymer solution is
obtained by plotting the relation of viscosity versus concen-
tration and extrapolating the obtained straight line to zero
concentration. Intrinsic viscosity shows the degree of molecu-
lar expansion of a polymer substance in a given solvent (sam-
ple solution) and is also a measure of the average molecular
mass of the polymer substance.
The downflowing time t (s) for a polymer solution, whose
concentration is c (g/dL), and t (s) for the solvent used for
dissolving the polymer, are measured by using the same vis-
cometer, and then the intrinsic viscosity of a given polymer
substance, [//], is calculated according to the following equa-
tion:
M-
lim
c^0
t
to
1
In
or [n] ■■
■ lim
c^0
When the concentration dependency of {(t/t ) — 1} /c is not
large, the value of {(t/t ) — 1} jc at a concentration directed in
the respective monograph can be assumed to be the intrinsic
viscosity for a given substance.
Unless otherwise specified, the viscosity of a sample solu-
tion is measured with the following apparatus and procedure.
Apparatus
For measurement of the kinematic viscosity in the range of
1 to 100,000 mm 2 /s, the Ubbelohde-type viscometer illustrat-
ed in Fig. 2.53-1 can be used. The approximate relations be-
tween kinematic viscosity range and inside diameter of the
capillary tube suitable for the measurement of various liquids
with different viscosity, are given in Table 2.53-1. Although a
capillary tube viscometer other than the Ubbelohde-type one
specified in Table 2.53-1 can also be used, a viscometer
should be selected in which the downflowing time, t (s), of a
sample solution to be determined would be between 200 s and
1000 s.
Procedure
Place a sample solution in a viscometer from the upper end
of tube I, so that the meniscus of the solution is at a level be-
tween the two marked lines of bulb A. Place the viscometer
vertically in a thermostatted bath maintained at a specified
temperature within 0.1°C, until bulb C is fully immersed,
and let it stand for about 20 minutes to attain the specified
temperature. Close tube 3 with a finger and pull the sample
solution up to the middle part of bulb C by gentle suction
from the top of tube 2, taking care not to introduce any bub-
bles into tube 2, and stop the suction. Open the end of tube 3,
JPXV
General Tests / Viscosity Determination 57
17.7-18.2
The figures are in mm.
Fig. 2.53-1 Capillary tube viscometer
and immediately close the end of tube 2. After confirming
Table 2.53-1 Specifications of the Ubbelohde-type
viscometer
Inner diameter
Volume of
Measuring
range of
kinematic
viscosity
(mm 2 /s)
Viscometer
constant
K
of capillary
tube (mm)
Permissible
bulb B
(mL)
Permissible
(mm 2 /s 2 )
tolerance
+ 10%
tolerance
+ 10%
0.005
0.46
3.0
1- 5
0.01
0.58
4.0
2- 10
0.03
0.73
4.0
6- 30
0.05
0.88
4.0
10- 50
0.1
1.03
4.0
20 - 100
0.3
1.36
4.0
60 - 300
0.5
1.55
4.0
100 - 500
1.0
1.83
4.0
200- 1000
3.0
2.43
4.0
600 - 3000
5.0
2.75
4.0
1000 - 5000
10.0
3.27
4.0
2000- 10,000
30.0
4.32
4.0
6000- 30,000
50.0
5.20
5.0
10,000- 50,000
100
6.25
5.0
20,000 - 100,000
that the liquid column is cut off at the lowest end of the capil-
lary tube, open the end of tube 2 to make the sample solution
flow down through the capillary tube. Record the time, t (s),
required for the meniscus of the sample solution to fall from
the upper to the lower marked line of bulb B.
Determine the viscometer constant K previously, using the
Standard Liquids for Calibrating Viscometers under the same
conditions. The temperature at which the calibration is con-
ducted must be identical with that specified in the mono-
graph.
J \
Hi
Fig. 2.53-2a Coaxial double cylinder-type rotational
viscometer
Method II Viscosity measurement by rotational viscometer
A rotational viscometer is usually used for measuring the
viscosity of Newtonian or non-Newtonian liquids. The meas-
uring principle of a rotational viscometer generally consists in
the detection and determination of the force acting on a rotor
(torque), when it rotates at a constant angular velocity in a
liquid. The extent of torque generated by the rotation can be
detected in terms of the torsion of a spring and the liquid vis-
cosity is calculated from the scale-indicated value cor-
responding to the degree of torsion.
The viscosity of a sample solution is measured with the fol-
lowing apparatus and procedure.
Apparatus
Viscosity measurement is performed by using any one of
the following three types of rotational viscometers.
(1) Coaxial double cylinder-type rotational viscometer
(Couette type viscometer)
In the coaxial double cylinder-type rotational viscometer,
viscosity is determined by placing a liquid in the gap between
the inner and the outer cylinders, which share the same cen-
tral axis and rotate separately, and the generated torque act-
ing on one cylinder surface when the other cylinder is rotated,
and the corresponding angular velocity, are measured.
As shown in Fig. 2.53-2a, the inner cylinder is hung by a
wire whose twist constant is designated as k. In Fig. 2.53-2a,
half the outer diameter of the inner cylinder and inner di-
ameter of the outer cylinder are designated as i? ; and R a ,
respectively, and the length of the inner cylinder immersed in
a liquid is designated as /. When a liquid is introduced into
the gap between the two cylinders, and the outer cylinder is
made to rotate at a constant angular velocity, co, the inner
cylinder is also forced to rotate due to the viscosity of the liq-
uid. Consequently, torque, T, is generated by the forced rota-
tion in a viscous liquid, and in the steady state the torque is
balanced by the torsion of the wire, as indicated by the degree
of rotation 9. Then, the relationship can be expressed by T=
kO and the viscosity of a liquid, n, is determined from the fol-
lowing equation by measuring the relationship between co
and 9. Conversely, viscosity measurement can also be per
formed by rotating the inner cylinder, and the same relation-
ship holds.
n-
ioor
Anlco
1
Ri 2
1
R n :
58 Viscosity Determination / General Tests
JP XV
UP
torque T
±
Fig. 2.53-2b Single cylinder-type rotational viscometer
n: viscosity of a liquid (mPa-s)
n: circumference/diameter ratio
/ : length of the inner cylinder (cm)
co: angular velocity (rad/s)
T: torque acting on cylinder surface (10~ 7 N-m)
Ri'. 1/2 of outer diameter of the inner cylinder (cm)
R : 1/2 of inner diameter of the outer cylinder (cm)
(2) Single cylinder-type rotational viscometer (Brookfield
type viscometer)
In the single cylinder-type rotational viscometer, viscosity
is determined by measuring the torque acting on the cylinder
surface when the cylinder immersed in a liquid is rotated at a
given angular velocity. Use an apparatus of the type illustrat-
ed in Fig. 2.53-2b. If the apparatus constant K B is previously
determined experimentally by using the Standard Liquids for
Calibrating Viscometers, the viscosity of a liquid, n, can be
obtained from the following equation.
1 = K B
T
CO
where, n: viscosity of a liquid (mPa-s)
K B : apparatus constant of viscometer (rad/cm 3 )
co: angular velocity (rad/s)
T: torque acting on cylinder surface (10 ~ 7 N-m)
(3) Cone-flat plate-type rotational viscometer (Cone-
plate type viscometer)
In the cone-flat plate-type rotational viscometer, viscosity
is determined by placing a liquid in the gap between a fiat disc
and a cone with a large vertical angle sharing the same rota-
tional axis, and the torque and the corresponding angular
velocity are measured, when either the disc or the cone is ro-
tated in a viscous liquid.
As shown in Fig. 2.53-2c, a liquid is introduced to fill the
gap between a flat disc and a cone forming an angle a(rad).
When either the flat disc or the cone is rotated at a constant
angular velocity or a constant torque, the torque acting on
the disc or cone surface rotated by the viscous flow and the
corresponding angular velocity in the steady state, are meas-
ured. The viscosity of the liquid, n, can be calculated from
the following equation.
torque 7
Fig. 2.53-2c Cone-flat plate-type rotational viscometer
n-
3a
2nR 3
ioor
CO
n: viscosity of a liquid (mPa-s)
n: circumference/diameter ratio
R : radius of cone (cm)
a : angle between flat disc and cone (rad)
co: angular velocity (rad/s)
T: torque acting on flat disc or cone surface (10~ 7 N-
m)
Procedure
Set up the viscometer so that its rotational axis is perpen-
dicular to the horizontal plane. Place a sufficient quantity of
a sample solution in the viscometer, and allow the measuring
system to stand until a specified temperature is attained, as
directed in the monograph. Where it is desired to measure the
viscosity within a precision of 1%, measuring temperature
should be controlled within 0.1 C C. Next, after confirming
that the sample solution is at the designated temperature,
start operating the rotational viscometer. After the forced ro-
tation induced by the viscous flow has reached a steady state
and the indicated value on the scale, which corresponds to the
rotational frequency or the torque, has become constant,
read the value on the scale. Then, calculate the viscosity n by
using the respective equation appropriate to the type of vis-
cometer being used. Determination or confirmation of the
apparatus constant should be conducted beforehand by using
the Standard Liquids for Calibrating Viscometers, and the
validation of the apparatus and operating procedure should
also be performed by using those standard liquids.
In the case of a non-Newtonian liquid, repeat the proce-
dure for measuring the viscosity of the liquid with variation
of the rotation velocity or torque from one measurement to
another. From a series of such viscosity measurements, the
relationship between the slip velocity and the slip stress of a
non-Newtonian liquid, i.e., the flow characteristics of a non-
Newtonian liquid, can be obtained.
Calibration of a rotational viscometer is conducted by us-
ing water and the Standard Liquids for Calibrating Viscome-
ters. These standard liquids are used for the determination or
confirmation of the apparatus constant of the rotational vis-
cometer. They are also used for periodic recalibration of the
viscometer to confirm maintenance of a specified precision.
JPXV
General Tests / pH Determination
59
2.54 pH Determination
pH is defined as the reciprocal of the common logarithm of
hydrogen ion activity, which is the product of hydrogen ion
concentration and the activity coefficient. Conventionally it is
used as a scale of hydrogen ion concentration of a sample so-
lution.
pH of a sample solution is expressed by the following equa-
tion in relation to the pH of a standard solution (pHs), and
can be measured by a pH meter using a glass electrode.
pH = pHs +
E-E,
2.3026 RT/F
pHs: pH value of a pH standard solution.
E: Electromotive force (volt) induced on the following gal-
vanic cell composed of a glass electrode and suitable
reference electrode in a sample solution:
Glass electrode I sample solution | reference electrode
E s : Electromotive force (volt) induced on the following
galvanic cell composed of a glass electrode and suitable
reference electrode in a pH standard solution:
Glass electrode I standard pH solution | reference electrode
R : Gas constant
T: Absolute temperature
E: Faraday's constant
The value of 2.3026 RT/F (V) in the above equation means
the degree of electromotive force (V) per one pH unit and it is
dependent on the temperature as shown in Table 2.54-1:
Table 2.54-1 Temperature dependency of the electromotive
force (V)
Temperature
of solution
(°C)
2.3026 RT/F
(V)
Temperature
of solution
(°C)
2.3026 RT/F
(V)
5
0.05519
35
0.06114
10
0.05618
40
0.06213
15
0.05717
45
0.06313
20
0.05817
50
0.06412
25
0.05916
55
0.06511
30
0.06015
60
0.06610
pH Standard solution
The pH standard solutions are used as a standard of pH,
for standardization of a pH meter. To prepare water for
preparation of the pH standard solutions, distill purified
water, boil the distillate for more than 15 minutes, and cool
in a container fitted with a carbon dioxide-absorbing tube
(soda lime). Next, prepare individually 6 kinds of pH stan-
dard solutions shown in Table 2.54-2.
Store the pH standard solutions in hard glass or polyethy-
lene bottles. For storage of alkaline pH standard solutions, it
is preferable to use a bottle fitted with a carbon dioxide-ab-
sorbing tube. Since the pH may change gradually during
storage over a long period, it is necessary to acertain whether
the expected pH value is held or not by comparison with new-
ly prepared standard, when the solution is used after long
storage.
(1) Oxalate pH standard solution — Pulverize potassium
trihydrogen dioxalate dihydrate for pH determination, and
dry in a desiccator (silica gel). Weigh 12.71 g (0.05 mole) of it
accurately, and dissolve in water to make exactly 1000 mL.
(2) Phthalate pH standard solution — Pulverize potassi-
um hydrogen phthalate for pH determination, and dry at 110
°C to constant mass. Weigh 10.21 g (0.05 mole) of it ac-
curately, and dissolve in water to make exactly 1000 mL.
(3) Phosphate pH standard solution — Pulverize potassi-
um dihydrogen phosphate for pH determination and disodi-
um hydrogen phosphate for pH determination, and dry at
110°C to constant mass. Weigh 3.40 g (0.025 mole) of potas-
sium dihydrogen phosphate and 3.55 g (0.025 mole) of diso-
dium hydrogen phosphate dodecahydrate accurately, and
dissolve in water to make exactly 1000 mL.
(4) Borate pH standard solution — Allow sodium
tetraborate for pH determination to stand in a desiccator
(saturated sodium bromide aqueous solution) until it reaches
constant mass. Weigh 3.81 g (0.01 mole) of it accurately, and
dissolve in water to make exactly 1000 mL.
(5) Carbonate pH standard solution — Dry sodium hydro-
gen carbonate for pH determination in a desiccator (silica
gel) to constant mass, and weigh 2.10 g (0.025 mole) of it ac-
curately. Dry sodium carbonate for pH determination be-
tween 300°C and 500°C to constant mass, and weigh 2.65 g
(0.025 mole) of it accurately. Dissolve both reagents in water
to make exactly 1000 mL.
(6) Calcium hydroxide pH standard solution — Reduce
calcium hydroxide for pH determination to a fine powder,
transfer 5 g to a flask, add 1000 mL of water, shake well, and
allow the solution to become saturated at a temperature be-
tween 23 °C and 27 °C. Then filter the supernatant at the same
temperature and use the clear filtrate (about 0.02mol/L).
The pH values of these pH standard solutions at various
temperatures are shown in the Table below. pH values at an
arbitrary temperature not indicated in Table 2.54-2 can be
calculated by the interpolation method.
Apparatus
A pH meter generally consists of an electrode system of a
glass electrode and a reference electrode, an amplifier and an
indicating unit for controlling the apparatus and for dis-
playing the measured value of electromotive force. The in-
dicating unit is usually fitted with dials for zero and span
(sensitivity) adjustment. Sometimes a temperature compensa-
tion dial is included.
The reproducibility of a pH meter should be within 0.05
pH unit, when measurements for an arbitrary pH standard
solution are repeated five times, following the procedure
described below. After each measurement it is necessary to
wash the detecting unit well with water.
Procedure
Immerse the glass electrode previously in water for more
than several hours. Start the measurement after confirming
stable running of the apparatus. Rinse well the detecting unit
with water, and remove the remaining water gently with a
piece of filter paper.
To standardize the pH meter, two pH standard solutions
are usually used as follows. Immerse the detection unit in the
phosphate pH standard solution and adjust the indicated pH
to the pH value shown in the Table. Next, immerse the detec-
tion system in the second pH standard solution, which should
be selected so that the expected pH of the sample solution to
be determined is between the pH values of the two pH stan-
dard solutions, and measure the pH under the same condi-
60
Vitamin A Assay / General Tests
JP XV
Table 2.54-2 pH values of six pH standard solutions
Temper-
ature
(°0
Oxalate
pH
standard
solution
Phthalate
pH
standard
solution
Phosphate
pH
standard
solution
Borate
pH
standard
solution
Carbonate
pH
standard
solution
Calcium
hydroxide
pH
standard
solution
1.67
4.01
6.98
9.46
10.32
13.43
5
1.67
4.01
6.95
9.39
10.25
13.21
10
1.67
4.00
6.92
9.33
10.18
13.00
15
1.67
4.00
6.90
9.27
10.12
12.81
20
1.68
4.00
6.88
9.22
10.07
12.63
25
1.68
4.01
6.86
9.18
10.02
12.45
30
1.69
4.01
6.85
9.14
9.97
12.30
35
1.69
4.02
6.84
9.10
9.93
12.14
40
1.70
4.03
6.84
9.07
11.99
50
1.71
4.06
6.83
9.01
11.70
60
1.73
4.10
6.84
8.96
11.45
tions as used for the first pH standard solution. Adjust the in-
dicated pH to the defined pH value using the span adjustment
dial, when the observed pH is not identical with that tabulat-
ed. Repeat the above standardization procedure until both
pH standard solutions give observed pH values within 0.02
pH unit of the tabulated value without further adjustments.
When a pH meter is fitted with a temperature compensation
dial, the standardization procedure is done after the setting
of the temperature to that of the pH standard solution to be
measured.
In the case of using an apparatus having an auto-calibra-
tion function, it is necessary to confirm periodically that the
pH values of two pH standard solutions are identical with the
tabulated values within 0.05 pH unit.
After finishing the standardization procedure described
above, rinse well the electrodes with water, remove the at-
tached water using a filter paper, immerse the electrode sys-
tem in the sample solution, and read the indicated pH value
after confirming the value is stable. If necessary, a sample so-
lution can be agitated gently.
In the pH determination, the temperature of a sample solu-
tion must be controlled to be the same as that of the pH stan-
dard solutions with which the pH meter was standardized
(within 2°C). When a sample solution is alkaline, the meas-
urement should be done in a vessel with a cover and if neces-
sary, in a stream of inert gas such as nitrogen. Furthermore
for a strongly alkaline solution above pH 1 1 containing alkali
metal ions, an alkali error may be induced in the pH measure-
ment. Thus, in such a case, an electrode with less alkali error
should be used and an appropriate correction should be ap-
plied to the measured value.
Note: Construction and treatment in detail are different for
different pH meters.
2.55 Vitamin A Assay
Vitamin A Assay is a method to determine vitamin A in
Retinol Acetate, Retinol Palmitate, Vitamin A Oil, Cod
Liver Oil and other preparations. Method 1 is for the assay of
synthetic vitamin A esters, using the ultraviolet-visible spec-
trophotometry (Method 1-1) or the liquid chromatography
(Method 1-2). Method 2 is for the assay of vitamin A of
natural origin, containing many geometrical isomers, using
the ultraviolet-visible spectrophotometry to determine vita-
min A as vitamin A alcohol obtained by saponification in an
alkaline solution and extraction.
One Vitamin A Unit (equal to 1 vitamin A I.U.) is equiva-
lent to 0.300 /ug of vitamin A (all-trans vitamin A alcohol).
Procedure
All procedures should be carried out quickly and care
should be taken as far as possible to avoid exposure to light,
air, oxidants, oxidizing catalysts (e.g. copper, iron), acids
and heat. If necessary, light-resistant vessels may be used.
Generally, for synthetic vitamin A esters apply Method 1-1
or Method 1-2, but if the assay conditions required for
Method 1-1 are not suitable, apply Method 2.
Method 1-1
Weigh accurately about 0.1 g of the sample, and dissolve in
2-propanol for vitamin A assay to make exactly 50 mL. Di-
lute this solution with 2-propanol for vitamin A assay to
make a solution so that each mL contains 10 to 15 vitamin A
Units, and use this solution as the sample solution. Deter-
mine the absorption spectrum of the sample solution between
220 nm and 400 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24> to obtain the wavelength of the
maximum absorption and the absorbances at 300 nm, 310
nm, 320 nm, 326 nm, 330 nm, 340 nm and 350 nm. When the
maximum absorption lies between 325 nm and 328 nm, and
the ratios, Au/A }2 6, of each absorbance, A )A , at 300 nm, 310
nm, 320 nm, 330 nm, 340 nm and 350 nm to the absorbance,
Aw, at 326 nm are within the range of ±0.030 of the values
in the Table, the potency of vitamin A in Units per g of the
sample is calculated from the following equation.
Units of vitamin A in 1 g -
26 : Absorbance at 326 nm
W
100
X1900
V: Total volume (mL) of the sample solution
W: Amount (g) of sample in FmL of the sample solution
1900: Conversion factor from specific absorbance of
retinol ester to IU (Unit/g)
This method is applied to drugs or preparations containing
vitamin A esters (retinol acetate or retinol palmitate) as the
main component. However, when the wavelength of maxi-
mum absorption does not lie between 325 nm and 328 nm, or
when the absorbance ratio A^/A i26 is not within the range of
±0.030 of the values in Table 2.55-1, apply Method 2.
Table 2.55-1 Absorbance Ratio, Au/A 32 6> of retinol acetate
and retinol palmitate
A,
■JA
326
Retinol acetate
Retinol palmitate
300
0.578
0.590
310
0.815
0.825
320
0.948
0.950
330
0.972
0.981
340
0.786
0.795
350
0.523
0.527
Method 1-2
Proceed with an appropriate amount of sample as directed
under Liquid Chromatography <2.01>.
For the assay of retinol acetate and retinol palmitate use
Retinol Acetate Reference Standard and Retinol Palmitate
JPXV
General Tests / Determination of Specific Gravity and Density
61
Reference Standard, respectively, and fix appropriately the
operating procedure, the operating conditions and the system
suitability based on the characteristics of the substance to be
tested and the species and amount of coexisting substances.
Method 2
Unless otherwise specified, weigh accurately a sample con-
taining not less than 500 Units of vitamin A, and not more
than 1 g of fat, transfer to a flask, and add 30 mL of alde-
hyde-free ethanol and 1 mL of a solution of pyrogallol in
ethanol (95) (1 in 10). Then add 3 mL of a solution of potassi-
um hydroxide (9 in 10), attach a reflux condenser, and heat
on a water bath for 30 minutes to saponify. Cool quickly to
ordinary temperature, add 30 mL of water, transfer to a
separator A, wash the flask with 10 mL of water and then 40
mL of diethyl ether, transfer the washings to the separator A,
shake well, and allow to stand. Transfer the water layer so
obtained to a separator B, wash the flask with 30 mL of
diethyl ether, add the washing to the separator B, and extract
by shaking. Transfer the water layer to a flask, add the
diethyl ether layer to the separator A, transfer the water layer
in the flask to the separator B, add 30 mL of diethyl ether,
and extract by shaking. Transfer the diethyl ether layer so ob-
tained to the separator A, add 10 mL of water, allow the
separator A to stand after gentle turning upside-down 2 or 3
times, and remove the water layer. Wash the content of the
separator A with three 50-mL portions of water with increas-
ingly vigorous shaking as the washing proceeds. Further wash
with 50-mL portions of water until the washing no longer
shows a pink color with phenolphthalein TS, and allow to
stand for 10 minutes. Remove remaining water as far as pos-
sible, transfer the diethyl ether to an Erlenmeyer flask, wash
the separator with two 10-mL portions of diethyl ether, add
the washings to the flask, add 5 g of anhydrous sodium sul-
fate to the flask, mix by shaking, and transfer the diethyl
ether to a round-bottomed flask by decantation. Wash the
remaining sodium sulfate in the flask with two or more
10-mL portions of diethyl ether, and transfer the washings to
the flask. Evaporate the diethyl ether in a water bath at 45 °C
while swirling the flask, using an aspirator, to about 1 mL,
immediately add an exactly measured amount of 2-propanol
for vitamin A assay to make a solution containing 6 to 10
vitamin A Units per mL, and designate the solution as the
sample solution. Determine the absorbances, A il0 at 310 nm,
A i2 s at 325 nm, and A 334 at 334 nm, of the sample solution as
directed under Ultraviolet-visible Spectrophotometry.
Units of vitamin A in 1 g of the sample
W
f=6. 815-2. 555X
100
-4310
X/X1830
- 4.260 x
A 325 : Absorbance at 325 nm
V: Total volume (mL) of the sample solution
W: Amount (g) of sample in KmL of the sample solution
/: Correction factor
1830: Conversion factor from specific absorbance of
retinol alcohol to IU (Unit/g)
2.56 Determination of Specific
Gravity and Density
The density p (g/mL or g/cm 3 ) means the mass per unit
volume, and the relative density means the ratio of the mass
of a sample specimen to that of an equal volume of a stan-
dard substance. The relative density is also called the specific
gravity.
The specific gravity, d',', means the ratio of the mass of the
sample specimen at t'°C to that of an equal volume of water
(H 2 0) at t°C . Unless otherwise specified, the measurement is
to be performed by Method 1, Method 2 or Method 4. When
the specified value is accompanied with the term "about" in
the monograph, Method 3 is also available.
Method 1. Measurement using a pycnometer
A pycnometer is a glass vessel with a capacity of usually 10
mL to 100 mL, having a ground-glass stopper fitted with a
thermometer, and a side inlet-tube with a marked line and a
ground-glass cap.
Weigh a pycnometer, previously cleaned and dried, to de-
termine its mass W. Remove the stopper and the cap. Fill the
pycnometer with the sample solution, keeping them at a
slightly lower temperature by 1°C to 3 C C than the specified
temperature t'°C, and stopper them, taking care not to leave
bubbles. Raise the temperature gradually, and when the ther-
mometer shows the specified temperature, remove the por-
tion of the sample solution above the marked line through the
side tube, cap the side tube, and wipe the outside surface
thoroughly. Measure the mass W x of the pycnometer filled
with the sample solution. Perform the same procedure, using
the same pycnometer containing water, and note the mass W 2
at the specified temperature t°C. The specific gravity d' t ' can be
calculated by use of the following equation.
rff=
W,-W
w,-w
Further, when measurements for a sample solution and
water are performed at the same temperature (t°C = t'°C),
the density of the sample solution at the temperature t'°C
(p' T ) can be calculated from the measured specific gravity d' t ',
and the density of water at the temperature V °C (p' sl ) indicat-
ed in Table 2.56-1 by using the following equation.
p' T =p' si d<;,
Method 2. Measurement using a Sprengel-Ostwald pycno-
meter
A Sprengel-Ostwald pycnometer is a glass vessel with a
capacity of usually 1 mL to 10 mL. As shown in Fig. 2.56-1,
both ends are thick-walled fine tubes (inside diameter: 1-1.5
mm, outside diameter: 3-4 mm), one of which, tube A, has
a line C marked on it. Determine the mass of a pycnometer,
W, previously cleaned and dried, by hanging it on the arm of
a chemical balance with a platinum or aluminum wire D. Im-
merse the fine tube B in the sample solution, which is at a
lower temperature by 3°C to 5°C than the specified tempera-
ture t'°C. Attach rubber tubing or a ground-glass tube to the
end of A, and suck up the sample solution until the meniscus
is above the marked line C, taking care to prevent bubble for-
mation. Immerse the pycnometer in a water bath kept at the
specified temperature t'°C for about 15 minutes, and then,
62
Determination of Specific Gravity and Density / General Tests
JP XV
(density: p S i, Ps2> must be measured at a specified temperature
t'°C, and the cell constant ^ f (g-cm 3
determined by using the following equation.
2 ) must be
y
Fig. 2.56-1 Sprengel-Ostwald pycnometer
by attaching a piece of filter paper to the end of B, adjust the
level of the sample solution to the marked line C. Take the
pycnometer out of the water bath, wipe thoroughly the out-
side surface and determine the mass W x . By use of the same
pycnometer, perform the same procedure for the standard
solution of water. Weigh the pycnometer containing water at
the specified temperature t°C, and note the mass W 2 . Calcu-
late the specific gravity d' t ', according to the equation
described in Method 1.
Further, when measurements of specific gravity for a sam-
ple solution and water are performed at the same temperature
(f °C = t°C), the density of sample solution at temperature V
°C can be calculated by using the equation described in
Method 1.
Method 3. Masurement using a hydrometer
Clean a hydrometer with ethanol (95) or diethyl ether. Stir
the sample well with a glass rod, and float the hydrometer in
the well. When the temperature is adjusted to the specified
temperature t'°C and the hydrometer comes to a standstill,
read the specific gravity d\ or the density p' T at the upper brim
of the meniscus. Here the temperature t°C indicates the tem-
perature at which the hydrometer is calibrated. If specific in-
structions for reading the meniscus are supplied with the
hydrometer, the reading must be in accordance with the in-
structions.
Further, when measurement of the specific gravity for a
sample solution is performed at the same temperature (t'°C
= t°C), at which the hydrometer is calibrated, the density of
a sample solution at V °C, p' T , can be calculated by using the
specific gravity df and the equation shown in Method 1.
Method 4. Measurement using an oscillator-type density
meter
Density measurement with an oscillator-type density meter
is a method for obtaining the density of liquid or gas by
measuring the intrinsic vibration period T (s) of a glass tube
cell filled with sample specimen. When a glass tube conta-
ining a sample is vibrated, it undergoes a vibration with an in-
trinsic vibration period T in proportion to the mass of the
sample specimen. If the volume of the vibrating part of the
sample cell is fixed, the relation of the square of intrinsic
oscillation period and density of the sample specimen shall be
linear.
Before measuring a sample density, the respective intrinsic
oscillation periods T SI and T S2 for two reference substances
K,
Psi Psi
Usually, water and dried air are chosen as reference sub-
stances. Here the density of water at t'°C, p' sl , is taken from
the attached Table, and that of dried air p' S2 is calculated by
using the following equation, where the pressure of dried air
is at p kPa.
/4 = 0.0012932 x {273.15/(273.15 + ?')} x (p/101.325)
Next, introduce a sample specimen into a sample cell hav-
ing a cell constant K r , the intrinsic vibration period, T T , for
the sample under the same operation conditions as employed
for the reference substances. The density of a sample speci-
men at t'°C, p' T , is calculated by use of the following equa-
tion, by introducing the intrinsic oscillation period T si and
the density of water at a specified temperature t'°C, p' sl! into
the equation.
p> T =p> sl +K t ,{TJ-T^)
Further, the specific gravity of a sample specimen d' t '
against water at a temperature t°C can be obtained by using
the equation below, by introducing the density of water at a
temperature t°C, p' sl , indicated in Table 2.56-1.
dl'=
P\
P'si
Apparatus
An oscillator-type density meter is usually composed of a
glass tube cell of about 1 mL capacity, the curved end of
which is fixed to the vibration plate, an oscillator which ap-
plies an initial vibration to the cell, a detector for measuring
the intrinsic vibration period, and a temperature controlling
system.
A schematic illustration of the apparatus is depicted in
Fig. 2.56-2.
Procedure
A sample cell, water, and a sample specimen are previously
adjusted to a specified temperature t'°C, Wash the sample
cell with water or an appropriate solvent, and dry it
thoroughly with a flow of dried air. Stop the flow of dried air,
confirm that the temperature is at the specified value, and
then measure the intrinsic oscillation period T S2 given by the
dried air. Separately, the atmospheric pressure p (kPa) must
be measured at the time and place of the examination. Next,
introduce water into the sample cell and measure the intrinsic
oscillation period T sl given by water. Using these values of
the intrinsic oscillation period and the atmospheric pressure,
the sample cell constant K r can be determined by use of the
above-mentioned equation.
Next, introduce a sample specimen into the glass cell, con-
firm the specified temperature, and measure the intrinsic
oscillation period T T given by the sample specimen. Using the
intrinsic oscillation periods for water and the sample speci-
men, the density of water p' si , and the cell constant K f , the
density of the sample specimen p' T can be obtained by use of
the above equation. If necessary, the specific gravity of the
sample specimen d' t ' against water at a temperature t°C, can
be calculated by using the density of water p' sl shown in
General Tests / Boiling Point and Distilling Range Test
63
I
D
J
1
A: Thermometer
temp . constant water B: Sample cell
C: Vibration plate
D: Amplifier
E: Detector
F: Vibrator
Fig. 2.56-2 Oscillator-type density meter
Table 2.56-1.
In this measurement, avoid the occurrence of bubble for-
mation in the sample cell, when a sample specimen or water is
introduced into the cell.
Table 2.56-1
Density of water
Temp.
Density
Temp.
Density
Temp.
Density
Temp.
Density
°C
g/mL
°C
g/mL
°C
g/mL
°C
g/mL
0.999 84
10
0.999 70
20
0.998 20
30
0.995 65
1
0.999 90
11
0.999 61
21
0.997 99
31
0.995 34
2
0.999 94
12
0.999 50
22
0.997 77
32
0.995 03
3
0.999 96
13
0.999 38
23
0.997 54
33
0.994 70
4
0.999 97
14
0.999 24
24
0.997 30
34
0.994 37
5
0.999 96
15
0.999 10
25
0.997 04
35
0.994 03
6
0.999 94
16
0.998 94
26
0.996 78
36
0.993 68
7
0.999 90
17
0.998 77
27
0.996 51
37
0.993 33
8
0.999 85
18
0.998 60
28
0.996 23
38
0.992 97
9
0.999 78
19
0.998 41
29
0.995 94
39
0.992 59
10
0.999 70
20
0.998 20
30
0.995 65
40
0.992 22
In this Table, although the unit of density is represented by g/mL
in order to harmonize with the unit expression in the text, it
should be expressed in g/cm 3 seriously.
2.57 Boiling Point and
Distilling Range Test
The boiling point and distilling range are determined by
Method 1 or Method 2 as described herein, unless otherwise
specified. Boiling point is the temperature shown between
when the first 5 drops of distillate leave the tip of the con-
denser and when the last liquid evaporates from the bottom
of the flask. Distilling range test is done to determine the
volume of the distillate which has been collected in the range
of temperature directed in the monograph.
Method 1 This method is applied to a sample for which the
permissible range of boiling temperature is smaller than 5°C.
(1) Apparatus
Use the apparatus illustrated in Fig. 2.57-1.
(2) Procedure
Measure 25 mL of the sample, whose temperature is previ-
ously noted, using a volumetric cylinder G graduated in 0.1
A: Distilling flask
B: Thermometer with an immersion
line
C: Immersion line
D: Cork stopper
E: Condenser
F: Adapter
G: Volumetric cylinder
(25 mL, graduated to 0.1 mL)
The figures are in mm.
Fig. 2.57-1
mL, and transfer it to a distilling flask A of 50- to 60-mL
capacity. Use this cylinder as the receiver for the distillate
without rinsing out any of the adhering liquid. Put boiling
chips into the distilling flask A, insert a thermometer B with
an immersion line so that its immersion line C is on a level
with the lower end of cork stopper D and the upper end of its
mercury bulb is located in the center of the delivery tube, and
connect condenser E with the distilling flask A and adapter F
with the condenser E. Insert the open end of F into the mouth
of cylinder G (receiver) so that air can pass through slightly.
Use a hood with a height sufficient to shield A, and heat A
with a suitable heat source. When direct flame is applied as
the heat source, put A on a hole of a fire-resistant, heat-in-
sulating board [a board consisting of a fire-resistant, heat-in-
sulating material, 150 mm square and about 6 mm thick (or a
wire gauge of 150 mm square bonded to fire-resistant, heat-
insulation materials in about 6 mm thickness), having an its
center a round hole 30 mm in diameter].
Unless otherwise specified, distil the liquid sample by the
application of heat, at a rate of 4 to 5 mL per minute of distil-
late in the case of liquids whose boiling temperature to be de-
termined is lower than 200°C and at a rate of 3 to 4 mL per
minute in the case of liquids whose boiling temperature is 200
°C or over, and read the boiling point. For the distilling
range test, bring the temperature of distillate to the tempera-
ture at which the volume was originally measured, and meas-
ure the volume of distillate.
Liquids that begin to distil below 80°C are cooled to be-
tween 10°C and 15°C before measuring the volume, and the
receiving cylinder is kept immersed in ice up to a point 25 mm
from the top during the distillation.
Correct the observed temperature for any variation in the
barometric pressure from the normal (101.3 kPa), by allow-
ing 0.1 degree for each 0.36 kPa of variation, adding if the
pressure is lower, or subtracting if higher than 101.3 kPa.
Method 2 This method is applied to the sample for which
the permissible range of boiling temperature is 5°C or more.
(1) Apparatus
The same apparatus as described in Method 1 is used.
64
X-Ray Powder Diffraction Method / General Tests
JP XV
However, use a 200-mL distilling flask A with a neck 18 to 24
mm in inside diameter having a delivery tube 5 to 6 mm in in-
side diameter. The fire-resistant, heat-insulating board used
for direct flame heating should have in its center a round hole
50 mm in diameter.
(2) Procedure
Measure 100 mL of the sample, whose temperature is
previously noted, using a volumetric cylinder graduated in 1
mL, and carry out the distillation in the same manner as in
Method 1.
2.58 X-Ray Powder Diffraction
Method
X-Ray Powder Diffraction Method is a method for meas-
uring characteristic X-ray diffraction angles and intensities
from randomly oriented powder crystallites irradiated by a
monochromated X-ray beam. In this diffraction process,
electrons in the component atoms of crystallites are forced to
vibrate by the incident X-ray beam, and make sharp coherent
scatters of the X-ray. The diffraction pattern from a crystal-
line specimen is specific for the crystal form of the specimen.
Therefore the X-ray powder diffraction method can be used
for qualitative and quantitative evaluation of crystal forms,
formation of polymorphism, and crystalline solvates. The
random arrangement of molecules in non-crystalline sub-
stances makes them poor interference scatters of X-rays,
resulting in broad, diffuse maxima in the diffraction patterns.
Thus, for non-crystalline substances and/or materials with
poor crystallinity, the utility of this method is limited.
However, the X-ray diffraction method can be used for es-
timating the crystallinity of materials like plastics.
In crystalline materials, the molecular and atomic species
are ordered in a three-dimensional array, called a lattice. The
smallest lattice is designated the unit cell in crystallography.
A plane in the crystal can be defined by three arbitrary lattice
points and there is a family of planes separated by a certain
interplanar spacing from each other. Thus a family of planes
is called a crystalline plane and is usually indexed by using
Miller indices (hkl). These indices are the reciprocals,
reduced to smallest integers, of the intercepts that a plane
makes along the three axes composing the unit cell. Interpla-
nar spacing for a set of parallel planes (hkl) is denoted by
"hkl-
The occurrence of X-ray diffraction and the direction of
the diffracted beam by a crystallographic plane obey the
Bragg's law. According to this law, X-ray diffraction occurs
only when the scattered beams in a specific direction travel
distances that differ by integral multiples («), the order of the
reflection, of the incident X-ray wavelength (X), This condi-
tion is described by the equation,
2d,
= nX
in which d hkl denotes the interplanar spacing and 9 is the an-
gle of diffraction. The diffracted angle 2 9 due to a plane (hkl)
is specified by the intrinsic interplanar spacing, defined by the
lattice constants, and the wavelength of the incident X-ray
beam.
The intensity of a coherent X-ray diffraction is dependent
upon structure factors, temperature factor, degree of crystal-
linity, volume and density of the powdered specimen, absorp-
tion characteristics, intensity and wavelength of the incident
X-ray beam, polarization factor, multiplicity, Lorentz fac-
tor, etc. Among these factors, the polarization factor is de-
pendent upon the monochromatizing method of the incident
X-ray beam, the Lorentz factor upon geometrical factors of
the apparatus, multiplicity factor upon the crystalline sys-
tems, absorption factor upon the component atoms of the
sample, temperature factor and crystallinity upon experimen-
tal temperature and physical properties of the specimen, and
structural factor upon the position of each atom in the unit
cell and atomic species.
Apparatus
Usually an X-ray diffractometer equipped with a radiation
detector is used for X-ray powder diffraction measurement.
The diffractometer is composed of an X-ray source, goniome-
ter, radiation detector, and the combined controller and data
processor.
X-Ray radiation sources are usually vacuum tubes, in
which heated electrons are emitted from a cathode, and im-
pinge violently against an anode under a high electric field.
Since the wavelength of the generated X-rays depend upon
the type of metal utilized as the anode, an appropriate anode
has to be selected for the specimen to be analyzed. In general,
since the generated X-rays have a wide spectrum of
wavelength, an appropriate filter element or crystal
monochromator must be chosen so that a monochromated
beam is practically used for the diffraction analysis.
The goniometer is an angle scanning apparatus for adjust-
ing two angles, that between the X-ray beam and the speci-
men surface, and that between the specimen surface and the
detector. It is usually scanned with the above two angles
being held equal, which is called the symmetrical reflection
technique. The goniometer is equipped with a filter or a
monochromator for selecting a specific X-ray beam. Further
equipment may be included for heating or cooling sample
specimens.
A counting apparatus is composed of a detector and a
computing apparatus, of which the former converts the in-
tensity of scattered X-rays to electrical signals, while the lat-
ter converts the obtained signals to diffraction intensity
values. A proportional counter or a scintillation counter is
usually used as the detector.
A combination of controller and data processor is used for
controlling the goniometer angle, for recording diffraction in-
tensity, and for data processing.
Operation Procedure
The following variables have to be selected and/or deter-
mined before performing a diffraction measurement for a
given specimen: anode type, electric current and voltage for
the X-ray vacuum tube, scanning speed and range of the
goniometer, time constant and so on. A copper anode is most
commonly employed for organic substances and polymers.
A powdered specimen is usually packed and prepared in a
specimen holder made of aluminum or glass. As a rule, the
orientation of sample crystallites have to be randomized be-
fore packaging. The specimen may be ground in an agate
mortar to a fine powder in order to randomize the orientation
of crystallites. However, this grinding method is sometimes
inappropriate due to the physical characteristics of a speci-
men or the measurement object.
In setting up the specimen and apparatus, coplanarity of
JPXV
General Tests / Test for Total Organic Carbon
65
the specimen surface with the specimen holder surface and
the setting of the specimen holder at the position of symmet-
ric reflection geometry have to be assured. Further it should
be noted that the grinding procedure may affect the crystallin-
ity of the specimen and the packaging pressure on the speci-
men holder may induce orientation of the crystallites.
Identification and/or Judgement
Identification of the specimen with the standard material
can be accomplished by comparing the X-ray powder diffrac-
tion patterns with each other. Judgement of polymorphism
and crystalline solvates can be done by comparison of the
diffraction pattern obtained for the specimen with that of the
reference material or the same material measured previously.
Comparison of two X-ray diffraction patterns should be
based on the intensity ratio of diffracted peaks, and the inter-
planar spacings d. The intensity ratio is defined by the ratio
of the peak intensity at a particular diffraction angle to the in-
tensity of the standard peak, for which the strongest maxi-
mum on the diffraction pattern is usually selected. However,
the diffraction angle 2 9 can be used as a basis for the identifi-
cation, where the same wavelength of the radiation beam is
utilized for the diffraction measurement of the sample and
reference material. The scanning angle range for diffraction
measurement is usually between 5° and 40° for ordinary or-
ganic substances, unless otherwise specified in Monographs.
Based on the obtained X-ray diffraction patterns, the identifi-
cation of a specimen with a standard material can be con-
firmed, if the diffraction pattern for the specimen gives
diffraction peaks of the same intensity at the same diffraction
angle 2 9, as those of the standard. If two powder crystallites
ascribed to the same substance have the same crystal form,
the X-ray diffraction angles should agree within ± 0.2°.
Assay
A quantitative analysis by X-ray powder diffraction does
not give a sufficiently precise result. Thus, the quantitative
application of this method is limited to a few analytical
problems: numerical estimation of degree of polymorphism,
solvation number for crystalline solvates, and degree of crys-
tallinity.
For a quantitative analysis of polymorphism and/or sol-
vate, an appropriate diffraction peak has to be selected.
Usually, the calibration curve method can be applied to the
quantitative estimation by the X-ray analysis. Before meas-
urement of the diffraction intensity for a sample specimen at
a selected diffraction peak, a calibration curve must be pre-
pared under the same conditions, using a series of standard
samples containing known amounts of the objective sub-
stance.
Alternatively the internal standard method can also be
effective in place of the above standard method. A known
amount of the internal standard is usually added to weighed
amounts of a sample to be analyzed. Diffraction intensity ra-
tios of the specimen to the internal standard are measured.
Separately, a calibration curve for the intensity ratio against
the mixing ratio of the reference material to the internal stan-
dard are prepared under the same conditions. By using the
calibration curve, a quantitative analysis is possible in X-ray
powder diffraction measurement. If more than two diffrac-
tion peaks ascribed to different lattice planes (hkl) are used,
the influence of orientation of crystallites can be detected.
The internal standard should have approximately the same
density as the specimen and similar absorption characteristics
with regard to the X-ray beam. Further the diffraction peak
given by the standard should not overlap with that of the
specimen to be analyzed.
Caution: Handle the apparatus with great care since X-ray
may affect the human health.
2.59 Test for
Total Organic Carbon
Test for Total Organic Carbon is a method for measuring
the amount of organic carbon, which forms organic com-
pounds, in water. Normally, organic carbon can be oxidized
to carbon dioxide by a dry decomposition method, where or-
ganic compounds are oxidized by combustion, or by a wet
decomposition method, where organic compounds are oxi-
dized by applying ultraviolet rays or by adding oxidizing a-
gent. The amount of carbon dioxide generated in the decom-
position process is measured using an appropriate method
such as infrared gas analysis, electric conductivity measure-
ment, or resistivity measurement. The amount of organic car-
bon in water can be calculated from the amount of carbon di-
oxide measured in one of the above methods.
There are two types of carbon in water: organic carbon and
inorganic carbon. For measuring the amount of organic car-
bon, two approaches can be taken. One method is to measure
the amount of total carbon in water, then to subtract the
amount of inorganic carbon from that of total carbon. The
other method is to remove inorganic carbon from the test
water, then to measure the amount of remaining organic
carbon.
Instrument
The instrument consists of a sample injection port, a
decomposition device, a carbon dioxide separation block, a
detector, and a data processor or a recorder. The instrument
should be capable of measuring the amount of organic car-
bon down to 0.050 mg/L.
The sample injection port is designed to be able to accept a
specific amount of sample injected by a microsyringe or other
appropriate sampling devices. The decomposition device for
the dry decomposition method consists of a combustion tube
and an electric furnace to heat the sample. Both devices are
adjusted to operate at specified temperatures. The decompo-
sition device for the wet decomposition method consists of an
oxidizing reaction box, an ultraviolet ray lamp, a decomposi-
tion aid injector, and a heater. The decomposition device for
either method should be capable of generating not less than
0.450 mg/L of organic carbon when using a solution of sodi-
um dodecylbenzenesulfonate (theoretical value of total or-
ganic carbon in this solution is 0.806 mg/L) as the sample.
The carbon dioxide separation block removes water from
carbon dioxide formed in the decomposition process or
separates carbon dioxide from the decomposed gas. An in-
frared gas analyzer, electric conductivity meter or specific
resistance meter is used as the detector which converts the
concentration of carbon dioxide into electric signal. The data
processor calculates the concentration of the total organic
carbon in the sample based on the electric signal converted by
the detector. The recorder records the electric signal intensity
converted by the detector.
66
Melting Point Determination / General Tests
JP XV
Reagents and standard solutions
Water used for measuring organic carbon (water for meas-
urement): This water is used for preparing standard solutions
or decomposition aid or for rinsing the instrument. The
amount of organic carbon in this water, when collected into a
sample container, should be not more than 0.250 mg/L.
Standard potassium hydrogen phthalate solution: The con-
centration of this standard solution is determined as specified
for the instrument. Dry potassium hydrogen phthalate (stan-
dard reagent) at 105 C C for 4 hours, and allow it to cool in a
desiccator (silica gel). Weigh accurately a prescribed amount
of dried potassium hydrogen phthalate, and dissolve it in the
water for measurement to prepare the standard solution.
Standard solution for measuring inorganic carbon: The
concentration of this standard solution is determined as spe-
cified for the instrument. Dry sodium hydrogen carbonate in
a desiccator (sulfuric acid) for not less than 18 hours. Dry so-
dium carbonate decahydrate separately between 500°C and
600°C for 30 minutes, and allow to cool in a desiccator (silica
gel). Weigh accurately prescribed amounts of these com-
pounds so that the ratio of their carbon content is 1:1, and
dissolve them in the water for measurement to prepare the
standard solution.
Decomposition aid: Dissolve a prescribed amount of
potassium peroxodisulfate or other substances that can be
used for the same purpose, in the water for measurement up
to the concentration as specified for the instrument.
Gas for removing inorganic carbon or carrier gas: Nitro-
gen, oxygen, or other gases that can be used for the same pur-
pose.
Acid for removing inorganic carbon: Dilute hydrochloric
acid, phosphoric acid or other acids that can be used for the
same purpose, with the water for measurement down to the
concentration as specified for the instrument.
Apparatus
Sample container and reagent container: Use a container
made of the material which does not release organic carbon
from its surface, such as hard glass. Soak the container be-
fore use in a mixture of diluted hydrogen peroxide solution (1
in 3) and dilute nitric acid (1:1), and wash well with the water
for measurement.
Microsyringe: Wash a microsyringe with a mixture of a so-
lution of sodium hydroxide (1 in 20) and ethanol (99.5) (1:1),
or diluted hydrochloric acid (1 in 4), and rinse well with the
water for measurement.
Procedure
Employ an analytical method suitable for the instrument
used. Calibrate the instruments using the standard potassium
hydrogen phthalate solution with the test procedure specified
for the instrument.
It is recommended that this instrument be incorporated
into the manufacturing line of the water to be tested.
Otherwise, this test should be performed in a clean circum-
stance where the use of organic solvents or other substances
that may affect the result of this test is prohibited, using a
large sample container to collect a large volume of the water
to be tested. The measurement should be done immediately
after the sample collection.
(1) Measurement of organic carbon by subtracting inor-
ganic carbon from total carbon
According to the test procedure specified for the instru-
ment used, inject a suitable volume of the sample for measur-
ing the expected amount of total carbon into the instrument
from sample injection port, and decompose organic and inor-
ganic carbon in the sample. Detect the generated carbon di-
oxide with the detector, and calculate the amount of total
carbon in the sample using a data processor or a recorder.
Change the setting of the instrument for measuring inorganic
carbon exclusively, and measure the amount of inorganic car-
bon in the same manner as total carbon. The amount of or-
ganic carbon can be obtained by subtracting the amount of
inorganic carbon from that of total carbon.
(2) Measurement of organic carbon after removing inor-
ganic carbon
Remove inorganic carbon by adding the acid for removing
inorganic carbon to the sample, followed by bubbling the gas
for removing inorganic carbon (e.g. nitrogen) into the sam-
ple. According to the test procedure specified for the instru-
ment used, inject a suitable volume of the sample for measur-
ing the expected amount of organic carbon into the instru-
ment from sample injection port, and decompose the sample.
Detect the generated carbon dioxide with the detector, and
calculate the amount of organic carbon in the sample using a
data processor or a recorder.
For the instrument where the removal of inorganic carbon
is performed in the instrument, first inject a suitable volume
of the sample for measuring the expected amount of organic
carbon into the instrument from sample injection port, ac-
cording to the test procedure specified for the instrument
used. Then, remove inorganic carbon by adding the acid for
removing inorganic carbon to the sample in the decomposi-
tion device, followed by bubbling the gas for removing inor-
ganic carbon into the sample. Decompose organic carbon,
detect the generated carbon dioxide with the detector, and
calculate the amount of organic carbon using a data proces-
sor or a recorder.
2.60 Melting Point Determination
The melting point is defined to be the temperature at which
a crystalline substance melts during heating, when the solid
phase and the liquid phase are in an equilibrium. However, in
this Pharmacopoeia it is conventionally defined to be the tem-
perature at which the remaining solid sample melts complete-
ly when it is subjected to continuous heating and the change
of the sample state that accompanies heating is accurately ob-
served. Since a pure substance has an intrinsic melting point,
it is used for the identification and/or confirmation of a sub-
stance and also as an indicator of the purity of a substance.
The melting point is determined by either of the following
methods: Method 1 is applied to those substances of which
the purity is comparably high and which can be pulverized,
Method 2 to those substances which are insoluble in water
and can not be readily pulverized, and Method 3 to petrola-
tums.
Unless otherwise specified, measurement is performed by
Method 1.
Method 1
This method is applied to those substances of which the
purity is comparably high and which can be pulverized.
Apparatus
Use the apparatus illustrated in the Fig. 2.60-1.
JPXV
General Tests / Melting Point Determination
67
Alternatively, apparatus in which some of the procedures,
such as stirring, heating, and cooling are automated, can be
used.
Bath fluid: Usually use clear silicone oil having a viscosity
of 50 to 100 mmVs at an ordinary temperature.
Thermometer with an immersion line: There are six types
of thermometers, Type 1 — Type 6, which are specified by an
appropriate measuring temperature range. For melting points
lower than 50°C, use a thermometer Type 1; for 40°C to
100°C, Type 2; for 90°C to 150°C, Type 3; for 140°C to
200°C, Type 4; for 190°C to 250°C, Type 5; for 240°C to
320°C, Type 6.
Capillary tube: Use a hard glass capillary tube 120 mm
long, 0.8 to 1.2 mm in inner diameter and 0.2 to 0.3 mm
thick, with one end closed.
Procedure
Pulverize the sample to a fine powder, and, unless other-
wise specified, dry in a desiccator (silica gel) for 24 hours.
When it is specified to do the test after drying, dry the sample
under the conditions specified in the monograph before meas-
urement. Place the sample in a dried capillary tube H, and
pack it tightly so as to form a layer about 2.5 - 3.5 mm high
by dropping the capillary repeatedly, with the closed end of
H down, through a glass tube, about 70 cm long, held verti-
cally on a glass or porous plate.
Heat the bath fluid B until the temperature rises to about
10°C below the expected melting point, place the thermome-
ter D in the bath with the immersion line at the same level as
the meniscus of the bath fluid, and insert capillary tube H
into a coil spring G so that the packed sample is placed in a
position corresponding to the center of the mercury bulb of
the thermometer D. Continue heating to raise the tempera-
ture at a rate of approximately 3°C per minute until the
temperature rises to 5°C below the expected melting point,
then carefully regulate the rate of temperature increase to
1°C per minute.
Read the thermometer indication of the instantaneous tem-
perature at which the sample liquefies completely and no
solid is detectable in the capillary, and designate the indicated
temperature as the melting point of the sample specimen.
System suitability test — Confirmation of the system
suitability of the apparatus should be done periodically by
using the Melting Point Standards. The Reference Standard
is prepared for the suitability test of the apparatus when it is
used with Type 2 — Type 5 thermometers, and consists of 6
highly purified substances: acetanilide, acetophenetidine,
caffeine, sulfanilamide, sulfapyridine, and vanillin. The label
shows the certified melting points of the respective substances
(the end point of the melting change), MP t .
After selecting one of the thermometers and the appropri-
ate Melting Point Standard based upon the expected melting
point of a sample specimen, perform a melting point meas-
urement of the selected Reference Standard, according to the
above procedure. When the value of the obtained melting
point of the Reference Standard is within MP f ± 0.5°C in the
case of vanillin and acetanilide, within MP t ±0.8°C in the
case of acetophenetidine and sulfanilamide, and within MP f
± 1 .0°C in the case of sulfapyridine and caffeine, the appara-
tus is assumed to be suitable.
The above-mentioned measurement is repeated 3 times and
the average is determined to be the melting point of the
Reference Standard tested. When the above suitability test
immersion line -^
7
|
( ° \ 6 j
■ o (Jo •
t
(it
10
10
,
- 52 -
lilt 1 figures are in mm.
A: Heating vessel of liard glass
B: Bath fluid
C: Teflon stopper
D: Thermometer with an immersion line
E: Thermometer-fastening spring
I-": Vent for adjustment of the bath fluid volume
G: Coil spring
H: Capillary tube
J: Spring for fastening Teflon stopper
Fig. 2.60-1 Melting point determination apparatus
criteria are not met in a certain melting point measurement
system of an apparatus and a Reference Standard, do the test
again, after checking the packing of the sample specimen into
the capillary tube, the locations and positioning of the ther-
mometer and the capillary tube, the heating and stirring of
the bath fluid, and the control of the temperature increasing
rate. When a melting point measurement system does not
meet the suitability test criteria again after checking these
measuring conditions, the thermometer with an immersion
line should be calibrated again or replaced with a new one.
Method 2
This method is applied to substances such as fats, fatty
acids, paraffins or waxes.
Apparatus
Instead of the apparatus specified in Method 1, use a
water-containing beaker as a bath fluid and a heating vessel.
In this measurement, total immersion mercury-filled ther-
mometers can also be used in place of the thermometer with
an immersion line. Furthermore, the capillary tube should be
68
Determination of Bulk and Tapped Densities / General Tests
JP XV
the same as specified in Method 1, except that both ends of
the tube are open.
Procedure
Carefully melt the sample at as low a temperature as possi-
ble, and, taking care to prevent bubbles, introduce it into a
capillary tube to a height of about 10 mm. Allow the capillary
containing the sample to stand for 24 hours at below 10°C, or
for at least 1 hour in contact with ice, holding the capillary so
that the sample can not flow out. Then attach the capillary to
the thermometer by means of a rubber band so that the
absorbed sample is located at a position corresponding to the
center of the mercury bulb. Adjust the capillary tube in a
water-containing beaker to such a position that the lower
edge of the sample is located 30 mm below the water surface.
Heat the beaker with constant stirring until the temperature
rises to 5°C below the expected melting point. Then regulate
the rate of temperature increase to 1 C C per minute. The
temperature at which the sample begins floating in the capil-
lary is taken as the melting point of the sample specimen.
Method 3
This method is applied to petrolatums.
Apparatus
Instead of the apparatus specified in Method 1, use a
water-containing beaker as a bath fluid and a heating vessel.
In this measurement, total immersion mercury-filled ther-
mometers can also be used in place of the thermometer with
an immersion line.
Procedure
Melt the sample slowly by heating, with thorough stirring,
until the temperature reaches 90 - 92°C. Discontinue the
heating, and allow the sample to cool to 8 - 10°C above the
expected melting point. Chill the bulb of the thermometer to
5°C, wipe and dry, and, while still cold, stick half of the
thermometer bulb into the melted sample. Withdraw it
immediately, hold vertically, cool until the attached sample
becomes turbid, then dip the sample-bearing bulb for 5
minutes in water having a temperature below 16°C. Next, fix
the thermometer securely in a test tube by means of a cork
stopper so that the lower end is located 15 mm above the
bottom. Suspend the test tube in a water-containing beaker
held at a temperature about 16°C, and raise the temperature
of the water bath to 30°C at a rate of 2°C per minute, then
continue heating carefully at a rate of 1 °C per minute until it
reaches the melting point. Read the thermometer indication
of the instantaneous temperature at which the first drop of
the sample leaves the thermometer. If the variations between
three repeated determinations are not more than 1°C, take
the average of the three as the melting point. If any variation
is greater than 1°C, make two additional measurements, and
take the average of the five as the melting point.
3. Powder Property
Determinations
3.01 Determination of Bulk and
Tapped Densities
Determination of Bulk and Tapped Densities is a method
to determine the bulk densities of powdered drugs under
loose and tapped packing conditions respectively. Loose
packing is defined as the state obtained by pouring a powder
sample into a vessel without any consolidation, and tapped
packing is defined as the state obtained when the vessel con-
taining the powder sample is to be repeatedly dropped a spe-
cified distance at a constant drop rate until the apparent
volume of sample in the vessel becomes almost constant. The
bulk density is expressed in mass per unit apparent volume of
powder (g/mL). Because the bulk density is one of the meas-
ures of packing properties, compressibility and flow proper-
ties, and is dependent on the "history' ' of the powder, it is es-
sential to report the bulk density to specify how the determi-
nation was made.
Bulk density
The bulk density is an apparent density obtained by pour-
ing a powder sample into a vessel without any consolidation.
The determination of bulk density is achieved by measuring
the apparent volume of a powder sample having a known
mass in a graduated cylinder (Method 1) or by measuring the
mass of powder in a vessel having a known volume (Method
2).
Method 1 (Constant mass method)
Unless otherwise specified, pass a quantity of sample
sufficient to complete the test through a 1000-//m (No. 16)
sieve to break up agglomerates that may have formed during
storage. Weigh accurately about 30 g of test sample, and
pour it into a dry 100-mL graduated glass cylinder (readable
to 1 mL). Carefully level the powder without consolidation,
if necessary, and read the unsettled apparent volume, V , to
the nearest graduated unit. Calculate the bulk density p B by
the formula:
p B = M/V a
p B : Bulk density by constant mass method (g/mL)
M: Mass of powder sample (g)
V : Apparent volume of powder sample (mL)
Record the average of 3 determinations using 3 different
powder samples. If a 30-g sample is too large to determine,
adjust the mass of sample so as to provide an apparent
volume of 60 - 100 mL.
Method 2 (Constant volume method)
Unless otherwise specified, pass a quantity of sample
sufficient to complete the test through a 1000-//m (No. 16)
sieve to break up agglomerates that may have formed during
storage. Allow an excess of sample powder to pour into the
measuring vessel having the volume of V and mass of M .
Carefully scrape excess powder from the top of the vessel us-
ing the edge of a slide glass or other tool by smoothly moving
across it. Remove any material from the sides of the vessel,
and determine the total mass M t . Calculate the bulk density p
B by the formula:
p B = (M t -M )/V
p B : Bulk density by constant volume method (g/mL)
M t : Total mass of powder and measuring vessel (g)
M : Mass of measuring vessel (g)
V: Volume of measuring vessel (mL)
Record the average of 3 determinations using 3 different
powder samples.
JPXV
General Tests / Specific Surface Area by Gas Adsorption
69
Tapped density
Tapped density is an apparent density obtained by mechan-
ically tapping a measuring vessel containing a powder sam-
ple. The determination of tapped density is achieved by meas-
uring the apparent volume of a powder sample having a
known mass in a vessel after tapping (Method 1) or by meas-
uring the mass of powder in a vessel having a known volume
after tapping (Method 2).
Method 1 (Constant mass method)
Unless otherwise specified, pass a quantity of sample
sufficient to complete the test through a lOOO-^m (No. 16) or a
710-//m (No. 22) sieve to break up agglomerates that may
have formed during storage. Weigh accurately about 100 g of
test sample, and pour it into a 250-mL graduated glass cylin-
der (readable to 2 mL) without consolidation. If it is not pos-
sible to use 100 g, proceed according to the same procedure as
that described above by using a 100-mL graduated glass
cylinder (readable to 1 mL). It is essential to select appropri-
ate masses of the cylinder support, holder and cylinder so as
to ensure the dynamic stability of the apparatus during tap-
ping. After attaching the glass cylinder containing the pow-
der sample to the tapping apparatus, carry out tapping under
the measuring conditions (tapping rate and drop height) spe-
cified for each apparatus.
Unless otherwise specified, repeat increments of 50 taps or
1 minute until the difference between succeeding measure-
ments is less than 2%, and determine the final apparent
volume, V t , Calculate the tapped density p T by the formula:
p T = M/V f
PY- Tapped density by constant mass method (g/mL)
M: Mass of powder sample (g)
V t : Final apparent volume of sample after tapping (mL)
Record the average of 3 determinations using 3 different
powder samples.
Method 2 (Constant volume method)
Unless otherwise specified, pass a quantity of sample
sufficient to complete the test through a 1000-//m (No. 16)
sieve to break up agglomerates that may have formed during
storage. Attach a supplementary cylinder to the stainless steel
measuring vessel having a known mass of M and a volume of
V (Fig. 3.01-1), and then pour an excess of the sample into
the vessel. After setting up the vessel in an adequate tapping
apparatus with a fixed drop height, carry out tapping at the
rate and cumulative tap number specified for each apparatus.
Then remove the supplementary cylinder from the vessel and
carefully scrape excess powder from the top of the vessel by
smoothly moving across it the edge of a slide glass or other
tool. Remove any material from the sides of the vessel, and
determine the total mass M t . Calculate the tapped density p T
by the formula:
p T = (M-M Q )/V
Pi\ Tapped density by constant volume method (g/mL)
M t : Total mass of powder and measuring vessel (g)
M : Mass of measuring vessel (g)
V: Volume of measuring vessel (mL)
Record the average of 3 determinations and the relative
standard deviation using 3 different powder samples. If the
relative standard deviation is not less than 2%, repeat the test
54.0
52.0
2.0 :
ft
L
Measuring vessel
57.0
505
°Zf
54.0
50.U
Supplementary cylinder
The figures are in mm used for a 100-mL measuring vessel and
supplementary cylinder for example.
Fig. 3.01-1 Measuring vessel (upper) and supplementary
cylinder (down)
with further tapping.
Balances: Use balances readable to the nearest 0.1 g.
3.02 Specific Surface Area
by Gas Adsorption
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* »).
*The specific surface area determination method is a
method to determine specific surface area (the total surface
area of powder per unit mass) of a pharmaceutical powder
sample by using gas adsorption method. » The specific sur-
face area of a powder is determined by physical adsorption of
a gas on the surface of the solid and by calculating the
amount of adsorbate gas corresponding to a monomolecular
layer on the surface. Physical adsorption results from rela-
tively weak forces (van der Waals forces) between the adsor-
bate gas molecules and the adsorbent surface of the test pow-
der. The determination is usually carried out at the tempera-
ture of liquid nitrogen. The amount of gas adsorbed can be
measured by a volumetric or continuous flow procedure.
70
Specific Surface Area by Gas Adsorption / General Tests
JP XV
MULTI-POINT MEASUREMENT
When the gas is physically adsorbed by the powder sample,
the following relationship (Brunauer, Emmett and Teller
(BET) adsorption isotherm) holds when the relative pressure
(P/P ) is in the range of 0.05 to 0.30 for pressure P of the ad-
sorbate gas in equilibrium for the volume of gas adsorbed,
l/[K a {(P„/P)-l}]
= {(C- l)/K m C} x (P/P ) + (UV m Q
(1)
P: Partial vapour pressure of adsorbate gas in equilibrium
with the surface at — 195. 8°C (b.p. of liquid nitrogen ), in
pascals,
P : Saturated pressure of adsordate gas, in pascals,
F a : Volume of gas adsorbed at standard temperature and
pressure (STP) [0°C and atmospheric pressure (1.013 x 10 5
Pa)], in milliliters,
V m : Volume of gas adsorbed at STP to produce an apparent
monolayer on the sample surface, in milliliters,
C: Dimensionless constant that is related to the enthalpy of
adsorption of adsorbate gas on the powder sample.
A value of F a is measured at each of not less than 3 values
of P/P . Then the BET value, l/[F a {(P /P)- 1}], is plotted
against P/P according to equation (1). This plot should yield
a straight line usually in the approximate relative pressure
range 0.05 to 0.3. The data are considered acceptable if the
correlation coefficient, r, of the linear regression is not less
than 0.9975; that is, r 1 is not less than 0.995. From the result-
ing linear plot, the slope, which is equal to (C— \)/V m C, and
the intercept, which is equal to l/V m C, are evaluated by
linear regression analysis. From these values, V m is calculated
as 1/ '{slope + intercept), while C is calculated as (slope/ ' inter-
cept) -\-\, From the value of V m so determined, the specific
surface area, S, in m 2 g _1 , is calculated by the equation:
S = (V m Na)/(mx 22400)
(2)
N: Avogadro constant (6.022 x 10 23 mol -1 ),
a: Effective cross-sectional area of one adsorbate molecule, in
square metres (0.162 nm 2 for nitrogen and 0.195 nm 2 for
krypton),
in: Mass of test powder, in grams,
Specific surface area is generally expressed in units of m 2 g~'.
SINGLE-POINT MEASUREMENT
Normally, at least 3 measurements of F a each at different
values of P/Pg are required for the determination of specific
surface area by the dynamic flow gas adsorption technique
(Method I) or by volumetric gas adsorption (Method II).
However, under certain circumstances described below, it
may be acceptable to determine the specific surface area of a
powder from a single value of K a measured at a single value
of P/P such as 0.30, using the following equation for cal-
culating V m :
V m =Vjl-(P/P )}
(3)
The single-point method may be employed directly for a s-
eries of powder samples of a given material for which the
material constant C is much greater than unity. Close similar-
ity between the single-point values and multiple-point values
suggests that 1/C approaches zero. The error on V m can be
reduced by using the multiple-point method to evaluate C for
one of the samples of the series on which the material con-
stant C is expected to be large. Then V m is calculated from the
single value of F a measured at a single value of P/P by the
equation:
V m = VJ(P /P) - 1 } [(1/Q + {(C- 1)/C} x (P/P )] (4)
SAMPLE PREPARATION
Before the specific surface area of the sample can be deter-
mined, it is necessary to remove gases and vapors that may
have become physically adsorbed onto the surface during
storage and handling. If outgassing is not achieved, the
specific surface area may be reduced or may be variable be-
cause an intermediate area of the surface is covered with
molecules of the previously adsorbed gases or vapors. The
outgassing conditions are critical for obtaining the required
precision and accuracy of specific surface area measurements
on pharmaceuticals because of the sensitivity of the surface
of the materials. The outgassing conditions must be demon-
strated to yield reproducible BET plots, a constant weight of
test powder, and no detectable physical or chemical changes
in the test powder.
The outgassing conditions defined by the temperature,
pressure and time should be so chosen that the original sur-
face of the solid is reproduced as closely as possible.
Outgassing of many substances is often achieved by ap-
plying a vacuum, by purging the sample in a flowing stream
of a non-reactive, dry gas, or by applying a desorption-ad-
sorption cycling method. In either case, elevated tempera-
tures are sometimes applied to increase the rate at which the
contaminants leave the surface. Caution should be exercised
when outgassing powder samples using elevated temperatures
to avoid affecting the nature of the surface and the integrity
of the sample.
If heating is employed, the recommended temperature and
time of outgassing are as low as possible to achieve
reproducible measurement of specific surface area in an ac-
ceptable time. For outgassing sensitive samples, other out-
gassing methods such as the desorption-adsorption cycling
method may be employed.
The standard technique is the adsorption of nitrogen at liq-
uid nitrogen temperature.
For powders of low specific surface area (<0.2 m 2 g _1 ) the
proportion adsorbed is low. In such cases the use of krypton
at liquid nitrogen temperature is preferred because the low
vapor pressure exerted by this gas greatly reduces error.
The use of larger sample quantities where feasible (equiva-
lent to 1 m 2 or greater total surface area using nitrogen) may
compensate for the errors in determining low surface areas.
All gases used must be free from moisture.
Accurately weigh a quantity of the test powder such that
the total surface of the sample is at least 1 m 2 when the adsor-
bate is nitrogen and 0.5 m 2 when the adsorbate is krypton.
Lower quantities of sample may be used after appropriate
validation.
Adsorption of gas should be measured either by Method I
or Method II.
Method I: the dynamic flow method
In the dynamic flow method (see Fig. 3.02-1), the recom-
mended adsorbate gas is dry nitrogen or krypton, while heli-
um is employed as a diluent gas, which is not adsorbed under
the recommended conditions. A minimum of 3 mixtures of
the appropriate adsorbate gas with helium are required wi-
thin the P/P range 0.05 to 0.30.
JPXV
General Tests / Powder Particle Density Determination
71
A: How control valve
B: Differential (low controller
C:On-off valve
D: Gas inlet
F: ring seals
F: Cold trap
G: Thermal equilibration tube
H: Detector
I: Digital display
J: Calibrating septum
K: Sample cell
L: Self seals quick connection
M: Short path ballast
N: Detector
0: Path selection valve
P:I.ong pass ballast
Q: How meter
R: Oulgassing station
S: Diffusion baffle
T: Vent
Fig. 3.02-1 Schematic diagram of the dynamic flow
method apparatus
The gas detector-integrator should provide a signal that is
approximately proportional to the volume of the gas passing
through it under defined conditions of temperature and pres-
sure. For this purpose, a thermal conductivity detector with
an electronic integrator is one among various suitable types.
A minimum of 3 data points within the recommended range
of 0.05 to 0.30 for P/P is to be determined.
A known mixture of the gases, usually nitrogen and heli-
um, is passed through a thermal conductivity cell, through
the sample again through the thermal conductivity cell and
then to a recording potentiometer.
Immerse the sample cell in liquid nitrogen, then the sample
adsorbs nitrogen from the mobile phase. This unbalances the
thermal conductivity cell, and a pulse is generated on a
recorder chart.
Remove from the coolant; this gives a desorption peak
equal in area and in the opposite direction to the adsorption
peak.
Since this is better defined than the adsorption peak, it is
the one used for the determination.
To effect the calibration, inject a known quantity of adsor-
bate into the system, sufficient to give a peak of similar mag-
nitude to the desorption peak and obtain the proportion of
gas volume per unit peak area.
Method II: the volumetric method
In the volumetric method (see Figure 3.02-2), the recom-
mended adsorbate gas is nitrogen is admitted into the
evacuated space above the previously outgassed powder sam-
ple to give a defined equilibrium pressure, P, of the gas. The
use of a diluent gas, such as helium, is therefore unnecessary,
although helium may be employed for other purposes, such
as to measure the dead volume.
Admit a small amount of dry nitrogen into the sample tube
A Vacuum gauge
B: Nitrogen reservoir
C: Helium reservoir
D: Vapor pressure manometer
E: Vacuum and air
F: Cold traps and vacuum pumps
Fig. 3.02-2 Schematic diagram of the volumetric
method apparatus
to prevent contamination of the clean surface, remove the
sample tube, insert the stopper, and weigh it. Calculate the
weight of the sample. Attach the sample tube to the volumet-
ric apparatus. Cautiously evacuate the sample down to the
specified pressure (e.g. between 2 Pa and 10 Pa).
If the principle of operation of the instrument requires the
determination of the dead volume in the sample tube, this
procedure is carried out at this point, followed by evacuation
of the sample. Raise a Dewar vessel containing liquid nitro-
gen up to a defined point on the sample cell. Admit a
sufficient volume of adsorbate gas to give the lowest desired
relative pressure. Measure the volume adsorbed, K a . For mul-
tipoint measurements, repeat the measurement of V a at suc-
cessively higher P/P values. When nitrogen is used as the ad-
sorbate gas, P/P values of 0.10, 0.20, and 0.30 are often
suitable.
REFERENCE MATERIALS
Periodically verify the functioning of the apparatus using
appropriate reference materials of known surface area, such
as or-alumina for specific surface area determination, which
should have a specific surface area similar to that the sample
to be examined.
3.03 Powder Particle Density
Determination
Powder Particle Density Determination is a method to
determine particle density of powdered pharmaceutical drugs
or raw materials of drugs, and the gas displacement pyenom-
eter is generally used. The powder density by this method is
determined with an assumption that the volume of the gas
displaced by the powder in a closed system is equal to the
volume of the powder. The bulk density at loose packing or
the tapped density at tapping express the apparent densities
of the powder, since interparticulate void volume of the pow-
der is assumed to contribute a part of the volume of the pow-
der. On the contrary, the pyenometric particle density ex-
presses the powder density nearly equal to the crystal density,
72
Particle Size Determination / General Tests
JP XV
since the volume of the powder, that is deducted with void
volume of open pores accessible to gas, is counted.
Powder particle density is expressed in mass per unit
volume (kg/m 3 ), and generally expressed in g/cm 3 .
Apparatus
The schematic diagram of particle density apparatus for
gas displacement pycnometric measurement is shown in Fig.
3.03-1. The apparatus consists of a test cell in which the sam-
ple is placed, a reference cell and a manometer.
Generally, helium is used as the measurement gas. The
apparatus has to be equipped with a system capable of
pressuring the test cell to the defined pressure through the
manometer.
Calibration of apparatus The volumes of the test cell (V c )
and the reference cell (F r ) must be accurately determined to
the nearest 0.001 cm 3 , and to assure accuracy of the results of
volume obtained, calibration of the apparatus is carried out
as follows using a calibration ball of known volume for parti-
cle density measurement. The final pressures (P t ) are deter-
mined for the initial empty test cell followed by the test cell
placed with the calibration ball for particle density measure-
ment in accordance with the procedures, and V c and V r are
calculated using the equation described in the section of
Procedure. Calculation can be made taking into account that
the sample volume (V s ) is zero in the first run.
Procedure
The measurement of the particle density is carried out
between 15 and 30°C, and temperature must not vary by
more than 2°C during the course of measurement.
Firstly, weigh the mass of the test cell and record it. After
weighing out the amount of the sample as described in the in-
dividual monograph and placing it in the test cell, seal the cell
in the pycnometer. Secondly, introduce the measurement gas
(helium) into the test cell, and remove volatile contaminants
in the powder. If necessary, keep the sample powder under
reduced pressure to remove the volatile contaminants in ad-
vance and use it as the test sample for measurement.
Open the valve which connects the reference cell with the
test cell, confirm with the manometer that the pressure inside
the system is stable, and then read the system reference
pressure (P r ). Secondly, close the valve that connects to the
two cells, and introduce the measurement gas into the test cell
to achieve positive pressure. Confirm with the manometer
that the pressure inside the system is stable, and then read the
initial pressure (Pi). Open the valve to connect the test cell
with the reference cell. After confirming that the indicator of
the manometer is stable, read the final pressure (P t ), and cal-
culate the sample volume (V s ) with the following equation.
Pi-Pr
-1
Pt-P,
V T : Reference cell volume (cm 3 )
V c : Test cell volume (cm 3 )
V s : Sample volume (cm 3 )
Pj-. Initial pressure (kPa)
P t : Final pressure (kPa)
P r : Reference pressure (kPa)
Repeat the measurement sequence for the same powder
sample until consecutive measurements of the sample volume
agree to within 0.5%, and calculate the mean of sample
Vr Reference cell volume (cm 3 )
Vc: Test cell volume (cm 3 )
Vs: Sample volume (cm 3 )
M: Manometer
Fig. 3.03-1 Schematic diagram of a gas displacement
pycnometer
volumes (V s ). Finally, unload the test cell, weigh the mass of
test cell, and calculate the final sample mass by deducting the
empty cell mass from the test cell mass. The powder particle
density p is calculated by the following equation.
p = m/ V s
p: Powder particle density (g/cm 3 )
m: Final sample mass (g)
V s : Sample volume (cm 3 )
3.04 Particle Size Determination
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* »).
*Particle Size Determination is a method to determine di-
rectly or indirectly morphological appearance, shape, size
and its distribution of powdered pharmaceutical drugs and
excipients to examine their micromeritic properties. Optical
microscopy and analytical sieving method may be used de-
pending on the measuring purpose and the properties of test
specimen.*
Method 1. Optical Microscopy
*The optical microscopy is used to observe the morpholog-
ical appearance and shape of individual particle either di-
rectly with the naked eye or by using a microscopic photo-
graph, in order to measure the particle size. The particle size
distribution can also be determined by this method. It is also
possible with this method to measure the size of the individ-
ual particle even when different kinds of particles mingle if
they are optically distinguishable. Data processing tech-
niques, such as image analysis, can be useful for determining
the particle size distribution. ♦
This method for particle characterization can generally be
applied to particles 1 pm and greater. The lower limit is im-
posed by the resolving power of the microscope. The upper
limit is less definite and is determined by the increased
difficulty associated with the characterization of larger parti-
cles. Various alternative techniques are available for particle
characterization outside the applicable range of optical
microscopy. Optical microscopy is particularly useful for
characterizing particles that are not spherical. This method
may also serve as a base for the calibration of faster and more
routine methods that may be developed.
JPXV
General Tests / Particle Size Determination
73
Apparatus — Use a microscope that is stable and protected
from vibration. The microscope magnification (product of
the objective magnification, ocular magnification, and addi-
tional magnifying components) must be sufficient to allow
adequate characterization of the smallest particles to be clas-
sified in the test specimen. The greatest numerical aperture of
the objective should be sought for each magnification range.
Polarizing filters may be used in conjunction with suitable
analyzers and retardation plates. Color filters of relatively
narrow spectral transmission should be used with achromatic
objectives and are preferable with apochromats and are re-
quired for appropriate color rendition in photomicrography.
Condensers corrected for at least spherical aberration should
be used in the microscope substage and with the lamp. The
numerical aperture of the substage condenser should match
that of the objective under the condition of use; this is affect-
ed by the actual aperture of the condenser diaphragm and the
presence of immersion oils.
Adjustment — The precise alignment of all elements of the
optical system and proper focusing are essential. The focus-
ing of the elements should be done in accordance with the
recommendations of the microscope manufacturer. Critical
axial alignment is recommended.
Illumination — A requirement for good illumination is a
uniform and adjustable intensity of light over the entire field
of view; Kohler illumination is preferred. With colored parti-
cles, choose the color of the filters used so as to control the
contrast and detail of the image.
Visual Characterization — The magnification and numeri-
cal aperture should be sufficiently high to allow adequate
resolution of the images of the particles to be characterized.
Determine the actual magnification using a calibrated stage
micrometer to calibrate an ocular micrometer. Errors can be
minimized if the magnification is sufficient that the image of
the particle is at least 10 ocular divisions. Each objective must
be calibrated separately. To calibrate the ocular scale, the
stage micrometer scale and the ocular scale should be aligned.
In this way, a precise determination of the distance between
ocular stage divisions can be made.
*When the particle size is measured, an ocular micrometer
is inserted at the position of the ocular diaphragm, and a
calibrated stage micrometer is placed at the center of the
microscope stage and fixed in place. The ocular is attached to
the lens barrel and adjusted to the focus point of the stage
micrometer scale. Then, the distance between the scales of
the two micrometers is determined, and the sample size e-
quivalent 1 division of the ocular scale is calculated using the
following formula:
The particle size equivalent 1 division on the ocular scale
(urn) = Length on the stage micrometer (Mm)/Number of
scale divisions on the ocular micrometer
The stage micrometer is removed and the test specimen is
placed on the microscope stage. After adjusting the focus,
the particle sizes are determined from the number of scale di-
visions read through the ocular. ♦
Several different magnifications may be necessary to
characterize materials having a wide particle size distribution.
Photographic Characterization — If particle size is to be de-
termined by photographic methods, take care to ensure that
the object is sharply focused at the plane of the photographic
emulsion. Determine the actual magnification by pho-
tographing a calibrated stage micrometer, using photograph-
ic film of sufficient speed, resolving power, and contrast. Ex-
posure and processing should be identical for photographs of
both the test specimen and the determination of magnifica-
tion. The apparent size of a photographic image is influenced
by the exposure, development, and printing processes as well
as by the resolving power of the microscope.
Preparation of the Mount — The mounting medium will
vary according to the physical properties of the test specimen.
Sufficient, but not excessive, contrast between the specimen
and the mounting medium is required to ensure adequate de-
tail of the specimen edge. The particles should rest in one
plane and be adequately dispersed to distinguish individual
particles of interest. Furthermore, the particles must be
representative of the distribution of sizes in the material and
must not be altered during preparation of the mount. Care
should be taken to ensure that this important requirement is
met. Selection of the mounting medium must include a con-
sideration of the analyte solubility.
Crystallinity Characterization — The crystallinity of a
material may be characterized to determine compliance with
the crystallinity requirement where stated in the individual
monograph of a drug substance. Unless otherwise specified in
the individual monograph, mount a few particles of the speci-
men in mineral oil on a clean glass slide. Examine the mixture
using a polarizing microscope: the particles show birefringen-
ce(interference colors) and extinction positions when the
microscope stage is revolved.
Limit Test of Particle Size by Microscopy — Weigh a suita-
ble quantity of the powder to be examined (for example, 10
to 100 mg), and suspend it in 10 mL of a suitable medium in
which the powder does not dissolve, adding, if necessary, a
wetting agent. A homogeneous suspension of particles can be
maintained by suspending the particles in a medium of simi-
lar or matching density and by providing adequate agitation.
Introduce a portion of the homogeneous suspension into a
suitable counting cell, and scan under a microscope an area
corresponding to not less than 10 fig of the powder to be ex-
amined. Count all the particles having a maximum dimension
greater than the prescribed size limit. The size limit and the
permitted number of particles exceeding the limit are defined
for each substance.
Particle Size Characterization — The measurement of parti-
cle size varies in complexity depending on the shape of the
particle and the number of particles characterized must be
sufficient to insure an acceptable level of uncertainty in the
measured parameters 1 '. For spherical particles, size is defined
by the diameter. For irregular particles, a variety of defini-
tions of particle size exist. In general, for irregularly shaped
particles, characterization of particle size must also include
information on the type of diameter measured as well as in-
formation on particle shape. Several commonly used mea-
surements of particle size are defined below (see Fig. 3.04-1):
Feret's Diameter — The distance between imaginary
parallel lines tangent to a randomly oriented particle and per-
pendicular to the ocular scale.
Martin's Diameter — The diameter of the particle at the
point that divides a randomly oriented particle into two equal
projected areas.
Projected area Diameter — The diameter of a circle that has
the same projected are as the particle.
Length — The longest dimension from edge to edge of a
particle oriented parallel to the ocular scale.
Width — The longest dimension of the particle measured at
74
Particle Size Determination / General Tests
JP XV
Table 3.04-1. Sizes of Standard Sieve Series in Range of Interest
ISO Nominal Aperture
Principal
Supplement
ary
US Sieve
Recommended
European
Japan
sizes
sizes
No.
USP Sieves
(mesh)
Sieve No.
Sieve No.
R20/3
R 20
R40/3
11.20 mm
11.20 mm
10.00 mm
9.00 mm
11.20 mm
9.50 mm
11200
8.00 mm
8.00 mm
7.10 mm
6.30 mm
8.00 mm
6.70 mm
5.60 mm
5.60 mm
5.00 mm
4.50 mm
5.60 mm
4.75 mm
5600
3.5
4
4.00 mm
4.00 mm
3.55 mm
3.15 mm
4.00 mm
3.35 mm
5
6
4000
4000
4.7
5.5
2.80 mm
2.80 mm
2.50 mm
2.24 mm
2.80 mm
2.36 mm
7
8
2800
2800
6.5
7.5
2.00 mm
2.00 mm
1.80 mm
1.60 mm
2.00 mm
1.70 mm
10
12
2000
2000
8.6
10
1.40 mm
1.40 mm
1.25 mm
1.12 mm
1.40 mm
1.18 mm
14
16
1400
1400
12
14
1 .00 mm
1.00 mm
900 ftm
800 ftm
1.00 mm
850 ftm
18
20
1000
1000
16
18
710 ftm
l\0ftm
630 ftm
560 ftm
l\0ftm
600 ftm
25
30
710
710
22
26
500 ftm
500 ftm
450 ftm
400 ftm
500 ftm
425 ftm
35
40
500
500
30
36
355 ftm
355 ftm
315 ftm
280 ftm
355 ftm
300 ftm
45
50
355
355
42
50
250 ftm
250 ftm
224 ftm
200 ftm
250 ftm
2\2fim
60
70
250
250
60
70
ISO ftm
\80 ftm
\60ftm
140 ftm
\%0 ftm
\50ftm
80
100
180
180
83
100
125 ftm
\25ftm
\l2ftm
\00ftm
125 ftm
106 ftm
120
140
125
125
119
140
90 ftm
90 ftm
SO ftm
71 ftm
90 ftm
15 ftm
170
200
90
90
166
200
63 ftm
63 ftm
56 ftm
50 ftm
63 ftm
53 ftm
230
270
63
63
235
282
45 ftm
45 ftm
40 ftm
45 ftm
38 ftm
325
45
45
38
330
391
right angles to the length.
Particle Shape Characterization — For irregularly shaped
particles, characterization of particle size must also include
information on particle shape. The homogeneity of the pow-
der should be checked using appropriate magnification. The
following defines some commonly used descriptors of particle
shape (see Fig. 3.04-2):
Acicular — Slender, needle-like particle of similar width
JPXV
General Tests / Particle Size Determination
75
Feret's diameter
Martin's cliainctcr
Projected HYVA diameter
Maximum horizontal intercept
Equant
i,' Oriat
Hake
Fig. 3.04-2 Commonly used descriptions of particle
shape
Fig. 3.04-1 Commonly used measurements of particle
size
and thickness.
Columnar — Long, thin particle with a width and thickness
that are greater than those of an acicular particle.
Flake — Thin, flat particle of similar length and width.
Plate — Flat particles of similar length and width but with
greater thickness than flakes.
Lath — Long, thin, and blade-like particle.
Equant — Particles of similar length, width, and thickness;
both cubical and spherical particles are included.
General Observations — A particle is generally considered
to be the smallest discrete unit. A particle may be a liquid or
semisolid droplet; a single crystal or polycrystalline; amor-
phous or an agglomerate. Particles may be associated. This
degree of association may be described by the following
terms:
Lamellar — Stacked plates.
Aggregate — Mass of adhered particles.
Agglomerate — Fused or cemented particles.
Conglomerate — Mixture of two or more types of particles.
Spherulite — Radial cluster.
Drusy — Particle covered with tiny particles.
Particle condition may be described by the following
terms:
Edges — Angular, rounded, smooth, sharp, fractured.
Optical — Color (using proper color balancing filters),
transparent, translucent, opaque.
Defects — Occlusions, inclusions.
Surface characteristics may be described as:
Cracked — Partial split, break, or fissure.
Smooth — Free of irregularities, roughness, or projections.
Porous — Having openings or passageways.
Rough — Bumpy, uneven, not smooth.
Pitted — Small indentations.
Method 2. Analytical Sieving Method
*The analytical sieving method is a method to estimate the
particle size distribution of powdered pharmaceutical drugs
by sieving. The particle size determined by this method is
shown as the size of a minimum sieve opening through which
the particle passes. "Powder" here means a gathering of
numerous solid particles. »
Sieving is one of the oldest methods of classifying powders
and granules by particle size distribution. When using a
woven sieve cloth, the sieving will essentially sort the particles
by their intermediate size dimension(i.e., breadth or width).
Mechanical sieving is most suitable where the majority of the
particles are larger than about 75 [im. For smaller particles,
the light weight provides insufficient force during sieving to
overcome the surface forces of cohesion and adhesion that
cause the particles to stick to each other and to the sieve, and
thus cause particles that would be expected to pass through
the sieve to be retained. For such materials other means of
agitation such as air-jet sieving or sonic sifting may be more
appropriate. Nevertheless, sieving can sometimes be used for
some powders or granules having median particle sizes
smaller than 75 ^m where the method can be validated. In
pharmaceutical terms, sieving is usually the method of choice
for classification of the coarser grades of single powders or
granules. It is a particularly attractive method in that pow-
ders and granules are classified only on the basis of particle
size, and in most cases the analysis can be carried out in the
dry state.
Among the limitations of sieving method are the need for
an appreciable amount of sample (normally at least 25 g, de-
pending on the density of the powder or granule, and the di-
ameter of test sieves) and difficulty in sieving oily or other co-
hesive powders or granules that tend to clog the sieve open-
ings. The method is essentially a two-dimensional estimate of
size because passage through the sieve aperture is frequently
more dependent on maximum width and thickness than on
length.
This method is intended for estimation of the total particle
size distribution of a single material. It is not intended for de-
termination of the proportion of particles passing or retained
on one or two sieves.
Estimate the particle size distribution as described under
Dry Sieving Method, unless otherwise specified in the individ-
ual monograph. Where difficulty is experienced in reaching
the endpoint (i.e., material does not readily pass through the
sieves) or when it is necessary to use the finer end of the siev-
ing range (below 75 //m), serious consideration should be
given to the use of an alternative particle-sizing method.
Sieving should be carried out under conditions that do not
cause the test sample to gain or lose moisture. The relative
humidity of the environment in which the sieving is carried
out should be controlled to prevent moisture uptake or loss
by the sample. In the absence of evidence to the contrary,
analytical test sieving is normally carried at ambient humidi-
ty. Any special conditions that apply to a particular material
should be detailed in the individual monograph.
Principles of Analytical Sieving — Analytical test sieves are
constructed from a woven-wire mesh, which is of simple
76
Particle Size Determination / General Tests
JP XV
weave that is assumed to give nearly square apertures and is
sealed into the base of an open cylindrical container. The bas-
ic analytical method involves stacking the sieves on top of
one another in ascending degrees of coarseness, and then
placing the test powder on the top sieve.
The nest of sieves is subjected to a standardized period of
agitation, and then the weight of material retained on each
sieve is accurately determined. The test gives the weight per-
centage of powder in each sieve size range.
This sieving process for estimating the particle size distri-
bution of a single pharmaceutical powder is generally intend-
ed for use where at least 80% of the particles are larger than
75 fim. The size parameter involved in determining particle
size distribution by analytical sieving is the length of the side
of the minimum square aperture through which the particle
will pass.
TEST SIEVES
Test sieves suitable for pharmacopoeial tests conform to
the most current edition of International Organisation for
Standardization (ISO) Specification ISO 3310-1; Test
sieves — Technical requirements and testing (see Table
3.04-1). Unless otherwise specified in the monograph, use
those ISO sieves listed in the Table as recommended in the
particular region.
Sieves are selected to cover the entire range of particle sizes
present in the test specimen. A nest of sieves having a ^2
progression of the area of the sieve openings is recommend-
ed. The nest of sieves is assembled with the coarsest screen at
the top and the finest at the bottom. Use micrometers or mil-
limeters in denoting test sieve openings. [Note — Mesh num-
bers are provided in the table for conversion purposes only.]
Test sieves are made from stainless steel or, less preferably,
from brass or other suitable non-reactive wire.
Calibration and recalibration of test sieves is in accordance
with the most current edition of ISO 3310-1 2) . Sieves should
be carefully examined for gross distortions and fractures, es-
pecially at their screen frame joints, before use. Sieves may
be calibrated optically to estimate the average opening size,
and opening variability, of the sieve mesh. Alternatively, for
the valuation of the effective opening of test sieves in the size
range of 212 to 850 ^m, Standard Glass Spheres are availa-
ble. Unless otherwise specified in the individual monograph,
perform the sieve analysis at controlled room temperature
and at ambient relative humidity.
Cleaning Test Sieves — Ideally, test sieves should be cleaned
using only an air jet or a liquid stream. If some apertures
remain blocked by test particles, careful gentle brushing may
be used as a last resort.
Test Specimen — If the test specimen weight is not given in
the monograph for a particular material, use a test specimen
having a weight between 25 and 100 g, depending on the bulk
density of the material, and test sieves having a 200 mm di-
ameter. For 76 mm sieves the amount of material that can be
accommodated is approximately l/7 th that which can be ac-
commodated on a 200 mm sieve. Determine the most ap-
propriate weight for a given material by test sieving accurate-
ly weighed specimens of different weights, such as 25, 50, and
100 g, for the same time period on a mechanical shaker.
[Note — If the test results are similar for the 25-g and 50-g
specimens, but the 100-g specimen shows a lower percentage
through the finest sieve, the 100-g specimen size is too large.]
Where only a specimen of 10 to 25 g is available, smaller di-
ameter test sieves conforming to the same mesh specifications
may be substituted, but the endpoint must be re-determined.
The use of test samples having a smaller mass (e.g. down to 5
g) may be needed. For materials with low apparent particle
density, or for materials mainly comprising particles with a
highly iso-diametrical shape, specimen weights below 5 g for
a 200 mm screen may be necessary to avoid excessive block-
ing of the sieve. During validation of a particular sieve analy-
sis method, it is expected that the problem of sieve blocking
will have been addressed.
If the test material is prone to picking up or losing sig-
nificant amounts of water with varying humidity, the test
must be carried out in an appropriately controlled environ-
ment. Similarly, if the test material is known to develop an
electrostatic charge, careful observation must be made to en-
sure that such charging is not influencing the analysis. An an-
tistatic agent, such as colloidal silicon dioxide and/or alumi-
num oxide, may be added at a 0.5 percent (m/m) level to
minimize this effect. If both of the above effects cannot elimi-
nated, an alternative particle-sizing technique must be select-
ed.
Agitation Methods — Several different sieve and powder
agitation devices are commercially available, all of which
may be used to perform sieve analyses. However, the differ-
ent methods of agitation may give different results for sieve
analyses and endpoint determinations because of the different
types and magnitude of the forces acting on the individual
particles under test. Methods using mechanical agitation or
electromagnetic agitation, and that can include either a verti-
cal oscillation or a horizontal circular motion, or tapping or a
combination of both tapping and horizontal circular motion
are available. Entrainment of the particles in an air stream
may also be used. The results must indicate which agitation
method was used and the agitation parameters used (if they
can be varied), since changes in the agitation conditions will
give different results for the sieve analysis and endpoint deter-
minations, and may be sufficiently different to give a failing
result under some circumstances.
Endpoint Determination — The test sieving analysis is com-
plete when the weight on any of the test sieves does not
change by more than 5% or 0.1 g (10% in the case of 76 mm
sieves) of the previous weight on that sieve. If less than 5% of
the total specimen weight is present on a given sieve, the en-
dpoint for that sieve is increased to a weight change of not
more than 20% of the previous weight on that sieve.
If more than 50% of the total specimen weight is found on
any one sieve, unless this is indicated in the monograph, the
test should be repeated, but with the addition to the sieve nest
of a more coarse sieve intermediate between that carrying the
excessive weight and the next coarsest sieve in the original
nest, i.e., addition of the ISO series sieve omitted from the
nest of sieves.
SIEVING METHODS
Mechanical agitation
Dry Sieving Method — Tare each test sieve to the nearest
0.1 g. Place an accurately weighed quantity of test specimen
on the top (coarsest) sieve, and replace the lid. Agitate the
nest of sieves for 5 minutes. Then carefully remove each from
the nest without loss of material. *If there is some fine pow-
der on the down surface of each sieve, take if off by the brush
gently, and combine it with the sieve fraction retained on
each next down sieve. » Reweigh each sieve, and determine
JPXV
General Tests / Bacterial Endotoxins Test
77
the weight of material on each sieve. Determine the weight of
material in the collecting pan in a similar manner. Reassem-
ble the nest of sieves, and agitate for 5 minutes. Remove and
weigh each sieve as previously described. Repeat these steps
until the endpoint criteria are met (see Endpoint Determina-
tion under Test Sieves), Upon completion of the analysis,
reconcile the weights of material. Total losses must not ex-
ceed 5% of the weight of the original test specimen.
Repeat the analysis with a fresh specimen, but using a sin-
gle sieving time equal to that of the combined times used
above. Confirm that this sieving time conforms to the re-
quirements for endpoint determination. When this endpoint
has been validated for a specific material, then a single fixed
time of sieving may be used for future analyses, providing the
particle size distribution falls within normal variation.
If there is evidence that the particles retained on any sieve
are aggregates rather than single particles, the use of mechan-
ical dry sieving is unlikely to give good reproducibility, a
different particle size analysis method should be used.
Air Entrainment Methods
Air Jet and Sonic Shifter Sieving — Different types of com-
mercial equipment that use a moving air current are available
for sieving. A system that uses a single sieve at a time is
referred to as air jet sieving. It uses the same general sieving
methodology as that described under the Dry Sieving
Method, but with a standardized air jet replacing the normal
agitation mechanism. It requires sequential analyses on in-
dividual sieves starting with the finest sieve to obtain a parti-
cle size distribution. Air jet sieving often includes the use of
finer test sieves than used in ordinary dry sieving. This tech-
nique is more suitable where only oversize or undersize frac-
tions are needed.
In the sonic sifting method, a nest of sieves is used, and the
test specimen is carried in a vertically oscillating column of
air that lifts the specimen and then carries it back against the
mesh openings at a given number of pulses per minute. It
may be necessary to lower the sample amount to 5 g, when
sonic shifting is employed.
The air jet sieving and sonic sieving methods may be useful
for powders or granules when mechanical sieving techniques
are incapable of giving a meaningful analysis.
These methods are highly dependent upon proper disper-
sion of the powder in the air current. This requirement may
be hard to achieve if the method is used at the lower end of
the sieving range (i.e., below 75 fim), when the particles tend
to be more cohesive, and especially if there is any tendency
for the material to develop an electrostatic charge. For the
above reasons endpoint determination is particularly critical,
and it is very important to confirm that the oversize material
comprises single particles and is not composed of aggregates.
INTERPRETATION
The raw data must include the weight of test specimen, the
total sieving time, and the precise sieving methodology and
the set values for any variable parameters, in addition to the
weights retained on the individual sieves and in the pan. It
may be convenient to convert the raw data into a cumulative
weight distribution, and if it is desired to express the distribu-
tion in terms of a cumulative weight undersize, the range of
sieves used should include a sieve through which all the
material passes. If there is evidence on any of the test sieves
that the material remaining on it is composed of aggregates
formed during the sieving process, the analysis is invalid.
"Additional information on particle size measurement, sample size,
and data analysis is available, for example, in ISO 9276.
^International Organization for Standardization (ISO) Specification
ISO 3310-1; Test sieves-Technical requirements and testing
4. Biological Tests/Biochemical
Tests/Microbial Tests
4.01 Bacterial Endotoxins Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* »).
Bacterial Endotoxins Test is a test to detect or quantify
bacterial endotoxins of gram-negative bacterial origin using a
lysate reagent prepared from blood corpuscle extracts of
horseshoe crab (Limulus polyphemus or Tachypleus tridenta-
tus). There are two types of techniques for this test: the gel-
clot techniques, which are based on gel formation by the
reaction of the lysate TS with endotoxins, and the photomet-
ric techniques, which are based on endotoxin-induced optical
changes of the lysate TS. The latter include turbidimetric
techniques, which are based on the change in lysate TS tur-
bidity during gel formation, and chromogenic techniques,
which are based on the development of color after cleavage
of a synthetic peptide-chromogen complex.
Proceed by any one of these techniques for the test. In the
event of doubt or dispute, the final decision is made based on
the gel-clot techniques, unless otherwise indicated.
The test is carried out in a manner that avoids endotoxin
contamination.
Apparatus
Depyrogenate all glassware and other heat-stable materials
in a hot-air oven using a validated process. Commonly used
minimum time and temperature settings are 30 minutes at
250°C. If employing plastic apparatus, such as multi-well
plates and tips for micropipettes, use only that which has
been shown to be free of detectable endotoxin and which
does not interfere with the test.
Preparation of Standard Endotoxin Stock Solution
*Prepare Standard Endotoxin Stock Solution by dissolv-
ing Endotoxin 10000 Reference Standard or Endotoxin 100
Reference Standard in water for bacterial endotoxins test
(BET).» Endotoxin is expressed in Endotoxin Units (EU).
One EU is equal to one International Unit (IU) of endotoxin.
Preparation of Standard Endotoxin Solution
After mixing Standard Endotoxin Stock Solution
thoroughly, prepare appropriate serial dilutions of Standard
Endotoxin Solution, using water for BET. Use dilutions as
soon as possible to avoid loss of activity by adsorption.
Preparation of sample solutions
Unless otherwise specified, prepare sample solutions by
dissolving or diluting drugs, using water for BET. *Sample
solutions for containers for medicines should be prepared
according to other specified procedures. » If necessary, adjust
78
Bacterial Endotoxins Test / General Tests
JP XV
the pH of the solution to be examined so that the pH of the
mixture of the lysate TS and sample solution falls within the
specified pH range for the lysate reagent to be used. This
usually applies to a sample solution with a pH in the range of
6.0 to 8.0. TSs or solutions used for adjustment of pH may
be prepared using water for BET, and then stored in contain-
ers free of detectable endotoxin.
Determination of Maximum Valid Dilution
The Maximum Valid Dilution (MVD) is the maximum
allowable dilution of a sample solution at which the endotox-
in limit can be determined.
Determine the MVD from the following equation:
MVD =
Endotoxin limit
x Concentration of sample solution
Endotoxin limit:
The endotoxin limit for injections, defined on the basis
of dose, equals K/M, where K is a minimum pyrogenic
dose of endotoxin per kg body mass (EU/kg), and M is
equal to the maximum dose of product per kg of body
mass in a single hour period.
Concentration of sample solution:
mg/mL in the case of endotoxin limit specified by mass
(EU/mg)
*mEq/mL in the case of endotoxin limit specified by
equivalent (EU/mEq)»
Units/mL in the case of endotoxin limit specified by bio-
logical unit (EU/Unit)
mL/mL in the case of endotoxin limit specified by
volume (EU/mL)
A : the labeled lysate reagent sensitivity in the gel-clot tech-
niques (EU/mL) or the lowest point used (EU/mL) in the
standard regression curve of the turbidimetric or chro-
mogenic techniques
Gel-clot techniques
The gel-clot techniques detect or quantify endotoxins
based on clotting of the lysate TS in the presence of endotox-
in. To ensure both the precision and validity of the test, per-
form the tests for confirming the labeled lysate reagent sen-
sitivity and for interfering factors as described under
Preparatory testing.
(1) Preparatory testing
(i) Test for confirmation of labeled lysate reagent sen-
sitivity
The labeled sensitivity of lysate reagent is defined as the
lowest concentration of endotoxin that is needed to cause the
lysate TS to clot under the conditions specified for the lysate
reagent to be used.
The test for confirmation of the labeled lysate reagent sen-
sitivity is to be carried out when each new lot of lysate reagent
is used or when there is any change in the experimental condi-
tions which may affect the outcome of the test. Perform the
test by the following procedures.
Prepare standard solutions having four concentrations
equivalent to 2 A, A, 0.5 A and 0.25 A by diluting the Standard
Endotoxin Stock Solution with water for BET. Prepare the
lysate TS by dissolving the lysate reagent with water for BET
or a suitable buffer. Mix a volume of the lysate TS with an
equal volume of one of the standard solutions (usually, 0.1
mL aliquots) in each test tube. When single test vials or
ampoules containing lyophilized lysate reagent are used, add
solutions directly to the vial or ampoule.
Keep the tubes (or containers such as vials or ampoules)
containing the reaction mixture usually at 37 ± 1 °C for 60 ±
2 minutes, avoiding vibration. To test the integrity of the gel
after incubation, invert each tube or container through ap-
proximately 180° in one smooth motion. If a firm gel has
formed that remains in place upon inversion, record the
result as positive. A result is negative if either a firm gel is not
formed, or if a fragile gel has formed but flows out upon
inversion.
Making the standard solutions of four concentrations one
set, test four replicates of the set.
The test is not valid unless 0.25 A of the standard solution
shows a negative result in each set of tests. If the test is not
valid, repeat the test after verifying the test conditions.
The endpoint is the last positive test in the series of decreas-
ing concentrations of endotoxin. Calculate the geometric
mean endpoint concentration using the following formula:
Geometric Mean Endpoint Concentration = antilog (Ee/f)
Ee = the sum of the log endpoint concentrations of the
dilution series used
/=the number of replicates
If the geometric mean endpoint concentration is not less
than 0.5 A and not more than 2.0 A, the labeled sensitivity is
confirmed.
(ii) Test for interfering factors
This test is performed to check for the presence of enhanc-
ing or inhibiting factors for the reaction in sample solutions.
Following Table 4.01-1, prepare solutions A and B using a
sample solution under test, and solutions C and D using
water for BET. Test solutions A and B and solutions C and D
in quadruplicate and in duplicate, respectively. Concerning
the incubation temperature, incubation time, and procedure
for the confirmation of gel formation, follow the procedure
under (i) Test for confirmation of labeled lysate reagent
sensitivity of (1) Preparatory testing.
The geometric mean endpoint concentrations of B and C
solutions are determined by using the formula described in (i)
Test for confirmation of labeled lysate reagent sensitivity of
(1) Preparatory testing.
This test must be repeated when there is any change in the
experimental conditions which may affect the outcome of the
test.
Table 4.01-1
Solution
Concentration of added
endotoxin in each solution/
Solution to which
endotoxin is added
Diluent
Dilution
factor
Concentration of
added endotoxin
after dilution
Number of
replicates
A
O/Sample solution
-
-
-
4
B
2A/Sample solution
Sample
solution
1
2
4
8
11
U
0.5A
0.25A
4
C
2A/Water for BET
Water for
BET
1
2
4
8
11
U
0.5A
0.25A
2
D
O/Water for BET
-
-
-
2
JPXV
General Tests / Bacterial Endotoxins Test
79
The test is valid if solutions A and D show no reaction and
the result for solution C confirms the labeled sensitivity.
If the geometric mean endpoint concentration of solution
B is not less than 0.5 A and not greater than 2.0 A, the sample
solution being examined does not contain interfering factors
and complies with the test for interfering factors. Otherwise
the sample solution interferes with the test.
If the sample under test does not comply with the test at a
dilution less than the MVD, repeat the test using a greater di-
lution, not exceeding the MVD. Furthermore, interference of
the sample solution or diluted sample solution may be elimi-
nated by suitable treatment, such as filtration, neutralization,
dialysis or heat treatment.
(2) Limit test
Based on the formation of a firm gel in the presence of
endotoxin at above labeled lysate reagent sensitivity, this
method tests whether a sample solution contains endotoxin
not greater than the endotoxin limit.
(i) Procedure
Prepare solutions A, B, C and D according to Table
4.01-2. Making these four solutions one set, test two repli-
cates of the set.
In preparing solutions A and B, use the sample solutions
complying with (ii) Test for interfering factors of (1) Prepara-
tory testing. Concerning the test conditions including the in-
cubation temperature, incubation time, and procedure for
the confirmation of gel formation, follow the procedure un-
der (i) Test for confirmation of labeled lysate reagent sensitiv-
ity of (1) Preparatory testing.
Table 4.01-2
Concentration of added endotoxin in
Solution each solution/Solution to which
endotoxin is added
A 0/Sample solution
Number of
replicates
B
C
D
2A/Sample solution
2A/Water for BET
0/Water for BET
(ii) Interpretation
The test is valid when both replicates of solutions B and C
are positive and those of solution D are negative.
The sample meets the endotoxin limit requirement of the
test when a negative result is found for both replicates of
solution A.
Repeat the test in duplicate when the test results are posi-
tive for one test but negative for the other one. The sample
meets the endotoxin limit requirement of the test when a
negative result is found for both replicates of solution A in
the repeat test.
The sample does not meet the endotoxin limit requirement
of the test when a positive result is found for both replicates
of the solution A at a dilution equal to the MVD. If the test is
positive for the sample at a dilution less than the MVD, the
test may be performed at a dilution not greater than the
MVD.
(3) Assay
The test measures endotoxin concentrations of sample
solutions by titration to an endpoint of gel formation.
(i) Procedure
Prepare solutions A, B, C and D according to Table
4.01-3. Making these four solutions one set, test two repli-
cates of the set. When preparing solutions A and B, use sam-
ple solutions complying with (ii) Test for interfering factors
of (1) Preparatory testing. Concerning the test conditions,
follow the procedure under (i) Test for confirmation of la-
beled lysate reagent sensitivity of (1) Preparatory testing.
Table 4.01-3
Solution
Concentration of added
endotoxin in each solution/
Solution to which
endotoxin is added
Diluent
Dilution
factor*
Concentration of
added endotoxin
after dilution
Number of
replicates
A
0/Sample solution
Water for
BET
1
2
4
8
-
2
B
2A/Sample solution
-
1
n
2
C
2A/Water for BET
Water for
BET
1
2
4
8
u
0.5A
0.25A
2
D
0/Water for BET
-
-
-
2
* The dilution range of the dilution series of solution A may be changed as appropriate,
but not exceeding the MVD.
(ii) Calculation and interpretation
The test is valid when the following three conditions are
met: (a) both replicates of the negative control solution D are
negative, (b) both replicates of the positive product control
solution B are positive and (c) the geometric mean endpoint
concentration of solution C is in the range of 0.5 A to 2 A.
The endpoint is defined as the maximum dilution showing
the last positive test in the dilution series of solution A, and
the endotoxin concentration of solution A is calculated by
multiplying the endpoint dilution factor by A.
Calculate the geometric mean endotoxin concentration of
the two replicates, using the formula given under (i) Test for
confirmation of labeled lysate reagent sensitivity of (1)
Preparatory testing.
If none of the dilutions of solution A is positive, report the
endotoxin concentration solution A as less than A x the
lowest dilution factor of solution A.
If all dilutions are positive, the endotoxin concentration of
solution A is reported as equal to or greater than the greatest
dilution factor of solution A multiplied by A.
Calculate the endotoxin concentration (in EU per mL, in
EU per mg or mEq or in EU per Unit) of the sample, based
on the mean endotoxin concentration of solution A. The
sample complies with the Bacterial Endotoxins Test <4.01> if
the endotoxin concentration of the sample meets the require-
ment for the endotoxin limit (in EU per mL, in EU per mg or
mEq or in EU per Unit) specified in the individual mono-
graph.
Photometric techniques
(1) Turbidimetric technique
This technique measures the endotoxin concentrations of
sample solutions based on the measurement of turbidity
change accompanying gel formation of the lysate TS. This
technique is classified as either endpoint-turbidimetric or
kinetic-turbidimetric .
The endpoint-turbidimetric technique is based on the
80
Bacterial Endotoxins Test / General Tests
JP XV
quantitative relationship between the concentration of en-
dotoxins and the turbidity of the reaction mixture at a speci-
fied reaction time.
The kinetic-turbidimetric technique is based on the quan-
titative relationship between the concentration of endotoxins
and either the time needed to reach a predetermined turbidity
of the reaction mixture or the rate of turbidity development.
The test is usually carried out at 37 ± 1 °C, and turbidity is
expressed in terms of either absorbance or transmission.
(2) Chromogenic technique
This technique measures the endotoxin concentrations of
sample solutions based on the measurement of chromophore
released from a synthetic chromogenic substrate by the reac-
tion of endotoxins with the lysate TS. This technique is classi-
fied as either endpoint-chromogenic or kinetic-chromogenic.
The endpoint-chromogenic technique is based on the quan-
titative relationship between the concentration of endotoxins
and the release of chromophore at the end of an incubation
period.
The kinetic-chromogenic technique is based on the quan-
titative relationship between the concentration of endotoxins
and either the time needed to reach a predetermined absor-
bance (or transmittance) of the reaction mixture or the rate of
color development.
The test is usually carried out at 37 ± 1°C.
(3) Preparatory testing
To assure the precision and validity of the turbidimetric or
chromogenic techniques, perform both Test for assurance of
criteria for the standard curve and Test for interfering fac-
tors, as indicated below.
(i) Test for assurance of criteria for the standard curve
♦The test must be carried out for each lot of lysate
reagent.*
Using the Standard Endotoxin Solution, prepare at least
three endotoxin concentrations to generate the standard
curve within the range of endotoxin concentrations indicated
by the instructions for the lysate reagent used. Perform the
test using at least three replicates of each standard endotoxin
concentration according to the optimal conditions for the
lysate reagent used (with regard to volume ratios, incubation
time, temperature, pH, etc.). If the desired range is greater
than two logs, additional standards should be included to
bracket each log increase in the range of the standard curve.
The absolute value of the correlation coefficient, \r\ , must
be greater than or equal to 0.980 for the range of endotoxin
concentrations set up.
If the test is not valid, repeat the test after verifying the test
conditions.
The test for assurance of criteria for the standard curve
must be repeated when any condition changes, which is likely
to influence the result of the test.
(ii) Test for interfering factors
Prepare solutions A, B, C and D according to Table
4.01-4. Perform the test on these solutions following the op-
timal conditions for the lysate reagent used (with regard to
volume of sample solution and lysate TS, volume ratio of
sample solution to lysate TS, incubation time, etc.).
The test for interfering factors must be repeated when any
condition changes, which is likely to influence the result of
the test.
Table 4.01-4
Solution
Concentration of added
Solution to which
endotoxin in each solution endotoxin is added
Sample solution* 1
Middle concentration of the
standard curve* 2
Sample solution* 1
At least 3 concentrations* 3
Water for BET
D
Water for BET Not less than 2
Number of test
tubes or wells
Not less than 2
Not less than 2
Each not less than 2
*1 The sample solution may be diluted not to exceed the MVD.
*2 Add the Standard Endotoxin Solution to the sample solution to make an endotoxin
concentration at or near the middle of the standard curve.
*3 The concentrations used in the test for the (i) Assurance of criteria for the standard
curve of (3) Preparatory testing.
*The test is not valid unless the following conditions are
met.
1 : The absolute value of the correlation coefficient of the
standard curve generated using solution C is greater
than or equal to 0.980.
2: The result with solution D does not exceed the limit of
the blank value required in the description of the lysate
reagent employed, or it is less than the endotoxin de-
tection limit of the lysate reagent employed.*
Calculate the recovery of the endotoxin added to solution
B from the concentration found in solution B after subtract-
ing the endotoxin concentration found in solution A.
When the recovery of the endotoxin added to solution B is
within 50% to 200%, the sample solution under test is consi-
dered to be free of interfering factors.
*When the endotoxin recovery is out of the specified
range, the sample solution under test is considered to contain
interfering factors. If the sample under test does not comply
with the test, repeat the test using a greater dilution, not ex-
ceeding the MVD. Furthermore, interference of the sample
solution or diluted sample solution not to exceed the MVD
may be eliminated by suitable treatment, such as filtration,
neutralization, dialysis or heat treatment.*
(4) Assay
(i) Procedure
Prepare solutions A, B, C and D according to Table
4.01-4, and follow the procedure described in (ii) Test for in-
terfering factors of (3) Preparatory testing.
(ii) Calculation of endotoxin concentration
Calculate the endotoxin concentration of solution A using
the standard curve generated with solution C. The test is not
valid unless all the following requirements are met.
1 : The absolute value of the correlation coefficient of the
standard curve generated using solution C is greater
than or equal to 0.980.
2: The endotoxin recovery, calculated from the concen-
tration found in solution B after subtracting the con-
centration of endotoxin found in solution A, is within
the range of 50% to 200%.
3: The result with solution D does not exceed the limit of
the blank value required in the description of the lysate
reagent employed, *or it is less than the endotoxin de-
tection limit of the lysate reagent employed.*
(iii) Interpretation
The sample complies with the Bacterial Endotoxins Test if
the endotoxin concentration of the sample calculated from
the endotoxin concentration of solution A meets the require-
ment of the endotoxin limit (in EU per mL, in EU per mg or
JP XV
General Tests / Microbial Assay for Antibiotics
81
mEq or in EU per Unit) specified in the individual mono-
graph.
4.02 Microbial Assay for
Antibiotics
Microbial Assay for Antibiotics is a method to determine
the antimicrobial potency of antibiotics based on their an-
timicrobial activities. There are three methods for this test:
the cylinder-plate, perforated plate, and turbidimetric
methods. The former two are based on the measurement of
the size of the zones of microbial growth inhibition in a
nutrient agar medium, and the turbidimetric method is based
on the measurement of the inhibition of turbidity develop-
ment in a fluid medium with microbial growth. Unless other-
wise specified in the individual monograph, tests specified to
be carried out by the cylinder-plate method may be conduct-
ed under the same test conditions using the perforated plate
method instead. If necessary, first sterilize water, isotonic so-
dium chloride solution, buffer solutions, reagents, test solu-
tions and essential parts of measuring instruments and appli-
ances to be used for the test. In performing the test, precau-
tions must be taken to prevent biohazard.
I. Cylinder-plate method
The cylinder-plate method is a method to determine the an-
timicrobial potency of the antibiotic to be tested, and is based
on the measurement of the size of the zone of growth inhibi-
tion of a test organism by the use of cylinder-agar plates.
1. Test organisms
Use the test organism specified in the individual mono-
graph.
2. Culture media
Unless otherwise specified, use media with the following
compositions. When 'peptone' is indicated as an ingredient
of a medium, either meat peptone or casein peptone is applic-
able. Use sodium hydroxide TS or 1 mol/L hydrochloric acid
TS to adjust the pH of the medium to obtain the specified
value after sterilization. In the case of the medium for Bacil-
lus subtilis ATCC 6633, adjust the pH using ammonia TS,
potassium hydroxide TS or 1 mol/L hydrochloric acid TS. A
different medium to the one specified for each test organism
may be used if it has both a similar composition and an equal
or better growth efficiency of the test organism in comparison
with the specified medium. Unless otherwise specified, steri-
lize the media to be used in an autoclave.
(1) Agar media for seed and base layer
1) Medium for test organism Bacillus subtilis ATCC 6633
i. Peptone 5.0 g
Meat extract 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 7.8 to 8.0 after sterilization,
ii. Peptone 5.0 g
Meat extract 3.0 g
Trisodium citrate dihydrate 10.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
2) Medium for test organism Saccharomyces cerevisiae
ATCC 9763
Glucose 10.0 g
Peptone 9.4 g
Meat extract 2.4 g
Yeast extract 4.7 g
Sodium chloride 10.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.0 to 6.2 after sterilization.
3) Medium for other organisms
i. Glucose 1.0 g
Peptone 6.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
ii. Glucose 1.0 g
Meat peptone 6.0 g
Casein peptone 4.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
iii. Peptone 10.0 g
Meat extract 5.0 g
Sodium chloride 2.5 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
(2) Agar media for transferring test organisms
1) Medium for test organism Saccharomyces cerevisiae
ATCC 9763
Glucose 15.0 g
Peptone 5.0 g
Yeast extract 2.0 g
Magnesium sulfate heptahydrate 0.5 g
Potassium dihydrogen phosphate 1.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.0 to 6.2 after sterilization.
2) Medium for other organisms
i. Glucose 1.0 g
Meat peptone 6.0 g
Casein peptone 4.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
ii. Peptone 10.0 g
Meat extract 5.0 g
Sodium chloride 2.5 g
Agar 15.0 g
82
Microbial Assay for Antibiotics / General Tests
JP XV
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
3. Preparation of agar slant or plate media
Unless otherwise specified, dispense approximately 9 mL
of melted agar medium in each test tube (approximately 16
mm in inside diameter), and make them as slant media, or
dispense approximately 20 mL of melted agar medium in
each Petri dish (approximately 90 mm in inside diameter),
and make them as plate media.
4. Preparation of stock suspensions of test spores or organ-
isms
Unless otherwise specified, prepare stock suspensions of
test spore or organism cultures as follows. Check the aspects
of the test spores or organisms as occasion demands.
(1) Preparation of a stock spore suspension of test organ-
ism Bacillus subtilis ATCC 6633
Inoculate the test organism onto the slant or plate of the
agar medium which was prepared for transferring the test or-
ganisms specified in 2 (2) 2) i. Incubate at 32 to 37°C for 16 to
24 hours. Inoculate the subcultured test organism onto a suit-
able volume of slant or plate of the agar medium (described
above), which was prepared for transferring the test organ-
isms specified in 2 (2) 2) ii. Then incubate at 32 to 37 °C for
not less than 1 week to prepare spores. Suspend the spores in
isotonic sodium chloride solution, heat at 65 °C for 30
minutes, and then centrifuge. Wash the spore sediment three
times with isotonic sodium chloride solution by means of cen-
trifugation. Re-suspend the spore sediment in water or iso-
tonic sodium chloride solution, and heat again at 65 °C for 30
minutes to prepare the stock spore suspension. The concen-
tration of the test organism is confirmed with the turbidity or
absorbance, as occasion demands. Store the stock spore sus-
pension at a temperature not exceeding 5°C, and use within 6
months. If the stock spore suspension shows a clear and
definite zone of growth inhibition in an antibiotics potency
test using adequate antibiotics, it may be used for further 6
months.
(2) Preparation of a stock suspension of the test organ-
ism Saccharomyces cerevisiae ATCC 9763
Inoculate test organism onto the slant or plate agar medi-
um which has been prepared for transferring test organism
specified in 2 (2) 1). Incubate at 25 to 26°C for 40 to 48 hours.
The subculture should be performed at least three times. In-
oculate the subcultured test organism onto another slant or
plate of the agar medium (described above), and incubate at
25 to 26°C for 40 to 48 hours. Scrape away and suspend the
resulting growth from the agar surface in isotonic sodium
chloride solution, and use this as a stock suspension of the
test organism. The concentration of the test organism is con-
firmed with the turbidity or absorbance, as occasion de-
mands. Store the stock suspensions of the test organisms at a
temperature not exceeding 5°C, and use within 30 days.
(3) Preparation of a stock suspension of other test organ-
isms
Inoculate the test organism onto the slant or the plate of
the agar medium which has been prepared for transferring
the test organisms specified in 2 (2) 2) i. Incubate the inoculat-
ed slant at 32 to 37 °C for 16 to 24 hours. The subculture
should be performed at least three times. Inoculate the sub-
cultured test organism onto another slant or plate agar medi-
um (described above), and incubate the slant at 32 to 37°C
for 16 to 24 hours. Scrape away and suspend the resulting
growth from the agar surface in isotonic sodium chloride so-
lution, and use this as a stock suspension of the test organ-
ism. The concentration of the test organism is confirmed with
the turbidity or absorbance, as occasion demands. Store the
stock suspensions of the test organisms at a temperature not
exceeding 5°C, and use within 5 days.
5. Preparation of agar base layer plates
Unless otherwise specified, dispense 20 mL of the melted
agar medium for the base layer into each Petri dish, and in
the case of a large dish, dispense a quantity of the agar medi-
um to form a uniform layer 2 to 3 mm thick. Distribute the
agar evenly in each dish on a flat, level surface, and allow it to
harden.
6. Preparation of seeded agar layers
Unless otherwise specified, determine the volume of the
stock suspension of the spore or the test organism with which
the employed standard solution shows a clear and definite
zone of growth inhibition. Prepare the seeded agar layer by
mixing thoroughly the previously determined volume of
stock suspension of spore or test organism with agar medium
for the seed layer kept at 48 to 51°C. Usually, the rate of a
stock spore suspension and a stock suspension of the test or-
ganism to add to the agar medium for the seed layer are 0.1 to
1.0 vol% and 0.5 to 2.0 vol%, respectively.
7. Preparation of cylinder-agar plates
Dispense 4 to 6 mL of the seeded agar layer, which is speci-
fied in the individual monograph, on an agar base layer plate
in a Petri dish. In the case of large dishes, dispense a quantity
of the agar medium to form a uniform layer 1.5 to 2.5 mm
thick, and spread evenly over the surface before hardening.
After coagulating the agar, allow the plate to stand under a
clean atmosphere to exhale moisture vapor of the inside of
Petri or large dishes and water on the agar surface. Place 4
cylinders on an agar plate in a Petri dish so that the individual
cylinders are equidistant from the center of the plate and
equally spaced from one another (the cylinders are set on the
circumference of a circle of 25 to 28 mm radius). When large
dish plates are used, place cylinders on each plate according
to the method of preparation for Petri dish agar plates. A set
of 4 cylinders on each large dish plate is considered to be
equivalent to one Petri dish plate.
Use stainless steel cylinders with the following dimensions:
outside diameter 7.9 to 8.1 mm; inside diameter 5.9 to 6.1
mm; length 9.9 to 10.1 mm. The cylinders should not inter-
fere with the test. Prepare the cylinder-agar plates before use.
8. Standard solutions
Use both a standard solution of high concentration and
one of low concentration, as specified in the individual mono-
graph. Unless otherwise specified, prepare the standard solu-
tions before use.
9. Sample solutions
Use both a sample solution of high concentration and one
of low concentration, as specified in the individual mono-
graph. Unless otherwise specified, prepare the sample solu-
tions before use.
10. Procedure
Unless otherwise specified, use 5 cylinder-agar plates as one
assay set when Petri dishes are employed. When large dishes
are employed, the number of cylinders for one assay set
JP XV
General Tests / Microbial Assay for Antibiotics
83
should be equal to that defined when using Petri dishes. App-
ly the standard solution of high concentration and that of low
concentration to a pair of cylinders set opposite each other on
each plate. Apply the high and low concentration sample so-
lutions to the remaining 2 cylinders. The same volume of
these solutions must be added to each cylinder. Incubate the
plates at 32 to 37°C for 16 to 20 hours. Using a suitable meas-
uring tool, measure the diameters of circular inhibition zones
with a precision that can discriminate differences of at least
0.25 mm. Each procedure should be performed quickly un-
der clean laboratory conditions.
11. Estimation of potency
The following correlation between the potency (P) of solu-
tion in a cylinder and the diameter (d) of zone of inhibition is
established.
d = a\og P + fi
where, a and /? are constants.
If necessary, ascertain the values in the above equation.
Based on this equation, estimate the potency of the sample
solutions by application of the following equation:
Amount (potency) of sample
=A x Potency of S H per mL x Dilution factor of U H
where:
\o%A-
IV
W
7=log (potency of S H /potency of S L )
V=ZU H +ZU L -ZS H -ZS L
w=zu u +zs H -zu L -z:s L
The sum of the diameter (mm) of the inhibitory zone meas-
ured in each plate is designated as follows:
for standard solution of high concentration (ShJ^^Sh
for standard solution of low concentration (S L )=2'S L
for sample solution of high concentration (U H )=2U H
for sample solution of low concentration (U L )=27U L
II. Perforated plate method
The perforated plate method is a method to determine the
antimicrobial potency of an antibiotic, based on the measure-
ment of the size of the zone of growth inhibition of a test or-
ganism by the use of perforated agar plates.
This method is carried out by the use of perforated agar
plates in lieu of cylinder-agar plates used in Cylinder-plate
method.
Proceed as directed below, but comply with the require-
ments of Cylinder-plate method, such as test organisms, me-
dia, preparation of agar slant or plate media, preparation of
stock suspensions of spores or test organisms, preparation of
agar base layer plates, preparation of seeded agar layers,
standard solutions, sample solutions, and estimation of
potency.
1. Preparation of perforated agar plates
Dispense 4 to 6 mL of the seeded agar layer specified in the
individual monograph on each agar base layer plate of the
Petri dish. In the case of large dishes, dispense a quantity of
the agar medium to form a uniform layer 1.5 to 2.5 mm
thick, and spread evenly over the surface before hardening.
After coagulating the agar, allow the plate to stand under a
clean atmosphere to exhale moisture vapor of the inside of
Petri or large dishes and water on the agar surface. Using a
suitable tool, prepare 4 circular cavities having a diameter of
7.9 to 8.1 mm on a Petri dish agar plate so that the individual
cavities are equidistant from the center of the plate. The cavi-
ties spaced equally from one another on the circumference of
a circle with radius 25 to 28 mm, and are deep enough to
reach the bottom of dish. When large dish plates are used,
prepare the circular cavities on each plate according to the
method of preparation for Petri dish agar plates. A set of 4
cavities on each large dish plate is considered to be equivalent
to one Petri dish plate.
Prepare the perforated agar plates before use.
2. Procedure
Unless otherwise specified, use 5 perforated agar plates as
one assay set when Petri dishes are employed. When large
dishes are employed, the number of cavities for one assay set
should be equal to that defined when using Petri dishes. App-
ly the high and low concentration standard solutions to a pair
of cavities prepared opposite each other on each plate, and
apply the high and low concentration sample solutions to the
remaining 2 cavities. The same volume of these solutions
must be added to each cavity. Incubate the plates at 32 to
37°C for 16 to 20 hours. Using a suitable measuring tool,
measure the diameters of the circular inhibition zones with a
precision that can discriminate differences of at least 0.25
mm. Each procedure should be performed quickly under
clean laboratory conditions.
III. Turbidimetric method
The turbidimetric method is a method to determine the an-
timicrobial potency of an antibiotic, based on the measure-
ment of the inhibition of growth of a microbial culture in a
fluid medium. The inhibition of growth of a test organism is
photometrically measured as changes in turbidity of the
microbial culture.
1. Test organisms
Use the test organism specified in the individual mono-
graph.
2. Culture media
Unless otherwise specified, use media with the following
compositions. When peptone is indicated as an ingredient of
a medium, either meat peptone or casein peptone is applica-
ble. Use sodium hydroxide TS or 1 mol/L hydrochloric acid
TS to adjust the pH of the medium to obtain the specified
value after sterilization. A different medium to the one speci-
fied for each test organism may be used if it has both a similar
composition and an equal or better growth efficiency of the
test organism in comparison with the specified medium. Un-
less otherwise specified, sterilize the media to be used in an
autoclave.
(1) Agar media for transferring test organisms
Glucose 1.0 g
Peptone 6.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Sodium chloride 2.5 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
(2) Liquid media for suspending test organisms
Glucose 1.0 g
Peptone 5.0 g
84
Digestion Test / General Tests
JP XV
Meat extract 1.5 g
Yeast extract 1.5 g
Sodium chloride 3.5 g
Potassium dihydrogen phosphate 1.32 g
Disodium hydrogen phosphate* 3.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 7.0 to 7.1 after sterilization.
*Dipotassium hydrogen phosphate (3.68 g) may be used in
lieu of disodium hydrogen phosphate (3.0 g).
3. Preparation of agar slant or plate media
Unless otherwise specified, proceed as directed in Prepara-
tion of agar slant or plate media under Cylinder-plate
method.
4. Preparation of stock suspensions of test organisms
Unless otherwise specified, inoculate the test organism
onto the slant or plate of the agar medium which was pre-
pared for transferring the specified test organism. Incubate
the inoculated medium at 32 to 37°C for 16 to 24 hours. The
subculture should be performed at least three times. Check
the aspects of the test spores or organisms as occasion de-
mands. Inoculate the subcultured test organism onto another
slant or plate of the agar medium (described above), and in-
cubate the slant at 32 to 37 °C for 16 to 24 hours. After incu-
bation, suspend the test organism in the liquid medium for
suspending the test organism, and use as the suspension of
the test organism. The concentration of the test organism is
confirmed with the turbidity or absorbance, as occasion de-
mands.
5. Standard solutions
Use the standard solutions specified in the individual
monograph. Unless otherwise specified, prepare the standard
solutions before use.
6. Sample solutions
Use the sample solutions specified in the individual mono-
graph. Unless otherwise specified, prepare the sample solu-
tions before use.
7. Procedure
Unless otherwise specified, proceed as follows:
Distribute 1.0 mL of each concentration of the standard
solution, the sample solution, and water used as a control,
into each set composed of 3 test tubes (about 14 mm in inside
diameter and about 13 cm in length). Add 9.0 mL of the sus-
pension of the test organism to each tube, and then incubate
in a water bath maintained at 35 to 37°C for 3 to 4 hours. Af-
ter incubation, add 0.5 mL of dilute formaldehyde (1 in 3) to
each tube, and read each transmittance or absorbance at a
wavelength of 530 nm.
8. Estimation of potency
Average the transmittance or absorbance values of each
concentration of the standard solution, the sample solution
and water used as a control, respectively. Generate the stan-
dard curve based on the average values of transmittance or
absorbance of each concentration of the standard solution,
and estimate the potency of the sample solution from its
average value of transmittance or absorbance using the ob-
tained standard curve.
If the standard dilutions of five concentrations in geomet-
ric progression are used, calculate the L and H values from
the following equations. Plot point L and point H on graph
paper and construct a straight line for the standard curve.
. _ (3a + 2b + c-e)
H-
5
(3e + 2d+c-a)
5
where:
L = calculated value of transmittance or absorbance for the
lowest concentration of the standard curve.
H= calculated value of transmittance or absorbance for
the highest concentration of the standard curve.
a, b, c, d, e = average transmittance or absorbance values
for each standard dilution, where a is the value from
the lowest concentration standard solution, b, c and d
are the values from each geometrically increased con-
centration standard solution, respectively, and e is the
value from the highest concentration standard solu-
tion.
4.03 Digestion Test
Digestion Test is a test to measure the activity of digestive
enzymes, as crude materials or preparations, on starch, pro-
tein and fat.
(1) Assay for Starch Digestive Activity
The assay for starch digestive activity is performed through
the measurement of starch saccharifying activity, dextriniz-
ing activity, and liquefying activity.
(i) Measurement of starch saccharifying activity
The starch saccharifying activity can be obtained by meas-
uring an increase of reducing activity owing to the hydrolysis
of the glucoside linkages when amylase acts on the starch.
Under the conditions described in Procedure, one starch sac-
charifying activity unit is the amount of enzyme that cata-
lyzes the increase of reducing activity equivalent to 1 mg of
glucose per minute.
Preparation of Sample Solution
Dissolve the sample in an appropriate amount of water, or
a buffer or salts solution specified in the monograph so that
the reducing activity increases in proportion to the concentra-
tion of the sample solution, when measuring under the condi-
tions described in Procedure. The concentration is normally
0.4 to 0.8 starch saccharifying activity unit/mL. Filter if
necessary.
Preparation of Substrate Solution
Use potato starch TS for measuring the starch digestive
activity. If necessary, add 10 mL of buffer or salts solution
specified in the monograph, instead of 10 mL of 1 mol/L
acetic acid-sodium acetate buffer solution, pH 5.0.
Procedure
Pipet 10 mL of the substrate solution, stand at 37 ± 0.5°C
for 10 minutes, add exactly 1 mL of the sample solution, and
shake immediately. Allow this solution to stand at 37 ±
0.5°C for exactly 10 minutes, add exactly 2 mL of alkaline
tartrate solution of the Fehling's TS for amylolytic activity
test, and shake immediately. Then, add exactly 2 mL of cop-
per solution of the Fehling's TS for amylolytic activity test,
shake gently, heat the solution in a water bath for exactly 15
JPXV
General Tests / Digestion Test
85
minutes, and then immediately cool to below 25°C. Then,
add exactly 2 mL of concentrated potassium iodide TS and 2
mL of diluted sulfuric acid (1 in 6), and titrate <2.50> the
released iodine with 0.05 mol/L sodium thiosulfate VS to the
disappearance of the blue color produced by addition of 1 to
2 drops of soluble starch TS (a mL). Separately, pipet 10 mL
of water instead of the substrate solution and titrate <2.50> in
the same manner (b mL).
Starch saccharifying activity (unit/g)
= amount (mg) of glucose x — — x — -
Amount (mg) of glucose = (b — a) x 1.6
W: Amount (g) of sample in 1 mL of sample solution
(ii) Measurement of starch dextrinizing activity
The starch dextrinizing activity can be obtained by measur-
ing a decrease in starch coloration by iodine resulting from
hydrolysis of the straight chain component (amylose) in
starch when amylase acts on the starch. Under the conditions
described in Procedure, one starch dextrinizing activity unit
is the amount of enzyme required to reduce the coloration of
potato starch by iodine by 10% per minute.
Preparation of Sample Solution
Dissolve the sample in an appropriate amount of water or a
buffer or salts solution specified in the monograph so that the
coloration of starch by iodine decreases in proportion to the
concentration of the sample solution, when measuring under
the conditions described in Procedure. The concentration is
normally 0.2 to 0.5 starch dextrinizing activity unit/mL.
Filter if necessary.
Preparation of Substrate Solution
Prepare the substrate solution in the same manner as the
substrate solution in the measurement of starch saccharifying
activity.
Procedure
Pipet 10 mL of the substrate solution, stand at 37 ± 0.5°C
for 10 minutes, add exactly 1 mL of the sample solution, and
shake immediately. Allow this solution to stand at 37 ±
0.5°C for exactly 10 minutes. Pipet 1 mL of this solution,
add it to 10 mL of 0.1 mol/L hydrochloric acid TS, and
shake immediately. Pipet 0.5 mL of this solution, add exactly
10 mL of 0.0002 mol/L iodine TS, and shake. Determine the
absorbance A T of this solution at the wavelength of 660 nm
as directed under Ultraviolet-visible Spectrophotometry
<2.24>. Separately, using 1 mL of water instead of the sample
solution, determine the absorbance A B in the same manner.
Starch dextrinizing activity (unit/g)
(,4b " A T )
1
W
W: Amount (g) of sample in 1 mL of sample solution
(hi) Measurement of starch liquefying activity
The starch liquefying activity can be obtained by measur-
ing a decrease in the viscosity of starch solution resulting
from the hydrolysis of molecules when amylase acts on the
starch. Under the conditions described in Procedure, one
starch liquefying activity unit is the amount of enzyme
required to reduce the viscosity of the substrate solution
equivalent to 1 g of potato starch from 200% to 100% of that
of the 50% sucrose standard solution.
Preparation of Sample Solution
Dissolve the sample in an appropriate amount of water, or
a buffer or salts solution specified in the monograph so that
the viscosity decreases in proportion to the concentration of
the sample solution, when measuring under the conditions
described in Procedure. The concentration is normally 0.15
to 0.25 starch liquefying activity unit/mL. Filter if necessary.
Preparation of Substrate Solution
Weigh accurately about 1 g of potato starch, and measure
the loss of drying at 105°C for 2 hours. Weigh exactly potato
starch equivalent to 15.00 g calculated on the dried basis, add
300 mL of water, then add gradually 25 mL of 2 mol/L sodi-
um hydroxide TS under thorough shaking, until the mixture
forms a paste. Heat the mixture in a water bath for 10
minutes, shaking it occasionally. After cooling, neutralize the
mixture with 2 mol/L hydrochloric acid TS, and add 50 mL
of the buffer solution specified in the monograph and water
to make exactly 500 g. Prepare before use.
Preparation of 50% Standard Sucrose Solution
Dissolve 50.0 g of sucrose in 50.0 mL of water.
Procedure
Put 50 mL of the 50% standard sucrose solution in a
100-mL conical flask, and allow it to stand in a thermostat at
37 ± 0.5 C C for 15 minutes. Fix a viscometer shown in Fig.
4.03-1 so that its lower end almost touches the bottom of the
flask and that the water in the thermostat circulates around
the outer cylinder of the viscometer. After slowly pulling up
the 50% standard sucrose solution by suction to the middle
of the upper bulb of the viscometer, let it flow down by gravi-
ty, measuring the time taken for the solution to fall from the
upper to the lower indicators (t l seconds). Take exactly 50 g
of the substrate solution in another 100-mL conical flask, and
stand it in another thermostat at 37 ± 0.5 °C for 20 minutes.
Add exactly 1 mL of the sample solution to it, and shake the
flask immediately. Fix a viscometer vertically so that its lower
end almost touches the bottom of the flask and that the water
in the thermostat circulates around the outer cylinder of the
viscometer. Occasionally pull the reaction solution up by suc-
tion to the middle of the upper bulb slowly, then let it flow
down by gravity, measuring the time taken for the solution to
fall from the upper to the lower indicators (t seconds).
Repeat this operation until t becomes shorter than t,. At
each measurement, record the time (T' seconds) from the
moment that the sample solution is added to the moment that
the solution surface in the flask passes the upper indicator.
(T' + t/2) is the reaction time (T) corresponding to t. Draw
a curve for both t and T, Obtain T, and T 2 that correspond to
tj and (2 x t,) by interpolation.
Starch liquefying activity (unit/g)
60
{T, - T 2 )
1.5
W
W: Amount (g) of sample in 1 mL of sample solution
(2) Assay for Protein Digestive Activity
The protein digestive activity can be obtained by the colori-
metric measurement, making use of Folin's reaction, of the
amount of acid-soluble low-molecular products, which is in-
creased owing to the hydrolysis of the peptide linkages when
protease acts on casein. One protein digestive activity unit is
86 Digestion Test / General Tests
JP XV
r
)
jj=
^
"X
£
<c
\
cr
i
C .
:Z:
3^
3
2
r,
L
'
^
E-
"■
>
|
The figures are in mm.
A: Bulb volume: 5 ml.
R: Indicators
C: Outside diameter: 30 mm
D: Capillary inside diameter;
1.25-1.30 mm
E: Outside diameter 8 mm
Fig. 4.03-1
the amount of enzymes that produces Folin's TS-colorable
substance equivalent to 1 /xg of tyrosine per minute under the
conditions described in Procedure.
Preparation of Sample Solution
Dissolve the sample in an appropriate amount of water, or
a buffer or salts solution specified in the monograph so that
the amount of non-protein, Folin's TS-colorable substances
increase in proportion to the concentration of the sample
solution, when measuring under the conditions described in
Procedure. The concentration is normally 15 to 30 protein
digestive activity unit/mL.
Tyrosine Calibration Curve
Weigh exactly 50 mg of Tyrosine Reference Standard,
previously dried at 105 °C for 3 hours, and dissolve in 0.2
mol/L hydrochloric acid TS to make exactly 50 mL. Pipet 1
mL, 2 mL, 3 mL and 4 mL of this solution separately, and
add 0.2 mol/L hydrochloric acid TS to each solution to make
them exactly 100 mL. Pipet 2 mL of each solution, and add
exactly 5 mL of 0.55 mol/L sodium carbonate TS and 1 mL
of diluted Folin's TS (1 in 3) to each solution, shake im-
mediately, then stand them at 37 ± 0.5°C for 30 minutes.
Determine the absorbances, A u A 2 , Aj, and^4 4 , of these solu-
tions at 660 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using a solution prepared with exactly
2 mL of 0.2 mol/L hydrochloric acid TS in the same manner
as the blank. Then, draw a calibration curve with the absor-
bances, A,, A 2 , A 3 and A 4 as the ordinate, and with the
amount (jig) of tyrosine in 2 mL of each solution as the ab-
scissa. Obtain the amount (jig) of tyrosine for the absorbance
1.
Preparation of Substrate Solution
Substrate solution 1: Weigh accurately about 1 g of milk
casein, and measure the loss on drying at 105°C for 2 hours.
Weigh exactly an amount of milk casein equivalent to 1.20 g
calculated on the dried basis, add 12 mL of lactic acid TS and
150 mL of water, and warm to dissolve in a water bath. After
cooling in running water, adjust to the pH specified in the
monograph with 1 mol/L hydrochloric acid TS or sodium
hydroxide TS, and add water to make exactly 200 mL. Pre-
pare before use.
Substrate solution 2: Weigh accurately about 1 g of milk
casein, and measure the loss on drying at 105°C for 2 hours.
Weigh exactly an amount of milk casein equivalent to 1.20 g
calculated on the dried basis, add 160 mL of 0.05 mol/L dis-
odium hydrogenphosphate TS, and warm to dissolve in a
water bath. After cooling in running water, adjust to the pH
specified in the monograph with the 1 mol/L hydrochloric
acid TS or sodium hydroxide TS, and add water to make
exactly 200 mL. Prepare before use.
Preparation of Precipitation Reagent
Trichloroacetic acid TS A: Dissolve 7.20 g of trichloroacet-
ic acid in water to make 100 mL.
Trichloroacetic acid TS B: Dissolve 1.80 g of trichloroacet-
ic acid, 1.80 g of anhydrous sodium acetate and 5.5 mL of
6 mol/L acetic acid TS in water to make 100 mL.
Procedure
Pipet 5 mL of the substrate solution specified in the mono-
graph, stand at 37 ± 0.5 C C for 10 minutes, add exactly 1 mL
of the sample solution, and shake immediately. After stand-
ing this solution at 37 ± 0.5°C for exactly 10 minutes, add
exactly 5 mL of trichloroacetic acid TS A or B as specified in
the monograph, shake, stand it at 37 ± 0.5 °C for 30
minutes, and then filter. Discard the first 3 mL of the filtrate,
exactly measure the subsequent 2 mL of the filtrate, add ex-
actly 5 mL of 0.55 mol/L sodium carbonate TS and 1 mL of
diluted Folin's TS (1 in 3) to the solution, shake well, and
stand it at 37 ± 0.5°C for 30 minutes. Determine the absor-
bance A-y of this solution at 660 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
blank. Separately, pipet 1 mL of the sample solution, add ex-
actly 5 mL of trichloroacetic acid TS A or B to the solution as
specified in the monograph, and shake. To this solution add
exactly 5 mL of the substrate solution specified in the mono-
graph, shake immediately, and stand it at 37 ± 0.5°C for 30
minutes. Follow the same procedure for the sample solution,
and determine the absorbance A B at 660 nm.
Protein digestive activity (unit/g)
= (Aj - A B )xFx
11
2
1
10
1
w
W: Amount (g) of sample in 1 mL of sample solution
F : Amount (/ug) of tyrosine for absorbance 1 determined
from Tyrosine Calibration Curve
(3) Assay for Fat Digestive Activity
The fat digestive activity can be obtained by back titration
of the amount of fatty acid produced from the hydrolysis of
the ester linkage, when lipase acts on olive oil. One fat diges-
tive activity unit is the amount of enzymes that produces 1
//mole of fatty acid per minute under the conditions described
in Procedure.
JP XV
General Tests / Pyrogen Test
87
Preparation of Sample Solution
Dissolve or suspend the sample in an appropriate amount
of cold water, or a buffer or salts solution specified in the
monograph so that the amount of fatty acid increases in
proportion to the concentration of the sample solution, when
measuring under the conditions described in Procedure. The
concentration is normally 1 to 5 fat digestive activity unit/
mL.
Preparation of Substrate Solution
Take 200 to 300 mL of a mixture of emulsifier and olive oil
(3:1) in a blender (see Fig. 4.03-2), and emulsify it at 12,000
to 16,000 revolutions per minute for 10 minutes, while cool-
ing the solution to a temperature below 10°C. Stand this so-
lution in a cool place for 1 hour, and make sure before use
that the oil does not separate.
Preparation of Emulsifier
Dissolve 20 g of polyvinyl alcohol specified in the mono-
graph in 800 mL of water by heating between 75°C and 80°C
for 1 hour while stirring. After cooling, filter the solution if
necessary, and add water to make exactly 1000 mL.
Procedure
Pipet 5 mL of the substrate solution and 4 mL of the buffer
solution specified in the monograph, transfer them to a
conical flask, and shake. After standing the mixture at 37 ±
0.5°C for 10 minutes, add exactly 1 mL of the sample
solution, and shake immediately. Stand this solution at 37 ±
0.5°C for exactly 20 minutes, add 10 mL of a mixture of
ethanol (95) and acetone (1:1), and shake. Then add exactly
10 mL of 0.05 mol/L sodium hydroxide VS, add 10 mL of a
mixture of ethanol (95) and acetone (1:1), and shake. Titrate
<2.50> the excess sodium hydroxide with 0.05 mol/L
hydrochloric acid VS (b mL) (indicator: 2 to 3 drops of
phenolphthalein TS). Separately, pipet 5 mL of the substrate
solution and 4 mL of buffer solution specified in the mono-
graph, transfer them to a conical flask, and shake. After
standing it at 37 ± 0.5°C for 10 minutes, add 10 mL of a
mixture of ethanol (95) and acetone (1:1), then add exactly 1
mL of the sample solution, and shake. Add exactly 10 mL of
0.05 mol/L sodium hydroxide VS, and titrate <2.50> in the
same manner (a mL).
Fat digestive activity (unit/g)
= 50x (a -6)x 1 Lx-i r
W: Amount (g) of sample in 1 mL of sample solution
4.04 Pyrogen Test
Pyrogen Test is a method to test the existence of pyrogens
by using rabbits.
Test animals
Use healthy mature rabbits, each weighing not less than 1.5
kg, which have not lost body mass when kept on a constant
diet for not less than one week. House the rabbits individual-
ly in an area free from disturbances likely to excite them.
Keep the temperature of the area constant between 20°C and
27°C for at least 48 hours before and throughout the test. Be-
fore using a rabbit that has not previously been used for a
pyrogen test, condition it 1 to 3 days prior to the test by con-
H Motor holder
A: Motor case
R: Inside column
C: Outside column
D: Cooling bath mounting
base
E: Motor top
F: Motor shaft
G: Motor ascend /descend
lever
H: Rotation control lever
I: Cup mounting base
J: Cooling bath
K: Knob
L: Cup lid
M: Spout shield
N: Blade
O: Screw
Cm I it Cup Cooling balh
Fig. 4.03-2 Blender
ducting a sham test omitting the injection. Do not use a rab-
bit for pyrogen tests more frequently than once every 48
hours, or after it has been given a test sample that was ad-
judged pyrogen-positive or that contained an antigen present
commonly in the test sample to be examined.
Apparatus, instruments
(1) Thermometer — Use a rectal thermometer or tempera-
ture-measuring apparatus with an accuracy of ±0.1°C or
less.
(2) Syringe and injection needle — Depyrogenate the
syringes and needles in a hot-air oven using a validated proc-
ess, usually by heating at 250°C for not less than 30 minutes.
Sterilized syringes with needles are also available provided
that they have been validated to assure that they are free of
detectable pyrogens and do not interfere with the test.
Test procedures
(1) Quantity of injection — Unless otherwise specified, in-
ject 10 mL of the sample per kg of body mass of each rabbit.
(2) Procedure — Perform the test in a separate area at an
environmental temperature similar to that of the room
wherein the animals were housed and free from disturbances
likely to excite them. Withhold food from the rabbits for
several hours before the first record of the temperature and
throughout the testing period. The test animals are usually
restrained with loosely fitting neck stocks that allow the
rabbits to assume a natural resting posture. Determine the
temperature of each rabbit by inserting the thermometer or
temperature-measuring probe into the rectum of the test
animal to a constant depth within the range of 60 mm to
90 mm. The "control temperature" of each rabbit is the
mean of two temperature readings recorded for that rabbit at
an interval of 30 min in the 40 min immediately preceding the
injection of the sample to be examined. Rabbits showing a
88
Microbial Limit Test / General Tests
JP XV
temperature variation greater than 0.2°C between the two
successive temperature readings or rabbits having an initial
temperature higher than 39.8°C are withdrawn from the test.
Warm the test solution to a temperature of 37 ± 2°C before
injection, and inject the solution slowly into the marginal
vein of the ear of each rabbit over a period not exceeding 10
min. Hypotonic test sample may be made isotonic by the ad-
dition of pyrogen-free sodium chloride. Record the tempera-
ture of each rabbit during a period of 3 hours after the injec-
tion, taking the measurements at intervals of not more than
30 min. The difference between the control temperature and
the maximum temperature of each rabbit is taken to be the
rise in body temperature. Consider any temperature
decreases as zero rise.
Interpretation of results
The test is carried out on a group of three rabbits and the
result is judged on the basis of the sum of the three tempera-
ture rises. Repeat if necessary on further groups of three rab-
bits to a total of three groups, depending on the results
obtained. If the summed response of the first group does not
exceed 1.3 C C, the sample is judged to be pyrogen-negative. If
the summed response exceeds 2.5 °C, the sample is judged to
be pyrogen-positive. If the summed response exceed 1.3°C
but does not exceed 2.5 °C, repeat the test on another group
of three rabbits. If the summed response of the first and sec-
ond group does not exceed 3.0°C, the sample is judged to be
pyrogen-negative. If the summed response of the 6 rabbits ex-
ceeds 4.2°C, the sample is judged to be pyrogen-positive. If
the summed response exceeds 3.0°C but does not exceed 4.2°
C, repeat the test on one more group of three rabbits. If the
summed response of the 9 rabbits does not exceed 5.0°C, the
sample is judged to be pyrogen-negative. If the summed
response exceeds 5.0°C, the sample is judged to be pyrogen-
positive.
When the test sample is judged to be pyrogen-negative, the
sample passes the pyrogen test.
4.05 Microbial Limit Test
This chapter provides tests for the qualitative and quantita-
tive estimation of viable microorganisms present in phar-
maceutical articles. It includes tests for total viable count
(bacteria and fungi) and specified microbial species (Es-
cherichia coli, Salmonella, Pseudomonas aeruginosa and
Staphylococcus aureus). Microbial limit test must be carried
out under conditions designed to avoid accidental microbial
contamination of the preparation during the test. When test
specimens have antimicrobial activity, or contain an-
timicrobial substances, any such antimicrobial properties
must be eliminated by means of procedures such as dilution,
filtration, neutralization or inactivation. For the test, use a
mixture of several portions selected at random from the bulk
or from the contents of a sufficient number of containers. If
test specimens are diluted with fluid medium, the test should
be performed quickly. In performing the test, precautions
must be taken to prevent biohazard.
1. Total viable aerobic count
This test determines the mesophilic bacteria and fungi
which grow under aerobic conditions. Psychrophilic, ther-
mophilic, basophilic and anaerobic bacteria, and microor-
ganisms which require specific ingredients for growth, may
give a negative result, even if a significant number exists in
the test specimens. The test may be carried out using one of
the following 4 methods, i.e., membrane filtration method,
pour plate method, spread plate method or serial dilution
method (most probable number method). Use an appropriate
method depending on the purpose. An automated method
may be used for the test presented here, provided it has been
properly validated as giving equivalent or better results.
Different culture media and temperature are required for the
growth of bacteria and fungi (molds and yeasts). Serial
dilution method is applicable only to the enumeration of bac-
teria.
Preparation of the test solution
Phosphate Buffer (pH 7.2), Buffered Sodium Chloride-
Peptone Solution or fluid medium used for the test is used to
dissolve or dilute the test specimen. Unless otherwise speci-
fied, 10 g or 10 mL of the test specimen is used for the test,
but another quantity or volume may be used according to the
nature of the test specimen. The pH of the solution is adjust-
ed to between 6 and 8. The test solution must be used within
an hour after preparation.
Fluid specimens or soluble solids: Take 10 g or 10 mL of
the test specimen, mix with the buffer or fluid medium speci-
fied to make 100 mL, and use this as the test fluid. A fluid
specimen containing insoluble materials must be shaken well,
just prior to mixing, to effect fine suspension.
Insoluble solids: Take 10 g or 10 mL of the test specimen,
reduce the substance to a fine powder, suspend it in the buffer
or fluid medium specified to make 100 mL, and use this as the
test fluid. A larger volume of the buffer or fluid medium than
indicated may be used for the suspension, depending on the
nature of the test specimen. The suspension may be dispersed
well using, if necessary, a mechanical blender. A suitable sur-
face-active agent (such as 0.1 w/v% polysorbate 80) may be
added to aid dissolution.
Fatty products: For water-immiscible fluids, ointments,
creams, waxes, and lotions which consist mainly of lipid,
take 10 g or 10 mL of the test specimen, emulsify it in the
buffer or fluid medium specified with the aid of a suitable sur-
face-active agent such as polysorbate 20 or 80 to make 100
mL, and use this as the test fluid. An emulsion may be made
by warming to a temperature not exceeding 45 °C, but do not
maintain this temperature for more than 30 minutes.
Test procedures
(1) Membrane Filtration Method
This method is applicable especially to specimens which
contain antimicrobial substances. Use membrane filters of
appropriate materials, having a normal pore size not greater
than 0.45 ftm. Filter discs about 50 mm in diameter are
recommended, but filters of a different diameter may also be
used. Filters, the filtration apparatus, media, etc., should be
sterilized well. Usually, take 20 mL of the test fluid (contain-
ing 2g of test specimen), transfer 10 mL of the solution to
each of two membrane filters, and filter. If necessary, dilute
the pretreated preparation so that a colony count of 10 to 100
may be expected. After the filtration of the test fluid, wash
each membrane by filtering through it three or more times
with a suitable liquid such as Buffered Sodium Chloride-Pep-
tone Solution, Phosphate Buffer, or the fluid medium to be
used. The volume of the washings to be used is approximately
100 mL each, but in case filter disc is significantly different
JPXV
General Tests / Microbial Limit Test
89
from 50 mm in diameter, the volume may be adjusted accord-
ing to the size of the filter. For fatty substances, the washings
may contain a suitable surface-active agent such as polysor-
bate 80. Put one of the membrane filters, intended primarily
for the enumeration of bacteria, on the surface of a plate of
Soybean-Casein Digest Agar and the other, intended primari-
ly for the enumeration of fungi, on the surface of a plate of
one of Sabouraud Glucose Agar, Potato Dextrose Agar, or
GP Agar Medium (each contains antibiotics). After incuba-
tion of the plates at least for 5 days, between 30°C and 35 °C
in the test for the detection of bacteria and between 20°C and
25 °C in the test for fungi, count the number of colonies that
are formed. If a reliable count is obtained in a shorter incuba-
tion time than 5 days, this may be adopted.
(2) Pour Plate Method
Use petri dishes 9 to 10 cm in diameter. Use at least two
petri dishes for each dilution. Pipet 1 mL of the test fluid or
its diluted solution onto each petri dish aseptically. Promptly
add to each dish 15 to 20 mL of sterilized agar medium that
has previously been melted and kept below 45 °C, and mix.
Primarily for the detection of bacteria, use Soybean-Casein
Digest Agar Medium, and, primarily for the detection of
fungi, use one of Sabouraud Glucose Agar, Potato Dextrose
Agar, and GP Agar Medium (each contains antibiotics).
After the agar solidifies, incubate the plates for at least 5
days, between 30°C and 35°C for bacteria and between 20°C
and 25 °C for fungi. If too many colonies are observed, dilute
the fluid as described above so that a colony count of not
more than 300 per plate may be expected in the case of bac-
teria, and not more than 100 per plate in the case of fungi. If
a reliable count is obtained in a shorter incubation time than
5 days, this may be adopted.
(3) Spread Plate Method
On the solidified and dried surface of the agar medium,
pipet 0.05 to 0.2 mL of the test fluid and spread it on the sur-
face with a spreader. The diameter of petri dishes, the kind
and volume of the medium to be used, the temperature and
time of incubation, and the method for calculation of total
viable count are the same as described in the Pour Plate
Method section.
(4) Serial Dilution Method (Most Probable Number
Method)
Prepare a series of 12 tubes each containing 9 to 10 mL of
Fluid Soybean-Casein Digest Medium. Use three tubes for
each dilution. To each of the first three tubes add 1 mL of the
test fluid (containing 0.1 g or 0.1 mL of specimen), resulting
in 1 in 10 dilution. To the next three tubes add 1 mL of a 1 in
10 dilution of the fluid, resulting in 1 in 100 dilution. To the
next three tubes add 1 mL of a 1 in 100 dilution of the fluid,
resulting in 1 in 1000 dilution. To the last three tubes add 1
mL of the diluent as a control. Incubate the tubes between
30°C and 35 °C for not less than 5 days. The control tubes
should show no microbial growth. If the reading of the
results is difficult or uncertain, transfer about 0.1 mL to a liq-
uid or solid medium and read the results after a further
period of incubation between 30°C and 35 C C for 24 to 72
hours. Determine the most probable number of microorgan-
isms per g or mL of the specimen from Table 4.05-1.
Table 4.05-1
Most probable number of microorganisms
Number of tubes in which microbial
growth is observed for each
quantity of the specimen
Most probable
number of micro-
0.1 g or
0.1 mL
per tube
0.01 g or
0.01 mL
per tube
1 mg or
per tube
organisms per
g or per mL
3
3
3
> 1100
3
3
2
1100
3
3
1
500
3
3
200
3
2
3
290
3
2
2
210
3
2
1
150
3
2
90
3
1
3
160
3
1
2
120
3
1
1
70
3
1
40
3
3
95
3
2
60
3
1
40
3
23
If, for the first column (containing 0.1 g or 0.1 mL of
specimen), the number of tubes showing microbial growth is
two or less, the most probable number of microorganisms per
g or per mL is likely to be less than 100.
Effectiveness of culture media and confirmation of an-
timicrobial substances
Use microorganisms of the following strains or their
equivalent. Grow them in Fluid Soybean-Casein Digest Medi-
um between 30°C and 35°C for bacteria and between 20°C
and 25 °C for Candida albicans.
Escherichia coli, such as ATCC 8739, NCIMB 8545, NBRC
3972
Bacillus subtilis, such as ATCC 6633, NCIMB 8054, NBRC
3134, JCM 2499
Staphylococcus aureus, such as ATCC 6538, NCIMB 8625,
NBRC 13276
Candida albicans, such as ATCC 2091, ATCC 10231, NBRC
1594, JCM 2085
Dilute portions of each of the cultures using Buffered Sodi-
um Chloride-Peptone Solution, or Phosphate Buffer to pre-
pare test suspensions containing about 50 to 200 CFU per
mL. Growth-promoting qualities are tested by inoculating 1
mL of each microorganism into each medium. The test media
are satisfactory if clear evidence of growth appears in all in-
oculated media after incubation at indicated temperature for
5 days. When a count of the test organisms with a test speci-
men differs by more than a factor of 5 from that without the
test specimen, any such effect must be eliminated by dilution,
filtration, neutralization or inactivation. To confirm the
sterility of the medium and of the diluent and the aseptic per-
formance of the test, carry out the total viable count method
using sterile Buffered Sodium Chloride-Peptone Solution or
Phosphate Buffer as the control.
2. Test for the detection of specified microorganisms
Escherichia coli, Salmonella, Pseudomonas aeruginosa
and Staphylococcus aureus are included as the target strains
of the test. These four species of microorganisms are im-
90
Microbial Limit Test / General Tests
JP XV
portant for the evaluation of microbial contamination not
only in the finished products, but also in the bulk or inter-
mediate of the production process, and are representative of
the microorganisms which should not exist in these materials.
Preparation of the test fluid
If necessary, refer to the paragraph on Preparation of the
Test Solution in Total viable aerobic count. When test speci-
mens are dissolved in or diluted with a fluid medium, use the
medium designated in each test, unless otherwise specified.
Test procedure
(1) Escherichia coli
To 10 g or 10 mL of the test specimen add a volume of
Fluid Lactose Medium to make 100 mL, and incubate be-
tween 30°C and 35 °C for 24 to 72 hours. Examine the medi-
um for growth, and if growth is present, mix by gentle shak-
ing, take a portion by means of an inoculating loop, and
streak it on the surface of MacConkey Agar Medium. Incu-
bate between 30°C and 35°C for 18 to 24 hours. If brick-red
colonies of Gram-negative rods surrounded by a reddish
precipitation zone are not found, the specimen meets the re-
quirements of the test for absence of Escherichia coli. If colo-
nies matching the above description are found, transfer the
suspect colonies individually to the surface of EMB Agar
Medium, and incubate between 30°C and 35°C for 18 to 24
hours. Upon examination, if none of the colonies exhibits
both a characteristic metallic sheen and a blue-black appear-
ance under transmitted light, the specimen meets the require-
ments of the test for the absence of Escherichia coli. Confirm
any suspect colonies on the plate by means of the IMViC test
(Indole production test, Methyl red reaction test, and Voges-
Proskauer test, and Citrate utilization test), and the colonies
which exhibit the pattern of either [+ H ] or [ — I ]
are judged as Escherichia coli. Rapid detection kits for Es-
cherichia coli may also be used.
(2) Salmonella
As in the case of the detection of Escherichia coli, to 10 g
or 10 mL of the test specimen add a volume of Fluid Lactose
Medium to make 100 mL, and incubate between 30°C and
35°C for 24 to 72 hours. Examine the medium for growth,
and if growth is present, mix by gentle shaking, pipet 1 mL
portions, respectively, into 10 mL of Fluid Selenite-Cystine
Medium and Fluid Tetrathionate Medium, and incubate for
12 to 24 hours. 10 mL of Fluid Selenite-Cystine Medium may
be replaced by the same volume of Fluid Rappaport Medium.
After the incubation, streak portions from both the fluid me-
dia on the surface of at least two of Brilliant Green Agar
Medium, XLD Agar Medium, and Bismuth Sulfite Agar
Medium, and incubate between 30 C C and 35 °C for 24 to 48
hours. Upon examination, if none of the colonies conforms
to the description given in Table 4.05-2, the specimen meets
the requirements of the test for the absence of the genus
Salmonella. If colonies of Gram-negative rods matching the
description in Table 4.05-2 are found, transfer suspect colo-
nies individually, by means of an inoculating wire, to a slant
of TSI Agar Medium using both surface and deep inocula-
tion. Incubate between 35°C and 37°C for 18 to 24 hours.
The presence of genus Salmonella is confirmed if, in the deep
culture but not in the surface culture, there is a change of
color from red to yellow and usually a formation of gas with
or without production of hydrogen sulfide. Precise identifica-
tion and typing of genus Salmonella may be carried out by
using appropriate biochemical and serological tests addition-
ally including an identification kit.
Table 4.05-2. Morphologic characteristics of Salmonella
species on selective agar media
Medium
Description of colony
Brilliant Green Agar Small, transparent and colorless, or
Medium opaque, pink or white (often surround-
ed by a pink to red zone)
XLD Agar Medium Red, with or without black centers
Bismuth Sulfite Agar Black or green
Medium
(3) Pseudomonas aeruginosa
To 10 g or 10 mL of the specimen add Fluid Soybean-
Casein Digest Medium, or another suitable fluid medium
without antimicrobial activity, to make 100 mL. Fluid Lac-
tose Medium is not suitable. Incubate between 30°C and
35°C for 24 to 48 hours. If, upon examination, growth is
present, use an inoculating loop to streak a portion of the
medium on the surface of Cetrimide Agar Medium or NAC
Agar Medium and incubate between 30°C and 35°C for 24 to
48 hours. If no growth of microorganisms is detected, the
specimen passes the test. If growth of colonies of Gram-nega-
tive rods with a greenish fluorescence occurs, streak suspect
colonies from the agar surface of Cetrimide Agar Medium on
the agar surfaces of Pseudomonas Agar Medium for Detec-
tion of Fluorescein and Pseudomonas Agar Medium for De-
tection of Pyocyanin, and incubate between 30°C and 35°C
for 24 to 72 hours. The production of yellowish fluorescence
on the surface of the former medium shows the production of
fluorescein, and the production of blue fluorescence on the
latter medium indicates the production of pyocyanin. Con-
firm any suspect colonies as Pseudomonas aeruginosa by
means of the Oxidase test. Transfer each of the suspect colo-
nies to filter paper that has previously been impregnated with
/V,/V-dimethyl-j9-phenylenediammonium dichloride. If the
colony changes to purple within 5 to 10 seconds, the oxidase
test is judged as positive. If the oxidase test is judged to be
negative, the specimen meets the requirements of the test for
the absence of Pseudomonas aeruginosa. The presence of
Pseudomonas aeruginosa may also be confirmed by using ap-
propriate biochemical tests including an identification kit.
(4) Staphylococcus aureus
To 10 g or 10 mL of the specimen add Fluid Soybean-
Casein Digest Medium, or another suitable fluid medium
without antimicrobial activity to make 100 mL. Incubate the
fluid containing the specimen between 30°C and 35 °C for 24
to 48 hours. If, upon examination, growth is present, use an
inoculating loop to streak a portion of the medium on the
surface of one of the Vogel-Johnson Agar Medium, Baird-
Parker Agar Medium, or Mannitol-Salt Agar Medium, and
incubate between 30°C and 35°C for 24 to 48 hours. Upon
examination, if no colonies of Gram-positive cocci having the
characteristics listed in Table 4.05-3 are found, the specimen
meets the requirements of the test for the absence of
Staphylococcus aureus. Confirm any suspect colonies as
Staphylococcus aureus by means of the coagulase test. With
the aid of an inoculating loop, transfer suspect colonies to in-
dividual tubes, each containing 0.5 mL of mammalian,
preferably rabbit or horse, plasma with or without suitable
additives. Incubate in a thermostat at 37 ± 1°C. Examine the
coagulation after 3 hours and subsequently at suitable inter-
JP XV
General Tests / Microbial Limit Test
91
vals up to 24 hours. Test positive and negative controls simul-
taneously. If no coagulation is observed, the specimen meets
the requirements of the test for the absence of Staphylococ-
cus aureus.
Table 4.05-3. Morphologic characteristics of Staphylococ-
cus aureus on selective agar media
Medium
Colonial characteristics
Vogel- Johnson Agar Black surrounded by yellow zone
Medium
Baird-Parker Agar Black, shiny, surrounded by clear zones
Medium
Mannitol-Salt Agar Yellow colonies with yellow zones
Medium
Effectiveness of culture media and confirmation of an-
timicrobial substances
Grow the test strains listed in Table 4.05-4 in the media in-
dicated between 30°C and 35°C for 18 to 24 hours. Dilute
portions of each of the cultures using Buffered Sodium Chlo-
ride-Peptone Solution, Phosphate Buffer, or medium indicat-
ed for each bacterial strain to make test suspensions contain-
ing about 1000 CFU per mL. As occasion demands, using a
mixture of 0.1 mL of each suspension of Escherichia coli,
Salmonella, Pseudomonas aeruginosa and Staphylococcus
aureus containing about 1000 CFU, the validity of the medi-
um and the presence of antimicrobial substances are tested in
the presence or absence of the specimen.
Table 4.05-4. Bacterial strains and media used for confir-
mation of the effectiveness of culture medium and validity of
the test for specified microorganisms
Microorganisms
Strain number
Media
Escherichia coli
Salmonella
ATCC 8739, NCIMB
8545, NBRC 3972 or
equivalent strains
No strain number is
recommended*
Fluid Lactose
Medium
Fluid Lactose
Medium
Pseudomonas
aeruginosa
Staphylococcus
aureus
ATCC 9027, NCIMB
8626, NBRC 13275 or
equivalent strains
ATCC 6538, NCIMB
8625, NBRC 13276 or
equivalent strains
Fluid Soybean-
Casein Digest
Medium
Fluid Soybean-
Casein Digest
Medium
* Salmonella strains of weak or no pathogenicity may be used.
Salmonella Typhi may not be used.
Retest
For the purpose of confirming a doubtful result, a retest is
conducted using 25 g or 25 mL of test specimen. Proceed as
directed under Test procedure, but make allowance for the
larger specimen size, for example by adjusting the volume of
the medium.
3. Buffer solution and media
Buffer solution and media used for the microbial limit test
are described below. Other media may be used if they have
similar nutritive ingredients, and selective and growth-
promoting properties for the microorganisms to be tested.
(1) Buffer solution
(i) Phosphate Buffer (pH 7.2)
Stock Solution: Dissolve 34 g of potassium dihydrogen-
phosphate in about 500 mL of water. Adjust to pH 7.1 to 7.3
by the addition of 175 mL of sodium hydroxide TS, add
water to make 1000 mL, and use this solution as the stock so-
lution. After sterilization by heating in an autoclave, store
under refrigeration. For use, dilute the Stock Solution with
water in the ratio of 1 to 800, and sterilize at 121 °C for 15 to
20 minutes.
(ii) Buffered Sodium Chloride-Peptone Solution (pH 7.0)
Potassium dihydrogen phosphate 3.56 g
Disodium hydrogen phosphate
dodecahydrate 18.23 g
Sodium chloride 4.30 g
Peptone 1.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.9-7.1. Polysorbate 20 or 80 (0.1 to 1.0 w/v%) may be ad-
ded.
(2) Media
(i) Soybean-Casein Digest Agar Medium
Casein peptone 15.0 g
Soybean peptone 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
7.1 -7.3.
(ii) Fluid Soybean-Casein Digest Medium
Casein peptone 17.0 g
Soybean peptone 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose 2.5 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
7.1 -7.5.
(iii) Sabouraud Glucose Agar Medium with Antibiotics
Peptones (animal tissue and casein) 10.0 g
Glucose 40.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.4-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium.
(iv) Potato Dextrose Agar Medium with Antibiotics
Potato extract 4.0 g
Glucose 20.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.4-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium.
(v) GP (Glucose-peptone) Agar Medium with Antibiotics
Glucose 20.0 g
Yeast extract 2.0 g
92
Microbial Limit Test / General Tests
JP XV
Magnesium sulfate heptahydrate 0.5 g
Peptone 5.0 g
Potassium dihydrogen phosphate 1.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.6-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium,
(vi) Fluid Lactose Medium
Meat extract 3.0 g
Gelatin peptone 5.0 g
Lactose monohydrate 5.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.7 - 7.1. After sterilization, cool immediately.
(vii) MacConkey Agar Medium
Gelatin peptone 17.0 g
Casein peptone 1.5 g
Animal tissue peptone 1.5 g
Lactose monohydrate 10.0 g
Sodium desoxycholate 1.5 g
Sodium chloride 5.0 g
Agar 13.5 g
Neutral red 0.03 g
Crystal violet 1.0 mg
Water 1000 mL
Mix all the components, boil for 1 minute, mix, and steri-
lize by heating in an autoclave at 121 °C for 15 to 20 minutes.
pH after sterilization: 6.9 - 7.3.
(viii) EMB (Eosin-Methylene Blue) Agar Medium
Gelatin peptone 10.0 g
Dipotassium hydrogen phosphate 2.0 g
Lactose monohydrate 10.0 g
Agar 15.0 g
Eosin Y 0.40 g
Methylene blue 0.065 g
Water 1000 mL
Mix all the components, and sterilize by heating in an
autoclave at 121 °C for 15 to 20 minutes. pH after steriliza-
tion: 6.9-7.3.
(ix) Fluid Selenite-Cystine Medium
Gelatin peptone 5.0 g
Lactose monohydrate 4.0 g
Trisodium phosphate dodecahydrate 10.0 g
Sodium selenite 4.0 g
L-Cystine 0.010 g
Water 1000 mL
Mix all the components, and heat to effect solution. Final
pH: 6.8 - 7.2. Do not sterilize,
(x) Fluid Tetrathionate Medium
Casein peptone 2.5 g
Animal tissue peptone 2.5 g
Sodium desoxycholate 1.0 g
Calcium carbonate 10.0 g
Sodium thiosulfate pentahydrate 30.0 g
Water 1000 mL
Heat the solution of solids to boiling. On the day of use,
add a solution prepared by dissolving 5 g of potassium iodide
and 6 g of iodine in 20 mL of water. Then add 10 mL of a so-
lution of brilliant green (1 in 1000), and mix. Do not heat the
medium after adding the brilliant green solution,
(xi) Fluid Rappaport Medium
Soybean peptone 5.0 g
Sodium chloride 8.0 g
Potassium dihydrogen phosphate 1.6 g
Malachite green oxalate 0.12 g
Magnesium chloride hexahydrate 40.0 g
Water 1000 mL
Dissolve malachite green oxalate and magnesium chloride
hexahydrate, and the remaining solids separately in the
water, and sterilize by heating in an autoclave at 121 °C for 15
to 20 minutes. For the use, mix the both solutions after
sterilization. Final pH: 5.4 - 5.8.
(xii) Brilliant Green Agar Medium
Peptones (animal tissue and casein) 10.0 g
Yeast extract 3.0 g
Sodium chloride 5.0 g
Lactose monohydrate 10.0 g
Sucrose 10.0 g
Phenol red 0.080 g
Brilliant green 0.0125 g
Agar 20.0 g
Water 1000 mL
Mix all the components, and boil for 1 minute. Sterilize
just prior to use by heating in an autoclave at 121 °C for 15 to
20 minutes. pH after sterilization: 6.7 - 7.1. Cool to about 50
°C and pour to petri dishes.
(xiii) XLD (Xylose-Lysine-Desoxycholate) Agar Medium
D-Xylose 3.5 g
L-Lysine monohydrochloride 5.0 g
Lactose monohydrate 7.5 g
Sucrose 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol red 0.080 g
Sodium desoxycholate 2.5 g
Sodium thiosulfate pentahydrate 6.8 g
Ammonium iron (III) citrate 0.80 g
Agar 13.5 g
Water 1000 mL
Mix all the components, and boil to effect solution. pH
after boiling: 7.2 - 7.6. Do not sterilize in an autoclave or
overheat. Cool to about 50°C and pour to petri dishes,
(xiv) Bismuth Sulfite Agar Medium
Meat extract 5.0 g
Casein peptone 5.0 g
Animal tissue peptone 5.0 g
Glucose 5.0 g
Trisodium phosphate dodecahydrate 4.0 g
Iron (II) sulfate heptahydrate 0.30 g
Bismuth sulfite indicator 8.0 g
Brilliant green 0.025 g
Agar 20.0 g
Water 1000 mL
Mix all the components, and boil to effect solution. pH
after boiling: 7.4-7.8. Do not sterilize in an autoclave or
overheat. Cool to about 50°C and pour to petri dishes,
(xv) TSI (Triple Sugar Iron) Agar Medium
Casein peptone 10.0 g
Animal tissue peptone 10.0 g
Lactose monohydrate 10.0 g
Sucrose 10.0 g
JPXV
General Tests / Sterility Test
93
Glucose
1.0 g
Ammonium iron (II) sulfate hexa-
hydrate
0.20 g
Sodium chloride
5.0 g
Sodium thiosulfate pentahydrate
0.20 g
Phenol red
0.025 g
Agar
13.0 g
Water
1000 mL
Mix all the components, and boil to effect solution. Dis-
tribute in small tubes and sterilize by heating in an autoclave
at 121 °C for 15 to 20 minutes. pH after sterilization: 7.1 -
7.5. Use as a slant agar medium. The medium containing 3 g
of meat extract or yeast extract additionally, or the medium
containing ammonium iron (III) citrate instead of ammoni-
um iron (II) sulfate hexahydrate may be used,
(xvi) Cetrimide Agar Medium
Gelatin peptone 20.0 g
Magnesium chloride hexahydrate 3.0 g
Potassium sulfate 10 g
Cetrimide 0.30 g
Glycerin 10 mL
Agar 13.6 g
Water 1000 mL
Dissolve all solid components in the water, and add the
glycerin. Heat, with frequent agitation, boil for 1 minute,
and sterilize by heating in an autoclave at 121 °C for 15 to 20
minutes. pH after sterilization: 7.0 - 7.4.
(xvii) NAC Agar Medium
Peptone 20.0 g
Dipotassium hydrogen phosphate 0.3 g
Magnesium sulfate heptahydrate 0.2 g
Cetrimide 0.2 g
Nalidixic acid 0.015 g
Agar 15.0 g
Water 1000 mL
Final pH: 7.2 - 7.6. Do not sterilize. Dissolve by warming.
(xviii) Pseudomonas Agar Medium for Detection of
Fluorescein
Casein peptone 10.0 g
Animal tissue peptone 10.0 g
Dipotassium hydrogen phosphate 1.5 g
Magnesium sulfate heptahydrate 1.5 g
Glycerin 10 mL
Agar 15.0 g
Water 1000 mL
Dissolve all the solid components in the water, and add the
glycerin. Heat, with frequent agitation, boil for 1 minute,
and sterilize by heating in an autoclave at 121 °C for 15 to 20
minutes. pH after sterilization: 7.0 - 7.4.
(xix) Pseudomonas Agar Medium for Detection of Pyoc-
yanin
Gelatin peptone 20.0 g
Magnesium chloride hexahydrate 3.0 g
Potassium sulfate 10.0 g
Glycerin 10 mL
Agar 15.0 g
Water 1000 mL
Dissolve all the solid components in the water, and add the
glycerin. Heat, with frequent agitation, boil for 1 minute,
and sterilize by heating in an autoclave at 121 °C for 15 to 20
minutes. pH after sterilization: 7.0 - 7.4.
(xx) Vogel-Johnson Agar Medium
Casein peptone 10.0 g
Yeast extract
5.0 g
D-Mannitol
10.0 g
Dipotassium hydrogen
phosphate
5.0 g
Lithium chloride
5.0 g
Glycine
10.0 g
Phenol red
0.025 g
Agar
16.0 g
Water
1000 mL
Mix all the components, and boil for 1 minute to effect so-
lution. Sterilize by heating in an autoclave at 121 °C for 15 to
20 minutes, and cool to between 45°C and 50°C. pH after
sterilization: 7.0 - 7.4. To this solution add 20 mL of sterile
potassium tellurite solution (1 in 100), and mix.
(xxi) Baird-Parker Agar Medium
Casein peptone 10.0 g
Meat extract 5.0 g
Yeast extract 1.0 g
Lithium chloride 5.0 g
Glycine 12.0 g
Sodium pyruvate 10.0 g
Agar 20.0 g
Water 950 mL
Mix all the components. Heat the mixture with frequent
agitation, and boil for 1 minute. Sterilize by heating in an
autoclave at 121 °C for 15 to 20 minutes, and cool to between
45°C and 50°C. pH after sterilization: 6.6 - 7.0. To this solu-
tion add 10 mL of sterile potassium tellurite solution (1 in
100) and 50 mL of egg-yolk emulsion. Mix gently, and pour
into petri dishes. Prepare the egg-yolk emulsion by mixing
egg-yolk and sterile saline with the ratio of about 30% to
70%.
(xxii) Mannitol-Salt Agar Medium
Casein peptone 5.0 g
Animal tissue peptone 5.0 g
Meat extract 1.0 g
D-Mannitol 10.0 g
Sodium chloride 75.0 g
Phenol red 0.025 g
Agar 15.0 g
Water 1000 mL
Mix all the components. Heat with frequent agitation, and
boil for 1 minute. Sterilize by heating in an autoclave at 121
C C for 15 to 20 minutes. pH after sterilization: 7.2 - 7.6.
4.06 Sterility Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( 4
>)•
Test for sterility is the method to establish the presence or
absence of viable microorganisms (bacteria and fungi) using
the defined culturing method. Unless otherwise specified, the
test is carried out by I. Membrane filtration method or II.
Direct inoculation method. Water, reagents, test solutions,
equipment, materials and all other requisites for the test
should be pre-sterilized. The test for sterility is carried out
under aseptic conditions. In order to achieve such conditions,
the test environment has to be adapted to the way in which
the sterility test is performed. The precautions taken to avoid
contamination are such that they do not affect any microor-
ganisms which are to be revealed in the test. The working
94
Sterility Test / General Tests
JP XV
conditions in which the tests are performed are monitored
regularly by appropriate sampling of the working area and by
carrying out appropriate controls.
Media and rinsing fluids
Fluid thioglycolate medium, soybean-casein digest medium
are used, unless otherwise specified. *When it is difficult to
use fluid thioglycolate medium due to turbidity or viscosity of
samples, alternative thioglycolate medium can be used, pro-
vided it is heated on a water bath just prior to use and
incubated under anaerobic conditions. ♦ Other products of
suitable quality yielding similar formulations may be used ac-
cording to the indications on the label.
(1) Fluid thioglycolate medium
L-Cystine 0.5 g
Agar 0.75 g
Sodium chloride 2.5 g
Glucose, monohydrate/anhydrate 5.5/5.0 g
Yeast extract (water-soluble) 5.0 g
Pancreatic digest of casein 15.0 g
Sodium thioglycolate or 0.5 g
Thioglycolic acid 0.3 mL
Resazurin sodium solution (1 in 1000),
freshly prepared 1 .0 mL
Water 1000 mL
(pH after sterilization 7.1 ±0.2)
Mix the L-cystine, agar, sodium chloride, glucose, water-
soluble yeast extract and pancreatic digest of casein with the
water, and heat until solution is effected. Dissolve the sodium
thioglycolate or thioglycolic acid in the solution and, if neces-
sary, add sodium hydroxide TS so that, after sterilization, the
solution will have a pH of 7.1 ±0.2. If filtration is necessary,
heat the solution again without boiling and filter while hot
through moistened filter paper. Add the resazurin sodium so-
lution, mix and place the medium in suitable vessels which
provide a ratio of surface to depth of medium such that not
more than the upper half of the medium has undergone a
color change indicative of oxygen uptake at the end of the in-
cubation period. Sterilize using a validated process. Store the
medium at a temperature between 2 - 25 °C. If more than the
upper one-third of the medium has acquired a pink color, the
medium may be restored once by heating the containers in a
water-bath or in free-flowing steam until the pink color disap-
pears and cooling quickly, taking care to prevent the in-
troduction of non-sterile air into the container.
*(2) Alternative thioglycolate medium
L-Cystine 0.5 g
Sodium chloride 2.5 g
Glucose, monohydrate/anhydrate 5.5/5.0 g
Yeast extract (water-soluble) 5.0 g
Pancreatic digest of casein 15.0 g
Sodium thioglycolate or 0.5 g
Thioglycolic acid 0.3 mL
Water 1000 mL
(pH after sterilization 7.1 ±0.2)
The methods for preparation follow those of fluid
thioglycolate medium. »
(3) Soybean-casein digest medium
Casein peptone 17.0 g
Soybean peptone 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose, monohydrate/anhydrous 2.5/2.3 g
Water 1000 mL
(pH after sterilization 7.3 ±0.2)
Mix all the ingredients and heat until solution is effected. If
necessary, add sodium hydroxide TS so that, after steriliza-
tion, the solution will have a pH of 7.3 ±0.2. Filter, if neces-
sary, to clarify, distribute into suitable vessels and sterilize
using a validated process. Store at a temperature between 2 -
25 °C in a sterile container.
(4) Rinsing fluids
Meat or casein peptone 1.0 g
Water 1000 mL
(pH after sterilization 7.1 ±0.2)
Dissolve animal tissue or casein peptone in water and
adjust the pH of the solution so that, after sterilization, it will
show 7.1 ±0.2. Filter, if necessary, to clarify, distribute into
suitable vessels and sterilize using a validated process. Store
at a temperature between 2 - 25 °C in a sterile container.
To rinsing fluid to be used for antibiotics or pharmaceuti-
cal products containing an antimicrobial agent, a suitable
neutralizer or inactive agent at concentration shown to be ap-
propriate in the validation of the test can be added. To rins-
ing fluid to be used for oils, oily solutions, ointments or
creams, suitable emulsifying agent at a concentration shown
to be appropriate in the validation of the test, for example
polysorbate 80 at a concentration of 10 g/L can be added.
Suitability of media
The media used comply with the following tests, carried
out before or in parallel with the test on the product to be
examined.
(1) Sterility of media
Confirm the sterility of each sterilized batch of medium by
incubating a portion of the media at the specified incubation
temperature for 14 days. No growth of microorganisms
occurs.
(2) Growth promotion test
Test each batch of ready-prepared medium and each batch
(lot)of medium prepared either from dehydrated medium or
from ingredients*. Inoculate a small number (not more than
100 CFU) of microorganism listed in Table 4.06-1 *or other
strains considered to be equivalent to these strains* in con-
tainers of each medium. Each of the test organisms should
show clearly visible growth in all inoculated media within 3
days for bacteria and within 5 days for fungi.
Effective period of media
*If prepared media are stored in unsealed containers, they
can be used for one month, provided that they are tested for
growth promotion within two weeks of the time of use and
that color indicator requirements are met. If stored in tight
containers, the media can be used for one year, provided that
they are tested for growth promotion within 3 months of the
time of use and that the color indicator requirements are
met.»
Validation test
The validation may be performed simultaneously with the
Test for sterility of the product to be examined in the
following cases,
a) When the test for sterility has to be carried out on a new
product.
**In appropriate cases periodic testing of the different batches pre-
pared from the same lot of dehydrated medium is acceptable..
JPXV
General Tests / Sterility Test
95
Table 4.06-1. Microorganisms for growth promotion test
and the validation test
Medium Test microorganisms
Staphylococcus aureus
(ATCC 6538, NBRC 13276,
CIP 4.83, NCTC 10788,
NCIMB 9518)
Fluid Pseudomonas aeruginosa
thioglycolate (ATCC 9027, NBRC 13275,
medium NCIMB 8626, CIP 82.118)
Clostridium sporogenes
(ATCC 19404, CIP 79.3,
NCTC 532, or ATCC 11437,
NBRC 14293)
*Alternative
thioglycolate
medium
Soybean-casein
digest medium
Clostridium sporogenes
(ATCC 19404, CIP 79.3,
NCTC 532, or ATCC 11437,
NBRC 14293)
Bacillus subtilis
(ATCC 6633, NBRC 3134,
CIP 52.62, NCIMB 8054)
Candida albicans
(ATCC 10231, NBRC 1594,
IP 48.72, NCPF 3179)
Aspergillus niger
(ATCC 16404, NBRC 9455,
IP 1431.83, IMI 149007)
Incubation
conditions
Aerobic
Anaerobic*
Aerobic
Seed lot culture maintenance techniques (seed-lot systems) are used
so that the viable microorganisms used for inoculation are not more
than five passages removed from the original master seed-lot.
b) Whenever there is a change in the experimental conditions
of the test.
Carry out the test as described below under Test for sterili-
ty of the product to be examined using exactly the same
methods except for the following modifications.
Membrane filtration After transferring the content of the
container or containers to be tested to the membrane add an
inoculum of a small number of viable micro-organisms (not
more than 100 CFU) to the final portion of sterile diluent
used to rinse the filter.
Direct inoculation After transferring the contents of the
container or containers to be tested to the culture medium
add an inoculum of a small number of viable micro-organ-
isms (not more than 100 CFU) to the medium.
In both cases use the same micro-organisms as those
described above under Growth promotion test. Perform a
growth promotion test as a positive control. Incubate all the
containers containing medium for not more than 5 days. If
clearly visible growth of micro-organisms is obtained after
the incubation, visually comparable to that in the control ves-
sel without product, either the product possesses no
antimicrobial activity under the conditions of the test or such
activity has been satisfactorily eliminated. The test for sterili-
ty may then be carried out without further modification. If
clearly visible growth is not obtained in the presence of the
product to be tested, visually comparable to that in the con-
trol vessels without product, the product possesses an-
timicrobial activity that has not been satisfactorily eliminated
under the conditions of the test. Modify the conditions in
order to eliminate the antimicrobial activity and repeat the
validation test.
In the membrane filtration, the antimicrobial activity should
be suppressed by suitable means such as replacement of the
membrane filters with less adsorptive ones, increase of the
amount of rinsing fluid, or addition of a suitable inactivating
agent to the rinsing fluid. Do not exceed a washing cycle of
5 times *100mL» per filter, even if during validation it has
been demonstrated that such a cycle does not fully eliminate
the antimicrobial activity.
In the direct inoculation, use a suitable inactivating agent
which does not affect the growth of microorganisms or in-
crease the volume of medium irrespective of the prescription
in II-2 so that no antimicrobial activity remains.
Test for sterility of the products to be examined
Number of articles to be tested
Items to be used for the test are taken from the lot accord-
ing to an appropriate sampling plan prepared by referring to
the numbers specified in Table 4.06-2.
Testing methods
The test may be carried out using the technique of mem-
brane filtration or by direct inoculation of the culture media
with the product to be examined. Appropriate negative
controls are included. The technique of membrane filtration
is used whenever the nature of the product permits, that is,
for filterable aqueous preparations, for alcoholic or oily
preparations and for preparations miscible with or soluble in
aqueous or oily solvents provided these solvents do not have
an antimicrobial effect in the conditions of the test.
I. Membrane filtration
By this method, a test article is filtered through a mem-
brane filter, and the filter is rinsed and incubated by being
transferred to a medium or by adding a medium to the filtra-
tion apparatus. Use membrane filter made from suitable
material having a nominal pore size of 0.45 //m or smaller.
Use a filter funnel sterilizable by the moist heat method or
other methods and free from any leakage or back flow when
filtration is performed with the membrane in place. The tech-
nique described below assumes that membranes about 50 mm
in diameter will be used. If filters of a different diameter are
used the volumes of the dilutions and the washings should be
adjusted accordingly.
1-1. Preparation of sample solution
a) Liquid medicine: Use as it is, as the sample solution.
*b) Solid medicine: In the case of a solid medicine, to be
administered after dissolving or suspending, the sample
solution is prepared with the provided solvent, isotonic
sodium chloride solution or water to give the concentra-
tion of use.*
c) Oils and oily solutions: Oils and oily solutions of
sufficiently low viscosity may be filtered without dilution
through a dry membrane. Viscous oils may be diluted as
necessary with a suitable sterile diluent such as isopropyl
myristate shown not to have antimicrobial activity in the
conditions of the test.
d) Ointments and creams: Ointments in a fatty base and
emulsions of the water-in-oil type may be diluted by using
sterile isopropyl myristate that has previously been filtered
through a sterilizing membrane filter or by using other sol-
vents not affecting the growth of microorganisms. Heat
96 Sterility Test / General Tests
Table 4.06-2. *Number of items to be taken from the lot*
v, _ u c u ■ .u i * Minimum number of items to
Number of items in the lot , . . , t , ,. *,
be tested tor each medium '
Injections 10% or 4 containers, which-
Not more than 100 containers ever is greater
JP XV
Table 4.06-3. Minimum quantity to be used for each
medium
More than 100 but not more
than 500 containers
More than 500 containers
♦For large-volume products
(more than 100 mL) of more
than 500 containers*
Ophthalmic and other
non-injectable products
Not more than 200 containers
More than 200 containers
If the product is presented in
the form of single-dose con-
tainers, apply the scheme
shown above for preparations
for parenteral use
Bulk solid products* 2
Up to 4 containers
More than 5 containers but
not more than 50 containers
More than 50 containers
10 containers
2% or 20 containers, which-
ever is less
*2% or 10 containers, which-
ever is less*
5% or 2 containers, which-
ever is greater
10 containers
Each container
20% or 4 containers, which-
ever is greater
2% or 10 containers, which-
ever is greater
Antibiotic Solids
Pharmacy bulk packages (>5 g)* J
♦Pharmacy bulk packages (<5
6 containers
♦20 containers*
*'If the contents of one container are enough to inoculate the two
media, this column gives the number of containers needed for both
the media together.
* 2 Bulk solid product is a sterile bulk powder product to be able to
prepare plural injections.
* 3 Pharmacy bulk package is an antibiotic solid to be able to prepare
plural injections. It should be dispensed in an infusion set at a time
after dissolving under a clean atmosphere.
the sample preparation, if necessary, to not more than 40 °C.
In exceptional cases it may be necessary to heat to not more
than 44°C.
1-2. Quantities of sample solution to be tested
Use for each medium not less than quantity of the product
prescribed in Table 4.06-3, unless otherwise specified. If the
contents of one container are insufficient to inoculate the two
media, twice or more containers shown in Table 4.06-2 are
used. When using the technique of membrane filtration, use,
whenever possible, the whole contents of the container, but
not less than the quantities indicated in Table 4.06-3, diluting
where necessary to about 100 mU with a suitable sterile
rinsing fluid.
1-3. Procedures
Usually complete the filtration of the sample solution with
one or two separate filter funnels. Transfer the contents of
the container or containers to be tested to the membrane or
Quantity per container
Liquids
Less than 1 mL
1 - 40 mL
Greater than 40 mL and
not greater than 100 mL
Greater than 100 mL
Antibiotic liquids
Other preparations soluble
in water or in isopropyl
myristate
Insoluble preparations, creams
and ointments to be suspended
or emulsified
Solids
Less than 50 mg
50 mg or more but less than
300 mg
300 mg - 5 g
Greater than 5 g
Minimum quantity to be
used for each medium
The whole contents of each
container
Half the contents of each
container but not less than
lmL
20 mL
10% of the contents of the
container but not less than
20 mL
lmL
The whole contents of each
container to provide not less
than 200 mg
Use the contents of each
container to provide not less
than 200 mg
The whole contents of each
container
Half the contents of each con-
tainer but not less than 50 mg
150 mg
500 mg
membranes. If the sample solution is not readily filterable, it
may be further diluted with rinsing fluid and thereafter
filtered. Rinse the membrane(s) with each 100-mL of rinsing
fluid per filter for established cycles in the validation test.
Provided the sample does not have antimicrobial activity, the
rinsing procedure can be omitted. Employ either of the two
methods described below for incubation of the membrane(s).
Use the same volume of each medium as in the validation
test.
(1) The processed membrane is aseptically transferred
from the apparatus and cut into two equal parts, or half the
volume of sample solution is filtered into an entire mem-
brane. Transfer each half of the cut membrane, or each
whole membrane into the medium.
(2) After filtration of sample solution into the apparatus
to which the membrane filters are fitted, each medium is
added to the apparatus itself.
II. Direct inoculation of the culture medium
This is the method by which the entire content or a portion
of the content of a sample container is transferred directly to
the culture medium and incubated. Usually, this method is
applied for medicines to which the membrane filtration
method cannot be applied or for which the application of the
direct transfer method, rather than the membrane filtration
method, is rational.
♦For products containing a mercurial preservative that
cannot be tested by the membrane-filtration method, fluid
thioglycolate medium incubated at 20 - 25 °C may be used in-
stead of Soybean-casein digest medium.*
JPXV
General Tests / Crude Drugs Test
97
11-1. Preparation of sample solution
Usually, proceed as directed for the membrane nitration
method. In the case of an insoluble medicine, the product is
suspended or crushed in a suitable manner and used as a sam-
ple.
a) Oily liquids. Use media to which have been added a suita-
ble emulsifying agent at a concentration shown to be
appropriate in the validation of the test, for example,
polysorbate 80 at a concentration of 10 g/L.
b) Ointments and creams. Prepare by diluting to about 1 in
10 by emulsifying with the chosen emulsifying agent in a
suitable sterile diluent such as a 1 g/L neutral solution of
meat or casein peptone. Transfer the diluted product to a
medium not containing an emulsifying agent.
1 1-2. Quantities of sample solution to be tested
Transfer the quantity of the preparation to be examined
prescribed in Table 4.06-3, by using pipette, syringe or other
suitable inoculation devices, directly into the culture medium
so that the volume of the product is not more than 10% of
the volume of the medium, unless otherwise prescribed.
Shake cultures containing oily products gently each observa-
tion day. However when thioglycolate medium is used for the
detection of anaerobic microorganisms keep shaking or mix-
ing to a minimum in order to maintain anaerobic conditions.
Cultivation and observation
Fluid thioglycolate medium *and Alternative thioglycolate
medium* are to be incubated at 30-35°C and Soybean-
casein digest medium is to be incubated at 20 - 25 C C for not
less than 14 days. Observe the cultures several times during
the incubation period. If the material being tested renders the
medium turbid so that the presence or absence of microbial
growth cannot be readily determined by visual examination,
14 days after the beginning of incubation transfer ♦ suitable
portions of» the medium to fresh vessels of the same medium
and then incubate the original and transfer vessels for not less
than 4 days.
Observation and interpretation of results
If no evidence of microbial growth is found, the product to
be examined complies with the test for sterility. If evidence of
microbial growth is found the product examined does not
comply with the test for sterility, unless it can be clearly
demonstrated that the test was invalid for causes unrelated to
the product to be examined. *However, provided that the
test itself was inadequate, the test is repeated. » If no evidence
of microbial growth is found in the repeat test the product
complies with the Sterility Test. If microbial growth is found
in the repeat test the product does not comply with the
Sterility Test.
5. Tests for Crude Drugs
5.01 Crude Drugs Test
Crude Drugs Test is applied to the crude drugs mentioned
in the General Rules for Crude Drugs.
Sampling
Unless otherwise specified, sample should be taken by the
following methods. If necessary, preserve the samples in tight
containers.
(1) When crude drugs to be sampled are small-sized, cut
or powdered, 50 to 250 g of sample should be taken after
mixing thoroughly.
(2) When crude drugs to be sampled are large-sized, 250
to 500 g of sample should be taken after mixing thoroughly.
(3) When the mass of each single piece of the crude drugs
is not less than 100 g, not less than 5 pieces should be taken
for a sample, or not less than 500 g of the sample should be
taken after cutting to a suitable size and mixing thoroughly.
Preparation of the test sample for analysis
Preparations are to be made by mixing the sample well.
Powdered drugs should be used as they are, and in the case of
unpowdered drugs, unless otherwise specified, grind the sam-
ple into powder. If the sample cannot be ground into powder,
reduce it as finely as possible, spread it out in a thin layer, and
withdraw a typical portion for analysis. If necessary, preserve
the test sample in a tight container.
Microscopic examination
(1) Apparatus
Use an optical microscope with objectives of 10 and 40
magnifications, and an ocular of 10 magnifications.
(2) Preparation for microscopic examination
(i) Section: To a section on a slide glass add 1 to 2 drops
of a mounting agent, and put a cover glass on it, taking
precaution against inclusion of bubbles. Usually use a section
10 to 20 ^m in thickness.
(ii) Powder: Place about 1 mg of powdered sample on a
slide glass, add 1 to 2 drops of a swelling agent, stir well with
a small rod preventing inclusion of bubbles, and allow to
stand for a while to swell the sample. Add 1 drop of the
mounting agent, and put a cover glass on it so that the tissue
sections spread evenly without overlapping each other, taking
precaution against inclusion of bubbles. In the case where the
tissue sections are opaque, place about 1 mg of powdered
sample on a slide glass, add 1 to 2 drops of chloral hydrate
TS, heat to make the tissues clear while stirring with a small
glass rod to prevent boiling. After cooling, add 1 drop of
mounting agent, and put a cover glass on it in the same man-
ner as above.
Unless otherwise specified, use a mixture of glycerin and
water (1:1) or a mixture of water, ethanol (95) and glycerin
(1:1:1) as the mounting agent and swelling agent.
(3) Observation of components in the Description
In each monograph, description is usually given of the out-
er portion and the inner portion of a section in this order, fol-
lowed by a specification of cell contents. Observation should
be made in the same order. In the case of a powdered sample,
description is given of a characteristic component or a matter
present in large amount, rarely existing matter, and cell con-
tents in this order. Observation should be made in the same
order.
Purity
(1) Foreign matter — Unless otherwise specified, weigh 25
to 500 g of the sample, spread out in a thin layer, and
separate the foreign matter by inspecting with the naked eye
or with the use of a magnifying glass of 10 magnifications.
Weigh, and determine the percentage of foreign matter.
(2) Total BHC's and total DDT's— Sodium chloride, an-
hydrous sodium sulfate and synthetic magnesium silicate for
98
Crude Drugs Test / General Tests
JP XV
column chromatography used in this procedure are used after
drying by heating at about 130°C for more than 12 hours and
cooling in a desiccator (silica gel). Chromatographic column
is prepared as follows: Place 20 g of synthetic magnesium sili-
cate for column chromatography in a 200-mL flask, add 50
mL of hexane for Purity of crude drug, shake vigorously,
and immediately pour the mixture into a chromatographic
tube about 2 cm in inside diameter and about 30 cm in length.
Drip until the depth of hexane layer at the upper part is about
5 cm, introduce 8 g of anhydrous sodium sulfate from the
top, and further drip until a small quantity of hexane is left at
the upper part.
Weigh accurately about 5 g of pulverized sample, place in a
glass-stoppered centrifuge tube, add 30 mL of a mixture of
acetone for Purity of crude drug and water (5:2), stopper
tightly, shake for 15 minutes, centrifuge, and separate the su-
pernatant liquid. Repeat the same procedure twice with the
residue using two 30-mL portions of the mixture of acetone
for Purity of crude drug and water (5:2). Combine all the su-
pernatant liquids, and concentrate under reduced pressure at
a temperature not higher than 40°C until the order of acetone
is faint. Transfer the concentrated solution to a separator
containing 100 mL of sodium chloride TS, and shake twice
with two 50-mL portions of hexane for Purity of crude drug
for 5 minutes each. Combine the hexane layers, transfer to a
separator containing 50 mL of sodium chloride TS, and
shake for 5 minutes. Take the hexane layer, dry with 30 g of
anhydrous sodium sulfate, and filter. Wash the residue on the
filter paper with 20 mL of hexane for Purity of crude drug.
Combine the filtrate and the washings, and concentrate under
reduced pressure at a temperature not higher than 40 °C to
about 5 mL. Transfer this solution to the chromatographic
column and allow to pass with 300 mL of a mixture of hexane
for Purity of crude drug and diethyl ether for Purity of crude
drug (17:3) at a rate of not more than 5 mL per minute. After
concentrating the eluate under reduced pressure at a tempera-
ture not higher than 40°C, add hexane for Purity of crude
drug to make exactly 5 mL. Transfer this solution to a glass-
stoppered test tube, add 1 mL of sulfuric acid, and shake
carefully. Take 4 mL of the upper layer, transfer to a
separate glass-stoppered test tube, add 2 mL of water, and
shake gently. Take 3 mL of the upper layer so obtained,
transfer to a glass-stoppered centrifuge tube, dry with 1 g of
anhydrous sodium sulfate, centrifuge, and use the super-
natant liquid as the sample solution. Separately, weigh ac-
curately about 10 mg each of a-BHC, /?-BHC, y-BHC, d-
BHC, o,p' -DDT, p,p' -DDT, p,p' -DDD and p,p' -DDE, dis-
solve in 5 mL of acetone for Purity of crude drug, and add
hexane for Purity of crude drug to make exactly 100 mL.
Pipet 10 mL of this solution, and add hexane for Purity of
crude drug to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add hexane for Purity of crude drug to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with 1 /xL each of the sample solution and the stan-
dard solution as directed under Gas Chromatography <2.02>
according to the following conditions, and determine the
peak areas corresponding to a-BHC, /?-BHC, y-BHC, 5-
BHC, o,p' -DDT, p,p' -DDT, p,p' -DDD and p,p' -DDH, A TA
andyl SA ;y4 TB and^4 SB ; A TC and^sci^TD and^4 SD ;^4 TE and A
SE ; A TF and A S¥ ; A TG and A SG ; ^4th and ^4 SH - Calculate the
content of each of a-BHC, fi-BUC, y-BHC, <5-BHC, o,p'-
DDT, p,p' -DDT, p,p' -DDD and/?,/?'-DDE by means of the
following equations.
Content (ppm) of a-BHC
amount (g) of a-BHC x A JX x 50
W
Content (ppm) of/?-BHC
= amount (g) of /?-BHC
W
Content (ppm) of y-BHC
_ amount (g) of y-BHC
W
Content (ppm) of <5-BHC
= amount (g) of 8 - BHC
W
Content (ppm) of o,p' -DDT
amount (g) of o,p' -DDT
W
x 4^X50
x 4^X50
x 4^ X50
X50
X50
X50
Content (ppm) of p,p' -DDT
amount (g) of p,p' -DDT
W
Content (ppm) of p,p' - DDD
amount (g) of p,p' —DDD
" W
Content (ppm) of p,p' -DDTL
= amount (g) of p,p' -DDE x A TU x
W A SH
W: Amount (g) of pulverized sample
Content (ppm) of total BHC's
= content (ppm) of a-BHC + content (ppm) of
/3-BHC + content (ppm) of y-BHC +
content (ppm) of J-BHC
Content (ppm) of total DDT's
= content (ppm) of o,p'-DDT + content (ppm) of
p,p' -DDT + content (ppm) of p,p' -DDD +
content (ppm) of p,p' -DDE
Operating conditions —
Detector: An electron capture detector
Sample injection system: A splitless injection system
Column: A fused silica capillary column about 0.3 mm in
inside diameter and about 30 m in length, coated the inside
wall with 7% cyanopropyl-7% phenylmethylsilicone polymer
for gas chromatography in a thickness of 0.25 to 1.0 /urn.
Column temperature: Maintain the temperature at 60°C
for 2 minutes after injection, program to increase the temper-
ature at a rate of 10°C per minute to 200°C, and then pro-
gram to increase the temperature at a rate of 2°C per minute
to 260 °C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention times
of the objective compounds are between 10 and 30 minutes.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add hexane to make exactly 10 mL. Confirm
that the peak area of each objective compound obtained with
1 /uL of this solution is equivalent to 5 to 15% of that of cor-
JPXV
responding compound with 1 fiL of the standard solution.
System performance: When the procedure is run with 1 /xL
of the standard solution under the above operating condi-
tions, the peaks of the object compounds separate completely
each other.
System repeatability: Repeat the test 6 times with 1 /uL of
the standard solution under the above operating conditions,
the relative standard deviation of the peak area of each object
compound is not more than 10%.
Loss on drying
Unless otherwise specified, transfer 2 to 6 g of the test sam-
ple for analysis to a tared weighing bottle, and weigh ac-
curately. Dry at 105°C for 5 hours, allow to cool in a desicca-
tor (silica gel), and weigh accurately. Continue the drying at
105 °C, and weigh accurately at 1-hour intervals. When the
mass of the sample becomes constant, the loss of mass
represents the percentage of loss on drying (%). When the
period of time for drying is specified, weigh accurately after
drying for the period of time specified, and determine the loss
on drying (%).
Total ash
Ignite previously a crucible of platinum, quartz or por-
celain between 500°C and 550°C for 1 hour. Cool, and weigh
accurately the crucible. Unless otherwise specified, weigh ac-
curately 2 to 4 g of the test sample for analysis in this cruci-
ble, take off the lid or keep it open a little if necessary, heat
the crucible at a low temperature at first, then gradually heat
to a temperature between 500°C and 550°C, ignite to inciner-
ate the residue for more than 4 hours until no carbonized sub-
stance remains in the ash, cool, and weigh accurately the ash.
Incinerate repeatedly to constant mass, cool, weigh accurate-
ly, and determine the amount (%) of total ash. If a car-
bonized substance remains and a constant mass cannot be ob-
tained in the above-mentioned method, extract the charred
mass with hot water, collect the insoluble residue on filter
paper for assay, and incinerate the residue and filter paper
until no carbonized substance remains in the ash. Then add
the filtrate, evaporate it to dryness, and incinerate. Cool,
weigh accurately, and determine the mass (%) of the total
ash. If a carbon-free ash cannot be obtained even in this way,
moisten the ash with a small amount of ethanol (95), break
up the ash with a glass rod, wash the rod with a small amount
of ethanol (95), evaporate carefully, and determine the mass
of the total ash as described above. A desiccator (silica gel) is
used for cooling.
Acid-insoluble ash
Add carefully 25 mL of dilute hydrochloric acid to the
total ash, boil gently for 5 minutes, collect the insoluble mat-
ter on filter paper for assay, and wash thoroughly with hot
water. Dry the residue together with the filter paper, and ig-
nite to incinerate in a tared crucible of platinum, quartz or
porcelain for 3 hours. Cool in a desiccator (silica gel), weigh,
and determine the amount (%) of acid-insoluble ash. When
the amount determined exceeds the limit specified, incinerate
repeatedly to a constant mass.
Extract content
The test for the extract content in crude drugs is performed
as directed in the following methods:
(1) Dilute ethanol-soluble extract — Unless otherwise spe-
cified, weigh accurately about 2.3 g of the sample for analy-
sis, extract with 70 mL of dilute ethanol in a suitable flask
General Tests / Crude Drugs Test 99
155
The figures are in mm.
Fig. 5.01-1 Fig. 5.01-2
with occasional shaking for 5 hours, and allow to stand for
16 to 20 hours. Filter, and wash the flask and residue with
small portions of dilute ethanol until the filtrate measures 100
mL. Evaporate a 50 mL aliquot of the filtrate to dryness, dry
at 105°C for 4 hours, and cool in a desiccator (silica gel).
Weigh accurately the amount, multiply it by 2, and determine
the amount of dilute ethanol-soluble extract. Calculate the
extract content (%) with respect to the amount of the sample
on the dried basis, obtained under the loss on drying.
(2) Water-soluble extract - Proceed as directed in (1),
using water instead of dilute ethanol, weigh accurately the
amount, multiply by 2, and determine the amount of water-
soluble extract. Calculate the extract content (%) with respect
to the amount of the sample on the dried basis, obtained un-
der the loss on drying.
(3) Diethyl ether-soluble extract — Unless otherwise speci-
fied, dry the test sample for analysis in a desiccator (silica gel)
for 48 hours, weigh accurately about 2 g of it, and place in a
suitable flask. Add 70 mL of diethyl ether, attach a reflux
condenser to the flask, and boil gently on a water bath for 4
hours. Cool, filter, and wash the flask and the residue with
small portions of diethyl ether until the filtrate measures 100
mL. Evaporate a 50 mL aliquot of the filtrate to dryness on a
water bath, dry in a desiccator (silica gel) for 24 hours, weigh
accurately the amount, multiply it by 2, determine the
amount of diethyl ether-soluble extract, and calculate the ex-
tract content (%).
Essential oil content
The test of essential oil content in crude drugs is performed
as directed in the following method:
Essential oil determination: Weigh the quantity of the test
sample for analysis directed in the monograph in a 1-L hard
glass-stoppered flask, and add from 5 to 10 times as much
water as the drug. Set up an apparatus for essential oil deter-
mination (Fig. 5.01-1), inserting a reflux condenser (Fig.
100
Microbial Limit Test for Crude Drugs / General Tests
JP XV
5.01-2) in the upper mouth of it, and heat the content of the
flask in an oil bath between 130°C and 150 C C to boiling. The
graduated tube of the apparatus is to be previously filled with
water to the standard line, and 2.0 mL of xylene is added to
the graduated tube. Unless otherwise specified, continue boil-
ing for 5 hours, allow to stand for some time, and open the
stopper of the apparatus. Draw off the water slowly until the
surface of the oil layer corresponds to the preparation line,
and allow it to stand for more than 1 hour at ordinary tem-
perature. Then lower the surface of the oil layer to the zero
line, and read the volume (mL) of the oil at ordinary temper-
ature. Subtract the volume (mL) of xylene from the volume
of the total oil.
5.02 Microbial Limit Test for
Crude Drugs
This chapter provides tests for the qualitative and quantita-
tive estimation of viable microorganisms present in crude
drugs. It includes tests for total viable count (aerobic bacteria
and fungi) and specified microbial species (Enterobacteria
and other gram-negative bacteria, Escherichia coli,
Salmonella, and Staphylococcus aureus). Microbial limit test
must be carried out under conditions designed to avoid ac-
cidental microbial contamination of the preparation during
the test. When test specimens have antimicrobial activity, or
contain antimicrobial substances, any such antimicrobial
properties must be eliminated by means of procedures such as
dilution, filtration, neutralization or inactivation. For the
test, use a mixture of several portions selected at random
from the bulk or from the contents of a sufficient number of
containers. If test specimens are diluted with fluid medium,
the test should be performed quickly. In performing the test,
precautions must be taken to prevent biohazard.
1. Total viable aerobic count
This test determines mesophilic aerobic bacteria and fungi
(molds and yeasts) which grow under aerobic conditions.
Psychrophilic, thermophilic, basophilic, and anaerobic bac-
teria, and microorganisms which require specific ingredients
for growth, may give a negative result, even if a significant
number exists in the test speciments. The test may be carried
out using one of the following 4 methods, i.e., pour plate
method, spread plate method, serial dilution method (most
probable number method) or membrane filtration method.
Use an appropriate method depending on the purpose. An
automated method may be used for the test presented here,
provided it has been properly validated as giving equivalent
or better results. Different culture media and temperature are
required for the growth of bacteria and fungi (molds and
yeasts). The serial dilution method is applicable only to the
enumeration of bacteria.
Sampling and Preparation of the test specimens
Unless otherwise specified, samples should be taken by the
following methods.
(1) When crude drugs to be sampled are small-sized, cut
or powdered, 50 to 250 g of sample should be taken after
mixing thoroughly.
(2) When crude drugs to be sampled are large-sized, 250
to 500 g of sample should be taken after mixing thoroughly
and cutting.
(3) When the mass of each single piece of the crude drug
is not less than 100 g, not less than 5 pieces should be taken
for a sample, or not less than 500 g of the sample should be
taken after cutting to a suitable size and mixing thoroughly.
If necessary, cut more for use.
(4) When crude drugs to be sampled are in the form of a
solution or a preparation, the sample should be taken after
mixing thoroughly.
(5) An insoluble solid should be taken after reducing the
substance to a moderately fine powder.
Preparation of the test fluid
Phosphate Buffer, pH 7.2, Buffered Sodium Chloride-Pep-
tone Solution, pH 7.0 or fluid medium used for the test is
used to suspend or dilute the test specimen. Unless otherwise
specified, usually take 10 g or 10 mL of the test specimen, and
suspend or dissolve it in 90 mL of the buffer or fluid medium
specified. A test specimen as a suspension must be shaken for
10 minutes. If necessary, for crude drugs to which microor-
ganisms might adhere, repeat the same method and use this
as the test fluid. A different quantity or volume may be used if
the nature of the test specimen requires it. The pH of the test
fluid is adjusted to between 6 and 8. The test fluid must be
used within an hour after preparation.
Fluid specimen: Take 10 mL of the test specimen, and sus-
pend or dissolve it in 90 mL of the buffer or fluid medium spe-
cified. A different quantity or volume may be used if the na-
ture of the test specimen requires it.
Insoluble solids: Pulverize the substance to a moderately
fine powder, take 10 g of the test specimen, and suspend it in
90 mL of the buffer or fluid medium specified. A different
quantity or a larger volume of buffer and fluid medium than
indicated may be used for the suspension, if the nature of the
test specimen requires it. The suspension may be dispersed
well using, if necessary, a mechanical blender. A suitable sur-
face active agent (such as 0.1 w/v% Polysorbate 80) may be
added to aid dissolution.
Test procedures
(1) Pour Plate Method
Use petri dishes 9 to 10 cm in diameter. Use at least two
petri dishes for each dilution. Pipet 1 mL of the test fluid or
its diluted solution onto each petri dish aseptically. Promptly
add to each dish 15 to 20 mL of sterilized agar medium that
has previously been melted and kept below 45 °C, and mix.
Primarily for the detection of aerobic microbes, use Soybean-
Casein Digest Agar Medium. For specimens that consist of
fragments of crude drugs, or to control the growth of fungi,
2,3,5-triphenyl-2//-tetrazolium chloride (TTC) TS for aero-
bic bacterial strains and amphotericin B TS as an antimycotic
may be added to the agar. Just prior to use, add 2.5-5 mL of
TTC TS or 2 mL of amphotericin B TS per liter of sterile
medium and mix. Primarily for the detection of fungi, use
one of Sabouraud Glucose Agar with antibiotics, Potato
Dextrose Agar with antibiotics, and GP Agar Medium with
antibiotics. For an agar medium that is suffused with fungi,
Rose Bengal TS may be added to the agar. Add 5 mL of Rose
Bengal TS per liter of agar medium, mix and sterilize by heat-
ing in an autoclave at 121 °C for 15 to 20 minutes. After the
agar solidifies, incubate the plates for at least 5 days at be-
tween 30°C and 35°C for aerobic bacteria, and between 20°C
and 25 °C for fungi. If too many colonies are observed, dilute
the fluid as described above so that a colony count of not
JPXV
General Tests / Microbial Limit Test for Crude Drugs
101
more than 300 per plate may be expected in the case of aero-
bic bacteria, and not more than 100 per plate in the case of
fungi. If a reliable count is obtained in a shorter incubation
time than 5 days, this may be adopted.
(2) Spread Plate Method
On the solidified and dried surface of the agar medium,
pipet 0.05 to 0.2 mL of the test fluid and spread it on the sur-
face with a spreader. The diameter of petri dishes, the kind
and volume of the medium to be used, TS to be added, tem-
perature and time of incubation, and the method for calcula-
tion of total viable count are the same as described in the
Pour Plate Method section.
(3) Serial Dilution Method (Most Probable Number
Method)
Prepare tubes each containing 9 to 10 mL of Fluid Soy-
bean-Casein Digest Medium. To each of the first three tubes
add 1 mL of the test fluid (containing 0.1 g or 0.1 mL of
specimen), resulting in 1 in 10 dilution. To the next three
tubes add 1 mL of a 1 in 10 dilution of the fluid, resulting in 1
in 100 dilution. To the next three tubes add 1 mL of a 1 in 100
dilution of the fluid, resulting in 1 in 1000 dilution. If neces-
sary, dilute further. To the last three tubes add 1 mL of the
diluent as a control. Incubate the tubes between 30°C and 35
°C for not less than 5 days. The control tubes should show no
microbial growth. If the reading of the results is difficult or
uncertain, transfer about 0.1 mL to a liquid or solid medium
and read the results after a further period of incubation be-
tween 30°C and 35°C for 24 to 72 hours. Determine the most
probable number of microorganisms per g or per mL of the
specimen from Table 5.02-1.
Table 5.02-1. Most probable number of microorganisms
Number of tubes in which microbial
growth is observed for each
quantity of the specimen
Most probable
number of micro-
0.1 g or
0.1 mL
0.01 g or
0.01 mL
1 mg or
1//L
organisms per
g or per mL
per tube
per tube
per tube
3
3
3
> 1100
3
3
2
1100
3
3
1
500
3
3
200
3
2
3
290
3
2
2
210
3
2
1
150
3
2
90
3
1
3
160
3
1
2
120
3
1
1
70
3
1
40
3
3
95
3
2
60
3
1
40
3
23
If, for the first column (0.1 g or 0.1 mL of specimen), the
number of tubes showing microbial growth is two or less, the
most probable number of microorganisms per g or per mL is
likely to be less than 100.
(4) Membrane Filtration Method
This method employs membrane filters of appropriate
materials, having a normal pore size not greater than 0.45
/urn. Filter discs about 50 mm in diameter are recommended,
but filters of a different diameter may also be used. Filters,
the filtration apparatus, media, etc., should be well sterilized.
Usually, take 20 mL of the test fluid (containing 2 g of test
specimen), transfer 10 mL of the solution to each of two
membrane filters, and filter. If necessary, dilute the pretreat-
ed preparation so that a colony count of 10 to 100 may be ex-
pected. After the filtration of the test fluid, wash each mem-
brane by filtering through it three or more times with a suita-
ble liquid such as Buffered Sodium Chloride-Peptone Solu-
tion, pH 7.0, Phosphate Buffer, pH 7.2, or the fluid medium
to be used. The volume of the washing to be used is approxi-
mately 100 mL each time, but if the filter disc is not about 50
mm in diameter, the volume may be adjusted according to
the size of the filter. For fatty substances, the washings may
contain a suitable surface-active agent such as Polysorbate
80. Put one of the membrane filters, intended primarily for
the enumeration of aerobic bacteria, on the surface of a plate
of Soybean-Casein Digest Agar and the other, intended
primarily for the enumeration of fungi, on the surface of a
plate of one of Sabouraud Glucose Agar with antibiotics,
Potato Dextrose Agar with antibiotics, and GP Agar Medi-
um with antibiotics. After incubation of the plates for at least
5 days, at between 30°C and 35 °C in the test for the detection
of aerobic bacteria and between 20°C and 25 °C in the test for
fungi, count the number of colonies that are formed. If a
reliable count is obtained in a shorter incubation time than 5
days, this may be adopted.
Effectiveness of culture media and confirmation of an-
timicrobial substances
Use microorganisms of the following strains or their
equivalent. Grow them in Fluid Soybean-Casein Digest Medi-
um between 30°C and 35 °C for aerobic bacteria and between
20°C and 25 °C for Candida albicans.
Escherichia coli, NBRC 3972, ATCC 8739,
NCIMB 8545, etc.
NBRC 3134, ATCC 6633,
NCIMB 8054, etc.
NBRC 13276, ATCC 6538,
NCIMB 9518, etc.
NBRC 1393, NBRC 1594,
ATCC 2091, ATCC 10231, etc.
Dilute portions of each of the cultures using Buffered
Sodium Chloride-Peptone Solution, pH 7.0, or Phosphate
Buffer, pH 7.2 to prepare test suspensions containing 50 to
200 cfu per mL. Growth-promoting qualities are tested by in-
oculating 1 mL of each microorganism into each medium.
The test media are satisfactory if clear evidence of growth ap-
pears in all inoculated media after incubation at the indicated
temperature for 5 days. When a count of test organisms with
a test specimen is less than 1/5 of that without the test speci-
men, any such effect must be eliminated by dilution, filtra-
tion, neutralization or inactivation. To confirm the sterility
of the medium and of the diluent and the aseptic perfor-
mance of the test, carry out the total viable count method us-
ing sterile Buffered Sodium Chloride-Peptone Solution, pH
7.0 or Phosphate Buffer, pH 7.2 as the control.
2. Test for the detection of specified microorganisms
Enterobacteria and certain other gram-negative bacteria,
Escherichia coli, Salmonella and Staphylococcus aureus, are
included as target strains of the test.
Bacillus subtilis,
Staphylococcus aureus,
Candida albicans,
102
Microbial Limit Test for Crude Drugs / General Tests
JP XV
Sampling and Preparation of the test specimens
Refer to the paragraph on Sampling and Preparation of
the Test Solution in Total viable aerobic count.
Preparation of the test fluid
If necessary, refer to the paragraph on Preparation of the
Test Fluid in Total viable aerobic count. When test specimens
are prepared, use the medium designated in each test, unless
otherwise specified. If necessary, eliminate antimicrobial sub-
stances so as to permit growth of the inocula, and adjust the
quantity of test specimen or increase the volume of medium
to suitable values.
Test Procedure
(1) Enterobacteria and certain other gram-negative bac-
teria
(i) Detection of bacteria
To 10 g or 10 mL of the test specimen add 90 mL of Fluid
Lactose Medium to form a suspension or solution. Transfer
10 mL to 90 mL of Enterobacteria enrichment broth Mossel
and incubate at between 35°C and 37°C for 18 to 24 hours.
Mix by gently shaking the container, take a portion by means
of an inoculating loop, and streak it on the surface of Crystal
violet, Neutral red, Bile Agar with glucose. Incubate at be-
tween 35°C and 37°C for 18 to 24 hours. If red or reddish
colonies are found, the specimen may contain Enterobacteria
and certain other gram-negative bacteria.
(ii) Quantitative evaluation
If Enterobacteria and certain other gram-negative bacteria
are found, to 10 g or 10 mL of the test specimen add 90 mL
of Fluid Lactose Medium to form a suspension or solution.
Transfer 1 mL of the test fluid (containing 0. 1 g or 0. 1 mL of
specimen) to a tube containing 9 mL of the fluid, and mix.
Next, transfer 1 mL of the test fluid (containing 0.01 g or 0.01
mL of specimen) to a tube containing 9 mL of the fluid, and
mix. Furthermore, transfer 1 mL of the test fluid (containing
1 mg or 1 fxL of specimen) to a tube containing 9 mL of the
fluid, and mix. Incubate the tubes at between 35°C and 37°C
for 18 to 24 hours, take a portion by means of an inoculating
loop, and streak it on the surface of Crystal violet, Neutral
red, Bile Agar with glucose. Incubate at between 35 °C and
37°C for 18 to 24 hours. If red or reddish colonies are found,
this constitutes a positive result. Note the smallest quantity of
the product which gives a positive result and the largest quan-
tity that gives a negative result. Determine from Table 5.02-2
the probable number of microorganisms.
Table 5.02-2. Most probable number of microorganisms
Results for each quantity of product
0.1 g or
0.1 mL
0.01 g or
0.01 mL
1 mg or
Probable number of
microorganisms
(cfu) per g or per mL
more than 10 3
less than 10 3 and
more than 10 2
less than 10 2 and
more than 10 1
less than 10 1
(2) Escherichia coli
(i) Detection of bacteria
To 10 g or 10 mL of the test specimen add 90 mL of Fluid
Lactose Medium to make a suspension or solution. Transfer
1 mL to a fermentation tube containing 9 to 10 mL of EC
broth and incubate the tube at 44.5±0.2 C C for 24 ±2 hours
in a water bath. If gas bubbles are not found, the specimen
meets the requirements of the test for absence of Escherichia
coli. If gas bubbles are found, take a portion by means of an
inoculating loop, and streak it on the surface of EMB Agar
Medium. Incubate at between 30°C and 35°C for 18 to 24
hours. Upon examination, if none of the colonies exhibits
both a characteristic metallic sheen and a blue-black appear-
ance under transmitted light, the specimen meets the require-
ments of the test for absence of Escherichia coli. Confirm any
suspect colonies on the plate by means of the IMViC test.
(Indole production test, Methyl red reaction test, Voges-
Proskauer test, and Citrate utilization test); colonies which
exhibit the pattern of either [ + H ] or [ — I ] are
judged as Escherichia coli. Rapid detection kits for Es-
cherichia coli may also be used.
(ii) Quantitative evaluation
If Escherichia coli is found, prepare tubes each containing
9 to 10 mL of EC broth. Use three tubes for each dilution. To
10 g or 10 mL of the test specimen add 90 mL of Fluid Lac-
tose Medium and suspend or dissolve. To each of the first
three fermentation tubes add 1 mL of the test fluid (contain-
ing 0.1 g or 0.1 mL of specimen), resulting in 1 in 10 dilution.
To the next three fermentation tubes add 1 mL of a 1 in 10 di-
lution of the fluid, resulting in 1 in 100 dilution. To the next
three fermentation tubes add 1 mL of a 1 in 100 dilution of
the fluid, resulting in 1 in 1000 dilution. To the last three fer-
mentation tubes add 1 mL of the diluent as a control. Incu-
bate the tubes at 44.5±0.2°C for 24 ±2 hours in a water
bath. If gas bubbles are found, take a portion by means of an
inoculating loop, and streak it on the surface of EMB Agar
Medium. Incubate at between 30°C to 35°C for 18 to 24
hours. Upon examination, colonies of Gram-negative organ-
isms show both a characteristic metallic sheen and a blue-
black appearance under transmitted light. Determine the
most probable number of microorganisms per g or per mL of
the specimen from Table 5.02-1.
(3) Salmonella
As in the case of the detection of Escherichia coli, to 10 g
or 10 mL of the test specimen add 90 mL of Fluid Lactose
Medium to form a suspension or solution. Incubate at be-
tween 30°C to 35 C C for 24 to 72 hours. Examine the medium
for growth, and if growth is apparent, mix by gentle shaking,
then pipet 1 mL portions, respectively, into 10 mL of Fluid
Selenite-Cystine Medium and Fluid Tetrathionate Medium,
and incubate for 12 to 24 hours. 10 mL of Fluid Selenite-
Cystine Medium may be replaced by the same volume of
Fluid Rappaport Medium. After the incubation, streak por-
tions from both the fluid media on the surface of at least two
of Brilliant Green Agar Medium, XLD Agar Medium, and
Bismuth Sulfite Agar Medium, and incubate at between 30°C
and 35°C for 24 to 48 hours. Upon examination, if none of
the colonies conforms to the description given in Table
5.02-3, the specimen meets the requirements of the test for
the absence of the genus Salmonella. If colonies of Gram-
negative rods matching the description in Table 5.02-3 are
found, transfer suspect colonies individually, by means of an
inoculating wire, to a slant of TSI Agar Medium using both
surface and deep inoculation. Incubate at between 35°C and
37 C C for 18 to 24 hours. The presence of genus Salmonella is
confirmed if, in the deep culture but not in the surface cul-
ture, there is a change of color from red to yellow and usually
formation of gas with or without production of hydrogen sul-
JPXV
General Tests / Microbial Limit Test for Crude Drugs
103
fide. Precise identification and typing of genus Salmonella
may be carried out by using appropriate biochemical and
serological tests additionally, including an identification kit.
Table 5.02-3. Morphologic characteristics of Salmonella
species on selective agar media
Medium
Description of colony
Brilliant Green Agar Small, transparent and colorless, or
Medium opaque, pink or white (often
surrounded by a pink to red zone)
XLD Agar Medium Red, with or without a black center
Bismuth Sulfite Agar Black or green
Medium
(4) Staphylococcus aureus
To 10 g or 10 mL of the test specimen add 90 mL of Fluid
Soybean-Casein Digest Medium, or another suitable fluid
medium without antimicrobial activity, to form a suspension
or solution. Incubate the fluid containing the specimen at be-
tween 30°C and 35°C for 24 to 48 hours, and pipet 1 mL into
9 mL of Fluid Soybean-Casein Digest Medium with 7.5% so-
dium chloride. If, upon examination, growth is apparent, use
an inoculating loop to streak a portion of the medium on the
surface of one of Vogel-Johnson Agar Medium, Baird-Par-
ker Agar Medium, or Mannitol-Salt Agar Medium, and incu-
bate at between 30°C and 35 °C for 24 to 48 hours. Upon ex-
amination, if no colonies of Gram-positive rods having the
characteristics listed in Table 5.02-4 are found, the specimen
meets the requirements of the test for the absence of
Staphylococcus aureus. Confirm any suspect colonies as
Staphylococcus aureus by means of the coagulase test. With
the aid of an inoculating loop, transfer suspect colonies to in-
dividual tubes, each containing 0.5 mL of mammalian,
preferably rabbit or horse, plasma with or without suitable
additives. Incubate in a thermostat at 37 ± 1°C. Examine the
coagulation after 3 hours and subsequently at suitable inter-
vals up to 24 hours. Test positive and negative controls simul-
taneously. If no coagulation is observed, the specimen meets
the requirements of the test for the absence of Staphylococ-
cus aureus.
Table 5.02-4. Morphologic characteristics of Staphylococ-
cus aureus on selective agar media
Medium
Colonial characteristics
Vogel-Johnson Agar Black surrounded by a yellow zone
Medium
Baird-Parker Agar Black, shiny, surrounded by a clear zone
Medium
Mannitol-Salt Agar Yellow colonies surrounded by a yellow
Medium zone
Effectiveness of culture media and confirmation of an-
timicrobial substances
Grow the test strains listed in Table 5.02-5 in the media in-
dicated at between 30°C and 35°C for 18 to 24 hours. Dilute
portions of each of the cultures using Buffered Sodium Chlo-
ride-Peptone Solution, pH 7.0, Phosphate Buffer, pH 7.2, or
medium indicated for each bacterial strain to make test sus-
pensions containing about 1000 cfu per mL. As occasion de-
mands, using a mixture of 0.1 mL of each suspension of Es-
cherichia coli, Salmonella and Staphylococcus aureus con-
taining about 1000 cfu, test the validity of the medium and
the presence of antimicrobial substances in the presence or
absence of the specimen.
Table 5.02-5. Bacteria strains and media used for confirma-
tion of the effectiveness of culture medium and validity of the
test for specified microorganisms
Microorganism
Strain number
Media
Escherichia coli
NBRC 3972, ATCC
8739, NCIMB 8545 or
equivalent strains
Fluid Lactose
Medium
Salmonella
No strain number is
recommended*
Fluid Lactose
Medium
Staphylococcus
aureus
NBRC 13276, ATCC
6538, NCIMB 9518 or
equivalent strains
Fluid Soybean-
Casein Digest
Medium
"Salmonella strains of weak or no pathogenicity may be used.
Salmonella typhi may not be used.
Retest
For the purpose of confirming a doubtful result, a retest is
conducted using a test specimen 2.5 times larger than the first
test specimen. Proceed as directed under Test procedure, but
make allowance for the larger specimen size, for example by
adjusting the volume of the medium.
3. Buffer solution, media and test solution (TS)
Buffer solution, media and TS used for the microbial limit
test are described below. Other media may be used if they
have similar nutritive ingredients, and selective and growth-
promoting properties for the microorganisms to be tested.
(1) Buffer solution
(i) Phosphate Buffer, pH 7.2
Stock Solution: Dissolve 34 g of potassium dihydrogen
phosphate in about 500 mL of water. Adjust to pH 7.1 to 7.3
by the addition of 175 mL of sodium hydroxide TS, add
water to make 1000 mL, and use this solution as the stock so-
lution. After sterilization by heating in an autoclave, store
under refrigeration. For use, dilute the Stock Solution with
water in the ratio of 1 to 800, and sterilize at 121 °C for 15 to
20 minutes.
(ii) Buffered Sodium Chloride-Peptone Solution, pH 7.0
Potassium dihydrogen phosphate 3.56 g
Disodium hydrogen phosphate
dodecahydrate 18.23 g
Sodium chloride 4.30 g
Peptone 1.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.9-7.1. Polysorbate 20 or 80(0.1 to 1.0 w/v%) may be ad-
ded.
(2) Media
(i) Soybean-Casein Digest Agar Medium
Casein peptone 15.0 g
Soybean peptone 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
7.1 -7.3.
(ii) Fluid Soybean-Casein Digest Medium
104
Microbial Limit Test for Crude Drugs / General Tests
JP XV
Casein peptone 17.0 g
Soybean peptone 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose 2.5 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
7.1 -7.5.
(iii) Sabouraud Glucose Agar Medium with Antibiotics
Peptone (animal tissue and casein) 10.0 g
Glucose 40.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.4-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium.
(iv) Potato Dextrose Agar Medium with Antibiotics
Potato extract 4.0 g
Glucose 20.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.4-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium.
(v) GP (Glucose-peptone) Agar Medium with Antibio-
tics
Glucose 20.0 g
Yeast extract 2.0 g
Magnesium sulfate heptahydrate 0.5 g
Peptone 5.0 g
Potassium dihydrogen phosphate 1.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
5.6-5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloram-
phenicol per liter of medium,
(vi) Fluid Lactose Medium
Meat extract 3.0 g
Gelatin peptone 5.0 g
Lactose monohydrate 5.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.7 - 7.1. After sterilization, cool immediately.
(vii) Fluid EC Medium
Peptone 20.0 g
Lactose monohydrate 5.0 g
Bile salts 1.5 g
Dipotassium hydrogen phosphate 4.0 g
Potassium dihydrogen phosphate 1.5 g
Sodium chloride 5.0 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.8 - 7.0. After sterilization cool immediately. If gas remains
in durham tube after cool, do not use the tube.
(viii) EMB (Eosin-Methylene Blue) Agar Medium
Gelatin peptone 10.0 g
Dipotassium hydrogen phosphate 2.0 g
Lactose monohydrate 10.0 g
Agar 15.0 g
Eosin 0.40 g
Methylene blue 0.065 g
Water 1000 mL
Mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
6.9-7.3.
(ix) Fluid Enterobacteria Enrichment Broth Mossel
Medium
Gelatin peptone 10.0 g
Glucose 5.0 g
Bile salts 20.0 g
Potassium dihydrogen phosphate 2.0 g
Disodium hydrogen phosphate
dodecahydrate 8.0 g
Brilliant green 0.015 g
Water 1000 mL
Mix all the components, boil at 100°C for 30 minutes, and
cool immediately. pH after boiling: 7.0 - 7.4.
(x) VRB (Violet /Red /Bile) Agar with glucose
Yeast extract 3.0 g
Gelatin peptone 7.0 g
Bile salts 1.5 g
Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Glucose 10.0 g
Agar 15.0 g
Neutral red 0.030 g
Crystal violet 0.002 g
Water 1000 mL
Mix all the components, and boil to effect solution. pH
after boiling: 7.2 - 7.6. Do not sterilize in an autoclave.
(xi) Fluid Selenite-Cystine Medium
Gelatin peptone 5.0 g
Lactose monohydrate 4.0 g
Trisodium phosphate dodecahydrate 10.0 g
Sodium selenite 4.0 g
L-Cystine 0.010 g
Water 1000 mL
Mix all the components, and heat to dissolve. Final pH: 6.8
- 7.2. Do not sterilize.
(xii) Fluid Tetrathionate Medium
Casein peptone 2.5 g
Animal tissue peptone 2.5 g
Sodium desoxycholate 1.0 g
Calcium carbonate 10.0 g
Sodium thiosulfate pentahydrate 30.0 g
Water 1000 mL
Heat the solution of solids to boiling. On the day of use,
add a solution prepared by dissolving 5 g of potassium iodide
and 6 g of iodine in 20 mL of water. Then add 10 mL of a so-
lution of brilliant green (1 in 1000), and mix. Do not heat the
medium after adding the brilliant green solution.
(xiii) Fluid Rappaport Medium
Soybean peptone 5.0 g
Sodium chloride 8.0 g
JPXV
General Tests / Microbial Limit Test for Crude Drugs
105
Potassium dihydrogen phosphate 1.6 g
Malachite green oxalate 0.12 g
Magnesium chloride hexahydrate 40.0 g
Water 1000 mL
Dissolve malachite green oxalate and magnesium chloride
hexahydrate, and the remaining solids separately in the
water, and sterilize by heating in an autoclave at 121 °C for 15
to 20 minutes. For the use, mix the both solutions after
sterilization. Final pH: 5.4 - 5.8.
(xiv) Brilliant Green Agar Medium
Peptones (animal tissue and casein) 10.0 g
Yeast extract 3.0 g
Sodium chloride 5.0 g
Lactose monohydrate 10.0 g
Sucrose 10.0 g
Phenol red 0.080 g
Brilliant green 0.0125 g
Agar 20.0 g
Water 1000 mL
Mix all the components, and boil for 1 minute. Sterilize
just prior to use by heating in an autoclave at 121 °C for 15 to
20 minutes. pH after sterilization: 6.7 - 7.1. Cool to about 50
°C and pour to petri dishes.
(xv) XLD (Xylose-Lysine-Desoxycholate) Agar Medium
D-Xylose 3.5 g
L-Lysine monohydrochloride 5.0 g
Lactose monohydrate 7.5 g
Sucrose 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol red 0.080 g
Sodium desoxycholate 2.5 g
Sodium thiosulfate pentahydrate 6.8 g
Ammonium iron (III) citrate 0.80 g
Agar 13.5 g
Water 1000 mL
Mix all the components, and boil to effect solution. pH
after boiling: 7.2-7.6. Do not sterilize in an autoclave or
overheat. Cool to about 50°C and pour to petri dishes,
(xvi) Bismuth Sulfite Agar Medium
Meat extract 5.0 g
Casein peptone 5.0 g
Animal tissue peptone 5.0 g
Glucose 5.0 g
Trisodium phosphate dodecahydrate 4.0 g
Iron (II) sulfate heptahydrate 0.30 g
Bismuth sulfite indicator 8.0 g
Brilliant green 0.025 g
Agar 20.0 g
Water 1000 mL
Mix all the components, and boil to effect solution. pH
after boiling: 7.4-7.8. Do not sterilize in an autoclave or
overheat. Cool to about 50°C and pour to petri dishes.
(xvii) TSI (Triple Sugar Iron) Agar Medium
Casein peptone 10.0 g
Animal tissue peptone 10.0 g
Lactose monohydrate 10.0 g
Sucrose 10.0 g
Glucose 1.0 g
Ammonium iron (II) sulfate hexahy-
drate 0.20 g
Sodium chloride 5.0 g
Sodium thiosulfate pentahydrate 0.20 g
Phenol red 0.025 g
Agar 13.0 g
Water 1000 mL
Mix all the components, and boil to effect solution. Dis-
tribute in small tubes and sterilize by heating in an autoclave
at 121°C for 15 to 20 minutes. pH after sterilization: 7.1 -
7.5. Use as a slant agar medium. The medium containing 3 g
of meat extract or yeast extract additionally, or the medium
containing ammonium iron (III) citrate instead of ammoni-
um iron (II) sulfate hexahydrate may be used.
(xviii) Fluid Soybean-Casein Digest Medium with 7.5%
Sodium Chloride
Casein peptone 17.0 g
Soybean peptone 3.0 g
Sodium chloride 75.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose 2.5 g
Water 1000 mL
Add the sodium chloride 70.0 g for (ii) Fluid Soybean-
Casein Digest Medium (containing 5 g of sodium chloride),
mix all the components, and sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes. pH after sterilization:
7.1 -7.5.
(xix) Vogel-Johnson Agar Medium
Casein peptone 10.0 g
Yeast extract 5.0 g
D-Mannitol 10.0 g
Dipotassium hydrogen phosphate 5.0 g
Lithium chloride 5.0 g
Glycine 10.0 g
Phenol red 0.025 g
Agar 16.0 g
Water 1000 mL
Mix all the components, and boil for 1 minute to make so-
lution. Sterilize by heating in an autoclave at 121 °C for 15 to
20 minutes, and cool to between 45°C and 50°C. pH after
sterilization: 7.0 - 7.4. To this solution add 20 mL of sterile
potassium tellurite solution (1 in 100), and mix.
(xx) Baird-Parker Agar Medium
Casein peptone 10.0 g
Meat extract 5.0 g
Yeast extract 1.0 g
Lithium chloride 5.0 g
Glycine 12.0 g
Sodium pyruvate 10.0 g
Agar 20.0 g
Water 950 mL
Mix all the components. Heat the mixture with frequent
agitation, and boil for 1 minute. Sterilize by heating in an au-
toclave at 121 °C for 15 to 20 minutes, and cool to between 45
°C and 50°C. pH after sterilization: 6.6-7.0. To this solu-
tion add 10 mL of sterile potassium tellurite solution (1 in
100) and 50 mL of egg-yolk emulsion. Mix gently, and pour
into petri dishes. Prepare the egg-yolk emulsion by mixing
egg-yolk and sterile saline with the ratio of about 30% to
70%.
(xxi) Mannitol-Salt Agar Medium
Casein peptone 5.0 g
Animal tissue peptone 5.0 g
Meat extract 1.0 g
D-Mannitol 10.0 g
Sodium chloride 75.0 g
Phenol red 0.025 g
106
Test for Metal Particles in Ophthalmic Ointments / General Tests
JP XV
Agar 15.0 g
Water 1000 mL
Mix all the components. Heat with frequent agitation, and
boil for 1 minute. Sterilize by heating in an autoclave at
121 °C for 15 to 20 minutes. pH after sterilization: 7.2 - 7.6.
(3) Reagents -Test solutions
Amphotericin B powder Amphotericin B added sodium
deoxycholic acid, sterilized by y-ray.
Amphotericin B TS Dissolve 22.5 mg of amphotericin B
powder in 9 mL of sterile purified water.
Bile salts Yellow-brown powder made from dried bile of
animal, consist of sodium taurocholic acid and sodium
glycocholic acid, and containing not less than 45% of cholic
acid. pH of 5% solution: 5.5 - 7.5.
Rose bengal C2oH2Cl 4 I 4 Na205 [Special class] Red-
brown powder, purple-red solution in water.
Rose bengal TS Dissolve 1 g of rose bengal in water to
make 100 mL.
TTC TS (2,3,5-Triphenyl-2//-tetrazolium chloride TS)
Dissolve 0.8 g of 2,3,5-triphenyl-2//-tetrazolium chlorid in
water to make 100 mL, distribute in small tubes, and sterilize
by heating in an autoclave at 121 °C for 15 to 20 minutes.
Store in light-resistant containers.
(4) Preparation
Preparation of agar medium with TTC
Just prior to use, add 2.5 to 5 mL of TTC TS per liter of
sterile agar medium and mix.
Preparation of agar medium with amphotericin B
Just prior to use, add 2 mL of amphotericin B TS in a liter
of agar medium, previously sterilized in an autoclave at
121 °C for 15 to 20 minutes, and mix.
Preparation of agar medium with rose bengal
Add 5 mL of rose bengal TS in a liter of agar medium, and
mix. Sterilize in an autoclave at 121 °C for 15 to 20 minutes.
6. Tests for Preparations in
Ophthalmic Ointments
6.01 Test for Metal Particles in
Ophthalmic Ointments
Test of Metal Particles in Ophthalmic Ointments is a
method to test the existence of foreign metal particles in the
ophthalmic ointments described in General Rules for Prepa-
rations.
Preparation of test sample
The test should be carried out in a clean place. Take 10
ophthalmic ointments to be tested, and extrude the contents
as completely as practicable into separate flat-bottomed petri
dishes 60 mm in diameter when the amount of the content is 5
g or less. Weigh 5 g of the contents when the amount of the
content is more than 5 g, and proceed in the same manner as
described above. Cover the dishes, and heat between 85 °C
and 1 10°C for 2 hours to dissolve bases. Allow the samples to
cool to room temperature without agitation to solidify the
contents.
Procedure
Invert each dish on the stage of a suitable microscope
previously adjusted to provide more than 40 times magnifica-
tions and equipped with an eyepiece micrometer disk. Each
dish is illuminated from above 45° relative to the plane of the
dish. Examine the entire bottom of each dish for metal parti-
cles, and record the total number of particles, measuring 50
Hm. or more in any dimension.
Note: Use petri dishes with a clean bottom and free from
foams and scratches, and if possible, the walls are at right an-
gles with the bottom.
6.02 Uniformity of Dosage Units
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* ♦).
The term "Uniformity of dosage unit" is defined as the
degree of uniformity in the amount of the drug substance
among dosage units. Therefore, the requirements of this
chapter apply to each drug substance being comprised in
dosage units containing one or more drug substances, unless
otherwise specified elsewhere in this Pharmacopoeia.
To ensure the consistency of dosage units, each unit in a
batch should have a drug substance content within a narrow
range around the label claim. Dosage units are defined as
dosage forms containing a single dose or a part of a dose of a
drug substance in each dosage unit. The Uniformity of
Dosage Units specification is not intended to apply to suspen-
sions, emulsions, or gels in unit-dose containers intended for
external, cutaneous administration.
The uniformity of dosage units can be demonstrated by
either of two methods, Content uniformity or Mass variation
(see Table 6.02-1.). The test for Content Uniformity of
preparations presented in dosage units is based on the assay
of the individual contents of drug substance(s) of a number
of dosage units to determine whether the individual contents
are within the limits set. The Content Uniformity method
may be applied in all cases.
The test for Mass Variation is applicable for the following
dosage forms:
(1) solutions enclosed in unit-dose containers and into soft
capsules *in which all components are perfectly dis-
solved; »
(2) solids (including powders, granules and sterile solids)
that are packaged in single-unit containers and contain
no active or inactive added substances;
(3) solids (including sterile solids) that are packaged in sin-
gle-unit containers, with or without active or inactive
added substances, that have been prepared from true so-
lutions and freeze-dried in the final containers and are
labeled to indicate this method of preparation; and
(4) hard capsules, uncoated tablets, or film-coated tablets,
containing 25 mg or more of a drug substance compris-
ing 25% or more, by weight, of the dosage unit or, in
the case of hard capsules, the capsule contents, *or in
the case of film-coated tablets, the pre-coated tablets, »
except that uniformity of other drug substances present
in lesser proportions is demonstrated by meeting Con-
tent Uniformity requirements.
The test for Content Uniformity is required for all dosage
forms not meeting the above conditions for the Mass Varia-
JPXV
General Tests / Test for Metal Particles
107
Table 6.02-1
. Application of Content Uniformity (CU) and Mass Variation (MV) Test for Dosage Forms
Dosage Forms
Type
Sub-type
Dose and ratio of drug substance
>25mg& >25%
<25 mg or <25%
Tablets
uncoated
MV
CU
coated
Film
MV
CU
others
CU
CU
Capsules
hard
MV
CU
soft
Sus., eml., gel
CU
CU
solutions
MV
MV
Solids in single unit
containers * (divided
forms, lyophilized
forms, et al.)»
Single component
MV
MV
Multiple components
Solution freeze-dried
in final container
MV
MV
others
CU
CU
Solutions enclosed in
unit-dose containers
MV
MV
Others
CU
CU
Sus.: suspension; eml.: emulsion;
tion test. Alternatively, products listed in item (4) above that
do not meet the 25 mg/25% threshold limit may be tested for
uniformity of dosage units by Mass Variation instead of the
Content Uniformity test if the concentration relative stan-
dard deviation (RSD) of the drug substance in the final
dosage units is not more than 2%, based on process valida-
tion data and development data, and if there has been regula-
tory approval of such a change. The concentration RSD is the
RSD of the concentration per dosage unit (w/w or w/v),
where concentration per dosage unit equals the assay result
per dosage unit divided by the individual dosage unit weight.
See the RSD formula in Table 6.02-2.
CONTENT UNIFORMITY
Select not less than 30 units, and proceed as follows for the
dosage form designated.
Where different procedures are used for assay of the prepa-
ration and for the content uniformity test, it may be necessa-
ry to establish a correction factor to be applied to the results
of the latter.
Solid dosage forms — Assay 10 units individually using an
appropriate analytical method. Calculate the acceptance
value (see Table 6.02-2).
Liquid dosage forms — Carry out the assay on the amount
of well-mixed material that is removed from an individual
container in conditions of normal use and express the results
as delivered dose. Calculate the acceptance value (see
Table 6.02-2.).
Calculation of Acceptance Value
Calculate the acceptance value by the formula:
\M-X\ +ks,
in which the terms are as defined in Table 6.02-2.
MASS VARIATION
Mass Variation is carried out based on the assumption that
the concentration (mass of drug substance per mass of dosage
unit) is uniform in a lot.
Carry out an assay for the drug substance(s) on a represen-
tative sample of the batch using an appropriate analytical
method. This value is result A, expressed as % of label claim
(see Calculation of the Acceptance Value). Select not less
than 30 dosage units, and proceed as follows for the dosage
form designated.
Uncoated or film-coated Tablets. Accurately weigh 10
tablets individually. Calculate the content, expressed as % of
label claim, of each tablet from the mass of the individual
tablets and the result of the assay. Calculate the acceptance
value.
Hard Capsules. Accurately weigh 10 capsules individual-
ly, taking care to preserve the identity of each capsule. Re-
move the contents of each capsule by suitable means. Ac-
curately weigh the emptied shells individually, and calculate
for each capsule the net mass of its contents by subtracting
the mass of the shell from the respective gross mass. Calcu-
late the drug substance content of each capsule from the mass
of the individual capsules and the result of the assay. Calcu-
late the acceptance value.
Soft Capsules. Accurately weigh the 10 intact capsules in-
dividually to obtain their gross masses, taking care to
preserve the identity of each capsule. Then cut open the cap-
sules by means of a suitable clean, dry cutting instrument
such as scissors or a sharp open blade, and remove the con-
tents by washing with a suitable solvent. Allow the occluded
solvent to evaporate from the shells at room temperature
over a period of about 30 min, taking precautions to avoid
uptake or loss of moisture. Weigh the individual shells, and
calculate the net contents. Calculate the drug substance con-
tent in each capsule from the mass of product removed from
the individual capsules and the result of the assay. Calculate
the acceptance value.
Solid dosage forms other than tablets and capsules — Pro-
ceed as directed for Hard Capsules, treating each dosage unit
as described therein. Calculate the acceptance value.
Liquid dosage forms — Accurately weigh the amount of liq-
uid that is removed from each of 10 individual containers in
108 Test for Metal Particles in Ophthalmic Ointments / General Tests
Table 6.02-2.
JP XV
Variable
Definition
Conditions
Value
X
mean of individual contents
(xi, x 2 , " , x n ) expressed as a percentage
of the label claim
X\> %2i '") *n
individual contents of the dosage units
tested, expressed as a percentage of the
label claim
n
sample size (number of dosage units in
a sample)
k
acceptability constant
If « = 10, then
2.4
If « = 30, then
2.0
s
sample standard deviation
ji(x>-x) 2
V n-\
RSD
relative standard deviation (the sample
standard deviation expressed as a per-
centage of the mean)
lOOs
X
M (case 1)
To be applied
when
r< 101.5
reference value
If 98.5% <X< 101.5%,
then
M=X
(AV=ks)
If X< 98.5%, then
M=98.5%
(AV=9S.5-X+ks)
If X> 101.5%, then
M= 101.5%
(AV=X-\0\.5 + ks)
M (case 2)
To be applied
when
T> 101.5
reference value
If 98.5% <X< 7", then
M=X
(A V= ks)
If X< 98.5%, then
M=98.5%
(AV=9S.5-X+ks)
If X> T, then
M= T%
(AV=X-T+ks)
Acceptance
Value (A V)
general formula:
\M-X\ +ks
[Calculations are specified
above for the different cases.]
LI
maximum allowed acceptance value
LI = 15.0 unless otherwise
specified.
LI
maximum allowed range for deviation
of each dosage unit tested from the
calculated value of M
On the low side, no dosage
unit result can be less than
0.75M while on the high
side, no dosage unit result
can be greater than 1.25M
(This is based on an L2
value of 25.0.)
L2 = 25.0 unless otherwise
specified.
T
target test sample amount at time of
manufacture. Unless otherwise specified in
the individual monograph, 7" is 100.0%.
JPXV
General Tests / Test for Acid-neutralizing Capacity of Gastrointestinal Medicines
109
conditions of normal use. Calculate the drug substance con-
tent in each container from the mass of product removed
from the individual containers and the result of the assay.
Calculate the acceptance value.
Calculation of Acceptance Value
Calculate the acceptance value as shown in Content Unifor-
mity, except that *the value of Xis replaced with A, and that
♦ the individual contents of the dosage units are replaced
with the individual estimated contents defined below.
X\,x 2 ■■■x„ = individual estimated contents of the dosage units
tested, where
X\ = W; X
W
w l ,w 1 ---w„ = individual masses of the dosage units tested,
A - content of drug substance (% of label claim) obtained
using an appropriate analytical method.
W=mean of individual masses (w i ,w 2 --, w n ).
CRITERIA
Apply the following criteria, unless otherwise specified.
Solid and Liquid Dosage Forms — The requirements for
dosage uniformity are met if the acceptance value of the first
10 dosage units is less than or equal to Ll%. If the accep-
tance value is greater than Ll%, test the next 20 dosage units
and calculate the acceptance value. The requirements are met
if the final acceptance value of the 30 dosage units is less than
or equal to L\% and no individual content of the dosage unit
is less than (1 -L2x 0.01)M nor more than (1 +Z2x0.01)M
in Calculation of Acceptance Value under Content Uniformi-
ty or under Mass Variation. Unless otherwise specified, L\ is
15.0 andZ,2 is 25.0.
6.03 Particle Size Distribution
Test for Preparations
Particle Size Distribution Test for Preparations is a
method to determine the particle size distribution of the gran-
ules and powders described in General Rules for Prepara-
tions.
Procedure
(1) Granules
The test is performed employing No. 10 (1700 //m), No. 12
(1400 //m), and No. 42 (355 /urn) sieves with the inside di-
ameter of 75 mm.
Weigh accurately 20.0 g of granules to be tested, and place
on the uppermost sieve which is placed on the other sieves
described above and a close-fitting receiving pan and is co-
vered with a lid. Shake the sieves in a horizontal direction for
3 minutes, and tap slightly at intervals. Weigh the amount
remaining on each sieve and in the receiving pan.
(2) Powders
The test is performed employing No. 18 (850 /urn), No. 30
(500 //m), and No. 200 (75 pm) sieves with the inside di-
ameter of 75 mm.
Weigh accurately 10.0 g of powders to be tested, and place
on the uppermost sieve which is placed on the other sieves
described above and a close-fitting receiving pan and is co-
vered with a lid. Shake the sieves in a horizontal direc-tion
for 3 minutes, and tap slightly at intervals. Weigh the amount
remaining on each sieve and in the receiving pan.
6.04 Test for Acid-neutralizing
Capacity of Gastrointestinal
Medicines
Test for Acid-neutralizing Capacity of Gastrointestinal
Medicines is a test to determine the acid-neutralizing capacity
of a medicine, as a crude material or preparation, which
reacts with the stomach acid and exercises an acid control ac-
tion in the stomach. When performing the test according to
the following procedure, the acid-neutralizing capacity of a
crude material is expressed in terms of the amount (mL) of
0.1 mol/L hydrochloric acid VS consumed per g of the
material, and that of a preparation is expressed by the
amount (mL) of 0.1 mol/L hydrochloric acid VS consumed
per dose per day (when the daily dose varies, the minimum
dose is used).
Preparation of sample
A crude material and a solid preparation which conforms
to Powders in the General Rules for Preparations: may be
used, without any treatment, as the sample. Preparations in
dose-unit packages: weigh accurately the content of not less
than 20 packages, calculate the average mass of the content
for a daily dose, mix uniformly, and use the mixture as the
sample. Granules in dose-unit packages and other solid
preparations which do not conform to Powders in the Gener-
al Rules for Preparations: weigh accurately the content of not
less than 20 packages, calculate the average mass of the con-
tent for a daily dose, powder it, and use as the sample. Gran-
ules not in dose-unit packages and other solid preparations
which do not conform to Powders in the General Rules for
Preparations: take not less than 20 doses, powder it, and use
as the sample. Capsules and tablets: take not less than 20
doses, weigh accurately, calculate the average mass for a dai-
ly dose, powder it, and use as the sample. Liquid prepara-
tions: shake well, and use as the sample.
Procedure
Take an amount of the sample so that 'a' in the equation
falls between 20 mL and 30 mL, and perform the test.
Accurately weigh the sample of the crude material or
preparation, and place it in a glass-stoppered, 200-mL flask.
Add exactly 100 mL of 0.1 mol/L hydrochloric acid VS,
stopper tightly, shake at 37 ± 2°C for 1 hour, and filter.
Take precaution against gas to be generated on the addition
of 0.1 mol/L hydrochloric acid VS, and stopper tightly. Af-
ter cooling, filter the solution again, if necessary. Pipet 50 mL
of the filtrate, and titrate <2.50> the excess hydrochloric acid
with 0.1 mol/L sodium hydroxide VS (pH Determination
<2.54>, end point: pH 3.5). Perform a blank determination.
For liquid preparations, pipet the sample in a 100-mL volu-
metric flask, add water to make 45 mL, then add exactly 50
mL of 0.1 mol/L hydrochloric acid VS while shaking. Add
water again to make the solution 100 mL. Transfer the solu-
tion to a glass-stoppered, 200-mL flask, wash the residue with
20.0 mL of water, stopper tightly, shake at 37 ± 2°C for 1
hour, and filter. Pipet 60 mL of the filtrate, and titrate <2.50>
the excess hydrochloric acid with 0.1 mol/L sodium
hydroxide VS (pH Determination <2.54>, end point: pH 3.5).
Perform a blank determination.
Acid-neutralizing capacity (amount of 0.1 mol / L
110
Test for Extractable Volume of Parenteral Preparations / General Tests
JP XV
hydrochloric acid VS consumed per g or daily dose) (mL)
= {b - a)/x 2 x (f/s)
a: Amount (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed
b: Amount (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed in the blank determination
/: The molarity coefficient of 0.1 mol/L sodium hydroxide
VS
t: 1000 mg of crude material or daily dose of preparation
(in mg of solid preparation, mL of liquid preparation)
s: Amount of the sample (in mg of crude material and solid
preparation, mL of liquid preparation)
6.05 Test for Extractable Volume
of Parenteral Preparations
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( 4
►).
*Test for Extractable Volume of Parenteral Preparations
is performed to confirm that a slightly excess volume is filled
for the nominal volume to be withdrawn. Injections may be
supplied in single-dose containers such as ampoules or plastic
bags, or in multi-dose containers filled with a volume of in-
jection which is sufficient to permit administration of the
nominal volume declared on the label. The excess volume is
determined by the characteristics of the product.*
Suspensions and emulsions must be shaken before
withdrawal of the contents and before the determination of
the density. Oily and viscous preparations may be warmed
according to the instructions on the label, if necessary, and
thoroughly shaken immediately before removing the con-
tents. The contents are then cooled to 20 - 25°C before meas-
uring the volume.
(1) Single-dose containers
Select one container if the volume is 10 mL or more, 3 con-
tainers if the nominal volume is more than 3 mL and less than
10 mL, or 5 containers if the nominal volume is 3 mL or less.
Take up individually the total contents of each container
selected into a dry syringe of a capacity not exceeding three
times the volume to be measured, and fitted with a 21-gauge
needle not less than 2.5 cm in length. Expel any air bubbles
from the syringe and needle, then discharge the contents of
the syringe without emptying the needle into a standardised
dry cylinder (graduated to contain rather than to deliver the
designated volumes) of such size that the volume to be meas-
ured occupies at least 40% of its graduated volume. Alterna-
tively, the volume of the contents in milliliters may be calcu-
lated as the mass in grams divided by the density.
For containers with a nominal volume of 2 mL or less the
contents of a sufficient number of containers may be pooled
to obtain the volume required for the measurement provided
that a separate, dry syringe assembly is used for each contain-
er. The contents of containers holding 10 mL or more may be
determined by opening them and emptying the contents di-
rectly into the graduated cylinder or tared beaker.
The volume is not less than the nominal volume in case of
containers examined individually, or, in case of containers
with a nominal volume of 2 mL or less, is not less than the
sum of the nominal volumes of the containers taken collec-
tively.
(2) Multi-dose containers
For injections in multidose containers labeled to yield a
specific number of doses of a stated volume, select one con-
tainer and proceed as directed for single-dose containers us-
ing the same number of separate syringe assemblies as the
number of doses specified. The volume is such that each
syringe delivers not less than the stated dose.
(3) Cartridges and prefilled syringes
Select one container if the volume is 10 mL or more, 3 con-
tainers if the nominal volume is more than 3 mL and less than
10 mL, or 5 containers if the nominal volume is 3 mL or less.
If necessary, fit the containers with the accessories required
for their use (needle, piston, syringe) and transfer the entire
contents of each container without emptying the needle into a
dry tared beaker by slowly and constantly depressing the
piston. Determine the volume in milliliters calculated as the
mass in grams divided by the density.
The volume measured for each of the containers is not less
than the nominal volume.
(4) Parenteral infusions
Select one container. Transfer the contents into a dry
measuring cylinder of such a capacity that the volume to be
determined occupies at least 40% of the nominal volume of
the cylinder. Measure the volume transferred.
The volume is not less than the nominal volume.
6.06 Foreign Insoluble Matter
Test for Injections
Foreign Insoluble Matter Test for Injections is a test
method to examine foreign insoluble matters in injections.
Method 1. This method is applied to injections either in so-
lutions, or in solution constituted from sterile drug solids.
Clean the exterior of containers, and inspect with the un-
aided eyes at a position of light intensity of approximately
1000 lx under an incandescent lamp: Injections must be clear
and free from readily detectable foreign insoluble matters. As
to Injections in plastic containers for aqueous injections, the
inspection should be performed with the unaided eyes at a
position of light intensity of approximately 8000 to 10,000 lx,
with an incandescent lamp at appropriate distances above
and below the container.
Method 2. This method is applied to injections with con-
stituted solution.
Clean the exterior of containers, and dissolve the contents
with constituted solution or with water for injection careful-
ly, avoiding any contamination with extraneous foreign sub-
stances. The solution thus constituted must be clear and free
from foreign insoluble matters that is clearly detectable when
inspected with the unaided eyes at a position of light intensity
of approximately 1000 lx, right under an incandescent lamp.
JPXV
General Tests / Insoluble Particulate Matter Test for Injections
111
6.07 Insoluble Particulate Matter
Test for Injections
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* ♦).
The insoluble particulate matter test for injections or infu-
sions is the method to test for insoluble particulates, to con-
firm that they are not present in excess of specified levels in
the solutions. For the determination of particulate contami-
nation 2 procedures, Method 1 (Light Obscuration Particle
Count Test) and Method 2 (Microscopic Particle Count
Test), are specified hereinafter. When examining injections
and parenteral infusions for sub-visible particles Method 1 is
preferably applied. However, it may be necessary to test
some preparations by Method 2 followed by Method 1 to
reach a conclusion on conformance to the requirements.
Not all parenteral preparations can be examined for sub-
visible particles by one or both of these methods. When
Method 1 is not applicable, e.g. in case of preparations hav-
ing reduced clarity or increased viscosity, the test should be
carried out according to Method 2. Emulsions, colloids, and
liposomal preparations are examples. Similarly, products
that produce air or gas bubbles when drawn into the sensor
may also require microscopic particle count testing. If the vis-
cosity of the preparation to be tested is sufficiently high so as
to preclude its examination by either test method, a quantita-
tive dilution with an appropriate diluent may be made to
decrease viscosity, as necessary, to allow the analysis to be
performed.
The results obtained in examining a discrete unit or group
of units for particulate contamination cannot be extrapolated
with certainty to other units that remain untested. Thus,
statistically sound sampling plans must be developed if valid
inferences are to be drawn from observed data to characterise
the level of particulate contamination in a large group of
units.
Method 1. Light Obscuration Particle Count Test
Use a suitable apparatus based on the principle of light
blockage which allows an automatic determination of the size
of particles and the number of particles according to size. *It
is necessary to perform calibration, as well as to demonstrate
the sample volume accuracy, sample flow rate, particle size
response curve, sensor resolution, and counting accuracy, at
least once a year.»
* Calibration
Particles to be used for calibration should be subject to
particle size sensitivity measurement, using spherical polysty-
rene particles having at least 5, 10 and 25 /um in diameter
(PSL particles) in mono-dispersed suspension. The PSL par-
ticles should have either a domestic or international traceabil-
ity in terms of length, with a level of uncertainly at not great-
er than 3%. The particles to be used for calibration should be
dispersed in particle-free water.
Manual method
The particle size response of the system to be applied
should be determined using at least 3 channels for threshold-
voltage setting, according to the half counting method of
window moving type. The threshold-voltage window should
be ±20% of the measuring particle size. After measuring the
sensitivity of response for the designated particle size, the size
response curve is prepared by the method indicated by the
manufacturer from particle-response measuring point, and
threshold-voltage of 5, 10 and 25 /xm of the apparatus is ob-
tained.
Electronic method
In the use of multichannel peak height analyzer, the parti-
cle size response is measured by half-count method of moving
window system same as the manual method, and the particle
size response curve is prepared by the method designated by
the instrument manufacturer, then, the threshold-voltage of
5,10 and 25 /xm of the apparatus is obtained. In this case, the
instrument manufacturer or the user should validate the
obtainability of the same result as that of the manual
method.
Automated method
The particle size response curve of the apparatus may be
obtained by using the software developed by the user or sup-
plied by the instrument manufacturer, whereas, the manufac-
turer or the user should validate the obtainability of the same
result as that of the manual method.
Sample volume accuracy
Sample volume accuracy should fall within 5% of the
measuring value in case measuring the decrease of test solu-
tion by the mass method after measuring the test solution of
10 mL.
Sample flow rate
The flow rate of the sample indicated into the sensor
should be calculated from the observed sample volume and
time, and should be conformed within the range of the
manufacturer's specification for sensor used.
Sensor
There is a possibility of changes of particle size resolution
and counting rate of particle-detecting sensor in each sensor
by assembling accuracy and parts accuracy even in the same
type sensor. The threshold accuracy also needs to be con-
firmed. Testing should accordingly be performed for each of
particle size resolution, accuracy in counting and in threshold
setting, using Particle Count Reference Standard Suspension
(PSL spheres having mean diameter of approximately 10 /xm,
of a concentration at 1000 particles/mL ±10%, not more
than 5% of CV value).
During measurement, stirring should be made for assuring
the uniformity in sample concentration.
Sensor resolution (Particle size resolution of apparatus)
Measurement should be made by either one of the follow-
ing methods.
1. Manual method to obtain the spread of histogram pre-
pared from the counting value of the apparatus.
2. Electronic method to obtain the spread of histogram of
the classification of system-responding signal by using the
multichannel peak height analyzer.
3. Automated method to obtain the spread of histogram of
responsive signal of the test-particle by using the software
prepared by the manufacturer or the user.
The difference between the threshold of particle size count-
ing 16 and 84% of the total counts and the test-particle size
should be within 10%, whereas, electronic method and auto-
mated method should be both validated for obtaining the
1 12 Insoluble Particulate Matter Test for Injections / General Tests
JP XV
same result as that of the manual method.
Particle counting accuracy
Data obtained by counting particles of 5 /xm and greater
should be 763 to 1155 particles per 1 mL.
Threshold accuracy
Particle size calculated from a threshold corresponding to
50% counts for particles of 5 /xm and greater should fall wi-
thin ±5% of the mean diameter of the test particles.
Reagents
Particle-free water: The purified water containing not more
than 5 particles of 10 /um or greater size, and not more than 2
particles of 25 /xm or greater size in 10 mL of the insoluble
particle number measured by the light obscuration particle
counter. ♦
General precautions
The test is carried out under conditions limiting particulate
contamination, preferably in a laminar-flow cabinet.
Very carefully wash the glassware and filtration equipment
used, except for the membrane filters, with a warm detergent
solution and rinse with abundant amounts of water to re-
move all traces of detergent. Immediately before use, rinse
the equipment from top to bottom, outside and then inside,
with particle-free water.
Take care not to introduce air bubbles into the preparation
to be examined, especially when fractions of the preparation
are being transferred to the container in which the determina-
tion is to be carried out.
In order to check that the environment is suitable for the
test, that the glassware is properly cleaned and that the water
to be used is particle-free, the following test is carried out: de-
termine the particulate contamination of 5 samples of par-
ticle-free water, each of 5 mL, according to the method de-
scribed below. If the number of particles of 10 /xm or greater
size exceeds 25 for the combined 25 mL, the precautions
taken for the test are not sufficient. The preparatory steps
must be repeated until the environment, glassware and water
are suitable for the test.
Method
Mix the contents of the sample by slowly inverting the con-
tainer 20 times successively. If necessary, cautiously remove
the sealing closure. Clean the outer surfaces of the container
opening using a jet of particle-free water and remove the
closure, avoiding any contamination of the contents.
Eliminate gas bubbles by appropriate measures such as allow-
ing to stand for 2 min or sonicating.
For large-volume parenterals or for small-volume paren-
terals having a volume of 25 mL or more, single units are test-
ed. For small-volume parenterals less than 25 mL in volume,
the contents of 10 or more units are combined in a cleaned
container to obtain a volume of not less than 25 mL; where
justified and authorised, the test solution may be prepared by
mixing the contents of a suitable number of vials and diluting
to 25 mL with particle-free water or with an appropriate sol-
vent without contamination of particles when particle-free
water is not suitable.
Powders for parenteral use are reconstituted with particle-
free water or with an appropriate solvent without contamina-
tion of particles when particle-free water is not suitable.
The number of test specimens must be adequate to provide
a statistically sound assessment. For large-volume parenterals
Cralicule field of view circle
S *- Reference circle
Cross hairs
|l|!lll||||||l|j!ll|||||||||||lll||ll|||||||llll!ll|ll| |IN|ni|!||i|l| III ll| lll!llll| linear scale
Fig. 6.07-1 Circular diameter graticule
or for small-volume parenterals having a volume of 25 mL or
more, fewer than 10 units may be tested, based on an ap-
propriate sampling plan.
Remove 4 portions, each of not less than 5 mL, and count
the number of particles equal to or greater than 10 /xm and 25
/um. Disregard the result obtained for the first portion, and
calculate the mean number of particles for the preparation to
be examined.
Evaluation
If the average number of particles exceeds the limits, test
the preparation by Method 2 (Microscopic Particle Count
Test).
Test LA — Solutions for parenteral infusion or solutions for
injection supplied in containers with a nominal content of
*equal to or* more than 100 mL
The preparation complies with the test if the average number
of particles present in the units tested does not exceed 25 per
milliliter equal to or greater than 10 /xm and does not exceed 3
per milliliter equal to or greater than 25 ^m.
Test LB — Solutions for parenteral infusion or solutions for
injection supplied in containers with a nominal content of
less than 100 mL.
The preparation complies with the test if the average number
of particles present in the units tested does not exceed 6000
per container equal to or greater than 10 /um and does not ex-
ceed 600 per container equal to or greater than 25 /um.
Method 2. Microscopic Particle Count Test
Use a suitable binocular microscope, filter assembly for
retaining particulate contamination and membrane filter for
examination.
The microscope is equipped with an ocular micrometer
calibrated with an objective micrometer, a mechanical stage
capable of holding and traversing the entire filtration area of
the membrane filter, two suitable illuminators to provide
episcopic illumination in addition to oblique illumination,
and is adjusted to 100±10 magnifications. The ocular
micrometer is a circular diameter graticule (see Fig. 6.07-1)
and consists of a large circle divided by crosshairs into quad-
rants, transparent and black reference circles 10 /xm and 25
/xm in diameter at 100 magnifications, and a linear scale grad-
uated in 10 /xm increments. It is calibrated using a stage
micrometer that is certified by either a domestic or interna-
JPXV
General Tests / Insoluble Particulate Matter Test for Ophthalmic Solutions
113
tional standard institution. A relative error of the linear scale
of the graticule within ±2 per cent is acceptable. The large
circle is designated the graticule field of view (GFOV).
Two illuminators are required. One is an episcopic bright-
field illuminator internal to the microscope, the other is an
external, focussable auxiliary illuminator adjustable to give
reflected oblique illumination at an angle of 10° to 20°.
The filter assembly for retaining particulate contamination
consists of a filter holder made of glass or other suitable
material, and is equipped with a vacuum source and a suita-
ble membrane filter.
The membrane filter is of suitable size, black or dark gray
in color, non-gridded or gridded, and 1.0 fim or finer in
nominal pore size.
General precautions
The test is carried out under conditions limiting particulate
contamination, preferably in a laminar-flow cabinet.
Very carefully wash the glassware and filter assembly used,
except for the membrane filter, with a warm detergent solu-
tion and rinse with abundant amounts of water to remove all
traces of detergent. Immediately before use, rinse both sides
of the membrane filter and the equipment from top to bot-
tom, outside and then inside, with particle-free water.
In order to check that the environment is suitable for the
test, that the glassware and the membrane filter are properly
cleaned and that the water to be used is particle-free, the fol-
lowing test is carried out: determine the particulate contami-
nation of a 50 mL volume of particle-free water according to
the method described below. If more than 20 particles 10 //m
or larger in size or if more than 5 particles 25 /um or larger in
size are present within the filtration area, the precautions
taken for the test are not sufficient. The preparatory steps
must be repeated until the environment, glassware, mem-
brane filter and water are suitable for the test.
Method
Mix the contents of the samples by slowly inverting the
container 20 times successively. If necessary, cautiously re-
move the sealing closure. Clean the outer surfaces of the con-
tainer opening using a jet of particle-free water and remove
the closure, avoiding any contamination of the contents.
For large-volume parenterals, single units are tested. For
small-volume parenterals less than 25 mL in volume, the con-
tents of 10 or more units is combined in a cleaned container;
where justified and authorised, the test solution may be pre-
pared by mixing the contents of a suitable number of vials
and diluting to 25 mL with particle-free water or with an ap-
propriate solvent without contamination of particles when
particle-free water is not suitable. Small-volume parenterals
having a volume of 25 mL or more may be tested individual-
ly.
Powders for parenteral use are constituted with particle-
free water or with an appropriate solvent without contamina-
tion of particles when particle-free water is not suitable.
The number of test specimens must be adequate to provide
a statistically sound assessment. For large-volume parenterals
or for small-volume parenterals having a volume of 25 mL or
more, fewer than 10 units may be tested, based on an ap-
propriate sampling plan.
Wet the inside of the filter holder fitted with the membrane
filter with several milliliter of particle-free water. Transfer to
the filtration funnel the total volume of a solution pool or of
a single unit, and apply vacuum. If needed add stepwise a
portion of the solution until the entire volume is filtered. Af-
ter the last addition of solution, begin rinsing the inner walls
of the filter holder by using a jet of particle-free water. Main-
tain the vacuum until the surface of the membrane filter is
free from liquid. Place the filter in a petri dish and allow the
filter to air-dry with the cover slightly ajar. After the filter has
been dried, place the petri dish on the stage of the micro-
scope, scan the entire membrane filter under the reflected
light from the illuminating device, and count the number of
particles that are equal to or greater than 10 /um and the num-
ber of particles that are equal to or greater than 25 ^m. Alter-
natively, partial filter count and determination of the total
filter count by calculation is allowed. Calculate the mean
number of particles for the preparation to be examined.
The particle sizing process with the use of the circular di-
ameter graticule is carried out by transforming mentally the
image of each particle into a circle and then comparing it to
the 10 /um and 25 /um graticule reference circles. Thereby the
particles are not moved from their initial locations within the
graticule field of view and are not superimposed on the refer-
ence circles for comparison. The inner diameter of the trans-
parent graticule reference circles is used to size white and
transparent particles, while dark particles are sized by using
the outer diameter of the black opaque graticule reference
circles.
In performing the microscopic particle count test (Method
2) do not attempt to size or enumerate amorphous, semi-liq-
uid, or otherwise morphologically indistinct materials that
have the appearance of a stain or discoloration on the mem-
brane filter. These materials show little or no surface relief
and present a gelatinous or film-like appearance. In such
cases the interpretation of enumeration may be aided by test-
ing a sample of the solution by Method 1.
Evaluation
Test 2. A — Solutions for parenteral infusion or solutions for
injection supplied in containers with a nominal content of
*equal to or» more than 100 mL
The preparation complies with the test if the average number
of particles present in the units tested does not exceed 12 per
milliliter equal to or greater than 10 /um and does not exceed 2
per milliliter equal to or greater than 25 /um.
Test 2.B — Solutions for parenteral infusion or solutions for
injection supplied in containers with a nominal content of
less than 100 mL
The preparation complies with the test if the average number
of particles present in the units tested does not exceed 3000
per container equal to or greater than 10 ^m and does not ex-
ceed 300 per container equal to or greater than 25 /um.
6.08 Insoluble Particulate Matter
Test for Ophthalmic Solutions
Insoluble Particulate Matter Test for Ophthalmic Solu-
tions is to examine for the size and the number of insoluble
particulate matter in Ophthalmic Solutions.
Apparatus
Use a microscope, filter assembly for retaining insoluble
particulate matter and membrane filter for determination.
Microscope: The microscope is equipped with a microme-
ter system, a mobile stage and an illuminator, and is adjusted
114
Disintegration Test / General Tests
JP XV
to 100 magnifications.
Filter assembly for retaining insoluble particulate matter:
The filter assembly for retaining insoluble particulate matter
consists of a filter holder made of glass or a proper material
uncapable of causing any trouble in testing, and a clip. The
unit is capable of fitting with a membrane filter 25 mm or 13
mm in diameter and can be used under reduced pressure.
Membrane filter for testing: The membrane filter is white in
color, 25 mm or 13 mm in diameter, not more than 10 fim in
nominal pore size and is imprinted with about 3 mm grid
marks. Upon preliminary testing, the insoluble particulate
matter equal to or greater than 25 /xm in size should not be
found on the filter. When necessary, wash the filter with puri-
fied water for particulate matter test.
Reagents
Purified water for particulate matter test: Purified water
which contains not more than 10 particles of 10 /urn or greater
size in 100 mL. Prepare before use by filtering through a
membrane filter with a nominal pore size of 0.5 ^m or less.
Procedure
Aqueous ophthalmic solutions
Carry out all operations carefully in clean equipment and
facilities which are low in dust. Fit the membrane filter onto
the membrane filter holder, and fix them with the clip.
Thoroughly rinse the holder inside with the purified water for
particulate matter test, and filter under reduced pressure with
200 mL of the purified water for particulate matter test at a
rate of 20 to 30 mL per minute. Apply the vacuum until the
surface of the membrane filter is free from water, and remove
the membrane filter. Place the filter in a flat-bottomed petri
dish with the cover slightly ajar, and dry the filter fully at a
temperature not exceeding 50 °C. After the filter has been
dried, place the petri dish on the stage of the microscope. Un-
der a down-light from an illuminating device, adjust the grid
of the membrane filter to the coordinate axes of the micro-
scope, adjust the microscope so as to get the best view of the
insoluble particulate matter, then count the number of parti-
cles that are equal to or greater than 150 /um within the effec-
tive filtering area of the filter, moving the mobile stage, and
ascertain that the number is not more than 1. In this case the
particle is sized on the longest axis.
Fit another membrane filter to the filtration device, and fix
them with the clip, then wet the inside of the filter holder with
several mL of purified water for particulate matter test. Clean
the outer surface of the container, and mix the sample solu-
tion gently by inverting the container several times. Remove
the cap, clean the outer surface of the nozzle, and pour the
sample solution into a measuring cylinder which has been
rinsed well with purified water for particulate matter test.
Repeat the process to prepare 25 mL of the test solution.
Pour the test solution into the filter holder along the inner
wall of the holder. Apply the vacuum and filter mildly so as
to keep the solution always on the filter. As for viscous sam-
ple solution, dilute suitably with purified water for particu-
late matter test or suitable diluent and then filter as described
above. When the amount of the solution on the filter becomes
small, add 30 mL of purified water for particulate matter test
or suitable diluent in such manner as to wash the inner wall of
the filter holder. Repeat the process 3 times with 30 mL of the
water. Apply the vacuum gently until the surface of the mem-
brane filter is free from water. Place the filter in a petri dish,
and dry the filter at a temperature below 50°C with the cover
slightly ajar. After the filter has been dried, place the petri
dish on the stage of the microscope, and count the number of
particles which are equal to or larger than 300 //m within the
effective filtering area of the filter according to the same
procedure of the microscope as described above. In this case
the particle is sized on the longest axis.
Ophthalmic solutions which are dissolved before use
Proceed as directed in Aqueous Ophthalmic Solutions
after dissolving the sample with the constituted solution.
Suspension type ophthalmic solutions
Proceed as directed in Aqueous Ophthalmic Solutions.
Take 25 mL of the sample in a vessel, which has been rinsed
well with purified water for particulate matter test, add a suit-
able amount of a suspension-solubilizing solvent or an ade-
quate solvent, shake to dissolve the suspending particles, and
use this solution as the sample solution. Use a membrane
filter which is not affected by the solvent to be used.
Ophthalmic solutions contained in a single-dose container
Proceed as directed in Aqueous Ophthalmic Solutions,
using 10 samples for the test. A 13-mm diameter membrane
filter and a 4-mm diameter filter holder for retaining insoluble
particulate matter are used.
6.09 Disintegration Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* »).
Disintegration Test is provided to determine whether
tablets, capsules, *granules or pills*, disintegrate within the
prescribed time when placed in a liquid medium at the ex-
perimental conditions presented below.
For the purposes of this test, disintegration does not imply
complete solution of the unit or even of its active constituent.
Apparatus
The apparatus consists of a basket-rack assembly, a
1000-mL, low-form beaker, 138 to 160 mm in height and
having an inside diameter of 97 to 115 mm for the immersion
fluid, a thermostatic arrangement for heating the fluid be-
tween 35° and 39°, and a device for raising and lowering the
basket in the immersion fluid at a constant frequency rate be-
tween 29 and 32 cycles per minute through a distance of not
less than 53 mm and not more than 57 mm. The volume of
the fluid in the vessel is such that at the highest point of the
upward stroke the wire mesh remains at least 15 mm below
the surface of the fluid and descends to not less than 25 mm
from the bottom of the vessel on the downward stroke. At no
time should the top of the basket-rack assembly become sub-
merged. The time required for the upward stroke is equal to
the time required for the downward stroke, and the change in
stroke direction is a smooth transition, rather than an abrupt
reversal of motion. The basket-rack assembly moves vertical-
ly along its axis. There is no appreciable horizontal motion or
movement of the axis from the vertical.
Basket-rack assembly — The basket-rack assembly consists
of six open-ended transparent tubes, each 77.5 ±2.5 mm long
and having an inside diameter of 20.7 to 23 mm and a wall
1.0 to 2.8 mm thick; the tubes are held in a vertical position
by two plates, each 88 to 92 mm in diameter and 5 to 8.5 mm
JPXV
General Tests / Disintegration Test
115
Basket-rack assembly
Disk
M±02
■ Top view
L -£±01
Side view
Bottom view
All dimensions are expressed in ram.
Fig. 6.09-1 Disintegration apparatus
in thickness, with six holes, each 22 to 26 mm in diameter, e-
quidistant from the center of the plate and equally spaced
from one another. Attached to the under surface of the lower
plate is a woven stainless steel wire cloth, which has a plain
square weave with 1.8- to 2.2-mm apertures and with a wire
diameter of 0.57 to 0.66 mm. The parts of the apparatus are
assembled and rigidly held by means of three bolts passing
through the two plates. A suitable means is provided to sus-
pend the basket-rack assembly from the raising and lowering
device using a point on its axis. The basket-rack assembly
conforms to the dimensions found in Fig. 6.09-1. The design
of the basket-rack assembly may be varied somewhat pro-
vided the specifications for the glass tubes and the screen
mesh size are maintained: *for example, in order to secure
the glass tubes and the upper and the lower plastic plates in
position at the top or the bottom, an acid-resistant metal
plate, 88 - 92 mm in diameter and 0.5 - 1 mm in thickness,
having 6 perforations, each about 22 to 26 mm in diameter,
may be used which coincide with those of the upper plastic
plate and upper open ends of the glass tubes. ♦
Disks — The use of disks is permitted only where specified
or allowed. Each tube is provided with a cylindrical disk 9.5
±0.15 mm thick and 20.7 ±0.1 5 mm in diameter. The disk is
made of a suitable, transparent plastic material having a
specific gravity of between 1.18 and 1 .20. Five parallel 2 ± 0. 1
mm holes extend between the ends of the cylinder. One of the
holes is centered on the cylindrical axis. The other holes are
centered 6 ± 0.2 mm from the axis on imaginary lines perpen-
dicular to the axis and parallel to each other. Four identical
trapezoidal-shaped planes are cut into the wall of the cylin-
der, nearly perpendicular to the ends of the cylinder. The
trapezoidal shape is symmetrical; its parallel sides coincide
with the ends of the cylinder and are parallel to an imaginary
line connecting the centers of two adjacent holes 6 mm from
the cylindrical axis. The parallel side of the trapezoid on the
bottom of the cylinder has a length of 1.6 ±0.1 mm, and its
bottom edges lie at a depth of 1.6 ±0.1 mm from the cylin-
der's circumference. The parallel side of the trapezoid on the
top of the cylinder has a length of 9.4 ±0.2 mm, and its cen-
ter lies at a depth of 2.6 ± 0. 1 mm from the cylinder's circum-
2.5-4.5
2.5-4.5
12 ±0.2
A : Plastic ring
B: Acid-resistant wire gauze; openings: 0.42
mm; wire diameter: 0.29 mm
C: Acid-resistant wire handle
D: Plastic tube
All dimensions are expressed in mm.
>Fig. 6.09-2 Auxiliary tube*
ference. All surfaces of the disk are smooth. If the use of dis-
ks is specified, add a disk to each tube, and operate the ap-
paratus as directed under Procedure. The disks conform to
dimensions found in Fig. 6.09-1. The use of automatic detec-
tion employing modified disks is permitted where the use of
disks is specified or allowed. Such disks must comply with the
requirements for density and dimension given in this chapter.
♦Auxiliary tube — The auxiliary tube, as illustrated in Fig.
6.09-2, consists of a plastic tube D, 12 ±0.2 mm in inside di-
ameter, 17 ±0.2 mm in outside diameter, 20 ± lmm in
length, having both outside ends screw-cut, and two plastic
rings A, each 12 ± 0.2 mm in inside diameter, 17 ± 0.2 mm in
outside diameter, 2.5 - 4.5 mm in length, having one inside
end screw-cut. Acid-resistant woven wire gauze having
0.42-mm openings and 0.29-mm wire diameter is placed in
each plastic ring and the rings are attached by screws to each
end of the plastic tube. The distance between two wire gauzes
is 20 ± 1 mm. A handle of an acid-resistant wire, 1 mm in di-
ameter and 80 ±5 mm in length, is attached to the mid por-
tion of the plastic tube. The auxiliary tube is used for the test
of granules and capsules containing enteric coated granules. ♦
Procedure
1) Immediate-release preparation
In case of tablets, capsules *and pills (except for pills con-
taining crude drugs), ♦ place 1 dosage unit in each of the six
tubes of the basket, and if prescribed add a disk. *Unless
otherwise specified, operate the apparatus, using water as the
immersion fluid, » maintained at 37±2°C as the immersion
fluid. *Unless otherwise specified, carry out the test for 20
minutes for capsules, 30 minutes for plain tablets, and 60
minutes for coated tablets and pills. » Lift the basket from
the fluid, and observe the dosage units. * Complete disin-
tegration is defined as that state in which any residue of the
unit, except fragments of insoluble coating or capsule shell,
remaining on the screen of the test apparatus or adhering to
the lower surface of the disks, if used, is a soft mass having
no palpably firm core.» The test is met if all of the dosage u-
nits have disintegrated completely. If 1 or 2 dosage units fail
to disintegrate, repeat the test on 12 additional dosage units.
The test is met if not less than 16 of the total of 18 dosage
units tested are disintegrated.
*For pills containing crude drugs, carry out the test for 60
116
Dissolution Test / General Tests
JP XV
minutes in the same manner, using 1st fluid for disintegration
test as the immersion fluid. When any residue of the unit is
observed, carry out the test successively for 60 minutes, using
2nd fluid for disintegration test.*
*In case of granules, shake granules on a No. 30 (500 /am)
sieve as directed in (1) Granules under Particle Size Distribu-
tion Test for Preparations <6.03>, transfer 0.10 g of the
residue on the sieve to each of the 6 auxiliary tubes, secure the
6 tubes to the bottom of the basket tightly, and operate the
apparatus, using water as the immersion fluid, maintained at
37±2°C as the immesion fluid, unless otherwise specified.
Observe the samples after 30 minutes of operation for plain
granules and after 60 minutes for coated granules, unless
otherwise specified. Complete disintegration is defined as that
state in which any residue of the granules, except fragments
of insoluble coating in the auxiliary tube, is a soft mass hav-
ing no palpably firm core. The test is met if all of 6 samples in
the auxiliary tubes have disintegrated completely. If 1 or 2
samples fail to disintegrate, repeat the test on 12 additional
samples. The test is met if not less than 16 of the total of 18
samples tested are disintegrated.*
♦2) Enteric coated preparations
Unless otherwise specified, perform the following two
tests, (a) the test with 1st fluid for disintegration test and (b)
the test with the 2nd fluid for disintegration test, separately.
(1) Enteric coated tablet and capsule
(a) The test with 1st fluid for disintegration test
Carry out the test for 120 minutes, using 1st fluid for disin-
tegration test according to the procedure described in im-
mediate release preparations. Disintegration is defined as that
state in which the tablet or capsule is broken or the enteric
coating film is ruptured or broken. The test is met if none of
six dosage units is disintegrated. If 1 or 2 dosage units are dis-
integrated, repeat the test on additional 12 dosage units. The
test is met if not less than 16 of the total of 18 dosage units
tested are not disintegrated.
(b) The test with 2nd fluid for disintegration test
According to the procedure described in immediate-release
preparations, carry out the test with new dosage units for 60
minutes, using 2nd fluid for disintegration test and determine
if the test is met or not.
(2) Enteric coated granules and capsules containing the
enteric coated granules
Shake granules or contents taken out from capsules on a
No. 30 (500 /im) sieve as directed in (1) Granules under Parti-
cle Size Distribution Test for Preparations <6.03>, transfer
0.10 g of the residue on the sieve to each of the 6 auxiliary
tubes, secure the 6 tubes to the bottom of the basket tightly,
and operate the apparatus, using the 1st and 2nd fluids for
disintegration test.
(a) The test with 1st fluid for disintegration test
According to the procedure described in immediate-release
preparations, carry out the test for 60 minutes, using 1st fluid
for disintegration test. The test is met if particles fallen from
the openings of the wire gauze number not more than 15.
(b) The test with 2nd fluid for disintegration test
According to the procedure described in immediate-release
preparations, carry out the test with new samples for 30
minutes, using 2nd fluid for disintegration test and determine
if test is met or not.*
6.10 Dissolution Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols (* *).
Dissolution Test is provided to determine compliance with
the dissolution requirements for dosage forms administered
orally. *This test also aims at preventing significant bioin-
equivalence.* In this test, a dosage unit is defined as 1 tablet
or 1 capsule or the amount specified equivalent to minimum
dose.
Apparatus
Apparatus for Basket Method (Apparatus 1) — The assem-
bly consists of the following: a vessel, which may be covered,
made of glass or other inert, transparent material* 1 ; a motor;
a drive shaft; and a cylindrical basket. The vessel is partially
immersed in a suitable water bath of any convenient size or
heated by a suitable device such as a heating jacket. The
water bath or heating device permits holding the temperature
inside the vessel at 37±0.5°C during the test and keeping the
bath fluid in constant, smooth motion. No part of the assem-
bly, including the environment in which the assembly is
placed, contributes significant motion, agitation, or vibration
beyond that due to the smoothly rotating stirring element.
Make the apparatus to permit observation of the specimen
and stirring element during the test. The vessel is cylindrical,
with a hemispherical bottom and a capacity of 1 liter. Its
height is 160 mm to 210 mm and its inside diameter is 98 mm
to 106 mm. Its sides are flanged at the top. Use a fitted cover
to retard evaporation.* 2 The shaft is positioned so that its
axis is not more than 2 mm at any point from the vertical axis
of the vessel and rotates smoothly and without significant
wobble that could affect the results. Adjust a speed-regulat-
ing device to maintaine the shaft rotation speed at a specified
rate, within ±4%.
Shaft and basket components of the stirring element shown
in Fig. 6.10-1 are fabricated of stainless steel (SUS316) or
other inert material. A basket having a gold coating of about
0.0001 inch (2.5 //m) thick may be used. The dosage unit is
placed in a dry basket at the beginning of each test. The dis-
tance between the inside bottom of the vessel and the bottom
of the basket is maintained at 25 ±2 mm during the test.
Apparatus for Paddle Method (Apparatus 2) — Use the as-
sembly from Apparatus 1 , except that a paddle formed from
a blade and a shaft is used as the stirring element. The shaft is
positioned so that its axis is not more than 2 mm from the
vertical axis of the vessel, at any point, and rotates smoothly
without significant wobble that could affect the results. The
vertical center line of the blade passes through the axis of the
shaft so that the bottom of the blade is flush with the bottom
of the shaft. The paddle conforms to the specifications shown
in Fig. 6.10-2. The distance of 25 ±2 mm between the bottom
of the blade and the inside bottom of the vessel is maintained
during the test. The metallic or suitably inert, rigid blade and
shaft comprise a single entity. A suitable two-part detachable
design may be used provided the assembly remains firmly en-
gaged during the test. The paddle blade and shaft may be
coated with a suitable coating so as to make them inert. The
dosage unit is allowed to sink to the bottom of the vessel be-
fore rotation of the blade is started. *When specified in the
individual monograph,* a small, loose piece of nonreactive
JPXV
General Tests / Dissolution Test
117
Vein hole
<j> 2.0 ±0.5
Retention spring with 'A clasps
opened on 120 centers
Clc
ii izu centers -v \
Cli'ar open i ng [r-f 1
20.2±1.G " It*
— <t> 6.3 to 6.5 or 9.4 to 10.1
m
5.1 ±0.5
-Screen OD 222 ±1.0
25.0 ±3.0
All dimensions arc expressed in mm.
$=diameter
A: Note-Maximum allowable runout at "A" is ±1.0 mm when the part
is rotated on center line axis will) basket mounted.
B: Screen with welded seam, 0,25-0.31 mm wire diameter with wire
openings of 0,36-0.44 mm [Note-After welding, the screen may be
slightly altered.]
_ ; __ 4 9.4-io.t
rati! us 1.2 ±0.2
1
All dimensions are expressed in mm.
0=diameter
Notes:
(1) A and B dimensions are not to vary more than 0.5 mm when part
is rotated on centering axis.
(2) Tolerances are ±1.0 mm unless otherwise stated.
Fig. 6.10-2 Apparatus 2, Paddle stirring element
Fig. 6.10-1 Apparatus 1, Basket stirring element
material, such as not more than a few turns of wire helix,
may be attached to dosage units that would otherwise float.
An alternative sinker device is shown in Fig. 6.10-2a. Other
validated sinker devices may be used.
Apparatus for Flow-Through Cell Method (Apparatus
3) — The assembly consists of a reservoir and a pump for the
dissolution medium; a flow-through cell; a water bath that
maintains the dissolution medium at 37±0.5°C. Use the cell
size specified in the individual monograph.
The pump forces the dissolution medium upwards through
the flow-through cell. The pump has a delivery range between
4 and 16 mL per minute, with standard flow rates of 4, 8, and
16 mL per minute. It must deliver a constant flow (±5 per
cent of the nominal flow rate); the flow profile should be
sinusoidal with a pulsation of 120 ± 10 pulses per minute. *A
pump without the pulsation can also be used.*
The flow-through cell (see Figures 6.10-3 and 6.10-4), of
transparent and inert material, is mounted vertically with a
filter system (specified in the individual monograph) that pre-
vents escape of undissolved particles from the top of the cell;
standard cell diameters are 12 and 22.6 mm; the bottom cone
is usually filled with small glass beads of about 1-mm di-
ameter with one bead of about 5 mm positioned at the apex
to protect the fluid entry tube; a tablet holder (see Figures
6.10-3 and 6.10-4) is available for positioning of special
dosage forms. The cell is immersed in a water bath, and the
temperature is maintained at 37±0.5°C.
The apparatus uses a clamp mechanism of two O-rings to
assemble the cell. The pump is separated from the dissolution
3.5 - 4.0
A 3.0-3.5 B
3.5-4.0
3,5-4.0
12.0 ±0.2
All dimensions are expressed in mm.
A: Acid-resistant wire clasp
B: Acid-resistant wire support
Fig. 6.10-2a Alternative sinker
unit in order to shield the latter against any vibrations
originating from the pump. The position of the pump should
not be on a level higher than the reservoir flasks. Tube con-
nections are as short as possible. Use suitably inert tubing,
such as polytef, with about 1.6-mm inner diameter and inert
flanged-end connections.
Apparatus Suitability — The determination of suitability of
a test assembly to perform dissolution testing must include
conformance to the dimensions and tolerances of the appara-
tus as given above. In addition, critical test parameters that
have to be monitored periodically during use include volume
and temperature of the dissolution medium, rotation speed
(Basket Method and Paddle Method), and flow rate of medi-
um (Flow-Through Cell Method).
Determine the acceptable performance of the dissolution
118
Dissolution Test / General Tests
-A
JP XV
— JL— 4, 0,8 ±0,05
A: Filter chamber
B: No. 36 sieve (0.425 mm) or wire gauze
(d: 0.2 mm, w: 0.45 mm)
C: Score for the sample holder
Fig. 6.10-3 Apparatus 3
Large cell for tablets and capsules (top); tablet holder for
the large cell (bottom)
(All dimensions are expressed in mm unless otherwise noted.)
test assembly periodically.
Procedure
Basket Method or Paddle Method
IMMEDIATE-RELEASE DOSAGE FORMS
Procedure — Place the stated volume of the dissolution
medium (±1%) in the vessel of the specified apparatus, as-
semble the apparatus, equilibrate the dissolution medium to
37 ± 0.5 °C , and remove the thermometer. Place 1 dosage unit
in the apparatus, taking care to exclude air bubbles from the
surface of the dosage unit, and immediately operate the ap-
paratus at the specified rate. Within the time interval speci-
fied, or at each of the times stated, withdraw a specimen from
a zone midway between the surface of the Dissolution Medi-
um and the top of the rotating basket or blade, not less than
10 mm from the vessel wall. [NOTE — Where multiple sam-
pling times are specified, replace the aliquots withdrawn for
analysis with equal volumes of fresh Dissolution Medium at
37°C or, where it can be shown that replacement of the medi-
ia5 - 14.0
^=diam<:ter
Fig. 6.10-4 Apparatus 3
Small cell for tablets and capsules (top); tablet holder for the
small cell (bottom)
(All dimensions are expressed in mm unless otherwise noted.)
um is not necessary, correct for the volume change in the cal-
culation. Keep the vessel covered for the duration of the test,
and verify the temperature of the mixture under test at suita-
ble times.] Perform the analysis using an indicated assay
method.* 3 Repeat the test with additional dosage units.
If automated equipment is used for sampling or the ap-
paratus is otherwise modified, verification that the modified
apparatus will produce results equivalent to those obtained
with the standard apparatus described in this chapter, is
necessary.
Dissolution Medium — A specified dissolution medium is
used. If the dissolution medium is a buffered solution, adjust
the solution so that its pH is within 0.05 unit of the specified
pH. [NOTE — Dissolved gases can cause bubbles to form,
which may change the results of the test. If dissolved gases in-
fluence the dissolution results, remove dissolved gases prior
testing.* 4 ]
Time — Where a single time specification is given, the test
may be concluded in a shorter period if the requirement for
minimum amount dissolved is met. Specimens are to be
withdrawn only at the stated times, within a tolerance of ±2
JP XV
General Tests / Dissolution Test
119
Stage
SI
S2
S3
Number
Tested
6
6
12
Acceptance Table 6.10-1
Acceptance Criteria Level
Each value is not less than Q + 5%. Al
Average value of the 12 dosage units (SI + S2) is A2
equal to or greater than g, and no value is less
than 0-15%.
Average value of the 24 dosage units (SI + S2 + A3
S3) is equal to or greater than g, not more than
2 values are less than g- 15%, and no value is
less than g-25%.
Number
Tested
12
Acceptance Table 6.10-3
Criteria
No individual value exceeds 10% dissolved.
The average value of the 12 dosage units (Al +
A2) is not more than 10% dissolved, and no
value is greater than 25% dissolved.
The average value of the 24 dosage units (Al +
A2 + A3) is not more than 10% dissolved, and
no value is greater than 25% dissolved.
Acceptance Table 6.10-2
Acceptance Table 6.10-4
Level
Number
Tested
Criteria
LI 6 No individual value lies outside each of the
stated ranges and no individual value is less
than the stated amount at the final test time.
L2 6 The average value of the 12 dosage units (LI +
L2) lies within each of the stated ranges and is
not less than the stated amount at the final test
time; no value is more than 10% of labeled
content outside each of the stated ranges; and
no value is more than 10% of labeled content
below the stated amount at the final test time.
L3 12 The average value of the 24 dosage units (LI +
L2 + L3) lies within each of the stated ranges,
and is not less than the stated amount at the
final test time; not more than 2 of the 24 values
are more than 10% of labeled content outside
each of the stated ranges; not more than 2 of the
24 values are more than 10% of labeled content
below the stated amount at the final test time;
and no value is more than 20% of labeled
content outside each of the stated ranges or
more than 20% of labeled content below the
stated amount at the final test time.
%.
EXTENDED-RELEASE DOSAGE FORMS
Procedure — Proceed as described for Immediate-Release
Dosage Forms.
Dissolution Medium — Proceed as directed under Im-
mediate-Release Dosage Forms.
Time — The test-time points, generally three, are expressed
in hours.
DELAYED-RELEASE DOSAGE FORMS
*Procedure — Unless otherwise specified, proceed the acid
stage test and buffer stage test separately as described for Im-
mediate-Release Dosage Forms. ♦
*Dissolution Medium — Acid stage: Unless 1st fluid for dis-
solution test is used, proceed as directed under Immediate-
Release Dosage Forms. Buffer stage: Unless 2nd fluid for dis-
solution test is used, proceed as directed under Immediate-
Release Dosage Forms. ♦
*Time — Acid stage: Generally, test time is 2 hours for
tablets and capsules, and 1 hour for granules. Buffer stage:
The same as directed under Immediate-Release Dosage
Forms. ♦ All test times stated are to be observed within a
tolerance of ±2%, unless otherwise specified.
Level
Bl
B2
B3
Number
Tested
12
Criteria
No value is less than Q + 5%.
The average value of the 12 dosage units (Bl +
B2) is equal to or greater than g, and no value is
less than Q-15%.
The average value of the 24 dosage units (Bl +
B2 + B3) is equal to or greater than Q, not more
than 2 values are less than Q-15%, and no
value is less than g-25%.
Flow-Through Cell Method
IMMEDIATE-RELEASE DOSAGE FORMS
Procedure — Place the glass beads into the cell specified in
the individual monograph. Place 1 dosage unit on top of the
beads or, if specified, on a wire carrier. Assemble the filter
head and fix the parts together by means of a suitable clamp-
ing device. Introduce by the pump the dissolution medium
warmed to 37±0.5 C C through the bottom of the cell to ob-
tain the flow rate specified and measured with an accuracy of
5%. Collect the eluate by fractions at each of the times stat-
ed. Perform the analysis as directed. Repeat the test with ad-
ditional dosage units.
Dissolution Medium — Proceed as directed under Im-
mediate-Release Dosage Forms under Basket Method and
Paddle Method.
Time — Proceed as directed under Immediate-Release
Dosage Forms under Basket Method and Paddle Method.
EXTENDED-RELEASE DOSAGE FORMS
Procedure — Proceed as described for Immediate-Release
Dosage Forms under Flow-Through Cell Method.
Dissolution Medium — Proceed as described for Im-
mediate-Release under Flow-Through Cell Method.
Time — The test-time points, generally three, are expressed
in hours.
Interpretation
IMMEDIATE-PELEASE DOSAGE FORMS
* Follow Interpretation 1 when the value Q is specified in the
individual monograph, otherwise follow Interpretation 2.»
Interpretation 1:
Unless otherwise specified, the requirements are met if the
quantities of active ingredient dissolved from the dosage
units tested conform to Acceptance Table 6.10-1. Continue
testing through the three stages unless the results conform at
either SI or S2. The quantity, Q, *is the specified amount of
dissolved active ingredient, ♦ expressed as a percentage of the
120
Test for Glass Containers and Packing Materials / General Tests
JP XV
labeled content of the dosage unit; the 5%, 15%, and 25%
values in the Acceptance Table are percentage of the labeled
content so that three values and Q are in the same terms.
* Interpretation 2:
Unless otherwise specified, perform the test on 6 dosage
forms: if the individual dissolution rate meet the require-
ments specified in the individual monograph, the dosage
forms conform to the test. When individual dissolution rates
of 1 or 2 dosage forms fail to meet the requirements, repeat
the test on another 6 dosage forms: if individual dissolution
rates of not less than 10 dosage forms out of 12 meet the re-
quirements, the dosage forms conform to the test.»
EXTENDED-RELEASE DOSAGE FORMS
* Interpretation 1:»
Unless otherwise specified, the requirements are met it the
quantities of active ingredient dissolved from the dosage u-
nits tested conform to Acceptance Table 6.10-2. Continue
testing through the three levels unless the results conform at
either LI or L2. Limits on the amounts of active ingredient
dissolved are expressed in terms of the percentage of labeled
content. The limits embrace each value of Q\, the amount dis-
solved at each specified fractional dosing interval. Where
more than one range is specified, the acceptance criteria apply
individually to each range.
* Interpretation 2:
Unless otherwise specified, perform the test on 6 dosage
forms: if the individual dissolution rate meet the require-
ments specified in the individual monograph, the dosage
forms conform to the test. When individual dissolution rates
of 1 or 2 dosage forms fail to meet the requirements, repeat
the test on another 6 dosage forms: if individual dissolution
rates of not less than 10 dosage forms out of 12 meet the re-
quirements, the dosage forms conform to the test. Where
more than one range is specified, the acceptance criteria apply
individually to each range. ♦
DELAYED-RELEASE DOSAGE FORMS
* Follow Interpretation 1 when the value Q is specified in
the test using 2nd fluid for dissolution test in the individual
monograph, otherwise follow Interpretation 2.
Interpretation 1:
Test using 1st fulid for dissolution test — Unless otherwise
specified, the requirements of this portion of the test are met
if the quantities, based on the percentage of the labeled con-
tent, of active ingredient dissolved from the units tested con-
form to Acceptance Table 6.10-3. Continue testing through
the three levels unless the result conforms at A2.»
*Test using 2nd fluid for dissolution test* — Unless otherwise
specified, the requirements are met it the quantities of active
ingredient dissolved from the units tested conform to Accep-
tance Table 6.10-4. Continue testing through the three levels
unless the results of both stages conform at an earlier level.
The value of Q in Acceptance Table 6.10-4 is the amount *
specified in monograph* of active ingredient dissolved, ex-
pressed as a percentage of the labeled content. The 5% and
15% and 25% values in Acceptance Table 6.10-4 are percen-
tages of the labeled contest so that these values and Q are in
the same terms.
* Interpretation 2:
Unless otherwise specified, both the tests using 1st fluid for
dissolution test and 2nd fluid for dissolution test in acid and
buffer stages, perform the test on 6 dosage forms: if the in-
dividual dissolution rate meet the requirements specified in
the individual monograph, the dosage forms conform to the
test. When individual dissolution rates of 1 or 2 dosage forms
fail to meet the requirements, repeat the test on another 6
dosage forms: if individual dissolution rates of not less than
10 dosage forms out of 12 meet the requirements, the dosage
forms conform to the test.»
*'The materials should not sorb, react, or interfere with the specimen
being tested.
* 2 If a cover is used, it provides sufficient openings to allow ready in-
sertion of the thermometer and withdrawal of specimens.
* 3 Test specimens are filtered immediately upon sampling unless filtra-
tion is demonstrated to be unnecessary. Use an inert filter that does
not cause adsorption of the ingredient or contain extractable sub-
stances that would interfere with the analysis.
* 4 One method of deaeration is as follows: Heat the medium, while
stirring gently, to about 41 °C, immediately filter under vacuum using
a filter having a porosity of 0.45 jum or less, with vigorous stirring,
and continue stirring under vacuum for about 5 minutes. Other vali-
dated deaeration techniques for removal of dissolved gases may be
used.
7. Tests for Containers and
Packing Materials
7.01 Test for Glass Containers for
Injections
The glass containers for injections do not interact physical-
ly or chemically with the contained medicament to alter any
property or quality, can protect the contained medicament
from the invasion of microbes by means of perfect sealing or
other suitable process, and meet the following requirements.
The surface-treated container for aqueous infusion is made
from glass which meets the requirements for the soluble alka-
li test for a container not to be fused under method 1.
(1) The containers are colorless or light brown and trans-
parent, and have no bubbles which interfere the test of the
Foreign Insoluble Matter Test for Injections <6.06>.
(2) Multiple-dose containers are closed by rubber stop-
pers or any other suitable stoppers. The stoppers permit
penetration of an injection needle without detachment of
fragments, and upon withdrawal of the needle, they reclose
the containers immediately to prevent external contamina-
tion, and also do not interact physically or chemically with
the contained medicaments.
Containers intended for aqueous infusions are closed by
rubber stoppers meeting the requirements of the test for Rub-
ber Closure for Aqueous Infusions <7.03>.
(3) Soluble alkali test — The testing methods may be
divided into the following two methods according to the type
of container or the dosage form of the medicament.
(i) Method 1: This method is applied to containers to be
fused, or containers not to be fused except containers for
aqueous infusions with a capacity exceeding 100 mL.
Rinse thoroughly the inside and outside of the containers
to be tested with water, dry, and roughly crush, if necessary.
Transfer 30 to 40 g of the glass to a steel mortar, and crush.
Sieve the crushed glass through a No. 12 (1400 /xm) sieve.
Transfer the portion retained on the sieve again to the steel
JPXV
General Tests / Test Methods for Plastic Containers
121
mortar, and repeat this crushing procedure until 2/3 of the
amount of powdered glass has passed through a No. 12 (1400
/um) sieve. Combine all portions of the glass powder passed
through a No. 12 (1400 ^m) sieve, shake the sieve in a
horizontal direction for 5 minutes with slight tapping at inter-
vals using No. 18 (850 /um) and No. 50 (300 ^m) sieves.
Transfer 7 g of the powder, which has passed through a No.
18 (850 //m) sieve but not through a No. 50 (300 ^m) sieve to
a No. 50 (300 /um) sieve, immerse it in a suitable container
filled with water, and wash the contents with gentle shaking
for 1 minute. Rinse again with ethanol (95) for 1 minute, dry
the washed glass powder at 100°C for 30 minutes, and allow
to cool in a desiccator (silica gel). Transfer exactly 5.0 g of
the powder thus prepared to a 200-mL conical flask of hard
glass, add 50 mL of water, and gently shake the flask so that
the powder disperses on the bottom of the flask evenly. Cover
the flask with a small beaker of hard glass or a watch glass of
hard glass, then heat it in boiling water for 2 hours, and im-
mediately cool to room temperature. Decant the water from
the flask into a 250-mL conical flask of hard glass, wash well
the residual powdered glass with three 20-mL portions of
water, and add the washings to the decanted water. Add 5
drops of bromocresol green-methyl red TS and titrate <2.50>
with 0.01 mol/L sulfuric acid VS until the color of the solu-
tion changes from green through slightly grayish blue to
slightly grayish red-purple. Perform a blank determination in
the same manner, and make any necessary correction.
The quantity of 0.01 mol/L sulfuric acid VS consumed
does not exceed the following quantity, according to the type
of containers.
Containers to be fused 0.30 mL
Containers not to be fused (including injection
syringes used as containers) 2.00 mL
(ii) Method 2: This method is applied to containers not to
be fused for aqueous infusions with a capacity exceeding 100
mL.
Rinse thoroughly the inside and outside of the containers
to be tested with water, and dry. Add a volume of water
equivalent to 90% of the overflow capacity of the container,
cover it with a small beaker of hard glass or close tightly with
a suitable stopper, heat in an autoclave at 121 °C for 1 hour,
and allow to stand until the temperature falls to room tem-
perature, measure exactly 100 mL of the this solution, and
transfer to a 250-mL conical flask of hard glass. Add 5 drops
of bromocresol green-methyl red TS, and titrate <2.50> with
0.01 mol/L sulfuric acid VS until the color of the solution
changes from green through slightly grayish blue to slightly
grayish red-purple. Measure accurately 100 mL of water,
transfer to a 250-mL conical flask of hard glass, perform a
blank determination in the same manner, and make any
necessary correction. The quantity of 0.01 mol/L sulfuric
acid VS consumed does not exceed 0.10 mL.
(4) Soluble iron test for light-resistant containers — Rinse
thoroughly five or more light-resistant containers to be tested
with water, and dry at 105°C for 30 minutes. Pour a volume
of 0.01 mol/L hydrochloric acid VS corresponding to the
labeled volume of the container into individual containers,
and fuse them. In the case of containers not to be fused,
cover them with small beakers of hard glass or watch glasses
of hard glass. Heat them at 105 C C for 1 hour. After cooling,
prepare the test solution with 40 mL of this solution accord-
ing to Method 1 of the Iron Limit Test <1.10>, and perform
the test according to Method B. Prepare the control solution
with 2.0 mL of the Standard Iron Solution.
(5) Light transmission test for light-resistant con-
tainers — Cut five light-resistant containers to be tested, pre-
pare test pieces with surfaces as flat as possible, and clean the
surfaces. Fix a test piece in a cell-holder of a spectrophotome-
ter to allow the light pass through the center of the test piece
perpendicularly to its surface. Measure the light transmit-
tance of the test piece with reference to air between 290 nm
and 450 nm and also between 590 nm and 610 nm at intervals
of 20 nm each. The percent transmissions obtained between
290 nm and 450 nm are not more than 50% and that between
590 nm and 610 nm are not less than 60% . In the case of con-
tainers not to be fused having a wall thickness over 1.0 mm,
the percent transmissions between 590 nm and 610 nm are
not less than 45%.
7.02 Test Methods for
Plastic Containers
Test methods for plastic containers may be used for design-
ing and quality assurance of plastic containers. Not all tests
described here will be necessary in any phases for any con-
tainers. On the other hand, the set does not include sufficient
number and kinds of tests needed for any design verification
and quality assurance of any containers. Additional tests may
be considered if necessary.
1. Combustion Tests
1.1 Residue on ignition
Weigh accurately about 5 g of cut pieces of the container
and perform the test according to the Residue on Ignition
<2.44>.
1.2 Heavy metals
Place an appropriate amount of cut pieces of the container
in a porcelain crucible, and perform the test according to
Method 2 of the Heavy Metals Limit Test <1.07>. Prepare the
control solution with 2.0 mL of Standard Lead Solution.
1.3 Lead
Method 1: Place 2.0 g of cut pieces of a container in a
crucible of platinum or quartz, moisten with 2 mL of sulfuric
acid, heat slowly to dryness, then heat to combustion at be-
tween 450°C and 500°C. Repeat this procedure, if necessary.
After cooling, moisten the residue with water, add 2 to 4 mL
of hydrochloric acid, evaporate to dryness on a water bath,
then add 1 to 5 mL of hydrochloric acid, and warm to dis-
solve. Then add 0.5 to 1 mL of a mixture of a solution of
citric acid monohydrate (1 in 2) and hydrochloric acid (1:1),
and add 0.5 to 1 mL of a warmed solution of ammonium
acetate (2 in 5). Filter through a glass filter if insoluble matter
remains. To the obtained filtrate add 10 mL of a solution of
diammonium hydrogen citrate (1 in 4), 2 drops of
bromothymol blue TS and ammonia TS until the color of the
solution changes from yellow to green. Then add 10 mL of a
solution of ammonium sulfate (2 in 5) and water to make 100
mL. Add 20 mL of a solution of sodium 7V,7V-diethyl-
dithiocarbamate trihydrate (1 in 20) to this solution, mix,
allow to stand for a few minutes, then add 20.0 mL of 4-
methyl-2-pentanone, and shake vigorously. Allow to stand to
separate the 4-methyl-2-pentanone layer, filter if necessary,
and use the layer as the sample solution. Separately, to 2.0
122
Test Methods for Plastic Containers / General Tests
JP XV
mL of Standard Lead Solution add water to make exactly 10
mL. To 1.0 mL of this solution add 10 mL of a solution of
diammonium hydrogen citrate (1 in 4) and 2 drops of
bromothymol blue TS, then proceed in the same manner as
for the sample solution, and use the solution so obtained as
the standard solution. Perform the test with the sample solu-
tion and the standard solution according to Atomic Absorp-
tion Spectrophotometry <2.23> under the following condi-
tions, and determine the concentration of lead in the sample
solution.
Gas: Combustible gas — Acetylene or hydrogen
Supporting gas — Air
Lamp: Lead hollow-cathode lamp
Wavelength: 283.3 nm
Method 2: Cut a container into pieces smaller than 5-mm
square, take 2.0 g of the pieces into a glass beaker, add 50 mL
of 2-butanone and 0.1 mL of nitric acid, and warm to dis-
solve. To this solution add 96 mL of methanol gradually to
precipitate a resinous substance, and filter by suction. Wash
the beaker and the resinous substance with 12 mL of
methanol followed by 12 mL of water, combine the washings
and the filtrate, and concentrate to about 10 mL under
reduced pressure. Transfer into a separator, add 10 mL of
ethyl acetate and 10 mL of water, shake vigorously, and
allow to stand to separate the water layer. Evaporate the
water layer to dryness, add 5 mL of hydrochloric acid to the
residue, and warm to dissolve. Then add 1 mL of a mixture
of a solution of citric acid monohydrate (1 in 2) and
hydrochloric acid (1:1), and add 1 mL of a warmed solution
of ammonium acetate (2 in 5). Filter through a glass filter
(G3) if insoluble matter remains. To the solution so obtained
add 10 mL of a solution of diammonium hydrogen citrate (1
in 4) and 2 drops of bromothymol blue TS, and then add am-
monia TS until the color of the solution changes from yellow
to green. Further add 10 mL of a solution of ammonium sul-
fate (2 in 5) and water to make 100 mL. Add 20 mL of a solu-
tion of sodium A^ 7V-diethyldithiocarbamate trihydrate (1 in
20) to this solution, mix, allow to stand for a few minutes,
then add 20.0 mL of 4-methyl-2-pentanone, and shake
vigorously. Allow to stand to separate the 4-methyl-2-penta-
none layer, filter the layer if necessary, and use the layer as
the sample solution. Separately, pipet 5 mL of Standard
Lead Solution, add water to make exactly 50 mL, and to 2.0
mL of this solution add 10 mL of a solution of diammonium
hydrogen citrate (1 in 4) and 2 drops of bromothymol blue
TS, then proceed in the same manner as for the sample solu-
tion, and use the solution so obtained as the standard solu-
tion. Perform the test with the sample solution and the stan-
dard solution according to Atomic Absorption Spec-
trophotometry <2.23> under the conditions described in
Method 1, and determine the concentration of lead in the
sample solution.
1.4 Cadmium
Method 1: To 2.0 mL of Standard Cadmium Solution add
10 mL of a solution of diammonium hydrogen citrate (1 in 4)
and 2 drops of bromothymol blue TS, and proceed in the
same manner as for the sample solution in Method 1 under
1.3, and use the solution so obtained as the standard solu-
tion. Perform the test with the sample solution obtained in
Method 1 under 1.3 and the standard solution according to
Atomic Absorption Spectrophotometry <2.23> under the fol-
lowing conditions, and determine the concentration of cad-
mium in the sample solution.
Gas: Combustible gas — Acetylene or hydrogen
Supporting gas — Air
Lamp: Cadmium hollow-cathode lamp
Wavelength: 228.8 nm
Method 2: To 2.0 mL of Standard Cadmium Solution add
10 mL of a solution of diammonium hydrogen citrate (1 in 4)
and 2 drops of bromothymol blue TS, and proceed in the
same manner as for the sample solution in Method 2 under
1.3, and use the solution so obtained as the standard solu-
tion. Perform the test with the sample solution obtained in
Method 2 under 1.3 and the standard solution according to
Atomic Absorption Spectrophotometry <2.23> under the con-
ditions described in Method 1, and determine the concentra-
tion of cadmium in the sample solution.
1.5 Tin
Cut a container into pieces smaller than 5-mm square,
place 5.0 g of the pieces in a Kjeldahl flask, add 30 mL of a
mixture of sulfuric acid and nitric acid (1:1), and decompose
by gentle heating in a muffle furnace, occasionally adding
dropwise a mixture of sulfuric acid and nitric acid (1:1) until
the content changes to a clear, light brown solution. Then
heat until the color of the solution changes to a clear, light
yellow, and heat to slowly concentrate to practical dryness.
After cooling, dissolve the residue in 5 mL of hydrochloric
acid by warming, and after cooling, add water to make exact-
ly 10 mL. Pipet 5 mL of this solution into a 25-mL volumet-
ric flask (A). Transfer the remaining solution to a 25-mL
beaker (B) by washing out with 10 mL of water, add 2 drops
of bromocresol green TS, neutralize with diluted ammonia
solution (28) (1 in 2), and measure the volume consumed for
neutralization as a mL. To the volumetric flask, A, add
potassium permanganate TS dropwise until a slight pale red
color develops, and add a small amount of L-ascorbic acid to
decolorize. Add 1.5 mL of 1 mol/L hydrochloric acid TS, 5
mL of a solution of citric acid monohydrate (1 in 10), a mL
of diluted ammonia solution (28) (1 in 2), 2.5 mL of poly-
vinyl alcohol TS, 5.0 mL of phenylfluorone-ethanol TS and
water to make 25 mL. Shake well, then allow to stand for
about 20 minutes, and use this solution as the sample solu-
tion. Separately, pipet 1.0 mL of Standard Tin Solution, add
5 mL of water, add potassium permanganate TS dropwise
until a slight pale red color develops, proceed in the same
manner as for the sample solution, and use this solution as
the standard solution. Determine the absorbances of the sam-
ple solution and the standard solution according to Ultrav-
iolet-visible Spectrophotometry <2.24> at 510 nm, using water
as the blank.
2. Extractable substances
Cut the container at homogeneous regions of low curva-
ture and preferably the same thickness, gather pieces to make
a total surface area of about 1200 cm 2 when the thickness is
0.5 mm or less, or about 600 cm 2 when the thickness is great-
er than 0.5 mm, and subdivide in general into strips approxi-
mately 0.5 cm in width and 5 cm in length. Wash them with
water, and dry at room temperature. Place these strips in a
300-mL hard glass vessel, add exactly 200 mL of water, and
seal the opening with a suitable stopper. After heating the
vessel in an autoclave at 121 °C for 1 hour, take out the ves-
sel, allow to stand until the temperature falls to room temper-
ature, and use the content as the test solution.
For containers made of composite plastics, the extraction
may be performed by filling a labeled volume of water in the
JPXV
General Tests / Test Methods for Plastic Containers
123
container. In this case, it is necessary to record the volume of
water used and the inside area of the container.
When containers are deformed at 121 °C, the extraction
may be performed at the highest temperature which does not
cause deformation among the following conditions: at 100 ±
2°C for 2 ±0.2 hours, at 70±2°C for 24 ±2 hours, at 50 ±
2°C for 72 ± 2 hours or at 37 ± 1 °C for 72 ± 2 hours.
Prepare the blank solution with water in the same manner.
For containers made of composite plastics, water is used as
the blank solution.
Perform the following tests with the test solution and the
blank solution:
(i) Foaming test: Place 5 mL of the test solution in a
glass-stoppered test tube about 15 mm in inside diameter and
about 200 mm in length, shake vigorously for 3 minutes, and
measure the time needed for almost complete disappearance
of the foam thus generated.
(ii) pH <2.54>: To 20 mL each of the test solution and the
blank solution add 1.0 mL of a solution of potassium chlo-
ride (1 in 1000), and obtain the difference in the reading of
pH between these solutions.
(iii) Potassium permanganate-reducing substances: Place
20 mL of the test solution in a glass-stoppered, conical flask,
add 20.0 mL of 0.002 mol/L potassium permanganate VS
and 1 mL of dilute sulfuric acid, and boil for 3 minutes. After
cooling, add 0.10 g of potassium iodide, stopper tightly,
shake, then allow to stand for 10 minutes, and titrate <2.50>
with 0.01 mol/L sodium thiosulfate VS (indicator: 5 drops of
starch TS). Perform the test in the same manner, using 20.0
mL of the blank solution, and obtain the difference of the
consumption of 0.002 mol/L potassium permanganate VS
between these solutions.
(iv) UV spectrum: Read the maximum absorbances be-
tween 220 nm and 240 nm and between 241 nm and 350 nm
of the test solution against the blank solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
(v) Residue on evaporation: Evaporate 20 mL of the test
solution on a water bath to dryness, and weigh the residue af-
ter drying at 105 C C for 1 hour.
3. Test for fine particles
Rinse thoroughly with water the inside and outside of con-
tainers to be used for the tests, fill the container with the la-
beled volume of 0.9 w/v% sodium chloride solution, adjust
so that the amount of air in the container is about 50 mL per
500 mL of the labeled volume, stopper tightly, and heat at
121 °C for 25 minutes in an autoclave. After allowing to cool
for 2 hours, take out the container, and then allow to stand at
ordinary temperature for about 24 hours. If the containers
are deformed at 121 °C, employ a suitable temperature-time
combination as directed under Extractable substances. Clean
the outside of the container, mix by turning upside-down 5 or
6 times, insert immediately a clean needle for filterless infu-
sion into the container through the rubber closure of the con-
tainer, take the effluent while mixing gently in a clean con-
tainer for measurement, and use as the test solution. Perform
the test with the solution according to the following fine par-
ticle test, and count the numbers of fine particles with di-
ameters of 5 - 10 [im, 10-25//m and larger than 25 /um in
1.0 mL of the test solution.
Fine particle test — Counting of the fine particles must be
performed in dustless, clean facilities or apparatus, using a
light-shielded automatic fine particle counter. The sensor of
the counter to be used must be able to count fine particles of
1.5 /um or more in diameter. The volume to be measured is 10
mL. Adjust the counter before measurement. For calibration
of the diameter and number of particles, the standard parti-
cles for calibration of the light-shielded automatic fine parti-
cle counter should be used in suspension in water or 0.9 w/v
% sodium chloride solution.
Count five times the numbers of particles with diameters of
5-10 ium, 10 - 25 fim and more than 25 fim while stirring the
test solution, and calculate the average particle numbers of
four counts, excluding the first, as the number of particles in
1.0 mL of the test solution.
Note: Water and 0.9 w/v% sodium chloride solution to be
used for the tests should not contain more than 0.5 particles
of 5 - 10//m in size per 1.0 mL.
4. Transparency test
Method 1 : This can be applied to containers which have a
smooth and not embossed surface and rather low curvature.
Cut the container at homogeneous regions of low curvature
and preferably the same thickness to make 5 pieces of about
0.9 x 4 cm in size, immerse each piece in water filled in a cell
for determination of the ultraviolet spectrum, and determine
the transmittance at 450 nm as directed under Ultraviolet-
visible Spectrophotomety <2.24> using a cell filled with water
as a blank.
Method 2: Sensory test — This can be applied to containers
which have a rough or embossed surface. It can also be ap-
plied to testing of the transparency of containers in case
where the turbidity of their pharmaceutical contents must be
checked.
Test solutions
Hexamethylenetetramine TS Dissolve 2.5 g of hex-
amethylenetetramine in 25 mL of water.
Hydrazinium sulfate TS Dissolve 1.0 g of hydrazinium
sulfate in water to make 100 mL.
Formadin stock suspension To 25 mL of hex-
amethylenetetramine TS add 25 mL of hydrazinium sulfate
TS, mix, and use after standing at 25 ± 3°C for 24 hours.
This suspension is stable for about 2 months after prepara-
tion, provided it is stored in a glass bottle free from inside
surface defects. Mix well before use.
Standard suspension: Dilute 15 mL of the formadin stock
suspension with water to make 1000 mL. Prepare before use
and use within 24 hours.
Reference suspension: Dilute 50 mL of the standard sus-
pension with water to make 100 mL.
Tests
(i) Method 2A (with control) Take 2 containers to be
tested, and place in one of them the labeled volume of the
reference suspension and in the other, the same volume of
water. Show the two containers to five subjects, separately,
who do not know which one is which, ask which one seems to
be more turbid, and calculate the rate of correct answers.
(ii) Method 2B (without control) Take 6 numbered con-
tainers to be tested, and place in three of them the labeled
volume of the reference suspension and in the others, the
same volume of water. Show each one of the containers at
random order to five subjects, separately, who do not know
which one is which, ask if it is turbid or not, and calculate the
percentage of the answer that it is turbid (100 X/15, X: num-
ber of containers judged as "being turbid") in each group.
124
Test Methods for Plastic Containers / General Tests
JP XV
5. Water vapor permeability test
Method 1 : This test method is applicable to containers for
aqueous injection. Fill the container with the labeled volume
of water. After closing it hermetically, accurately weigh the
container and record the value. Store the container at 65 ±
5% relative humidity and a temperature of 20 ± 2°C for 14
days, and then accurately weigh the container again and
record the value. Calculate the mass loss during storage.
Method 2 : This test method is provided for evaluating
moisture permeability of containers for hygroscopic drugs.
Unless otherwise specified, perform the test according to the
following procedure.
Desiccant — Place a quantity of calcium chloride for water
determination in a shallow container, taking care to exclude
any fine powder, then dry at 1 10°C for 1 hour, and cool in a
desiccator.
Procedure — Select 12 containers, clean their surfaces with
a dry cloth, and close and open each container 30 times in the
same manner. Ten among the 12 containers are used as "test
containers" and the remaining two, as "control containers".
A torque for closing screw-capped containers is specified in
Table 7.02-1. Add desiccant to 10 of the containers, designat-
ed test containers, filling each to within 13 mm of the closure
if the container volume is 20 mL or more, or filling each to
two-thirds of capacity if the container volume is less than 20
mL. If the interior of the container is more than 63 mm in
depth, an inert filler or spacer may be placed in the bottom to
minimize the total mass of the container and desiccant; the
layer of desiccant in such a container shall be not less than 5
cm in depth. Close each container immediately after adding
desiccant, applying the torque designated in the table. To
each of the control containers, add a sufficient number of
glass beads to attain a mass approximately equal to that of
each of the test containers, and close, applying the torque
designated in the table. Record the mass of the individual
containers so prepared to the nearest 0.1 mg if the container
volume is less than 20 mL, to the nearest 1 mg if the container
volume is 20 mL or more but less than 200 mL, or to the
nearest 10 mg if the container volume is 200 mL or more, and
store the containers at 75 ± 3% relative humidity and a tem-
perature of 20 ± 2°C. After 14 days, record the mass of the
individual containers in the same manner. Completely fill 5
empty containers with water or a non-compressible, free-
flowing solid such as fine glass beads, to the level indicated by
the closure surface when in place. Transfer the contents of
each to a graduated cylinder, and determine the average con-
tainer volume, in mL. Calculate the rate of moisture permea-
bility, in mg per day per liter, by use of the formula:
(1000/14 V) [(Tf-Td-iQ-Q)]
V: average volume (mL)
T f — 7j: difference between the final and initial masses of
each test container (mg)
C t — C t : average of the differences between the final and ini-
tial masses of the two controls (mg)
6. Leakage test
Fill a container with a solution of fluorescein sodium (1 in
1000), stopper tightly, place filter papers on and under the
container, and apply a pressure of 6.9 N (0.7 kg)/cm 2 at 20°C
for 10 minutes. Judge the leakiness by observing the color of
the paper.
Table 7.02-1 Torque Applicable to Screw-Type Container
Closure Diameter (mm)
Torque (N-cm)
8
59
10
60
13
88
15
59-98
18
78-118
20
88-137
22
98-157
24
118-206
28
137-235
30
147-265
33
167-284
38
196-294
43
196-304
48
216-343
53
235-402
58
265-451
63
284-490
66
294-510
70
314-569
83
363-735
86
451-735
89
451-794
100
510-794
110
510-794
120
618-1069
132
677-1069
7. Cytotoxicity test
The following test methods are designed to detect cytotoxic
substances in plastic materials by evaluating the cytotoxicity
of the culture medium extracts from plastic containers for
pharmaceutical products. Other appropriate standard
methods of cytotoxicity testing may be used for the evalua-
tion, if appropriate. However, the final decision shall be
made based upon the test methods given here, if the test
results obtained according to the other methods are question-
able.
Cell lines
The recommended cell lines are L929 (American Type Cul-
ture Collection-ATCC CCL1) and V79 (Health Science
Research Resources Bank-JCRB0603). In addition, other es-
tablished cell lines may be used when it is confirmed that they
form well-defined colonies reproducibly, with characteristics
comparable to those of L929 and V79 cells.
Culture medium
Eagle's minimum essential medium prepared as follows
shall be used. Dissolve the chemicals listed below in 1000 mL
of water. Sterilize the solution by autoclaving at 121 °C for 20
minutes. Cool the solution to room temperature, and add 22
mL of sterilized sodium hydrogen carbonate TS and 10 mL
of sterilized glutamine TS. To the resultant solution add fetal
calf serum (FCS) to make 10 vol% FCS in the medium,
sodium chloride 6.80 g
potassium chloride 400 mg
sodium dihydrogen phosphate (anhydrous) 115 mg
magnesium sulfate (anhydrous) 93.5 mg
calcium chloride (anhydrous) 200 mg
glucose 1.00 g
L-arginine hydrochloride 126 mg
L-cysteine hydrochloride monohydrate 31.4 mg
JPXV
General Tests / Test Methods for Plastic Containers
125
L-tyrosine 36.0 mg
L-histidine hydrochloride monohydrate 42.0 mg
L-isoleucine 52.0 mg
L-leucine 52.0 mg
L -lysine hydrochloride 73.0 mg
L-methionine 15.0 mg
L-phenylalanine 32.0 mg
L-threonine 48.0 mg
L-tryptophan 10.0 mg
L-valine 46.0 mg
succinic acid 75.0 mg
succinic acid hexahydrate 100 mg
choline bitartrate 1.8 mg
folic acid 1.0 mg
myo-inositol 2.0 mg
nicotinamide 1.0 mg
calcium D-pantothenate 1.0 mg
pyridoxal hydrochloride 1.0 mg
riboflavin 0.1 mg
thiamine hydrochloride 1.0 mg
biotin 0.02 mg
phenol red 6.0 mg
Reagents
(i) Sodium hydrogen carbonate TS: Dissolve 10 g of sodi-
um hydrogen carbonate in water to make 100 mL. Sterilize
the solution either by autoclaving in a well-sealed container at
121 °C for 20 minutes or by filtration through a membrane
filter with a nominal pore diameter of 0.22 tun or less.
(ii) Glutamine TS: Dissolve 2.92 g of L-glutamine in
water to make 100 mL. Sterilize the solution by passing it
through a membrane filter of pore size equal to or less than
0.22 //m.
(iii) Phosphate buffer solution (PBS): Dissolve 0.20 g of
potassium chloride, 0.20 g of potassium dihydrogen-
phosphate, 8 .00 g of sodium chloride, and 1.15 g of disodium
hydrogenphosphate (anhydrous) in water to make 1000 mL.
Sterilize the solution by autoclaving at 121 °C for 20 minutes.
(iv) Trypsin TS: Dissolve 0.5 g of trypsin and 0.2 g of dis-
odium dihydrogen ethylenediamine tetraacetate dihydrate in
phosphate buffer solution to make 1000 mL. Sterilize the so-
lution by passing it through a membrane filter of pore size
equal to or less than 0.22 iim.
(v) Formaldehyde solution: Dilute formaldehyde solu-
tion with water by a factor of ten.
(vi) Giemsa's stain solution: Dilute a commercially avail-
able Giemsa's test solution with the diluent by a factor of fif-
ty. Prepare before use.
(vii) Diluent: Dissolve 4.54 g of potassium dihydrogen-
phosphate and 4.75 g of disodium hydrogen phosphate (an-
hydrous) in water to make 1000 mL.
Devices and instruments
It is recommended to use the following devices and instru-
ments for testing.
(i) Pipets : Pasteur pipet, pipet for partial delivery, meas-
uring pipet for partial delivery, and dispenser with microtip.
(ii) Screw-capped glass bottles : 50 - 1000 mL volume.
(iii) Sterile disposable centrifuge tubes: 15 and 50 mL
volume.
(iv) Sterile disposable tissue culture flasks with a flat
growth area of approximately 25 or 75 cm 2 .
(v) Sterile disposable multiple well plates (24 wells)
(vi) Inverted microscope and stereomicroscope
(vii) Humidified incubator — maintain the conditions as
follows: temperature, 37°C; C0 2 gas concentration, 5%.
Control materials and substances
(i) Negative control material: polyethylene film
(ii) Positive control material (A): polyurethane film con-
taining 0.1% zinc diethyldithiocarbamate
(iii) Positive control material (B): polyurethane film con-
taining 0.25% zinc dibutyldithiocarbamate
(iv) Control substances: zinc diethyldithiocarbamate
(reagent grade) and zinc dibutyldithiocarbamate (reagent
grade)
Test procedure
(i) Sample preparation: When the material of the con-
tainer consists of a single homogeneous layer, subdivide the
cut pieces of a container into pieces of the size of approxi-
mately 2x15 mm and subject the pieces to the test. When the
material of the container has multiple layers, such as laminat-
ed and coated materials, prepare cut pieces with a surface
area of one side of 2.5 cm 2 and subject the pieces to the test
without subdividing them into smaller pieces.
(ii) Preparation of test solutions: Transfer an appropriate
amount of the sample to a screw-capped glass bottle or a
sterile disposable centrifuge tube. Cap the bottle or tube
loosely and cover the cap with clean aluminum foil. Sterilize
the bottle or tube by autoclaving at 121 °C for 20 minutes.
When the material of the sample is not resistant to heat dur-
ing autoclaving, gas sterilization with ethylene oxide (EO)
may be used. In the case of EO sterilization, sufficient aera-
tion should be achieved to avoid an additional toxic effect of
residual EO in the test results. To the bottle or tube add the
culture medium in a proportion of 10 mL to one gram or 1
mL to 2.5 cm 2 (one side) of the sample, loosely cap the bottle
or tube, and allow to stand in a humidified incubator for 24
hours. Transfer the culture medium extract, which is desig-
nated 100% test solution, to a sterilized screw-capped glass
bottle or a sterile disposable centrifuge tube. Dilute the 100%
test solution with culture medium using a dilution factor of
two to prepare serial dilutions having extract concentrations
of 50%, 25%, 12.5%, 6.25%, 3.13% and so on.
(iii) Preparation of cell suspension: Remove the culture
medium from the maintained cell culture flask and rinse the
cells with an appropriate volume of PBS by gentle rotation of
the flask two or three times, and discard the PBS. Add a
sufficient volume of trypsin solution to cover the cell layer.
Cap the flask and place the flask in a humidified incubator for
one or two minutes. After confirming detachment of the cell
layer from the bottom of the flask by using a microscope, add
an appropriate volume of the fresh culture medium and gent-
ly pipet the cells completely out of the flask by using a
Pasteur pipet. Transfer the pipetted cell suspension into a
sterile disposable centrifuge tube and centrifuge the tube at
800 - 1000 revolutions per minute for 2-5 minutes. Discard
the supernatant, resuspend the cells in an appropriate volume
of PBS by pipetting, using a Pasteur pipet, and centrifuge the
tube again. Discard the PBS, and add an appropriate volume
of fresh culture medium to the flask. Resuspend the cells by
pipetting and make a single cell suspension. Determine the
cell concentration using a hemocytometer.
(iv) Cytotoxicity testing: Dilute the cell suspension pre-
pared according to procedure (iii) with culture medium to ad-
just the cell concentration to 100 cells/mL. Place a 0.5 mL
aliquot of the diluted cell suspension on each well of a sterile
126
Test Methods for Plastic Containers / General Tests
JP XV
disposable multiple well plate. Incubate the plate in the humi-
dified incubator for 4-6 hours to attach the cells to the bot-
tom surface of the well. Discard the medium from each well,
and add a 0.5 mL aliquot of the test solution or fresh medium
to quadruplicate wells. Place the plate immediately in the hu-
midified incubator and incubate the plate for the appropriate
period: 7-9 days for L929 cells; 6-7 days for V79 cells. Af-
ter the incubation, discard the medium from the plate, add an
appropriate volume of dilute formaldehyde TS to each well
and allow the plate to stand for 30 minutes to fix the cells.
Discard the dilute formaldehyde TS from each well and add
an appropriate volume of dilute Giemsa's TS to each well.
After ensuring good staining of the colonies, discard the stain
solution from the wells and count the number of colonies in
each well. Calculate a mean number of colonies for each con-
centration of the test solution, and divide the mean by the
mean number of colonies for the fresh medium to obtain the
colony formation rate (%) for each extract concentration of
the test solution. Plot the extract concentration (%) of the
test solution on a logarithmic scale and the colony formation
rate on an ordinary scale on semi-logarithmic graph paper to
obtain a colony formation inhibition curve of the container.
Read the % extract concentration which inhibits colony for-
mation to 50%, IC 50 (%), from the inhibition curve. It is
recommended to check the sensitivity and the reproducibility
of the test system by the use of suitable control materials or
substances in the test system, if necessary.
Plastic Containers for Aqueous Injections
Plastic containers for the aqueous injections do not inter-
act with pharmaceuticals contained therein to alter the effica-
cy, safety or stability, and do not permit the contamination
with microorganisms. The containers meet the following re-
quirements.
1. Polyethylene or polypropylene containers for aqueous
injections
The containers are made of polyethylene or polypropylene
and free from any adhesive.
(1) Transparency — The containers have a transmittance
of not less than 55%, when tested as directed in Method 1 un-
der the Transparency test. When Method 1 can not be ap-
plied, test according to the Method 2B of the Transparency
test. In this case, the rate that the water-containing container
is judged as "being turbid" is not more than 20%, and the
rate that the reference suspension-containing container is
judged as "being turbid" is not less than 80%.
(2) Appearance — The containers do not have strips,
cracks, bubbles, or other faults which cause difficulties in
practical use.
(3) Water vapor permeability — Proceed as directed in
Method 1 of the Water vapor permeability test. The loss of
mass is not more than 0.20%.
(4) Heavy metals <1.0T> — The turbidity of the test solu-
tion is not greater than that of the control solution when the
amount of the sample taken is 1.0 g.
(5) Lead — Perform the test as directed in Method 1. The
absorbance of the sample solution is not more than that of
the standard solution.
(6) Cadmium — Perform the test as directed in Method 1.
The absorbance of the sample solution is not more than that
of the standard solution.
(7) Residue on ignition — The residue is not more than 0.1
%.
(8) Extractable substances —
(i) Foaming test: the foam formed almost disappears wi-
thin 3 minutes.
(ii) pH: the difference in the reading of pH between the
test solution and the blank solution is not more than 1.5.
(iii) Potassium permanganate-reducing substances: The
difference in the consumption of 0.002 mol/L potassium per-
manganate VS between the test solution and the blank solu-
tion is not more than 1.0 mL.
(iv) UV spectrum: The maximum absorbance between
220 nm and 240 nm is not more than 0.08, and that between
241 nm and 350 nm is not more than 0.05.
(v) Residue on evaporation: Not more than 1 .0 mg.
(9) Cytotoxicity— IC 50 (%) is not less than 90%. The
result obtained by the other standard methods is negative.
2. Polyvinyl chloride containers for aqueous injections
The containers are composed of homopolymer of vinyl
chloride, free from any adhesive, and the plasticizer added to
the material should be di(2-ethylhexyl)phthalate. The con-
tainers may be covered with easily removable material to pre-
vent the permeation of water vapor. In this case, perform the
water vapor permeability test on the covered containers.
(1) Thickness — Measure the thickness of a container at
five different locations. The difference between the maximum
and minimum values of thickness is 0.05 mm or less.
(2) Transparency — Proceed as directed in (1) under Poly-
ethylene or polypropylene containers for aqueous injections.
(3) Appearance — Proceed as directed in (2) under Poly-
ethylene or polypropylene containers for aqueous injections.
(4) Leakage — Proceed with the test according to Leakage
test. The solution contained does not leak.
(5) Flexibility — Insert the spike needle for infusion
through a rubber closure of the container used in (4) Leak-
age. The contained solution is almost completely discharged
without displacement by air.
(6) Water vapor permeability — Proceed as directed in (3)
under Polyethylene or polypropylene containers for aqueous
injections.
(7) Heavy metals <1.07> — The turbidity of the test solu-
tion is not greater than that of the control solution when the
amount of the sample taken is 1.0 g.
(8) Lead — Perform the test as directed in Method 2. The
absorbance of the sample solution is not more than that of
the standard solution.
(9) Cadmium — Perform the test as directed in Method 2.
The absorbance of the sample solution is not more than that
of the standard solution.
(10) Tin — The absorbance of the sample solution is not
more than that of the standard solution.
(11) Vinyl chloride — Wash a cut piece of a container with
water, wipe thoroughly with a filter paper, subdivide into
pieces smaller than 5-mm square, and place 1 .0 g of them in a
20-mL volumetric flask. Add about 10 mL of tetrahydrofu-
ran for gas chromatography, dissolve by occasional shaking
in a cold place, add tetrahydrofuran for gas chro-
matography, previously cooled in a methanol-dry ice bath, to
make 20 mL while cooling in a methanol-dry ice bath, and
use this solution as the sample solution. Perform the tests as
directed under Gas Chromatography <2.02> according to the
operating conditions 1 and 2, using 10 /uL each of the sample
solution and Standard Vinyl Chloride Solution. Under either
operating condition, the peak height of vinyl chloride from
JPXV
General Tests / Test for Rubber Closure for Aqueous Infusions
127
the sample solution is not more than that from the Standard
Vinyl Chloride Solution.
Operating conditions 1 —
Detector: A hydrogen fiame-ionization detector.
Column: A column about 3 mm in inside diameter and 2 to
3 m in length, packed with 150 to 180 /am siliceous earth for
gas chromatography coated with 15% to 20% polyalkylene
glycol monoether for gas chromatography.
Column temperature: A constant temperature of between
60°C and 70°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
vinyl chloride is about 1.5 minutes.
Selection of column: Proceed with 10 iuL of Standard Vinyl
Chloride Solution under the above operating conditions. Use
a column from which vinyl chloride and ethanol are eluted in
that order, with a good resolution between their peaks.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height from 10 fiL of the Standard Vinyl Chlo-
ride Solution is 5 to 7 mm.
Operating conditions 2 —
Detector: A hydrogen flame-ionization detector. Column:
A column about 3 mm in inside diameter and about 1.5 m in
length, packed with 150 to 180 ^m porous acrylonitrile-
divinylbenzene copolymer for gas chromatography (pore
size: 0.06 - 0.08 /an; 100 - 200 m 2 /g).
Column temperature: A constant temperature of about
120°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
vinyl chloride is about 3 minutes.
Selection of column: Proceed with 10 fiL of Standard Vinyl
Chloride Solution under the above operating conditions. Use
a column from which vinyl chloride and ethanol are eluted in
that order, with a good resolution between their peaks.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height from 10 fiL of the Standard Vinyl Chlo-
ride Solution is 5 to 7 mm.
(12) Fine particles — The number of fine particles in 1 .0
mL of the test solution is counted as not more than 100 of 5
to 10 fim, not more than 10 of 10 to 25 fim and not more than
1 of 25 fim or more.
(13) Residue on ignition — The residue is not more than
0.1%.
(14) Extractable substances — Proceed as directed in (8)
under Polyethylene or polypropylene containers for aqueous
injections.
(15) Cytotoxicity — Proceed as directed in (9) under Poly-
ethylene or polypropylene containers for aqueous injections.
3. Plastic containers for aqueous injections being not
described above
The containers meet the following specifications and other
necessary specifications for their materials with regard to
heavy metals, residue on ignition and extractable substances,
etc.
(1) Transparency — Proceed as directed in (1) under Poly-
ethylene or polypropylene containers for aqueous injections.
(2) Appearance — Proceed as directed in (2) under Poly-
ethylene or polypropylene containers for aqueous injections.
(3) Vapor permeability — Proceed as directed in (3) under
Polyethylene or polypropylene containers for aqueous injec-
tions.
(4) Cytotoxicity — Proceed as directed in (9) under Poly-
ethylene or polypropylene containers for aqueous injections.
7.03 Test for Rubber Closure for
Aqueous Infusions
The rubber closure for aqueous infusions means a rubber
closure (containing material coated or laminated with the
stuff like plastics) used for a container for aqueous infusion
having a capacity of 100 mL or more, and is in direct contact
with the contained aqueous infusion. The rubber closure
when in use does not interact physically or chemically with
the contained medicament to alter any property or quality,
does not permit the invasion of microbes, does not disturb
the use of the contained infusion, and meets the following re-
quirements.
(1) Cadmium — Wash the rubber closures with water, dry
at room temperature, cut into minute pieces, mix well, place
2.0 g of them in a crucible of platinum or quartz, moisten
them with 2 mL of sulfuric acid, heat gradually to dryness,
and ignite between 450°C and 500°C until the residue is in-
cinerated. When incineration was insufficient, moisten the
residue with 1 mL of sulfuric acid, heat to dryness, and ignite
again. Repeat the above-mentioned procedure if necessary.
Cool the crucible, moisten the residue with water, add 2 to 4
mL of hydrochloric acid, heat on a water bath to dryness,
add 1 to 5 mL of hydrochloric acid, and dissolve by heating.
Then add 0.5 to 1 mL of a mixture of a solution of citric acid
monohydrate (1 in 2) and hydrochloric acid (1:1) and 0.5 to 1
mL of a warmed solution of ammonium acetate (2 in 5).
When any insoluble residue remains, filter through a glass
filter. To the solution thus obtained add 10 mL of a solution
of diammonium hydrogen citrate (1 in 4), 2 drops of
bromothymol blue TS and ammonium TS until the color of
the solution changes from yellow to green. Then add 10 mL
of ammonium sulfate solution (2 in 5) and water to make 100
mL. Next, add 20 mL of a solution of sodium 7V,7V-diethyl-
dithiocarbamate trihydrate (1 in 20), mix, allow to stand for a
few minutes, add 20.0 mL of 4-methyl-2-pentanone, and mix
by vigorous shaking. Allow to stand to separate the 4-methyl-
2-pentanone layer from the solution, filter if necessary, and
use as the sample solution. On the other hand, to 10.0 mL of
Standard Cadmium Solution add 10 mL of a solution of di-
ammonium hydrogen citrate (1 in 4) and 2 drops of
bromothymol blue TS, proceed in the same manner as for the
sample solution, and use this solution as the standard solu-
tion. Perform the tests according to the Atomic Absorption
Spectrophotometry <2.23> under the following conditions,
using the sample solution and the standard solution. The
absorbance of the sample solution is not more than that of
the standard solution.
Gas: Combustible gas — Acetylene or hydrogen
Supporting gas — Air
Lamp: Cadmium hollow-cathode lamp
Wavelength: 228.8 nm
(2) Lead — To 1.0 mL of the Standard Lead Solution add
10 mL of a solution of diammonium hydrogen citrate (1 in 4)
and 2 drops of bromothymol blue TS, proceed as directed for
the sample solution under (1), and use this solution as the
128
Sterilization and Aseptic Manipulation, etc. / General Tests
JP XV
standard solution. Perform the tests according to the Atomic
Absorption Spectrophotometry <2.23> under the following
conditions, using the sample solution obtained in (1) and the
standard solution. The absorbance of the sample solution is
not more than that of the standard solution.
Gas: Combustible gas — Acetylene or hydrogen
Supporting gas — Air
Lamp: Lead hollow-cathode lamp
Wavelength: 283.3 nm
(3) Extractable substances — Wash the rubber closures
with water, and dry at room temperature. Place them in a
glass container, add water exactly 10 times the mass of the
test material, close with a suitable stopper, heat at 121 °C for
1 hour in an autoclave, take out the glass container, allow to
cool to room temperature, then take out immediately the rub-
ber closures, and use the remaining solution as the test solu-
tion. Prepare the blank solution with water in the same man-
ner. Perform the following tests with the test solution and the
blank solution.
(i) Description: The test solution is clear and colorless.
Read the transparency of the test solution at 430 nm and 650
nm (10 mm), using the blank solution as the blank. Both of
them are not less than 99.0%.
(ii) Foam test: Place 5 mL of the test solution in a glass-
stoppered test tube of about 15 mm in inner diameter and
about 200 mm in length, and shake vigorously for 3 minutes.
The foam arisen disappears almost completely within 3
minutes.
(iii) pH <2.54>: To 20 mL each of the test solution and the
blank solution add 1.0 mL each of potassium chloride solu-
tion, prepared by dissolving 1 .0 g of potassium chloride in
water to make 1000 mL. The difference of pH between the
two solutions is not more than 1.0.
(iv) Zinc: To 10.0 mL of the test solution add diluted di-
lute nitric acid (1 in 3) to make 20 mL, and use this solution
as the sample solution. Further, to 1.0 mL of Standard Zinc
Solution for atomic absorption spectrophotometry add dilut-
ed nitric acid (1 in 3) to make exactly 20 mL, and use this so-
lution as the standard solution. Perform the tests according
to the Atomic Absorption Spectrophotometry <2.23>, using
these solutions, under the following conditions. The absor-
bance of the sample solution is not more than that of the
standard solution.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
Standard Zinc Solution for atomic absorption spec-
trophotometry: Measure exactly 10 mL of the Standard Zinc
Stock Solution, and add water to make exactly 1000 mL. Pre-
pare before use. One mL of this solution contains 0.01 mg of
zinc (Zn).
(v) Potassium Permanganate-reducing substances: Meas-
ure 100 mL of the test solution in a glass-stoppered, Erlen-
myer flask, add 10.0 mL of 0.002 mol/L potassium perman-
ganate VS and 5 mL of dilute sulfuric acid, and boil for 3
minutes. After cooling, add 0.10 g of potassium iodide, stop-
per, mix by shaking, then allow to stand for 10 minutes, and
titrate <2.50> with 0.01 mol/L sodium thiosulfate VS (indica-
tor: 5 drops of starch TS). Perform the blank test in the same
manner, using 100 mL of the blank solution. The difference
in mL of 0.002 mol/L potassium permanganate VS required
between the tests is not more than 2.0 mL.
(vi) Residue on evaporation: Measure 100 mL of the test
solution, evaporate on a water bath to dryness, and dry the
residue at 105 °C for 1 hour. The mass of the residue is not
more than 2.0 mg.
(vii) UV spectrum: Read the absorbance of the test solu-
tion between 220 nm and 350 nm against the blank solution
as directed under Ultraviolet-visible Spectrophotometry
<2.54>: it is not more than 0.20.
(4) Acute systemic toxicity — The test solution meets the
requirements, when examined under the following conditions
against the blank solution.
Preparation of the test solution and the blank solution:
Wash the rubber closures with water and Water for Injection
successively, and dry under clean conditions at room temper-
ature. Transfer the rubber closures to a glass container. Add
isotonic sodium chloride solution 10 times the mass of the
test material, stopper adequately, heat in an autoclave at 121
C C for 1 hour, take out the glass container, and allow to cool
to room temperature. The solution thus obtained is used as
the test solution. The blank solution is prepared in the same
manner.
(i) Test procedures
Test animals: Use healthy male mice of inbred strain or
from a closed colony, weighing 17 to 23 g.
Procedure: Separate the animals into two groups of 10
mice, and inject intravenously 50 mL each of the solutions
per kg body mass.
(ii) Interpretation
Observe the animals for 5 days after injection: During the
observation period, none of the animals treated with the test
solution show any abnormality or death.
(5) Pyrogen test — The test solution specified in (4) meets
the requirements of the Pyrogen Test <4.04> as does the blank
solution.
(6) Hemolysis test — When 0.1 mL of defibrinated blood
of rabbit is added to 10 mL of the test solution specified in (4)
and the mixture is allowed to stand at 37°C for 24 hours,
hemolysis is not observed. Perform the blank test in the same
manner, using 10 mL of the blank solution.
8. Other Methods
8.01 Sterilization and Aseptic
Manipulation, and Reverse
Osmosis-Ultrafiltration
(1) Sterilization and Aseptic
Manipulation
1. Sterilization
Sterilization means a process whereby the killing or
removal of all living microorganisms is accomplished. Gener-
ally, the sterilization process requires the choice of appropri-
ate procedure and accurately controlled operation and condi-
tions depending on the kind of microorganism, the condi-
tions of contamination and the quality and nature of the sub-
JPXV
General Tests / Reference Standards etc.
129
stance to be sterilized.
The adequacy of sterilization is decided by means of the
Sterility Test <4.06>.
The procedure for sterilization should be carried out after
confirming that the temperature, pressure, etc. are adequate
for the desired sterilization.
For the choice of the conditions for sterilization or verifica-
tion of the integrity of sterilization, biological indicators suit-
able for individual conditions of sterilization may be used.
2. Aseptic manipulation
Aseptic manipulation is a technique used for processing the
sterile drug products which are not terminally sterilized in
their final containers, and applied to a series of aseptic
processing of the sterile products which are prepared by the
filtration sterilization and/or with sterile raw materials.
Generally, aseptic manipulation requires the presteriliza-
tion of all equipments and materials used for processing the
sterile products, and then the products are processed in a way
to give a defined sterility assurance level in the aseptic
processing facilities where microbial and particulate levels are
adequately maintained.
(2) Reverse Osmosis-Ultrafiltration
Reverse Osmosis-Ultrafiltration is a water filtration
method by means of crucial flow filtration utilizing either a
reverse osmotic membrane or an ultrafilter, or an apparatus
combining both.
When Water for Injection is prepared by the reverse os-
mosis-ultrafiltration, pretreatment facilities, facilities for
preparation of water for injection, and facilities for sup-
plying water for injection are usually used. The pretreatment
facilities, placed before the preparation facilities, are used to
remove solid particles, dissolved salts and colloids in original
water, so as to reduce load on the preparation facilities. They
are assemblies having a cohesion apparatus, precipitation-
separation apparatus, filtration apparatus, chlorine steriliza-
tion apparatus, oxidation-reduction apparatus, residual chlo-
rine removing apparatus, precise filtration apparatus, reverse
osmosis apparatus, ultrafiltration apparatus, ion exchange
apparatus, etc., which are combined properly depending
upon the quality of original water. The facilities for prepar-
ing water for injection consist of a pretreatment water sup-
plying apparatus, ultraviolet sterilization apparatus, heat ex-
change apparatus, membrane module, cleansing-sterilization
apparatus, etc. The facilities for supplying water for injection
consist of a reservoir with a capacity to meet changing de-
mand, tubes for distributing Water for Injection, heat ex-
change apparatus, circulation pump, pressure control ap-
paratus, etc. Usually, Water for Injection prepared by the
reverse osmosis-ultrafiltration circulates in the facilities at a
temperature not lower than 80°C for prevention of microbial
proliferation.
For preparing water for Injection by means of the reverse
osmosis-ultrafiltration, use a membrane module which re-
moves microorganisms and substances of molecular masses
approximately not less than 6000.
9. Reference Standards;
Standard Solutions; Reagents,
Test Solutions; Measuring
Instruments, Appliances, etc.
Reference Standards
9.01 Reference Standards
Reference Standards are the reference substances pre-
pared to a specified quality necessary with regard to their in-
tended use as prescribed in monographs of the Phar-
macopoeia.
The Japanese Pharmacopoeia Reference Standards are as
follows:
* A: Assay
AF: Anti-factor Ha activity
AH: Potency test for anti-heparin
B: Bacterial Endotoxins Test <4.01>
C: Content ratio of active principle
D: Dissolution
DG: Digestion Test <4.03>
I: Identification
IS: Isomer ratio
M: Melting Point Determination <2.60>
P: Purity
T: Thermal Analysis <2.52>
U: Uniformity of dosage units
V: Vitamin A Assay <2.55>
Reference Standard
Intended Use*
Aceglutamide
I, P, A
Acetaminophen
I, A
Aclarubicin
A
Actinomycin D
I, A
Adrenaline Bitartrate
P
Alprostadil
I, P, A
Amikacin Sulfate
I, A
p-Aminobenzoyl Glutamic Acid
P
Amitriptyline Hydrochloride
I, D, A
Amoxicillin
I, A
Amphotericin B
I, P, A
Ampicillin
I, P, A
Anhydrous Lactose
I
Arbekacin Sulfate
I, A
Ascorbic Acid
A
Aspirin
A
Aspoxicillin
I, P, A
Astromicin Sulfate
I, A
Atropine Sulfate
I, A
Azathioprine
I, A
Azithromycin
I, A
Aztreonam
I, P, A
130
Reference Standards etc. / General Tests
JP XV
Bacampicillin Hydrochloride
I, A
Bacitracin
I, A
Baclofen
I, D, A
Baicalin
I, A
Beclometasone Dipropionate
I, A
Bekanamycin Sulfate
I, A
Benzylpenicillin Potassium
I, A
Berberine Chloride
I, A
Betamethasone
I, P, U, D, A
Betamethasone Sodium Phosphate
I, A
Betamethasone Valerate
I, A
Bisacodyl
I, A
Bleomycin A 2 Hydrochloride
A
Caffeine
A
Calcium Folinate
I, A
Calcium Oxalate Monohydrate
T
Camostat Mesilate
I, A
(/-Camphor
A
(//-Camphor
A
Carbidopa
I, P, A
Carumonam Sodium
I, P, A
Cefaclor
I, P, U, D, A
Cefadroxil
I, A
Cefalexin
A
Cefalotin Sodium
I, P, A
Cefapirin Sodium
I, A
Cefatrizine Propylene Glycolate
I, A
Cefazolin
P, A
Cefbuperazone
A
Cefcapene Pivoxil Hydrochloride
I, U, A
Cefdinir
I, P, D, A
Cefditoren Pivoxil
I, U, D, A
Cefepime Dihydrochloride
I, A
Cefixime
I, P, A
Cefmenoxime Hydrochloride
I, P, A
Cefmetazole
A
Cefminox Sodium
I, A
Cefodizime Sodium
I, P, A
Cefoperazone
A
Cefotaxime
P, A
Cefotetan
I, P, A
Cefotiam Hexetil Hydrochloride
I, P, IS, A
Cefotiam Hydrochloride
I, P, A
Cefozopran Hydrochloride
I, A
Cefpiramide
P, A
Cefpirome Sulfate
I, A
Cefpodoxime Proxetil
I, IS, A
Cefroxadine
I, A
Cefsulodin Sodium
I, P, A
Ceftazidime
I, A
Cefteram Pivoxil Mesitylene Sulfonate
A
Ceftibuten Hydrochloride
A
Ceftizoxime
P, A
Ceftriaxone Sodium
I, A
Cefuroxime Axetil
I, P, IS, A
Cefuroxime Sodium
I, A
Cellacefate
I
Chloramphenicol
I, A
Chloramphenicol Palmitate
I, A
Chloramphenicol Succinate
A
Chlordiazepoxide
I, P, A
Chlormadinone Acetate
I, A
Chlorpheniramine Maleate
I, U, A
A
A
A
D, A
D, A
D, A
Cholecalciferol I, A
Ciclacillin I, A
Ciclosporin I, P, A
Cilostazol I, U, D, A
Cisplatin I, A
Clarithromycin I, P, U, D, A
Clindamycin Hydrochloride I, U, D, A
Clindamycin Phosphate I, P, A
Clofibrate I, A
Clomifene Citrate I, A
Cloxacillin Sodium I, P, A
Colistin Sodium Methanesulfonate I, A
Colistin Sulfate A
Cortisone Acetate I, A
Cyanocobalamin I, P, A
Cycloserine I, A
Daunorubicin Hydrochloride I, A
Deferoxamine Mesilate I, A
Demethylchlortetracycline Hydrochlo-
ride I, P,
Deslanoside I, P,
Dexamethasone I, A
Dibekacin Sulfate I, P,
Diclofenamide I, P,
Dicloxacillin Sodium I, A
Diethanolammonium Fusidate A
Diethylcarbamazine Citrate A
Digitoxin I, U,
Digoxin I, U,
Dihydroergotoxine Mesilate A
Dobutamine Hydrochloride I, A
Doxorubicin Hydrochloride I, A
Doxycycline Hydrochloride I, A
Edrophonium Chloride I, A
Elcatonin A
Endotoxin 100 B
Endotoxin 10000 B
Enviomycin Sulfate A
Epirubicin Hydrochloride I, P, A
Epitiostanol P, A
Ergocalciferol I, A
Ergometrine Maleate P, U, A
Erythromycin I, P, A
Estradiol Benzoate I, P, A
Estriol I, U, D, A
Ethenzamide A
Ethinylestradiol I, U, D, A
Ethyl Aminobenzoate A
Ethyl Icosapentate I, P, A
Etoposide I, A
Faropenem Sodium I, U, A
Flomoxef Triethylammonium P, A
Fluocinolone Acetonide I, A
Fluocinonide I, A
Fluorometholone I, A
Fluoxymesterone I, A
Folic Acid I, A
Fosfestrol I, D, A
Fosfomycin Phenethylammonium A
Fradiomycin Sulfate I, A
Furosemide I, U, D, A
Fursultiamine Hydrochloride I, A
Gabexate Mesilate I, P, A
JPXV
General Tests / Reference Standards etc.
131
Gentamicin Sulfate
Ginsenoside Rbj
Ginsenoside Rg!
Gitoxin
Glycyrrhizinic Acid
Gonadorelin Acetate
Gramicidin
Griseofulvin
Guaifenesin
Heparin Sodium
High-molecular Mass Urokinase
Human Chorionic Gonadotrophin
Human Insulin
Human Menopausal Gonadotrophin
Hydrochlorothiazide
Hydrocortisone
Hydrocortisone Acetate
Hydrocortisone Sodium Phosphate
Hydrocortisone Succinate
Hypromellose Phthalate
Idarubicin Hydrochloride
Idoxuridine
Imipenem
Imipramine Hydrochloride
Indomethacin
Insulin
Interleukin-2
Isepamicin Sulfate
Isoflurane
Josamycin
Josamycin Propionate
Kallidinogenase
Kanamycin Monosulfate
Lactose
Lactulose
Lanatoside C
Latamoxef Ammonium
Lenampicillin Hydrochloride
Leucomycin A 5
Limaprost
Lincomycin Hydrochloride
Lithium Clavulanate
Low-molecular Mass Heparin
Loxoprofen
Lysozyme
Maltose
Mecobalamin
Melting Point Standard-Acetanilide
Melting Point Standard-Acetopheneti-
dine
Melting Point Standard-Caffeine
Melting Point Standard-Sulfanilamide
Melting Point Standard-Sulfapyridine
Melting Point Standard-Vanillin
Menatetrenone
Meropenem
Mestranol
Methotrexate
Methoxsalen
Methyldopa
Methylergometrine Maleate
Methylprednisolone Succinate
Methyltestosterone
I, A
Metildigoxin
I, A
I, A
Mexiletine Hydrochloride
I, P, A
I, A
Micronomicin Sulfate
I, A
P
Midecamycin
I, A
I, A
Midecamycin Acetate
I, A
I, A
Minocycline Hydrochloride
I, P, A
I, A
Mitomycin C
I, A
I, P, A
Mupirocin Lithium
P, A
I, A
Neostigmine Methylsulfate
I, A
AH, A
Netilmicin Sulfate
I, A
A
Nicotinamide
I, A
A
Nicotinic Acid
I, A
I, A
Nilvadipine
I, U, D,
A
P, A
Noradrenaline Bitartrate
P, A
I, A
Norgestrel
I, U, D,
A
I, P, A
Nystatin
I, P, A
LA
Oxytetracycline Hydrochloride
I, A
I, A
Oxytocin
P, A
I, A
Paeonifiorin
I, A
I
Panipenem
A
I, U, A
Pentobarbital
P, A
I,A
Peplomycin Sulfate
I, A
I, U, A
Perphenazine
I, U, D,
A
I, D, A
Phenethicillin Potassium
A
I, P, D, A
Phytonadione
A
P, A
Pimaricin
I, A
A
Piperacillin
A
I, P, A
Pirarubicin
I, P, A
I, P, A
Pivmecillinam Hydrochloride
I, P, A
I, C, A
Polymixin B Sulfate
I, A
LA
Potassium Sucrose Octasulfate
P, A
A
Povidone
I
I, P, A
Pravastatin 1,1,3,3-tetramethylbutylam-
I
monium
I, A
P, A
Prednisolone
I, U, D,
A
I, P, U, D, A
Prednisolone Acetate
I, A
P, A
Prednisolone Succinate
I, A
I, A
Primidone
A
C, A
Probenecid
I, D, A
P, A
Prochlorperazine Maleate
I, A
I, P, A
Progesterone
I, A
A
Protamine Sulfate
P
AF, A
Puerarin
I, A
A
Pyridoxine Hydrochloride
I, A
A
Pyrrolnitrin
I, A
A
Ranitidine Hydrochloride
I, A
I, A
Reserpine
I, U, D,
A
M
Retinol Acetate
I, v
Retinol Palmitate
I, v
M
Riboflavin
I, A
M
Ribostamycin Sulfate
I, A
M
Rifampicin
I, P, A
M
Ritodrine Hydrochloride
I, U, D,
A
M
Rokitamycin
I, A
I, P, A
Roxatidine Acetate Hydrochloride
I, U, D,
A
I, A
Roxithromycin
I, P, A
I, A
Saccharated Pepsin
A
LA
Scopolamine Hydrobromide
I, A
I, A
Sennoside A
I, A
I, A
Sennoside B
A
I, U, D, A
Serum Gonadotrophin
A
I, P, A
Siccanin
I, A
I, U, A
Sisomicin Sulfate
I, A
132
Standard Solutions for Volumetric Analysis / General Tests
JP XV
Spectinomycin Hydrochloride
I, A
Spiramycin Acetate II
C, A
Spironolactone
I, A
Streptomycin Sulfate
I, A
Sulbactam
P, A
Sulbenicillin Sodium
I, A
Sulfadiazine Silver
I, A
Sulfinpyrazone
I, A
Sultamicillin Tosilate
I, A
Swertiamarin
I, A
Talampicillin Hydrochloride
I, A
Teicoplanin
I, A
Testosterone Propionate
I, A
Tetracycline Hydrochloride
I, P, A
Thiamine Chloride Hydrochloride
I, P, A
Thiamylal
A
Thrombin
A
Tobramycin
I, A
Tocopherol
I, P, A
Tocopherol Acetate
I, A
Tocopherol Nicotinate
I, A
Tocopherol Succinate
A
Tolazamide
I, A
Tolbutamide
D
Tolnaftate
I, A
Tranexamic Acid
I, P, D, A
Triamcinolone
I, A
Triamcinolone Acetonide
I, A
Trichlormethiazide
I, U, D, A
Trichomycin
A
Trihexyphenidyl Hydrochloride
I, U, D, A
Tubocurarine Chloride Hydrochloride
I, A
Tyrosine
A, DG
Ubidecarenone
I, A
Ulinastatin
A
Vancomycin Hydrochloride
I, A
Vasopressin
A
Vinblastine Sulfate
I, U, A
Warfarin Potassium
I, U, A
Zinostatin Stimalamer.
I, A
Standard Solutions
9.21 Standard Solutions
for Volumetric Analysis
Standard Solutions for Volumetric Analysis are the solu-
tions of reagent with an accurately known concentration,
mainly used for the volumetric analysis. They are prepared to
a specified molar concentration. A 1 molar solution is a solu-
tion which contains exactly 1 mole of a specified substance in
each 1000 mL of the solution and is designated as 1 mol/L. If
necessary, these solutions are diluted to other specified molar
concentrations and the diluted solutions are also used as stan-
dard solutions. For example, 0.1 mol/L solution is obtained
by diluting 1 mol/L solution 10 times by volume.
Unless otherwise directed, standard solutions for volumet-
ric analysis should be stored in colorless or light-resistant,
glass-stoppered bottles.
Preparation and Standardization
A volumetric standard solution is prepared according to
one of the following methods. The degree of difference from
a specified concentration n (mol/L) is expressed as a factor
(molar concentration coefficient) /. Usually, standard solu-
tions are prepared so that the factor is in the range of 0.970 -
1.030. The determination procedure of the factor is called
standardization of the standard solution.
(1) Weigh accurately a quantity equivalent to about 1
mole or its multiple or a fractional mole number of the pure
substance, and dissolve it in the specified solvent to make ex-
actly 1000 mL to prepare a standard solution having a con-
centration close to the specified molarity n (mol/L). In this
case, the factor / of the standard solution is obtained by
dividing the mass of the pure substance taken (g) by the
molecular mass of the substance (g) and the specified molari-
ty number n.
When a pure substance is not obtainable, it is permissible
to use a highly purified substance whose purity has been ex-
actly determined and certified.
(2) In the case where a pure substance or a highly purified
substance is not obtainable, weigh a quantity equivalent to
about 1 mole or its multiple or a fractional mole number of
the substance specified for each standard solution and dis-
solve it in the specified solvent to make about 1000 mL to pre-
pare a standard solution having a concentration close to the
specified molarity n (mol/L). The factor /of this solution is
determined by applying the standardization procedure de-
scribed for the respective standard solution. The procedure is
classified into direct and indirect methods, as follows:
a) Direct method
Weigh accurately a standard reagent or an indicated sub-
stance specified for each standard solution, dissolve it in the
specified solvent, then titrate with the prepared standard so-
lution to be standardized, and determine the factor /by ap-
plying the following equation.
/=
1000m
VMn
M: Molecular mass equivalent to 1 mole of the standard
reagent or the specified substance (g)
m: Mass of the standard reagent or the specified substance
taken (g)
V: Volume of the prepared standard solution consumed
for the titration (mL)
n: Arithmetical mole number of the specified molar con-
centration of the standard solution to be standardized
(e.g. « = 0.02 for 0.02 mol/L standard solution)
b) Indirect method
When an appropriate standard reagent is not available, ti-
trate a defined volume V 2 (mL) of a standard solution to be
standardized with the specified standard solution having a
known factor (/), and calculate the factor (/ 2 ) by applying
the following equation.
/:
_ ^x/i
/: Factor of the titrating standard solution having a
known factor
/ 2 : Factor of the prepared standard solution to be stan-
dardized
JPXV
General Tests / Standard Solutions for Volumetric Analysis
133
V\. Volume of the titrating standard solution consumed
(mL)
V 2 : Volume of the prepared standard solution taken (mL)
(3) Standard solutions may be prepared by diluting ex-
actly an accurately measured volume of a standard solution
having a known factor, according to the specified dilution
procedure. During this dilution procedure, the original factor
of the standard solution is assumed to remain constant.
Ammonium Thiocyanate, 0.1 mol/L
1000 mL of this solution contains 7.612 g of ammonium
thiocyanate (NH 4 SCN: 76.12).
Preparation — Dissolve 8 g of ammonium thiocyanate in
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Measure exactly 25 mL of the 0.1 mol/L
silver nitrate VS, and add 50 mL of water, 2 mL of nitric acid
and 2 mL of ammonium iron (III) sulfate TS. Titrate <2.50>
the solution with the prepared ammonium thiocyanate solu-
tion to the first appearance of a persistent red-brown color
with shaking. Calculate the molarity factor.
Note: Store protected from light.
Ammonium Thiocyanate, 0.02 mol/L
1000 mL of this solution contains 1.5224 g of ammonium
thiocyanate (NH 4 SCN: 76.12).
Preparation — Before use, dilute 0.1 mol/L ammonium
thiocyanate VS with water to make exactly 5 times the initial
volume.
Ammonium Iron (III) Sulfate, 0.1 mol/L
1000 mL of this solution contains 48.22 g of ammonium
iron (III) sulfate dodecahydrate [FeNH 4 (S0 4 ) 2 .12H 2 0:
482.19].
Preparation — Dissolve 49 g of ammonium iron (III) sulfa-
te dodecahydrate in a cooled mixture of 6 mL of sulfuric acid
and 300 mL of water, add water to make 1000 mL, and stan-
dardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
ammonium iron (III) sulfate solution into an iodine flask,
add 5 mL of hydrochloric acid, and shake the mixture. Dis-
solve 2 g of potassium iodide, and stopper the flask. After al-
lowing the mixture to stand for 10 minutes, add 50 mL of
water, and titrate <2.50> the liberated iodine with 0.1 mol/L
sodium thiosulfate VS. When the solution assumes a pale yel-
low color as the end point is approached, add 3 mL of starch
TS. Continue the titration, until the blue color disappears.
Perform a blank determination. Calculate the molarity fac-
tor.
Note: Store protected from light. This solution, if stored
for a long period of time, should be restandardized.
Ammonium Iron (II) Sulfate, 0.1 mol/L
1000 mL of this solution contains 39.214 g of ammonium
iron (II) sulfate hexahydrate [Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 0:
392.14].
Preparation — Dissolve 40 g of ammonium iron (II) sulfate
hexahydrate in a cooled mixture of 30 mL of sulfuric acid
and 300 mL of water, dilute with water to make 1000 mL,
and standardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
ammonium iron (II) sulfate solution, and add 25 mL of water
and 5 mL of phosphoric acid. Titrate <2.50> the solution with
0.02 mol/L potassium permanganate VS. Calculate the
molarity factor.
Note: Prepare before use.
Ammonium Iron (II) Sulfate, 0.02 mol/L
1000 mL of this solution contains 7.843 g of ammonium
iron (II) sulfate hexahydrate [Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 0:
392.14].
Preparation — Before use, dilute 0.1 mol/L ammonium
Iron (II) sulfate VS with diluted sulfuric acid (3 in 100) to
make exactly 5 times the initial volume.
Barium chloride, 0.1 mol/L
1000 mL of this solution contains 24.426 g of barium chlo-
ride dihydrate (BaCl 2 .2H 2 0: 244.26).
Preparation — Dissolve 24.5 g of barium chloride dihydrate
in water to make 1000 mL, and standardize the solution as
follows:
Standardization — Measure exactly 20 mL of the prepared
solution, add 3 mL of hydrochloric acid, and warm the mix-
ture. Add 40 mL of diluted sulfuric acid (1 in 130), previously
warmed, heat the mixture on a water bath for 30 minutes,
and allow it to stand overnight. Filter the mixture, wash the
precipitate on the filter paper with water until the last wash-
ing shows no turbidity with silver nitrate TS, transfer the
precipitate together with the filter paper to a tared crucible,
and then heat strongly to ashes. After cooling, add 2 drops of
sulfuric acid, and heat again at about 700°C for 2 hours. Af-
ter cooling, weigh accurately the mass of the residue, and cal-
culate the molarity factor as barium sulfate (BaS0 4 ).
Each mL of 0.1 mol/L barium chloride VS
= 23.34 mg of BaS0 4
Barium Chloride, 0.02 mol/L
1000 mL of this solution contains 4.885 g of barium chlo-
ride dihydrate (BaCl 2 .2H 2 0: 244.26).
Preparation — Dissolve 4.9 g of barium chloride dihydrate
in water to make 1000 mL, and standardize the solution as
follows:
Standardization — Measure exactly 100 mL of the prepared
barium chloride solution, add 3 mL of hydrochloric acid,
and warm the mixture. Add 40 mL of diluted sulfuric acid (1
in 130), warmed previously, heat the mixture on a water bath
for 30 minutes, and allow to stand overnight. Filter the mix-
ture, wash the collected precipitate of filter paper with water
until the last washing shows no turbidity with silver nitrate
TS, transfer the precipitate together with the filter paper to a
tared crucible, and then heat strongly to ashes. After cooling,
add 2 drops of sulfuric acid, and heat strongly again at about
700°C for 2 hours. After cooling, weigh accurately the
residue as barium sulfate (BaS0 4 ), and calculate the molarity
factor.
Each mL of 0.02 mol/L barium chloride VS
= 4.668 mg of BaS0 4
Barium Chloride, 0.01 mol/L
1000 mL of this solution contains 2.4426 g of barium chlo-
ride dihydrate (BaCl 2 .2H 2 0: 244.26).
Preparation — Before use, dilute 0.02 mol/L barium chlo-
ride VS with water to make exactly twice the initial volume.
134
Standard Solutions for Volumetric Analysis / General Tests
JP XV
Barium Perchlorate, 0.005 mol/L
1000 mL of this solution contains 1.6812 g of barium per-
chlorate [Ba(C10 4 ) 2 : 336.23].
Preparation — Dissolve 1.7 g of barium perchlorate in 200
mL of water, dilute with 2-propanol to make 1000 mL, and
standardize the solution as follows:
Standardization — Measure exactly 20 mL of the prepared
barium perchlorate solution, add 55 mL of methanol and
0.15 mL of arsenazo III TS. Titrate <2.50> the solution with
0.005 mol/L sulfuric acid VS until its purple color changes
through red-purple to red. Calculate the molarity factor.
Bismuth Nitrate, 0.01 mol/L
1000 mL of this solution contains 4.851 g of bismuth
nitrate pentahydrate [Bi(N0 3 ) 3 .5H 2 0: 485.07].
Preparation— Dissolve 4.86 g of bismuth nitrate pentahy-
drate in 60 mL of dilute nitric acid, add water to make 1000
mL, and standardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
bismuth nitrate solution, add 50 mL of water and 1 drop of
xylenol orange TS, and titrate <2.50> the solution with 0.01
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
until the red color changes to yellow. Calculate the molarity
factor.
Bromine, 0.05 mol/L
1000 mL of this solution contains 7.990 g of bromine (Br:
79.90).
Preparation — Dissolve 2.8 g of potassium bromate and 15
g of potassium bromide in water to make 1000 mL, and stan-
dardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
solution into an iodine flask. Add 120 mL of water, quickly
add 5 mL of hydrochloric acid, stopper the flask immedi-
ately, and shake it gently. Then add 5 mL of potassium
iodide TS, re-stopper immediately, shake the mixture gently,
and allow to stand for 5 minutes. Titrate <2.50> the liberated
iodine with 0.1 mol/L sodium thiosulfate VS. When the solu-
tion assumes a pale yellow color as the end point is ap-
proached, add 3 mL of starch TS. Continue the titration, un-
til the blue color disappears. Perform a blank determination.
Calculate the molarity factor.
Cerium (IV) Tetraammonium Sulfate, 0.1 mol/L
1000 mL of this solution contains 63.26 g of cerium (IV)
tetraammonium sulfate dihydrate [Ce(NH 4 )4(S04)4.2H 2 0:
632.55].
Preparation — Dissolve 64 g of cerium (VI) tetraammoni-
um sulfate dihydrate in 0.5 mol/L sulfuric acid VS to make
1000 mL, allow to stand for 24 hours, filter the solution
through a glass filter (G3 or G4), if necessary, and stan-
dardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
cerium (IV) tetraammonium sulfate solution into an iodine
flask. Add 20 mL of water and 20 mL of dilute sulfuric acid,
then dissolve 1 g of potassium iodide in the mixture. Immedi-
ately titrate <2.50> the solution with 0.1 mol/L sodium thio-
sulfate VS. When the solution assumes a pale yellow color as
the end point is approached, add 3 mL of starch TS. Con-
tinue the titration, until the blue color disappears. Perform a
blank determination. Calculate the molarity factor.
Note: Store protected from light. This solution, if stored
for a long period of time, should be restandardized.
Cerium (IV) Tetraammonium Sulfate, 0.01 mol/L
1000 mL of this solution contains 6.326 g of cerium (IV)
tetraammonium sulfate dihydrate [Ce(NH 4 )4(S04)4.2H 2 0:
632.55].
Preparation — Before use, dilute 0.1 mol/L cerium (IV)
tetraammonium sulfate VS with 0.5 mol/L sulfuric acid VS
to make exactly 10 times the initial volume.
Ceric Ammonium Sulfate, 0.1 mol/L
See cerium (IV) tetraammonium sulfate, 0.1 mol/L.
Ceric Ammonium Sulfate, 0.01 mol/L
See cerium (IV) tetraammonium sulfate, 0.01 mol/L.
Disodium Dihydrogen Ethylenediamine Tetraacetate,
0.1 mol/L
1000 mL of this solution contains 37.224 g of disodium di-
hydrogen ethylenediamine tetraacetate dehydrate
(C 10 H 14 N 2 Na 2 O 8 .2H 2 O: 372.24).
Preparation — Dissolve 38 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in water to make 1000
mL, and standardize the solution as follows:
Standardization — Wash zinc (standard reagent) with dilute
hydrochloric acid, water and then acetone, dry at 110°C for 5
minutes, and allow to cool in a desiccator (silica gel). Weigh
accurately about 1.3 g of this zinc, add 20 mL of dilute
hydrochloric acid and 8 drops of bromine TS, and dissolve it
by gentle warming. Expel any excess of bromine by boiling,
and add water to make exactly 200 mL. Pipet 25 mL of this
solution, and neutralize with sodium hydroxide solution (1 in
50). Add 5 mL of ammonia-ammonium chloride buffer solu-
tion, pH 10.7, and 0.04 g of eriochrome black T-sodium
chloride indicator. Titrate <2.50> the solution with the pre-
pared disodium dihydrogen ethylenediamine tetraacetate so-
lution until the red-purple color changes to blue-purple. Cal-
culate the molarity factor.
Each mL of 0.1 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS = 6.541 mg of Zn
Note: Store in polyethylene bottles.
Disodium Dihydrogen Ethylenediamine Tetraacetate,
0.05 mol/L
1000 mL of this solution contains 18.612 g of disodium di-
hydrogen ethylenediamine tetraacetate dehydrate
(C 10 H 14 N 2 Na 2 O 8 .2H 2 O: 372.24).
Preparation — Dissolve 19 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in water to make 1000
mL, and standardize the solution as follows:
Standardization — Wash zinc (standard reagent) with dilute
hydrochloric acid, water and then acetone, dry at 110°C for 5
minutes, and allow to cool in a desiccator (silica gel). Weigh
accurately about 0.8 g of this zinc, add 12 mL of dilute
hydrochloric acid and 5 drops of bromine TS, and dissolve it
by gentle warming. Expel any excess of bromine by boiling,
and add water to make exactly 200 mL. Measure exactly 20
mL of this solution, and neutralize with sodium hydroxide
solution (1 in 50). Add 5 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.7, and 0.04 g of eriochrome black
T-sodium chloride indicator. Titrate <2.50> the solution with
the prepared disodium dihydrogen ethylenediamine tetraa-
JP XV
General Tests / Standard Solutions for Volumetric Analysis
135
cetate solution until the red-purple color changes to blue-pur-
ple. Calculate the molarity factor.
Each mL of 0.05 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS = 3.271 mg of Zn
Note: Store in polyethylene bottles.
Disodium Dihydrogen Ethylenediamine Tetraacetate,
0.02 mol/L
1000 mL of this solution contains 7.445 g of disodium di-
hydrogen ethylenediamine tetraacetate dihydrate
(C 10 H 14 N 2 Na 2 O 8 .2H 2 O: 372.24).
Preparation — Dissolve 7.5 g of disodium dihydrogen eth-
ylenediamine tetraacetate dihydrate in water to make 1000
mL, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS, but weigh accurately 0.3 g of zinc (standard
reagent), previously washed with dilute hydrochloric acid,
with water and with acetone, and cooled in a desiccator (silica
gel) after drying at 1 10°C for 5 minutes, and add 5 mL of di-
lute hydrochloric acid and 5 drops of bromine TS.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 1.308 mg of Zn
Note: Store in polyethylene bottles.
Disodium Dihydrogen Ethylenediamine Tetraacetate,
0.01 mol/L
1000 mL of this solution contains 3.7224 g of disodium di-
hydrogen ethylenediamine tetraacetate dihydrate (CioH 14 N 2
Na 2 8 .2H 2 0: 372.24).
Preparation — Before use, dilute 0.02 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS with water to
make exactly twice the initial volume.
Disodium Dihydrogen Ethylenediamine Tetraacetate,
0.001 mol/L
1000 mL of this solution contains 0.37224 g of disodium
dihydrogen ethylenediamine tetraacetate dihydrate
(C 10 H 14 N 2 Na 2 O 8 .2H 2 O: 372.24).
Preparation — Before use, dilute 0.01 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS with water to
make exactly 10 times the initial volume.
Ferric Ammonium Sulfate, 0.1 mol/L
See Ammonium Iron (III) Sulfate, 0.1 mol/L.
Ferrous Ammonium Sulfate, 0.1 mol/L
See Ammonium Iron (II) Sulfate, 0.1 mol/L.
Ferrous Ammonium Sulfate, 0.02 mol/L
See Ammonium Iron (II) Sulfate, 0.02 mol/L.
Hydrochloric Acid, 2 mol/L
1000 mL of this solution contains 72.92 g of hydrochloric
acid (HC1: 36.46).
Preparation — Dilute 180 mL of hydrochloric acid with
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Proceed as directed for standardization
under 1 mol/L hydrochloric acid VS, but weigh about 1.5 g
of sodium carbonate (standard reagent) accurately, and dis-
solve in 100 mL of water.
Each mL of 2 mol/L hydrochloric acid VS
= 106.0 mg of Na 2 C0 3
Hydrochloric Acid, 1 mol/L
1000 mL of this solution contains 36.461 g of hydrochloric
acid (HC1: 36.46).
Preparation — Dilute 90 mL of hydrochloric acid with
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization— -W eigh accurately about 0.8 g of sodium
carbonate (standard reagent), previously heated between 500
°C and 650°C for 40 to 50 minutes and allowed to cool in a
desiccator (silica gel). Dissolve it in 50 mL of water, and ti-
trate <2.50> with the prepared hydrochloric acid to calculate
the molarity factor (Indicator method: 3 drops of methyl red
TS; or potentiometric titration). In the indicator method,
when the end-point is approached, boil the content carefully,
stopper the flask loosely, allow to cool, and continue the
titration until the color of the solution changes to persistent
orange to orange-red. In the potentiometric titration, titrate
with vigorous stirring, without boiling.
Each mL of 1 mol/L hydrochloric acid VS
= 52.99 mg of Na 2 C0 3
Hydrochloric Acid, 0.5 mol/L
1000 mL of this solution contains 18.230 g of hydrochloric
acid (HC1: 36.46).
Preparation — Dilute 45 mL of hydrochloric acid with
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Proceed as directed for standardization
under 1 mol/L hydrochloric acid VS, but weigh accurately
about 0.4 g of sodium carbonate (standard reagent), and dis-
solve in 50 mL of water.
Each mL of 0.5 mol/L hydrochloric acid VS
= 26.50 mg of Na 2 C0 3
Hydrochloric Acid, 0.2 mol/L
1000 mL of this solution contains 7.292 g of hydrochloric
acid (HC1: 36.46).
Preparation — Dilute 18 mL of hydrochloric acid with
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Proceed as directed for standardization
under 1 mol/L hydrochloric acid VS, but weigh accurately
about 0.15 g of sodium carbonate (standard reagent), and
dissolve in 30 mL of water.
Each mL of 0.2 mol/L hydrochloric acid VS
= 10.60 mg of Na 2 C0 3
Hydrochloric Acid, 0.1 mol/L
1000 mL of this solution contains 3.6461 g of hydrochloric
acid (HC1: 36.46).
Preparation — Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly twice the initial volume.
Hydrochloric Acid, 0.05 mol/L
1000 mL of this solution contains 1.8230 g of hydrochloric
acid (HC1: 36.46).
136
Standard Solutions for Volumetric Analysis / General Tests
JP XV
Preparation — Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 4 times the initial volume.
Hydrochloric Acid, 0.02 mol/L
1000 mL of this solution contains 0.7292 g of hydrochloric
acid (HC1: 36.46).
Preparation — Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 10 times the initial
volume.
Hydrochloric Acid, 0.01 mol/L
1000 mL of this solution contains 0.36461 g of hydro-
chloric acid (HC1: 36.46).
Preparation — Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 20 times the initial
volume.
hydrate in freshly boiled and cooled water to make 1000 mL,
and standardize the solution as follows:
Standardization — Measure exactly 25 mL of the prepared
magnesium chloride solution. Add 50 mL of water, 3 mL of
pH 10.7 ammonia-ammonium chloride buffer solution and
0.04 g of eriochrome black T-sodium chloride indicator, and
titrate <2.50> with 0.05 mol/L disodium dihydrogen ethylene-
diamine tetraacetate VS until the red-purple color of the solu-
tion changes to blue-purple. Calculate the molarity factor.
Magnesium Chloride, 0.01 mol/L
1000 mL of this solution contains 2.0330 g of magnesium
chloride hexahydrate (MgCl 2 .6H 2 0: 203.30).
Preparation — Before use, dilute 0.05 mol/L magnesium
chloride VS with water to make exactly 5 times the initial
volume.
Hydrochloric Acid, 0.001 mol/L
1000 mL of this solution contains 0.036461 g of hydro-
chloric acid (HC1: 36.46).
Preparation — Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 200 times the initial
volume.
Iodine, 0.05 mol/L
1000 mL of this solution contains 12.690 g of iodine (I:
126.90).
Preparation — Dissolve 13 g of iodine in 100 mL of a solu-
tion of potassium iodide (2 in 5), add 1 mL of dilute
hydrochloric acid and water to make 1000 mL, and stan-
dardize the solution as follows:
Standardization — Measure exactly 15 mL of the iodine so-
lution, and titrate <2.50> with 0.1 mol/L sodium thiosulfate
VS (Indicator method: starch TS; or potentiometric titration:
platinum electrode). In the indicator method, when the solu-
tion assumes a pale yellow color as the end point is ap-
proached, add 3 mL of starch TS, and continue the titration
until the blue color disappears. Calculate the molarity factor.
Note: Store protected from light. This solution, if stored
for a long period, should be restandardized before use.
Iodine, 0.01 mol/L
1000 mL of this solution contains 2.5381 g of iodine (I:
126.90).
Preparation — Before use, dilute 0.05 mol/L iodine VS
with water to make exactly 5 times the initial volume.
Iodine, 0.005 mol/L
1000 mL of this solution contains 1.2690 g of iodine (I:
126.90).
Preparation — Before use, dilute 0.05 mol/L iodine VS with
water to make exactly 10 times the initial volume.
Iodine, 0.002 mol/L
1000 mL of this solution contains 0.5076 g of iodine (I:
126.90).
Preparation — Before use, dilute 0.05 mol/L iodine VS
with water to make exactly 25 times the initial volume.
Magnesium Chloride, 0.05 mol/L
1000 mL of this solution contains 10.165 g of magnesium
chloride hexahydrate (MgCl 2 .6H 2 0: 203.30).
Preparation — Dissolve 10.2 g of magnesium chloride hexa-
Oxalic Acid, 0.05 mol/L
1000 mL of this solution contains 6.303 g of oxalic acid
(C 2 H 2 4 .2H 2 0: 126.07).
Preparation — Dissolve 6.3 g of oxalic acid dihydrate in
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Measure exactly 25 mL of the prepared
oxalic acid solution in a 500-mL conical flask, and add 200
mL of diluted sulfuric acid (1 in 20), previously boiled for 10
to 15 minutes and then cooled to 27 ± 3°C. Transfer
freshly standardized 0.02 mol/L potassium permanganate
VS to a burette. Add quickly 22 mL of the 0.02 mol/L potas-
sium permanganate VS to the oxalic acid solution from the
burette under gentle stirring, and allow to stand until the red
color of the mixture disappears. Heat the solution up to be-
tween 55 °C and 60°C, and complete the titration <2.50> by
adding 0.02 mol/L potassium permanganate VS until a faint
red color persists for 30 seconds. Add the last 0.5 to 1 mL
dropwise, being particularly careful to allow the solution to
become decolorized before the next drop is added. Calculate
the molarity factor.
Note: Store protected from light.
Oxalic Acid, 0.005 mol/L
1000 mL of this solution contains 0.6303 g of oxalic acid
dihydrate (C 2 H 2 4 .2H 2 0: 126.07).
Preparation — Before use, dilute 0.05 mol/L oxalic acid VS
with water to make exactly 10 times the initial volume.
Perchloric Acid, 0.1 mol/L
1000 mL of this solution contains 10.046 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Add slowly 8.7 mL of perchloric acid to
1000 mL of acetic acid (100) while keeping the temperature at
about 20 °C. Allow the mixture to stand for about 1 hour.
Perform quickly the test as directed under Water Determina-
tion with 3.0 mL of the mixture, and designate the water con-
tent as A (g/dL). To the rest mixture add slowly [(A - 0.03) x
52.2] mL of acetic anhydride with shaking at about 20°C. Al-
low the solution to stand for 24 hours, and standardize it as
follows:
Standardization — Weigh accurately about 0.3 g of potassi-
um hydrogen phthalate (standard reagent), previously dried
at 105 °C for 4 hours and allowed to cool in a desiccator (sili-
ca gel). Dissolve it in 50 mL of acetic acid (100), and titrate
<2.50> the solution with the prepared perchloric acid solution
JPXV
General Tests / Standard Solutions for Volumetric Analysis
137
(Indicator method: 3 drops of crystal violet TS; or potentio-
metric titration). In the indicator method, titrate until the so-
lution acquires a blue color. Perform a blank determination.
Calculate the molarity factor.
Each mL of 0.1 mol/L perchloric acid VS
= 20.42 mg of KHC 6 H 4 (COO) 2
Note: Store protected from moisture.
Perchloric Acid, 0.05 mol/L
1000 mL of this solution contains 5.023 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Before use, dilute 0.1 mol/L perchloric acid
VS with acetic acid for nonaqueous titration to make exactly
twice the initial volume. Perform quickly the test as directed
under Water Determination with 8.0 mL of acetic acid for
nonaqueous titration, and designate the water content as A
(g/dL). If A is not less than 0.03, add [(A -0.03) x 52.2] mL
of acetic anhydride to 1000 mL of acetic acid for nonaqueous
titration, and use it for the preparation.
Perchloric Acid, 0.02 mol/L
1000 mL of this solution contains 2.0092 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Before use, dilute 0.1 mol/L perchloric acid
VS with acetic acid for nonaqueous titration to make exactly
5 times the initial volume. Perform quickly the test as direct-
ed under Water Determination with 8.0 mL of acetic acid for
nonaqueous titration, and designate the water content as A
(g/dL). If A is not less than 0.03, add [(A -0.03) x 52.2] mL
of acetic anhydride to 1000 mL of acetic acid for nonaqueous
titration, and use it for the preparation.
Perchloric Acid-l,4-Dioxane, 0.1 mol/L
1000 mL of this solution contains 10.046 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Dilute 8.5 mL of perchloric acid with 1,4-di-
oxane to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Weigh accurately about 0.5 g of potassi-
um hydrogen phthalate (standard reagent), previously dried
at 105 °C for 4 hours and allowed to cool in a desiccator (sili-
ca gel). Dissolve it in 80 mL of acetic acid for nonaqueous
titration, and add 3 drops of crystal violet TS. Titrate <2.50>
the solution with the prepared perchloric acid-1 ,4-dioxane so-
lution until it acquires a blue color. Perform a blank determi-
nation. Calculate the molarity factor.
Each mL of 0.1 mol/L perchloric acid-1, 4-dioxane VS
= 20.42 mg of KHC 6 H 4 (COO) 2
Note: Store in a cold place, protected from moisture.
Perchloric Acid-1, 4-Dioxane, 0.05 mol/L
1000 mL of this solution contains 5.023 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Before use, dilute 0.1 mol/L perchloric acid-
1, 4-dioxane VS with 1, 4-dioxane to make exactly twice the in-
itial volume.
Perchloric Acid-1, 4-Dioxane, 0.004 mol/L
1000 mL of this solution contains 0.4018 g of perchloric
acid (HC10 4 : 100.46).
Preparation — Before use, dilute 0.1 mol/L perchloric acid-
1, 4-dioxane VS with 1, 4-dioxane to make exactly 25 times the
initial volume.
Potassium Bichromate, 1/60 mol/L
See Potassium Dichromate, 1/60 mol/L
Potassium Bromate, 1/60 mol/L
1000 mL of this solution contains 2.7833 g of potassium
bromate (KBr0 3 : 167.00).
Preparation — Dissolve 2.8 g of potassium bromate in
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Measure exactly 25 mL of the prepared
potassium bromate solution into an iodine flask. Add 2 g of
potassium iodide and 5 mL of dilute sulfuric acid, stopper
the flask, and allow the solution to stand for 5 minutes. Add
100 mL of water, and titrate <2.50> the liberated iodine with
0.1 mol/L sodium thiosulfate VS. When the solution as-
sumes a pale yellow color as the end point is approached, add
3 mL of starch TS. Continue the titration until the blue color
disappears. Perform a blank determination. Calculate the
molarity factor.
Potassium Dichromate, 1/60 mol/L
1000 mL of this solution contains 4.903 g of potassium
dichromate (K 2 Cr 2 7 : 294.18).
Preparation — Weigh accurately about 4.903 g of potassi-
um dichromate (standard reagent), previously powdered,
dried between 100°C and 110°C for 3 to 4 hours and allowed
to cool in a desiccator (silica gel), dissolve it in water to make
exactly 1000 mL, and calculate the molarity factor.
Potassium Ferricyanide, 0.1 mol/L
See Potassium Hexacyanoferrate (III), 0.1 mol/L
Potassium Ferricyanide, 0.05 mol/L
See Potassium Hexacyanoferrate (III), 0.05 mol/L.
Potassium Hexacyanoferrate (III), 0.1 mol/L
1000 mL of this solution contains 32.924 g of potassium
hexacyanoferrate (III) [K 3 Fe(CN) 6 : 329.24].
Preparation — Dissolve 33 g of potassium hexacyanoferrate
(III) in water to make 1000 mL, and standardize the solution
as follows:
Standardization — Measure exactly 25 mL of the prepared
potassium hexacyanoferrate (III) solution into an iodine
flask. Add 2 g of potassium iodide and 10 mL of dilute hy-
drochloric acid, stopper the flask, and allow to stand for 15
minutes. Add 15 mL of zinc sulfate TS, and titrate <2.50> the
liberated iodine with 0.1 mol/L sodium thiosulfate VS.
When the solution assumes a pale yellow color as the end
point is approached, add 3 mL of starch TS. Continue the
titration, until the blue color disappears. Perform a blank de-
termination. Calculate the molarity factor.
Note: Store protected from light. This solution, if stored
for a long period, should be restandardized.
Potassium Hexacyanoferrate (III), 0.05 mol/L
1000 mL of this solution contains 16.462 g of potassium
hexacyanoferrate (III) [K 3 Fe(CN) 6 : 329.24].
Preparation — Before use, dilute 0.1 mol/L potassium hex-
acyanoferrate (III) VS with water to make exactly twice the
initial volume.
138
Standard Solutions for Volumetric Analysis / General Tests
JP XV
Potassium Hydroxide, 1 moI/L
1000 mL of this solution contains 56.11 g of potassium hy-
droxide (KOH: 56.11).
Preparation — Dissolve 65 g of potassium hydroxide in 950
mL of water. Add a freshly prepared, saturated solution of
barium hydroxide octahydrate until no more precipitate is
produced. Shake the mixture thoroughly, and allow it to
stand for 24 hours in a tightly stoppered bottle. Decant the
supernatant liquid or filter the solution through a glass filter
(G3 or G4), and standardize the solution as follows:
Standardization — Weigh accurately about 2.5 g of
amidosulfuric acid (standard reagent), previously dried in a
desiccator (in vacuum, silica gel) for about 48 hours. Dissolve
it in 25 mL of freshly boiled and cooled water, and add 2
drops of bromothymol blue TS. Titrate <2.50> the solution
with the prepared potassium hydroxide solution until it ac-
quires a green color. Calculate the molarity factor.
Each mL of 1 mol/L potassium hydroxide VS
= 97.09 mg of HOS0 2 NH 2
Note: Store in tightly stoppered bottles or in containers
provided with a carbon dioxide-absorbing tube (soda-lime).
This solution, if stored for a long period, should be restan-
dardized.
Potassium Hydroxide, 0.5 mol/L
1000 mL of this solution contains 28.053 g of potassium
hydroxide (KOH: 56.11).
Preparation — Weigh 32 g of potassium hydroxide, proceed
as directed for preparation under 1 mol/L potassium hydrox-
ide VS, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 1 mol/L potassium hydroxide VS, but weigh accurate-
ly about 1.3 g of amidosulfuric acid (standard reagent).
Each mL of 0.5 mol/L potassium hydroxide VS
= 48.55 mg of HOS0 2 NH 2
Note: Store as directed under 1 mol/L potassium hydrox-
ide VS. This solution, if stored for a long period, should be
restandardized.
Potassium Hydroxide, 0.1 mol/L
1000 mL of this solution contains 5.611 g of potassium hy-
droxide (KOH: 56.11).
Preparation — Weigh 6.5 g of potassium hydroxide, pro-
ceed as directed for preparation under 1 mol/L potassium
hydroxide VS, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 1 mol/L potassium hydroxide VS, but weigh accurate-
ly about 0.25 g of amidosulfuric acid (standard reagent).
Each mL of 0.1 mol/L potassium hydroxide VS
= 9.709 mg of HOS0 2 NH 2
Note: Store as directed under 1 mol/L potassium hydrox-
ide VS. This solution, if stored for a long period, should be
restandardized.
Potassium Hydroxide-Ethanol, 0.5 mol/L
1000 mL of this solution contains 28.053 g of potassium
hydroxide (KOH: 56.11).
Preparation — Dissolve 35 g of potassium hydroxide in 20
mL of water, and add aldehyde-free ethanol to make 1000
mL. Allow the solution to stand for 24 hours in a tightly stop-
pered bottle. Then quickly decant the supernatant liquid, and
standardize the solution as follows:
Standardization — Measure exactly 15 mL of 0.25 mol/L
sulfuric acid VS, add 50 mL of water, and titrate with the
prepared potassium hydroxide-ethanol solution to calculate
the molarity factor (Indicator method: 2 drops of
phenolphthalein TS; or potentiometric titration). In the indi-
cator method, titrate <2.50> until the solution acquires a pale
red color.
Note: Store in tightly stoppered bottles, protected from
light. Standardize before use.
Potassium Hydroxide-Ethanol, 0.1 mol/L
1000 mL of this solution contains 5.611 g of potassium hy-
droxide (KOH: 56.11).
Preparation — Weigh 7 g of potassium hydroxide, proceed
as directed for preparation under 0.5 mol/L potassium hy-
droxide-ethanol VS, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 0.5 mol/L potassium hydroxide-ethanol VS, but meas-
ure exactly 15 mL of 0.05 mol/L sulfuric acid VS.
Note: Store as directed under 0.5 mol/L potassium
hydroxide-ethanol VS. Standardize before use.
Potassium Iodate, 0.05 mol/L
1000 mL of this solution contains 10.700 g of potassium io-
date (KI0 3 : 214.00).
Preparation — Weigh accurately about 10.700 g of potassi-
um iodate (standard reagent), previously dried between 120°C
and 140°C for 1.5 to 2 hours and allowed to cool in a
desiccator (silica gel), and dissolve it in water to make exactly
1000 mL. Calculate the molarity factor.
Potassium Iodate, 1/60 mol/L
1000 mL of this solution contains 3.567 g of potassium io-
date (KI0 3 : 214.00).
Preparation — Weigh accurately about 3.567 g of potassi-
um iodate (standard reagent), previously dried between
120°C and 140°C for 2 hours and allowed to cool in a desic-
cator (silica gel), and dissolve it in water to make exactly 1000
mL. Calculate the molarity factor.
Potassium Iodate, 1/1200 mol/L
1000 mL of this solution contains 0.17833 g of potassium
iodate (KI0 3 : 214.00).
Preparation — Weigh accurately about 0.17833 g of potas-
sium iodate, previously dried between 120°C and 140°C for
1.5 to 2 hours and allowed to cool in a desiccator (silica gel),
and dissolve it in water to make exactly 1000 mL. Calculate
the molarity factor.
Potassium Permanganate, 0.02 mol/L
1000 mL of this solution contains 3.1607 g of potassium
permanganate (KMn0 4 : 158.03).
Preparation — Dissolve 3.2 g of potassium permanganate
in water to make 1000 mL, and boil the solution for 15
minutes. Allow the solution to stand for at least 48 hours in a
tightly stoppered flask, and filter it through a glass filter (G3
or G4). Standardize the solution as follows:
Standardization — Weigh accurately about 0.3 g of sodium
oxalate (standard reagent), previously dried between 150°C
and 200°C for 1 to 1.5 hours and allowed to cool in a desicca-
JPXV
General Tests / Standard Solutions for Volumetric Analysis
139
tor (silica gel), transfer it to a 500 mL conical flask, dissolve
in 30 mL of water, add 250 mL of diluted sulfuric acid (1 in
20), and warm the mixture between 30°C and 35 °C. Transfer
the prepared potassium permanganate solution to a buret,
add quickly 40 mL of the solution under gentle stirring from
the buret, and allow to stand until the red color of the mix-
ture disappears. Warm the solution between 55°C and 60°C,
and complete the titration <2.50> with the potassium per-
manganate solution until a faint red color persists for 30 sec-
onds. Add the last 0.5 to 1 mL dropwise before the end point,
being particularly careful to allow the solution to be decolo-
rized before the next drop is added. Calculate the molarity
factor.
Each mL of 0.02 mol/L potassium permanganate VS
= 6.700 mg of Na 2 C 2 4
Note: Store protected from light. This solution, if stored
for a long period, should be restandardized.
Potassium Permanganate, 0.002 mol/L
1000 mL of this solution contains 0.31607 g of potassium
permanganate (KMn0 4 : 158.03).
Preparation — Before use, dilute 0.02 mol/L potassium per-
manganate VS with water to make exactly 10 times the initial
volume.
Silver Nitrate, 0.1 mol/L
1000 mL of this solution contains 16.987 g of silver
nitrate (AgN0 3 : 169.87).
Preparation — Dissolve 17.0 g of silver nitrate in water to
make 1000 mL, and standardize the solution as follows:
Standardization — Weigh accurately about 80 mg of sodi-
um chloride (standard reagent), previously dried between
500°C and 650°C for 40 to 50 minutes and allowed to cool in
a desiccator (silica gel), dissolve it in 50 mL of water, and ti-
trate <2.50> under vigorous stirring with the prepared silver
nitrate solution to calculate the molarity factor (Indicator
method: 3 drops of fluorescein sodium TS; or potentiometric
titration: silver electrode). In the indicator method, titrate
until the color of the solution changes from yellow-green to
orange through yellow.
Each mL of 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
Note: Store protected from light.
Silver Nitrate, 0.02 mol/L
1000 mL of this solution contains 3.3974 g of silver
nitrate (AgN0 3 : 169.87).
Preparation — Before use, dilute 0.1 mol/L silver nitrate
VS with water to make exactly 5 times the initial volume.
Silver Nitrate, 0.01 mol/L
1000 mL of this solution contains 1.6987 g of silver
nitrate (AgN0 3 : 169.87).
Preparation — Before use, dilute 0.1 mol/L silver nitrate
VS with water to make exactly 10 times the initial volume.
Silver Nitrate, 0.005 mol/L
1000 mL of this solution contains 0.8494 g of silver
nitrate (AgN0 3 : 169.87).
Preparation — Before use, dilute 0.1 mol/L silver nitrate
VS with water to make exactly 20 times the initial volume.
Silver Nitrate, 0.001 mol/L
1000 mL of this solution contains 0.16987 g of silver ni-
trate (AgN0 3 : 169.87).
Preparation — Dilute 0.1 mol/L silver nitrate VS with
water to make exactly 100 times of the initial volume before
Sodium Acetate, 0.1 mol/L
1000 mL of this solution contains 8.203 g of sodium
acetate (CH 3 COONa: 82.03).
Preparation — Dissolve 8.20 g of anhydrous sodium
acetate in acetic acid (100) to make 1000 mL, and standardize
the solution as follows:
Standardization — Pipet 25 mL of the prepared sodium ac-
etate solution, add 50 mL of acetic acid (100) and 1 mL of p-
naphtholbenzeine TS, and titrate <2.50> with 0.1 mol/L per-
chloric acid VS until a yellow-brown color changes through
yellow to green. Perform a blank determination. Calculate
the molarity factor.
Sodium Hydroxide, 1 mol/L
1000 mL of this solution contains 39.997 g of sodium hy-
droxide (NaOH: 40.00).
Preparation — Dissolve 42 g of sodium hydroxide in 950
mL of water. Add a freshly prepared, saturated solution of
barium hydroxide octahydrate until no more precipitate is
produced. Mix well the mixture, and allow to stand for 24
hours in a tightly stoppered bottle. Decant the supernatant
liquid or filter the solution through a glass filter (G3 or G4),
and standardize the solution as follows:
Standardization— Weigh accurately about 1.5 g of
amidosulfuric acid (standard reagent), previously dried in a
desiccator (in vacuum, silica gel) for about 48 hours. Dissolve
it in 25 mL of freshly boiled and cooled water, and titrate
<2.50> the solution with the prepared sodium hydroxide solu-
tion to calculate the molarity factor (Indicator method: 2
drops of bromothymol blue TS; or potentiometric titration).
In the indicator method, titrate until the solution acquires a
green color.
Each mL of 1 mol/L sodium hydroxide VS
= 97.09 mg of HOS0 2 NH 2
Note: Store in tightly stoppered bottles or in containers
provided with a carbon dioxide-absorbing tube (soda lime).
This solution, if stored for a long period, should be restan-
dardized.
Sodium Hydroxide, 0.5 mol/L
1000 mL of this solution contains 19.999 g of sodium
hydroxide (NaOH: 40.00).
Preparation — Weigh 22 g of sodium hydroxide, proceed as
directed for preparation under 1 mol/L sodium hydroxide
VS, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 1 mol/L sodium hydroxide VS, but weigh accurately
about 0.7 g of amidosulfuric acid (standard reagent).
Each mL of 0.5 mol/L sodium hydroxide VS
= 48.55 mg of HOS0 2 NH 2
Note: Store as directed under 1 mol/L sodium hydroxide
VS. This solution, if stored for a long period, should be
restandardized.
140
Standard Solutions for Volumetric Analysis / General Tests
JP XV
Sodium Hydroxide, 0.2 mol/L
1000 mL of this solution contains 7.999 g of sodium
hydroxide (NaOH: 40.00).
Preparation — Weigh 9 g of sodium hydroxide, proceed as
directed for preparation under 1 mol/L sodium hydroxide
VS, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 1 mol/L sodium hydroxide VS, but weigh accurately
about 0.3 g of amidosulfuric acid (standard reagent).
Each mL of 0.2 mol/L sodium hydroxide VS
= 19.42 mg of HOS0 2 NH 2
Note: Store as directed under 1 mol/L sodium hydroxide
VS. This solution, if stored for a long period, should be
restandardized.
Sodium Hydroxide, 0.1 mol/L
1000 mL of this solution contains 3.9997 g of sodium
hydroxide (NaOH: 40.00).
Preparation — Weigh 4.5 g of sodium hydroxide, proceed
as directed for preparation under 1 mol/L sodium hydroxide
VS, and standardize the solution as follows.
Standardization — Proceed as directed for standardization
under 1 mol/L sodium hydroxide VS, but weigh accurately
about 0.15 g of amidosulfuric acid (standard reagent).
Each mL of 0.1 mol/L sodium hydroxide VS
= 9.709 mg of HOS0 2 NH 2
Note: Store as directed under 1 mol/L sodium hydroxide
VS. This solution, if stored for a long period, should be
restandardized.
Sodium Hydroxide, 0.05 mol/L
1000 mL of this solution contains 1.9999 g of sodium
hydroxide (NaOH: 40.00).
Preparation— Before use, dilute 0.1 mol/L sodium
hydroxide VS with freshly boiled and cooled water to make
exactly twice the initial volume.
Sodium Hydroxide, 0.02 mol/L
1000 mL of this solution contains 0.7999 g of sodium
hydroxide (NaOH: 40.00).
Preparation — Before use, dilute 0.1 mol/L sodium
hydroxide VS with freshly boiled and cooled water to make
exactly 5 times the initial volume.
Sodium Hydroxide, 0.01 mol/L
1000 mL of this solution contains 0.39997 g of sodium
hydroxide (NaOH: 40.00).
Preparation — Before use, dilute 0.1 mol/L sodium
hydroxide VS with freshly boiled and cooled water to make
exactly 10 times the initial volume.
Sodium Lauryl Sulfate, 0.01 mol/L
1000 mL of this solution contains 2.8838 g of sodium
lauryl sulfate (C 12 H 25 Na0 4 S: 288.38).
Preparation — Dissolve 2.9 g of sodium lauryl sulfate in
water to make 1000 mL, and standardize the solution as fol-
lows:
Standardization — Weigh accurately about 0.3 g of papa-
verine hydrochloride for assay, previously dried, and dissolve
in water to make exactly 100 mL. Pipet 10 mL of this solu-
tion into a glass-stoppered conical flask, add 5 mL each of
water and dilute sulfuric acid and 60 mL of dichloromethane,
then add 5 to 6 drops of a solution of methyl yellow in dichlo-
romethane (1 in 500) as indicator, and titrate <2.50>, while
vigorous shaking, with the sodium lauryl sulfate solution
prepereed above, using a buret with a minimum graduation
of 0.02 mL. End point is reached when the color of the
dichloromethane layer changes from yellow to orange-red af-
ter dropwise addition of the sodium lauryl sulfate solution,
vigorous shaking and standing for a while.
Each mL of 0.01 mol/L sodium lauryl sulfate VS
= 3.759 mg of C 20 H 21 NO 4 .HCl
Sodium Methoxide, 0.1 mol/L
1000 mL of this solution contains 5.402 g of sodium meth-
oxide (CH 3 ONa: 54.02).
Preparation — Add little by little 2.5 g of freshly cut sodi-
um pieces to 150 mL of methanol cooled in ice-water. After
the sodium has dissolved, add benzene to make 1000 mL, and
standardize the solution as follows:
Standardization — Weigh accurately about 0.3 g of benzoic
acid, previously dried for 24 hours in a desiccator (silica gel),
dissolve it in 80 mL of /V,/V-dimethylformamide, and add 3
drops of thymol blue-/V,/V-dimethylformamide TS. Titrate
<2.50> the solution with the prepared sodium methoxide solu-
tion until a blue color appears. Perform a blank determina-
tion. Calculate the molarity factor.
Each mL of 0.1 mol/L sodium methoxide VS
= 12.21 mg of C 6 H 5 COOH
Note: Store in a cold place, protected from moisture. Stan-
dardize before use.
Sodium Methoxide-Dioxane, 0.1 mol/L
See Sodium Methoxide-l,4-Dioxane, 0.1 mol/L.
Sodium Methoxide-l,4-Dioxane, 0.1 mol/L
1000 mL of this solution contains 5.402 g of sodium meth-
oxide (CH 3 ONa: 54.02).
Preparation — Add little by little 2.5 g of freshly cut sodi-
um pieces to 150 mL of methanol cooled in ice-water. After
the sodium has dissolved, add 1,4-dioxane to make 1000 mL,
and standardize the solution as follows:
Standardization — Weigh accurately about 0.3 g of benzoic
acid, previously dried in a desiccator (silica gel) for 24 hours,
dissove it in 80 mL of /V,/V-dimethylformamide, and add 3
drops of thymol blue-A^TV-dimethylformamide TS. Titrate
<2.50> the solution with the prepared sodium methoxide-1,4-
dioxane solution until a blue color appears. Perform a blank
determination. Calculate the molarity factor.
Each mL of 0.1 mol/L sodium methoxide-l,4-dioxane VS
= 12.21 mg of QH5COOH
Note: Store in a cold place, protected from moisture. Stan-
dardize before use.
Sodium Nitrite, 0.1 mol/L
1000 mL of this solution contains 6.900 g of sodium
nitrite (NaN0 2 : 69.00).
Preparation — Dissolve 7.2 g of sodium nitrite in water to
make 1000 mL, and standardize the solution as follows:
JPXV
General Tests / Standard Solutions for Volumetric Analysis
141
Standardization — Weigh accurately about 0.44 g of sulfa-
nilamide for titration of diazotization, previously dried at
105 °C for 3 hours and allowed to cool in a desiccator (silica
gel), dissolve in 10 mL of hydrochloric acid, 40 mL of water
and 10 mL of a solution of potassium bromide (3 in 10), cool
below 15°C, and titrate with the prepared sodium nitrite so-
lution as directed in the potentiometric titration or ampero-
metric titration under Endpoint Detection Methods in Titri-
metry <2.50>. Calculate the molarity factor.
Each mL of 0.1 mol/L sodium nitrite VS
= 17.22 mg of H 2 NC 6 H 4 S0 2 NH 2
Note: Store protected from light. This solution, if stored
for a long period, should be restandadized.
Sodium Oxalate, 0.005 mol/L
1000 mL of this solution contains 0.6700 g of sodium oxa-
late (Na 2 C 2 4 : 134.00).
Preparation— Weigh accurately about 0.6700 g of sodium
oxalate (standard reagent), previously dried between 150°C
and 200°C for 2 hours and allowed to cool in a desiccator (sil-
ica gel), dissolve it in water to make exactly 1000 mL, and cal-
culate the molarity factor.
Sodium Tetraphenylborate, 0.02 mol/L
1000 mL of this solution contains 6.844 g of sodium tetra-
phenylborate [NaB(C 6 H 5 ) 4 : 342.22].
Preparation — Dissolve 7.0 g of sodium tetraphenylborate
in water to make 1000 mL, and standardize the solution as
follows:
Standardization — Weigh 0.5 g of potassium hydrogen
phthalate (standard reagent), dissolve it in 100 mL of water,
add 2 mL of acetic acid (31), and warm to 50°C in a water
bath. Add slowly 50 mL of the prepared sodium tetra-
phenylborate solution under stirring from a buret, then cool
the mixture quickly, and allow to stand for 1 hour at room
temperature. Transfer the precipitate to a tared glass filter
(G4), wash with three 5 mL portions of potassium tetra-
phenylborate TS, dry at 105 °C for 1 hour, and weigh ac-
curately the glass filter. Calculate the molarity factor from
the mass of potassium tetraphenylborate [KB(C 6 H 5 ) 4 :
358.32].
Each mL of 0.02 mol/L sodium tetraphenylborate VS
= 7.167 mg of KB(QH 5 ) 4
Note: Prepare before use.
Sodium Tetraphenylboron, 0.02 mol/L
See Sodium Tetraphenylborate, 0.02 mol/L.
Sodium Thiosulfate, 0.1 mol/L
1000 mL of this solution contains 24.818 g of sodium thio-
sulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Dissolve 25 g of sodium thiosulfate and 0.2
g of anhydrous sodium carbonate in freshly boiled and
cooled water to make 1000 mL, allow to stand for 24 hours,
and standardize the solution as follows:
Standardization — Weigh accurately about 50 mg of potas-
sium iodate (standard reagent), previously dried between
120°C and 140°C for 1.5 to 2 hours and allowed to cool in a
desiccator (silica gel), and transfer to an iodine flask. Dis-
solve it in 25 mL of water, add 2 g of potassium iodide and 10
mL of dilute sulfuric acid, and stopper the flask. After allow-
ing the mixture to stand for 10 minutes, add 100 mL of water,
and titrate <2.50> the liberated iodine with the prepared sodi-
um thiosulfate solution (Indicator method; or potentiometric
titration: platinum electrode). In the indicator method, when
the solution assumes a pale yellow color as the end point is
approached, add 3 mL of starch TS. Continue the titration,
until the blue color disappears. Perform a blank determina-
tion. Calculate the molarity factor.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 3.567 mg of KI0 3
Note: This solution, if stored for a long period, should be
restandardized.
Sodium Thiosulfate, 0.05 mol/L
1000 mL of this solution contains 12.409 g of sodium thio-
sulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Before use, dilute 0.1 mol/L sodium thio-
sulfate VS with freshly boiled and cooled water to make ex-
actly 2 times the initial volume.
Sodium Thiosulfate, 0.02 mol/L
1000 mL of this solution contains 4.964 g of sodium thio-
sulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Before use, dilute 0.1 mol/L sodium thio-
sulfate VS with freshly boiled and cooled water to make ex-
actly 5 times the initial volume.
Sodium Thiosulfate, 0.01 mol/L
1000 mL of this solution contains 2.4818 g of sodium thio-
sulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Before use, dilute 0.1 mol/L sodium thio-
sulfate VS with freshly boiled and cooled water to make ex-
actly 10 times the initial volume.
Sodium Thiosulfate, 0.005 mol/L
1000 mL of this solution contains 1.2409 g of sodium thio-
sulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Before use, dilute 0.1 mol/L sodium thio-
sulfate VS with freshly boiled and cooled water to make ex-
actly 20 times the initial volume.
Sodium Thiosulfate, 0.002 mol/L
1000 mL of this solution contains 0.4964 g of sodium
thiosulfate pentahydrate (Na 2 S 2 3 .5H 2 0: 248.18).
Preparation — Before use, dilute 0.1 mol/L sodium thiosul-
fate VS with freshly boiled and cooled water to make exactly
50 times the initial volume.
Sulfuric Acid, 0.5 mol/L
1000 mL of this solution contains 49.04 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Add slowly, under stirring, 30 mL of sulfu-
ric acid to 1000 mL of water, allow to cool, and standardize
the solution as follows:
Standardization — Weigh accurately about 0.8 g of sodium
carbonate (standard reagent), previously heated between
500°C and 650°C for 40 to 50 minutes and allowed to cool in
a desiccator (silica gel). Dissolve it in 50 mL of water, and ti-
trate <2.50> the solution with the prepared sulfuric acid (Indi-
cator method: 3 drops of methyl red TS; or potentiometric
titration). In the indicator method, when the end point is ap-
142
Standard Solutions for Volumetric Analysis / General Tests
JP XV
proached, boil the solution carefully, stopper the flask loose-
ly, allow to cool, and continue the titration, until the color of
the solution changes to persistent orange to orange-red. Cal-
culate the molarity factor. In the potentiometric titration, ti-
trate with vigorous stirring without boiling.
Each mL of 0.5 mol/L sulfuric acid VS
= 52.99 mg of Na 2 C0 3
Sulfuric Acid, 0.25 mol/L
1000 mL of this solution contains 24.520 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Add slowly, under stirring, 15 mL of sulfu-
ric acid to 1000 mL of water, allow to cool, and standardize
the solution as follows:
Standardization — Proceed as directed for standardization
under 0.5 mol/L sulfuric acid VS, but weigh accurately about
0.4 g of sodium carbonate (standard reagent), and dissolve in
50 mL of water.
Each mL of 0.25 mol/L sulfuric acid VS
= 26.50 mg of Na 2 C0 3
Sulfuric Acid, 0.1 mol/L
1000 mL of this solution contains 9.808 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Add slowly, under stirring, 6 mL of sulfuric
acid to 1000 mL of water, allow to cool, and standardize the
solution as follows:
Standardization — Proceed as directed for standardization
under 0.5 mol/L sulfuric acid VS, but weigh accurately about
0.15 g of sodium carbonate (standard reagent), and dissolve
in 50 mL of water.
Each mL of 0.1 mol/L sulfuric acid VS
= 10.60 mg of Na 2 C0 3
Sulfuric Acid, 0.05 mol/L
1000 mL of this solution contains 4.904 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Add slowly, under stirring, 3 mL of sulfuric
acid to 1000 mL of water, allow to cool, and standardize the
solution as follows:
Standardization — Proceed as directed for standardization
under 0.5 mol/L sulfuric acid VS, but weigh accuretely about
80 mg of sodium carbonate (standard reagent), and dissolve
in 30 mL of water.
Each mL of 0.05 mol/L sulfuric acid VS
= 5.299 mg of Na 2 C0 3
Sulfuric Acid, 0.025 mol/L
1000 mL of this solution contains 2.4520 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Before use, dilute 0.05 mol/L sulfuric acid
VS with water to make exactly twice the initial volume.
Sulfuric Acid, 0.01 mol/L
1000 mL of this solution contains 0.9808 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Before use, dilute 0.05 mol/L sulfuric acid
VS with water to make exactly 5 times the initial volume.
Sulfuric Acid, 0.005 mol/L
1000 mL of this solution contains 0.4904 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Before use, dilute 0.05 mol/L sulfuric acid
VS with water to make exactly 10 times the initial volume.
Surfuric Acid, 0.0005 mol/L
1000 mL of this solution contains 0.04904 g of sulfuric acid
(H 2 S0 4 : 98.08).
Preparation — Before use, dilute 0.05 mol/L sulfuric acid
VS with water to make exactly 100 times the initial volume.
Tetrabutylammonium Hydroxide, 0.1 mol/L
1000 mL of this solution contains 25.947 g of tetrabutyl
ammonium hydroxide [(C 4 H 9 ) 4 NOH: 259.47].
Preparation — Before use, dilute a volume of 10% tetra-
butylammonium hydroxide-methanol TS, equivalent to 26.0
g of tetrabutylammonium hydroxide, with 2-propanol to
make 1000 mL, and standardize the solution as follows:
Standardization — Weigh accurately about 0.3 g of benzoic
acid, previously dried in a desiccator (silica gel) for 24 hours,
dissolve it in 50 mL of acetone, and titrate <2.50> the solution
with the prepared tetrabutylammonium hydroxide solution
(potentiometric titration). Perform a blank determination in
the same manner.
Each mL of 0.1 mol/L tetrabutylammonium hydroxide VS
= 12.21 mg of C 6 H 5 COOH
Note: Store in tightly stoppered bottles. This solution, if
stored for a long period, should be restandardized.
Tetramethylammonium Hydroxide, 0.2 mol/L
1000 mL of this solution contains 18.231 g of tetramethyl-
ammonium hydroxide [(CH 3 ) 4 NOH: 91.15].
Preparation — Before use, dilute a volume of tetramethyl-
ammonium hydroxide-methanol TS, equivalent to 18.4 g of
tetramethylammonium hydroxide, with water to make 1000
mL, and standardize the solution as follows:
Standardization — Weigh accurately about 0.4 g of benzoic
acid, previously dried in a desiccator (silica gel) for 24 hours,
dissolve it in 60 mL of iV,./V-dimethylformamide, and titrate
<2.50> the solution with the prepared 0.2 mol/L tetramethyl
ammonium hydroxide solution (Indicator method: 3 drops of
thymol blue-/V,7V-dimethylformamide TS; or potentiometric
titration). In the indicator method, titrate until a blue color is
produced. Perform a blank determination in the same man-
ner. Calculate the molarity factor.
Each mL of 0.2 mol/L tetramethylammonium
hydroxide VS
= 24.42 mg of C 6 H 5 COOH
Note: Store in tightly stoppered bottles. This solution, if
stored for a long period, should be restandardized.
Tetramethylammonium Hydroxide, 0.1 mol/L
1000 mL of this solution contains 9.115 g of tetramethyl-
ammonium hydroxide [(CH 3 ) 4 NOH: 91.15].
Preparation — Before use, dilute a volume of tetramethyl-
ammonium hydroxide-methanol TS, equivalent to 9.2 g of
tetramethylammonium hydroxide, with water to make 1000
mL, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 0.2 mol/L tetramethylammonium hydroxide VS.
Weigh accurately about 0.2 g of benzoic acid and titrate
<2.50>.
JPXV
General Tests / Standard Solutions
143
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 12.21 mg of C 6 H 5 COOH
Note: Store in tightly stoppered bottles. This solution, if
stored for a long period, should be restandardized.
Tetramethylammonium Hydroxide, 0.02 mol/L
1000 mL of this solution contains 1.8231 g of tetramethyl-
ammonium hydroxide [(CH 3 ) 4 NOH: 91.15].
Preparation — Before use, dilute 0.1 mol/L tetramethyl-
ammonium hydroxide VS with freshly boiled and cooled
water to make exactly 5 times the initial volume.
Tetramethylammonium Hydroxide-Methanol, 0.1 mol/L
1000 mL of this solution contains 9.115 g of tetramethyl-
ammonium hydroxide [(CH 3 ) 4 NOH: 91.15].
Preparation — Before use, dilute a volume of tetramethyl-
ammonium hydroxide-methanol TS, equivalent to 9.2 g of
tetramethylammonium hydroxide, with methanol to make
1000 mL, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 0.1 mol/L tetramethylammonium hydroxide VS.
Note: Store in tightly stoppered bottles. This solution, if
stored for a long period, should be restandardized.
Titanium (III) Chloride, 0.1 mol/L
1000 mL of this solution contains 15.423 g of titanium (III)
chloride (TiCl 3 : 154.23).
Preparation — Add 75 mL of hydrochloric acid to 75 mL of
titanium (III) chloride, and dilute with freshly boiled and
cooled water to make 1000 mL. Transfer the solution into a
buret provided with a reservoir protected from light, replace
the air with hydrogen, and allow to stand for 48 hours. Be-
fore use, standardize the solution as follows:
Standardization — Weigh 3 g of ammonium iron (II) sulfa-
te hexahydrate in a wide-mouthed, 500 mL conical flask.
Passing carbon dioxide through the flask, dissolve it in 50 mL
of freshly boiled and cooled water, and add 25 mL of diluted
sulfuric acid (27 in 100). Rapidly add exactly 40 mL of 0.02
mol/L potassium permanganate VS to the mixture, while
passing carbon dioxide through the flask. Titrate <2.50> with
the prepared titanium (III) chloride solution until the calcu-
lated end point is approached, then add 5 g of ammonium
thiocyanate immediately, and continue the titration with the
prepared titanium (III) chloride solution until the color of the
solution disappears. Perform a blank determination. Calcu-
late the molarity factor.
Note: Store after the air has been displaced with hydrogen.
Titanium Trichloride, 0.1 mol/L
See Titanium (III) Chloride, 0.1 mol/L.
Zinc, 0.1 mol/L
1000 mL of this solution contains 6.541 g of zinc (Zn:
65.41).
Preparation — To 6.541 g of zinc (standard reagent), previ-
ously washed with dilute hydrochloric acid, with water and
then acetone, and cooled in a desiccator (silica gel) after
drying at 110°C for 5 minutes, add 80 mL of dilute
hydrochloric acid and 2.5 mL of bromine TS, dissolve by
gentle warming, evaporate excess bromine by boiling, and
add water to make exactly 1000 mL.
Zinc Acetate, 0.05 mol/L
1000 mL of this solution contains 10.977 g of zinc acetate
dihydrate [Zn(CH 3 COO) 2 .2H 2 0: 219.53].
Preparation — Dissolve 11.1 g of zinc acetate dihydrate in
40 mL of water and 4 mL of dilute acetic acid, add water to
make 1000 mL, and standardize the solution as follows:
Standardization — Measure exactly 20 mL of 0.05 mol/L
disodium dihydrogen ethylenediamine tetraacetate VS, and
add 50 mL of water, 3 mL of ammonia-ammonium chloride
buffer solution, pH 10.7, and 0.04 g of eriochrome black T-
sodium chloride reagent. Titrate <2.50> the solution with the
prepared zinc acetate solution, until the blue color changes to
blue-purple. Calculate the molarity factor.
Zinc Acetate, 0.02 mol/L
1000 mL of this solution contains 4.391 g of zinc acetate
dihydrate [Zn(CH 3 COO) 2 .2H 2 0: 219.53].
Preparation — Dissolve 4.43 g of zinc acetate dihydrate in
20 mL of water and 2 mL of dilute acetic acid, add water to
make 1000 mL, and standardize the solution as follows:
Standardization — Proceed as directed for standardization
under 0.05 mol/L zinc acetate VS, but measure exactly 20
mL of 0.02 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS.
Zinc Sulfate, 0.1 mol/L
1000 mL of this solution contains 28.758 g of zinc sulfate
heptahydrate (ZnS0 4 .7H 2 0: 287.58).
Preparation — Dissolve 28.8 g of zinc sulfate heptahydrate
in water to make 1000 mL, and standardize the solution as
follows:
Standardization — Pipet 25 mL of the prepared zinc sulfate
solution, add 5 mL of ammonia-ammonium chloride buffer
solution, pH 10.7, and 0.04 g of eriochrome black T-sodium
chloride indicator, and titrate <2.50> with 0.1 mol/L disodi-
um dihydrogen ethylenediamine tetraacetate VS until the
color of the solution changes from red-purple to blue-purple.
Calculate the molarity factor.
9.22 Standard Solutions
Standard Solutions are used as the standard for the com-
parison in a text of the Pharmacopoeia.
Borate pH Standard Solution See pH Determination
<2.54>.
Calcium Hydroxide pH Standard Solution See pH Deter-
mination <2.54>.
Carbonate pH Standard Solution See pH Determination
<2.54>.
Oxalate pH Standard Solution See pH Determination
<2.54>.
pH Standard Solution, Borate See pH Determination
<2.54>.
pH Standard Solution, Calcium Hydroxide See pH De-
termination <2.54>.
144
Standard Solutions / General Tests
JP XV
pH Standard Solution, Carbonate See pH Determination
<2.54>.
pH Standard Solution, Oxalate See pH Determination
<2.54>.
pH Standard Solution, Phosphate See pH Determination
<2.54>.
pH Standard Solution, Phthalate See pH Determination
<2.54>.
Phosphate pH Standard Solution See pH Determination
<2.54>.
Phthalate pH Standard Solution See pH Determination
<2.54>.
Standard Aluminum Stock Solution Weigh 1.0 g of alu-
minum, add 60 mL of diluted hydrochloric acid (1 in 2), dis-
solve by heating, cool, add water to make 1000 mL. Pipet 10
mL of this solution, add 30 mL of water and 5 mL of acetic
acid-ammonium acetate buffer solution, pH 3.0, and adjust
the pH to about 3 with ammonia TS added dropwise. Then,
add 0.5 mL of Cu-PAN TS, and titrate <2.50> with 0.01 mol/
L disodium dihydrogen ethylenediamine tetraacetate VS
while boiling until the color of the solution changes from red
to yellow lasting for more than 1 minute. Perform a brank
determination.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 0.2698 mg of Al
Standard Ammonium Solution Dissolve 2.97 g of ammo-
nium chloride, exactly weighed, in purified water for ammo-
nium limit test to make exactly 1000 mL. Measure exactly 10
mL of this solution, and add purified water for ammonium
limit test to make exactly 1000 mL. Each mL of this solution
contains 0.01 mg of ammonium (NH 4 + ).
Standard Arsenic Stock Solution See Arsenic Limit Test
<7.77>.
Standard Arsenic Solution See Arsenic Limit Test <1.11>.
Standard Boron Solution Weigh exactly 0.286 g of boric
acid, previously dried in a desiccator (silica gel) to constant
mass, and dissolve in water to make exactly 1000 mL. Pipet
10 mL of this solution, and add water to make exactly 1000
mL. Each mL of this solution contains 0.5 fig of boron (B).
Standard Cadmium Stock Solution Dissolve 1.000 g of
cadmium ground metal, exactly weighed, in 100 mL of dilute
nitric acid by gentle heating, cool, and add dilute nitric acid
to make exactly 1000 mL.
Standard Cadmium Solution Measure exactly 10 mL of
Standard Cadmium Stock Solution, and add diluted nitric
acid (1 in 3) to make exactly 1000 mL. Pipet 10 mL of this so-
lution, and add diluted nitric acid (1 in 3) to make 100 mL.
Each mL of this solution contains 0.001 mg of cadmium
(Cd). Prepare before use.
Standard Calcium Solution Weigh exactly 0.250 g of cal-
cium carbonate, add 5 mL of dilute hydrochloric acid and 25
mL of water, and dissolve by heating. After cooling, add
water to make exactly 1000 mL. Each mL of this solution
contains 0.1 mg of calcium (Ca).
Standard Calcium Solution for Atomic Absorption Spec-
trophotometry Weigh accurately 0.250 g of calcium
carbonate, and add 1 mol/L hydrochloric acid TS to make
exactly 100 mL. Each mL of this solution contains 1.00 mg of
calcium (Ca).
Standard Copper Solution Pipet 10 mL of Standard
Copper Stock Solution, and dilute with water to make exactly
1000 mL. Each mL of this solution contains 0.01 mg of cop-
per (Cu). Prepare before use.
Standard Copper Stock Solution Weigh exactly 1.000 g
of copper (standard reagent), add 100 mL of dilute nitric
acid, and dissolve by heating. After cooling, add water to
make exactly 1000 mL.
Standard Cyanide Stock Solution Dissolve 2.5 g of po-
tassium cyanide in water to make exactly 1000 mL. Measure
exactly 100 mL of this solution, add 0.5 mL of 4-dimeth-
ylaminobenzylidene rhodanine TS, and titrate <2.50> with 0.1
mol/L silver nitrate VS until the solution shows a red color.
Each mL of 0.1 mol/L silver nitrate VS = 5.204 mg of CN
Standard Cyanide Solution Measure exactly a volume of
Standard Cyanide Stock Solution, equivalent to 10 mg of
cyanide (CN), add 100 mL of sodium hydroxide TS and
water to make exactly 1000 mL. Each mL of this solution
contains 0.01 mg of cyanide (CN). Prepare before use.
Standard Fluorine Solution See Oxygen Flask Combus-
tion Method <1.06>.
Standard Gold Stock Solution Dissolve 0.209 g of hydro-
gen tetrachloroaurate (III) tetrahydrate, exactly weighed, in 2
mL of aqua regia, heat on a water bath for 10 minutes, and
add 1 mol/L hydrochloric acid TS to make exactly 100 mL.
Each mL of this solution contains 1.00 mg of gold (Au).
Standard Gold Solution for Atomic Absorption Spectro-
photometry To 25 mL of Standard Gold Stock Solution,
exactly measured, add water to make exactly 1000 mL. Each
mL of this solution contains 0.025 mg of gold (Au).
Standard Iron Solution Weigh exactly 86.3 mg of ammo-
nium iron (III) sulfate dodecahydrate, dissolve in 100 mL of
water, and add 5 mL of dilute hydrochloric acid and water to
make exactly 1000 mL. Each mL of this solution contains
0.01 mg of iron (Fe).
Standard Lead Stock Solution Weigh exactly 159.8 mg of
lead (II) nitrate, dissolve in 10 mL of dilute nitric acid, and
add water to make exactly 1000 mL. Prepare and store this
solution using glass containers, free from soluble lead salts.
Standard Lead Solution Measure exactly 10 mL of Stan-
dard Lead Stock Solution, and add water to make exactly 100
mL. Each mL of this solution contains 0.01 mg of lead (Pb).
Prepare before use.
Standard Liquids for Calibrating Viscosimeters [JIS,
Standard Liquids for Calibrating Viscosimeters (Z 8809)]
Standard Mercury Solution Weigh exactly 13.5 mg of
mercury (II) chloride, previously dried for 6 hours in a desic-
cator (silica gel), dissolve in 10 mL of dilute nitric acid, and
add water to make exactly 1000 mL. Pipet 10 mL of this solu-
tion, and add 10 mL of dilute nitric acid and water to make
exactly 1000 mL. Each mL of this solution contains 0.1 fig of
JPXV
General Tests / Matching Fluids for Color
145
mercury (Hg). Prepare before use.
Standard Methanol Solution See Methanol Test <1.12>.
Standard Nickel Solution Dissolve 6.73 g of ammonium
nickel (II) sulfate hexahydrate, exactly weighed, in water to
make exactly 1000 mL. Pipet 5 mL of this solution, add
water to make exactly 1000 mL. Each mL of this solution
contains 0.005 mg of nickel (Ni).
Standard Nitric Acid Solution Weigh exactly 72.2 mg of
potassium nitrate, dissolve in water to make exactly 1000
mL. Each mL of this solution contains 0.01 mg of nitrogen
(N).
Standard Phosphoric Acid Solution Weigh exactly 0.358
g of potassium dihydrogen phosphate, previously dried to
constant mass in a desiccator (silica gel), and add 10 mL of
diluted sulfuric acid (3 in 10) and water to make exactly 1000
mL. Pipet 10 mL of this solution, and add water to make ex-
actly 100 mL. Each mL of this solution contains 0.025 mg of
phosphoric acid (as P0 4 ).
Standard Potassium Stock Solution Weigh exactly 9.534
g of potassium chloride, previously dried at 130°C for 2
hours, and dissolve in water to make exactly 1000 mL. Each
mL of this solution contains 5.00 mg of potassium (K).
Standard Selenium Solution To exactly 1 mL of Stan-
dard Selenium Stock Solution add water to make exactly
1000 mL. Prepare before use. It contains 1.0 jx% of selenium
(Se) per mL.
Standard Selenium Stock Solution Dissolve exactly 1 .405
g of selenium dioxide in 0.1 mol/L nitric acid to make exactly
1000 mL.
Standard Silver Stock Solution Dissolve 1.575 g of silver
nitrate, exactly weighed, in water to make exactly 1000 mL.
Each mL of this solution contains 1.00 mg of silver (Ag).
Standard Silver Solution for Atomic Absorption Spec-
trophotometry Measure exactly 10 mL of Standard Silver
Stock Solution, and add water to make exactly 1000 mL.
Each mL of this solution contains 0.01 mg of silver (Ag). Pre-
pare before use.
Standard Sodium Dodecylbenzene Sulfonate Solution
Weigh exactly 1.000 g of sodium dodecylbenzene sulfonate,
and dissolve in water to make exactly 1000 mL. Pipet 10 mL
of this solution, and add water to make exactly 1000 mL.
Each mL of this solution contains 0.01 mg of sodium dode-
cylbenzene sulfonate [CH3(CH 2 ) u C 6 H4S03Na].
Standard Sodium Stock Solution Weigh exactly 2.542 g
of sodium chloride (standard reagent), previously dried at
130°C for 2 hours, and dissolve in water to make exactly 1000
mL. Each mL of this solution contains 1.00 mg of sodium
(Na).
Standard Tin Solution Weigh exactly 0.250 g of tin, and
dissolve in 10 mL of sulfuric acid by heating. After cooling,
transfer this solution with 400 mL of diluted hydrochloric
acid (1 in 5) to a 500-mL volumetric flask, and add diluted hy-
drochloric acid (1 in 5) to make 500 mL. Pipet 10 mL of this
solution, and add diluted hydrochloric acid (1 in 5) to make
exactly 1000 mL. Each mL of this solution contains 0.005 mg
of tin (Sn). Prepare before use.
Standard Vinyl Chloride Solution Transfer about 190
mL of ethanol for gas chromotography into a 200-mL volu-
metric flask, and stopper with a silicone rubber stopper.
Cooling this volumetric flask in a methanol-dry ice bath, in-
ject 0.20 g of vinyl chloride, previously liquidized, through
the silicone rubber stopper, and then inject ethanol for gas
chromatography, previously cooled in a methanol-dry ice
bath, through the silicone rubber stopper to make 200 mL.
Then pipet 1 mL of this solution, add ethanol for gas chro-
matography, previously cooled in a methanol-dry ice bath, to
make exactly 200 mL. Pipet 1 mL of this solution, add etha-
nol for gas chromatography, cooled previously in a metha-
nol-dry ice bath to make exactly 100 mL. Preserve in a her-
metic container, at a temperature not exceeding — 20°C.
Standard Water-Methanol Solution See Water Determi-
nation <2.48>.
Standard Zinc Stock Solution Dissolve exactly 1.000 g of
zinc (standard reagent), in 100 mL of water and 5 mL of hy-
drochloric acid with the aid of gentle heating, cool, and add
water to make exactly 1000 mL.
Standard Zinc Solution Measure exactly 25 mL of Stan-
dard Zinc Stock Solution, and add water to make exactly
1000 mL. Prepare before use. Each mL of this solution con-
tains 0.025 mg of zinc (Zn).
Standard Zinc Solution for Atomic Absorption Spectro-
photometry See Test for Rubber Closure for Aqueous Infu-
sions <7.03>.
9.23 Matching Fluids for Color
Matching Fluids for Color are used as the reference for the
comparison of color in a text of the Pharmacopoeia.
They are prepared from the following colorimetric stock
solutions. Colorimetric stock solutions are prepared by the
following procedures and stored in glass-stoppered bottles.
When the color of the solution is compared with Matching
Fluids for Color, unless otherwise specified, transfer both so-
lutions and fluids to Nessler tubes and view transversely
against a white background.
Cobalt (II) Chloride Colorimetric Stock Solution Weigh
65 g of cobalt (II) chloride hexahydrate, and dissolve in 25
mL of hydrochloric acid and water to make 1000 mL. Meas-
ure exactly 10 mL of this solution, and add water to make ex-
actly 250 mL. Measure exactly 25 mL of the solution, add 75
mL of water and 0.05 g of mulexide-sodium chloride indica-
tor, and add dropwise diluted ammonia solution (28) (1 in 10)
until the color of the solution changes from red-purple to yel-
low. Titrate <2.50> with 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS until the color of the solu-
tion changes, after the addition of 0.2 mL of diluted ammo-
nia solution (28) (1 in 10) near the endpoint, from yellow to
red-purple.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.379 mg of CoCl 2 .6H 2
According to the titrated value, add diluted hydrochloric
acid (1 in 40) to make a solution containing 59.5 mg of cobalt
146
Reagents, Test Solutions / General Tests
JP XV
(II) chloride hexahydrate (CoCl 2 .6H 2 0: 237.93) in each mL,
and use this solution as the colorimetric stock solution.
Cobaltous Chloride Colorimetric Stock Solution
Cobalt (II) Chloride Colorimetric Stock Soluiton.
See
Copper (II) Sulfate Colorimetric Stock Solution Weigh
65 g of copper (II) sulfate pentahydrate, and dissolve in 25
mL of hydrochloric acid and water to make 1000 mL. Meas-
ure exactly 10 mL of this solution, and add water to make ex-
actly 250 mL. Measure exactly 25 mL of this solution, add 75
mL of water, 10 mL of a solution of ammonium chloride (3
in 50), 2 mL of diluted ammonia solution (28) (1 in 10) and
0.05 g of mulexide-sodium chloride indicator. Titrate <2.50>
with 0.01 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS until the color of the solution changes from
green to purple.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.497 mg of CuS0 4 .5H 2
According to the titrated value, add diluted hydrochloric
acid (1 in 40) to make a solution containing 62.4 mg of cop-
per (II) sulfate pentahydrate (CuS0 4 .5H 2 0: 249.69) in each
mL, and use this solution as the colorimetric stock solution.
Copper Sulfate Colorimetric Stock Solution See Copper
(II) Sulfate Colorimetric Stock Solution.
Iron (III) Chloride Colorimetric Stock Solution Weigh 55
g of iron (III) chloride hexahydrate, and dissolve in 25 mL of
hydrochloric acid and water to make 1000 mL. Measure ex-
actly 10 mL of this solution, transfer to an iodine flask, add
15 mL of water and 3 g of potassium iodide, stopper tightly,
and allow to stand in a dark place for 15 minutes. Add 100
mL of water to the mixture, and titrate <2.50> the liberated
iodine with 0.1 mol/L sodium thiosulfate VS (indicator: 1
mL of starch TS).
Each mL of 0.1 mol/L sodium thiosulfate VS
= 27.03 mg of FeCl 3 .6H 2
According to the titrated value, add diluted hydrochloric
acid (1 in 40) to make a solution containing 45.0 mg of iron
(III) chloride hexahydrate (FeCl 3 .6H 2 0: 270.30) in each mL,
and use this solution as the colorimetric stock solution.
Matching Fluids for Color Measure exactly the volume
of colorimetric stock solutions and water shown in the fol-
lowing table with a buret or a pipet graduated to less than 0.1
mL, and mix.
Table 9.23-1 Matching fluid for color
Match-
ing fluid
for color
Parts of
cobalt
(II) chloride
colorimetric
stock solution
(mL)
Parts of iron
(III) chloride
colorimetric
stock solution
(mL)
Parts of copper
(II) sulfate
colorimetric
stock solution
(mL)
Parts of
water
(mL)
A
0.1
0.4
0.1
4.4
B
0.3
0.9
0.3
3.5
C
0.1
0.6
0.1
4.2
D
0.3
0.6
0.4
3.7
E
0.4
1.2
0.3
3.1
F
0.3
1.2
—
3.5
G
0.5
1.2
0.2
3.1
H
0.2
1.5
—
3.3
I
0.4
2.2
0.1
2.3
J
0.4
3.5
0.1
1.0
K
0.5
4.5
—
—
L
0.8
3.8
0.1
0.3
M
0.1
2.0
0.1
2.8
N
—
4.9
0.1
—
O
0.1
4.8
0.1
—
P
0.2
0.4
0.1
4.3
Q
0.2
0.3
0.1
4.4
R
0.3
0.4
0.2
4.1
S
0.2
0.1
—
4.7
T
0.5
0.5
0.4
3.6
Reagents, Test Solutions, etc.
9.41 Reagents, Test Solutions
Reagents are the substances used in the tests of the Phar-
macopoeia. The reagents that are described as "Standard
reagent for volumetric analysis", "Special class", "First c-
lass", "For water determination", etc. in square brackets
meet the corresponding requirements of the Japan Industrial
Standards (JIS). The tests for them are performed according
to the test methods of JIS. In the case where the reagent name
in the Pharmacopoeia differs from that of JIS, the JIS name
is given in the brackets. The reagents for which a
monograph's title is given in the brackets meet the require-
ments of the corresponding monograph. In the case of the
reagents that are described merely as test items, the cor-
responding test method of the Pharmacopoeia is applied.
Test Solutions are the solutions prepared for use in the
tests of the Pharmacopoeia.
Acenaphthene C 12 H 10 White to pale yellowish white
crystals or crystalline powder, having a characteristic aroma.
Freely soluble in diethyl ether and in chloroform, soluble in
acetonitrile, sparingly soluble in methanol, and practically in-
soluble in water.
Identification — Determine the infrared absorption spec-
trum of acenaphthene according to the paste method under
Infrared Spectrophotometry <2.25>, with 5 mg of
acenaphthene: it exhibits absorption at the wave numbers of
about 1605 cm" 1 , 840 cm" 1 , 785 cm 1 and 750 cm" 1 .
Melting point <2.60>: 93 - 96°C
Purity — Dissolve 0.1 g of acenaphthene in 5 mL of chloro-
JPXV
General Tests / Reagents, Test Solutions
147
form, and use this solution as the sample solution. Perform
the test with 2 /uL of the sample solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions. Measure each peak area by the automatic integration
method, and calculate the amount of acenaphthene by the
area percentage method: it shows a purity of not less than
98.0%.
Operating conditions
Detector: Hydrogen flame-ionization detector
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with 150- to 180-//m siliceous
earth for gas chromatography coated with 10% of poly-
ethylene glycol 20 M.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
acenaphthene is about 8 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of acenaphthene obtained from 2 /uL of
the solution prepared by adding chloroform to 1.0 mL of the
sample solution to make 100 mL is 5% to 15% of the full
scale.
Time span of measurement: About 3 times as long as the
retention time of acenaphthene beginning after the solvent
peak.
Residue on ignition <2.44> — Not more than 0.1% (1 g).
Acetal C 6 H 14 2 A clear and colorless volatile liquid.
Miscible with water and with ethanol (95).
Refractive index <2.45> n™: about 1.382
Specific gravity <2.56> df a : about 0.824
Boiling point <2.57>: about 103°C
Acetaldehyde CH 3 CHO [K 8030, First class]
Acetaldehyde for assay Distil 100 mL of acetaldehyde
under reduced pressure, discard the first 20 mL of the distil-
late, and use the subsequent. Prepare before use.
Acetaldehyde for gas chromatography C 2 H 4 A clear
and colorless, flammable liquid. Miscible with water and with
ethanol (95).
Refractive index <2.45> n™: about 1.332
Specific gravity <2.56> d§: about 0.788
Boiling point <2.57>: about 21 °C
2-Acetamidoglutarimide C 7 H 10 N 2 O 3 : 170.17
Identification — Determine the infrared absorption spec-
trum of 2-acetamidoglutarimide as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3350 cm- 1 , 1707 cm- 1 , 1639 cm-' and 1545 cm-'.
Purity Related substances — Dissolve 10 mg of 2-
acetamidoglutarimide in 100 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Proceed
with exactly 20 /uL each of the sample solution and standard
solution as directed in the Purity (3) under Aceglutamide
Aluminum: the total of the peak areas other than 2-
acetamidoglutarimide from the sample solution is not more
than the peak area from the standard solution.
Content: not less than 98.0%. Assay — Weigh accurately
about 20 mg of 2-acetamidoglutarimide, and perform the test
as directed under Nitrogen Determination <1.08>.
Each mL of 0.01 mol/L sulfuric acid VS
= 0.8509 mg of C 7 H I0 N 2 O 3
Acetaminophen C 8 H 9 N0 2 [Same as the namesake
monograph]
Acetanilide C 8 H 9 N0 2 White, crystals or crystalline
powder.
Melting point <2.60>: 114 - 117°C
p-Acetanisidide C 9 H u N0 2 White to purplish white,
crystals or crystalline powder, having a characteristic odor.
It is freely soluble in ethanol (95) and in acetonitrile, and
very slightly soluble in water.
Melting point <2.60>: 126 - 132°C
Content: not less than 98.0%. Assay — Dissolve 0.1 g of
j9-acetanisidide in 5 mL of ethanol (95). Perform the test with
2,uL of this solution as directed under Gas Chromatography
<2.02> according to the following conditions, and determine
the area of each peak by the automatic integration method.
Content 1
peak area of p-acetanisidide ...
total of all peak areas
Operating conditions
Detector: Hydrogen flame-ionization detector
Column: A glass tube 3 mm in inside diameter and 2 m in
length, packed with acid-treated and silanized siliceous earth
for gas chromatography coated with alkylene glycol phtha-
late ester for gas chromatography in 1% (177-250 [im in par-
ticle diameter).
Column temperature: A constant temperature of about
210°C
Carrier gas: Nitrogen
Flow rate: Adjust to a constant flow rate of between 30 and
50 mL per minute and so that the retention time of
/?-acetanisidide is between 11 and 14 minutes.
Time span of measurement: About 3 times as long as the
retention time of p-acetanisidide beginning after the solvent
peak.
Acetate buffer solution, pH 3.5 Dissolve 50 g of ammoni-
um acetate in 100 mL of 6 mol/L hydrochloric acid TS, ad-
just to pH 3.5 with ammonia TS or 6 mol/L hydrochloric
acid TS, if necessary, and add water to make 200 mL.
Acetate buffer solution, pH 4.5 Dissolve 63 g of anhy-
drous sodium acetate in a suitable amount of water, add 90
mL of acetic acid (100) and water to make 1000 mL.
Acetate buffer solution, pH 5.4 To 5.78 mL of acetic acid
(100) add water to make 1000 mL (solution A). Dissolve 8.2 g
of anhydrous sodium acetate in water to make 1000 mL (so-
lution B). Mix 176 mL of the solution A and 824 mL of the
solution B, and adjust, if necessary, the pH to 5.4 with the
solution A or the solution B.
0.01 mol/L Acetate buffer solution, pH 5.0 Dissolve 385
g of ammonium acetate in 900 mL of water, add acetic acid
(31) to adjust the pH to 5.0, and then add water to make 1000
mL.
Acetate buffer solution, pH 5.5 Dissolve 2.72 g of sod-
uim acetate trihydrate in water to make 1000 mL, and adjust
the pH to 5.5 with diluted acetic acid (100) (3 in 2500).
Acetic acid See acetic acid (31).
148
Reagents, Test Solutions / General Tests
JP XV
Acetic acid-ammonium acetate buffer solution, pH 3.0
Add acetic acid (31) to ammonium acetate TS, and adjust the
pH to 3.0.
Acetic acid-ammonium acetate buffer solution, pH 4.5
Dissolve 77 g of ammonium acetate in about 200 mL of
water, adjust the pH to 4.5 by adding acetic acid (100), and
add water to make 1000 mL.
Acetic acid-ammonium acetate buffer solution, pH 4.8
Dissolve 77 g of ammonium acetate in about 200 mL of
water, and add 57 mL of acetic acid (100) and water to make
1000 mL.
Acetic acid, dilute Dilute 6 g of acetic acid (100) with
water to make 100 mL (1 mol/L).
Acetic acid for nonaqueous titration [K 8355, Special
class, meeting with following requirement.]
Purity Acetic anhydride — Dissolve 1.0 g of aniline in
acetic acid for nonaqueous titration to make 100 mL, and use
this solution as the sample solution. Pipet 25 mL of the sam-
ple solution, titrate <2.50> with 0.1 mol/L perchloric acid VS,
and designate the consumed volume as A (mL). A is not less
than 26 mL. Pipet 25 mL of the sample solution, add 75 mL
of acetic acid for nonaqueous titration, and titrate <2.50>
with 0.1 mol/L perchloric acid VS, and designate the con-
sumed volume as B (mL) (potentiometric titration). A — B is
not more than 0.1 (mL) (not more than 0.001 g/dL).
Acetic acid, glacial See acetic acid (100).
Acetic acid-potassium acetate buffer solution, pH 4.3
Dissolve 14 g of potassium acetate in 20.5 mL of acetic acid
(100), and add water to make 1000 mL.
Acetic acid-sodium acetate buffer solution, 0.05 mol/L,
pH 4.0 To 3.0 g of acetic acid (100) add water to make 1000
mL. Adjust to pH 4.0 with a solution prepared by dissolving
3.4 g of sodium acetate trihydrate in water to make 500 mL.
0.05 mol/L Acetic acid-sodium acetate buffer solution, pH
4.0 To 3.0 g of acetic acid (100) add water to make 1000
mL. To this solution add a sufficient amount of a solution of
sodium acetate trihydrate (3.4 in 500) to adjust to pH 4.0.
Acetic acid-sodium acetate buffer solution, pH 4.5 To 80
mL of sodium acetate TS add 120 mL of dilute acetic acid
and water to make 1000 mL.
Acetic acid-sodium acetate buffer solution, pH 4.5, for
iron limit test Dissolve 75.4 mL of acetic acid (100) and 111
g of sodium acetate trihydrate in 1000 mL of water.
Acetic acid-sodium acetate buffer solution, pH 4.7 Dis-
solve 27.2 g of sodium acetate trihydrate in 900 mL of water,
adjust the pH to 4.7 by adding acetic acid (100) dropwise,
and add water to make 1000 mL.
Acetic acid-sodium acetate buffer solution, pH 5.0 To 140
mL of sodium acetate TS add 60 mL of dilute acetic acid and
water to make 1000 mL.
Acetic acid-sodium acetate buffer solution, pH 5.5
Dissolve 20 g of sodium acetate trihydrate in 80 mL of water,
adjust the pH to 5.5 by adding acetic acid (100) dropwise,
and add water to make 100 mL.
Acetic acid-sodium acetate buffer solution, pH 5.6
Dissolve 12 g of sodium acetate trihydrate in 0.66 mL of
acetic acid (100) and water to make 100 mL.
1 mol/L Acetic acid-sodium acetate buffer solution, pH
5.0 To sodium acetate TS add dilute acetic acid, and adjust
the pH to 5.0.
0.1 mol/L Acetic acid-sodium acetate buffer solution, pH
4.0 Dissolve 13.61 g of soduim acetate trihydrate in 750 mL
of water, adjust the pH to 4.0 with acetic acid (100), and add
water to make 1000 mL.
0.05 mol/L Acetic acid-sodium acetate buffer solution, pH
4.6 Dissolve 6.6 g of sodium acetate trihydrate in 900 mL of
water, and add 3 mL of acetic acid and water to make 1000
mL.
Acetic acid-sodium acetate TS Mix 17 mL of 1 mol/L so-
dium hydroxide VS with 40 mL of dilute acetic acid, and add
water to make 100 mL.
0.02 mol/L Acetic acid-sodium acetate TS Dissolve 2.74
g of sodium acetate trihydrate in a suitable amount of water,
and add 2 mL of acetic acid (100) and water to make 1000
mL.
6 mol/L Acetic acid TS Dilute 36 g of acetic acid (100)
with water to make 100 mL.
0.25 mol/L Acetic acid TS To 3 g of acetic acid (100) add
water to make 200 mL.
Acetic acid (100) CH 3 COOH [K 8355, Acetic Acid,
Special class]
Acetic acid (100)-sulfuric acid TS To 5 mL of acetic acid
(100) add cautiously 5 mL of sulfuric acid while cooling in an
ice bath, and mix.
Acetic acid (31) Dilute 31.0 g of acetic acide (100) with
water to make 100 mL (5 mol/L).
Acetic anhydride (CH 3 CO) 2 [K 8886, Special class]
Acetic anhydride-pyridine TS Place 25 g of acetic an-
hydride in a 100 mL volumetric flask, add pyridine to make
100 mL, and mix well. Preserve in light-resistant containers,
protected from air. This solution may be used even if it
becomes colored during storage.
Acetone CH 3 COCH 3 [K 8034, Special class]
Acetone for nonaqueous titration Add potassium per-
manganate to acetone in small portions, and shake. When the
mixture keeps its purple color after standing for 2 to 3 days,
distil, and dehydrate with freshly ignited anhydrous potassi-
um carbonate. Distil by using a fractionating column under
protection from moisture, and collect the fraction distilling at
56°C.
Acetone for purity of crude drug [K 8034, Special class]
Use acetone meeting the following additional specification.
Evaporate 300.0 mL of acetone to be tested in vacuum at a
temperature not higher than 40°C, add the acetone to make
exactly 1 mL, and use this solution as the sample solution.
Separately, dissolve 2.0 mg of y-BHC in hexane for purity of
crude drug to make exactly 100 mL. Pipet 1 mL of this solu-
tion, and add hexane for purity of crude drug to make exactly
100 mL. Further pipet 2 mL of this solution, add hexane for
purity of crude drug to make exactly 100 mL, and use this so-
JPXV
General Tests / Reagents, Test Solutions
149
lution as the standard solution (1). Perform the test with ex-
actly 1 /uL each of the sample solution and standard solution
(1) as directed under Gas Chromatography <2.02> according
to the following operating conditions, and determine each
peak area by the automatic integration method: the total area
of peaks other than the solvent peak from the sample solu-
tion is not larger than the peak area of y-BHC beginning
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Purity (2) under
Crude Drugs Test <5.01> except detection sensitivity and time
span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add hexane for purity of crude drug to make exactly 20
mL, and use this solution as the standard solution (2). Adjust
the detection sensitivity so that the peak area of y-BHC ob-
tained from 1 /uL of the standard solution (2) can be meas-
ured by the automatic integration method, and the peak
height of y-BHC from 1 ftL of the standard solution (1) is
about 20% of the full scale.
Time span of measurement: About three times as long as
the retention time of y-BHC beginning after the solvent peak.
Acetonitrile CH 3 CN [K 8032, Special class]
Acetonitrile for liquid chromatography CH 3 CN Color-
less and clear liquid. Mixable with water.
Purity Ultraviolet light absorbing substances — Deter-
mine the absorbances at the following wavelengths as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, using
water as the control: not more than 0.07 at 200 nm, not more
than 0.046 at 210 nm, not more than 0.027 at 220 nm, not
more than 0.014 at 230 nm and not more than 0.009 at 240
nm.
Acetrizoic acid C 9 H 6 I 3 N0 3 White powder.
Purity Related substances — Dissolve 0.06 g of acetrizoic
acid in a solution of meglumine (3 in 1000) to make 100 mL.
To 10 mL of this solution add water to make 100 mL, and use
this solution as the sample solution. Proceed the test with 5
/uL of the sample solution as directed in the Assay under
Meglumine Sodium Amidotrizoate Injection: any peaks
other than the principal peak are not observed.
Acetylacetone
class]
CH 3 COCH 2 COCH 3 [K 8027, Special
Acetylacetone TS Dissolve 150 g of ammonium acetate in
a sufficient quantity of water, and add 3 mL of acetic acid
(100), 2 mL of acetylacetone and water to make 1000 mL.
Prepare before use.
Acetylene See dissolved acetylene.
Acidic ferric chloride TS See iron (III) chloride TS, acid-
ic.
Acidic potassium chloride TS See potassium chloride TS,
acidic.
Acidic potassium permanganate TS See potassium per-
manganate TS, acidic.
Acidic stannous chloride TS See tin (II) chloride TS,
acidic.
Acid-treated gelatin See gelatin, acid-treated.
Aconitine for purity C 34 H 47 NO u White, crystals or crys-
talline powder. Sparingly soluble in acetonitrile and in
ethanol (99.5), slightly soluble in diethyl ether, and practical-
ly insoluble in water. Melting point: about 185°C (with
decomposition).
Identification — Determine the infrared absorption spec-
trum of aconitine for purity as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3500 cm" 1 , 1718 cm" 1 , 1278 cm" 1 , 1111cm" 1 , 1097 cm" 1
and 717 cm" 1 .
Absorbance <2.24> E{ v ° m (230 nm): 211 - 243 [5 mg dried
for not less than 12 hours in a desiccator (reduced pressure
not exceeding 0.67 kPa, phosphorus (V) oxide, 40°C),
ethanol (99.5), 200 mL].
Purity Related substances —
(1) Dissolve 5.0 mg of aconitine for purity in 2 mL of
acetonitrile, and use as the sample solution. Pipet 1 mL of the
sample solution, add acetonitrile to make exactly 50 mL, and
use as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, and
proceed the test as directed in the Identification in Processed
Aconite Root: the spot other than the principal spot obtained
with the sample solution is not more intense than the spot
with the standard solution.
(2) Dissolve 5.0 mg of aconitine for purity in 5 mL of
acetonitrile, and use as the sample solution. Pipet 1 mL of the
sample solution, add acetonitrile to make exactly 50 mL, and
use as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peaks of aconitine and the solvent obtained
with the sample solution is not larger than the peak area of
aconitine with the standard solution.
Operating conditions
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity under Proc-
essed Aconitine Root.
Mobile phase: A mixture of phosphate buffer solution for
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust the flow rate so that the retention time of
aconitine is about 26 minutes.
Time span of measurement: About 3 times as long as the
retention time of aconitine.
System suitability
Test for required detectability: Pipet 1 mL of the standard
solution, and add acetonitrile to make exactly 20 mL.
Confirm that the peak area of aconitine obtained from 10 /uL
of this solution is equivalent to 3.5 to 6.5% of that obtained
from 10 /xL of the standard solution.
System performance: Dissolve 1 mg each of aconitine for
purity, hypaconitine for purity and mesaconitine for purity,
and 8 mg of jesaconitine for purity in 200 mL of acetonitrile.
When the procedure is run with lO/iL of this solution under
the above operating conditions, mesaconitine, hypaconitine,
aconitine and jesaconitine are eluted in this order, and each
resolution between these peaks is not less than 1.5, respec-
tively.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
150
Reagents, Test Solutions / General Tests
JP XV
conditions, the relative standard deviation of the peak area of
aconitine is not more than 1.5%.
Water <2.48>: not more than 1.0% [5 mg dried for not less
than 12 hours in a desiccator (reduced pressure not exceeding
0.67 kPa, phosphorus (V) oxide, 40°C), coulometric titra-
tion].
Aconitum diester alkaloids standard solution for purity
It is a solution containing 10 mg of aconitine for purity,
10 mg of jesaconitine for purity, 30 mg of hypaconitine for
purity and 20 mg of mesaconitine for purity in 1000 mL of a
mixture of phosphate buffer solution for processed aconite
root and acetonitrile (1:1). When proceed the test with 20 fiL
of this solution as directed in the Purity under Processed
Aconite Root at the detection wavelength 231 nm, the peaks
of aconitine, jesaconitine, hypaconitine and mesaconitine are
observed, and the ratio of their peak heights is about
10:1:35:30. When proceed the test at the detection
wavelength 254 nm, the peaks of aconitine, jesaconitine,
hypaconitine and mesaconitine are observed, and the ratio of
their peak heights is about 2:8:7:6.
Acrinol C 15 H 1 5N30.C 3 H 6 3 .H 2 [Same as the mono-
graph Acrinol Hydrate]
Acrylamide CH 2 CHCONH 2 Pale yellow crystalline
powder.
Melting point <2.60>: 83 - 86°C
Content: not less than 97.0%.
Activated alumina Aluminum oxide with specially strong
adsorptive activity.
Activated charcoal [Same as the monograph Medicinal
Carbon]
Activated thromboplastin-time assay reagent It is pre-
pared by lyophilization of phospholipid (0.4 mg/mL) which
is suspended in 1 mL of a solution of 2-[4-(2-hydroxymethyl)-
l-piperazinyl]propanesulfonic acid (61 in 5000), mixed with
both silica-gel (4.3 mg/mL) and dextran after the extraction
and purificaton from rabbit brain. Activated thromboplastin-
time: 25 - 45 seconds (as assayed with human normal plas-
ma).
Activated thromboplastin-time assay solution Dissolve
an aliquot of activated thromboplastin-time assay reagent
equivalent to 0.4 mg of phospholipid in 1 mL of water.
Adipic acid C 4 H 8 (COOH) 2 White crystals or crystalline
powder. Freely soluble in ethanol (95), and sparingly soluble
in water.
Melting point <2.60>: 151 - 154°C
Content: not less than 98.0%. Assay — Weigh accurately
about 1 g of adipic acid, and 100 mL of water, dissolve by
warming, cool, and titrate <2.50> with 1 mol/L sodium hy-
droxide VS (indicator: 2 drops of phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 73.07 mg of C 6 H 10 O 4
Agar [K 8263, Special class. Same as the monograph
Agar or Agar Powder. Loss on drying is not more than 15%.]
Agar medium, ordinary See ordinary agar medium.
Agar slant Dispense portions of about 10 mL of ordinary
agar medium into test tubes, and sterilize by autoclaving. Be-
fore the medium congeals, allow to stand in a slanting
position, and solidify. When the coagulating water is lost,
reprepare by dissolving with the aid of heat.
Ajmaline for assay C 20 H 26 N 2 O 2 [Same as the mono-
graph Ajmaline. When dried, it contains not less than 99.0%
of ajmaline (C 20 H 26 N 2 O 2 ).]
Alacepril C 20 H 26 N 2 O 5 S [Same as the namesake mono-
graph]
Alacepril for assay [Same as the monograph Alacepril.
When dried, it contains not less than 99.0% of alacepril (C 20
H 26 N 2 5 S).]
L-Alanine C 3 H 7 N0 2 [K 9101, Special class]
Albiflorin C 2 3H 28 On.xH 2 Colorless powder having no
odor. Freely soluble in water and in methanol, and practical-
ly insoluble in diethyl ether.
Purity — Dissolve 1 mg in 10 mL of diluted methanol (1 in
2), and use this solution as the sample solution. Perform the
test with 10 /uL of the sample solution as directed in the Assay
under Peony Root; when measure the peak areas about 2
times as long as the retention time of peoniflorin, the total
area of the peaks other than albiflorin and other than the sol-
vent is not larger than 1/10 of the total area of the peaks
other than the solvent peak.
Albumin TS Carefully separate the white from the yolk
of a fresh hen's egg. Shake the white with 100 mL of water
until the mixture is thoroughly mixed, and filter. Prepare be-
fore use.
Aldehyde dehydrogenase Each mg contains not less than
2 enzyme activity units. White powder.
Assay — Dissolve about 20 mg of aldehyde dehydrogenase,
accurately weighed, in 1 mL of water, add ice-cold solution
of bovine serum albumin (1 in 100) to make exactly 200 mL,
and use this solution as the sample solution. In a spec-
trophotometric cell, place 2.50 mL of pyrophosphate buffer
solution, pH 9.0, 0.20 mL of a solution prepared by dissolv-
ing 20.0 mg of /^-nicotinamide adenine dinucleotide (NAD)
to make exactly 1 mL, 0.10 mL of a pyrazole solution (17 in
2500) and 0.10 mL of the sample solution, stir, stopper tight-
ly, and allow to stand at 25 ± 1°C for 2 minutes. To this so-
lution add 0.01 mL of an acetaldehyde solution (3 in 1000),
stir, stopper tightly, determine every 30 seconds the absor-
bance at 340 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and calculate a change (AA) in absor-
bance per minute starting from the spot where the relation of
time and absorbance is shown with a straight line. One en-
zyme activity unit means an amount of enzyme which oxi-
dizes 1 ^mol of acetaldehyde per minute when the test is con-
ducted under the conditions of the Procedure.
Enzyme activity unit (unit/mg) of aldehyde dehydrogenase
2.91 XAA X2Q0
6.3 x 1^x0. 10x1000
W: Amount (g) of sample
Aldehyde dehydrogenase TS Dissolve an amount
equivalent to 70 aldehyde dehydrogenase units in 10 mL of
water. Prepare before use.
Aldehyde-free ethanol See ethanol, aldehyde-free.
Alisol A for thin-layer chromatography C3 H 50 O 5 A
JPXV
General Tests / Reagents, Test Solutions
151
white to pale yellow powder. Very soluble in methanol, freely
soluble in ethanol (99.5), and practically insoluble in water.
Optical rotation <2.49> [a]c <■
+ 106° (5 mg previ-
ously dried on silica gel for 24 hours, methanol, 1 mL, 50
mm).
Purity Related substances — Dissolve 1 mg in 1 mL of
methanol. Proceed the test with 5 /xL of this solution as
directed in the Identification (6) under Saireito Extract: no
spot appears other than the principal spot of around Rf 0.3.
Alizarin complexone C 19 H 15 N0 8 (1,2-Dihydroxyan-
thra-quino-3-ylmethylamine-./V,iV-diacetate) A yellow-
brown powder. Soluble in ammonia TS, and practically in-
soluble in water, in ethanol (95) and in diethl ether.
Sensitivity — Dissolve 0.1 g of alizarin complexone by add-
ing 2 drops of ammonia solution (28), 2 drops of ammonium
acetate TS and 20 mL of water. To 10 mL of this solution
add acetic acid-potassium acetate buffer solution, pH 4.3, to
make 100 mL. Place 1 drop of this solution on a white spot
plate, add 1 drop of a solution of sodium fluoride (1 in
100,000) and 1 drop of cerium (III) nitrate hexahydrate TS,
stir, and observe under scattered light after 1 minute: a blue-
purple color is produced, and the color of the control solu-
tion is red-purple. Use a solution prepared in the same man-
ner, to which 1 drop of water is added in place of a solution
of sodium fluoride, as the control solution.
Alizarin complexone TS Dissolve 0.390 g of alizarin
complexone in 20 mL of a freshly prepared solution of sodi-
um hydroxide (1 in 50), then add 800 mL of water and 0.2 g
of sodium acetate trihydrate, and dissolve. Adjust the pH to
4 to 5 with 1 mol/L hydrochloric acid VS, and add water to
make 1000 mL.
Alizarin red S C 14 H 7 Na0 7 S.H 2 [K 8057, Special
class]
Alizarin red S TS Dissolve 0.1 g of alizarin red S in water
to make 100 mL, and filter if necessary.
Alizarin S See alizarin red S.
Alizarin S TS See alizarin red S TS.
Alizarin yellow GG C 13 H 8 N 3 Na05
class]
[K 8056, Special
Alizarin yellow GG-thymolphthalein TS Mix 10 mL of
alizarin GG TS with 20 mL of thymolphthalein TS.
Alizarin yellow GG TS Dissolve 0.1 g of alizarin yellow
GG in 100 mL of ethanol (95), and filter if necessary.
Alkali copper TS Dissolve 70.6 g of disodium hydrogen
phosphate dodecahydrate, 40.0 g of potassium sodium tar-
trate tetrahydrate and 180.0 g of anhydrous sodium sulfate in
600 mL of water, and add 20 mL of a solution of sodium hy-
droxide (1 in 5). To this mixture add, with stirring, 100 mL of
a solution of copper (II) sulfate (2 in 25), 33.3 mL of 0.05
mol/L potassium iodate VS and water to make 1000 mL.
Alkaline blue tetrazolium TS See blue tetrazolium TS,
alkaline.
Alkaline copper solution Dissolve 0.8 g of sodium
hydroxide in water to make 100 mL, and dissolve 4 g of anhy-
drous sodium carbonate in this solution to make solution A.
Combine 1 mL of a solution of copper (II) sulfate pentahy-
drate (1 in 50) and 1 mL of a solution of sodium tartrate de-
hydrate (1 in 25) to make solution B. Mix 50 mL of freshly
prepared solution A and 1 mL of freshly prepared solution B.
Alkaline copper TS Dissolve 2 g of anhydrous sodium
carbonate in 100 ml of 0.1 mol/L sodium hydroxide TS. To
50 mL of this solution add 1 mL of a mixture of a solution of
copper (II) sulfate pentahydrate (1 in 100) and a solution of
potassium tartrate (1 in 50) (1:1), and mix.
Alkaline copper TS for protein content determination
Dissolve 0.8 g of sodium hydroxide in water to make 100 mL.
Dissolve 4 g of anhydrous sodium carbonate in this solution
to make solution A. Combine 1 mL of copper (II) sulfate
pentahydrate solution (1 in 50) and 1 mL of sodium tartrate
dihydrate solution (1 in 25) to make solution B. Mix 50 mL of
solution A and 1 mL of solution B. Prepare at the time of
use.
Alkaline copper (II) sulfate solution See copper (II) sul-
fate solution, alkaline.
Alkaline glycerin TS To 200 g of glycerin add water to
make 235 g, and add 142.5 mL of sodium hydroxide TS and
47.5 mL of water.
Alkaline hydroxylamine TS See hydroxylamine TS,
alkaline.
Alkaline m-dinitrobenzene TS See 1,3-dinitrobenzene
TS, alkaline.
Alkaline picric acid TS See 2,4,6-trinitrophenol TS, alka-
line.
Alkaline potassium ferricyanide TS See potassium hexa-
cyanoferrate (III) TS, alkaline.
Alkylene glycol phthalate ester for gas chromatography
Prepared for gas chromatography.
Alternative thioglycolate medium See Sterility Test
<4.06> under the General Tests, Processes and Apparatus.
Aluminon C22H23N3O9 [K 8011, Special class]
Aluminon TS Dissolve 0.1 g of aluminon in water to
make 100 mL, and allow this solution to stand for 24 hours.
Aluminum Al [K 8069, Special class]
Aluminum chloride See aluminum (III) chloride hexahy-
drate.
Aluminum chloride TS See Aluminum (III) chloride TS.
Aluminum (III) chloride TS Dissolve 64.7 g of aluminum
(III) chloride hexahydrate in 71 mL of water, add 0.5 g of ac-
tivated charcoal, then shake for 10 minutes, and filter. Ad-
just the pH of the filtrate to 1.5 with a solution of sodium
hydroxide (1 in 100) with stirring, and filter if necessary.
Aluminum (III) chloride hexahydrate A1C1 3 .6H 2
[K 8114, Special class]
Aluminum oxide A1 2 3 White crystals, crystalline
powder, or powder. Boiling point: about 3000°C. Melting
point: about 2000°C.
Aluminum potassium sulfate dodecahydrate A1K(S0 4 ) 2 .-
12H 2 [K 8255, Special class]
Amidosulfuric acid (standard reagent) HOS0 2 NH 2
[K 8005, Standard substance for volumetric analysis]
152
Reagents, Test Solutions / General Tests
JP XV
Amidotrizoic acid for assay CnH 9 I 3 N 2 4 [Same as the
monograph Amidotrizoic Acid] It contains not less than
99.0% of amidotrizoic acid (C u H 9 I 3 N 2 4 ), calculated on the
dried basis.
Aminoacetic acid See glycine.
p-Aminoacetophenone See 4-aminoacetophenone.
p-Aminoacetophenone TS See 4-aminoacetophenone TS.
4-Aminoacetophenone H 2 NC 6 H 4 COCH 3 Light yellow,
crystals or crystalline powder, having a characteristic odor.
Melting point <2.60>: 105 - 108°C
4-Aminoacetophenone TS Dissolve 0.100 g of 4-amino-
acetophenone in methanol to make exactly 100 mL.
4-Aminoantipyrine C u H 13 N 3 [K 8048, Special class]
4-Aminoantipyrine hydrochloride CnHn^O.HCl
Light yellow crystalline powder. It dissolves in water. Melting
point: 232 - 238°C (decomposition).
Purity Clarity of solution — Dissolve 1 g of 4-amino-
antipyrine hydrochloride in 25 mL of water: the solution is
almost clear.
Content: 100.6 - 108.5%. Assay— Weigh accurately about
0.5 g of 4-aminoantipyrine hydrochloride, dissolve in 50 mL
of water, and, if necessary, neutralize with 0.1 mol/L sodium
hydroxide VS (indicator: red litmus paper). Add 4 drops of
dichlorofiuorescein TS, and titrate <2.50> with 0.1 mol/L sil-
ver nitrate VS.
Each mL of 0.1 mol/L silver nitrate VS
= 23.97 mg of C„H 13 N 3 0.HC1
4-Aminoantipyrine hydrochloride TS Dissolve 1 g of 4-
aminoantipyrine hydrochloride in water to make 50 mL.
4-Aminoantipyrine TS Dissolve 0.1 g of 4-aminoanti-
pyrine in 30 mL of water, add 10 mL of a solution of sodium
carbonate decahydrate (1 in 5), 2 mL of sodium hydroxide TS
and water to make 100 mL. Prepare before use.
p-Aminobenzoic acid See 4-aminobenzoic acid.
4-Aminobenzoic acid C 7 H 7 N0 2 White to very pale yel-
low crystalline powder. A solution of 4-aminobenzoic acid in
ethanol (95) (1 in 100) is clear.
2-Amino-l-butanoI CH 3 CH 2 CH(NH 2 )CH 2 OH
Clear, colorless to light yellow liquid. Miscible with water
and dissolves in methanol.
Refractive index <2. 45> n™: 1.450-1.455
Specific gravity <2.56> df : 0.944 - 0.950
Purity Related substances — Dissolve 50 mg of 2-amino-
1-butanol in 10 mL of methanol, measured exactly, and per-
form the test with 2 fiL of this solution as directed in the Puri-
ty (4) under Ethambutol Hydrochloride: any spot other than
the principal spot at the Rf value of about 0.3 does not ap-
pear.
2-Amino-5-chlorobenzophenone for thin-layer chromato-
graphy C 13 HioClNO Yellow, crystalline powder.
Melting point <2.60>: 97 - 101 °C
Purity Related substances — Dissolve 10 mg of 2-amino-
5-chlorobenzophenone for thin-layer chromatography in
methanol to make exactly 200 mL, and perform the test with
this solution as directed in the purity (3) under Chlor-
diazepoxide: any spot other than the principal spot at the Rf
value about 0.7 does not appear.
4-Amino-iV,/V-diethylaniline sulfate H 2 NC 6 H 4 N(C 2 H 5 ) 2 .
H 2 S0 4 .H 2 White to slightly colored powder. It dissolves
in water.
Melting point <2.60>: 173 - 176°C
Residue on ignition <2.44>: not more than 0.1% (1 g).
4-Amino-iV,./V-diethylaniline sulfate TS Dissolve 0.2 g of
4-amino-Af,A r -diethylaniline sulfate in water to make 100 mL.
Prepare before use, protected from light.
2-AminoethanethioI hydrochloride H 2 NCH 2 CH 2 SH.HC1
White crystal or granule.
Melting point <2.60>: 65 - 71 °C
2-Aminoethanol NH 2 CH 2 CH 2 OH [K 8109, Special class]
/V-Aminohexamethyleneimine (CH 2 ) 6 NNH 2 Clear,
colorless to pale yellow liquid.
Refraction index <2.45> n™: 1.482 - 1.487
Specific gravity <2.56> df : 0.936 - 0.942
2-Amino-2-hydroxymethyl-l,3-propanediol C 4 H u N0 3
[K 9704, Special class]
2-Amino-2-hydroxymethyl-l,3-propanediol hydrochloride
C 4 H U N0 3 .HC1 White crystals or crystalline powder
4-(AminomethyI)benzoic acid C 8 H 9 N0 2 A white powder.
Purity — Dissolve 10 mg of 4-(aminomethyl)benzoic acid in
100 mL of water, and use this as the sample solution. Pipet 1
mL of the sample solution, add water to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with exactly 20 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the operating conditions as directed in the
Purity (5) under Tranexamic Acid, and determine each peak
area by the automatic integration method: each area of the
peak other than 4-(aminomethyi)benzoic acid obtained from
the sample solution is not more than the peak area of 4-
(aminomethyl)benzoic acid from the standard solution.
3-(2-Aminoethyl)indole Ci Hi 2 N 2 Yellowish-brown
crystals.
Melting point <2.60>: about 118 C C.
l-Amino-2-naphthol-4-sulfonic acid
[K 8050, Special class]
C 10 H 9 NO 4 S
l-Amino-2-naphthol-4-sulfonic acid TS Mix thoroughly
5 g of anhydrous sodium sulfite, 94.3 g of sodium bisulfite
and 0.7 g of l-amino-2-naphthol-4-sulfonic acid. Before use,
dissolve 1.5 g of this mixture in water to make 10 mL.
?n-Aminophenol See 3-aminophenol.
3-Aminophenol H 2 NC 6 H 4 OH White, crystals or crys-
talline powder.
Melting point <2.60>: 121 - 125°C
Content: not less than 97.0%. Assay — Weigh accurately
about 0.2 g, dissolve in 50 mL of acetic acid for nonaqueous
titration, and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 10.91 mg of H 2 NC 6 H 4 OH
JPXV
General Tests / Reagents, Test Solutions
153
p-Aminophenol hydrochloride
hydrochloride.
See 4-aminophenol
4-Aminophenol hydrochloride H0C 6 H 4 NH 2 .HC1
White to pale colored crystals. Freely soluble in water and in
ethanol (95). Melting point: about 306°C (with decomposi-
tion).
Content: not less than 99.0%. Assay — Weigh accurately
about 0.17 g of 4-aminophenol hydrochloride, dissolve in 50
mL of acetic acid for nonaqueous titration and 5 mL of mer-
cury (II) acetate TS for nonaqueous titration, and titrate
<2.50> with 0.1 mol/L perchloric acid-l,4-dioxane VS (indi-
cator: 1 mL of a-naphtholbenzeine TS). Perform a blank de-
termination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid-l,4-dioxane VS
= 14.56 mg of C 6 H 8 NOCl
Storage — Preserve in tight, light-resistant containers.
Aminopropylsilanized silica gel for pretreatment Pre-
pared for pretreatment.
L-2-Aminosuberic acid C 8 H 15 N0 4 White, crystals or
crystalline powder. Odorless.
Optical rotation <2.49> [ a ]g>: +19.1 -+20.1° (after
drying, 0.1 g, 5 mol/L hydrochloric acid TS, 100 mm).
Loss on drying <2.41>: not more than 0.3% (1 g, 105°C, 2
hours).
Assay — Weigh accurately about 0.3 g of L-2-aminosuberic
acid, previously dried, add exactly 6 mL of formic acid to dis-
solve, then add exactly 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.92 mg of C 8 H 15 N0 4
Ammonia-ammonium acetate buffer solution, pH 8.0 To
ammonium acetate TS add ammonia TS dropwise to adjust
the pH to 8.0.
Ammonia-ammonium acetate buffer solution, pH 8.5
Dissolve 50 g of ammonium acetate in 800 mL of water and
200 mL of ethanol (95), and add ammonia solution (28) to
adjust the pH to 8.5.
Ammonia-ammonium chloride buffer solution, pH 8.0
Dissolve 1.07 g of ammonium chloride in water to make 100
mL, and adjust the pH to 8.0 by adding diluted ammonia TS
(1 in 30).
Ammonia-ammonium chloride buffer solution,
pH 10.0 Dissolve 70 g of ammonium chloride in water, add
100 mL of ammonia solution (28), dilute with water to make
1000 mL, and add ammonia solution (28) dropwise to adjust
the pH to 10.0.
Ammonia-ammonium chloride buffer solution,
pH 10.7 Dissolve 67.5 g of ammonium chloride in water,
add 570 mL of ammonia solution (28), and dilute with water
to make 1000 mL.
Ammonia-ammonium chloride buffer solution,
pH 11.0 Dissolve 53.5 g of ammonium chloride in water,
add 480 mL of ammonia solution (28), and dilute with water
to make 1000 mL.
Ammonia copper TS To 0.5 g of cupric carbonate mono-
hydrate add 10 mL of water, triturate, and add 10 mL of am-
monia solution (28).
Ammonia-ethanol TS To 20 mL of ammonia solution
(28) add 100 mL of ethanol (99.5).
Ammonia gas NH 3 Prepare by heating ammonia solu-
tion (28).
Ammonia-saturated 1-butanol TS To 100 mL of 1-
butanol add 60 mL of diluted ammonia solution (28) (1 in
100), shake vigorously for 10 minutes, and allow to stand.
Use the upper layer.
Ammonia solution (28) NH 4 OH [K 8085, Ammonia
Water, Special class, Specific gravity: about 0.90, Density:
0.908 g/mL, Content: 28-30%]
Ammonia TS To 400 mL of ammonia solution (28) add
water to make 1000 mL (10%).
Ammonia water See ammonia TS.
1 mol/L Ammonia water To 65 mL of ammonia solution
(28) add water to make 1000 mL.
13.5 mol/L Ammonia water To exactly 9 mL of water
add ammonia solution (28) to make exactly 50 mL.
Ammonia water, strong See ammonia solution (28).
Ammonium acetate CH 3 COONH 4 [K 8359, Special
class]
Ammonium acetate TS Dissolve 10 g of ammonium
acetate in water to make 100 mL.
0.5 mol/L Ammonium acetate TS Dissolve 38.5 g of am-
monium acetate in water to make 1000 mL.
Ammonium amidosulfate NH 4 OS0 2 NH 2 [K 8588,
Special class]
Ammonium amidosulfate TS Dissolve 1 g of ammonium
amidosulfate in water to make 40 mL.
Ammonium amminetrichloroplatinate for liquid chro-
matography Cl3H 7 N 2 Pt To 20 g of cisplatin add 600 mL
of 6 mol/L hydrochloric acid TS, and heat under a reflux
condenser for 4-6 hours to boil while stirring. After cool-
ing, evaporate the solvent, and dry the orange residue at
room temperature under reduced pressure. To the residue so
obtained add 300 mL of methanol, and heat at about 50°C to
dissolve. Filter, separate insoluble yellow solids, and wash
the solids with 10 mL of methanol. Combine the filtrate and
the washing, heat at about 50°C, and add slowly 100 mL of
ethyl acetate while stirring. Cool the mixture to room temper-
ature avoiding exposure to light, and allow to stand at — 10°
C for 1 hour. Filter the mixture to take off the formed crys-
tals, wash the crystals with 100 mL of acetone, combine the
washing to the filtrate, and evaporate to dryness to obtain
orange crystals. If necessary, repeat the purification proce-
dure described above to take off the insoluble crystals. To the
orange crystals obtained add 300 to 500 mL of a mixture of
acetone and methanol (5:1), and heat at about 50°C while
stirring to dissolve. Filter while hot to take off the insoluble
crystals, wash the crystals with the mixture, and combine the
filtrate and washing. Repeat the procedure several times, and
evaporate to dryness. Suspense the crystals so obtained in 50
154
Reagents, Test Solutions / General Tests
JP XV
mL of acetone, filter, wash the crystals with 20 mL of ace-
tone, and dry the crystals at room temperature under reduced
pressure. It is a yellow-brown crystalline powder.
Identification — Determine the infrared absorption spec-
trum of the substance to be examined, previously dried at
80°C for 3 hours, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>: it exhibits
absorption at the wave numbers of about 3480 cm -1 ,
3220 cm" 1 , 1622 cm" 1 , 1408 cm" 1 and 1321 cm" 1 .
Related substances — Cisplatin Conduct this procedure
using light-resistant vessels. Dissolve 10 mg in 7V,7V-dimethyl-
formamide to make exactly 10 mL, and use this solution as
the sample solution. Separately, dissolve 10 mg of Cisplatin
in iV,./V-dimethylformamide to make exactly 50 mL. Pipet 5
mL of this solution, add iV,iV-dimethylformamide to make
exactly 20 mL, and use this solution as the standard solution.
Perform the test with exactly 40 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak area of cisplatin by the automatic in-
tegration method: the peak area from the sample solution is
not more than that from the standard solution.
Operating conditions
Proceed as directed in the operating conditions in the
Assay under Cisplatin.
System suitability
System performance: When the procedure is run with
40 /xL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of cisplatin are not less than 2500 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
40 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cisplatin is not more than 5.0%.
Ammonium aurintricarboxylate See aluminon.
Ammonium carbonate [K 8613, Special class]
Ammonium carbonate TS Dissolve 20 g of ammonium
carbonate in 20 mL of ammonia TS and water to make 100
mL.
Ammonium chloride NH 4 C1 [K 8116, Special class]
Ammonium chloride-ammonia TS To ammonia solution
(28) add an equal volume of water, and saturate this solution
with ammonium chloride.
Ammonium chloride buffer solution, pH 10 Dissolve 5.4
g of ammonium chloride in water, and add 21 mL of ammo-
nia solution (28) and water to make 100 mL.
Ammonium chloride TS Dissolve 10.5 g of ammonium
chloride in water to make 100 mL (2 mol/L).
Ammonium citrate See diammonium hydrogen citrate.
Ammonium dihydrogenphosphate NH 4 H 2 P0 4 [K 9006,
Special class]
0.02 mol/L Ammonium dihydrogenphosphate TS
Dissolve 2.30 g of ammonium dihydrogen phosphate in water
to make 1000 mL.
Melting point <2.60>: 116 - 119°C
0.05 mol/L Ammonium formate buffer solution, pH 4.0
Dissolve 3.5 g of ammonium formate in about 750 mL of
water, adjust the pH to 4.0 with formic acid, and add water
to make 1000 mL.
Ammonium hydrogen carbonate NH 4 HC0 3 White or
semi-transparency crystals, crystalline powder or masses,
having an ammonia odor.
Ammonium iron (II) sulfate hexahydrate
FeS0 4 (NH 4 ) 2 S04.6H 2 [K 8979, Special class]
Ammonium iron (III) citrate [Same as the monograph
Ferric Ammonium Citrate in the Japanese Standards of Food
Additives]
Ammonium iron (III) sulfate TS Dissolve 8 g of ammo-
nium iron (III) sulfate dodecahydrate in water to make 1 00 mL .
Ammonium iron (III) sulfate TS, acidic Dissolve 20 g of
ammonium iron (III) sulfate dodecahydrate in a suitable
amount of water, add 9.4 mL of sulfuric acid, and add water
to make 100 mL.
Ammonium iron (III) sulfate TS, dilute To 2 mL of am-
monium iron (III) sulfate TS add 1 mL of 1 mol/L hydro-
chloric acid TS and water to make 100 mL.
Ammonium iron (III) sulfate dodecahydrate
FeNH 4 (S0 4 ) 2 .12H 2 [K 8982, Special class]
Ammonium molybdate See hexaammonium hep-
tamolybdate tetrahydrate.
Ammonium molybdate-sulfuric acid TS See hexaammo-
nium heptamolybdate-sulfuric acid TS
Ammonium molybdate TS See hexaammonium hep-
tamolybdate TS.
Ammonium nickel (II) sulfate hexahydrate
NiS0 4 (NH 4 ) 2 S0 4 .6H 2 [K 8990, Special class]
Ammonium nitrate NH 4 N0 3 [K 8545, Special class]
Ammonium oxalate See ammonium oxalate monohy-
drate.
Ammonium oxalate monohydrate (NH 4 ) 2 C 2 4 .H 2 [K
8521, Special class]
Ammonium oxalate TS Dissolve 3.5 g of ammonium
oxalate monohydrate in water to make 100 mL (0.25 mol/L).
Ammonium peroxodisulfate (NH 4 ) 2 S 2 8 [K 8252,
Special class]
10% Ammonium peroxodisulfate TS Dissolve 1 g of
ammonium peroxodisulfate in water to make 10 mL.
Ammonium persulfate See ammonium peroxodisulfate.
Ammonium polysulflde TS (NH 4 ) 2 S, [K 8943, Ammo-
nium Sulfide Solution (yellow), First class]
Ammonium sodium hydrogenphosphate tetrahydrate
NaNH 4 HP0 4 . 4H 2 [K 9013, Special class]
Ammonium sulfamate See ammonium amidosulfate.
Ammonium formate HCOONH 4 Colorless crystals.
Very soluble in water.
Ammonium sulfamate TS See ammonium amidosulfate
TS.
JPXV
General Tests / Reagents, Test Solutions
155
Ammonium sulfate (NH 4 ) 2 S0 4 [K 8960, Special class]
Ammonium sulfate buffer solution Dissolve 264 g of am-
monium sulfate in 1000 mL of water, add 1000 mL of 0.5
mol/L sulfuric acid TS, shake, and filter. The pH of this
solution is about 1.
Ammonium sulfide TS (NH 4 ) 2 S [K 8943, Ammonium
Sulfide Solution, (colorless), First class] Store in small,
well-filled containers, protected from light.
Ammonium tartrate See L-ammonium tartrate.
L- Ammonium tartrate C 4 H I2 N 2 6
monium tartrate, Special class]
[K 8534, ( + ) Am-
Ammonium thiocyanate NH 4 SCN [K 9000, Special
class]
Ammonium thiocyanate-cobalt (II) nitrate TS Dissolve
17.4 g of ammonium thiocyanate and 2.8 g of cobalt (II) ni-
trate hexahydrate in water to make 100 mL.
Ammonium thiocyanate TS Dissolve 8 g of ammonium
thiocyanate in water to make 100 mL (1 mol/L).
Ammonium vanadate See ammonium vanadate (V).
Ammonium vanadate (V) NH 4 V0 3 [K 8747, Special c-
lass]
Amoxicillin Ci 6 H 19 N 3 05S.3H 2 [Same as the namesake
monograph]
Amphoteric electrolyte solution for pH 3 to 10 Extremely
pale yellow liquid. Mixture consisting of multiple types of
molecules, buffer capacity is 0.35 mmol/pH-mL. Forms a pH
gradient over a pH range of 3 to 10 when mixed with poly-
acrylamide gel and placed in an electric field.
Amphoteric electrolyte solution for pH 6 to 9 Forms a
pH gradient over a pH range of 6 to 9 when mixed with poly-
acrylamide gel and placed in an electric field. Prepare by
diluting a 0.35 mmol/pH-mL buffer capacity solution about
20-fold with water. Almost colorless.
Amphoteric electrolyte solution for pH 8 to 10.5 Ex-
tremely pale yellow liquid. Mixture consisting of multiple
types of molecules, buffer capacity is 0.35 mmol/pH-mL.
Forms a pH gradient over a pH range of 8 to 10.5 when mix-
ed with polyacrylamide gel and placed in an electric field.
Amygdalin for thin-layer chromatography C 20 H 27 NOii
A white, odorless powder. Freely soluble in water, sparingly
soluble in methanol, and practically insoluble in diethyl
ether.
Purity Related substances — Dissolve 20 mg of amygdalin
for thin-layer chromatography in 5 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with 10 /xh
each of the sample solution and standard solution as directed
in the Identification under Apricot Kernel: any spot other
than the principal spot at the Rf value of about 0.5 obtained
from the sample solution is not more intense than the spot
from the standard solution.
M-Amyl alcohol CH 3 (CH 2 ) 4 OH Clear, colorless liquid,
having a characteristic odor. Sparingly soluble in water, and
miscible with ethanol (95) and with diethyl ether.
Refractive index <2.45> n„: 1.409 - 1.411
Specific gravity <2.56> df: 0.810 - 0.820
Distilling range <2.57>: 135 - 140°C, not less than 95 vol%.
r-Amyl alcohol (CH 3 ) 2 C(OH)CH 2 CH 3 Clear, colorless
liquid, having a characteristic odor. Miscible with tert-
butanol and with 2-butanone, and freely soluble in water.
Specific gravity <2.56> d*: 0.808 -0.815
Purity Acid and ester — To 20 mL of ?-amyl alcohol add
20 mL of ethanol (95) and 5.0 mL of 0.1 mol/L sodium hy-
droxide VS, and heat gently under a reflux condenser in a
water bath for 10 minutes. Cool, add 2 drops of phenol-
phthalein TS, and titrate <2.50> with 0.1 mol/L hydrochloric
acid VS. Perform a blank determination: not more than 1.25
mL of 0.1 mol/L sodium hydroxide VS is consumed.
Nonvolatile residue — Evaporate 50 mL of ^-arnyl alcohol,
and dry at 105°C for 1 hour: the residue is not more than 1.6
mg.
Distilling range <2.57>: 100 - 103°C, not less than 95 vol%.
tert-Amy\ alcohol See t-amyl alcohol.
Amyl alcohol, iso See 3-methyl-l-butanol.
Anesthetic ether See ether, anesthetic.
Anhydrous caffeine See caffeine, anhydrous.
Anhydrous cupric sulfate See copper (II) sulfate (an-
hydrous).
Anhydrous dibasic sodium phosphate See disodium
hydrogen phosphate.
Anhydrous dibasic sodium phosphate for pH determina-
tion See disodium hydrogen phosphate for pH deter-
mination.
Anhydrous hydrazine for amino acid analysis Prepared
for amino acid analysis.
Anhydrous lactose Ci 2 H 22 O u [Same as the monograph
Anhydrous Lactose]
Anhydrous potassium carbonate See potassium car-
bonate.
Anhydrous sodium acetate See sodium acetate, anhy-
drous.
Anhydrous sodium carbonate See sodium carbonate, an-
hydrous.
Anhydrous sodium sulfate See sodium sulfate, anhy-
drous.
Anhydrous sodium sulfite See sodium sulfite, anhy-
drous.
Aniline C 6 H 5 NH 2 [K 8042, Special class]
Animal tissue peptone See peptone, animal tissue.
p-Anisaldehyde See 4-methoxybenzaldehyde.
p-Anisaldehyde-acetic acid TS See 4-methoxyhenzalde-
hyde-acetic acid TS.
p-Anisaldehyde-sulfuric acid TS See 4-methoxybenzalde-
hyde-sulfuric acid TS.
Anisole C 7 H 8 [K 8041, Special class]
Anthrone C 14 H 10 O [K 8082, Special class]
156
Reagents, Test Solutions / General Tests
JP XV
Anthrone TS Dissolve 35 mg of anthrone in 100 mL of
sulfuric acid.
Anti-A type antibody for blood typing Conforms to the
requirements of antibody for blood typing.
Anti-B type antibody for blood typing Conforms to the
requirements of antibody for blood typing.
Antibody fragment (Fab') Purify E. coli protein an-
tibody by affinity chromatography using Staphylococcus
aureus protein A as a ligand, and fractionate IgG. Digest this
fraction using pepsin, remove the pepsin and Fc fragment by
gel filtration chromatography, and obtain F(ab')2 fragment
after removing undigested IgG by affinity chromatography
with protein A as ligand. Reduce this with 2-mercaptoethyla-
mine.
Anti-bradykinin antibody A colorless to light brown,
clear solution prepared by dissolving rabbit origin anti-
bradykinin antibody in 0.04 mol/L phosphate buffer solu-
tion, pH 7.0 containing 1 mg/mL of bovine serum albumin.
Performance test — To a suitable amount of anti-bradyki-
nin antibody to be tested add 0.04 mol/L phosphate buffer
solution, pH 7.0 containing 1 mg/mL bovine serum albumin
to make a 1 vol% solution. Perform the test with 0.1 mL of
this solution as directed in the Purity (2) under Kal-
lidinogenase, and determine the absorbances at 490 - 492
nm, A! and A 2 , of the standard solution (1) and the standard
solution (7): the value, A 2 -A lt is not less than 1.
Anti-bradykinin antibody TS To 0.15 mL of anti-
bradykinin antibody, 15 mg of bovine serum albumin, 2.97
mg of sodium dihydrogen phosphate dihydrate, 13.5 mg of
disodium hydrogen phosphate dodecahydrate and 13.5 mg of
sodium chloride add water to make 15 mL, and lyophilize.
Dissolve this in 15 mL of water. Prepare before use.
Anti-E. coli protein antibody stock solution Taking E.
coli protein stock solution as the immunogen, mix with
Freund's complete adjuvant, and immunize rabbits by sub-
cutaneous injection at 3 week intervals to obtain antiserum.
Treat the antiserum obtained by ammonium sulfate precipi-
tation.
Protein concentration: Dilute anti-E. coli protein antibody
stock solution with 0.05 mol/L tris hydrochloride buffer so-
lution (pH 7.5), measure the absorbance at 280 nm using 0.05
mol/L tris hydrochloride buffer solution (pH 7.5) as a control
as direct under Ultraviolet-visible Spectrophotometry <2.24>,
and determine the protein concentration (absorbance 1.0 =
0.676 mg/mL).
Antimony (III) chloride SbCL, [K 8400, Special class]
Antimony (III) chloride TS Wash chloroform with an
equal volume of water twice or three times, add freshly ignit-
ed and cooled potassium carbonate, and allow to stand over-
night in a well-closed container protected from light.
Separate the chloroform layer, and distil it, preferably with
protection from light. With this chloroform, wash the sur-
face of antimony (III) chloride until the rinsing solution
becomes clear, add the chloroform to this antimony (III)
chloride to make a saturated solution, and place in light-
resistant, glass-stoppered bottles. Prepare before use.
Antimony trichlorid See antimony (III) chloride.
Antimony trichlorid TS See antimony (III) chlorid TS.
Antipyrine CnH^IS^O [Same as the namesake mono-
graph]
Anti-rabbit antibody-coated wells Wells of a polystyrene
microplate coated with goat origin anti-rabbit IgG antibody.
Anti-thrombin III A white powder.
Water <2.4S>: not more than 5%.
Content: not less than 80% and not more than 130% of the
labeled amount.
Anti-thrombin III TS Dissolve 10 unit of anti-thrombin
III in 10 mL of water.
Anti-ulinastatin rabbit serum To a suitable amount of
Ulinastatin having the specific activity of more than 3000
Units per mg protein add isotonic sodium chloride solution
so that each mL of the solution contains about 1 mg of
protein. To 1 mL of this solution add 1 mL of Freund's
complete adjuvant, and emulsify completely. Intracutane-
ously, inject the emulsion so obtained into a rabbit weighing
about 2 kg. Repeat the injection at least 4 times at one-week
intervals, and draw the blood of the animal from the carotid
artery after the antibody titer reaches 16 times or more.
Separate the serum after the blood has coagulated. Preserve
at below -20°C.
Anti-urokinase serum Take Urokinase containing not
less than 140,000 Unit per mg of protein, dissolve in isotonic
sodium chloride solution to make a solution containing 1 mg
of protein per mL, and emulsify with an equal volume of
Freund's complete adjuvant. Inject intracutaneously three
2-mL portions of the emulsion to a healthy rabbit weighed
between 2.5 kg and 3.0 kg in a week interval. Collect the
blood from the rabbit at 7 to 10 days after the last injection,
and prepare the anti-serum.
Performance test — Dissolve 1.0 g of agar in 100 mL of
boric acid-sodium hydroxide buffer solution, pH 8.4, by
warming, and pour the solution into a Petri dish to make a
depth of about 2 mm. After cooling, bore three of a pair -well
2.5 mm in diameter with a space of 6 mm each other. In
one of the wells of each pair-well, place 10 fiL of anti-uro-
kinase serum, and in each another well, place 10 /xL of a solu-
tion of Urokinase containing 30,000 Units per mL in isotonic
sodium chloride solution, 10 /xL of human serum and 10 /uL
of human urine, respectively, and allow to stand for over-
night: a precipitin line appears between anti-urokinase serum
and urokinase, and not appears between anti-urokinase se-
rum and human serum or human urine.
a-Apooxytetracydine C 2 2H 2 2N 2 8 Yellow-brown to
green powder.
Melting point <2.60>: 200- 205 °C
jS-Apooxytetracycline C22H22N2O8 Yellow-brown to
brown powder.
Purity Related substances — Dissolve 8 mg of /?-apoox-
ytetracycline in 5 mL of 0.01 mol/L sodium hydroxide TS,
add 0.01 mol/L hydrochloric acid TS to make 100 mL, and
use this solution as the sample solution. Proceed the test with
20 fiL of the sample solution as directed in the Purity (2) un-
der Oxytetracycline Hydrochloride, determine each peak area
by the automatic integration method, and calculate the
amounts of them by the area percentage method: the total
amount of the peaks other than /3 -apooxytetracycline is not
more than 10%.
JPXV
General Tests / Reagents, Test Solutions
157
Aprotinin A clear and colorless liquid containing aproti-
nin extracted from the lung or parotid gland of a healthy cat-
tle. The pH is between 5.0 and 7.0.
Content: not less than 15,000 KIE Units and not more than
25,000 KIE Units of aprotinin per mL. Assay — (i) Trypsin
solution: Weigh an amount of crystallized trypsin equivalent
to about 250 FIP Units of trypsin according to the labeled
FIP Units, and dissolve in 0.001 mol/L hydrochloric acid TS
to make exactly 10 mL. Prepare before use, and preserve in
ice. (ii) Sample solution: Dilute a suitable quantity of aproti-
nin with sodium tetraborate-calcium chloride buffer solution,
pH 8.0 so that each mL of the solution contains 800 KIE U-
nits of aprotinin, and use this solution as the sample solution,
(hi) Apparatus: Use a glass bottle as a reaction reservoir, 20
mm in inside diameter and 50 mm in height, equipped with a
rubber stopper for attachment to a glass /silver-silver chloride
electrode, a nitrogen-induction tube and an exhaust port. Fix
the reaction reservoir in a thermostat, and keep the tempera-
ture of the bath at 25 ± 0.1 °C by means of a precise ther-
moregulator. (iv) Procedure: To 5.0 mL of TV-a-benzoyl-L-
arginine ethyl ester TS add 45.0 mL of sodium tetraborate-
calcium chloride buffer solution, pH 8.0, and use this solu-
tion as the substrate solution. Pipet 1 mL of the trypsin solu-
tion, add sodium tetraborate-calcium chloride buffer solu-
tion, pH 8.0 to make exactly 10 mL, and use this solution as
the test solution I. Transfer 10.0 mL of the substrate solution
to the reaction reservoir, adjust the pH of the solution to 8.00
by adding dropwise 0.1 mol/L sodium hydroxide VS while
stirring and passing a current of nitrogen, add exactly 1 mL
of the test solution I previously allowed to stand at 25 ± 0.1°
C for 10 minutes, then immediately add dropwise 0.1 mol/L
sodium hydroxide VS by a 50-,mL micropipet (minimum
graduation of 1 fiL), while stirring, to keep the reaction solu-
tion at pH 8.00, and read the amount of 0.1 mol/L sodium
hydroxide VS consumed and the reaction time when the pH
reached 8.00. Continue this procedure up to 6 minutes.
Separately, pipet 2 mL of the trypsin solution and 1 mL of
the sample solution, add sodium tetraborate-calcium chloride
buffer solution, pH 8.0 to make exactly 10 mL, and use this
solution as the test solution II. Transfer 10.0 mL of the sub-
strate solution to the reaction reservoir, adjust the pH of the
solution to 8.00, while stirring and passing a current of nitro-
gen, add exactly 1 mL of the test solution II, previously al-
lowed to stand at 25 ± 0.1 °C for 10 minutes, and proceed in
the same manner. Separately, transfer 10.0 mL of the sub-
strate solution to the reaction reservoir, adjust the pH of the
solution to 8.00, while stirring and passing a current of nitro-
gen, add 1 mL of sodium tetraborate-calcium chloride buffer
solution, pH 8.0, previously allowed to stand at 25 ± 0.1 °C
for 10 minutes, and perform a blank determination in the
same manner, (v) Calculation: Plot the amount of consump-
tion (fiL) of 0.1 mol/L sodium hydroxide VS against the
reaction time (minutes), select linear reaction times, t x and t 2 ,
designate the corresponding consumption amount of 0.1 mol
/L sodium hydroxide VS as V] and v 2 , respectively, and desig-
nate ,Mmol of sodium hydroxide consumed per minute as M.
M i/umol NaOH/min)
h-U
10 J
f: Factor of 0.1 mol/L sodium hydroxide VS
KIE Units per mL of aprotinin to be tested
_ 2(M A -M )-(M B -M„)
L
L: Amount (mL) of the sample solution added to the test
solution II
«: Dilution coefficient of aprotinin to be tested
M A : //mol of sodium hydroxide consumed in 1 minute
when the test solution I is used
M B : fimol of sodium hydroxide consumed in 1 minute
when the test solution II is used
M : /imol of sodium hydroxide consumed in 1 minute when
the solution for blank determination is used
32.5: Equivalent coefficient for calculation of KIE Units
from FIP Units
One KIE Unit means an amount of aprotinin making a
reduction of 50% off the potency of 2 Units of kal-
lidinogenase at pH 8.0 and room temperature for 2 hours.
Storage — Preserve in a light-resistant, hermetic container
and in a cold place.
Aprotinin TS Measure an appropriate amount of aproti-
nin, and dissolve in 0.05 mol/L phosphate buffer solution,
pH 7.0 to prepare a solution containing 50 KIE Units per mL.
Aqua regia Add 1 volume of nitric acid to 3 volumes of
hydrochloric acid. Prepare before use.
L-Arabinose C 5 H 10 O 5 [K 8054: 1991, L( + )-Arabinose,
Special class]
Arbutin for component determination Use arbutin for
thin-layer chromatography meeting the following additional
specifications.
Absorbance <2.24> E\ 0/ ° m (280 nm): 70-76 [4 mg, previ-
ously dried in a desiccator (in vaccum, silica gel), 12 hours,
water, 100 mL].
Purity Related substances — Dissolve 40 mg of arbutin
for component determination in 100 mL of water, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add water to make exactly 100 mL, and use this so-
lution as the standard solution (1). Perform the test with ex-
actly 10 fiL each of the sample solution and standard solution
(1) as directed under Liquid Chromatography <2.01> accord-
ing to the following conditions, and measure each peak area
of the both solutions by the automatic integration method:
the total area of the peaks other than that of arbutin from the
sample solution is not larger than the peak area of arbutin
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Component deter-
mination under Bearberry Leaf except detection sensitivity
and time span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add water to make exactly 20 mL, and use this solution as
the standard solution (2). Adjust the detection sensitivity so
that the peak area of arbutin obtained from 10 fiL of the
standard solution (2) can be measured by the automatic in-
tegration method and the peak height of arbutin obtained
from 10 fiL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement: About 3 times as long as the
retention time of arbutin beginning after the solvent peak.
Arbutin for thin-layer chromatography Ci2H 16 7 .«H 2
Colorless to white crystals or crystalline powder, and odor-
less. Freely soluble in water, soluble in methanol, sparingly
soluble in ethanol (95), and practically insoluble in ethyl ace-
x«x32.5
158
Reagents, Test Solutions / General Tests
JP XV
tate and in chloroform.
Melting point <2.60>: 199 - 201 °C
Purity Related substances — Dissolve 1.0 mg of arbutin
for thin-layer chromatography in exactly 1 mL of a mixture
of ethanol (95) and water (7:3). Perform the test with 20 /uL
of this solution as directed in the Identification (2) under
Bearberry Leaf: any spot other than the main spot at the Rf
value of about 0.4 does not appear.
Arecoline hydrobromide for thin-layer chromatography
C 8 H 13 N0 2 .HBr White crystals. Freely soluble in water,
soluble in methanol, and practically insoluble in diethyl
ether.
Melting point <2.60>: 169 - 171 °C
Purity Related substances — Dissolve 50 mg of arecoline
hydrobromide for thin-layer chromatography in exactly 10
mL of methanol. Perform the test with 10 fiL of this solution
as directed in the Identification under Areca: any spot other
than the principal spot at the Rf value of about 0.4 does not
appear.
L-Arginine C 6 H I4 N 4 2 White, crystals or crystalline
powder. It has a characteristic odor.
Optical rotation <2.49> [alp : +26.9 - +27.9° (After
drying, 4 g, 6 mol/L hydrochloric acid TS, 50 mL, 200 mm).
Loss on drying <2.41>: not more than 0.50% (1 g, 105 C C, 3
hours).
Content: not less than 98.0% and not more than 102.0%.
Assay — Weigh accurately about 0.15 g of L-arginine, previ-
ously dried, dissolve in 3 mL of formic acid, add 50 mL of
acetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the color of the solution changes to green
through yellow (indicator: 10 drops of p-naphtholbenzein
TS). Perform a blank determination in the same manner and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 8.710 mg of C 6 H 14 N 4 2
L-Arginine hydrochloride C 6 H 14 N 4 2 .HC1 [Same as the
namesake monograph]
Aristolochic acid I for crude drugs purity test
C 17 H u N0 7 Yellow crystalline powder. Melting point: about
280°C (with decomposition).
Absorbance <2.24> E\°^ (318 nm): 384-424 (1 mg,
methanol, 100 mL).
Purity Related substances — Dissolve 1 .0 mg of aristo-
lochic acid I for crude drugs purity test in 100 mL of diluted
methanol (3 in 4), and use this solution as the sample solu-
tion. Pipet 1 mL of this solution, add diluted methanol (3 in
4) to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 10 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine each peak area by the automatic in-
tegration method: the total area of the peaks other than
aristolochic acid I obtained from the sample solution is not
more than the peak area of aristolochic acid I from the stan-
dard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (3) under Asiasarum Root.
Time span of measurement: About 3 times as long as the
retention time of aristolochic acid I beginning after the sol-
vent peak.
System suitability
Proceed as directed in the system suitability in the Purity
(3) under Asiasarum Root.
Arsenazo III C 22 Hi 8 As 2 N 4 14 S 2 [K 9524]
Arsenazo III TS Dissolve 0.1 g of arsenazo III in water to
make 50 mL.
Arsenic-free zinc See zinc for arsenic analysis.
Arsenic (III) trioxide As 2 3 [K 8044, Arsenic (III) tri-
oxide, Special class]
Arsenic (III) trioxide TS Add 1 g of arsenic (III) trioxide
to 30 mL of a solution of sodium hydroxide (1 in 40), dissolve
by heating, cool, and add gently acetic acid (100) to make 100
mL.
Arsenic trioxide See arsenic (III) trioxide.
Arsenic trioxide TS See arsenic (III) trioxide TS.
Ascorbic acid See L-ascorbic acid.
L-Ascorbic acid C 6 H 8 6 [K 9502, L( + )-Ascorbic
Acid, Special class]
Ascorbic acid for iron limit test See L-ascorbic acid.
0.012 g/dL L-Ascorbic acid-hydrochloric aicd TS Dis-
solve 15 mg of L-ascorbic acid in 25 mL of methanol, add
carefully 100 mL of hydrochloric acid, and mix. Prepare be-
fore use.
0.02 g/dL L-Ascorbic acid-hydrochloric acid TS Dissolve
25 mg of L-ascorbic acid in 25 mL of methanol, add carefully
100 mL of hydrochloric acid, and mix. Prepare before use.
0.05 g/dL L-Ascorbic acid-hydrochloric acid TS Dissolve
0.05 g of L-ascorbic acid in 30 mL of methanol, add carefully
hydrochloric acid to make 100 mL. Prepare before use.
DL-Aspartic acid C 4 H 7 N0 4 A white crystalline powder
that is sparingly soluble in water. Melting point: 270 to
271°C.
L-Aspartic acid C 4 H 7 N0 4 [K 9045, Special class]
Aspartic acid See L-aspartic acid.
Aspirin C 9 H 8 4 [Same as the namesake monograph]
Astragaloside IV for thin-layer chromatography
C 41 H 68 14 A white powder. Sparingly soluble in methanol,
very slightly soluble in ethanol (99.5), and practically insolu-
ble in water.
Optical rotation <2.49> [a] d°: + 19 - +26° (10 mg dried
with silica gel for 24 hours, methanol, 2 mL, 50 mm).
Purity Related substances — Dissolve 1 mg in 1 mL of
methanol. Proceed the test with 5 fiL of the standard solution
as directed in the Identification (4) under Hochuekkito Ex-
tract: no spot appears other than the principal spot of around
Rf 0.5.
Atractylenolide III for thin-layer chromatography
C 15 H 20 O 3 White crystals or crystalline powder. Very soluble
in methanol, freely soluble in ethanol (99.5), and practically
insoluble in water. Melting point: 193 - 196°C
Identification — Determine the absorption spectrum of a
JP XV
General Tests / Reagents, Test Solutions
159
solution in methanol (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 217 nm and 221 nm.
Purity Related substances — Dissolve 1 mg in 10 mL of
methanol. Proceed the test with 2 fiL of the standard solution
as directed in the Identification (3) under Kamishoyosan Ex-
tract: no spot appears other than the principal spot of around
Rf 0.5
Atropine sulfate (C 17 H 2 3N03) 2 .H 2 S04.H 2 [Same as the
monograph Atropin sulfate Hydrate]
Atropine sulfate for assay [Same as the monograph Atro-
pine Sulfate Hydrate. When dried, it contains not less than
99.0% of atropine sulfate [(C 17 H 23 N0 3 ) 2 .H 2 S0 4 ].]
Atropine sulfate for thin-layer chromatography Use
atropine sulfate for assay meeting the following additional
specifications. Weigh accurately about 50 mg of atropine sul-
fate for assay, dissolve in ethanol (95) to make exactly 10 mL,
and use this solution as the sample solution. Perform the test
with the sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 50,mL of the solution on a plate of
silica gel for thin-layer chromatography, develop the plate
with a mixture of chloroform and diethylamine (9:1) to a dis-
tance of about 10 cm, air-dry the plate, and spray evenly
chloroplatinic acid-potassium iodide TS on the plate: any
spot other than the spot at the Rf value of about 0.4 does not
appear.
A-type erythrocyte suspension Prepare a suspension con-
taining 1 vol% of erythrocyte separated from human A-type
blood in isotonic sodium chloride solution.
Baicalin for thin-layer chromatography
C 21 H 18 O u .H 2 Light yellow odorless powder. Slightly
soluble in methanol, and practically insoluble in water and in
diethyl ether. Melting point: about 206 °C (with decomposi-
tion).
Purity Related substance — Dissolve l.Omg of baicalin
for thin-layer chromatography in exactly 1 mL of methanol.
Perform the test with 10 iiL of this solution as directed in the
Identification (2) under Scutellaria Root: any spot other than
the principal spot at the Rf value of about 0.4 does not
appear.
Balsam Canada balsam for microscopy. Before use,
dilute to a suitable concentration with xylene.
Bamethan sulfate (C 12 H 19 N0 2 ) 2 .H 2 S0 4 [Same as the
namesake monograph]
Barbaloin for component determination Use barbaloin
for thin-layer chromatography meeting the following addi-
tional specifications.
Absorbance <2.24> £}% (360 nm): 260 - 290 [10 mg dried
in a desiccator (in vacuum, phosphorus (V) oxide) for not less
than 24 hours, methanol, 500 mL.]
Purity Related substances — Dissolve 10 mg of the sub-
stance to be tested in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add methanol to make exactly 100 mL, and use this
solution as the standard solution (1). Perform the test with
exactly 20 /uL each of the sample solution and standard solu-
tion (1) as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and measure each peak
area of the both solutions by the automatic integration
method: the total area of the peaks other than barbaloin
from the sample solution is not larger than the peak area of
barbaloin from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Component deter-
mination under Aloe except wavelength, detection sensitivity
and time span of measurement.
Wavelength: 300 nm
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add methanol to make exactly 20 mL, and use this solu-
tion as the standard solution (2). Adjust the detection sen-
sitivity so that the peak area of barbaloin obtained from 20
fiL of the standard solution (2) can be measured by the auto-
matic integration method and the peak height of barbaloin
obtained from 20 fiL of the standard solution (1) shows
about 20% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of barbaloin beginning after the solvent peak.
Barbaloin for thin-layer chromatography C 21 H 22 O s
Light yellow, crystalline powder. Freely soluble in methanol,
practically insoluble in water and in diethyl ether.
Melting point <2.60>: 148°C
Purity Related substances — Dissolve 1.0 mg of barbaloin
for thin-layer chromatography in exactly 1 mL of methanol.
Perform the test with 20 /xL of this solution as directed in the
Identification (2) under Aloe: any spot other than the prin-
cipal spot at the Rf value of about 0.6 does not appear.
Barbital C 8 H 12 N 2 3 [Same as the namesake mono-
graph]
Barbital buffer solution Dissolve 15 g of barbital sodium
in 700 mL of water, adjust the pH to 7.6 with dilute hydro-
chloric acid, and filter.
Barbital sodium C 8 H u N 2 Na0 3 White, odorless crystals
of crystalline powder, having a bitter taste. Freely soluble in
water, slightly soluble in ethanol (95), and practically insolu-
ble in diethyl ether.
pH <2.54> — The pH of a solution of barbital sodium (1 in
200) is between 9.9 and 10.3.
Loss on drying <2.41>: not more than 1.0% (1 g, 105 °C, 4
hours).
Content: not less than 98.5%. Assay — Weigh accurately
about 0.5 g of barbital sodium, previously dried, transfer to a
separator, dissolve in 20 mL of water, add 5 mL of ethanol
(95) and 10 mL of dilute hydrochloric acid, and extract with
50 mL of chloroform. Then extract with three 25-mL por-
tions of chloroform, combine the total extract, wash with
two 5-mL portions of water, and extract the washings with
two 10-mL portions of chloroform. Combine the chloroform
extracts, and filter into a conical flask. Wash the filter paper
with three 5-mL portions of chloroform, combine the filtrate
and the washings, add 10 mL of ethanol (95), and titrate
<2.50> with 0.1 mol/L potassium hydroxide-ethanol VS until
the color of the solution changes from yellow to purple
through light purple (indicator: 2 mL of alizarin yellow GG-
thymolphthalein TS). Perform a blank determination in the
same manner.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 20.62 mg of C 8 H„N 2 Na0 3
Barium chloride See barium chloride dihydrate.
Barium chloride dihydrate BaCl 2 .2H 2 Q [K 8155, Spec-
160
Reagents, Test Solutions / General Tests
JP XV
ial class]
Barium chloride TS Dissolve 12 g of barium chloride
dihydrate in water to make 100 mL (0.5 mol/L).
Barium hydroxide See barium hydroxide octahydrate.
Barium hydroxide octahydrate Ba(OH) 2 .8H 2
[K 8577, Special class] Store in tightly stoppered containers.
Barium hydroxide TS Saturate barium hydroxide octahy-
drate in freshly boiled and cooled water (0.25 mol/L). Pre-
pare before use.
Barium nitrate Ba(N0 3 ) 2 [K 8565, Special class]
Barium nitrate TS Dissolve 6.5 g of barium nitrate in
water to make 100 mL (0.25 mol/L).
Barium oxide BaO A white to yellowish or grayish white,
powder.
Identification — (1) Dissolve 0.5 g in 15 mL of water and 5
mL of hydrochloric acid, and add 10 mL of dilute sulfuric
acid: white precipitates appear.
(2) Perform the test as directed under Flame Coloration
Test (1) <].04>: a green color appears.
Barium perchlorate Ba(C10 4 ) 2 [K 9551, Special class]
Becanamycin sulfate [Same as the namesake monograph]
Beclometasone dipropionate C 28 H 37 C107 [Same as the
namesake monograph]
Benidipine hydrochloride C 28 H 3 iN 3 6 .HCl [Same as
the namesake monograph]
Benidipine hydrochloride for assay C 28 H 31 N 3 06.HC1
[Same as the monograph Benidipine Hydrochloride. When d-
ried, it contains not less than 99.5% of benidipine hydrochlo-
ride (C 28 H 31 N 3 6 .HC1)]
Benzaldehyde C 6 H 5 CHO [K 8857, First class]
Benzalkonium chloride [Same as the namesake mono-
graph]
Benzalphthalide Ci 5 H 10 O 2 Yellow crystalline powder.
Melting point: 99 - 102°C.
Benzene C 6 H 6 [K 8858, Special class]
Benzethonium chloride for assay C 27 H 42 C1N0 2 [Same
as the monograph Benzethonium Chloride. When dried, it
contains not less than 99.0% of benzethonium chloride
(C 27 H 42 C1N0 2 ).]
Benzoic acid C 6 H 5 COOH [K 8073, Special class]
Benzoin C 6 H 5 CH(OH)COC 6 H 5 White to pale yellow,
crystals or powder.
Melting point <2.60>: 132- 137°C
Benzophenone C 6 H 5 COC 6 H 5 Colorless crystals, having
a characteristic odor.
Melting point <2.60>: 48 - 50°C
p-Benzoquinone C 6 H 4 2 Yellow to yellow-brown,
crystals or crystalline powder, having a pungent odor. Solu-
ble in ethanol (95) and in diethyl ether, slightly soluble in
water. It is gradually changed to a blackish brown color by
light.
Melting point <2.60>: 111 - 116 C C
Content: not less than 98.0%. Assay — Weigh accurately
about 0.1 g of /?-benzoquinone, place in an iodine bottle, add
exactly 25 mL of water and 25 mL of diluted sulfuric acid (1
in 15), dissolve 3 g of potassium iodide by shaking, and ti-
trate <2.50> with 0.1 mol/L sodium thiosulfate VS (indicator:
3 mL of starch TS). Perform a blank determination.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 5.405 mg of C 6 H 4 2
p-Benzoquinone TS Dissolve 1 g of p-benzoquinone in 5
mL of acetic acid (100), and add ethanol (95) to make 100
mL.
/V-a-Benzoyl-L-arginine ethyl ester hydrochloride
C 15 H 22 N 4 3 .HC1 White crystals or crystalline powder.
Freely soluble in water and in ethanol (95), and slightly solu-
ble in diethyl ether.
Melting point <2.60>: 129 - 133°C
Optical rotation <2.49> [a] 2 ^: -15.5 --17.0° (2.5 g,
water, 50 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.1 g
of /V-a-benzoyl-L-arginine ethyl ester hydrochloride in 20 mL
of water: the solution is clear and colorless.
(2) Related substances — Weigh 0.10 g of 7V-a-benzoyl-L-
arginine ethyl ester hydrochloride, dissolve in 6 mL of water,
add 4 mL of hydrochloric acid, heat in a boiling water bath
for 5 minutes to decompose, and use this solution as the sam-
ple solution. Perform the test with the sample solution as
directed under Paper Chromatography. Spot 5 /xL of the
sample solution on a chromatographic filter paper. Develop
with a mixture of water, acetic acid (100) and 1-butanol
(5:4:1) to a distance of about 30 cm, and air-dry the paper.
Spray evenly a solution of ninhydrin in acetone (1 in 50) upon
the paper, and heat at 90°C for 10 minutes: only one purple
spot appears.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.6 g of ,/V-a-benzoyl-L-arginine ethyl ester hydrochlo-
ride, dissolve in 50 mL of water, neutralize with 0.1 mol/L
sodium hydroxide VS, if necessary, and titrate <2.50> with 0.1
mol / L silver nitrate VS (indicator: 4 drops of
dichlorofluorescein TS).
Each mL of 0.1 mol/L silver nitrate VS
= 34.28 mg of C 15 H 22 N 4 3 .HC1
./V-a-Benzoyl-L-arginine ethyl ester TS Dissolve 0.07 g of
./V-a-benzoyl-L-arginine ethyl ester hydrochloride in freshly
boiled and cooled water to make exactly 10 mL.
./V-a-Benzoyl-L-arginine-4-nitroanilide hydrochloride
C 19 H 22 N 6 4 .HC1 Light yellow crystalline powder.
Optical rotation <2.49> [ a ]g>: +45.5 -+48.0° (after
drying, 0.5 g, /V,7V-dimethylformamide, 25 mL, 100 mm).
Purity Related substances — Dissolve 0.20 g of 7V-a-ben-
zoyl-L-arginine-4-nitroanilide hydrochloride in 10 mL of
/V,7V-dimethylformamide, and use this solution as the sample
solution. Perform the test with this solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL of the sample
solution on a plate of silica gel for thin-layer chro-
matography, develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (4:1:1) to a distance of about 10
cm, and air-dry the plate. Exposure the plate to a vapor of io-
dine: only one spot appears.
./V-a-Benzoyl-L-arginine-4-nitroanilide TS Dissolve 0.1 g
JPXV
General Tests / Reagents, Test Solutions
161
of A^-a-benzoyl-L-arginine-4-nitroanilide hydrochloride in
water to make 100 mL.
Benzoyl chloride C 6 H 5 COCl [K 8158, Special class]
iV-Benzoyl-L-isoleucyl-L-glutamyUy-ORJ-glycyl-L-arginyl-
p-nitroanilide hydrochloride An equal amount mixture of
two components, R = H and R = CH 3 . A white powder.
Slightly soluble in water.
Absorbance <2.24> E{°£ m (316 nm): 166 - 184 (10 mg, water,
300 mL).
Benzoylmesaconine hydrochloride for thin-layer chro-
matography C 3I H 43 NO 10 .HCl.xH 2 O White, crystals or
crystalline powder. Soluble in water and in ethanol (99.5).
Melting point: about 250°C (with decomposition).
Purity Related substances — Dissolve 1.0 mg of ben-
zoylmesaconine hydrochloride for thin-layer chro-
matography in exactly 10 mL of ethanol (99.5), and use this
solution as the sample solution. Perform the test with this so-
lution as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL of the sample solution on a plate of silica gel for
thin-layer chromatography. Proceed the test as directed in
the Identification under Processed Aconite Root: no spot
other than the principal spot at around Rf 0.4 appears.
Benzoyl peroxide, 25% water containing (C 6 H 5 CO) 2 2
White moist crystals or powder. Soluble in diethyl ether and
in chloroform, and very slightly soluble in water and in etha-
nol (95). Melting point: 103 - 106°C (dried substance) (with
decomposition).
Loss on drying <2.4]>: not more than 30% (0.1 g, in vacu-
um, silica gel, constant mass).
Benzyl alcohol C 6 H 5 CH 2 OH [K 8854, Special class]
Benzyl benzoate C 6 H 5 COOCH 2 C 6 H 5 [K 8079, Special
class]
Benzyl parahydroxybenzoate HOC 6 H 4 COOCH 2 C 6 H 5
White, odorless, fine crystals or crystalline powder. Freely
soluble in ethanol (95), in acetone and in diethyl ether, and
very slightly soluble in water.
Melting point <2.60>: 109 - 112°C
Residue on ignition <2.44>: not more than 0.1%.
Content: not less than 99.0%. Assay — Proceed as direct-
ed in the Assay under Ethyl Parahydroxybenzoate.
Each mL of 1 mol/L sodium hydroxide VS
= 228.2 mg of Ci 4 H 12 3
Benzylpenicillin Bezyathine C 48 H 64 N 6 12 S 2 [same as the
monograph Benzylpenicillin Benzathine Hydrate]
Benzylpenicillin potassium Ci 6 H I7 KN 2 4 S [Same as the
monograph Benzylpenicillin Potassium]
Benzyl p-hydroxybenzoate See benzyl parahydroxyben-
zoate.
p-Benzylphenol C 6 H 5 CH 2 C 6 H 4 OH White to pale yel-
lowish white crystals or crystalline powder.
Melting point <2.60>: 80 - 85 °C
Berberine chloride C 20 H I8 ClNO 4 .xH 2 O [Same as the
monograph Berberine chloride Hydrate]
Berberine chloride for thin-layer chromatography [Same
as the monograph Berberine Chloride Hydrate. Use the ber-
berine chloride meeting the following additional spec-
ifications.]
Purity Related substances — Dissolve 10 mg of berberine
chloride for thin-layer chromatography in 10 mL of metha-
nol, and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add methanol to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with 10 fiL each of the sample solution and standard
solution as directed in the Identification (2) under Phelloden-
dron Bark: any spot other than the principal spot from the
sample solution is not more intense than the spot from the
standard solution.
Bergenin for thin-layer chromatography
C 14 H 16 9 xH 2 White crystals or crystalline powder. Free-
ly soluble in ethanol (95), soluble in water, and insoluble in
diethyl ether.
Melting point <2.60>: 131 - 133°C, 234-236°C (double
melting points).
Purity Related substances — Dissolve 1.0 mg of bergenin
for thin-layer chromatography in exactly 1 mL of methanol.
Perform the test with 20 /uL of this solution as directed in the
Identification under Mallotus Bark: any spot other than the
principal spot at the Rf value of about 0.5 does not appear.
Betahistine mesilate C 8 H 12 N 2 .2CH 4 3 S [Same as the
namesake monograph]
Betahistine mesilate for assay [Same as the monograph
Betahistine Mesilate. When dried, it contains not less than
99.0% of betahistine mesilate (C 8 H 12 N 2 .2CH 4 3 S).]
Betanidine sulfate for assay (C 10 Hi 5 N 3 ) 2 .H 2 SO 4 [Same
as the monograph Betanidine Sulfate. Calculated on the
dried basis, it contains not less than 99.0% of betanidine sul-
fate (C 10 H 15 N 3 ) 2 .H 2 SO 4 .]
Bezafibrate for assay C 19 H 20 ClNO 4 [Same as the mono-
graph Bezafibrate. When dried it contains not less than 99.0
% of bezafibrate (C 19 H 20 ClNO 4 ).]
BGLB Dissolve 10 g of peptone and 10 g of lactose
monohydrate in 500 mL of water, add 200 mL of fresh ox
bile or a solution prepared by dissolving 20 g of dried ox bile
powder in 200 mL of water and adjusted the pH to between
7.0 and 7.5, then add water to make 975 mL, and again ad-
just to pH 7.4. Then add 13.3 mL of a solution of brilliant
green (1 in 1000) and water to make 1000 mL in total volume,
and filter through absorbent cotton. Dispense 10 mL portions
of the filtrate into tubes for fermentation, and sterilize by au-
toclaving at 121 C C for not more than 20 minutes, then cool
quickly, or sterilize fractionally on each of three successive
days for 30 minutes at 100°C.
a-BHC (a-Hexachlorocyclohexane) C 6 H 6 C1 6
Melting point <2.60>: 157 - 159°C
Purity Related substances — Dissolve 10 mg of a-BHC in
5 mL of acetone for purity of crude drug, and add hexane for
purity of crude drug to make exactly 100 mL. Pipet 1 mL of
this solution, add hexane for purity of crude drug to make ex-
actly 100 mL, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add hexane for purity of
crude drug to make exactly 100 mL, and use this solution as
the standard solution (1). Perform the test with exactly 1 ftL
each of the sample solution and standard solution (1) as
directed under Gas Chromatography <2.02> according to the
following conditions, and measure each peak area from these
162
Reagents, Test Solutions / General Tests
JP XV
solutions by the automatic integration method: the total peak
area other than a-BHC from the sample solution is not larger
than the peak area of a-BHC from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Purity (2) under
Crude Drugs Test <5.01> except detection sensitivity and time
span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add hexane for purity of crude drug to make 20 mL, and
use this solution as standard solution (2). Adjust the detec-
tion sensitivity so that the peak area of a-BHC obtained from
1 mL of the standard solution (2) can be measured by the au-
tomatic integration method, and the peak height of a-BHC
from 1 /uL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement: About twice as long as the
retention time of a-BHC beginning after the peak of solvent.
/?-BHC 06-Hexachlorocyclohexane) C 6 H 6 C1 6
Melting point <2.60>: 308 - 310°C
Purity Related substances — Proceed as directed in the
Purity under a-BHC using the following standard solution
(1).
Standard solution (1): Pipet 2 mL of the sample solution,
and add hexane for purity of crude drug to make exactly 100
mL.
y-BHC (y-Hexachlorocyclohexane) C 6 H 6 C1 6
Melting point <2.60>: 112- 114°C
Purity Related substances — Proceed as directed in the
Purity under a-BHC.
<5-BHC (<5-Hexachlorocyclohexane) C 6 H 6 C1 6
Melting point <2.60>: 137 - 140°C
Purity Related substances — Proceed as directed in the
Purity under a-BHC using the following standard solution
(1).
Standard solution (1): Pipet 5 mL of the sample solution,
and add hexane for purity of crude drug to make exactly 100
mL.
2-(4-Biphenylyl)propionic acid Ci 5 H I4 2 Light yellow-
ish white powder.
Melting point <2.60>: 145 - 148°C
Purity — Dissolve 1 mg of 2-(4-biphenylyl) propionic acid
in a mixture of water and acetonitrile (11:9) to make 50 mL.
Perform the test with 20 /xL of this solution as directed under
Liquid Chromatography <2.01> according to the operating
conditions of the Related substances in the Purity (3) under
Flurbiprofen. Determine each peak area of the solution in
about twice as long as the retention time of the main peak by
the automatic integration method, and calculate the amount
of 2-(4-biphenylyl)propionic acid by the area percentage
method: it is not less than 98.0%.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.5 g of 2-(4-biphenilyl)propionic acid, previously
dried in vacuum over silica gel for 4 hours, and titrate <2.50>
with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 22.63 mg of C 15 H 14 2
2,2'-Bipyridyl C 10 H 8 N 2 [K 8486, Special class]
4,4'-Bis(diethylamino)benzophenone
(C 2 H 5 ) 2 NC 6 H 4 ] 2 CO Light yellow crystals.
Content: not less than 98%. Assay — Weigh accurately
0.25 g of 4,4'-bis(diethylamino)benzophenone, dissolve in 50
mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank titration in the same manner, and make any necessary
correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.22 mg of C 21 H 28 N 2
/V,iV'-Bis[2-hydroxy-l-(hydroxymethyl)ethyl]-5-hydroxy-
acetylamino-2,4,6-triiodoisophthalamide C^H^L^Og
White crystalline powder.
Identification — (1) Heat 0.1 g of 7V,/V'-bis[2-hydroxy-l-
(hydroxymethyl)ethyl] -5 -hydroxy acetylamino -2,4, 6-triio-
doisophthalamide over free flame: a purple colored gas
evolves.
(2) Determine the infrared absorption spectrum of
N,N' - bis [2 - hydroxy - 1 - (hydroxymethyl)ethyl] - 5 - hy droxya-
cetylamino-2,4,6-triiodoisophthalamide according to the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3390 cm- 1 , 3230 cm- 1 , 2882 cm- 1 , 1637
cm- 1 , 1540 cm- 1 , 1356 cm 1 and 1053 cm" 1 .
Purity — Dissolve 0.10 g of A r ,A r '-bis[2-hydroxy-l-
(hydroxymethyl)ethyl]-5-hydroxyacetylamino-2,4,6-triiodo-
isophthalamide in 10 mL of water, and use this solution as
the sample solution. Pipet 1 mL of this solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 fiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of both solutions by auto-
matic integration method: the total area of the peaks other
than the peak of 7V,/V'-bis[2-hydroxy-l-(hydrox-
ymethyl)ethyl]-5-hydroxyacetylamino-2,4,6-triio-
doisophthalamide obtained from the sample solution is not
more than 3 times of the peak area of /V,/V'-bis[2-hydroxy-l-
(hydroxymethyl)ethyl]-5 - hydroxyacetylamino - 2,4,6 - triio-
doisophthalamide obtained from the standard solution.
Operating conditions
Proceed the operating conditions in the Purity (6) under
Iopamidol.
System suitability
Proceed the system suitability in the Purity (6) under
Iopamidol.
Bismuth nitrate See bismuth nitrate pentahydrate.
Bismuth nitrate pentahydrate
[K 8566, Special class]
Bi(N0 3 ) 3 .5H 2
Bismuth nitrate-potassium iodide TS Dissolve 0.35 g of
bismuth nitrate pentahydrate in 4 mL of acetic acid (100) and
16 mL of water (solution A). Dissolve 8 g of potassium iodide
in 20 mL of water (solution B). To 20 mL of a mixture of so-
lution A and solution B (1:1) add 80 mL of dilute sulfuric
acid and 0.2 mL of hydrogen peroxide (30). Prepare before
use.
Bismuth nitrate TS Dissolve 5.0 g of bismuth nitrate pen-
tahydrate in acetic acid (100) to make 100 mL.
Bismuth potassium iodide TS Dissolve 10 g of l -tartaric
acid in 40 mL of water, add 0.85 g of bismuth subnitrate,
JPXV
General Tests / Reagents, Test Solutions
163
shake for 1 hour, add 20 mL of a solution of potassium
iodide (2 in 5), shake thoroughly, allow to stand for 24 hours,
and filter (solution A). Separately, dissolve 10 g of L-tartaric
acid in 50 mL of water, add 5 mL of solution A, and preserve
in a light-resistant, glass-stoppered bottle.
Bismuth sodium trioxide NaBi0 3 [K 8770, Special
class]
Bismuth subnitrate [Same as the namesake monograph]
Bismuth subnitrate TS Dissolve 10 g of L-tartaric acid in
40 mL of water, add 0.85 g of bismuth subnitrate, stir for 1
hour, then add 20 mL of a solution of potassium iodide (2 in
5), and shake well. After standing for 24 hours, filter, and
preserve the filtrate in a light-resistant bottle.
Bismuth sulfite indicator Prepared for microbial test.
Bis-(l-phenyl-3-methyI-5-pyrazolone) C 2 oH 18 B 4 02
White to pale yellow crystals or crystalline powder. It dis-
solves in mineral acids and in alkali hydroxides, and it does
not dissolve in water, in ammonia TS, or in organic solvents.
Melting point: not below 300°C.
Nitrogen content <1.08>: 15.5 - 16.5%
Residue on ignition <2.44>: not more than 0.1%.
Bis-trimethyl silyl acetamide CH 3 CON[Si(CH 3 ) 3 ] 2
Colorless liquid.
Boiling point <2.57>: 71 - 73°C
Refractive index <2.45> n™: 1.414 - 1.418
Specific gravity <2.56> df \ 0.825 - 0.835
Block buffer solution Dissolve 4 g of blocking agent in
100 mL of water, and add 100 mL of 0.01 mol/L phosphate
buffer-sodium chloride TS, pH 7.4.
Blocking agent Powder whose main ingredient is bovine-
derived lactoprotein. For immunological research purposes.
Blue tetrazolium C^H^C^NgC^ 3,3'-Dianisole-bis-
[4,4'-(3,5-diphenyl) tetrazolium chloride] Light yellow
crystals. Freely soluble in methanol, in ethanol (95) and in
chloroform, slightly soluble in water, and practically insolu-
ble in acetone and in ether. Melting point: about 245 °C (with
decomposition).
Absorbance <2.24> E\ 0/ ° m (252 nm): not less than 826
(methanol).
Blue tetrazolium TS, alkaline To 1 volume of a solution
of blue tetrazolium in methanol (1 in 200) add 3 volumes of a
solution of sodium hydroxide in methanol (3 in 25). Prepare
before use.
Borate-hydrochloric acid buffer solution, pH 9.0 Dis-
solve 19.0 g of sodium borate in 900 mL of water, adjust the
pH to exactly 9.0 with 1 mol/L hydrochloric acid TS, and
add water to make 1000 mL.
Borax See sodium tetraborate decahydrate.
Boric acid H 3 B0 3 [K 8863, Special class]
Boric acid-methanol buffer solution Weigh exactly 2.1 g
of boric acid, dissolve in 28 mL of sodium hydroxide TS, and
dilute with water to exactly 100 mL. Mix equal volumes of
this solution and methanol, and shake.
Boric acid-potassium chloride-sodium hydroxide buffer so-
lution, pH 9.0 To 50 mL of 0.2 mol/L boric acid-0.2 mol/
L potassium chloride TS for buffer solution add 21.30 mL of
0.2 mol/L sodium hydroxide VS and water to make 200 mL.
Boric acid-potassium chloride-sodium hydroxide buffer so-
lution, pH 9.2 To 50 mL of 0.2 mol/L boric acid-0.2 mol/
L potassium chloride TS for buffer solution add 26.70 mL of
0.2 mol/L sodium hydroxide VS and water to make 200 mL.
Boric acid-potassium chloride-sodium hydroxide buffer so-
lution, pH 9.6 To 50 mL of 0.2 mol/L boric acid-0.2 mol/
L potassium chloride TS for buffer solution add 36.85 mL of
0.2 mol/L sodium hydroxide VS and water to make 200 mL.
Boric acid-potassium chloride-sodium hydroxide buffer so-
lution, pH 10.0 To 50 mL of 0.2 mol/L boric acid-0.2 mol/
L potassium chloride TS for buffer solution add 43.90 mL of
0.2 mol/L sodium hydroxide VS and water to make 200 mL.
0.2 mol/L Boric acid-0.2 mol/L potassium chloride TS for
buffer solution Dissolve 12.376 g of bodic acid and 14.911 g
of potassium chloride in water to make 1000 mL.
Boric acid-sodium hydroxide buffer solution, pH 8.4 Dis-
solve 24.736 g of boric acid in 0.1 mol/L sodium hydroxide
VS to make exactly 1000 mL.
Boron trifluoride BF 3 Colorless gas, having an irritat-
ing odor.
Boiling point <2.57>: - 100. 3°C
Melting point <2.60>: - 127. 1 °C
Boron trifluoride-methanol TS A solution containing 14
w/v% of boron trifluoride (BF 3 : 67.81) in methanol.
Bovine activated blood coagulation factor X A protein
obtained from bovine plasma. It has an activity to decom-
pose prothrombin specifically and limitedly and produce
thrombin. It does not contain thrombin and plasmin. It
contains not less than 500 Units per mg protein. One unit
indicates an amount of the factor X which hydrolyzes 1 fimol
of AMjenzoyl-L-isoleucyl-L-glutamyl(y-OR)-glycyl-L-arginyl-
p-nitroanilide in 1 minute at 25 °C.
Bovine serum Serum obtained from blood of bovine. In-
terleukin-2 dependent cell growth suppression substance is re-
moved by heat at 56°C for 30 min before use
Bovine serum albumin Obtained from cattle serum as
Cohn's fifth fraction. Contains not less than 95% of albu-
min.
Bovine serum albumin for assay White or yellowish crys-
tals or crystalline powder.
Take 50 mg of bovine serum albumin containing 99% or
more albumin in glass ampoules and put them in the desicca-
tor, whose humidity is adjusted to 31%RH at 25 °C with cal-
cium chloride-saturated solution, for 2 weeks, and then take
out and seal them immediately.
Protein content: 88% or more. Assay — Weigh accurately
about 0.1 g of bovine serum albumin for assay, dissolve in
water, and add water to make exactly 20 mL. Put exactly 3
mL of the solution in the Kjeldahl frask, and determine pro-
tein content following Nitrogen Determination <1.08>.
Each mL of 0.005 mol/L sulfuric acid VS
= 0.8754 mg protein
Storage— Store at 4°C or lower.
164
Reagents, Test Solutions / General Tests
JP XV
Bovine serum albumin for test of ulinastatin White crys-
talline powder obtained from bovine serum by a purification
method which does not denature albumin and other serum
proteins. It contains not less than 99% of albumin.
Bovine serum albumin-isotonic sodium chloride solution
Dissolve 0.1 g of bovine serum albumin in isotonic sodium
chloride solution to make 100 mL. Prepare before use.
1 w/v% Bovine serum albumin-phosphate buffer-sodium
chloride TS Dissolve 1 g of bovine serum albumin in 100
mL of 0.01 mol/L phosphate buffer-sodium chloride TS, pH
7.4.
Bovine serum albumin-sodium chloride-phosphate buffer
solution, pH 7.2 Dissolve 10.75 g of disodium hydrogen
phosphate dodecahydrate, 7.6 g of sodium chloride and 1.0 g
of bovine serum albumin in water to make 1000 mL. Adjust
to pH 7.2 with dilute sodium hydroxide TS or diluted phos-
phoric acid (1 in 10) before use.
Bovine serum albumin TS for secretin Dissolve 0.1 g of
bovine serum albumin, 0.1 g of L-cysteine hydrochloride
monohydrate, 0.8 g of L-alanine, 0.01 g of citric acid mono-
hydrate, 0.14 g of disodium hydrogen phosphate dodecahy-
drate and 0.45 g of sodium chloride in 100 mL of water for
injection.
Bovine serum albumin TS for Secretin Reference Standard
Dissolve 0.1 g of bovine serum albumin, 0.8 g of L-alanine,
0.01 g of citric acid monohydrate, 0. 14 g of disodium hydro-
gen phosphate dodecahydrate and 0.45 g of sodium chloride
in 100 mL of water for injection.
Bradykinin CsoH^N^On A white powder. Freely solu-
ble in water and in acetic acid (31), and practically insoluble
in diethyl ether.
Optical rotation <2.49> [a]™: -80 - -90° (15 mg, water, 5
mL, 100 mm).
Purity Related substances — Dissolve 2.0 mg of bradyki-
nin in 0.2 mL of water, and use this solution as the sample so-
lution. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 5 fiL of the
sample solution on a plate of cellulose for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water, pyridine and acetic acid (31) (15:12:10:3) to a distance
of about 10 cm, and dry the plate at 60°C. Spray evenly a so-
lution of ninhydrin in 1-butanol (1 in 1000) on the plate, and
heat at 60°C for 30 to 60 minutes: any spot other than the
principal spot arisen from bradykinin does not appear.
Brilliant green C27H34N2O4S Fine, glistening, yellow
crystals. It dissolves in water and in ethanol (95). The wave-
length of absorption maximum: 623 nm.
Bromine Br [K 8529, Special class]
Bromine-acetic acid TS Dissolve 10 g of sodium acetate
trihydrate in acetic acid (100) to make 100 mL, add 5 mL of
bromine, and shake. Preserve in light-resistant containers,
preferably in a cold place.
Bromine-carbon tetrachloride TS To 0.1 g of bromine
add carbon tetrachloride to make 100 mL, and dilute a 2 mL
portion of this solution with carbon tetrachloride to make
100 mL. Prepare before use.
Bromine-cyclohexane TS Dissolve 0. 1 g of bromine in cy-
clohexane to make 100 mL. To 2 mL of this solution add cy-
clohexane to make 10 mL. Prepare before use.
Bromine-sodium hydroxide TS To 100 mL of a solution
of sodium hydroxide (3 in 100) add 0.2 mL of bromine. Pre-
pare before use.
Bromine TS Prepare by saturating water with bromine as
follows: Transfer 2 to 3 mL of bromine to a glass-stoppered
bottle, the stopper of which should be lubricated with
petrolatum, add 100 mL of cold water, insert the stopper,
and shake. Preserve in light-resistant containers, preferably
in a cold place.
Bromocresol green C2iH I4 Br 4 05S [K 8840, Special
class]
Bromocresol green-crystal violet TS Dissolve 0.3 g of
bromocresol green and 75 mg of crystal violet in 2 mL of eth-
anol (95), and dilute with acetone to make 100 mL.
Bromocresol green-methyl red TS Dissolve 0.15 g of bro-
mocresol green and 0.1 g of methyl red in 180 mL of ethanol
(99.5), and add water to make 200 mL.
Bromocresol green-sodium hydroxide-acetic acid-sodium
acetate TS To 0.25 g of bromocresol green add 15 mL of
water and 5 mL of dilute sodium hydroxide TS, then add a
small quantity of acetic acid-sodium acetate buffer solution,
pH 4.5, dissolve while shaking, and add acetic acid-sodium
acetate buffer solution, pH 4.5, to make 500 mL. Wash 250
mL of the solution with two 100 mL portions of dichloro-
methane. Filter if necessary.
Bromocresol green-sodium hydroxide-ethanol TS Dis-
solve 50 mg of bromocresol green in 0.72 mL of 0.1 mol/L
sodium hydroxide VS and ethanol (95), and add water to
make 100 mL.
Bromocresol green-sodium hydroxide TS Triturate 0.2 g
of bromocresol green with 2.8 mL of 0.1 mol/L sodium
hydroxide VS in a mortar, add water to make 200 mL, and
filter if necessary.
Bromocresol green TS Dissolve 0.05 g of bromocresol
green in 100 mL of ethanol (95), and filter if necessary.
Bromocresol purple C 21 H 16 Br 2 5 S [K 8841,
class]
Special
Bromocresol purple-dipotassium hydrogenphosphate-
citric acid TS Mix 30 mL of bromocresol purple-sodium hy-
droxide TS and 30 mL of dibasic potassium phosphate-citric
acid buffer solution, pH 5.3, and wash with three 60-mL por-
tions of chloroform.
Bromocresol purple-sodium hydroxide TS Triturate 0.4 g
of bromocresol purple with 6.3 mL of dilute sodium
hydroxide TS in a mortar, add water to make 250 mL, and
filter if necessary.
Bromocresol purple TS Dissolve 0.05 g of bromocresol
purple in 100 mL of ethanol (95), and filter if necessary.
Bromophenol blue C^HioB^OjS [K 8844, Special
class]
Bromophenol blue-potassium biphthalate TS Dissolve
0.1 g of bromophenol blue in potassium biphthalate buffer
solution, pH 4.6, to make 100 mL.
JPXV
General Tests / Reagents, Test Solutions
165
Bromophenol blue TS Dissolve 0.1 g of bromophenol
blue in 100 mL of dilute ethanol, and filter if necessary.
0.05% Bromophenol blue TS Dissolve 0.01 g of
bromophenol blue in water to make 20 mL.
Bromophenol blue TS, dilute Dissolve 0.05 g of bromo-
phenol blue in 100 mL of ethanol (99.5). Prepare before use.
Bromophenol blue TS, pH 7.0 Mix 10 mL of bromo-
phenol blue TS and 10 mL of ethanol (95), and adjust the pH
to 7.0 with dilute sodium hydroxide TS.
iV-Bromosuccinimide C 4 H 4 BrN0 2 [K 9553, Special
class]
iV-Bromosuccinimide TS Dissolve 1 g of N-bromosuc-
cinimide in 1000 mL of water.
Bromothymol blue C 27 H 28 Br 2 5 S [K 8842, Special
class]
Bromothymol blue-sodium hydroxide TS To 0.2 g of
powdered bromothymol blue add 5 mL of dilute sodium hy-
droxide TS and a small quantity of water, dissolve by shaking
in a water bath at 50°C, then add water to make 100 mL.
Bromothymol blue TS Dissolve 0.1 g of bromothymol
blue in 100 mL of dilute ethanol, and filter if necessary.
Bromovalerylurea C 6 H u BrN 2 2 [Same as the name-
sake monograph]
Brucine See brucine dihydrate.
Brucine dihydrate C 23 H 26 N 2 4 .2H 2 [K 8832, Brucine
tt-hydrate, Special class]
B-type erythrocyte suspension Prepare a suspension con-
taining 1 vol% of erythrocyte separated from human B-type
blood in isotonic sodium chloride solution.
Bucillamine C 7 H 13 N0 3 S 2 [Same as the namesake mono-
graph]
Bufalin for component determination C 24 H 34 4 .xH 2
White, odorless, crystalline powder.
Absorbance <2.24> E\°f m (300 nm): 143-153 (10 mg,
methanol, 250 mL). Use the sample dried in a desiccator (sili-
ca gel) for 24 hours for the test.
Purity Related substances — Dissolve 40 mg of bufalin
for component determination in 5 mL of chloroform and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add chloroform to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chromatogra-
phy. Develop the plate with a mixture of cyclohexane, ace-
tone and chloroform (4:3:3) to a distance of about 14 cm,
and air-dry. Spray evenly sulfuric acid, and heat at 100°C for
2 to 3 minutes: any spot other than the principal spot ob-
tained from the sample solution is not larger and not more in-
tense than the spot from the standard solution.
Content: not less than 99.0%. Component determina-
tion — Weigh accurately about 10 mg of bufalin for
component determination, previously dried in a desiccator
(silica gel) for 24 hours, dissolve in methanol to make exactly
10 mL, and use this solution as the sample solution. Perform
the test with 20 liL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions. Measure the peak area by the automatic integra-
tion method, and calculate the amount of bufalin by the area
percentage method.
Operating conditions
Detector: An ultraviolet absorption photometer (wave-
length: 300 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 /urn in
particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
bufalin is about 6 minutes.
Selection of column: Dissolve 0.01 g each of bufalin for
component determination, cinobufagin for component deter-
mination and resibufogenin for component determination in
methanol to make 200 mL. Proceed with 20 /xL of this solu-
tion according to the above conditions. Use a column giving
elution of bufalin, cinobufagin and resibufogenin in this ord-
er and completely resolving these peaks.
Detection sensitivity: Pipet 1 mL of the sample solution,
add methanol to make exactly 100 mL, and use this solution
as the standard solution (1). Pipet 1 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the
standard solution (2). Adjust the detection sensitivity so that
the peak area of bufalin obtained from 20 fiL of the standard
solution (2) can be measured by the automatic integration
method, and the peak height of bufalin from 20 liL of the
standard solution (1) is about 20% of the full scale.
Time span of measutrement: About twice as long as the
retention time of bufalin beginning after the solvent peak.
Bufexamac for assay C 12 H I7 N0 3 [Same as the mono-
graph Bufexamac. When dried, it contains not less than
99.0% of C 12 H 17 N0 3 . Proceed as directed in the Identifica-
tion under Bufexamac Ointment: any peak other than the
principal peak does not appear.]
Buffer solution for celmoleukin Combine 12.5 mL of 0.5
mol/L tris buffer solution, pH 6.8, 10 mL of sodium lauryl
sulfate solution (1->10), 10 mL of glycerin, and 17.5 mL of
water, shake, and then add and dissolve 5 mg of
bromophenol blue.
Storage — Store in a cool place, shielded from light.
n-Butanol See 1-butanol.
sec-ButanoI See 2-butanol.
r-Butanol (CH 3 ) 3 COH [K 8813, Special class]
tot-Butanol See ?-butanol.
1-Butanol CH 3 (CH 2 ) 2 CH 2 OH [K 8810, Special class]
2-Butanol CH 3 CH 2 CH(OH)CH 3 [K 8812, Special
class]
2-Butanone CH 3 COC 2 H 5 [K 8900, Special class]
./V-?-Butoxycarbonyl-L-glutamic acid-a-phenyl ester
C 16 H 21 N0 6 White powder.
Melting point <2.60>: 95-104°C
Purity Related substances — Dissolve 10 mg of N-t-butox-
166
Reagents, Test Solutions / General Tests
JP XV
ycarnonyl-L-glutamic acid-a-phenyl ester in 5 mL of dilute
ethanol, and use this solution as the sample solution. Pipet 1
mL of the sample solution, add dilute ethanol to make exact-
ly 50 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on three plates of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the first plate with a mixture of chloroform, ethyl acetate and
acetic acid (100) (25:25:1), the second plate with a mixture of
benzene, 1,4-dioxane and acetic acid (100) (95:25:4), and the
third plate with a mixture of chloroform, methanol and acet-
ic acid (100) (45:4:1) to a distance of about 12 cm, and air-dry
these plates. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
obtained from the sample solution are not more intense than
the spot from the standard solution in all plates.
B-Butyl acetate
Special class]
CH 3 COOCH 2 CH 2 CH 2 CH 3 [K 8377,
H-Butylamine CH 3 CH 2 CH 2 CH 2 NH 2 A colorless liquid,
having an amine-like, characteristic odor. Miscible with
water, with ethanol (95) and with diethyl ether. The solution
in water shows alkalinity and rapidly absorbs carbon dioxide
from the air.
Specific gravity <2.56> clf : 0.740 - 0.747
Distilling range <2.57>: 76.5 - 79°C, not less than 96 vol%.
w-Butyl chloride CH 3 (CH 2 ) 3 C1 Clear and colorless liq-
uid, miscible with ethanol (95) and with diethyl ether, practi-
cally insoluble in water.
Boiling point <2.57>: about 78°C
Refractive index <2.45> n™: 1.401 - 1.045
Specific gravity <2.56> df \ 0.884 - 0.890
M-Butyl formate HCOO(CH 2 ) 3 CH 3 Clear and colorless
liquid, having a characteristic odor.
Specific gravity <2.56> df : 0.884 - 0.904
terr-Butyl methyl ether (CH 3 ) 3 COCH 3 Clear colorless
liquid, having a specific odor.
Refractive index <2.45> n™: 1.3689
Specific gravity <2.56> df: 0.7404
Butyl parahydroxybenzoate
HOC 6 H 4 COOCH 2 CH 2 CH 2 CH 3 [Same as the namesake
monograph]
Butyrolactone C 4 H 6 2 Clear, colorless to practically
colorless liquid.
Boiling point <2.57>: 198 - 208°C
Specific gravity <2.56> df: 1.128-1.135
Cadmium acetate See cadmium acetate dihydrate.
Cadmium acetate dihydrate Cd(CH 3 COO) 2 .2H 2
[K 8362, Special class]
Cadmium ground metal Cd [H 2113, First class]
Cadmium-ninhydrin TS Dissolve 0.05 g of cadmium ace-
tate dihydrate in 5 mL of water and 1 mL of acetic acid (100),
add 2-butanone to make 50 mL, and dissolve 0.1 g of nin-
hydrin in this solution. Prepare before use.
Caffeine C 8 H I0 N 4 O 2 .H 2 O [Same as the monograph
Caffeine Hydrate]
Caffeine, anhydrous C 8 H I0 N 4 O 2 [Same as the namesake
monograph]
Calcium carbonate CaC0 3 [K 8617, Special class]
Calcium chloride See calcium chloride dihydrate.
Calcium chloride dihydrate CaCl 2 .2H 2 [K 8122,
Special class]
Calcium chloride for drying CaCl 2 [K 8124, For dry-
ing]
Calcium chloride for Karl Fischer method CaCl 2
[K 8125, For water determination]
Calcium chloride TS Dissolve 7.5 g of calcium chloride
dihydrate in water to make 100 mm (0.5 mol/L).
Calcium gluconate for thin-layer chromatography [Same
as the monograph Calcium Gluconate Hydrate. When the
test is performed as directed in the Identification (1) under
Calcium Gluconate Hydrate, no spot other than the principal
spot appears.]
Calcium hydroxide Ca(OH) 2 [K 8575, Special class]
Calcium hydroxide for pH determination Calcium
hydroxide prepared for pH determination.
Calcium hydroxide pH standard solution See pH
Determination <2.54>.
Calcium hydroxide TS To 3 g of calcium hydroxide add
1000 mL of cold distilled water, and occasionally shake the
mixture vigorously for 1 hour. Allow to stand, and use the
supernatant liquid (0.04 mol/L).
Calcium nitrate See calcium nitrate tetrahydrate.
Calcium nitrate tetrahydrate Ca(N0 3 ) 2 .4H 2
[K 8549, Special class]
Calcium oxide CaO [K 8410, Special class]
Camphor C 10 H 16 O [Same as the monograph c?-Cam-
phor or (//-Camphor]
rf-Camphorsulfonic acid C 10 H 16 O 4 S White crystals or
crystalline powder, having a characteristic odor. Very soluble
in water, and soluble in chloroform.
Purity Clarity and color of solution — Dissolve 1 .0 g of d-
camphorsulfonic acid in 10 mL of water: the solution is clear
and colorless or pale yellow.
Loss on drying <2.41>: not more than 2.0% (1 g, 105 °C, 5
hours).
Content: not less than 99.0%, calculated on the dried ba-
sis. Assay — Weigh accurately about 4 g of d-camphorsul-
fonic acid, dissolve in 50 mL of water, and titrate <2.50> with
1 mol/L sodium hydroxide VS (indicator: 3 drops of methyl
red TS). Perform a blank determination in the same manner.
Each mL of 1 mol/L sodium hydroxide VS
= 232.3 mg of C 10 H 16 O 4 S
Caprylic acid CH 3 (CH 2 ) 6 COOH A clear and colorless
oily liquid, having a slight unpleasant odor. Freely soluble in
ethanol (95) and in chloroform, and very slightly soluble in
water.
Distilling range <2.57>: 238 - 242 C C, not less than 95 vol%.
JP XV
General Tests / Reagents, Test Solutions
167
Refractive index <2.45>: n™ 1.426 - 1.430
Specific gravity <2.56> df: 0.908 - 0.912
Capsaicin for component determination Use capsaicin
for thin-layer chromatography meeting the following addi-
tional specifications.
Absorbance <2.24> £}* (281 nm): 97-105 (10 mg,
methanol, 200 mL). Use the sample dried in a desiccator (in
vacuum, phosphorus (v) oxide, 40°C) for 5 hours for the test.
Purity Related substances — Dissolve 10 mg of capsaicin
for component determination in 50 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and measure each peak area
from these solutions by the automatic integration method:
the total area of the peaks other than capsaicin from the sam-
ple solution is not larger than the peak area of capsaicin from
the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed the operating conditions in the Compo-
nent determination under Capsicum.
Time span of measurement: About 3 times as long as the
retention time of capsaicin beginning after the solvent peak.
System suitability
System performance, and system repeatability: Proceed
the system suitability in the Component determination under
Capsicum.
Test for required detectability: Pipet 1 mL of the standard
solution, and add methanol to make exactly 20 mL. Confirm
that the peak area of capsaicin from 20 /uL of this solution is
equivalent to 3.5 to 6.5% of that of capsaicin from the stan-
dard solution.
Capsaicin for thin-layer chromatography C 18 H 27 N03
White crystals, having a strong irritative odor. Very soluble
in methanol, freely soluble in ethanol (95) and in diethyl
ether, and practically insoluble in water.
Melting point <2.60>: 64.5 -66.5°C
Purity Related substances — Dissolve 20 mg of capsaicin
for thin-layer chromatography in 2 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with 10 /xL
each of the sample solution and standard solution as directed
in the Identification under Capsicum: any spot other than the
principal spot at the Rf value of about 0.5 from the sample
solution is not more intense than the spot from the standard
solution.
Carbazochrome C 10 H 12 N 4 O3 Yellow-red to red crystals
or crystalline powder. Melting point: about 222°C (with
decomposition).
Content: not less than 98.0%. Assay— Dissolve about 0.2
g of carbazochrome, previously weighed accurately, in 20 mL
of acetic acid (100) by heating, add 80 mL of acetic anhy-
dride, cool, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 23.62 mg of C 10 H 12 N 4 O 3
Carbazochrome sodium sulfonate for component determi-
nation [Same as the monograph Carbazochrome Sodium
Sulfonate Hydrate. It contains not less than 14.0% and not
more than 15.0% of water, and not less than 99.0% of
carbazochrome sodium sulfonate (CioH n N 4 Na0 5 S), calculat-
ed on the dehydrated basis.]
0.1 mol/L Carbonate buffer solution, pH 9.6 Dissolve
3.18 g of anhydrous sodium carbonate and 5.88 g of sodium
hydrogen carbonate in water to make 1000 mL.
Carbon dioxide C0 2 [Same as the namesake mono-
graph]
Carbon disulfide CS 2 [K 8732, Special class] Preserve
in tightly stoppered containers in a dark, cold place, remote
from fire.
Carbonic anhydrase White powder. Derived from bovine
RBC. Molecular weight about 29,000.
Carbon monoxide CO A toxic, colorless gas. Prepare
by passing the gas generated by reacting formic acid with sul-
furic acid through a layer of sodium hydroxide TS. Carbon
monoxide from a metal cylinder may be used.
Carbon tetrachloride CC1 4 [K 8459, Special class]
Casein, milk [K 8234, Special class]
Casein peptone See peptone, casein.
Castor oil [Same as the namesake monograph]
Catechol C 6 H 4 (OH) 2 [K 8240, Special class]
Cefadroxil C 16 Hi 7 N 3 05S [Same as the namesake mono-
graph]
Cefcapene pivoxil hydrochloide C 2 3H 29 N 5 8 S 2 .HC1.H 2
[Same as the monograph Cefcapene Pivoxil Hydrochloide
Hydrate]
Cefdinir lactam ring-cleavage lactones C 14 H 15 N 5 6 S 2 A
white to yellow powder. A mixture of 4 diastereoisomers.
Identification — Determine the infrared absorption spec-
trum of cefdinir lactam ring-cleavage lactones as directed in
the paste method under Infrared Spectrophotometry <2.25>:
it exhibits absorption at the wave numbers of about 1743
cm" 1 , 1330 cm- 1 , 1163 cm" 1 and 1047 cm- 1 .
Content: not less than 90%. Assay — Dissolve about 5 mg
of cefdinir lactam ring-cleavage lactones in 5 mL of 0.1
mol/L phosphate buffer solution, pH 7.0, and use this solu-
tion as the sample solution. Perform the test with 5 /uL of the
sample solution as directed in the operating conditions of the
Purity (2) Related substances under Cefdinir, and calculate
the areas of each peak by the automatic integration method.
Determine the percent of the total peak area of 4 cefdinir lac-
tam ring-cleavage lactones to the total area of all peaks.
Cefoselis-3-ene-isomer C 19 H 22 N 8 6 S 2 A white to yel-
lowish white powder.
Identification — After drying under reduced pressure at
60°C for 3 hours, determine the infrared absorption spec-
trum of cefoselis-3-ene-isomer according to the paste method
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 3300 cm- 1 , 1768 cm- 1 ,
1618 cm 1 , 1520 cm 1 and 865 cm 1 .
Content: not less than 90%. Assay — Dissolve about 2.5 mg
168
Reagents, Test Solutions / General Tests
JP XV
of cefoselis-3-ene-isomer in 5 mL of 0.1 mol/L phosphate
buffer solution, pH 7.0 and use this solution as the sample so-
lution. Perform the test with 5 /uL of the sample solution as
directed in the Assay under Cefoselis Sulfate, and calculate
the percentage of the peak area of cefoselis-3-ene-isomer to
the total peak area by the automatic integration method.
Cell suspension solution for teceleukin Centrifuge for 5
minutes at 1000 r.p.m culture medium of NK-7 cells that have
been cultured statically for 2 to 4 hours. Remove the super-
natant by aspiration, and add culture medium for assay of
teceleukin to a cell concentration of 2 to 4 x 10 5 cells/mL.
Celmoleukin for liquid chromatography
C 6 93H 1118 N 178 O 2 03S7 [Same as the monograph Celmoleukin
(Genetical Recombination). However, contains 0.5 to 1.5 mg
of protein per mL, polymers amount for 0.5% or less, and
conforms to the following test].
Identification: (1) When the amino acid sequence is inves-
tigated using the Edman technique and liquid chro-
matography, the amino acids are detected in the following se-
quence: alanine, proline, threonine, serine, serine, serine,
threonine, lysine, lysine, threonine, glutamine, leucine, gluta-
mine, leucine, and glutamic acid. Also, based on the results
of the protein content determination test, place an amount of
celmoleukin equivalent to about 0.3 mg in a hydrolysis tube,
evaporate to dryness under vacuum, and then add 100 /uL of
hydrazine anhydride for amino acid sequence analysis.
Reduce the internal pressure of the hydrolysis tube by heating
for 6 hours at about 100°C. After evaporating to dryness un-
der vacuum, add 250 fiL of water to dissolve the residue. To
this add 200 /uL of benzaldehyde, shake occasionally, leave
for one hour, centrifuge, and remove the aqueous layer. Add
250 fiL of water to the benzaldehyde layer, shake, centrifuge,
combine the aqueous layers, and evaporate to dryness under
vacuum. Threonine is detected when amino acid analysis is
conducted using the postcolumn technique with ninhydrin on
a solution of the residue dissolved by adding 100 ,mL of 0.02
mol/L hydrochloric acid TS.
(2) Add 1 mL of protein digestive enzyme solution to 1
mL of celmoleukin, shake, and leave for 18 to 24 hours at 37°
C. Pipet 1 mL of this solution and add 25 [iL of triffuoroacet-
ic acid (1 in 10). To another 1 mL, add 10 /xh of 2-mercap-
toethanol, leave for 30 minutes at 37°C, and then add 25 iiL
of trifiuoroacetic acid (1 in 10). Perform Liquid Chro-
matography <2.01> on these two solutions separately under
the conditions outlined in Celmoleukin (Genetical Recombi-
nation), Identification (4). Repeatedly pipet the celmoleukin
derived peak fraction that elutes and when the test is per-
formed according to Celmoleukin (Genetical Recombina-
tion), Identification (2), except for the lysines in positions 9
and 49 from the amino terminal amino acid, a peptide esti-
mated from the complete primary structure is detected.
Cephaeline hydrobromate C28H3gN2O4.2i-IBr.xH2O
A white or light-yellow crystalline powder.
Purity — Dissolve 10 mg in 10 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 10
mL, and use this solution as the standard solution. Perform
the test with exactly 10 fiL each of the sample solution and
standard solution as directed in the Component determina-
tion under Ipecac: when measure the peak areas 2 times as
long as the retention time of emetine, the total area of the
peaks other than cephaeline is not larger than the peak area
of cephaeline from the standard solution.
Ceric ammonium sulfate See cerium (IV) tetraammoni-
um sulfate dihydrate.
Ceric ammonium sulfate-phosphoric acid TS See cerium
(IV) tetraammonium sulfate-phosphoric acid TS.
Ceric ammonium sulfate TS See cerium (IV) tetraammo-
nium sulfate TS.
Cerium (III) nitrate hexahydrate Ce(N0 3 )3.6H 2 A
colorless or light yellow, crystalline powder. It dissolves in
water.
Purity (1) Chloride <1.03>: not more than 0.036%.
(2) Sulfate <1.14>: not more than 0.120%.
Content: not less than 98.0%. Assay— To about 1.5 g of
cerous nitrate, accurately weighed, add 5 mL of sulfuric acid,
and heat it until white fumes are evolved vigorously. After
cooling, add 200 mL of water, 0.5 mL of 0.1 mol/L silver ni-
trate VS, dissolve 5 g of ammonium peroxodisulfate, dis-
solve, and boil it for 15 minutes. After cooling, add 2 drops
of 1,10-phenanthroline TS, and titrate <2.50> with 0.1 mol/L
ferrous ammonium sulfate VS until the pale blue color of the
solution changes to red.
Each mL of 0.1 mol/L ferrous ammonium sulfate VS
= 43.42 mg of Ce(N0 3 )3-6H 2
Cerium (III) nitrate TS Dissolve 0.44 g of cerium (III)
nitrate hexahydrate in water to make 1000 mL.
Cerium (IV) diammonium nitrate Ce(NH 4 )2(N0 3 ) 6 [K
8556, Special class]
Cerium (IV) diammonium nitrate TS Dissolve 6.25 g of
cerium (IV) diammonium nitrate in 160 mL of diluted dilute
nitric acid (9 in 50). Use within 3 days.
Cerium (IV) sulfate tetrahydrate Ce(S0 4 ) 2 • 4H 2 [K8976 ,
Special class]
Cerium (IV) tetraammonium sulfate dihydrate
Ce(S0 4 )2.2(NH4)2S04.2H 2 [K 8977, Special class]
Cerium (IV) tetraammonium sulfate-phosphoric acid TS
Dissolve 0.1 g of cerium (IV) tetraammonium sulfate in dilut-
ed phosphoric acid (4 in 5) to make 100 mL.
Cerium (IV) tetraammonium sulfate TS Dissolve 6.8 g of
cerium (IV) tetraammonium sulfate in diluted sulfuric acid (3
in 100) to make 100 mL.
Cerous nitrate See cerium (III) nitrate hexahydrate.
Cerous nitrate TS See cerium (III) nitrate TS.
Cetanol [Same as the namesake monograph]
Cetrimide Ci7H 38 BrN White to pale yellowish white
powder, having a faint, characteristic odor.
Purity Clarity of solution — Dissolve 1 .0 g of cetrimide in
5 mL of water: the solution is clear.
Content: not less than 96.0%. Assay — Weigh accurately
about 2 g of cetrimide, previously dried, and dissolve in water
to make exactly 100 mL. Pipet 25 mL of this solution into a
separator, add 25 mL of chloroform, 10 mL of 0.1 mol/L so-
dium hydroxide VS and 10 mL of a freshly prepared solution
of potassium iodide (1 in 20), shake well, allow to stand, and
remove the chloroform layer. Wash the solution with three
JP XV
General Tests / Reagents, Test Solutions
169
10-mL portions of chloroform, take the water layer, and add
40 mL of hydrochloric acid. After cooling, titrate with 0.05
mol/L potassium iodide VS until the deep brown color of the
solution almost disappears, add 2 mL of chloroform, and ti-
trate <2.50> again until the red-purple color of the chloroform
layer disappears. The end point is reached when the red-pur-
ple color of the chloroform layer no more reappears within 5
minutes after the chloroform layer is decolorized. Perform a
blank determination with 20 mL of water, 10 mL of a solu-
tion of potassium iodide (1 in 20) and 40 mL of hydrochloric
acid.
Each mL of 0.05 mol/L potassium iodate VS
= 33.64 mg of C 17 H 38 BrN
Chenodeoxycholic acid for thin-layer chromatography
C24H40O4 White crystals or crystalline powder. Very soluble
in methanol and in acetic acid (100), freely soluble in ethanol
(95), soluble in acetone, sparingly soluble in ethyl acetate,
slightly soluble in chloroform, and practically insoluble in
water. Melting point: about 119°C (recrystallize from ethyl
acetate).
Purity Related substances — Dissolve 25 mg of cheno-
deoxycholic acid for thin-layer chromatography in a mixture
of chloroform and ethanol (95) (9 : 1 ) to make exactly 250 mL .
Perform the test with 10 /xh of this solution as directed in the
Purity (7) under Ursodeoxycholic Acid: any spot other than
the principal spot at the Rf value of about 0.4 does not ap-
pear.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.5 g of chenodeoxycholic acid for thin-layer chroma-
tography, previously dried under reduced pressure (phos-
phorus (V) oxide) at 80°C for 4 hours, and dissolve in 40 mL
of neutralized ethanol and 20 mL of water. Add 2 drops of
phenolphthalein TS, and titrate <2.50> with 0.1 mol/L sodi-
um hydroxide VS. Near the end point add 100 mL of freshly
boiled and cooled water, and titrate again.
Each mL of 0.1 mol/L sodium hydroxide VS
= 39.26 mg of C 2 4H 40 O4
Chikusetsusaponin IV for thin-layer chromatography
C 4 7H740 18 .«H20 White crystalline powder. Freely soluble
in methanol and in ethanol (95), and practically insoluble in
diethyl ether. Melting point: about 215°C (with decomposi-
tion).
Purity Related substances — Dissolve 2 mg of chikuset-
susaponin IV for thin-layer chromatography in 1 mL of
methanol, and perform the test with 5 fiL of this solution as
directed in the Identification under Panax Rhizome: any spot
other than the principal spot at the Rf value of about 0.4 does
not appear.
Chloral hydrate CC1 3 CH(0H) 2 [Same as the namesake
monograph]
Chloral hydrate TS Dissolve 5 g of chloral hydrate in 3
mL of water.
Chloramine See sodium toluensulfonchloramide trihy-
drate.
Chloramine TS See sodium toluensulfonchloramide TS.
Chloramphenicol CnH 12 Cl 2 N 2 05 [Same as the mono-
graph Chloramphenicol]
Chlorauric acid See hydrogen tetrachloroaurate (III)
tetrahydrate.
Chlorauric acid TS See hydrogen tetrachloroaurate (III)
tetrahydrate TS.
Chlordiazepoxide C 16 H I4 C1N 3 [Same as the namesake
monograph]
Chlordiazepoxide for assay Ci 6 H 14 ClN 3 [Same as the
monograph Chlordiazepoxide. When dried, it contains not
less than 99.0% of C 16 H 14 C1N 3 0].
Chlorinated lime [Same as the namesake monograph]
Chlorinated lime TS Triturate 1 g of chlorinated lime
with 9 mL of water, and filter. Prepare before use.
Chlorine Cl 2 A yellow-green gas, having a suffocating
odor. It is heavier than air, and dissolves in water. Prepare
from chlorinated lime with hydrochloric acid. Chlorine from
a metal cylinder may be used.
Chlorine TS Use a saturated solution of chlorine in
water. Preserve this solution in fully filled, light-resistant,
glass-stopered bottles, preferably in a cold place.
Chloroacetic acid C 2 H 3 C10 2 [K 8899, Special class]
p-Chloroaniline See 4-chloroaniline
4-Chloroaniline H 2 NC 6 H 4 C1 White crystals or crystal-
line powder. Freely soluble in ethanol (95) and in acetone,
and soluble in hot water.
Melting point <2.60>: 70 - 72°C
Residue on ignition <2.44>: not more than 0.1% (1 g).
4-Chlorobenzenediazonium TS Dissolve 0.5 g of 4-chlo-
roaniline in 1.5 mL of hydrochloric acid, and add water to
make 100 mL. To 10 mL of this solution add 10 mL of sodi-
um nitrite TS and 5 mL of acetone. Prepare before use.
p-Chlorobenzene sulfonamide See 4-chlorobenzene sul-
fonamide.
4-Chlorobenzene sulfonamide C1C 6 H 4 S0 2 NH 2 White
to pale yellow, odorless, crystalline powder. Dissolves in ace-
tone.
Purity Related substances — Dissolve 0.60 g of 4-chlo-
robenzene sulfonamide in acetone to make exactly 300 mL,
and perform the test with 5 /uL of this solution as directed in
the Purity (5) under Chlorpropamide: any spot other than the
principal spot at the Rf value of about 0.5 does not appear.
p-Chlorobenzoic acid See 4-chlorobenzoic acid.
4-Chlorobenzoic acid ClC 6 H 4 COOH White crystals or
powder. Sparingly soluble in ethanol (95), slightly soluble in
chloroform, and practically insoluble in water.
Melting point <2.60>: 238 - 242°C
Content: not less than 99.0%. Assay — Weigh accurately
about 0.3 g of 4-chlorobenzoic acid, dissolve in 30 mL of
neutralized ethanol, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (indicator: 2 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 15.66 mg of C 7 H 5 C10 2
Chlorobutanol C 4 H 7 C1 3 [Same as the namesake
monograph]
l-Chloro-2,4-dinitrobenzene C 6 H 3 (N0 2 ) 2 C1 [K 8478,
Special class]
170
Reagents, Test Solutions / General Tests
JP XV
Chloroform CHC1 3 [K 8322, Special class]
Chloroform, ethanol-free Mix 20 mL of chloroform with
20 mL of water, gently shake for 3 minutes, separate the
chloroform layer, wash the layer again with two 20-mL por-
tions of water, and filter it through dry filter paper. To the
filtrate add 5 g of anhydrous sodium sulfate, shake for 5
minutes, allow the mixture to stand for 2 hours, and filter
through dry filter paper. Prepare before use.
Chloroform for Karl Fischer method See Water
Determination <2.48>.
Chlorogenic acid for thin-layer chromatography
C 16 H 18 9 .xH 2 A white powder. Freely soluble in
methanol and in ethanol (99.5), and sparingly soluble in
water. Melting point: about 205°C (with decomposition).
Purity Related substances — Dissolve 1.0 mg of chloro-
genic acid for thin-layer chromatography in 2 mL of
methanol, and use this solution as the sample solution.
Perform the test with this solution as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL of the sample
solution on a plate of silica gel for thin-layer chro-
matography, develop the plate with a mixture of ethyl
acetate, water and formic acid (6:1:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): no spot other than the principal
spot at around Rf 0.5 appears.
p-Chlorophenol See 4-Chlorophenol.
4-Chlorophenol ClC 6 H 4 OH Colorless or pale red crys-
tals or crystalline mass, having a characteristic odor. Very
soluble in ethanol (95), in chloroform, in diethyl ether and in
glycerin, and sparingly soluble in water. Melting point: about
43°C.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.2 g of 4-chlorophenol, and dissolve in water to make
100 mL. Measure exactly 25 mL of this solution into an io-
dine flask, add exactly 20 mL of 0.05 mol/L bromine VS and
then 5 mL of hydrochloric acid, stopper immediately, shake
occasionally for 30 minutes, and allow to stand for 15
minutes. Add 5 mL of a solution of potassium iodide (1 in 5),
stopper immediately, shake well, and titrate <2.50> with 0.1
mol/L sodium thiosulfate VS (indicator: 1 mL of starch TS).
Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 3.214 mg of QH5CIO
Preserve in tight, light-resistant containers.
(2-Chlorophenyl)-diphenylmethanol for thin-layer chro-
matography C 19 H 15 C10 To 5 g of clotrimazole add 300
mL of 0.2 mol/L hydrochloric acid TS, boil for 30 minutes,
cool, and extract with 100 mL of diethyl ether. Wash the
diethyl ether extract with two 10 mL portions of 0.2 mol/L
hydrochloric acid TS, then with two 10-mL portions of
water. Shake the diethyl ether extract with 5 g of anhydrous
sodium sulfate, and filter. Evaporate the diethyl ether of the
filtrate, dissolve the residue in 200 mL of methanol by warm-
ing, and filter. Warm the filtrate, and add gradually 100 mL
of water by stirring. Cool in an ice bath, filter the separated
crystals, and dry in a desiccator (phosphorus (V) oxide) for
24 hours. A white crystalline powder. Very soluble in
dichloromethane, freely soluble in diethyl ether, soluble in
methanol, and practically insoluble in water.
Melting point <2.60>: 92 - 95 °C
Purity Related substances — Dissolve 10 mg of (2-chlo-
rophenyl)-diphenylmethanol in dichloromethane to make ex-
actly 20 mL, and perform the test with 10 /xh of this solution
as directed in the Purity (7) under Clotrimazole: any spot
other than the principal spot does not appear.
Chloroplatinic acid See hydrogen hexachloroplatinate
(IV) hexahydrate.
Chloroplatinic acid-potassium iodide TS See hydrogen
hexaclhoroplatinate (IV)-potassium iodide TS.
Chloroplatinic acid TS See hydrogen hexachloroplatinate
(IV) TS.
Chlorpheniramine maleate C16H19CIN2.C4H4O4 [Same
as the namesake monograph]
Chlorpromazine hydrochloride for assay
C 17 H 19 C1N 2 S.HC1 [Same as the monograph
promazine Hydrochloride]
Chlor-
Chlorpropamide for assay C 10 H I3 ClN 2 O 3 S [Same as the
monograph Chlorpropamide. When dried, it contains not
less than 99.0% of chlorpropamide (C 10 H 13 ClN 2 O 3 S).]
Cholesterol C27H45OH [Same as the namesake mono-
graph]
Cholesterol benzoate C34H 50 O2 White crystalline pow-
der.
Melting point <2.60>: 145 - 152°C
Cholic acid for thin-layer chromatography C24H 40 O 5
White, crystals or crystalline powder. Soluble in acetic acid
(100), sparingly soluble in acetone and in ethanol (95), and
very slightly soluble in water. Melting point: about 198°C
Purity Related substances — Dissolve 25 mg in acetone to
make exactly 250 mL. Proceed with 10 fiL of this solution as
directed in the Purity (7) under Ursodeoxycholic Acid: any
spot other than the principal spot, having Rf about 0.1, does
not appear.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.5 g, previously dried at 80°C for 4 hours (in vacuum,
phosphorous (V) oxide), dissolve in 40 mL of neutralized
ethanol and 20 mL of water, add 2 drops of phenolphthalein
TS, and titrate with 0.1 mol/L sodium hydroxide VS until
immediately before the end-point has been reached. Then
add 100 mL of freshly boiled and cooled water, and continue
the titration <2.50>. Perform a blank determination in the
same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.86 mg of C 2 4H 40 O5
Choline chloride [(CH 3 ) 3 NCH 2 CH 2 OH]Cl White cry-
stalline powder.
Melting point <2.60>: 303 - 305°C (decomposition).
Water <2.48>: less than 0.1%.
Chromic acid-sulfuric acid TS Saturate chromium (VI)
trioxide in sulfuric acid.
Chromium trioxide See chromium (VI) trioxide.
Chromium trioxide TS See chromium (VI) trioxide TS.
Chromium (VI) trioxide Cr0 3 A dark red-purple thin
JPXV
General Tests / Reagents, Test Solutions
171
needle-shaped or inner prism-like crystals, or light masses.
Identification — To 5 mL of a solution (1 in 50) add 0.2 mL
of lead (II) acetate TS: yellow precipitates appearwhich does
not dissolve on the addition of acetic acid.
Chromium (VI) trioxide TS Dissolve 3 g of chromium
(VI) trioxide in water to make 100 mL.
Chromogenic synthetic substrate Equal amount mixture
of A r -benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginyl-/>-
nitroanilid hydrochloride and /V-benzoyl-L-isoleucyl-y-
metoxy glutamyl-glycyl-L-arginyl-/?-nitroanilid hydrochlo-
ride. White or pale yellow masses or powder. It is slightly
soluble in water.
Identification: Perform the test with the solution (1 in
30,000) as directed under Untraviolet-visible Spectrophoto-
metry <2.24>: the absorption maximum at about 316 nm is
observed.
Purity: Free 4-nitroaniline (not more than 0.5%)
Loss on drying <2.41>: not more than 5% (0.2 g, reduced
pressure (0.3 kPa), calcium chloride, between 30 and 40°C,
18 hours)
Content: not less than 95% and not more than 105% of the
label.
Chromophore TS for teceleukin Mix 0.1 mL of diluted
hydrogen peroxide (30) (1 in 20) with 10 mL of 0.2mol/L
citric acid buffer, pH 3.8, containing 0.2 mmol/L 3, 3', 5,5'-
tetramethylbenzidine dihydrochloride dehydrate, and use im-
mediately.
Chromotropic acid See disodium chromotropate dihy-
drate.
Chromotropic acid TS Dissolve 0.05 g of disodium chro-
motropate dihydrate in the solution prepared by cautiously
adding 68 mL of sulfuric acid to 30 mL of water, cooling,
then adding water to make 100 mL. Preserve in light-resistant
containers.
Chromotropic acid TS, concentrated Suspend 0.5 g of
disodium chromotropate dihydrate in 50 mL of sulfuric acid,
centrifuge, and use the supernatant liquid. Prepare before
use.
Cilastatin ammonium for assay C 16 H 2 9N 3 5 S: 375.48
A white crystalline powder.
Water <2.48>: not more than 0.5% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44>: not more than 0.5% (1 g)
Purity Related substances — Dissolve 40 mg of the sub-
stance to be examined in 25 mL of water, and use this as the
sample solution. Pipet 3 mL of the sample solution, add
water to make exactly 100 mL, and use this as the standard
solution. Perform the test with exactly 20 /xL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the each peak area by the automatic in-
tegration method. Separately, perform the test with 20 jxL of
water in the same manner to correct any variance of the peak
area caused the variation of the baseline: the total area of the
peaks other than cilastatin is not larger than 1/6 times the
peak area of cilastatin from the standard solution.
Operating conditions
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase A: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (7:3).
Mobile phase B: Diluted phosphoric acid (1 in 1000).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phase A and B directed in the following table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-30
30-40
15^100
100
85->0
100
Flow rate: 2.0 mL per minute.
Time span of measurement: 40 minutes.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add water to make exactly 30 mL. Confirm that
the peak area of cilastatin obtained with 20 /xL of this solu-
tion is equivalent to 2.3 to 4.5% of that with 20 /uL of the
standard solution.
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions: the retention time of cilastatin is about 20 minutes, and
the number of theoretical plates and the symmetry factor of
the peak of cilastatin are not less than 10,000 and not more
than 2.5, respectively.
System repeatability: When the test is repeated 3 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cilastatin is not more than 3.0%.
Residual solvent — Weigh accurately about 1 g, dissolve in
water to make exactly 100 mL, and use this as the sample so-
lution. Separately, weigh accurately about 0.10 g of ethanol
(99.5), add water to make exactly 100 mL, and use this as the
standard solution. Perform the test with exactly 1 /xL each of
the sample solution and standard solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions. Determine the peak areas, A T and A s , of ethanol by
the automatic integration method, and calculate the amount
of ethanol (C 2 H 5 OH): not more than 0.5%.
Amount (%) of ethanol (C 2 H 5 OH)
W T
At
x ^ x 100
W s : Amount (mg) of ethanol (99.5)
W T : Amount (mg) of the sample
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A fused silica column 0.5 mm in inside diameter
and 30 m in length, coated the inside with 5% diphenyl-95%
dimethylpolysiloxane for gas chromatography in thickness of
5 /xm.
Column temperature: Inject the sample at a constant tem-
perature of about 50°C, keep on for 150 seconds, then raise
172
Reagents, Test Solutions / General Tests
JP XV
to 70°C at the rate of 8°C per minute, and keep this for 30
seconds.
Carrier gas: Helium
Flow rate: Adjust so that the retention time of ethanol is
about 1 minute.
Sprit ratio: 5:1
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add water to make exactly 10 mL, and desig-
nate this the solution for system suitability test. To exactly 1
mL of the solution for system suitability test add water to
make exactly 10 mL. Confirm that the peak area of ethanol
obtained with 1 /uL of this solution is equivalent to 7 to 13%
of that with 1 iiL of the solution for system suitability test.
System performance: When the procedure is run with 1 liL
of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of ethanol are not less than 1500 and not more
than 3.0, respectively.
System repeatability: When determine the peak area of
methanol by repeating 6 times with 1 /uL of the standard solu-
tion under the above operating conditions, the relative stan-
dard deviation of the peak area is not more than 2.0%.
Content: not less than 99.0% of cilastatin ammonium (Ci 6
H29N3O5S), calculated on the anhydrous basis and corrected
on the amount of ethanol. Assay — Weigh accurately about
0.5 g, dissolve in 30 mL of methanol, and add 5 mL of water.
Adjust to pH 3.0 with 0.1 mol/L hydrochloric acid TS, and
titrate <2.50> with 0.1 mol/L sodium hydroxide VS from the
first equivalence point to the second equivalence point
(potentiometric titration).
Each mL of 0.1 mol/L sodium hydroxide VS
= 37.55 mg of C 16 H 29 N 3 5 S
Cinchoiiidine C 19 H 2 2N 2 White crystals or crystalline
powder. Soluble in ethanol (95), in methanol and in chloro-
form, sparingly soluble in diethyl ether, and practically in-
soluble in water. A solution of cinchonidine in ethanol (95) (1
in 100) is levorotatory. Melting point: about 207°C
Content: not less than 98.0%. Assay — Weigh accurately
about 0.3 g of cinchonidine, dissolve in 20 mL of acetic acid
(100), add 80 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (indicator: 3 drops of crystal
violet). Perform a blank determination in the same manner.
Each mL of 0.1 mol/L perchloric acid VS
= 14.72 mg of C 19 H 22 N 2
Cinchonine C 19 H 22 N 2 White crystals or powder.
Identification — Dissolve 1 g in 20 mL of diluted
hydrochloric acid (1 in 4), and add 2 mL of potassium hexac-
yanoferrate (II) TS: yellow precipitates appear, which are dis-
solved by heating, and crystals are formed after allowing to
cool.
Purity Cinchonidine and quinine — To 1 g add 30 mL of
water, add diluted hydrochloric acid (2 in 3) dropwise until
the substance to be tested dissolves, and neutralize with am-
monia TS. To this solution add 10 mL of a solution of sodi-
um tartrate dihydrate (1 in 2), boil, and allow to stand for 1
hour: no precipitates appear.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.3 g, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 14.72 mg of C I9 H 22 N 2
Cineol for assay C 10 H 18 O Clear and colorless liquid,
having a characteristic aroma.
Refractive index <2.45> n 2 °: 1.457 - 1.459
Specific gravity <2.56> df : 0.920 - 0.930
Purity (1) Related substances (i) — Dissolve 0.20 g of
cineol for assay in 10 mL of hexane and use this solution as
the sample solution. Perform the test with the sample solu-
tion as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL of the sample solution on a plate of silica gel for
thin-layer chromatography, develop the plate with a mixture
of hexane and ethyl acetate (9:1) to a distance of about 10
cm, and air-dry. Spray evenly 4-methoxybenzaldehyde-sul-
furic acid TS, and heat at 105°C for 5 minutes: any spot
other than the principal spot does not appear.
(2) Related substances (ii) — Dissolve 0.10 g of cineol for
assay in 25 mL of hexane and use this solution as the sample
solution. Perform the test with 2 /xL of the sample solution as
directed under Gas Chromatography <2.02> according to the
following conditions. Measure each peak area by the auto-
matic integration method and calculate the amount of cineol
by the area percentage method: it is not less than 99.0%.
Operating conditions
Proceed the operating conditions in the Assay under Eu-
calyptus Oil except detection sensitivity and time span of
measurement.
Detection sensitivity: Measure 1 mL of the sample solution
and add hexane to make 100 mL. Adjust the detection sen-
sitivity so that the peak height of cineol obtained from 2 iiL
of this solution is 40% to 60% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of cineol beginning after the solvent peak.
Cinnamaldehyde for thin-layer chromatography C 9 H 8
A colorless or light yellow liquid, having a characteristic aro-
matic odor. Very soluble in methanol and in ethanol (99.5),
and practically insoluble in water.
Absorbance <2.24> E\°f m (285 nm): 1679-1943 (5 mg,
methanol, 2000 mL)
Purity Related substances — Dissolve 10 mg in 2 mL of
methanol. Perform the test with 1 itL of this solution as
directed in the Identification (3) under Kakkonto Extract: no
spot other than the principal spot (Rf value is about 0.4) ap-
pears.
Cinnamic acid C 9 H 8 2 White crystalline powder, hav-
ing a characteristic odor.
Melting point <2.60>: 132- 135°C
(£)-Cinnamic acid for component determination (E)-
Cinnamic acid for thin-layer chromatography. It meets the
following requirements.
Purity Related substances — Conduct this procedure
without exposure to day-light, using light-resistant vessels.
Dissolve 10 mg in 50 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 11L each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
JP XV
General Tests / Reagents, Test Solutions
173
cording to the following conditions, and determine each peak
area by the automatic integration method: the total area of
the peaks other than (£)-cinnamic acid and the solvent is not
more than the peak area of (E)-cinnamic acid obtained with
the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay (1) under Ryokeijutsukanto Extract.
Time span of measurement: About 6 times as long as the
retention time of (£)-cinnamic acid.
System suitability
Test for required detectability: To exactly measured 1 mL
of the standard solution add the mobile phase to make ex-
actly 20 mL. Confirm that the peak area of (£)-cinnamic acid
obtained with 10 fiL of this solution is equivalent to 3.5 to 6.5
% of that with 10 fiL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay (1) under
Ryokeijutsukanto Extract.
(E)-Cinnamic acid for thin-layer chromatography
C 9 H 8 2 White crystals or crystalline powder, having a char-
acteristic aromatic odor. Freely soluble in methanol and in
ethanol (99.5), and practically insoluble in water.
Melting point <2.60>: 132- 136°C
Absorbance <2.24> E\°f m (273 nm): 1307-1547 (5 mg
dried with silica gel for 24 hours, methanol, 1000 mL).
Purity Related substances — Conduct this procedure
without exposure to day-light, using light-resistant vessels.
Dissolve 10 mg in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Proceed the test with 10 fiL each of the
sample solution and standard solution as directed in the Iden-
tification (1) under Ryokeijutsukanto Extract: the spot other
than the principal spot of around Ri 0.5 is not more intense
than the spot obtained with the standard solution.
Cinobufagin for component determination
C 26 H3 4 6 .nH 2 White crystalline odorless powder.
Absorbance <2.24> E\ 0/ ° m (295 nm): 125-127 (10 mg,
methanol, 250 mL). Use the sample dried in a desiccator (sili-
ca gel) for 24 hours for the test.
Purity Related substances — Proceed with 40 mg of
cinobufagin for component determination as directed in the
Purity under bufalin for component determination.
Content: not less than 98.0%. Content deter-
mination — Weigh accurately about 10 mg of cinobufagin for
component determination, previously dried in a desiccator
(silica gel) for 24 hours, dissolve in methanol to make exactly
10 mL, and use this solution as the sample solution. Perform
the test with 20 fiL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions. Measure each peak area by the automatic integra-
tion method and calculate the amount of cinobufagin by the
area percentage method.
Operating conditions
Detector: Ultraviolet absorption photometer (wavelength:
295 nm).
Column: A stainless steel column 4 to 6 mm in inside
diameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to lO^m in
particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
cinobufagin is about 7 minutes.
Selection of column: Dissolve 10 mg each of bufalin for
component determination, cinobufagin for component deter-
mination and resibufogenin for component determination in
methanol to make 200 mL. Proceed with 20 fiL of this solu-
tion under the above operating conditions. Use a column giv-
ing elution of bufalin, cinobufagin and resibufogenin in this
order, and clearly dividing each peak.
Detection sensitivity: Pipet 1 mL of the sample solution,
add methanol to make exactly 100 mL, and use this solution
as the standard solution (1). Pipet 1 mL of the standard solu-
tion (1), add methanol to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of cinobufagin obtained from
20 /uL of the standard solution (2) can be measured by the au-
tomatic integration method, and the peak height of cinobufa-
gin from 20,mL of the standard solution (1) is about 20% of
the full scale.
Time span of measurement: About twice as long as the
retention time of cinobufagin beginning after the solvent
peak.
Cisplatin Cl 2 H 6 N 2 Pt [Same as the namesake mono-
graph]
Citric acid See citric acid monohydrate.
Citric acid-acetic acid TS To 1 g of citric acid monohy-
drate add 90 mL of acetic anhydride and 10 mL of acetic acid
(100), and dissolve under shaking.
Citric acid-acetic anhydride TS To 1 g of citric acid
monohydrate add 50 mL of acetic anhydride, and dissolve by
heating. Prepare before use.
Citric acid monohydrate C 6 H 8 7 .H 2 [K 8283, or
same as the monograph Citric Acid Hydrate]
Citric acid-phosphate-acetonitrile TS Dissolve 2.1 g of
citric acid monohydrate, 13.4 g of dipotassium hydrogen
phosphate and 3.1 g of potassium dihydrogen phosphate in
1000 mL of a mixture of water and acetonitrile (3:1).
0.01 mol/L Citric acid TS Dissolve 2.1 g of citric acid
monohydrate in water to make 1000 mL.
1 mol/L Citric acid TS for buffer solution Dissolve
210.14 g of citric acid monohydrate in water to make 1000
mL.
Clotrimazole C 22 H 17 C1N 2 [Same as the namesake mono-
graph]
Cloxazolam C 17 H 14 C1 2 N 2 2 [Same as the namesake
monograph]
Cobalt (II) chloride-ethanol TS Dissolve 0.5 g of cobalt
(II) chloride hexahydrate, previously dried at 105°C for 2
hours, in ethanol (99.5) to make 100 mL.
Cobalt (II) chloride hexahydrate CoCl 2 .6H 2
[K 8129, Special class]
Cobalt (II) chloride TS Dissolve 2 g of cobalt (II) chlo-
174
Reagents, Test Solutions / General Tests
JP XV
ride hexahydrate in 1 mL of hydrochloric acid and water to
make 100 mL (0.08 mol/L).
Cobalt (II) nitrate hexahydrate Co(N0 3 ) 2 .6H 2
[K 8552, Special class]
Cobaltous chloride See cobalt (II) chloride hexahydrate.
Cobaltous nitrate See cobalt (II) nitrate hexahydrate.
Codeine phosphate for assay C18H21NO3.H3PO4.V2H2O
[Same as the monograph Codeine Phosphate Hydrate. It
contains not less than 99.0% of codeine phosphate (C 18 H 21
NO3.H3PO4), calculated on the anhydrous basis.]
Collodion Clear, colorless, viscous liquid, having a
diethyl ether -like odor.
pH <2.54>: 5.0-8.0
Stir 5 g of collodion while warming, add 10 mL of water
gradually, and dry at 110°C after evaporating to dryness:
mass of the residue is 0.250-0.275 g.
Concentrated chromotropic acid TS See chromotropic
acid, concentrated.
Concentrated diazobenzenesulfonic acid TS See diazo-
benzenesulfonic acid TS, concentrated.
Congo red C 32 H 22 N 6 Na 2 6 S 2 [K 8352, Special class]
Congo red TS Dissolve 0.5 g of congo red in 100 mL of a
mixture of ethanol (95) and water (1:9).
Control anti-interleukin-2 antiserum solution Anti-inter-
leukin-2 antiserum is diluted with culture media for celmoleu-
kin, so that the diluted antiserum solution neutralizes the
same volume of about 800 unit/mL solution of Celmoleukin
(Genetical Recombination).
Coomassie brilliant blue G-250 C 4 7H4 8 N3Na0 7 S 2
A deep violet powder. A solution in ethanol (99.5) (1 in
100,000) exhibits an absorption maxima at a wavelength of
608 nm.
Coomassie brilliant blue R-250 C 4 5H44N3Na0 7 S 2 Deep
blue-purple powder. Odorless.
Content: not less than 50%.
Coomassie staining TS Dissolve 125 mg of Coomassie
brilliant blue R-250 in 100 mL of a mixture of water,
methanol and acetic acid (100) (5:4:1), and filter.
Copper Cu [K 8660, Special class]
Copper (II) acetate monohydrate Cu(CH 3 COO) 2 .H 2
[K 8370, Special class]
Copper (II) acetate TS, strong Dissolve 13.3 g of copper
(II) acetate monohydrate in a mixture of 195 mL of water and
5 mL of acetic acid.
Copper (II) chloride-acetone TS Dissolve 0.3 g of cop-
per (II) chloride dihydrate in acetone to make 10 mL.
Copper (II) chloride dihydrate CuCl 2 .2H 2 [K 8145,
Special class]
Copper (II) disodium ethylenediamine tetraacetate tetrahy-
drate C 10 H 12 CuN 2 Na 2 O 8 .4H 2 O A blue powder.
pH <2.54>: 7.0-9.0
Purity Clarity and color of solution — Add 0.10 g of cop-
per (II) disodium ethylenediamine tetraacetate tetrahydrate
to 10 mL of freshly boiled and cooled water: the solution is
blue in color and clear.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.45 g of coppor (II) disodium ethylenediamine tetraa-
cetate tetrahydrate, and add water to make exactly 100 mL.
Pipet 10 mL of this solution, adjust the pH of the mixture to
about 1.5 by adding 100 mL of water and dilute nitric acid,
then add 5 mL of a solution of 1,10-phenanthroline monohy-
drate in methanol (1 in 20), and titrate <2.50> with 0.01 mol/
L bismuth nitrate VS until the color of the solution changes
from yellow to red (indicator: 2 drops of xylenol orange TS).
Each mL of 0.01 mol/L bismuth nitrate VS
= 4.698 mg of C, H 12 CuN 2 Na 2 O 8 .4H 2 O
Copper (II) hydroxide Cu(OH) 2 Light blue powder.
Practically insoluble in water.
Content: not less than 95.0% as Cu(OH) 2 . As-
say — Weigh accurately about 0.6 g of Copper (II) hydroxide,
and dissolve in 3 mL of hydrochloric acid and water to make
exactly 500 mL. Pipet 25 mL of this solution, add 75 mL of
water, 10 mL of a solution of ammonium chloride (3 in 50), 3
mL of diluted ammonia solution (28) (1 in 10) and 0.05 g of
murexide-sodium chloride indicator, and titrate <2.50> with
0.01 mol/L disodium dihydrogen ethylenediamine tetra-
acetate VS until the color of the liquid is changed from yel-
low-green to red-purple.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 0.9756 mg of Cu(OH) 2
Copper (II) sulfate (anhydrous) CuS0 4
[K 8984, First class]
Copper (II) sulfate pentahydrate CuS0 4 .5H 2
[K 8983, Special class]
Copper (II) sulfate-pyridine TS Dissolve 4 g of copper
(II) sulfate pentahydrate in 90 mL of water, then add 30 mL
of pyridine. Prepare before use.
Copper (II) sulfate solution, alkaline Dissolve 150 g of
potassium bicarbonate, 101.4 g of potassium carbonate and
6.93 g of copper (II) sulfate pentahydrate in water to make
1000 mL.
Copper (II) sulfate TS Dissolve 12.5 g of copper (II) sul-
fate pentahydrate in water to make 100 mL (0.5 mol/L).
Copper (standard reagent) Cu [K 8005, Standard rea-
gent for quantitative analysis]
Corn oil [Same as the namesake monograph]
Cortisone acetate C 23 H3 O 6 [Same as the namesake
monograph]
Cottonseed oil A refined, nonvolatile fatty oil obtained
from the seed of plants of Gossypium hirsutum Linne (Gos-
sypium) or of other similar species. A pale yellow, odorless,
oily liquid. Miscible with diethyl ether, and with hexane.
Slightly soluble in ethanol (95).
Refractive index <2.45> n 2 °: 1.472 - 1.474
Specific gravity <2.56> d\\: 0.915 - 0.921
Acid value <1.13>: not more than 0.5.
Saponification value <1.13>: 190 - 198
Iodine value <1.13>: 103 - 116
JPXV
General Tests / Reagents, Test Solutions
175
Cresol CH 3 C 6 H 4 (OH) [Same as the namesake mono-
graph]
m-Cresol CH 3 C 6 H 4 (OH) [K 8305, Special class]
m-Cresol purple C 21 H 18 5 S [K8889, Special grade]
m-Cresol purple TS Dissolve 0. 10 g of m-cresol purple in
13 mL of 0.01 mo/L sodium hydroxide TS, and add water to
make 100 mL.
Cresol red C 21 H I8 5 S [K 8308, Special class]
Cresol red TS Dissolve 0.1 g of cresol red in 100 mL of
ethanol (95), and filter if necessary.
Crystalline trypsin for ulinastatin assay A proteolytic
enzyme prepared from bovine pancreas. White to light yellow
crystalline powder. Odorless. Sparingly soluble in water, and
dissolves in 0.001 mol/L hydrochloric acid TS.
Content: not less than 3200 trypsin Units per mg. As-
say — (i) Sample solution: Weigh accurately about 20 mg of
crystalline trypsin for ulinastatin assay, and dissolve in 0.001
mol/L hydrochloric acid TS so that each mL of the solution
contains about 3000 trypsin Units. Dilute this solution with
0.001 mol/L hydrochloric acid TS so that each mL of the so-
lution contains about 40 trypsin Units, and use this solution
as the sample solution, (ii) Diluent: Dissolve 4.54 g of potas-
sium dihydrogen phosphate in water to make exactly 500 mL
(Solution I). Dissolve 4.73 g of anhydrous disodium hydro-
gen phosphate in water to make exactly 500 mL (Solution II).
To 80 mL of Solution II add a suitable amount of Solution I
to adjust to pH 7.6. (hi) Substrate solution: Dissolve 85.7 mg
of ./V-a-benzoyl-L-arginine ethyl ester hydrochloride in water
to make exactly 100 mL, and use this solution as the substrate
stock solution. Pipet 10 mL of the stock solution, add the dil-
uent to make exactly 100 mL, and use this solution as the sub-
strate solution. The absorbance of the substrate solution de-
termined at 253 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24> using water as the blank is between
0.575 and 0.585. If the absorbance of the substrate solutionis
not in this range, adjust with the diluent or the substrate
stock solution, (iv) Procedure: Pipet 3 mL of the substrate
solution, previously warmed at 25 ± 0.1 °C, into a 1-cm
quartz cell, add exactly 0.2 mL of the sample solution, and
start the determination of the absorbance change at 253 nm
for 5 minutes at 25 ± 0.1 °C using a solution prepared by
adding exactly 0.2 mL of 0.001 mol/L hydrochloric acid TS
to exactly 3 mL of the substrate solution as the blank. Deter-
mine the difference of the absorbance change per minute, A,
when the difference has been constant for at least 3 minutes,
(v) Calculation: Trypsin Units per mg is obtained by use of
the following equation. One trypsin Unit is an amount of the
enzyme which gives 0.003 change in absorbance per minute
under the conditions described above.
A
Trypsin Units per mg
0.003 x W
W: Amount (mg) of the substance to be assayed in
0.2 mL of the sample solution
Storage — Preserve in a cold place.
Crystallized trypsin To trypsin obtained from bovine
pancreas gland add an appropriate amount of trichloroacetic
acid to precipitate the trypsin, and recrystallize in ethanol
(95). White to yellowish white crystals or powder. It is odor-
less. Freely soluble in water and in sodium tetraborate-calci-
um chloride buffer solution, pH 8.0.
Content: not less than 45 FIP Units of trypsin per mg.
Assay — (i) Sample solution: Weigh accurately an appropriate
amount of crystallized trypsin according to the labeled Units,
dissolve in 0.001 mol/L hydrochloric acid TS to prepare a so-
lution containing 50 FIP Units per mL, and use this solution
as the sample solution. Prepare before use, and preserve in
ice. (ii) Apparatus: Use a glass bottle as a reaction reservoir
20 mm in inside diameter and 50 mm in height, equipped with
a rubber stopper for attachment to a glass /silver-silver chlo-
ride electrode for pH determination, nitrogen-induction tube
and an exhaust port. Fix the reaction reservoir in a ther-
mostat, and keep the temperature at 25 ± 0.1 °C by means of
a precise thermoregulator. (iii) Procedure: Pipet 1.0 mL of
A^-a-benzoyl-L-arginine ethyl ester TS, transfer to the reac-
tion reservoir, and add 9.0 mL of sodium tetraborate-calci-
um chloride buffer solution, pH 8.0. Allow to stand in the
thermostat for 10 minutes to make the temperature of the
contents reach to 25 ± 0.1 °C, adjust the pH of the solution
to 8.00 by adding dropwise 0.1 mol/L sodium hydroxide VS
while stirring and passing a current of nitrogen, add exactly
0.05 mL of the sample solution previously allowed to stand at
25 ± 0.1 °C, then immediately add dropwise 0.1 mol/L sodi-
um hydroxide VS by a 50//L-micropipet (minimum gradua-
tion of 1 fiV) while stirring to keep the reaction solution at pH
8.00, and read the amount of 0.1 mol/L sodium hydroxide
VS consumed and the reaction time when the pH reached
8.00. Continue this procedure up to 8 minutes. Separately,
transfer 10 mL of sodium tetraborate-calcium chloride buffer
solution, pH 8.0, and perform a blank determination in the
same manner, (iv) Calculation: Plot the amount of consump-
tion (mL) of 0.1 mol/L sodium hydroxide VS against the
reaction time (minutes), select linear reaction times, t { and t 2 ,
designate the corresponding consumption amount of 0.1 mol
/L sodium hydroxide VS as Vj and v 2 , respectively, and desig-
nate fimol of sodium hydroxide consumed per minute as M
(FIP Unit).
M(umol NaOH/min)= Vl ~ Vl X -^- X /
<2 1 1 10
/: Factor of 0.1 mol/L sodium hydroxide VS
FIP Units per mg of crystallized trypsin to be tested
= (Mi-M )xr
LXW
M x : ^mol of sodium hydroxide consumed in 1 minute when
the sample solution is used
M : /miol of sodium hydroxide consumed in 1 minute when
the solution for blank determination is used
W: Amount (mg) of crystallized trypsin sampled
L: Amount (mL) of the sample solution put in the reaction
reservoir
T: Total volume (mL) of the sample solution prepared by
dissolving in 0.001 mol/L hydrochloric acid TS
One FIP Unit is an amount of enzyme which decomposes 1
//mol of 7V-a-benzoyl-L-arginine ethyl ester per minute under
the conditions described in the Assay.
Storage — Preserve in a cold place.
Crystal violet C 2 5H 3 oCN 3 .9H 2 [K 8294, Special class]
Crystal violet TS Dissolve 0.1 g of crystal violet 10 mL of
acetic acid (100).
176
Reagents, Test Solutions / General Tests
JP XV
Culture medium for assay of teceleukin Add 100 mL of
fetal calf serum to 1000 mL of medium for float culture.
Store at 4°C.
Culture medium for celmoleukin Take a specified
amount of RPMI-1640 powdered medium that contains
glutamate but does not contain sodium hydrogen carbonate,
add water to dissolve, and add iV-2-hydroxyethylpiperidine-
7V-2-ethansulfonic acid as a buffering agent to a concentra-
tion of 0.025 mol/L. To 1000 mL of this solution add 0.1 g
(potency) of streptomycin sulfate, 100,000 units of potassium
benzylpenicillin, and 2 g of sodium hydrogen carbonate, ad-
just the pH to 7.1 to 7.2 with sodium hydroxide TS, and then
sterilize by filtration. To this solution add fetal calf serum
heated at 56 °C for 30 minutes to 20 vol%.
Cu-PAN Prepare by mixing 1 g of l-(2-pyridylazo)-2-
naphthol (free acid) with 11.1 g of coppor (II) disodium eth-
ylenediamine tetraacetate tetrahydrate.
A grayish orange-yellow, grayish red-brown or light
grayish purple powder.
Absorbance — Dissolve 0.50 g of Cu-PAN in diluted 1,4-di-
oxane (1 in 2) to make exactly 50 mL. Pipet 1 mL of this solu-
tion, add methanol to make exactly 100 mL. Read the ab-
sorbance of this solution at 470 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
blank solution: the absorbance is not less than 0.48.
Purity Clarity and color of solution — Dissolve 0.50 g of
Cu-PAN in 50 mL of diluted 1,4-dioxane (1 in 2): the solu-
tion is clear and yellow-brown.
Cu-PAN TS Dissolve 1 g of Cu-PAN in 100 mL of dilut-
ed 1,4-dioxane (1 in 2).
Cupferron C 6 H 9 N 3 2 [K 8289, Special class]
Cupferron TS Dissolve 6 g of cupferron in water to make
100 mL. Prepare before use.
Cupric acetate See copper (II) acetate monohydrate.
Cupric acetate TS, strong See copper (II) acetate mono-
hydrate TS, strong.
Cupric carbonate See cupric carbonate monohydrate.
Cupric carbonate monohydrate CuC0 3 .Cu(OH) 2 .H 2
A blue to blue-green powder. It is insoluble in water,
and dissolves foamingly in dilute acid. It dissolves in ammo-
nia TS and shows a deep blue color.
Purity (1) Chloride <1.03>: not more than 0.036%.
(2) Sulfate <1.14>: not more than 0.120%.
(3) Iron — Dissolve 5.0 g of cupric carbonate monohy-
drate in excess ammonia TS and filter. Wash the residue with
ammonia TS, dissolve in dilute hydrochloric acid, add excess
ammonia TS and filter. Wash the residue with ammonia TS,
and dry to constant mass: the residue is not more than 10 mg.
Cupric chloride See copper (II) chloride dihydrate.
Cupric chloride-acetone TS See copper (II) chloride-ace-
tone TS.
Cupric sulfate See copper (II) sulfate pentahydrate.
Cupric sulfate, anhydrous See copper (II) sulfate (anhy-
drous).
Cupric sulfate-pyridine TS See copper (II) sulfate-pyri-
dine TS.
Cupric sulfate solution, alkaline See copper (II) sulfate
solution, alkaline.
Cupric sulfate TS See copper (II) sulfate TS.
1 mol/L Cupriethylenediamine TS Put 100 g of copper
(II) hydroxide in a 1-L thick-walled bottle marked a 500-mL
line, and add water to make 500 mL. Connect the bottle with
a liquid introducing funnel, a nitrogen introducing glass tube
and a gas removing glass tube. Adjust so that the lower end
of the nitrogen introducing tube is located at about 1.3 cm
above of the bottom of the bottle. Introduce the nitrogen for
about 3 hours to replacing the inside gas by adjusting the
pressure (about 14 kPa) to get a mild bubbling. Then add
gradually 160 mL of ethylenediamine TS through the funnel
while introducing the nitrogen and cooling the bottle with the
running water, and replace the funnel with a glass rod to
close tightly. After introducing the nitrogen for further 10
minutes, replace the gas removing tube with a glass rod to
close tightly. Keep the inside pressure with the nitrogen to
about 14 kPa. After allowing the bottle to stand for about 16
hours while occasional shaking, filter the content if necessary
using a glass-filter under reducing pressure, and reserve under
nitrogen atmosphere. The concentration of copper (II) ion of
this solution is about 1.3 mol/L. Determine the concentra-
tion of ethylenediamine of this solution X (mol/L) and cop-
per (II) ion Y (mol/L) by the following Assays, and adjust to
that X is 1.96-2.04, Y is 0.98-1.02 and X/Y is 1.96-2.04 by
adding water, copper (II) hydroxide or ethylenediamine TS,
then determine X and Y again in the same manner, and use
this solution as the test solution.
Assay (1) Ethylenediamine — Pipet 1 mL (V{) of the so-
lution to be assayed, add 60 mL of water, and titrate <2.50>
with 0.1 mol/L hydrochloric acid VS (pH Determination
<2.54>; End point is about pH 8.4).
X =
N,a
X: Concentration of ethylenediamine (mol/L)
a: Volume of 0.1 mol/L hydrochloric acid VS consumed
for the titration (mL)
N\\ Concentration of 0.1 mol/L hydrochloric acid VS
(mol/L)
(2) Copper (II) ion — Pipet 2 mL ( V 2 ) of the solution to be
assayed, add 20 mL of water, about 3 g of potassium iodide
and 50 mL of 2 mol/L sulfuric acid TS, shake for 5 minutes,
and titrate <2.50> the liberated iodine with 0.1 mol/L sodium
thiosulfate VS. When the solution turns light yellow at near
the end point add 3 mL of starch TS and 10 mL of a solution
of ammonium thiocyanate (2 in 10), and then titrate until the
blue color disappears.
Y=
N>b
Y: Concentration of copper (II) ion (mol/L)
b: Volume of 0.1 mol/L sodium thiosulfate VS consumed
for the titration (mL)
N 2 : Concentration of 0.1 mol/L sodium thiosulfate VS
(mol/L)
Curcumin C 2 iH 20 O 6 [K 8297, Special class]
Curcumin TS Dissolve 0.125 g of curcumin in acetic acid
(100) to make 100 mL. Prepare before use.
JPXV
General Tests / Reagents, Test Solutions
177
Cyanoacetic acid C 3 H 3 N0 2 White to light yellow crys-
tals. Very soluble in water.
Content: not less than 99%. Assay — Weigh accurately
about 300 mg of cyanoacetic acid, add 25 mL of water and 25
mL of ethanol (95) to dissolve, and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L sodium hydroxide VS
= 85.06 mg of C 3 H 3 N0 2
Cyanocobalamin C 63 H 88 CoN 14 I4 P
namesake monograph]
[Same as the
Cyanogen bromide TS To 100 mL of ice-cold water add 1
mL of bromine, shake vigorously, and add ice-cold potassi-
um cyanide TS dropwise until the color of bromine just dis-
appears. Prepare this test solution in a hood before use.
On handling this solution, be careful not to inhale its
vapors, which are very toxic.
1-Cyanoguanidine NH 2 C(NH)NHCN A white crystal-
line powder. Freely soluble in water.
Melting point <2.60>: 209 - 212°C
Loss on drying <2.41>: not more than 0.1% (1 g, 105°C, 3
hours)
Nitrogen content <I.08>: 66.0 - 67.3% (after drying)
6% Cyanopropyl-6% phenyl-methyl silicone polymer for
gas chromatography Prepaired for gas chromatography.
7% Cyanopropyl-7% phenylmethylsilicone polymer for
gas chromatography Prepared for gas chromatography.
Cyclohexane C 6 H 12 [K 8464, Special class]
Cyclohexylamine C 6 H U NH 2 A clear and colorless
liquid, having a characteristic amine-like odor. Miscible with
water, with ./V,iV-dimethylformamide and with acetone.
Purity Related substances — Use cyclohexylamine as the
sample solution. Separately, pipet 1 mL of cyclohexylamine,
add hexane to make exactly 100 mL, and use this as the stan-
dard solution. Perform the test as directed in Thin-layer
Chromatography <2.03>. Spot 5 /xL each of the sample solu-
tion and standard solution on a plate of silica gel for thin-lay-
er chromatography, develop the plate with a mixture of ethyl
acetate, methanol, ammonia water (28) and cyclohexane
(6:2:1:1) to a distance of about 10 cm, and air-dry the plate.
Allow the plate to stand in iodine vapor: the spot other than
the principal spot obtained with the sample solution is not
more intense than the spot with the standard solution.
Cyclohexylmethanol C 7 H 14 A liquid having slight
camphor odor. Soluble in ethanol (99.5).
Bioling point <2.57>: about 185°C
Refractive index <2.45> n™: about 1.464
Cyclosporine U
Optical rotation <2.49>
methonol, 20 mL 100 mm)
[u]d- about -190°C (0.1 g,
L-Cysteic acid C 3 H 7 N0 5 S White powder.
Melting point <2.60>: about 260°C.
Optical rotation <2.49> [a]™'- +7.5 - +9.0° (1.5 g, water,
20 mL, 100 mm)
L-Cysteine hydrochloride See L-cysteine hydrochloride
monohydrate.
L-Cysteine hydrochloride monohydrate
HSCH 2 CH(NH 2 )COOH.HCl.H 2 [K 8470, Special class]
L-Cystine HOOCCH(NH 2 )CH 2 SSCH 2 CH(NH 2 )COOH
[K 9048, L(-)-Cystine, Special class]
Cytochrome c An oxidase (molecular weight: 8000 -
13,000) derived from bovine cardiac muscle
Cytosine C 4 H 5 N 3 White, crystalline powder or pow-
der.
Absorbance <2.24> E\°^ (276 nm): not less than 800 (after
drying, 40 mg, 10,000 mL of 0.1 mol/L hydrochloric acid
TS).
Dacuronium Bromide for thin-layer chromatography
C 33 H 58 Br 2 N 2 3 White crystalline powder. Very soluble in
water, freely soluble in ethanol (95), and practically insoluble
in acetic anhydride and in diethyl ether. Hygroscopic.
Identification — Determine the infrared absorption spec-
trum of dacuronium bromide for thin-layer chromatography
according to the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits the absorptions
at the wave numbers at about 2938 cm -1 , 1737 cm 1 , 1630
cm" 1 , 1373 cm" 1 , 1233 cm 1 and 1031 cm" 1 .
Purity Related substances — Dissolve 10 mg of dacuroni-
um bromide for thin-layer chromatography in 2 mL of
ethanol (95), and use this solution as the sample solution.
Pipet 1 mL of this solution, add ethanol (95) to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with 10 fiL each of the sample solution and stan-
dard solution as directed in the Purity (2) Related substances
under Pancuronium Bromide: the spots other than the prin-
cipal spot from the sample solution do not show more intense
color than the spot from the standard solution.
Water <2.48>: not more than 1.0% (1 g, volumetric titra-
tion, direct titration).
Content: not less than 98.0%, calculated on the dehydrat-
ed basis. Assay — Weigh accurately about 0.2 g of dacuroni-
um bromide for thin-layer chromatography, dissolve in 50
mL of acetic anhydride by warming, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 34.53 mg of C 33 H 58 Br 2 N 2 3
p,p' -DDD (2,2-Bis(4-chlorophenyl)-l,l-dichloroethane)
Ci 4 H 10 Cl 4
Melting point <2.60>: 108 - 110°C
Purity Related substances — Dissolve 10 mg of p,p'-DDD
in hexane for purity of crude drug to make exactly 100 mL,
pipet 1 mL of this solution, add hexane for purity of crude
drug to make exactly 100 mL, and use this solution as the
sample solution. Pipet 2 mL of the sample solution, add
hexane for purity of crude drug to make exactly 100 mL, and
use this solution as the standard solution (1). Perform the test
with exactly 1 /uL each of the sample solution and standard
solution (1) as directed under Gas Chromatography <2.02>
according to the following conditions, and measure each
peak area from these solutions by the automatic integration
method: the total peak area other than p,p' -DDD from the
sample solution is not larger than the peak area of p,p'-DDD
178
Reagents, Test Solutions / General Tests
JP XV
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Purity (2) under
Crude Drugs Test <5.01> except detection sensitivity and time
span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add hexane for purity of crude drug to make exactly 20
mL, and use this solution as the standard solution (2). Adjust
the detection sensitivity so that the peak area of p,p' -DDD
obtained from 1 /nL of the standard solution (2) can be meas-
ured by the automatic integration method, and the peak
height of p,p' -DDD from 1 fiL of the standard solution (1) is
about 20% of the full scale.
Time span of measurement: About twice as long as the
retention time of p,p' -DDD beginning after the solvent peak.
p,p' -DDE (2,2-Bis(4-chlorophenyl)-l,l-dichloroethyl-
ene) C 14 H 8 C1 4
Melting point <2.60>: 88 - 90 C C
Purity Related substances — Proceed as directed in the
Purity of p,p'-DDD using the following standard solution
(1).
Standard solution (1): Pipet 1 mL of the sample solution,
and add hexane for purity of crude drug to make exactly 100
mL.
o,p' -DDT (1,1 , l-Trichloro-2-(2-chlorophenyl)-2-(4-
chlorophenyl)ethane) Ci 4 H 9 Cl 5
Melting point <2.60>: 73 - 75 °C
Purity Related substances — Proceed as directed in the
Purity of p,p' -DDD.
p,p' -DDT (1 , 1 , l-Trichloro-2,2-bis(4-chlorophenyl)eth-
ane) C I4 H 9 C1 5
Melting point <2.60>: 108 - 110°C
Purity Related substances — Proceed as directed in the
Purity of p,p'-DDD using the following standard solution
(1).
Standard solution (1): Pipet 1 mL of the sample solution,
and add hexane for purity of crude drug to make exactly 100
mL.
DEAE-Cross-linking dextran anion exchanger (CI type),
slightly alkaline Slightly alkaline anion exchanger prepared
by introducing diethylaminoethyl group into cross-linking
dextran of gel filtration carrier.
Decolorized fuchsin TS Add 1 g of fuchsin in 100 mL of
water, heat at about 50°C, then cool with occasional shaking.
After standing for 48 hours, mix and filter. To 4 mL of the
filtration add 6 mL of hydrochloric acid and water to make
100 mL. Use after standing for at least 1 hour. Prepare be-
fore use.
n-Decyl trimethylammonium bromide C 13 H 30 NBr
White powder. Melting point: about 232°C (with decomposi-
tion).
Content: not less than 99%. Assay — Weigh accurately
about 0.5 g of n-decyl trimethylammonuim bromide, dissolve
in 50 mL of water, and titrate <2.50> with 0.1 mol/L silver ni-
trate VS (indicator: 1 mL of potassium chromate TS). Per-
form a blank determination in the same manner and make
any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 28.03 mg of C 13 H 30 NBr
0.005 mol/L n-Decyl trimethylammonium bromide TS
Dissolve 6.94 g of potassium dihydrogen phosphate, 3.22 g
of disodium hydrogen phosphate dodecahydrate and 1.40 g
of M-decyl trimethylammonium bromide in water to make
1000 mL.
Defibrinated blood of rabbit Transfer 100 mL of blood
obtained from rabbit to a flask, put in about 20 glass balls 8
mm in diameter, shake for 5 minutes gently, and filter
through gauze. Prepare before use.
Dehydrated ethanol See ethanol (99.5).
Dehydrated ether See diethyl ether.
Dehydrated pyridine See pyridine, dehydrated.
Dehydrocorydaline nitrate for component determination
C22H 24 N 2 7 Yellow, crystals or crystalline powder. It is
sparingly soluble in methanol, slightly soluble in water, in
ethanol (95) and in acetonitrile, and practically insoluble in
diethyl ether. Melting point: about 240°C (with decomposi-
tion).
Absorbance <2.24> E\*, (333 nm): 577-642 [3 mg after
drying in a desiccator (silica gel) for not less than 1 hour,
water, 500 mL].
Purity Related substances — (1) Dissolve 5.0 mg of de-
hydrocorydaline nitrate for component determination in 1
mL of a mixture of water and methanol (1:1), and use this so-
lution as the sample solution. Pipet 0.5 mL of the sample so-
lution, add a mixture of water and methanol (1:1) to make
exactly 50 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /xL of the sample solu-
tion and standard solution on a plate of silica gel for thin-lay-
er chromatography. Develop immediately with a mixture of
methanol, water and acetic acid (100) (20:1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 365 nm) and then spray Dragendor-
ff's TS on the plate: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution in either case.
(2) Dissolve 5.0 mg of dehydrocorydaline nitrate for
component determination in 10 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 5 pL of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and measure each peak
area from these solutions by the automatic integration
method in each solution: the total area of peaks other than
dehydrocorydaline and the solvent from the sample solution
is not larger than the peak area of dehydrocorydaline from
the standard solution.
Operating conditions
Column, column temperature, mobile phase, and flow
rate: Proceed the operating conditions in the Component de-
termination under Corydalis Tuber.
Detector: Ultraviolet absorption photometer (wave-
length: 230 nm)
Time span of measurement: About 3 times as long as the
retention time of dehydrocorydaline beginning after the sol-
vent peak.
System suitability
JPXV
General Tests / Reagents, Test Solutions
179
System performance and system repeatability: Proceed as
directd in the system suitability in the Component determina-
tion under Corydalis Tuber.
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of dehydrocorydaline obtained
from 5 nL of this solution can be measured by the automatic
integration method, and that the peak height of de-
hydrocorydaline obtained from 5 /xL of the standard solution
is equivalent to around 20% of the full scale.
iV-Demethylroxithromycin C40H74N2O15 White powder.
Identification — Determine the infrared absorption spec-
trum of a solution of the substance to be tested in chloroform
(1 in 20) as directed in the solution method under Infrared
Spectrophotometry <2.25> using a 0.1 -mm cell made of potas-
sium bromide: it exhibits absorption at the wave numbers of
about 3600 cm- 1 , 3520 cm-', 3450 cm- 1 , 3340 cm- 1 , 1730
cm- 1 and 1627 cm- 1 .
iV-Demethylerythromycin C 36 H 65 N0 13 White to light
yellowish white powder.
2'-Deoxyuridine for liquid chromatography C 9 H 12 N 2 5
White, crystalline powder.
Melting point <2.60>: 162- 166°C
Purity — Dissolve 3.0 mg of 2'-deoxyuridine for liquid
chromatography in diluted methanol (1 in 25) to make 50
mL. Perform the test with 10 fiL of this solution as directed
under Liquid Chromatography <2.01> according to the oper-
ating conditions in the Purity under Idoxuridine Ophthalmic
Solution. Measure each peak area by the automatic integra-
tion method to the range about twice the retention time of 2'-
deoxyuridine, and calculate the amount of 2'-deoxyuridine
by the area percentage method: it shows a purity of not less
than 98.5%.
Content: not less than 98.5%. Assay — Weigh accurately
about 5 mg of 2'-deoxyuridine for liquid chromatography,
previously dried in vacuum at 60°C for 3 hours, and dissolve
in water to make exactly 250 mL. Pipet 10 mL of this solu-
tion, dilute with water to make exactly 20 mL. Perform the
test with this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and determine absorbance A at
the maximum wavelength at about 262 nm.
Amount (mg) of deoxyuridine (C9LL2N2O5)
A
447
X5000
Deuterated dimethylsulfoxide for nuclear magnetic
resonance spectroscopy (CD 3 ) 2 SO Prepared for nuclear
magnetic resonance spectroscopy.
Deuterated formic acid for nuclear magnetic resonance
spectroscopy DCOOD Prepared for nuclear magnetic
resonance spectroscopy.
Deuterated hydrochloric acid for nuclear magnetic
resonance spectroscopy DC1 Prepared for nuclear mag-
netic resonance spectroscopy.
Deuterated methanol for nuclear magnetic resonance spec-
troscopy CD3OD Prepared for nuclear magnetic
resonance spectroscopy.
Deuterated NMR solvents Prepared for nuclear magnetic
resonance spectroscopy. For example: deuterated dimethyl-
sulfoxide [(CD 3 ) 2 SO], deuterated pyridine (C 5 D 5 N), deu-
terochloroform (CDC1 3 ), heavy water (D 2 0), etc.
Deuterated pyridine for nuclear magnetic resonance spec-
troscopy C5D5N Prepared for nuclear magnetic resonance
spectroscopy.
Deuterochloroform for nuclear magnetic resonance spec-
troscopy CDCI3 Prepared for nuclear magnetic resonance
spectroscopy.
Devarda's alloy [K 8653, Special class]
Diacetyl CH 3 COCOCH 3 A yellow to yellow-green,
clear liquid, having a strong, pungent odor. Miscible with
ethanol (95) and with diethyl ether, and freely soluble in
water.
Boiling point <2.57>: 85 - 91 °C
Congealing point <2.42>: -2.0- -5.5°C
Refractive index <2.45> n™: 1.390- 1.398
Specific gravity <2.56> df : 0.98 - 1.00
Purity Clarity of solution — Dissolve 1.0 g of diacetyl in
10 mL of water: the solution is clear.
Content: not less than 95.0%. Assay — Weigh accurately
about 0.4 g of diacetyl, add exactly 75 mL of hydroxylamine
TS, and heat on a water bath for 1 hour under a reflux con-
denser. After cooling, titrate <2.50> the excess hydroxylamine
with 0.5 mol/L hydrochloric acid VS until the color of the so-
lution changes from blue to yellow-green through green (indi-
cator: 3 drops of bromophenol blue TS). Perform a blank de-
termination in the same manner.
Each mL of 0.5 mol/L hydrochloric acid VS
= 21.52 mg of C 4 H 6 2
Diacetyl TS Dissolve 1 mL of diacetyl in water to make
100 mL, and dilute 5 mL of this solution with water to make
100 mL. Prepare before use.
2,3-Diaminonaphthalene Ci H 10 N 2 Light yellow-
brown crystals or powder. Slightly soluble in ethanol (95) and
in diethyl ether, and practically insoluble in water.
Melting point <2.60>: 193 - 198°C
Sensitivity — Pipet separately 40 mL each of the selenium
standard solution and diluted nitric acid (1 in 60) as the blank
solution into beakers, and to these solutions add ammonia
solution (28) to adjust the pH to between 1.8 and 2.2. Dis-
solve 0.2 g of hydroxylammonium chloride in each of these
solutions under gentle shaking, add 5 mL of 2,3-di-
aminonaphthalene TS, mix by shaking, and allow to stand
for 100 minutes. Transfer these solutions to separators
separately, rinse the beakers with 10 mL of water, add these
rinsings to the separators, extract each with 5.0 mL of cyclo-
hexane by thorough shaking for 2 minutes, and centrifuge
the cyclohexane layers to remove moisture. When the absorb-
ance at 378 nm of cyclohexane extract obtained from seleni-
um standard solution is determined using the solution ob-
tained from the blank solution as the reference solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
it is not less than 0.08.
Selenium standard solution — Weigh accurately 40 mg of
selenium, dissolve in 100 mL of diluted nitric acid (1 in 2), by
heating on water bath if necessary, and add water to make ex-
actly 1000 mL. Pipet 5 mL of this solution, and add water to
make exactly 200 mL. Pipet 2 mL of this solution, and add
180
Reagents, Test Solutions / General Tests
JP XV
diluted nitric acid (1 in 60) to make exactly 50 mL. Prepare
before use. This solution contains 0.04 /xg of selenium (Se)
per mL.
2,3-Diaminonaphthalene TS Dissolve 0.10 g of 2,3-di-
aminonaphthalene and 0.5 g of hydroxylammonium chloride
in 0.1 mol/L hydrochloric acid TS to make 100 mL.
2,4-Diaminophenol hydrochloride C 6 H 8 N 2 0.2HC1
Pale yellow-brown to grayish yellow-green crystalline pow-
der. Freely soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
Purity Clarity of solution — Dissolve 1.0 g of 2,4-
diaminophenol hydrochloride in 20 mL of water: the solution
is clear or a slight turbidity is produced.
Loss on drying <2.41>: not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44>: not more than 0.5% (1 g).
Content: not less than 98.0%. Assay — Weigh accurately
about 0.2 g of 2,4-diaminophenol hydrochloride, dissolve in
50 mL of water, and titrate <2.50> with 0.1 mol/L silver ni-
trate VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 9.854 mg of C 6 H 8 N 2 0.2HC1
2,4-Diaminophenol hydrochloride TS Dissolve 1 g of
2,4-diaminophenol hydrochloride and 20 g of sodium bisul-
fite in 100 mL of water, and filter, if necessary.
Diammonium hydrogen citrate C 6 H 14 N 2 07
Special class]
[K 8284,
Diammonium hydrogenphosphate
[K 9016, Special class]
(NH 4 ) 2 HP0 4
Diazobenzenesulfonic acid TS Weigh 0.9 g of sulfanilic
acid, previously dried at 105 °C for 3 hours, dissolve it in 10
mL of dilute hydrochloric acid by heating, and add water to
make 100 mL. Pipet 3.0 mL of this solution, add 2.5 mL of
sodium nitrite TS, and allow to stand for 5 minutes while
cooling with ice. Then add 5 mL of sodium nitrite TS and
water to make 100 mL, and allow to stand in ice water for 15
minutes. Prepare before use.
Diazobenzenesulfonic acid TS, concentrated Weigh 0.2 g
of sulfanilic acid, previously dried at 105°C for 3 hours, dis-
solve it in 20 mL of 1 mol/L hydrochloric acid TS by warm-
ing. Cool this solution with ice, and add 2.2 mL of a solution
of sodium nitrite (1 in 25) dropwise under stirring. Allow to
stand in ice water for 10 minutes, and add 1 mL of a solution
of sulfaminic acid (1 in 20). Prepare before use.
Diazo TS Weigh accurately 0.9 g of sulfanilic acid, add
0.9 mL of hydrochloric acid and 20 mL of water, and dis-
solve by heating. After cooling, filter, and dilute the filtrate
with water to make exactly 100 mL. Pipet 1.5 mL of this so-
lution, cool in an ice bath, and add exactly 1 mL of sodium
nitrite solution (1 in 20) dropwise, while shaking. Cool in an
ice bath for 10 minutes, add cold water to make exactly 50
mL. Store in a cold place, and use within 8 hours.
Dibasic ammonium phosphate See diammonium hydro-
gen phosphate.
Dibasic potassium phosphate See dipotassium hydrogen
phosphate.
Dibasic potassium phosphate-citric acid buffer solution,
pH 5.3 See dipotassium hydrogen phosphate-citric acid
buffer solution, pH 5.3.
1 mol/L Dibasic potassium phosphate TS for buffer solu-
tion See 1 mol/L dipotassium hydrogen phosphate TS for
buffer solution.
Dibasic sodium ammonium phosphate See ammonium
sodium hydrogen phosphate tetrahydrate.
Dibasic sodium phosphate See disodium hydrogen phos-
phate dodecahydate.
Dibasic sodium phosphate TS See disodium hydrogen
phosphate TS.
Dibasic sodium phosphate, anhydrous See disodium
hydrogen phosphate.
Dibasic sodium phosphate, anhydrous, for pH determina-
tion See disodium hydrogen phosphate for pH determina-
tion.
Dibasic sodium phosphate-citric acid buffer solution, pH
4.5 See disodium hydrogen phosphate-citric acid buffer so-
lution, pH 4.5.
Dibasic sodium phosphate-citric acid buffer solution, pH
5.4 See disodium hydrogen phosphate-citric acid buffer so-
lution, pH 5.4.
Dibasic sodium phosphate-citric acid buffer solution, pH
6.0 See disodium hydrogen phosphate-citric acid buffer so-
lution, pH 6.0.
0.05 mol/L Dibasic sodium phosphate TS See 0.05 mol/
L disodium hydrogen phosphate TS.
0.5 mol/L Dibasic sodium phosphate TS See 0.5 mol/L
disodium hydrogen phosphate TS.
Dibekacin sulfate [Same as the namesake monograph]
Dibenzyl Ci 4 H 14 White crystals, freely soluble in
diethyl ether, soluble in methanol and in ethanol (95), and
practically insoluble in water.
Melting point <2.60>: 50 - 54°C
Purity — Dissolve 32 mg of dibenzyl in methanol to make
exactly 50 mL, and use this solution as the sample solution.
Perform the test with 20 /uL of the sample solution as directed
under Liquid Chromatography <2.01> according to the oper-
ating conditions in the Assay under Vinblastine Sulfate for
Injection: any peak other than the principal peak does not
appear. Adjust the detection sensitivity so that the peak
height of dibenzyl obtained from 20 /xh of the solution pre-
pared by adding methanol to 10 mL of the sample solution to
make 20 mL, is 3 to 5 cm, and the time span of measurement
is about 1 .2 times as long as the retention time of dibenzyl af-
ter the solvent peak.
./V,iV'-Dibenzylethylenediamine diacetate A white to
slightly pale yellow crystalline powder.
Identification — Determine the infrared absorption spec-
trum of the substance to be examined as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>: it exhibits absorption at the wave numbers of about
1530 cm" 1 , 1490 cm" 1 , 1460 cm" 1 , 1400 cm" 1 and 1290
JPXV
General Tests / Reagents, Test Solutions
181
Content: not less than 99.0%. Assay — Weigh accurately
about 25 mg of iV,iV'-dibenzylethylenediamine diacetate, dis-
solve in 25 mL of methanol, and add a solution containing
1.02 g of disodium hydrogen phosphate and 6.80 g of potas-
sium dihydrogen phosphate in 1000 mL of water to make ex-
actly 50 mL. Pipet 5 mL of this solution, add a mixture of the
solution containing 1.02 g of disodium hydrogen phosphate
and 6.80 g of potassium dihydrogen phosphate in 1000 mL of
water and methanol (1:1) to make exactly 20 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 8 mg of acetic acid (100), add 25 mL of methanol, and
add the solution containing 1.02 g of disodium hydrogen
phosphate and 6.80 g of potassium dihydrogen phosphate in
1000 mL of water to make exactly 50 mL. Pipet 5 mL of this
solution, add a mixture of the solution containing 1.02 g of
disodium hydrogen phosphate and 6.80 g of potassium di-
hydrogen phosphate in 1000 mL of water and methanol (1:1)
to make exactly 20 mL, and use this solution as the control
solution. Perform the test with exactly 20 /xL each of the sam-
ple solution and control solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method. After making correction for the peak areas
based on the valiance of the base-line and the peak of acetic
acid on the chromatogram obtained with the sample solution,
calculate the amount of ./V,/V-dibenzylethylenediamine by
the area percentage method.
Operating conditions
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, methanol and 0.25
mol/L potassium dihydrogen phosphate TS, pH 3.5 (11:7:2).
Flow rate: Adjust the flow rate so that the retention time of
TV.TV'-dibenzylethylenediamine is about 4 minutes.
Time span of measurement: About 5 times as long as the
retention time of 7V,7V'-dibenzylethylenediamine.
System suitability
System performance: Dissolve an amount of Benzylpenicil-
lin Benzathine, equivalent to about 85,000 Units, in 25 mL of
methanol, add a solution containing 1.02 g of disodium
hydrogen phosphate and 6.80 g of potassium dihydrogen
phosphate in 1000 mL of water to make exactly 50 mL. Pipet
5 mL of this solution, add a mixture of the solution contain-
ing 1.02 g of disodium hydrogen phosphate and 6.80 g of
potassium dihydrogen phosphate in 1000 mL of water and
methanol (1:1) to make exactly 20 mL. When the procedure is
run with 20 [iL of this solution under the above operating
conditions, A^/V'-dibenzylethylenediamine and benzyl-
penicillin are eluted in this order with the resolution between
these peaks being not less than 20.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
/V,/V'-dibenzylethylenediamine is not more than 2.0%.
2, 6-Dibromo-iV-chloro-l, 4-benzoquinone monoimine
C 6 H 2 Br 2 :ClNO [K 8491, Special class]
2,6-Dibromo-./V-chloro-l, 4-benzoquinone monoimine TS
Dissolve 0.5 g of 2,6-dibromo-A r -chloro-l,4-benzoquinone
monoimine in methanol to make 100 mL.
2,6-Dibromo-./V-chloro-l, 4-benzoquinone monoimine TS,
dilute Dissolve 0.2 g of 2,6-dibromo-A r -chloro-l,4-benzo-
quinone monoimine in methanol to make 100 mL.
2,6-Dibromoquinone chlorimide See 2,6-dibromo-A r -
chloro-1, 4-benzoquinone monoimine.
2,6-Dibromoquinone chlorimide TS See 2,6-dibromo-7V r -
chloro-1, 4-benzoquinone monoimine TS.
Dibucaine hydrochloride C20H29N3O2.HCI [Same as the
namesake monograph]
Di-n-butyl ether (C 4 H 9 ) 2 Clear colorless, water-non-
miscible liquid.
Specific gravity <2.56> df: 0.768 - 0.771
Di-n-butyl phthalate C 6 H 4 (COOC 4 H 9 ) 2 Clear colorless
liquid.
Purity Related substances — Dissolve 0.5 g of di-n-butyl
phthalate in 50 mL of methanol, and use this solution as the
sample solution. Perform the test with 10 /xL of the sample
solution as directed in the Assay under Nicardipine
Hydrochloride Injection, and determine the peak area by the
automatic integration method. Calculate the amount of di-n-
butyl phthalate by the area percentage method: the amount
of di-n-butyl phthalate is not less than 98.0%, and no peak
appears at the same position as nicardipine. Adjust the detec-
tion sensitivity so that the peak height of di-n-butyl phthalate
obtained from 10 /xL of the sample solution is 50 to 100% of
the full scale, and measure about 2 times as long as the reten-
tion time of di-n-butyl phthalate after the solvent peak.
1,2-Dichlorobenzene C 6 H 4 C1 2 A colorless liquid.
Boiling point <2.57>: 180 - 181 °C
Specific gravity <2.56> df: 1.306
1,2-Dichloroethane C1CH 2 CH 2 C1 [K 8465, Special
class]
Dichlorofluorescein C2oH 10 Cl 2 05 Orange to red-brown
powder.
Identification — (1) Dissolve 0.1 g in 10 mL of sodium
hydroxide TS: the solution is an orange-red color, and red-
orange precipitates appear by the addition of 10 mL of dilute
hydrochloric acid.
(2) Dissolve 0.1 g in 10 mL of sodium hydroxide TS, and
add 40 mL of water: a green-yellow fluorescence is exhibited.
Dichlorofluorescein TS Dissolve 0.1 g of dichlorofluores-
cein in 60 mL of ethanol (95), add 2.5 mL of 0.1 mol/L sodi-
um hydroxide VS, and dilute with water to make 100 mL.
2,6-Dichloroindophenol sodium dihydrate
C 12 H 6 Cl 2 NNa0 2 .2H 2 [K 8469, Special class]
2,6-Dichloroindophenol sodium TS Add 0.1 g of 2,6-
dichloroindophenol sodium dihydrate to 100 mL of water,
warm, and filter. Use within 3 days.
2,6-Dichloroindophenol sodium TS for titration See the
monograph Ascorbic Acid Powder.
Dichloromethane CH 2 C1 2 [K 8161, Special class]
2,6-Dichlorophenol C 6 H 4 C1 2 White to purplish white
182
Reagents, Test Solutions / General Tests
JP XV
crystals.
Melting point <2.60>: 65
67°C
2,6-DichIorophenol-indophenol sodium See 2,6-
dichloroindophenol sodium dihydrate.
2,6-Dichlorophenol-indophenol sodium TS See 2,6-
dichloroindophenol sodium TS.
2,6-Dichlorophenol-indophenol sodium TS for titration
See 2,6-dichloroindophenol sodium TS for titration.
Dicyclohexyl C 12 H 22
Boiling point <2.57>: about 227°C
Melting point <2.60>: about 4°C
Specific gravity <2.56> df : about 0.864
N,N' -Dicyclohexylcarbodiimide C 6 H n N = C = NC 6 H U
Colorless or white crystals or crystalline mass. Dissolves in
ethanol (95), but decomposes in water to produce a white pre-
cipitate.
Melting point <2.60>: 35 - 36°C
iV,./V'-Dicyclohexylcarbodiimide-dehydrated ethanol TS
See 7V,/V'-dicyclohexylcarbodiimide-ethanol (99.5) TS.
A^/V'-Dicyclohexylcarbodiimide-ethanol (99.5) TS Dis-
solve 6 g of /V,/V'-dicyclohexylcarbodiimide in ethanol (99.5)
to make 100 mL.
Storage — Preserve in tight containers, in a cold place.
Dicyclohexyl phthalate C 6 H 4 (COOC 6 H u ) 2 A white,
crystalline powder.
Melting point <2.60>: 63 - 66°C
Purity Clarity and color of solution — Dissolve 1.0 g of
dicyclohexyl phthalate in 20 mL of ethanol (95): the solution
is clear and colorless.
Dicyclohexylurea C 6 H H NHCONHC 6 H u A white crys-
talline powder, having no odor.
Purity Related substances — Dissolve 50 mg of dicyclo-
hexylurea in methanol to make 100 mL. Pipet 10 mL of this
solution, and add methanol to make 100 mL. Pipet 20 mL of
this solution, add 10 mL of 0.5 mol/L sodium hydroxide TS,
shake, then add 5 mL of diluted hydrochloric acid (1 in 10),
and shake. Perform the test with 50 /xh of this solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, determine the area of each peak by
the automatic integration method, and calculate the amount
by the area percentage method: the total amount of the peaks
other than dicyclohexylurea is not more than 3.0%.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (5) (ii) under Acetohexamide.
Time span of measurement: About 5 times as long as the
retention time of dicyclohexylurea beginning after the solvent
peak.
System suitability
Test for required detectability: To exactly 5 mL of the stan-
dard solution add water to make exactly 200 mL. Confirm
that the peak area of dicyclohexylurea obtained with 50 /uL of
this solution is equivalent to 1 . 8 to 3 . 3 % of that with 50 /uL of
the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Purity (5) (ii) under
Acetohexamide.
Diethanolamine C 4 H u N0 2 Colorless viscous liquid.
Melting point <2.60>: 27 - 30°C
Water <2.48>: less than 0.1%.
Diethanolamine hydrochloride C 4 H U N0 2 .HC1 A pale
yellow liquid.
Refractive index <2.45> n™: 1.515 - 1.519
Specific gravity <2.56> df : 1.259 - 1.263
Water <2.48>: less than 0.1%.
Diethylamine (C 2 H 5 ) 2 NH A clear, colorless liquid, hav-
ing an amine-like odor. Miscible with water and with ethanol
(95). The solution in water is alkaline, and readily absorbs
carbon dioxide in air.
Distilling range <2.57>: 54 - 58°C; not less than 96 vol%.
Specific gravity <2.56> d\°: 0.702 - 0.708
Content: not less than 99.0%. Assay — Weigh accurately
about 1.5 g of diethylamine in a flask containing exactly 30
mL of 0.5 mol/L sulfuric acid VS, and titrate <2.50> the ex-
cess of sulfuric acid with 1 mol/L sodium hydroxide VS (in-
dicator: 2 drops of methyl red TS). Perform a blank determi-
nation.
Each mL of 0.5 mol/L sulfuric acid VS
= 73.14 mg of (C 2 H 5 ) 2 NH
Diethylene glycol HO(CH 2 CH 2 0) 2 H Colorless and
odorless liquid. Miscible with water and with ethanol (95).
Specific gravity <2.56> df : 1.118-1.120
Diethylene glycol adipinate for gas chromatography Pre-
pared for gas chromatography.
Diethylene glycol dimethyl ether (CH 3 OCH 2 CH 2 ) 2
Clear and colorless liquid, miscible with water.
Specific gravity <2.56> df: 0.940 - 0.950
Distilling range <2.57>: 158 - 160°C, not less than 95 vol%.
Diethylene glycol monoethyl ether
C 2 H 5 (OCH 2 CH 2 ) 2 OH [2-(2-ethoxyethoxy)ethanol] Clear,
colorless liquid, of which boiling point is about 203°C. It
freely mixed with water.
Acid (as CH 3 COOH): less than 0.01%.
Refractive index <2.45> n™: 1.425 - 1.429
Specific gravity <2.56> df : 0.990 - 0.995
Diethylene glycol monoethyl ether for water determination
See Water Determination <2.48>.
Diethylene glycol succinate ester for gas chromato-
graphy Prepared for gas chromatography.
Diethylene glycol succinate polyester for gas chromato-
graphy Prepared for gas chromatography.
Diethyl ether C 2 H 5 OC 2 H 5 [K 8103, Special class]
Diethyl ether, dehydrated C 2 H 5 OC 2 H 5 [K 8103, Spec-
ial class. The water content is not more than 0.01%.]
Diethyl ether for purity of crude drug [K 8103, Special c-
lass] Use diethyl ether meeting the following additional
specification. Evaporate 300.0 mL of diethyl ether for purity
of crude drug in vacuum at a temperature not higher than 40°
C, add the diethyl ether to make exactly 1 mL, and use this
solution as the sample solution. Separately, dissolve 2.0 mg
JPXV
General Tests / Reagents, Test Solutions
183
of y-BHC in hexane for purity of crude drug to make exactly
100 mL. Pipet 1 mL of this solution, and add hexane for
purity of crude drug to make exactly 100 mL. Pipet 2 mL of
this solution, add hexane for purity of crude drug to make ex-
actly 100 mL, and use this solution as the standard solution I.
Perform the test with 1 fiL each of the sample solution and
standard solution I as directed under Gas Chromatography <
2.02> according to the following operating conditions, and
determine each peak area by the automatic integration
method: the total area of peaks other than the solvent peak
from the sample solution is not larger than the peak area of y-
BHC from the standard solution I.
Operating conditions
Proceed the operating conditions in the Purity (2) under
the Crude Drugs Test <5.01> except detection sensitivity and
time span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
I, add hexane for purity of crude drug to make exactly 20
mL, and use this solution as the standard solution II. Adjust
the detection sensitivity so that the peak area of y-BHC ob-
tained from 1 /uL of the standard solution II can be measured
by the automatic integration method, and the peak height of
y-BHC from 1 /uL of the standard solution I is about 20% of
the full scale.
Time span of measurement: About three times as long as
the retention time of y-BHC beginning after the peak of sol-
vent.
iV,./V-DiethyI-iV' -1-naphthylethylenediamine oxalate
C18H24N2O4 A white crystalline powder.
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3340 cm -1 , 2940 cm -1 , 1581
cm -1 , 1536 cm -1 , 1412 cm -1 , 789 cm -1 , 774 cm -1 and 721
cm -1 .
Purity Clarity of solution — To 0.1 g add 20 mL of water,
and dissolve by warming: the solution is clear.
iV,./V-DiethyI-iV ' -1-naphthylethylenediamine oxalate-ace-
tone TS Dissolve 1 g of iV, N-Diethyl-TV' -1-naphthylethy-
lenediamine oxalate in 100 mL of a mixture of acetone and
water (1:1). Prepare before use.
iV,./V-DiethyI-iV'-l-naphthyIethylenediamine oxalate TS
Dissolve 1 g of Af A r -Z)/eZ/!.y/-/V'-l-naphthylethylenediamine
oxalate in water to make 1000 mL.
Diethyl phthalate C 6 H 4 (COOC 2 H 5 ) 2 A colorless, clear
liquid.
Refractive index <2.45> n™: 1.500-1.505
Purity Related substances — To 1 mL of diethyl phthalate
add a solution of tetra M-heptylammonium bromide in a mix-
ture of water, acetonitrile and methanol (137:80:23) (2 in 625)
to make 100 mL. To 6 mL of this solution add a solution of
tetra »-heptylammonium bromide in a mixture of water,
acetonitrile and methanol (137:80:23) (2 in 625) to make 50
mL, and use this solution as the sample solution. Perform the
test with 10 /xL of the sample solution as directed in the Assay
under Cefetamet Pivoxil Hydrochloride: any peaks other
than peaks of diethyl phthalate and the solvent are not ob-
served.
Melting point <2.60>: 44 - 46 °C
Content: not less than 99%. Assay — Dissolve 100 mg of
diethyl terephthalate in 10 mL of methanol. Perform the test
with 2 /iL of this solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and determine the area of each peak by the automatic in-
tegration method.
Content 1
peak area of diethyl terephthalate
total of all peak areas
X100
Diethyl terephthalate C 6 H 4 (COOC 2 H 5 ) 2
pale brownish white, crystalline or mass.
White to
Operating conditions
Detector: Hydrogen flame-ionization detector.
Column: A glass tube 4 mm in inside diameter and 2 m in
length, packed with Shimalite W(AW, DMCS) coated with
SE-30 in 10% (177 - 250 /mi in particle diameter).
Column temperature: A constant temperature of about 200
°C
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
diethyl terephthalate is between 6 and 7 minutes.
Time span of measurement: About 5 times as long as the
retention time of diethyl terephthalate beginning after the
solvent peak.
Digitonin C 56 H 92 2 9 [K 8452, Special class]
Digoxin C 4 iH 64 I4 [Same as the namesake monograph]
Dihydrocodeine phosphate for assay
C 18 H 23 N0 3 .H 3 P0 4 [Same as the monograph Dihydro-
codeine Phosphate. It contains not less than 99.0% of di-
hydrocodeine phosphate (C I8 H 23 N0 3 .H 3 P0 4 ), calculated on
the dried basis.
Dihydroergocristine mesilate for thin-layer chromato-
graphy C 35 H 41 N 5 5 .CH 4 3 S A pale yellowish white pow-
der. Freely soluble in methanol, in ethanol (95) and in chlo-
roform, soluble in acetonitrile, sparingly soluble in water,
and practically insoluble in diethyl ether. Melting point:
about 190°C (with decomposition).
Purity Related substances — Dissolve 6 mg of dihydroer-
gocristine mesilate for thin-layer chromatography in exact
100 mL of a mixture of chloroform and methanol (9:1), and
perform the test with 5 /xL of this solution as directed in the
Purity (3) under Dihydroergotoxine Mesilate: any spot other
than the principal spot at the Rf value around 0.4 does not
appear.
3,4-Dihydro-6-hydroxy-2(LH)-quinolinone
C 9 H 9 N0 2 A white to light brown powder or granule. Melt-
ing point: about 240°C (with decomposition).
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3210 cm -1 , 1649 cm -1 , 1502
cm -1 , 1252 cm -1 and 816 cm -1 .
2,4-Dihydroxybenzoic acid C 7 H 6 4 White to pale
brown powder.
Purity Clarity of solution — Dissolve 1.0 g of 2,4-di-
hydroxybenzoic acid in 20 mL of ethanol (95): the solution is
clear.
Content: not less than 95%. Assay — Weigh accurately
about 1 g of 2,4-dihydroxybenzoic acid, dissolve in 50 mL of
ethanol (95) and 50 mL of water, and titrate <2.50> with 0.1
mol/L sodium hydroxide VS.
184
Reagents, Test Solutions / General Tests
JP XV
Each mL of 0.1 mol/L sodium hydroxide VS
= 15.41 mg of C 7 H 6 4
1,3-Dihydroxynaphthalene Ci H 6 (OH) 2 Purple-brown,
crystals or powder. Freely soluble in water and in ethanol
(95).
Melting point <2.60>: about 125°C
2,7-Dihydroxynaphthalene C 10 H 6 (OH) 2
Purity: not less than 97.0%.
2,7-Dihydroxynaphthalene TS Dissolve 0.10 g of 2,7-di-
hydroxynaphthalene in 1000 mL of sulfuric acid, and allow
to stand until the yellow color initially developed disappears.
If the solution is blackened notably, prepare freshly.
Diltiazem hydrochloride C22H26N2O4S.HCI [Same
as the namesake monograph]
Dilute acetic acid See acetic acid, dilute.
Dilute bismuth subnitrate-potassium iodide TS for spray
Dissolve 10 g of L -tartaric acid in 50 mL of water, and add 5
mL of bismuth subnitrate TS.
Dilute bromophenol blue TS See bromophenol blue TS,
dilute.
Diluted ethanol See ethanol, diluted.
Dilute ethanol See ethanol, dilute.
Dilute ferric ammonium sulfate TS See ammonium iron
(III) sulfate TS, dilute.
Dilute ferric chloride TS See iron (III) chloride TS, di-
lute.
Dilute hydrochloric acid See hydrochloric acid, dilute.
Dilute hydrogen peroxide TS See hydrogen peroxide TS,
dilute.
Dilute iodine TS See iodine TS, dilute.
Dilute iron-phenol TS See iron-phenol TS, dilute.
Dilute lead subacetate TS See lead subacetate TS, dilute.
Dilute methyl red TS See methyl red TS, dilute.
Dilute nitric acid See nitric acid, dilute.
Dilute p-dimethylaminobenzaldehyde-ferric chloride TS
See 4-dimethylaminobenzaldehyde-iron (III) chloride TS, di-
lute.
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2i/-tetrazolium
bromide C 18 H 16 BrN 5 S Yellow crystals. Melting point:
about 195°C (with decomposition).
3-(4,5-dimethylthiazol-2-yI)-2,5-diphenyltetrazolium
bromide C 18 Hi 6 N 5 SBr Yellowish crystals, Melting point:
195°C
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide TS Dissolve 5 g of 3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyl tetrazolium bromide in phosphate-buffered so-
dium chloride TS to make 1000 mL.
Dilute potassium hydroxide-ethanol TS See potassium
hydroxide-ethanol TS, dilute.
dilute.
Dilute sulfuric acid See sulfuric acid, dilute.
Dilute thymol blue TS See thymol blue TS, dilute.
Dilute vanadium pentoxide TS See vanadium (V) oxide
TS, dilute.
Dilute 4-dimethylaminobenzaldehyde-iron (III) chloride
TS See 4-dimethylaminobenzaldehyde-iron (III) chloride
TS, dilute.
Dimedon C 8 H 12 2 White to pale yellow, crystalline
powder.
Melting point <2.60>: 145-149°C
JV,iV-Dimethylacetamide CH 3 CON(CH 3 ) 2 Clear and
colorless liquid.
Boiling point <2.57>: 163 - 165°C
Specific gravity <2.56> d: 0.938 - 0.945 (Method 3).
Water <2.48>: not more than 0.2% (0.1 g, Coulometric
titration).
Purity — Perform the test with 3 /uh of N,N-
dimethylacetamide as directed under Gas Chromatography
<2.02> according to the following conditions, determine each
peak area by the automatic integration method, and calculate
the amount of _/V,./V-dimethylacetamide by the area percen-
tage method: not less than 98.0%.
Operating conditions
Detector: A hydrogen flame-ionization detector.
Column: A fused silica column 0.25 mm in inside diameter
and 30 m in length, coated the inside surface 0.5 fim in thick-
ness with polyethylene glycol 20 M for gas chromatography.
Column temperature: The sample is injected at a constant
temperature of about 70 °C, keep this temperature for 1
minute, then raise to 200°C in a rate of 10°C per minute, and
keep 200°C for 3 minutes.
Carrier gas: Helium
Flow rate (linear velocity): About 30 cm/sec.
Time span of measurement: About 2 times as long as the
retention time of iV,./V-dimethylacetamide.
System suitability
Test for required detection: To exactly 1.0 g of N,N-
dimethylacetamide add acetone to make exactly 100 mL.
Pipet 5 mL of this solution, and add acetone to make exactly
50 mL. Confirm that the peak area of N,N-
dimethylacetamide obtained from 3 /xL of this solution is
equivalent to 40 to 60% of the full-scale.
System repeatability: When the test is repeated with 3 /uL
of ./V,./V-dimethylacetamide under the above operating condi-
tions, the relative standard deviation of the peak area of
7V,./V-dimethylacetamide is not more than 2.0%.
Dimethylamine (CH 3 ) 2 NH Colorless, clear liquid, hav-
ing amine-like, characteristic odor. It is miscible with water
and with ethanol (99.5). It is alkaline.
Specific gravity <2.56> df : 0.85 - 0.93
Content: 38.0-45.0%. Assay— Weigh accurately about
1.0 g of dimethylamine, transfer to a flask containing exactly
20 mL of 0.5 mol/L sulfuric acid VS, and titrate <2.50> the
excess sulfuric acid with 1 mol/L sodium hydroxide VS (indi-
cator: 2 drops of methyl red TS). Perform a blank determina-
tion in the same manner.
Dilute sodium hydroxide TS See sodium hydroxide TS,
Each mL of 0.5 mol/L sulfuric acid VS
JPXV
General Tests / Reagents, Test Solutions
185
= 45.08 mg of (CH 3 ) 2 NH
4-Dimethylaminoantipyrine C 13 H 17 N 3 Colorless or
white crystals, or a white crystalline powder.
Purity — Proceed the test with 5 /xh of a solution of 4-
dimethylaminoantipyrine (1 in 2000) as directed in the Assay
under Cefpiramide Sodium, determine each peak area in a
range of about 2 times as long as the retention time of 4-
dimethylaminoantipyrine after the solvent peak by the auto-
matic integration method, and calculate the total amount of
the peaks other than 4-dimethylaminoantipyrine by the area
percentage method: not more than 1.0%.
p-Dimethylaminobenzaldehyde See 4-dimethylamino-
benzaldehyde.
4-Dimethylaminobenzaldehyde (CH 3 ) 2 NC 6 H 4 CHO [K
8496, p-Dimethylaminobenzaldechyde, Special class]
p-Dimethylaminobenzaldehyde-ferric chloride TS See 4-
dimethylaminobenzaldehyde-iron (III) chloride TS.
p-Dimethylaminobenzaldehyde-ferric chloride TS, dilute
See 4-dimethylaminobenzaldehyde-iron (III) chloride TS, di-
lute.
p-Dimethylaminobenzaldehyde-hydrochloric acid TS See
4-dimethylaminobenzaldehyde-hydrochloric acid TS.
4-Dimethylaminobenzaldehyde-hydrochloric acid TS
Dissolve 1.0 g of 4-dimethylaminobenzaldehyde in 50 mL of
hydrochloric acid while cooling, and add 50 mL of ethanol
(95).
4-Dimethylaminobenzaldehyde-iron (III) chloride TS
Dissolve 0.125 g of 4-dimethylaminobenzaldehyde in a cold
mixture of 65 mL of sulfuric acid and 35 mL of water, then
add 0.05 mL of iron (III) chloride TS. Use within 7 days.
4-Dimethylaminobenzaldehy de-iron (III) chloride TS,
dilute To 80 mL of water add carefully 100 mL of 4-
dimethylaminobenzaldehyde-iron (III) chloride TS and 0.15
mL of iron (III) chloride TS, while cooling with ice.
p-Dimethylaminobenzaldehyde TS See 4-dimethylamino-
benzaldehyde TS.
4-Dimethylaminobenzaldehyde TS Dissolve 10 g of 4-
dimethylaminobenzaldehyde in a cold mixture of 90 mL of
sulfuric acid and 10 mL of water. Prepare before use.
p-Dimethylaminobenzaldehyde TS for spraying See 4-
dimethylaminobenzaldehyde TS for spraying.
4-Dimethylaminobenzaldehyde TS for spraying
Dissolve 1.0 g of 4-dimethylaminobenzaldehyde in 20 mL of
dilute sulfuric acid. Prepare before use.
p-Dimethylaminobenzylidene rhodanine See 4-dimethyl-
aminobenzylidene rhodanine.
4-Dimethylaminobenzylidene rhodanine Ci2H 12 N 2 OS2
[K 8495, Special class]
p-Dimethylaminobenzylidene rhodanine TS See 4-
dimethylaminobenzylidene rhodanine TS.
4-Dimethylaminobenzylidene rhodanine TS Dissolve
0.02 g of 4-dimethylaminobenzylidene rhodanine in acetone
to make 100 mL.
p-Dimethylaminocinnamaldehyde See 4-dimethylamino-
cinnam aldehyde.
4-Dimethylaminocinnamaldehyde CnH 13 NO Orange
crystals or crystalline powder, having a characteristic odor.
Freely soluble in dilute hydrochloric acid, sparingly soluble in
ethanol (95) and in diethyl ether, and practically insoluble in
water.
Melting point <2.60>: 140 - 142°C
Purity Clarity of solution — Dissolve 0.20 g of 4-dimeth-
ylaminocinnamaldehyde in 20 mL of ethanol (95): the solu-
tion is clear.
Loss on drying <2.41>: not more than 0.5% (1 g, 105°C, 2
hours).
Residue on ignition <2.44>: not more than 0.1% (1 g).
Nitrogen content <1.08>: 7.8 - 8.1% (105°C, 2 hours, after
drying).
p-Dimethylaminocinnamaldehyde TS
aminocinnamaldehyde TS.
See 4-dimethyl
4-Dimethylaminocinnamaldehyde TS Before use, add 1
mL of acetic acid (100) to 10 mL of a solution of 4-
dimethylaminocinnamaldehyde in ethanol (95) (1 in 2000).
Dimethylaminophenol (CH 3 ) 2 NC 6 H 4 OH Dark purple,
crystals or crystalline mass.
Melting point <2.60>: 85°C
Dimethylaniline See 7V,7V-dimethylaniline.
JV,iV-Dimethylaniline C 6 H 5 N(CH 3 ) 2 Colorless or light
yellow liquid, having a characteristic odor.
Specific gravity <2.56> dl%: 0.955 - 0.960
Distilling range <2.57>: 192 - 195°C, not less than 95 vol%.
Dimethylformamide See iV,7V-dimethylformamide.
JV,ALDimethylformamide HCON(CH 3 ) 2 [K 8500,
Special class]
./V,iV-Dimethylformamide for liquid chromatography [K
8500, iV,./V-Dimethylformamide, Special class] Read absor-
bance as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24> (in a 1-cm cell, using water as the blank): the absor-
bance is not more than 0.60 at 270 nm, not more than 0.15 at
280 nm, and not more than 0.05 at 300 nm.
Dimethylglyoxime C 4 H 8 N 2 2 [K 8498, Special class]
Dimethylglyoxime-thiosemicarbazide TS Solution A: Di-
ssolve 0.5 g of dimethylglyoxime in hydrochloric acid to
make 100 mL. Prepare before use. Solution B: Dissolve 0.1 g
of thiosemicarbazide in 50 mL of water with the acid of
warming if necessary, and add diluted hydrochloric acid (1 in
2) to make 100 mL. Prepare before use.
Mix 10 mL each of solution A and solution B, add diluted
hydrochloric acid (1 in 2) to make 100 mL, and allow the mix-
ture to stand for 1 hour. Use within 24 hours.
Dimethylglyoxime TS Dissolve 1 g of dimethylglyoxime
in ethanol (95) to make 100 mL.
Dimethyl malonate C 5 H 8 4 Clear, colorless or pale yel-
low liquid.
Specific gravity <2.56> df: 1 . 152 - 1 . 162
Water <2.48>: not more than 0.3%.
Residue on ignition <2.44>: not more than 0.1%.
./V,iV-Dimethyl-rt-octylamine Ci H 23 N Colorless liquid.
Refractive index <2.45> n 2 £: 1.424
186
Reagents, Test Solutions / General Tests
JP XV
Dimethyl phthalate Ci 6 H 22 4 Colorless, clear liquid,
having a slight aroma.
Refractive index <2.45> n™: 1-491 - 1.493
Purity — To 6.0 mL of a solution of Dimethyl phthalate in
isooctane (1 in 100) add a solution of w-amyl alcohol in hex-
ane (3 in 1000) to make 50 mL, and perform the test with 10
[iL of this solution as directed under Liquid Chromatography
<2.01> according to the conditions described in the Assay un-
der Ergocalciferol or Cholecalciferol: any peak other than
the principal peak does not appear.
N, iV-Dimethyl-p-phenylenediammonium clichloride
H 2 NC 6 H 4 N(CH 3 ) 2 .2HC1 [K 8193, Ar,iV-Dimethyl-.p-phenyl
enediammonium dichloride, Special class]
iV,/V-Dimethyl-p-phenylenediammonium hydrochloride
See N, 7V-dimethyl-/?-phenylenediamine dichloride.
Dimethylsulfoxide (CH 3 ) 2 SO [K 9702, Special class]
Dimethylsulfoxide for ultraviolet-visible spectrophotomet-
ry Colorless crystals or clear colorless liquid, having a char-
acteristic odor. It is highly hygroscopic.
Congealing point <2.42>: not less than 18.3°C.
Purity — Read absorbance of dimethylsulfoxide for ultrav-
iolet-visible spectrophotometry, immediately after saturating
with nitrogen, using water as the blank as directed under
Ultraviolet-visible Spectrophotometry <2.24>: its value is not
more than 0.20 at 270 nm, not more than 0.09 at 275 nm, not
more than 0.06 at 280 nm, and not more than 0.015 at 300
nm. It exhibits no characteristic absorption between 260 nm
and 350 nm.
Water <2.48>: not more than 0.1%.
2,6-Dimethyl-4-(2-nitrosophenyl)3,5-pyridinedicarboxylic
acid dimethyl ester for thin-layer chromatography
Ci 7 H 16 N 2 5 Irradiate xenon light at 50,000 lx of illumina-
tion for 8 hours to a methanol solution of nifedipine (1 in
100), and evaporate the methanol on a water bath. Recrystal-
lize the residue 4 times from 1-propanol, and dry in a desicca-
tor (in vacuum, phosphorus pentoxide). Pale blue crystals.
Very soluble in chloroform, freely soluble in acetone, and
practically insoluble in water.
Melting point <2.60>: 93 - 95 °C
Content: not less than 99.0%. Assay — Weigh accurately
about 0.4 g of 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridine-
dicarboxylic acid dimethyl ester for thin-layer chromatogra-
phy, dissolve in 70 mL of acetic acid (100), and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination in the same manner.
Each mL of 0.1 mol/L perchloric acid VS
= 32.83 mg of C 17 H 16 N 2 5
3-(4,5-DimethyIthiazoIe-2-yl)-2,5-diphenyI-2iZ-tetrazoIium
bromide C 18 H 16 BrN 5 S Yellow crystals. Melting point:
about 195°C (with decomposion).
Dimorpholamine for assay [Same as the monograph
Dimorpholamine. When dried, it contains not less than
99.0% of dimorpholamine (C 20 H 38 N 4 O 4 ).
m-Dinitrobenzene See 1,3-dinitrobenzene.
m-Dinitrobenzene TS See 1,3-dinitrobenzene TS.
1,3-Dinitrobenzene TS Dissolve 1 g of 1,3-dinitroben-
zene in 100 mL of ethanol (95). Prepare before use.
m-Dinitrobenzene TS, alkaline See 1,3-dinitrobenzene
TS, alkaline.
1,3-Dinitrobenzene TS, alkaline Mix 1 mL of tetra-
methylammonium hydroxide and 140 mL of ethanol (99.5),
titrate a part of the mixture with 0.01 mol/L hydrochloric
acid VS, and dilute the remainder with ethanol (99.5) to give
a 0.008 mol/L solution. Before use, mix 40 mL of this solu-
tion with 60 mL of a solution of 1,3-dinitrobenzene in ben-
zene (1 in 20).
2,4-Dinitrochlorobenzene See l-chloro-2, 4-dinitroben-
zene.
2,4-Dinitrofluorobenzene See l-fluoro-2, 4-dinitroben-
zene.
2,4-Dinitrophenol C 6 H 3 OH(N0 2 ) 2 Yellow crystals or
crystalline powder.
Melting point <2.60>: 110 - 114°C
2,4-Dinitrophenol TS Dissolve 0.5 g of 2,4-dinitrophenol
in 100 mL of ethanol (95).
2 ,4-Dinitrophenylhydrazine
[K 8480, Special class]
(NQ 2 ) 2 C 6 H 3 NHNH 2
1,3-Dinitrobenzene C 6 H 4 (N0 2 ) 2
benzene, Special class]
[K 8482, m-Dinitro-
2,4-Dinitrophenylhydrazine-diethylene glycol dimethyl
ether TS Dissolve 3 g of 2,4-dinitrophenylhydrazine in 100
mL of diethylene glycol dimethyl ether while heating, cool,
and filter if necessary.
2,4-Dinitrophenylhydrazine-ethanol TS Dissolve 1.5 g of
2,4-dinitrophenylhydrazine in a cold mixture of 10 mL of sul-
furic acid and 10 mL of water, then add a mixture of 1
volume of aldehyde-free ethanol and 3 volumes of water to
make 100 mL, and filter if necessary.
2,4-Dinitrophenylhydrazine TS Dissolve 1.5 g of 2,4-
dinitrophenylhydrazine in a cold mixture of 10 mL of sulfu-
ric acid and 10 mL of water, then add water to make 100 mL,
and filter if necessary.
Dinonyl phthalate C 6 H 4 (COOC 9 H 19 ) 2 Colorless to
pale yellow, clear liquid.
Acid value <1.13>: not more than 2.
Specific gravity <2.56> df : 0.967 - 0.987
Dioxane See 1,4-dioxane.
1,4-Dioxane C 4 H 8 2 [K 8461, Special class]
Diphenhydramine Ci 7 H 21 NO [Same as the namesake
monograph]
Diphenhydramine tannate [Same as the namesake mono-
graph]
Diphenyl C 12 H I0 White crystals or crystalline powder,
having a characteristic odor. Freely soluble in acetone and in
diethyl ether, soluble in ethanol (95), and practically insolu-
ble in water.
Melting point <2.60>: 68 - 72°C
Purity — Dissolve 0.1 g of diphenyl in 5 mL of acetone and
use this solution as the sample solution. Perform the test with
JPXV
General Tests / Reagents, Test Solutions
187
2 /uL of this solution as directed under Gas Chromatography
<2.02> according to the following conditions. Measure each
peak area by the automatic integration method and calculate
the amount of diphenyl by the area percentage method: it
shows the purity of not less than 98.0%.
Operating conditions
Detector: Hydrogen fiame-ionization detector.
Column: A glass tube about 3 mm in inside diameter and
about 2 m in length, packed with 150 to 180//m mesh sil-
iceous earth for gas chromatography coated with 10% of
polyethylene glycol 20 M for thin-layer chromatography.
Column temperature: A constant temperature of about
180°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
diphenyl is about 8 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of diphenyl obtained from 2 /uL of the
slution prepared by adding acetone to 1.0 mL of the sample
solution to make 100 mL, is 5 % to 15% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of diphenyl beginning after the solvent peak.
Diphenylamine (C 6 H 5 ) 2 NH [K 8487, Special class]
Diphenylamine-acetic acid TS Dissolve 1.5 g of
diphenylamine in 1.5 mL of sulfuric acid and acetic acid (100)
to make 100 mL.
Diphenylamine-acetic acid (100) TS See diphenilamine-a-
cetic acid TS.
Diphenylamine TS Dissolve 1 g of diphenylamine in 100
mL of sulfuric acid. Use the colorless solution.
9,10-Diphenylanthracene C 2 6H 18 Yellow crystalline
powder. Soluble in diethyl ether, and practically insoluble in
water.
Melting point <2.60>: about 248°C
1,4-Diphenylbenzene Ci 8 H 14 White scaly crystals, hav-
ing a slight aromatic odor. It is freely soluble in ethanol
(99.5), and slightly soluble in water.
Identification — Determine the infrared absorption spec-
trum of 1,4-diphenylbenzene as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3050 cm-', 3020 cm-', 1585 cm-', 1565 cm-', 1476 cm-',
1450 cm-', 995 cm-', 834 cm-', 740 cm-' and 680 cm-'.
Diphenylcarbazide See 1,5-diphenilcarbonohydrazide.
Diphenylcarbazide TS See 1,5-diphenilcarbonohydrazide
TS.
Diphenylcarbazone [K 8489, Speical class]
Diphenylcarbazone TS Dissolve 1 g of diphenylcarba-
zone in ethanol (95) to make 1000 mL.
1,5-Diphenylcarbonohydrazide C 13 H 14 N 4 [K 8488,
Special
class]
1,5-Diphenylcarbonohydrazide TS Dissolve 0.2 g of 1,5-
diphenylcarbonohydrazide in 100 mL of a mixture of ethanol
(95) and acetic acid (100) (9:1).
5% Diphenyl-95% dimethylpolysiloxane for gas chro-
matography Prepared for gas chromatography.
Diphenyl ether C 12 H 10 O Colorless crystals, having a
geranium-like aroma. Dissolves in alcohol (95) and in diethyl
ether, and practically insoluble in water.
Boiling point <2.57>: 254 - 259°C
Melting point <2.60>: 28°C
Specific gravity <2.56> d™: 1.072 - 1.075
Diphenyl imidazole C 15 H I2 N 2 White crystals or crystal-
line powder, freely soluble in acetic acid (100), and sparingly
soluble in methanol.
Melting point <2.60>: 234 - 238°C
Loss on drying <2.41>: not more than 0.5% (0.5 g, 105 C C,
3 hours).
Content: not less than 99.0%. Assay — Dissolve about 0.3
g of diphenyl imidazole, previously dried and weighed ac-
curately, in 70 mL of acetic acid (100), and titrate <2.50> with
0.1 mol/L perchloric acid VS (indicator: 2 drops of crystal
vioret TS).
Each mL of 0.1 mol/L perchloric acid VS
= 22.03 mg of C 15 H I2 N 2
Diphenyl phthalate C 6 H 4 (COOC 6 H 5 ) 2 White crystalline
powder.
Melting point <2.60>: 71 - 76°C
Purity Related substances — Dissolve 0.06 g of diphenyl
phthalate in 50 mL of chloroform and use this solution as the
sample solution. Proceed with 10 /uL of the sample solution
as directed in the Assay under Tolnaftate Solution: any peak
other than the principal peak at the retention time of about 8
minutes and the peak of the solvent does not appear. Adjust
the detection sensitivity so that the peak height of diphenyl
phthalate obtained from 10 /xL of the sample solution is 50 to
100% of the full scale, and the time span of measurement is
about twice as long as the retention time of diphenyl phthal-
ate after the solvent peak.
l,l-Diphenyl-4-pyperidino-l-butene hydrochloride for
thin-layer chromatography C 2I H 25 N.HC1 To 1 g of di-
phenidole hydrochloride add 30 mL of 1 mol/L hydrochloric
acid TS, and heat under a reflux condenser for 1 hour. After
cooling, extract twice with 30 mL-portions of chloroform,
combine the chloroform extracts, wash twice with 10 mL por-
tions of water, and evaporate chloroform under reduced
pressure. Recrystallize the residue from a mixture of diethyl
ether and ethanol (95) (3:1), and dry in a desiccator (in vac-
cum, silica gel) for 2 hours. White crystals or crystalline pow-
der.
Absorbance <2.24> E\°f m (250 nm): 386-446 (10 mg,
water, 1000 mL).
Melting point <2.60>: 176 - 180°C
Content: not less than 99.0%. Assay — Dissolve about 0.2
g of l,l-diphenyl-4-pyperidino-l-butene hydrochloride for
thin-layer chromatography, previously weighed accurately,
in 20 mL of acetic acid (100), add 20 mL of acetic anhydride,
and titrate <2.50> with 0.05 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 16.40 mg of C 21 H 25 N.HC1
Dipotassium hydrogen phosphate K 2 HP0 4 [K 9017,
188
Reagents, Test Solutions / General Tests
JP XV
Special class]
Dipotassium hydrogen phosphate-citric acid buffer solu-
tion, pH 5.3 Mix 100 mL of 1 mol/L dipotassium
hydrogen phosphate TS for buffer solution and 38 mL of 1
mol/L citric acid TS for buffer solution, and add water to
make 200 mL.
1 mol/L Dipotassium hydrogen phosphate TS for buffer
solution Dissolve 174.18 g of dipotassium hydrogen phos-
phate in water to make 1000 mL.
Diprophylline CioH 14 N 4 4 A white, powder or grain.
Freely soluble in water, and slightly soluble in ethanol (95).
Identification — Determine the infrared absorption spec-
trum of the substance to be examined, previously dried at 105
°C for 4 hours, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>: it exhibits
absorption at the wave numbers of about 3460 cm -1 , 3330
cm" 1 , 1651 cm"', 1242 cm" 1 , 1059 cm" 1 and 1035 cm" 1 .
a,a'-Dipyridyl See 2,2'-bipyridyl.
Disodium chromotropate dihydrate
C 10 H 6 Na 2 O 8 S 2 .2H 2 O [K 8316, Special class] Preserve in
light-resistant containers.
Disodium dihydrogen ethylenediamine tetraacetate di-
hydrate C 10 H 14 N 2 Na 2 O 8 .2H 2 O [K 8107, Special class]
0.1 mol/L Disodium dihydrogen ethylenediamine tetra-
acetate TS Dissolve 37.2 g of disodium dihydrogen ethyl-
enediamine tetraacetate dihydrate in water to make 1000 mL.
0.04 mol/L Disodium dihydrogen ethylenediamine tetraa-
cetate TS Dissolve 14.890 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in water to make 1000
mL.
0.4 mol/L Disodium dihydrogen ethylenediamine tetra-
acetate TS, pH 8.5 Dissolve 148.9 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in about 800 mL of
water, adjust to pH 8.5 with 8 mol/L sodium hydroxide TS,
and add water to make 1000 mL.
Disodium ethylenediaminetetraacetate See disodium di-
hydrogen ethylenediamine tetraacetate dihydrate.
Disodium ethylenediaminetetraacetate copper See cop-
por (II) disodium ethylenediamine tetraacetate tetrahydrate.
0.1 mol/L Disodium ethylenediaminetetraacetate TS
See 0.1 mol/L disodium dihydrogen ethylenediamine tetra-
acetate TS.
Disodium hydrogen phosphate, anhydrous Na 2 HP0 4
[K 9020, Special class]
Disodium hydrogen phosphate-citric acid buffer solution,
pH 3.0 Dissolve 35.8 g of disodium hydrogenphosphate 12-
water in water to make 500 mL. To this solution add a solu-
tion of citric acid monohydrate (21 in 1000) to adjust the pH
to 3.0.
Disodium hydrogen phosphate-citric acid buffer solution,
pH 4.5 Dissolve 21 .02 g of citric acid monohydrate in water
to make 1000 mL, and adjust the pH to 4.5 with a solution
prepared by dissolving 35.82 g of disodium hydrogen-
phophate 12-water in water to make 1000 mL.
pH 5.4 Dissolve 1.05 g of citric acid monohydrate and 2.92
g of disodium hydrogen phosphate dodecahydrate in 200 mL
of water, and adjust the pH with phosphoric acid or sodium
hydroxide TS, if necessary.
Disodium hydrogen phosphate-citric acid buffer solution,
0.05 mol/L, pH 6.0 To 1000 mL of 0.05 mol/L disodium
hydrogen phosphate TS add a solution prepared by dissolv-
ing 5.25 g of citric acid monohydrate in water to make 1000
mL to adjust pH 6.0.
Disodium hydrogen phosphate-citric acid buffer solution,
pH 6.0 Dissolve 71.6 g of disodium hydrogen phosphate
dodecahydrate in water to make 1000 mL. To this solution
add a solution, prepared by dissolving 21.0 g of citric acid
monohydrate in water to make 1000 mL, until the pH
becomes 6.0 (ratio of volume: about 63:37).
Disodium hydrogen phosphate-citric acid buffer solution,
pH 7.2 Dissolve 7.1 g of disodium hydrogen phosphate in
water to make 1000 mL. Adjust this solution to pH 7.2 with a
solution prepared by dissolving 5.3 g of citric acid monohy-
drate in water to make 1000 mL.
Disodium hydrogen phosphate-citric acid buffer solution
for penicillium origin /?-galactosidase, pH 4.5 Dissolve 71.6
g of disodium hydrogen phosphate dodecahydrate in water to
make 1000 mL, and adjust the pH to 4.5 with a solution pre-
pared by dissolving 21 .0 g of citric acid monohydrate in water
to make 1000 mL (volume ratio: about 44:56).
Disodium hydrogen phosphate for pH determination
Na 2 HP0 4 [K 9020, for pH determination]
Disodium hydrogen phosphate TS Dissolve 12 g of diso-
dium hydrogen phosphate dodecahydrate in water to make
100 mL (0.3 mol/L).
0.05 mol/L Disodium hydrogen phosphate TS Dissolve
7.0982 g of disodium hydrogen phosphate in water to make
1000 mL.
0.5 mol/L Disodium hydrogen phosphate TS Dissolve
70.982 g of disodium hydrogen phosphate in water to make
1000 mL.
Disodium hydrogen phosphate dodecahydrate
Na 2 HP0 4 .12H 2 [K 9019, Special class]
Disodium l-nitroso-2-naphthol-3,6-disulfonate
C 10 H 5 NNa 2 O 8 S 2 [K 8714, Special class]
Dissolved acetylene C 2 H 2 [K 1902]
Distigmine bromide for assay [Same as the monograph
Distigmine Bromide. It contains not less than 99.0% of dis-
tigmine bromide (C 22 H 32 Br 2 N 4 4 ), calculated on the anhy-
drous basis.]
Distilled water for injection [Same as the monograph
Water for Injection. Prepared by distillation.]
2,6-Di-tert-butylcresol [(CH 3 ) 3 C] 2 C 6 H 2 (CH 3 )OH
A white, crystalline powder. Freely soluble in ethanol (95).
Melting point <2.60>: 69 - 71 °C
Residue on ignition <2.44>: not more than 0.05%.
2,6-Di-tert-butylcresol TS Dissolve 0.1 g of 2,6-di-tert-
butylcresol in ethanol (95) to make 10 mL.
Disodium hydrogen phosphate-citric acid buffer solution,
2,6-Di-tot-butyl-p-cresol See 2,6-di-fert-butylcresol.
JPXV
General Tests / Reagents, Test Solutions
189
2,6-Di-tert-butyl-p-cresol TS See 2,6-di-terf-butylcresol
TS.
1,3-Di (4-pyridyl) propane C 13 H 14 N 2 A pale yellow
powder.
Melting point <2.60>: 61 -62°C
Water <2.48>: less than 0.1%.
l,l'-[3,3'-Dithiobis(2-inethyl-l-oxopropyl)]-L-diproline
C 18 H 28 N 2 6 S 2 White, crystals or crystalline powder. Spar-
ingly soluble in methanol, and practically insoluble in water.
Identification — Determine the infrared absorption spec-
trum of l,l'-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-dipro-
line according to potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 2960 cm -1 , 1750 cm -1 , 1720
cm 1 , 1600 cm- 1 , 1480 cm- 1 , 1450 cm- 1 and 1185 cm- 1 .
Purity Related substances — Dissolve 0.10 g of 1 , 1 '-[3,3'-
dithiobis (2-methyl-l-oxopropyl)]-L-diproline in exactly 10
mL of methanol. Perform the test with this solution as direct-
ed in the Purity (3) under Captopril: any spot other than the
principal spot at the i?f value of about 0.2 does not appear.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.3 g of l,l'-[3,3'-dithiobis (2-methyl-l-oxopropyl)]-L-
diproline, dissolve in 20 mL of methanol, add 50 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS until the color of the solution changes from yellow
through bluish green to blue (indicator: 3 drops of
bromothymol blue TS). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 21.63 mg of C 18 H 28 N 2 6 S 2
Dithiothreitol C 4 H 10 O 2 S 2 Crystals.
Melting point <2.60>: about 42°C
Dithizone C 6 H 5 NHNHCSN:NC 6 H 5 [K 8490, Special c-
lass]
Dithizone solution for extraction Dissolve 30 mg of dithi-
zone in 1000 mL of chloroform, and add 5 mL of ethanol
(95). Store in a cold place. Before use, shake a suitable
volume of the solution with one-half of its volume of diluted
nitric acid (1 in 100), and use the chloroform layer after dis-
carding the water layer.
Dithizone TS Dissolve 25 mg of dithizone in ethanol (95)
to make 100 mL. Prepare before use.
Dopamine hydrochloride for assay C 8 H U N0 2 .HC1
[Same as the monograph Dopamine hydrochloride. When
dried, it contains not less than 99.0% of dopamine
hydrochloride (C 8 H„N0 2 .HC1).
Doxifluridine C 9 H u FN 2 5
monograph]
[Same as the namesake
Doxifluridine for assay C 9 H n FN 2 5 [Same as the
monograph Doxifluridine. When dried, it contains not less
than 99.5% of doxifluridine (C 9 H u FN 2 5 ).]
Dragendorff's TS Dissolve 0.85 g of bismuth subnitrate
in 10 mL of acetic acid (100) and 40 mL of water with
vigorous shaking (solution A). Dissolve 8 g of potassium
iodide in 20 mL of water (solution B). Immediately before
use, mix equal volumes of solution A, solution B and acetic
acid (100). Store solution A and solution B in light-resistant
containers.
Dragendorff's TS for spraying Add 20 mL of diluted
acetic acid (31) (1 in 5) to 4 mL of a mixture of equal volumes
of solution A and solution B of Dragendorff's TS. Prepare
before use.
Dried human normal plasma powder Freeze-dried
normal plasma obtained from healthy human.
Dried sodium carbonate Na 2 C0 3 [Same as the name-
sake monograph]
Dydrogesterone for assay C 2 iH 28 2 [Same as the mono-
graph Dydrogesterone. When dried, it contains not less than
99.0% of C 21 H 28 2 ]
E. coli protein Process E. coli cells (E. coli
N4830/pTB281) retaining a plasmid deficient in the celmoleu-
kin gene according to the celmoleukin purification process in
the following order; (i) extraction, (ii) butylated vinyl poly-
mer hydrophobic chromatography, (iii) carboxymethylated
vinyl polymer ion-exchange column chromatography, and
(iv) sulfopropyl-polymer ion-exchange chromatography, and
during process (iv) collect the fractions corresponding to the
celmoleukin elution position. Dialyze the fractions obtained
in (iv) against 0.01 mol/L acetate buffer solution, pH 5.0,
and take the dialysis solution as E.coli protein.
Description: Clear and colorless solution
Identification: When the absorption spectrum is deter-
mined using UV absorption photometry <2.24>, an absorp-
tion maximum is observed in the region of 278 nm.
Protein content: When determining the protein content us-
ing the Assay (1) Total protein content under Celmoleukin
(Genetical Recombination), the protein content per mL is 0.1
to 0.5 mg.
E. coli protein stock solution A solution obtained by cul-
turing a bacteria that contains a plasmid lacking the teceleu-
kin gene but is otherwise exactly identical to the teceleukin-
producing E. coli strain in every function except teceleukin
production, and then purified using a purification technique
that is more simple than that for teceleukin. Determine the
amount of protein by Brodford method using bovine serum
albumin as the standard substance. Store shielded from light
at -70°C.
ECP See E. coli protein.
Eleutheroside B for liquid chromatography
C 17 H 24 9 .xH 2 A white crystalline powder. Sparingly solu-
ble in methanol, slightly soluble in water, and very slightly
soluble in ethanol (99.5). Melting point: 190 - 194°C.
Identification — Determine the absorption spectrum of a
solution in methanol (1 in 200,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 261 nm and 265 nm.
Purity Related substances — Dissolve 1.0 mg in 10 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add methanol to make exactly
50 mL, and use this solution as the standard solution. Per-
form the test with exactly 10 jXL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Determine each peak area by the automatic integration
method: the total area of the peaks other than the peak of
eleutheroside B is not larger than the peak area of eleuthero-
190
Reagents, Test Solutions / General Tests
JP XV
side B obtained with the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Identification under Eleutherococcus Senticosus Rhi-
zome.
Time span of measurement: About 3 times as long as the
retention time of eleutheroside B beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add methanol to make exactly 20 mL. Confirm
that the peak area of eleutheroside B obtained with 10 /xL of
this solution is equivalent to 3.5 to 6.5% of that with 10 /uL of
the standard solution.
System performance: Proceed as directed in the system
suitability in the Identification under Eleutherococcus Sen-
ticosus Rhizome.
EMB plate medium Melt eosin methylene blue agar medi-
um by heating, and cool to about 50°C. Transfer about 20
mL of this medium to a Petri dish, and solidify horizontally.
Place the dish with the cover slightly opened in the incubator
to evaporate the inner vapor and water on the plate.
Emetine hydrochloride for component determination
C29H40N2O4.2HCl.xH2O A white or light-yellow crystalline
powder. Soluble in water.
Melting point <2.60>: about 250°C [with decomposition,
after drying in a desiccator (reduced pressure below 0.67 kPa,
phosphorus (V) oxide, 50°C) for 5 hours].
Absorbance <2.24> E\f m (283 nm) : 116-127 (10 mg,
diluted methanol (1 in 2), 400 mL) [after drying in a desicca-
tor (reduced pressure below 0.67 kPa, phosphorus (V) oxide,
50°C) for 5 hours.]
Purity Related substances — Dissolve 10 mg of emetine
hydrochloride for component determination in 10 mL of the
mobile phase, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add the mobile phase to
make exactly 100 mL, and use this solution as the standard
solution (1). Perform the test with exactly 10,mL each of the
sample solution and standard solution (1) as directed under
Liquid Chromatography <2.0I> according to the following
operating conditions. Determine the peak areas from both
solutions by the automatic integration method: the total area
of peaks other than emetine from the sample solution is not
larger than the peak of emetine from the standard solution
(1).
Operating conditions
Proceed the operating conditions in the Component deter-
mination under Ipecac except the detection sensitivity and
time span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the sensitivity so
that the peak area of emetine obtained from 10 /xL of the
standard solution (2) can be measured by the automatic in-
tegration method, and the peak height of emetine obtained
from 10 /uL of the standard solution (1) is about 20 % of the
full scale.
Time span of measurement: About 3 times as long as the
retention time of emetine beginning after the soluent peak.
Endo's medium Melt 1000 mL of the ordinary agar medi-
um by heating in a water bath, and adjust the pH to between
7.5 and 7.8. Add 10 g of lactose monohydrate previously dis-
solved in a small quantity of water, mix thoroughly, and add
1 mL of fuchsin-ethanol (95) TS. After cooling to about 50°
C, add dropwise a freshly prepared solution of sodium bis-
ulfite (1 in 10) until a light red color develops owing to reduc-
ing fuchsin, requiring about 10 to 15 mL of a solution of so-
dium sulfite heptahydrate (1 in 10). Dispense the mixture, and
sterilize fractionally on each of three successive days for 15
minutes at 100°C.
Endo's plate medium Melt Endo's medium by heating,
and cool to about 50°C. Transfer about 20 mL of this medi-
um to a Petri dish, and solidify horizontally. Place the dishes
with the cover slightly opened in the incubator to evaporate
the inner vapor and water on the surface of the agar.
Enzyme TS The supernatant liquid is obtained as fol-
lows: To 0.3 g of an enzyme preparation potent in amylolytic
and phosphorolytic activities, obtained from Aspergillus ory-
zae, add 10 mL of water and 0.5 mL of 0.1 mol/L hydro-
chloric acid TS, mix vigorously for a few minutes, and cen-
trifuge. Prepare before use.
Eosin See eosin Y.
Eosin Y C2oH 6 Br 4 Na 2 05 Red, masses or powder.
Identification — To 10 mL of a solution (1 in 1000) add 1
drop of hydrochloric acid: yellow-red precipitates appear.
Eosin methylene blue agar medium Dissolve by boiling
10 g of casein peptone, 2 g of dipotassium hydrogen-
phosphate and 25 to 30 g of agar in about 900 mL of water.
To this mixture add 10 g of lactose monohydrate, 20 mL of a
solution of eosin Y(l in 50), 1 3 mL of a solution of methylene
blue (1 in 200) and warm water to make 1000 mL. Mix thor-
oughly, dispense, sterilize by autoclaving at 121 °C for not
more than 20 minutes, and cool quickly by immersing in cold
water, or sterilize fractionally on each of three successive
days for 30 minutes at 100°C.
Ephedrine hydrochloride C IO H 15 NO.HC1 [Same as the
namesake monograph]
Ephedrine hydrochloride for assay C10H15NO.HCI
[Same as the monograph Ephedrine Hydrochloride meeting
the following additional specifications.]
Purity Related substances — Dissolve 50 mg of ephedrine
hydrochloride for assay in 50 mL of the mobile phase and use
this solution as the sample solution. Pipet 1 mL of this solu-
tion and add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution (1). Perform the test
with exactly 10 iuL of the sample solution and standard solu-
tion (1) as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and measure each peak
area from these solutions by the automatic integration
method: the total peak area other than ephedrine from the
sample solution is not larger than the peak area of ephedrine
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Assay under
Ephedra Herb except detection sensitivity and time span of
measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
JPXV
General Tests / Reagents, Test Solutions
191
sensitivity so that the peak area of ephedrine obtained from
10 /xL of the standard solution (2) can be measured by the au-
tomatic integration method, and the peak height of ephedrine
from 10 [iL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement : About 3 times as long as the
retention time of ephedrine beginning after the solvent peak.
6-Epidoxycycline hydrochloride C 2 2H 2 4N 2 8 .HC1
Yellow to dark yellow, crystals or crystalline powder.
Purity Related substances — Dissolve 20 mg of 6-epiox-
ycycline hydrochloride in 25 mL of 0.01 mol/L hydrochloric
acid TS, and use this solution as the sample solution. Proceed
the test with 20 [iL of the sample solution as directed in the
Purity (2) under Doxycycline Hydrochloride Hydrate, deter-
mine each peak area by the automatic integration method,
and calculate the amounts of them by the area percentage
method: the total area of the peaks other than 6-epidoxycy-
cline is not more than 10%.
4-Epioxytetracycline C22H24N2O9 Green-brown to bro-
wn powder.
Purity Related substances — Dissolve 20 mg of 4-epiox-
ytetracycline in 25 mL of 0.01 mol/L hydrochloric acid TS,
and use this solution as the sample solution. Proceed the test
with 20 fiL of the sample solution as directed in the Purity (2)
under Oxytetracycline Hydrochloride, determine each peak
area by the automatic integration method, and calculate the
amounts of them by the area percentage method: the total
amount of the peaks other than 4-epioxytetracycline is not
more than 10%.
Eriochrome black T C2oH 12 N 3 Na0 7 S [K 8736, Special
class]
Eriochrome black T-sodium chloride indicator Mix 0.1 g
of eriochrome black T and 10 g of sodium chloride, and tri-
turate until the mixture becomes homogeneous.
Eriochrome black T TS Dissolve 0.3 g of eriochrome
black T and 2 g of hydroxylammonium chloride in methanol
to make 50 mL. Use within 1 week. Preserve in light-resistant
containers.
Erythromycin B C 37 H 67 NOi2 White to light yellowish
white powder.
Purity Related substances — Dissolve 10 mg of erythro-
mycin B in 1 mL of methanol, add a mixture of phosphate
buffer solution, pH 7.0 and methanol (15:1) to make 5 mL,
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add a mixture of phosphate buffer solu-
tion, pH 7 . and methanol ( 1 5 : 1 ) to make exactly 20 mL , and
use this solution as the standard solution. Proceed with ex-
actly 100 /xL each of the sample solution and standard solu-
tion as directed in the Purity (3) Related substances under
Erythromycin, and determine each peak area from the solu-
tions by the automatic integration method: the total of areas
of the peaks other than erythromycin B from the sample solu-
tion is not more than the peak area of erythromycin B from
the standard solution.
Erythromycin C C 36 H 65 NOi 3 White to light yellowish
white powder.
Purity Related substances — Dissolve 10 mg of erythro-
mycin C in 1 mL of methanol, add a mixture of phosphate
buffer solution, pH 7.0 and methanol (15:1) to make 5 mL,
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add a mixture of phosphate buffer solu-
tion, pH 7.0 and methanol (15:1) to make exactly 20 mL, and
use this solution as the standard solution. Proceed with ex-
actly 100 /uL each of the sample solution and standard solu-
tion as directed in the Purity (3) Related substances under
Erythromycin, and determine each peak area from the solu-
tions by the automatic integration method: the total of areas
of the peaks other than erythromycin C from the sample so-
lution is not more than the peak area of erythromycin C from
the standard solution.
Essential oil Same as the essential oil under the mono-
graph.
Etacrynic acid for assay [Same as the monograph
Etacrynic acid. When dried, it contains not less than 99.0%
of etacrynic acid (C 13 H 12 Ci20 4 ).]
Ethanol See ethanol (95).
Ethanol, aldehyde-free Transfer 1000 mL of ethanol (95)
to a glass-stoppered bottle, add the solution prepared by dis-
solving 2.5 g of lead (II) acetate trihydrate in 5 mL of water,
and mix thoroughly. In a separate container, dissolve 5 g of
potassium hydroxide in 25 mL of warm ethanol (95), cool,
and add this solution gently, without stirring, to the first solu-
tion. After 1 hour, shake this mixture vigorously, allow to
stand overnight, decant the supernatant liquid, and distil the
ethanol.
Ethanol, dehydrated See ethanol (99.5).
Ethanol, dilute To 1 volume of ethanol (95) add 1 volume
of water. It contains 47.45 to 50.00 vol% of C 2 H 5 OH.
Ethanol, diluted Prepare by diluting ethanol (99.5).
Ethanol for alcohol number determination See Alcohol
Number Determination <1.01>.
Ethanol for disinfection [Same as the namesake mono-
graph]
Ethanol for gas chromatography Use ethanol prepared
by distilling ethanol (99.5) with iron (II) sulfate heptahy-
drate. Preserve in containers, in which the air has been dis-
placed with nitrogen, in a dark, cold place.
Ethanol-free chloroform See chloroform, ethanol-free.
Ethanol-isotonic sodium chloride solution To 1 volume
of ethanol (95) add 19 volumes of isotonic sodium chloride
solution.
Ethanol, methanol-free See ethanol (95), methanol- free.
Ethanol, neutralized To a suitable quantity of ethanol
(95) add 2 to 3 drops of phenolphthalein TS, then add 0.01
mol/L or 0.1 mol/L sodium hydroxide VS until a light red
color develops. Prepare before use.
Ethanol (95) C 2 H 5 OH [K 8102, Special class]
Ethanol (95), methanol-free Perform the test for metha-
nol, by using this methanol-free ethanol (95) in place of the
standard solution, as directed in Methanol Test <I.12>: it is
practically colorless.
Ethanol (99.5) C 2 H 5 OH [K 8101, Special class]
Ethenzamide C 9 H H N0 2 [Same as the namesake mono-
192
Reagents, Test Solutions / General Tests
JP XV
graph].
Ether See diethyl ether.
Ether, anesthetic C2H5OC2H5 [Same as the namesake
monograph]
Ether, dehydrated See diethyl ether, dehydrated.
Ether for purity of crude drug See diethyl ether for purity
of crude drug.
Ethinylestradiol
monograph]
C20H24O2 [Same as the namesake
3-Ethoxy-4-hydroxybenzaldehyde C9FL0O3 White to
pale yellowish white crystalline. Freely soluble in ethanol
(95), and slightly soluble in water.
Melting point <2.60>: 76 - 78°C
Content: not less than 98.0%. Assay — Weigh accurately
about 0.3 g of 3-ethoxy-4-hydroxybenzaldehyde, previously
dried in a desiccator (phosphorous (V) oxide) for 4 hours,
dissolve in 50 mL of ./V,7V-dimethylacetamide, and titrate
<2.50> with 0.1 mol/L sodium methoxide VS (indicator:
thymol blue TS).
Each mL of 0.1 mol/L sodium methoxide VS
= 16.62 mg of C 9 H 10 O 3
p-EthoxyphenoI See 4-ethoxyphenol.
4-EthoxyphenoI C 8 H 10 O2 White to light yellow-brown
crystals or crystalline powder. Freely soluble in ethanol (95),
and very slightly soluble in water.
Melting point <2.60>: 62 - 68°C
Purity — Dissolve 0.5 g of 4-Ethoxyphenol in 5 mL of etha-
nol (95), and use this solution as the sample solution. Per-
form the test as directed under Gas Chromatography <2.02>
according to the following conditions. Measure each peak
area by the automatic integration method and calculate the
amount of substance other than 4-ethoxyphenol by the area
percentage method: it is not more than 2.0%.
Operating conditions
Detector: Thermal conductivity detector.
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with 180- to 250-//m siliceous
earth for gas chromatography coated with methylsilicone
porymer for gas chromatography.
Column temperature: A constant temperature of about 150
°C.
Carrier gas: Herium
Flow rate: Adjust the flow rate so that the retention time of
4-ethoxyphenol is about 5 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of 4-ethoxyphenol obtained from 1 /uL
of the sample solution is not less than 50% of the full scale.
Time span of measurement: 3 times as long as the retention
time of 4-ethoxyphenol beginning after the solvent peak.
Ethyl acetate CH 3 COOC 2 H 5 [K 8361, Special class]
Ethyl aminobenzoate C 9 H u N0 2 [Same as the name-
sake monograph]
Ethylbenzene C 6 H 5 C2H 5 A colorless liquid. Freely solu-
ble in acetone and in ethanol (99.5), and practically insoluble
in water.
Specific gravity <2.56> df: 0.862-0.872
Boiling point <2.57>: about 135°C
Ethyl benzoate QH5COOC2H5 Clear, colorless liquid.
Refractive index <2.45> n™: 1.502 - 1.507
Specific gravity <2.56> df : 1.045 - 1.053
Ethyl carbamate H 2 NCOOC 2 H 5 White crystals or pow-
der.
Melting point <2.60>: 48 - 50°C
Purity Clarity of solution: Dissolve 5 g in 20 mL of
water: the solution is clear.
Ethyl cyanoacetate NCCH 2 COOC 2 H 5 Colorless or
light yellow, clear liquid, having an aromatic odor. Specific
gravity df : about 1.08
Identification — To 0.5 mL of a solution in ethanol (99.5) (1
in 10,000) add a mixture of 1 mL of a solution of quinhy-
drone in diluted ethanol (99.5) (1 in 2) (1 in 20,000) and 1
drop of ammonia solution (28): a light blue color develops.
Ethylenediamine C 2 H 8 N 2 [Same as the namesake mono-
graph]
Ethylenediamine TS Dissolve 70 g of ethylenediamine in
30 g of water.
Ethylene glycol
class]
HOCH 2 CH 2 OH
[K 8105, Special
Ethylene glycol for Karl Fischer method Distil ethylene
glycol, and collect the fraction distilling between 195°C and
198°C. The water content is not more than 1.0 mg per mL.
Ethyl iodide See iodoethane.
./V-Ethylmaleimide C 6 H 7 N02 White crystals, having a
pungent, characteristic odor. Freely soluble in ethanol (95),
and slightly soluble in water.
Melting point <2.60>: 43 - 46 °C
Purity Clarity and color of solution — Dissolve 1 g of N-
ethylmaleimide in 20 mL of ethanol (95): the solution is clear
and colorless.
Content: not less than 99.0%. Assay — Dissolve about 0.1
g of Af-ethylmaleimide, accurately weighed, in 20 mL of
ethanol (95), add exactly 20 mL of 0.1 mol/L sodium
hydroxide VS, and titrate <2.50> with 0.1 mol/L hydrochloric
acid VS (indicator: 2 drops of phenolphthalein TS). Perform
a blank determination.
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.51 mg of C 6 H 7 N0 2
Ethyl H-caprylate C 10 H 20 O2 Clear and colorless to
almost colorless liquid.
Specific gravity <2.56> d™: 0.864 - 0.871
Purity Related substances — Dissolve 0.1 g of ethyl n-
caprylate in 10 mL of dioxane and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
dichloromethane to make exactly 100 mL, and use this solu-
tion as the standard solution (1). Perform the test with exact-
ly 5 fiL each of the sample solution and standard solution (1)
as directed under Gas Chromatography <2.02> according to
the following conditions, and measure each peak area from
these solutions by the automatic integration method: the total
peak areas other than ethyl w-caprylate from the sample solu-
tion is not larger than the peak area of ethyl w-caprylate from
the standard solution (1).
Operating conditions
JPXV
General Tests / Reagents, Test Solutions
193
Proceed the operating conditions in the Assay under Men-
tha Oil except detection sensitivity and time span of measure-
ment.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add dichloromethane to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of ethyl w-caprylate obtained
from 5 //L of the standard solution (2) can be measured by
the automatic integration method, and the peak height of
ethyl M-caprylate from 5 /uL of the standard solution (1) is
about 20% of the full scale.
Time span of measurement: 3 times as long as the retention
time of ethyl M-caprylate beginning after the solvent peak.
Ethyl parahydroxybenzoate
as the namesake monograph]
HOC 6 H 4 COOC 2 H 5 [Same
Ethyl propionate CH 3 CH 2 COOC 2 H 5 Colorless, clear
liquid.
Specific gravity <2.56> df: 0.890 - 0.892
2-Ethyl-2-phenylmalondiamide CnH 14 02N 2 White,
odorless crystals. Soluble in ethanol (95), and very slightly
soluble in water. Melting point: about 120°C (with decompo-
sition).
Purity Related substances — To 5.0 mg of 2-ethyl-2-
phenylmalondiamide add 4 mL of pyridine and 1 mL of bis-
trimethylsilylacetamide, shake thoroughly, and heat at 100°C
for 5 minutes. After cooling, add pyridine to make exactly 10
mL, and use this solution as the sample solution. Perform the
test with 2 /uL of the sample solution as directed under Gas
Chromatography <2.02> according to the conditions in the
Purity (3) under Primidone: any peak other than the peaks of
2-ethyl-2-phenylmalondiamide and the solvent does not ap-
pear. Adjust the detection sensitivity so that the peak height
of 2-ethyl-2-phenylmalondiamide obtained from 2 /xL of the
sample solution is about 80% of the full scale, and the time
span of measurement is about twice as long as the retention
time of 2-ethyl-2-phenylmalondiamide beginning after the
solvent peak.
Etidronate disodium for assay C 2 H 6 Na 2 7 P 2 [Same as
the monograph Etidronate Disodium. When dried, it con-
tains not less than 99.0% of C 2 H 6 Na 2 7 P 2 ]
Etilefrine hydrochloride Ci H 15 NO 2 .HCl [Same as the
namesake monograph]
Etilefrine hydrochloride for assay Ci Hi 5 NO 2 .HCl [Same
as the monograph Etilefrine Hydrochloride. When dried, it
contains not less than 99.0% of etilefrine hydrochloride (C 10
H 15 N0 2 .HC1).]
Factor Xa It is prepared from lyophilization of Factor
Xa which has been prepared from bovine plasma. White or
pale yellow masses or powder.
Clarity and color of solution: Dissolve 7 lnto s _ 2222 of it in
10 mL water; the solution is clear and colorless or pale yel-
low.
Content: not less than 75% and not more than 125% of the
label.
Factor Xa TS Dissolve 7 \nkat s _ 2 222 of factor Xa in 10 mL
of water.
Famotidine for assay C 8 H 15 N 7 2 S3 [Same as the mono-
graph Famotidine. When dried, it contains not less than 99.0
% of famotidine (C 8 H 15 N 7 2 S3), and when proceed as direct-
ed in the Purity (3), the total related substance is not more
than 0.4%.]
Fatty oil Same as the fatty oil under the monograph.
Fehling's TS The copper solution — Dissolve 34.66 g of
copper (II) sulfate pentahydrate in water to make 500 mL.
Keep this solution in a glass-stoppered bottles in well-filled.
The alkaline tartrate solution — Dissolve 173 g of potassi-
um sodium tartrate tetrahydrate and 50 g of sodium hydrox-
ide in water to make 500 mL. Preserve this solution in a poly-
ethylene container.
Before use, mix equal volumes of both solutions.
Fehling's TS for amylolytic activity test
The copper solution — Dissolve 34.660 g of copper (II) sul-
fate pentahydrate, accurately weighed, in water to make ex-
actly 500 mL. Preserve this solution in well-filled, glass-stop-
pered bottles.
The alkaline tartrate solution — Dissolve 173 g of potassi-
um sodium tartrate tetrahydrate and 50 g of sodium
hydroxide in water to make exactly 500 mL. Preserve this
solution in polyethylene containers.
Before use, mix exactly equal volumes of both solutions.
Ferric ammonium citrate
citrate.
See ammonium iron (III)
Ferric ammonium sulfate See ammonium iron (III) sul-
fate dodecahydrate.
Ferric ammonium sulfate TS See ammonium iron (III)
sulfate TS.
Ferric ammonium sulfate TS, dilute See ammonium iron
(III) sulfate TS, dilute.
Ferric chloride See iron (III) chloride hexahydrate.
Ferric chloride-acetic acid TS See iron (III) chloride-
acetic acid TS.
Ferric chloride-iodine TS See iron (III) chloride-iodine
TS.
Ferric chloride-methanol TS See iron (III) chloride-
methanol TS.
Ferric chloride-pyridine TS, anhydrous See iron (III)
chloride-pyridin TS, anhydrous.
Ferric chloride TS See iron (III) chloride TS.
Ferric chloride TS, acidic See iron (III) chloride TS,
acidic.
Ferric chloride TS, dilute See iron (III) chloride TS,
dilute.
Ferric nitrate See iron (III) nitrate enneahydrate.
Ferric nitrate TS See iron (III) nitrate TS.
Ferric perchlorate See iron (III) perchlorate hexahydrate.
Ferric perchlorate-dehydrated ethanol TS See iron (III)
perchlorate-ethanol TS.
Ferric salicylate TS See Iron salicylate TS
Ferric sulfate See iron (III) sulfate w-hydrate.
Ferric sulfate TS See iron (III) sulfate TS.
194
Reagents, Test Solutions / General Tests
JP XV
Ferrous ammonium sulfate See ammonium iron (II) sul-
fate hexahydrate.
Ferrous sulfate See iron (II) sulfate heptahydrate.
Ferrous sulfate TS See iron (II) sulfate TS.
Ferrous sulfide See iron (II) sulfide.
Ferrous tartrate TS See iron (II) tartrate TS.
Ferrous thiocyanate TS See iron (II) thiocyanate TS.
Ferrous trisodium pentacyanoamine TS See iron (II)
trisodium pentacyanoamine TS.
(£)-Ferulic acid Ci H 10 O 4 White to light yellow, crys-
tals or crystalline powder. Freely soluble in methanol, soluble
in ethanol (99.5), and practically insoluble in water. Melting
point: 173 - 176°C.
Identification — Determine the absorption spectrum of a
solution in methanol (1 in 200,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 215 nm and 219 nm, between 231 nm and 235 nm,
and between 318 nm and 322 nm.
Purity Related substances — Dissolve 1 mg in 1 mL of
methanol. Proceed the test with 2//L of this solution as
directed in the Identification (11) under Hochuekkito Ex-
tract: no spot other than the principle spot of around Rf 0.6
appears.
Fetal calf serum Serum obtained from fetal calves. Inter-
leukin-2 dependent cell growth suppression substance is re-
moved by heat at 56 °C for 30 min before use.
Fibrinogen Fibrinogen is prepared from human or bo-
vine blood by fractional precipitation with ethanol or ammo-
nium sulfate. It may contain citrate, oxalate and sodium
chloride. A white, amorphous solid. Add 1 mL of isotonic
sodium chloride solution to 0.01 g of fibrinogen. It, when
warmed to 37°C, dissolves with a slight turbidity, and clots
on the subsequent addition of 1 unit of thrombin.
1st Fluid for disintegration test See 1st fluid for dissolu-
tion test.
1st Fluid for dissolution test Dissolve 2.0 g of sodium
chloride in 7.0 mL of hydrochloric acid and water to make
1000 mL. It is clear and colorless, and has a pH of about 1.2.
Fixed oil Same as the vegetale oils under the monograph.
Flopropione [Same as the namesake monograph]
Flopropione for assay [Same as the monograph
Flopropione. It contains not less than 99.0% of flopropione
(C 9 H 10 O 4 : 182.17), calculated on the dehydrated basis.]
Fluid thioglycolate medium See the Sterility Test <4.06>.
Fluocinolone acetonide C 24 H 3 oF 2 6 [Same as the
namesake monograph]
Fluorescein C 2 oH I2 5 An yellowish red powder.
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 1597 cm -1 , 1466 cm" 1 , 1389
cm" 1 , 1317 cm" 1 , 1264 cm" 1 , 1247 cm" 1 , 1213 cm" 1 , 1114
cm" 1 and 849 cm" 1 .
Fluorescein sodium C 2 oH 10 Na20 5 [Same as the name-
sake monograph].
Fluorescein sodium TS Dissolve 0.2 g of fluorescein
sodium in water to make 100 mL.
4-Fluorobenzoic acid C 7 H 5 F0 2 White, crystals or crys-
talline powder.
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 1684 cm" 1 , 1606 cm" 1 and 1231
Melting point <2.60>: 182 - 188°C
l-Fluoro-2,4-dinitrobenzene
Special class]
C 6 H 3 (N0 2 ) 2 F [K 8479,
Flurazepam for assay C 21 H 23 OFN 3 [Same as the
monograph Flurazepam. When drid, it contains not less than
99.0%of flurazepan (C 21 H 23 CIFN 3 0).]
Folic acid C 19 H I9 N 7 6 [Same as the namesake mono-
graph]
Folin's TS Place 20 g of sodium tungstate (VI) dihydrate,
5 g of sodium molybdate dihydrate and about 140 mL of
water in a 300-mL volumetric flask, add 10 mL of diluted
phosphoric acid (17 in 20) and 20 mL of hydrochloric acid,
and boil gently using a reflux condenser with ground-glass
joints for 10 hours. To the mixture add 30 g of lithium sulfate
monohydrate and 10 mL of water, and then add a very small
quantity of bromine to change the deep green color of the so-
lution to yellow. Remove the excess bromine by boiling for 15
minutes without a condenser, and cool. Add water to make
200 mL, and filter through a glass filter. Store it free from
dust. Use this solution as the stock solution, and dilute with
water to the directed concentration before use.
Folin's TS, dilute Titrate <2.50> Folin's TS with 0.1
mol/L sodium hydroxide VS (indicator: phenolphthalein
TS), and determine the acid concentration. Prepare by add-
ing water to Folin's TS so the acid concentration is 1 mol/L.
Formaldehyde solution HCHO [K 8872, Special
class]
Formaldehyde solution-sulfuric acid TS Add 1 drop of
formaldehyde solution to 1 mL of sulfuric acid. Prepare
before use.
Formaldehyde solution TS To 0.5 mL of formaldehyde
solution add water to make 100 mL.
Formaldehyde TS, dilute See Test Methods for Plastic
Containers <7.02>.
Formalin See formaldehyde solution.
Formalin TS See formaldehyde solution TS.
Formalin-sulfuric acid TS See formaldehyde solution-
sulfuric acid TS.
Formamide HCONH 2 [K 8873, Special class]
Formamide for Karl Fischer method HCONH 2
[K 8873, Special class; water content per g of formamide for
Karl Fischer method should be not more than 1 mg.]
Formic acid HCOOH [K 8264, Special class, specific
gravity: not less than 1.21].
JPXV
General Tests / Reagents, Test Solutions
195
2-FormyIbenzoic acid CHOC 6 H 4 COOH White crys-
tals. Melting point: 97 - 99°C
Content: not less than 99.0%. Assay — Weigh accurately
about 0.3 g of 2-formylbenzoic acid, previously dried (in
vacuum, phosphorus (V) oxide, 3 hours), dissolve in 50 mL
of freshly boiled and cooled water, and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (indicator: 3 drops of phenol
red TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 15.01 mg of C 8 H 6 3
Freund's complete adjuvant A suspension of 5 mg of
mycobacteria of Corynebacterium butyricum, killed by heat-
ing, in 10 mL of a mixture of mineral oil and aricel A (17:3).
Fructose C 6 H I2 6 [Same as the namesake mono-
graph]
Fuchsin A lustrous, green, crystalline powder or mass,
slightly soluble in water and in ethanol (95).
Loss on drying <2.41>: 17.5 - 20.0% (1 g, 105°C, 4 hours)
Residue on ignition <2.44>: not more than 0.1% (1 g).
Fuchsin -ethanol TS Dissolve 1 1 g of fuchsin in 100 mL of
ethanol (95).
Fuchsin-sulfurous acid TS Dissolve 0.2 g of fuchsin in
120 mL of hot water, and allow the solution to cool. Add a
solution prepared by dissolving 2 g of anhydrous sodium sul-
fite in 20 mL of water, then add 2 mL of hydrochloric acid
and water to make 200 mL, and allow to stand for at least 1
hour. Prepare before use.
Fumaric acid for thin-layer chromatography C 4 H 4 4
White, crystalline powder, odorless, and has a characteristic
acid taste.
Purity — Perform the test as directed in the Identification
(5) under Clemastine Fumarate: any spot other than the prin-
cipal spot at the Rf value of about 0.8 does not appear.
Fuming nitric acid See nitric acid, fuming.
Fuming sulfuric acid See sulfuric acid, fuming.
Furfural C 5 H 4 2 A clear, colorless liquid.
Specific gravity <2.56> df : 1.160 - 1.165
Distilling range <2.57>: 160 - 163 °C, not less than 95 vol%.
Galactose See D-galactose.
D-Galactose C 6 H I2 6 White crystals, granules or pow-
der.
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3390 cm -1 , 3210 cm -1 , 3140
cm -1 , 1151 cm -1 , 1068 cm -1 , 956 cm -1 , 836 cm -1 , 765 cm -1
and 660 cm -1 .
Optical rotation <2.49> [a]g>: +79 - +82° (desiccator (sili-
ca gel), 2.5 g after drying for 18 hours, diluted ammonia so-
lution (28) (1 in 300), 25 mL, 100 mm).
Gallic acid See gallic acid monohydrate.
Gallic acid monohydrate C 6 H 2 (OH) 3 COOH.H 2
White to pale yellowish white, crystals or powder.
Melting point <2.60>: about 260°C (with decomposition).
Gelatin [Same as the namesake monograph]
Gelatin, acid-treated [Same as the monograph Gelatin.
Its isoelectric point is at pH between 7.0 and 9.0]
Gelatin peptone See peptone, gelatin.
Gelatin-phosphate buffer solution Dissolve 13.6 g of
potassium dihydrogen phosphate, 15.6 g of sodium dihydro-
gen phosphate dihydrate and 1.0 g of sodium azide in water
to make 1000 mL, adjust the pH to 3.0 with diluted phos-
phoric acid (1 in 75) (solution A). Dissolve 5.0 g of acid-treat-
ed gelatin in 400 mL of the solution A by warming, after
cooling, adjust the pH to 3.0 with diluted phosphoric acid (1
in 75), and add the solution-A to make 1000 mL.
Gelatin-phosphate buffer solution, pH 7.0 Dissolve 1.15
g of sodium dihydrogen phosphate dihydrate, 5.96 g of diso-
dium hydrogen phosphate dodecahydrate and 5.4 g of sodi-
um chloride in 500 mL of water. Dissolve 1.2 g of gelatin to
this solution by heating, and after cooling add water to make
600 mL.
Gelatin-phosphate buffer solution, pH 7.4 To 50 mL of
0.2 mol/L potassium dihydrogen phosphate TS for buffer so-
lution add 39.50 mL of 0.2 mol/L sodium hydroxide VS and
50 mL of water. Dissolve 0.2 g of gelatin to this solution by
heating, then after cooling adjust to pH 7.4 with 0.2 mol/L
sodium hydroxide TS, and add water to make 200 mL.
Gelatin-tris buffer solution Dissolve 6.06 g of 2-amino-2-
hydroxymethyl-l,3-propanediol and 2.22 g of sodium chlo-
ride in 700 mL of water. Separately, dissolve 10 g of acid-
treated gelatin in 200 mL of water by warming. After cool-
ing, mix these solutions, and adjust the pH to 8.8 with dilute
hydrochloric acid, and add water to make 1000 mL.
Gelatin-tris buffer solution, pH 8.0 Dissolve 40 g of 2-
amino-2-hydroxymethyl-l,3-propanediol and 5.4 g of sodi-
um chloride in 500 mL of water. Add 1.2 g of gelatin to dis-
solve by heating, adjust to pH 8.0 with dilute hydrochloric
acid after cooling, and add water to make 600 mL.
Gelatin TS Dissolve 1 g of gelatin in 50 mL of water by
gentle heating, and filter if necessary. Prepare before use.
Geniposide for component determination Use geniposide
for thin-layer chromatography meeting the following addi-
tional specifications.
Absorbance <2.24> E\°^ (240 nm): 249 - 269 [10 mg dried
in a desiccator (reduced pressure of not exceeding 0.67 kPa,
phosphorus (V) oxide) for 24 hours, diluted methanol (1 in
2), 500 mL].
Purity Related substances — Dissolve 5 mg of geniposide
for component determination in 50 mL of diluted methanol
(1 in 2), and use this solution as the sample solution. Pipet 1
mL of the sample solution, add diluted methanol (1 in 2) to
make exactly 100 mL, and use this solution as the standard
solution (1). Perform the test with exactly 10 /xL each of the
sample solution and standard solution (1) as directed under
Liquid Chromatography <2.01> according to the following
conditions, and measure each peak area of the both solutions
by the automatic integration method: the total area of the
peaks other than geniposide from the sample solution is not
larger than the peak area of geniposide from the standard so-
lution (1).
Operating conditions
Proceed as directed in the Component determination under
196
Reagents, Test Solutions / General Tests
JP XV
Gardenia Fruit except detection sensitivity and time span of
measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add diluted methanol (1 in 2) to make exactly 20 mL, and
use this solution as the standard solution (2). Adjust the de-
tection sensitivity so that the peak area of geniposide ob-
tained from 10 /uL of the standard solution (2) can be meas-
ured by the automatic integration method and the peak
height of geniposide obtained from 10 /xL of the standard so-
lution (1) shows about 20% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of geniposide beginning after the solvent peak.
Geniposide for thin-layer chromatography C17H24O10
White crystals or crystalline powder. Melting point: 159 - 163
°C.
Purity Related substances — Dissolve 1.0 mg of genipo-
side for thin-layer chromatography in exactly 1 mL of
methanol, and perform the test with 20 /xL of this solution as
directed in the Identification (2) under Gardenia Fruit: any
spot other than the principal spot at the Rf value of about 0.3
does not appear.
Gentamicin B Ci9H 38 N 4 Oio White to pale yellowish
white powder. Very soluble in water, and practically insolu-
ble in ethanol (95).
Content: not less than 80.0% . Assay — Dissolve a suitable
amount of gentamicin B in 0.05 mol/L sulfuric acid TS to
make the solution containing 0.1 mg of gentamicin B per mL,
and use this solution as the sample solution. Perform the test
with 5 /xL of the sample solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and measure each peak area by the automatic integra-
tion method. Calculate the amount of gentamicin B by the
area percentage method.
Operating conditions
Apparatus, detector, column, column temperature, reac-
tion coil, mobile phase, reagent, reaction temperature, flow
rate of the mobile phase, and flow rate of the reagent: Pro-
ceed the operating conditions in the Assay under Isepamicin
Sulfate.
Time span of measurement: About 3 times as long as the
retention time of gentamicin B.
System suitability
Proceed the system suitability in the Assay under Isepami-
cin Sulfate.
Gentiopicroside for thin-layer chromatography
C16H20O9 A white powder. Freely soluble in water and in
methanol, and practically insoluble in diethyl ether. Melting
point: about 110°C (with decomposition).
Purity Related substances — Dissolve 10 mg of gentiopicro-
side for thin-layer chromatography in 1 mL of methanol, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, and methanol to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with 10 /xL each of the sample solution and standard solution
as directed in the Identification (2) under Gentian: the spots
other than the principal spot at the Rf value of about 0.4
from the sample solution are not more intense than the spot
from the standard solution.
Giemsa's TS Dissolve 3 g of azure Il-eosin Y and 0.8 g of
azure II in 250 g of glycerin by warming to 60°C. After cool-
ing, add 250 g of methanol, and mix well. Allow to stand for
24 hours, and filter. Store in tightly stoppered bottles.
Azure Il-eosin Y is prepared by coupling eosin Y to azure
II. Azure II is the mixture of equal quantities of methylene a-
zure (azure I), prepared by oxidizing methylene blue, and
methylene blue.
[6]-Gingerol for thin-layer chromatography Ci 7 H 2 60 4
A yellow-white to yellow, liquid or solid. Freely soluble in
methanol, in ethanol (99.5) and in diethyl ether, and practi-
cally insoluble in water.
Purity Related substances — Dissolve 1.0 mg of [6]-gin-
gerol for thin-layer chromatography in exactly 2 mL of
methanol. Perform the test with 10 fiL of this solution as
directed in the Identification under Ginger: any spot other
than the principal spot at the Rf value of about 0.3 does not
appear.
Ginsenoside Re C 5 3H9 O22.xH 2 O A white crystalline
powder. It is odorless.
Purity — Dissolve 1 mg in diluted methanol (3 in 5) to make
10 mL. Perform the test with 10 /xL of this solution as direct-
ed under Liquid Chromatography <2.01> according to the
conditions directed in the Assay under Ginseng: the total area
of the peak other than ginsenoside Re and solvent peak is not
more than 1/10 times the total peak area excluding the peak
area of the solvent.
Ginsenoside Re C 48 H 8 20i8.xH 2 A white crystalline
powder. It is odorless.
Purity — Dissolve 1 mg in diluted methanol (3 in 5) to make
10 mL. Perform the test with 10 /xL of this solution as direct-
ed under Liquid Chromatography <2.01> according to the
conditions directed in the Assay under Ginseng: the total area
of the peak other than ginsenoside Re and solvent peak is not
more than 1/10 times the total peak area excluding the peak
area of the solvent.
Ginsenoside Rgi for thin-layer chromatography
C42H72CV White, crystalline powder, having a slight, bitter
taste. Freely soluble in methanol and in ethanol (95), and
practically insoluble in diethyl ether and in chloroform.
Melting point <2.60>: 194 - 196. 5°C
Purity Related substances — Dissolve 1 mg of ginsenoside
Rgi for thin layer chromatography in 1 mL of methanol, and
perform the test with 20 /xL of this solution as directed in the
Identification (2) under Ginseng: any spot other than the
principal spot at the Rf value of about 0.4 does not appear.
Glacial acetic acid See acetic acid (100).
Glacial acetic acid for nonaqueous titration See acetic
acid for nonaqueous titration.
Glacial acetic acid-sulfuric acid TS See acetic acid (100)-
sulfuric acid TS.
y-Globulin A plasma protein obtained from human se-
rum as Cohn's II and III fractions. White crystalline powder.
It contains not less than 98% of y-globulin in the total pro-
tein.
Glucose C 6 H 12 6 [Same as the namesake monograph]
Glucose detection TS Dissolve 1600 units of glucose oxi-
dase, 16 mg of 4-aminoantipyrine, 145 units of peroxidase
and 0.27 g of p-hydroxybenzoic acid in tris buffer solution,
pH 7.0, to make 200 mL.
Glucose detection TS for penicillium origin /?-galactosidase
JPXV
General Tests / Reagents, Test Solutions
197
Dissolve glucose oxidase (not less than 500 units), peroxidase
(not less than 50 units), 0.01 g of 4-aminoantipyrine and 0.1 g
of phenol in phosphate buffer, pH 7.2 to make 100 mL.
Glucose oxidase Obtained from Aspergillus nigar.
White powder. It is freely soluble in water. It contains about
200 Units per mg. One unit indicates an amount of the en-
zyme which produces 1 ^mol of D-glucono-<5-lactone in 1
minute at 25 °C and pH 7.0 from glucose used as the sub-
strate.
Glucose-pepton medium for sterility test See soybean-
casein digest medium
Glucose TS Dissolve 30 g of glucose in water to make 100
mL. Prepare as directed under Injections.
L-Glutamic acid HOOC(CH 2 ) 2 CH(NH 2 )COOH
[K 9047, Special class]
L-Glutamine H 2 NCO(CH 2 ) 2 CH(NH 2 )COOH
[K 9103, Special class]
Glutamine TS See Test Methods for Plastic Containers
<7.02>.
7-(Glutarylglycyl-L-arginylamino)-4-methylcoumarin
C 23 H 30 N 6 O 7 White powder. It is freely soluble in acetic acid
(100), sparingly soluble in dimethylsulf oxide, and practically
insoluble in water.
Absorbance <2.24> E\ * (325 nm): 310 - 350 [2 mg, dilut-
ed acetic acid (100) (1 in 500), 200 mL].
Optical rotation <2.49> [a]™: -50 - -60° [0.1 g, diluted
acetic acid (100) (1 in 2), 10 mL, 100 mm].
Purity Related substances — Prepare the sample solution
by dissolving 5 mg of 7-(glutarylglycyl-L-arginylamino)-4-
methylcoumarin in 0.5 mL of acetic acid (100), and perform
the test as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL of the sample solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of 1-butanol, water, pyridine and acetic acid (100)
(15:12:10:3) to a distance of about 10 cm, air-dry the plate,
and dry more at 80°C for 30 minutes. After cooling, allow
the plate to stand for 30 minutes in a box filled with iodine
vapors: any observable spot other than the principal spot at
the Rf value of about 0.6 does not appear.
7-(Glutarylglycyl-L-arginylamino)-4-methylcoumarin TS
Dissolve 5 mg of 7-(glutarylglycyl-L-arginylamino)-4-methyl-
coumarin in 0.5 to 1 mL of acetic acid (100), lyophilize, dis-
solve this in 1 mL of dimethylsulfoxide, and use this solution
as solution A. Dissolve 30.0 g of 2-amino-2-hydroxymethyl-
1,3-propanediol and 14.6 g of sodium chloride in 400 mL of
water, adjust the pH to 8.5 with dilute hydrochloric acid, add
water to make 500 mL, and use this solution as solution B.
Mix 1 mL of the solution A and 500 mL of the solution B be-
fore use.
Glycerin C 3 H 8 3 [K 8295, Glycerol, Special class.
Same as the monograph Concentrated Glycerin]
85% Glycerin C 3 H 8 3 [Same as the monograph
Glycerin]
Glycine C 2 H 6 N0 2 [K8291, Special class]
Glycolic acid C 2 H 4 3 Purity: not less than 98.0%.
Glycyrrhizinic acid for thin-layer chromatography
C 42 H 62 16 .xH 2 Colorless or white, sweet, crystalline pow-
der. Freely soluble in hot water and in ethanol (95), and prac-
tically insoluble in diethyl ether. Melting point: 213 - 218°C
(with decomposition).
Purity Related substances — Dissolve 10 mg of glycyr-
rhizinic acid for thin-layer chromatography in 5 mL of dilute
ethanol, and use this solution as the sample solution. Pipet 1
mL of the sample solution, add dilute ethanol to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with 10 /xh each of the sample solution and
standard solution as directed in the Identification under Gly-
cyrrhiza: the spots other than the principal spot at the Rf
value of about 0.3 from the sample solution are not more in-
tense than the spot from the standard solution.
Goat anti-ECP antibody Combine 1 volume of ECP
standard substance (equivalent to about 1 mg of protein) and
1 volume of Freund's complete adjuvant, and immunize
goats subcutaneously in the back region with this solution 5
times at 2 week intervals. Harvest blood on the 10 th day after
completing the immunization to obtain goat antiserum. Goat
anti-ECP antibody is obtained by preparing an immobilized
ECP column in which ECP standard substance is bound to
sepharose 4B and then purifying by affinity column chro-
matography.
Description: Clear and colorless solution.
Identification: When sodium lauryl sulfate-supplemented
polyacrylamide gel electrophoresis is conducted under non-
reducing conditions, the molecular weight of the major band
is within the range of 1.30X 10 5 to 1.70X10 5 .
Protein content: When determining the protein content us-
ing Assay (1) under Celmoleukin (Genetical Recombination),
the protein content per mL is 0.2 to 1.0 mg.
Goat anti-ECP antibody TS Dilute goat anti-ECP an-
tibody with 0.1 mol/L carbonate buffer solution, pH 9.6 to
prepare a solution containing 50 /ug protein per mL.
Griess-Romijin's nitric acid reagent Triturate thoroughly
1 g of 1-naphthylamine, 10 g of sulfanilic acid and 1.5 g of
zinc dust in a mortar.
Storage — Preserve in tight, light-resistant containers.
Griess-Romijin's nitrous acid reagent Triturate thor-
oughly 1 g of 1-naphthylamine, 10 g of sulfanilic acid and 89
g of tartaric acid in a mortar.
Storage — Preserve in tight, light-resistant containers.
Guaiacol CH 3 OC 6 H 4 OH Clear, colorless to yellow liq-
uid or colorless crystals, having a characteristic aroma. Spar-
ingly soluble in water, and miscible with ethanol (95), with
diethyl ether and with chloroform. Melting point: about
28°C
Purity — Perform the test with 0.5 [iL of guaiacol as direct-
ed under Gas Chromatography <2.02> according to the fol-
lowing conditions. Measure each peak area by the automatic
integration method, and calculate the amount of guaiacol by
the area percentage method:It showed the purity of not less
than 99.0%.
Operating conditions
Detector: Hydrogen flame-ionization detector
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with siliceous earth for gas
chromatography, 150- to 180-^<m in particle diameter, coated
with polyethylene glycol 20 M at the ratio of 20%.
198
Reagents, Test Solutions / General Tests
JP XV
Column temperature: A constant temperature of about 200
°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
guaiacol is 4 to 6 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of guaiacol obtained from 0.5 /uh of
guaiacol is about 90% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of guaiacol beginning after the solvent peak.
Guaifenesin Ci H 14 O 4 [Same as the namesake mono-
graph]
Haloperidol for assay C21H23CIFNO2 [Same as the
monograph Haloperidol.]
Hanus' TS Dissolve 20 g of iodine monobromide in 1000
mL of acetic acid (100). Preserve in light-resistant, glass-stop-
pered bottles, in a cold place.
Heavy hydrogenated solvent for nuclear magnetic reso-
nance spectroscopy Prepared for nuclear magnetic reso-
nance spectroscopy. Heavy hydrogenated chloroform
(CDC1 3 ), heavy hydrogenated dimethyl sulfoxide [(CD 3 ) 2 SO],
heavy water (D 2 0), and heavy hydrogenated pyridine
(C5D5N) are available.
Heavy water for nuclear magnetic resonance spectroscopy
D 2 Prepared for nuclear magnetic resonance spec-
troscopy.
Helium He Not less than 99.995 vol%.
Hematoxylin C 16 H 14 6 .wH 2 White or light yellow to
brownish crystals or crystalline powder. It is soluble in hot
water and in ethanol (95), and sparingly soluble in cold
water.
Residue on ignition <2.44>: not more than 0.1% (1 g).
Hematoxylin TS Dissolve 1 g of hematoxylin in 12 mL of
ethanol (99.5). Dissolve 20 g of aluminum potassium sulfate
12-water in 200 mL of warm water, cool, and filter. After 24
hours, mix these two prepared solutions. Allow to stand for 8
hours in a wide-mouthed bottle without using a stopper, and
filter.
Heparin sodium [Same as the namesake monograph]
HEPES buffer solution, pH 7.5 Dissolve 2.38 g of N-2-
hydroxyethylpiperazine--/V'-2-ethanesulfonic acid in 90 mL
of water, adjust to pH 7.5 with 5 mol/L sodium hydroxide
TS, and add water to make 100 mL.
Heptane CH 3 (CH 2 ) 5 CH 3 [K 9701, Special class]
Hesperidin for component determination Hesperidin for
thin-layer chromatography. It meets the following require-
ments.
Optical rotation <2.49> [a]g>: - 100 - - 120° (5 mg dried
with silica gel for 24 hours, methanol, 50 mL, 100 mm).
Purity Related substances — Dissolve 2 mg in 10 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add the mobile phase to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with exactly 10,mL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the total area of the peaks other than the peak
of hesperidin and the solvent is not larger than the peak area
of hesperidin obtained with the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay (1) under Hochuekkito Extract.
Time span of measurement: About 6 times as long as the
retention time of hesperidin.
System suitability
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of hesperidin obtained with 10 /uL
of this solution is equivalent to3.5to6.5%of that with 1 /uL
of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay (1) under
Hochuekkito Extract.
Hesperidin for thin-layer chromatography C 28 H 34 15 A
white to light brown-yellow, crystalline powder or powder.
Very slightly soluble in methanol and in ethanol (99.5), and
practically insoluble in water. Melting point: about 245°C
(with decomposition).
Absorbance <2.24> E\ v ° m (284 nm): 310 - 340 (8 mg dried in
a desiccator (silica gel) for 24 hours, methanol, 500 mL).
Purity Related substances — Dissolve 1 mg in 2 mL of
methanol. Proceed the test with 20 fiL of this solution as
directed in the Identification (6) under Hochuekkito Extract:
no spot other than the principle spot of around Rf 0.3 ap-
pears.
Hexaammonium heptamolybdate-cerium (IV) sulfate TS
Dissolve 2.5 g of hexaammonium heptamolybdate tetrahy-
drate and 1.0 g of cerium (IV) sulfate tetrahydrate in diluted
sulfuric acid (3 in 50) to make 100 mL. Prepare before use.
Hexaammonium heptamohybdate-sulfuric acid TS Dis-
solve 1.0 g of nexaammonium heptamolybdate tetrahydrate
in diluted sulfuric acid (3 in 20) to make 40 mL. Prepare be-
fore use.
Hexaammonium heptamolybdate tetrahydrate
(NH 4 ) 6 Mo 7 24 .4H 2 [K 8905, Special class]
Hexaammonium heptamolybdate TS dissolve 21.2 g of
hexaammonium heptamolybdate tetrahydrate in water to
make 200 mL (10%). Prepare before use.
Hexamethylenetetramine (CH 2 ) 6 N 4 [K 8847, Special
class]
Hexamethylenetetramine TS See Test Methods for Plas-
tic Containers <7.02>.
Hexamine See hexamethylenetetramine.
Hexane C 6 H I4 [K 8848, Special class]
Hexane for liquid chromatography CH 3 (CH 2 ) 4 CH 3
Colorless, clear liquid. Miscible with ethanol (95), with
diethyl ether, with chloroform and with benzene.
Boiling point <2.57>: about 69°C
Purity (1) Ultraviolet absorptive substances — Read the
absorbances of hexane for liquid chromatography as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using
water as the blank: not more than 0.3 at the wavelength of
210 nm, and not more than 0.01 between 250 nm and 400
JPXV
General Tests / Reagents, Test Solutions
199
nm.
(2) Peroxide — To a mixture of 100 mL of water and 25
mL of dilute sulfuric acid add 25 mL of a solution of potassi-
um iodide (1 in 10) and 20 g of hexane for liquid chromatog-
raphy. Stopper tightly, shake, and allow to stand in a dark
place for 15 minutes. Titrate <2.50> this solution, while shak-
ing well, with 0.01 mol/L sodium thiosulfate (indicator: 1
mL of starch TS). Perform a blank determination in the same
manner.
n-Hexane for liquid chromatography See hexane for liq-
uid chromatography.
Hexane for purity of crude drug [K 8848, Special class]
Use hexane meeting the following additional specification.
Evaporate 300.0 mL of hexane for purity of crude drug in
vacuum at a temperature not higher than 40°C, add the
hexane to make exactly 1 mL, and use this solution as the
sample solution. Separately, dissolve 2.0 mg of y-BHC in
hexane to make exactly 100 mL. Pipet 1 mL of this solution,
and add hexane to make exactly 100 mL. Further pipet 2 mL
of this solution, add hexane to make exactly 100 mL, and use
this solution as the standard solution I. Perform the test with
exactly 1 juL each of the sample solution and standard solu-
tion I as directed under Gas Chromatography <2.02> accord-
ing to the following operating conditions, and determine each
peak area by the automatic integration method: the total area
of peak other than the solvent peak from the sample solution
is not larger than the peak area of y-BHC from the standard
solution I.
Operating conditions
Proceed the operating conditions in the Purity (2) under
Crude Drugs Test <5.01> except detection sensitivity and time
span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
I, add hexane to make exactly 20 mL, and use this solution as
the standard solution II. Adjust the detection sensitivity so
that the peak area of y-BHC obtained from 1 /uL of the stan-
dard solution II can be measured by the automatic integra-
tion method, and the peak height of y-BHC from 1 iiL of the
standard solution I is about 20% of the full scale.
Time span of measurement: About three times as long as
the retention time of y-BHC beginning after the solvent peak.
Hexane for ultraviolet-visible spectrophotometry
[K 8848, Special class]. When determining the absorbance of
hexane for ultraviolet-visible spectrophotometry as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using
water as the blank solution, its value is not more than 0.10 at
220 nm and not more than 0.02 at 260 nm, and it has no char-
acteristic absorption between 260 nm and 350 nm.
n-Hexane for ultraviolet-visible spectrophotometry See
hexane for ultraviolet-visible spectrophotometry.
Hirsutine C22H28N2O3 White to light yellow, crystals or
crystalline powder. Melting point: about 100°C.
Optical rotation <2.49> [ a ]^°: about +70° (10 mg,
methanol, 1 mL, 50 mm).
Absorbance <2.24> E\°^ (245 nm): 354 - 379 (5 mg calcu-
lated on the anhydrous basis, a mixture of methanol and di-
lute acetic acid (7:3), 500 mL).
Purity Related substances — Dissolve 5 mg of hirsutine in
100 mL of a mixture of methanol and dilute acetic acid (7:3),
proceed with 20 11L of this solution as directed in the Compo-
nent determination under Uncaria Thorn, and perform the
Liquid Chromatography <2.01>: the sum of the peak areas ex-
cept the areas of hirsutine and the solvent is not more than
1/10 of the sum of the peak areas except the solvent.
L-Histidine hydrochloride See L-histidine hydrochloride
monohydrate.
L-Histidine hydrochloride monohydrate
C 6 H 9 N 3 02.HC1.H 2 [K 9050, Special class]
Homatropine hydrobromide C 16 H 2 iN0 3 .HBr [Same as
the namesake monograph]
Honokiol Ci8H 18 2 .xH 2 Odorless white, crystals or
crystalline powder.
PunYy-Dissolve 1 mg of honokiol in the mobile phase to
make 10 mL, and use this solution as the sample solution.
Perform the Liquid Chromatography <2.01> with 10 [iL of
the sample solution as directed in the Component determina-
tion under Magnolia Bark: when measure the peak areas 2
times as long as the retention time of magnorole, the total
area of peaks other than honokiol is not larger than 1/10
times the total area of the peaks other than the solvent peak.
Horseradish peroxidase An oxidase (Molecular weight:
about 40,000) derived from horseradish
Horse serum Collect the blood from horse in a flask,
coagulate, and allow to stand at room temperature until the
serum is separated. Transfer the separated serum in glass
containers, and preserve at — 20°C.
Human antithrombin III Serine protease inhibition
factor obtained from normal plasma of health human. It is a
protein, which inhibits the activities of thrombin and
activated blood coagulation factor X. It contains not less
than 300 Units per mg protein. One unit indicates an amount
of the antithrombin III which inhibits 1 unit of thrombin at
25 °C under the existence of heparin.
Human chorionic gonadotrophin TS Weigh accurately a
suitable amount of Human Chorionic Gonadotrophin ac-
cording to the labeled amount, and dissolve in bovine serum
albumin-sodium chloride-phosphate buffer solution, pH 7.2
so that each mL contains 80 human chorionic gonadotrophin
Units.
Human insulin desamide substance-containing TS Dis-
solve 1 .5 mg of Insulin Human (Genetical Recombination) in
1 mL of 0.01 mol/L hydrochloric acid TS, allow to stand at
25 °C for 3 days, and when the procedure is run with this
solution according to the conditions as directed in the Purity
(1) under Insurin Human (Genetical Recombination), the so-
lution contains about 5% of the desamide substance.
Human insulin dimer-containing TS Allow to stand Insu-
lin Human (Genetical Recombination) at 25 °C for 10 days or
more, and dissolve 4 mg of this in 1 mL of 0.01 mol/L
hydrochloric acid TS.
Human normal plasma Dissolve an amount of dried hu-
man normal plasma powder, equivalent to 1 mL of the nor-
mal plasma of human, in 1 mL of water. Store between 2 and
10°C, and use within one week.
Human serum albumin for assay White to pale-yellow
powder. Albumin content is at least 99%. Convert to the de-
hydrate using the following water determination method.
200
Reagents, Test Solutions / General Tests
JP XV
Water content <2.48>: (0.20 g, direct titration). However,
in a dehydration solvent, use a mixture of pyridine for water
determination and ethylene glycol for water determination
(5:1).
Hydralazine hydrochloride C 8 H 8 N 4 .HC1 [Same as
the namesake monograph]
Hydralazine hydrochloride for assay C 8 H 8 N 4 .HC1
[Same as the monograph Hydralazine Hydrochloride. When
dried, it contains not less than 99.0% of hydralazine
hydrochloride (C 8 H 8 N 4 .HC1).]
Hydrazine hydrate See hydrazine monohydrate.
Hydrazine monohydrate NH 2 NH 2 .H 2 Colorless liq-
uid, having a characteristic odor.
Hydrazine sulfate See hydrazinum sulfate.
Hydrazinum sulfate N 4 H 6 S0 4 [K 8992, Special class]
Hydrazinum sulfate TS Dissolve 1.0 g of hydrazinum
sulfate in water to make 100 mL.
Hydrobromic acid HBr [K 8509, Special class]
Hydrochloric acid HC1 [K 8180, Special class]
Hydrochloric acid-ammonium acetate buffer solution, pH
3.5 Dissolve 25 g of ammonium acetate in 45 mL of 6 mol/
L hydrochloric acid TS, and add water to make 100 mL.
Hydrochloric acid, dilute Dilute 23.6 mL of hydrochloric
acid with water to make 100 mL (10%).
Hydrochloric acid-ethanol TS See hydrochloric acid-
ethanol (95) TS.
Hydrochloric acid-ethanol (95) TS Dilute 23.6 mL of hy-
drochloric acid with ethanol to make 100 mL.
0.01 mol/L Hydrochloric acid-methanol TS To 20 mL of
0.5 mol/L hydrochloric acid TS add methanol to make 1000
mL.
0.05 mol/L Hydrochloric acid-methanol TS To 100 mL
of 0.5 mol/L hydrochloric acid add methanol to make 1000
mL.
Hydrochloric acid-2-propanol TS Add 0.33 mL of
hydrochloric acid to 100 mL of 2-propanol, mix, and store in
a dark and cool place.
Hydrochloric acid-potassium chloride buffer solution, pH
2.0 To 10.0 mL of 0.2 mol/L hydrochloric acid VS add
88.0 mL of 0.2 mol/L potassium chloride TS, adjust the pH
to 2.0 ± 0.1 with 0.2 mol/L hydrochloric acid VS or 0.2 mol
/L potassium chloride TS, then add water to make 200 mL.
Hydrochloric acid, purified Add 0.3 g of potassium per-
manganate to 1000 mL of diluted hydrochloric acid (1 in 2),
distil, discard the first 250 mL of the distillate, and collect the
following 500 mL of the distillate.
0.001 mol/L Hydrochloric acid TS Dilute 10 mL of 0.1
mol/L hydrochloric acid TS with water to make 1000 mL.
0.01 mol/L Hydrochloric acid TS Dilute 100 mL of 0.1
mol/L hydrochloric acid TS with water to make 1000 mL.
0.02 mol/L Hydrochloric acid TS Dilute 100 mL of 0.2
mol/L hydrochloric acid TS with water to make 1000 mL.
0.05 mol/L Hydrochloric acid TS Dilute 100 mL of 0.5
mol/L hydrochloric acid TS with water to make 1000 mL.
0.1 mol/L Hydrochloric acid TS Dilute 100 mL of 1 mol
/L hydrochloric acid TS with water to make 1000 mL.
0.2 mol/L Hydrochloric acid TS Dilute 18 mL of
hydrochloric acid with water to make 1000 mL.
0.5 mol/L Hydrochloric acid TS Dilute 45 mL of
hydrochloric acid with water to make 1000 mL.
1 mol/L Hydrochloric acid TS Dilute 90 mL of hydro-
chloric acid with water to make 1000 mL.
2 mol/L Hydrochloric acid TS Dilute 180 mL of hydro-
chloric acid with water to make 1000 mL.
3 mol/L Hydrochloric acid TS Dilute 270 mL of hydro-
chloric acid with water to make 1000 mL.
5 mol/L Hydrochloric acid TS Dilute 450 mL of hydro-
chloric acid with water to make 1000 mL.
6 mol/L Hydrochloric acid TS Dilute 540 mL of hydro-
chloric acid with water to make 1000 mL.
7.5 mol/L Hydrochloric acid TS Dilute 675 mL of
hydrochloric acid with water to make 1000 mL.
10 mol/L Hydrochloric acid TS Dilute 900 mL of
hydrochloric acid with water to make 1000 mL.
Hydrocortisone C 21 H 3 o0 5 [Same as the namesake mon-
ograph]
Hydrocortisone acetate C 23 H 32 6 [Same as the name-
sake monograph]
Hydrocotarnine hydrochloride for assay
C12Hj5NO3.HCl.H2O [Same as the monograph Hydro-
cotarnine Hydrochloride Hydrate. When dried, it contains
not less than 99.0% of hydrocotarnine hydrochloride
(C I2 H 15 N0 3 .HC1).]
Hydrofluoric acid HF [K 8819, Special class] It con-
tains not less than 46.0% of HF.
Hydrogen H 2 [K 0512, Standard substance, Third
class] It contains not less than 99.99% of H 2 .
Hydrogen chloride-ethanol TS See hydrogen chloride-
ethanol (99.5) TS.
Hydrogen chloride-ethanol (99.5) TS Pass dry hydrogen
chloride, which is generated by slowly adding 100 mL of sul-
furic acid dropwise to 100 mL of hydrochloric acid and dried
by washing with sulfuric acid, through 75 g of ethanol (99.5)
cooled in an ice bath until the increase in mass has reached 25
g. Prepare before use.
Hydrogen hexachloroplatinate (IV) hexahydrate
H 2 PtCl 6 .6H 2 [K 8153, Special class]
Hydrogen hexachloroplatinate (IV)-potassium iodide TS
To 3 mL of hydrogen hexachloroplatinate (IV) TS add 97 mL
of water and 100 mL of a solution of potassium iodide (3 in
50). Prepare before use.
Hydrogen hexachloroplatinate (IV) TS Dissolve 2.6 g of
chloroplatinic acid in water to make 20 mL (0.25 mol/L).
Hydrogen peroxide-sodium hydroxide TS To a mixture
JP XV
General Tests / Reagents, Test Solutions
201
of water and hydrogen peroxide (30) (9:1) add 3 drops of
bromophenol blue TS, and then add 0.01 mol/L sodium
hydroxide TS until a purple-blue color develops. Prepare
before use.
Hydrogen peroxide TS Dilute 1 volume of hydrogen per-
oxide (30) with 9 volumes of water. Prepare before use (3%).
Hydrogen peroxide TS, dilute Dilute 1 mL of hydrogen
peroxide (30) with 500 mL of water, and dilute 5 mL of this
solution with water to make 100 mL. Prepare before use.
Hydrogen peroxide water, strong
oxide (30).
See hydrogen per-
Hydrogen peroxide (30) H 2 2 [K 8230, Hydrogen
peroxide, Special class, Concentration: 30.0 - 35.5%].
Hydrogen sulfide H 2 S Colorless, poisonous gas, heavi-
er than air. It dissolves in water. Prepare by treating iron (II)
sulfide heptahydrate with dilute sulfuric acid or dilute hydro-
chloric acid. Other sulfides yielding hydrogen sulfide with di-
lute acids may be used.
Hydrogen sulfide TS A saturated solution of hydrogen
sulfide. Prepare by passing hydrogen sulfide into cold water.
Preserve in well-filled, light-resistant bottles, in a dark, cold
place.
Hydrogen tetrachloroaurate (III) tetrahydrate
HAuCl 4 .4H 2 [K 8127, Special class]
Hydrogen tetrachloroaurate (III) tetrahydrate TS Dis-
solve 1 g of hydrogen tetrachloroaurate (III) tetrahydrate in
35 mL of water (0.2 mol/L).
Hydroiodic acid HI [K 8917, Special class]
Hydroquinone C 6 H 4 (OH) 2 [K 8738, Special class]
Hydroxocobalamin acetate C 62 H 89 CoN 13 Oi5P.C 2 H 4 2
Dark red crystals or powder.
Loss on drying <2.41>: not more than 12% (50 mg, in vacu-
um not exceeding 0.67 kPa, phosphorus (V) oxide, 100°C, 6
hours).
Content: not less than 98.0%. Assay — Proceed as direct-
ed in the Assay under Hydroxocobalamin Acetate.
m-Hydroxyacetophenone C 8 H 8 2 White to light yel-
lowish white crystalline powder.
Melting point <2.60>: about 96°C
Purity Related substances — Perform the test with 10 /uL
of a solution of m-hydroxyacetophenone in 0.1 mol/L phos-
phate buffer solution, pH 4.5 (1 in 15,000) as directed in the
Assay under Cefalexin: Any obstructive peaks for determina-
tion of cefalexin are not observed.
p-Hydroxyacetophenone C 8 H 8 2 White to pale yellow
crystals or crystalline powder. It is freely soluble in methanol.
Melting point <2.60>: 107 - 111°C
Purity — Weigh 1 mg of p-hydroxyacetophenone, add
methanol and dissolve to make exactly 10 mL, and use this
solution as the sample solution. Perform the test with 20 /xL
of the sample solution as directed under Liquid Chro-
matography <2.01> according to the Assay under Peony
Root: the total area of the peaks other than the peak of p-
hydroxyacetophenone from the sample solution is not larger
than 3/100 of the total area of the peaks other than the sol-
vent peak.
3-Hydroxybenzoic acid HOC 6 H 4 COOH White, crys-
tals or crystalline powder.
Identification — Determine the infrared absorption spec-
trum according to the paste method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3300 cm- 1 , 1690 cm- 1 , 1600 cm- 1 , 1307
cm- 1 , 1232 cm- 1 and 760 cm- 1 .
Melting point <2.60>: 203 - 206°C
Purity Clarity — Dissolve 1 g of 3-hydroxybenzoic acid in
20 mL of methanol: the solution is clear.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.2 g of 3-hydroxybenzoic acid, dissolve in 20 mL of
diluted ethanol (95) (1 in 2), and titrate <2.50> with 0.1 mol/L
sodium hydroxide VS (indicator: 3 drops of cresol red TS)
until the color of the solution changes from yellow to dark
orange-red. Perform a blank determination and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 13.81 mg of C 7 H 6 3
p-Hydroxybenzoic acid See parahydroxybenzoic acid.
./V-(2-HydroxyethyI)isonicotinamide nitric ester
C 8 H 9 N 3 4 A white crystalline powder.
Identification — Determine the infrared absorption spec-
trum of Af-(2-hydroxyethyl)isonicotinamide nitric ester as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3270 cm" 1 , 1653 cm -1 , 1546
cm -1 and 1283 cm -1 .
./V-2-HydroxyethyIpiperazine-./V' -2-ethanesulf onic acid
C 8 H I8 N 2 4 S White crystalline powder.
Purity Clarity and color of solution — Dissolve 11.9 g of
7V-2-hydroxyethylpiperazine-iV' -2-ethanesulf onic acid in 50
mL of water: the solution is clear and colorless.
Content: not less than 99.0%. Assay — Weigh accurately
about 1 g of _/V-2-hydroxyethylpiperazine-7V' -2-ethanesulf on-
ic acid, dissolve in 60 mL of water, and titrate <2.50> with 0.5
mol/L sodium hydroxide VS (Potentiometric titration).
Each mL of 0.5 mol/L sodium hydroxide VS
= 119.2mg of C 8 Hi 8 N 2 4 S
l-(2-Hydroxyethyl)-l/Metrazol-5-thiol C 3 H 6 N 4 OS
White, crystals or powder.
Melting point <2.60>: 136- 141 °C
Purity Related substances — Dissolve 0.10 g of l-(2-
hydroxyethyl)-l//-tetrazol-5-thiol in 1 mL of water, and use
this solution as the sample solution. Pipet 0.5 mL of the sam-
ple solution, add water to make exactly 25 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 1 [iL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography, develop with a mixture of ethyl
acetate, water, methanol and formic acid (60:10:7:6) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spot other
than the principal spot obtained from the sample solution is
not more intense than the spot from the standard solution.
d-3-Hydroxy-cir-2,3-dihydro-5-[2-(dimethylamino)ethyl]-
2-(4-methoxyphenyl)-l,5-benzothiazepine-4(5//)-one
hydrochloride C 20 H 24 N 2 O 3 S.HCl To 9 g of diltiazem hy-
202
Reagents, Test Solutions / General Tests
JP XV
drochloride add 50 mL of ethanol (99.5), and dissolve by
heating at 80°C. To this solution add slowly 50 mL of a solu-
tion of potassium hydroxide in ethanol (99.5) (33 in 500)
dropwise, and heat for 4 hours with stirring. Cool in an ice
bath, filter, and evaporate the filtrate to dryness. Dissolve the
residue in ethanol (99.5), add slowly a solution of hydro-
chloric acid in ethanol (99.5) (59 in 250) to make acidic, and
filter. Add diethyl ether slowly to the filtrate, and filter the
crystals produced. To the crystals add ethanol (99.5), heat to
dissolve, add 0.5 g of activated charcoal, allow to stand, and
filter. After cooling the filtrate in an ice-methanol bath, filter
the crystals formed, and wash with diethyl ether. Further,
add ethanol (99.5) to the crystals, and heat to dissolve. After
cooling, filter the crystals produced, and dry under reduced
pressure. White crystals or crystalline powder, having a
slight, characteristic odor.
Purity — Dissolve 50 mg of rf-3-hydroxy-c«-2,3-dihydro-
5 - [2 - (dimethylamino)ethyl] -2-(p - methoxyphenyl) -1,5 - ben-
zothiazepine-4-(5//)-one hydrochloride in chloroform to
make exactly 10 mL, and use this solution as the sample solu-
tion. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 20 juL of the
sample solution on a plate of silica gel for thin-layer chroma-
tography. Develop the plate with a mixture of ethanol (99.5),
chloroform, water and acetic acid (100) (12:10:3:1) to a dis-
tance of about 13 cm, and air-dry the plate. Spray evenly io-
dine TS on the plate: any spot other than the principal spot
does not appear.
Water <2.48>: not more than 1.0% (0.5 g).
Content: not less than 99.0%, calculated on the anhydrous
basis. Assay — Weigh accurately about 0.5 g of d-3-
hydroxy-cw-2,3-dihydro-5-[2-(dirnethylamino)ethyi]-2-(/?-
methoxyphenyl)-l ,5-benzothiazepine-4-(5//)-one hydrochlo-
ride, dissolve in 2.0 mL of formic acid, add 60 mL of acetic
anhydride, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determination
in the same manner.
Each mL of 0.1 mol/L perchloric acid VS
= 40.89 mg of C 20 H 24 N 2 O 3 S.HCl
tf-3-Hydroxy-m-2,3-dihydro-5-[2-(dimethylamino) ethyl]-
2-(p-inethoxyphenyI)-l,5-benzothiazepine-4 (5H)-one hydro-
chloride See rf-3-hydroxy-m-2,3-dihydro-5-[2-(dimethyl-
amino) ethyl]-2-(4-methoxyphenyl)-l ,5-benzothiazepine-4
(5//)-one hydrochloride.
2-Hydroxy-l-(2-hydroxy-4-sulfo-l-naphthyIazo)-3-naph-
thoic acid C 21 H 14 N 2 7 S [K 8776, Special class]
Hydroxylamine hydrochloride See hydroxylammonium
chloride.
Hydroxylamine hydrochloride-ferric chloride TS See
hydroxylammonium chloride-iron (III) chloride TS.
Hydroxylamine hydrochloride TS See hydroxylammoni-
um chloride TS.
Hydroxylamine perchlorate NH 2 OH.HC10 4 Hygro-
scopic, white crystals. Dissolves in water and in ethanol (95).
Melting point <2.60>: 87.5 -90°C
Hydroxylamine perchlorate-dehydrated ethanol TS See
hydroxylamine perchlorate-ethanol (99.5) TS.
Hydroxylamine perchlorate-ethanol (99.5) TS Dilute
2.99 mL of hydroxylamine perchlorate TS with ethanol
(99.5) to make 100 mL.
Storage — Preserve in tight containers, in a cold place.
Hydroxylamine perchlorate TS An ethanol (95) solution
which contains 13.4% of hydroxylamine perchlorate.
Storage — Preserve in tight containers, in a cold place.
Hydroxylamine TS Dissolve 10 g of hydroxylammonium
chloride in 20 mL of water, and add ethanol (95) to make 200
mL. To this solution add, with stirring, 150 mL of 0.5 mol/L
potassium hydroxide-ethanol VS, and filter. Prepare before
use.
Hydroxylamine TS, alkaline Mix equal volumes of a so-
lution of hydroxylammonium chloride in methanol (7 in 100)
and a solution of sodium hydroxide in methanol (3 in 25),
and filter. Prepare before use.
Hydroxylammine hydrochloride TS, pH 3.1 See hydrox-
ylammonium chloride TS, pH 3.1.
Hydroxylammonium chloride NH 2 OH.HCl [K 8201,
Special class]
Hydroxylammonium chloride-iron (III) chloride TS
Acidify 100 mL of a solution of iron (III) chloride hexahy-
drate in ethanol (95) (1 in 200) with hydrochloric acid, and
dissolve 1 g of hydroxylammonium chloride in the solution.
Hydroxylammonium chloride TS Dissolve 20 g of hy-
droxylammonium chloride in water to make 65 mL, transfer
it to a separator, add 2 to 3 drops of thymol blue TS, then
add ammonia solution (28) until the solution exhibits a yel-
low color. Shake well after adding 10 mL of a solution of so-
dium iV,./V-diethyklithiocarbamate trihydrate (1 in 25), allow
to stand for 5 minutes, and extract this solution with 10 to 15
mL portions of chloroform. Repeat the extraction until 5 mL
of the extract does not exhibit a yellow color, upon adding 5
drops of a solution of copper (II) sulfate pentahydrate (1 in
100) and shaking it. Add 1 to 2 drops of thymol blue TS, add
dropwise dilute hydrochloric acid to this aqueous solution
until it exhibits a red color, then add water to make 100 mL.
Hydroxylammonium chloride TS, pH 3.1 Dissolve 6.9 g
of hydroxylammonium chloride in 80 mL of water, adjust
the pH to 3.1 by adding dilute sodium hydroxide TS, and add
water to make 100 mL.
Hydroxylammonium chloride-ethanol TS Dissolve 34.8 g
of hydroxylammonium chloride in water to make 100 mL,
and use this solution as Solution A. Dissolve 10.3 g of sodi-
um acetate trihydrate and 86.5 g of sodium hydroxide in
water to make 1000 mL, and use this solution as Solution B.
Mix 1 volume of Solution A, 1 volume of Solution B and 4
volumes of ethanol (95).
4-Hydroxy-3-methoxybenzyl nonylic acid amide
C 17 H 27 N0 3 A white crystalline powder, having a faint,
characteristic odor.
Purity Related substances — Dissolve 10 mg in 50 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add methanol to make exactly
20 mL, and use this solution as the standard solution. Per-
form the test with exactly 20 /uL each of the sample solution
and standard solution as directed in the Component determi-
nation under Capsicum: when measure the peak areas 2 times
as long as the retention time of capsaicin, the total area of the
JPXV
General Tests / Reagents, Test Solutions
203
peaks other than 4-hydroxy-3-methoxybenzyl nonylic acid
amide is not larger than the peak area of 4-hydroxy-3-
methoxybenzyl nonylic acid amide from the standard solu-
tion.
iV-(3-Hydroxyphenyl)acetamide C 8 H 6 N0 2 White to
pale yellowish white crystals. It is freely soluble in ethanol
(95), and sparingly soluble in water.
Melting point <2.60>: 146 - 149 C C
Purity (1) Clarity and color of solution — Dissolve 0.5 g
of ./V-(3-hydroxyphenyi)acetamide in 50 mL of water: the so-
lution is clear and colorless.
(2) Related substances — Dissolve 0.1 g of JV-(3-hydrox-
yphenyl)acetamide in 1000 mL of water. Pipet 10 mL of this
solution, add 6.5 mL of acetonitrile and water to make exact-
ly 50 mL, and use this solution as the sample solution. Per-
form the test with 10 liL of the sample solution as directed in
the Assay under Aspoxicillin Hydrate: any peak other than
those of A r -(3-hydroxyphenyl)acetamide and the solvent does
not appear.
3-(3-Hydroxy-4-methoxyphenyl)-2-(E)-propenic acid
C 10 H 10 O 4 White to light yellow, crystals or crystalline pow-
der. Sparingly soluble in methanol and in ethanol (99.5), and
practically insoluble in water. Melting point: about 230°C
(with decomposition).
Identification — Determine the absorption spectrum of a
solution in methanol (1 in 200,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 215 nm and 219 nm, between 238 nm and 242 nm,
between 290 nm and 294 nm, and between 319 nm and 323
nm.
Purity Related substances — Dissolve 1 mg in 1 mL of
methanol. Proceed the test with 2 liL of this solution as
directed in the Identification (11) under Hochuekkito Ex-
tract: no spot other than the principle spot of around Rf 0.6
appears.
3-(3-Hydroxy-4-methoxyphenyl)-2-(E)-propenic acid-(E)-
ferulic acid TS for thin-layer chromatography Dissolve 1
mg of 3-(3-hydroxy-4-methoxyphenyl)-2-(ii)-propenic acid
and 1 mg of (E)-ferulic acid in 2 mL of methanol.
2-[4-(2-HydroxymethyI)-l-piperazinyl] propanesulfonic
acid C 8 H 18 N 2 4 S A white crystalline powder.
Residue on ignition <2.44>: not more than 0.1%.
Content: not less than 99%.
3-(/7-Hydroxyphenyl)propionic acid C9H10O3
Description — White to light yellow-brown crystals or crys-
talline powder, having a faint, characteristic odor.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.2 g of 3-(/>-hydroxyphenyl)propionic acid, previous-
ly dried (in vacuum, 60°C, 4 hours), dissolve in 5 mL of
methanol, add 45 mL of water, and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (indicator: 5 drops of
bromothymol blue TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 16.62 mg of C 9 H 10 O 3
Hypaconitine for purity C33H45NO10 White, crystals or
crystalline powder. Soluble in acetonitrile, sparingly soluble
in ethanol (99.5) and in diethyl ether, and practically insolu-
ble in water. Melting point: about 175°C (with decomposi-
tion).
Identification — Determine the infrared absorption spec-
trum of hypaconitine for purity as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3500 cm- 1 , 1728 cm" 1 , 1712 cm" 1 , 1278 cm" 1 , 1118 cm" 1 ,
1099 cm" 1 and 714 cm" 1 .
Absorbance <2.24> E\ a/ ° m (230 nm): 217 - 252 [5 mg dried
for not less than 12 hours in a desiccator (reduced pressure
not exceeding 0.67 kPa, phosphorus (V) oxide, 40°C),
ethanol (99.5), 200 mL].
Purity Related substances — (1) Dissolve 5.0 mg of
hypaconitine for purity in 2 mL of acetonitrile, and use as the
sample solution. Pipet 1 mL of the sample solution, add
acetonitrile to make exactly 50 mL, and use as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 [iL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography, and proceed the test as
directed in the Identification in Processed Aconite Root: the
spot other than the principal spot obtained with the sample
solution is not more intense than the spot with the standard
solution.
(2) Dissolve 5.0 mg of hypaconitine for purity in 5 mL of
acetonitrile, and use as the sample solution. Pipet 1 mL of the
sample solution, add acetonitrile to make exactly 50 mL, and
use as the standard solution. Perform the test with exactly 10
/XL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peaks of hypaconitine and the solvent
obtained with the sample solution is not larger than the peak
area of hypaconitine with the standard solution.
Operating conditions
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity under Proc-
essed Aconite Root.
Mobile phase: A mixture of phosphate buffer solution for
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust the flow rate so that the retention time of
hypaconitine is about 23 minutes.
Time span of measurement: About 3 times as long as the
retention time of hypaconitine.
System suitability
Test for required detectability: Pipet 1 mL of the standard
solution, and add acetonitrile to make exactly 20 mL.
Confirm that the peak area of hypaconitine obtained from 10
liL of this solution is equivalent to 3.5 to 6.5% of that
obtained from 10 liL of the standard solution.
System performance: Dissolve 1 mg each of aconitine for
purity, hypaconitine for purity and mesaconitine for purity,
and 8 mg of jesaconitine for purity in 200 mL of acetonitrile.
When the procedure is run with 10,mL of this solution under
the above operating conditions, mesaconitine, hypaconitine,
aconitine and jesaconitine are eluted in this order, and each
resolution between these peaks is not less than 1.5, respec-
tively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
hypaconitine is not more than 1.5%.
Water <2.48>: not more than 1.0% [5 mg dried for not less
than 12 hours in a desiccator (reduced pressure not exceeding
204
Reagents, Test Solutions / General Tests
JP XV
0.67 kPa, phosphorus (V) oxide, 40°C), coulometric titra-
tion].
Hypophosphorus acid See phosphinic acid.
Hypoxanthine C 5 H 4 N 4 White crystals or crystalline
powder. Freely soluble in ammonia TS, sparingly soluble in
dilute hydrochloric acid and in hot water, very slightly solu-
ble in water, and practically insoluble in methanol.
Purity Related substances — Dissolve 5.0 mg of hypo-
xanthine in 100 mL of a solution of ammonia solution (28) in
methanol (1 in 10) to make exactly 100 mL. Proceed with this
solution as directed in the Purity (4) under Mercaptopurine
Hydrate: any spot other than the principal spot at the Rf
value of about 0.2 does not appear.
Content: not less than 97.0% and not more than 103.0%.
Assay — Weigh accurately about 0.15 g of hypoxanthine,
previously dried at 105 °C for 3 hours, and dissolve in phos-
phate buffer solution, pH 7.0, to make exactly 1000 mL.
Pipet 10 mL of this solution, and dilute with phosphate
buffer solution, pH 7.0, to make exactly 250 mL. Read the
absorbance A of this solution at the wavelength of 250 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using phosphate buffer solution, pH 7.0, as the blank solu-
tion.
Amount (mg) of C 5 H 4 N 4 =
A
779
x 250,000
Ibuprofen C I3 H 18 2 [Same as the namesake mono-
graph]
Icariin for thin-layer chromatography C 33 H 40 O 15 Light
yellow crystals. Very slightly soluble in methanol and in
ethanol (99.5), and practically insoluble in water. Melting
point: about 234°C (with decomposition).
Purity Related substances — Dissolve 1.0 mg of icariin for
thin-layer chromatography in 1 mL of methanol. Perform
the test with 10 /xh of this solution as directed in the Identifi-
cation under Epimedium Herb: no spot other than the
principal spot having Rf value about 0.4 appears.
Imidazole C 3 H 4 N 2 White crystalline powder. Very solu-
ble in water and in methanol.
Melting point <2.60>: 89 - 92°C
Absorbance <2.24> E\°f m (313 nm): not more than 0.031
(8g, water, 100 mL).
Imidazole for Karl Fischer method See the Water Deter-
mination <2.48>.
Imidazole for thin-layer chromatography C 3 H 4 N 2
White, crystalline powder. Very soluble in water and in meth-
anol, and freely soluble in ethyl acetate and in
dichloromethane.
Melting point <2.60>: 89 - 92°C
Purity Related substances — Dissolve 10 mg of imidazole
for thin-layer chromatography in exactly 20 mL of
dichloromethane, and proceed with this solution as directed
in the Purity (6) under Clotrimazole: any spot other than the
principal spot does not appear.
Imidazole TS Dissolve 8.25 g of imidazole in 65 mL of
water, adjust the pH to 6.8 with 5 mol/L hydrochloric acid
TS, and add water to make 100 mL.
Iminodibenzyl Ci 4 H 13 N White to light brown crystals
or crystalline powder, having a slight, characteristic odor.
Melting point <2.60>: 104 - 110°C
Purity (1) Clarity of solution — Dissolve 1.0 g of imino-
dibenzyl in 20 mL of methanol by heating on a water bath:
the solution is clear.
(2) Related substances — Proceed as directed in the Purity
(6) under Carbamazepine: any spot other than the principal
spot at the Rf value of about 0.9 does not appear.
Nitrogen <1.08>: 6.8 - 7.3%.
Imipramine hydrochloride
the namesake monograph]
Ci 9 H 24 N 2 .HCl [Same as
Indigo carmine C 16 H 8 N 2 Na 2 8 S 2 [K 8092, Special class]
Indigo carmine TS Dissolve 0.20 g of indigo carmine in
water to make 100 mL. Use within 60 days.
2,3-Indolinedione C 8 H 5 N0 2 [K 8089, Special class]
Indometacin C 19 H 16 C1N0 4 [Same as the namesake
monograph]
Interleukin-2 dependent mouse natural killer cell (NKC3)
Fractionate using discontinuous concentration gradient
method cells obtained by removing adhesive cells and
phagocyteic cells from C3H/He mouse spleen cells. Then,
cultivate in soft agar containing interleukin-2 the cell fraction
with potent NK activity and obtain the colonies. From among
the cell lines obtained, one of the cell lines dependent on in-
terleukin-2 that grows in liquid medium and serially subcul-
tured in liquid medium containing interleukin-2 is identified
as NKC3.
Iodine I [K 8920, Special class]
Iodine for assay I [Same as the monograph Iodine]
Iodine monobromide IBr Blackish brown crystals or
masses.
It dissolves in water, in ethanol (95), in diethyl ether, in
carbon disulfide and in acetic acid (100).
Melting point <2.60>: 40°C
Storage — Preserve in light-resistant glass containers, in a
cold place.
Iodine-starch TS To 100 mL of starch TS add 3 mL of di-
lute iodine TS.
Iodine trichloride IC1 3 [K 8403, Special class]
Iodine TS Dissolve 14 g of iodine in 100 mL of a solution
of potassium iodide (2 in 5), add 1 mL of dilute hydrochloric
acid, and dilute with water to make 1000 mL (0.05 mol/L).
Storage — Preserve in light-resistant containers.
Iodine TS, dilute To 1 volume of iodine TS add 4
volumes of water.
0.0002 mol/L Iodine TS Measure exactly 1 mL of 0.5
mol/L iodine TS, add water to make exactly 250 mL, pipet
10 mL of the solution, and add water to make exactly 100
mL. Prepare before use.
0.5 mol/L Iodine TS To 12.7 g of iodine and 25 g of
potassium iodide add 10 mL of water, triturate, and add
water to make 100 mL.
Iodoethane C 2 H 5 I A colorless or a dark-brown, clear
liquid, having diethyl ether-like odor.
Distilling range <2.57>: 71.0 - 72.5°C, not less than 94 vol
JPXV
General Tests / Reagents, Test Solutions
205
Iodomethane CH 3 I [K 8919, Special class]
5-Iodouracil for liquid chromatography C4H3IN2O2
White, crystalline powder. Melting point: about 275°C (with
decomposition).
Purity — Dissolve 3 mg of 5-iodouracil for liquid chroma-
tography in a mixture of diluted methanol (1 in 25) to make
10 mL. Perform the test with 10 juL of this solution as direct-
ed under Liquid Chromatography <2.01>, according to the
operating conditions in the Purity under Idoxuridine Eye
Drops. Measure each peak area by the automatic integration
method over a time span of twice as long as the retention time
of the principal peak, and calculate the amount of 5-io-
douracil by the area percentage method: It shows the purity
of not less than 98.5%.
Content: not less than 98.5%. Assay — Weigh accurately
about 5 mg of 5-iodouracil for liquid chromatography, previ-
ously dried at 60°C for 3 hours under reduced pressure, dis-
solve in water to make exactly 250 mL. Perform the test with
this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and determine the absorbance^4 at the
wavelength of maximum absorption at about 282 nm.
Amount (mg) of 5-iodouracil (C4H3IN2O2)
A
265
X2500
Iotalamic acid for assay C11H9I3N2O4 [Same as the
monograph Iotalamic Acid]
Iron Fe Iron in the forms of strips, sheets, granules or
wires. Fe: not less than 97.7%. It is attracted by a magnet.
Iron (III) chloride-acetic acid TS Dissolve 0.1 g of iron
(III) chloride hexahydrate in diluted acetic acid (3 in 100) to
make 100 mL.
Iron (III) chloride hexahydrate FeCl 3 .6H 2 [K 8142,
Special class]
Iron (III) chloride-iodine TS Dissolve 5 g of iron (III)
chloride hexahydrate and 2 g of iodine in a mixture of 50 mL
of acetone and 50 mL of a solution of tartaric acid (1 in 5).
Iron (III) chloride-methanol TS Dissolve 1 g of iron (III)
chloride hexahydrate in methanol to make 100 mL.
Iron (III) chloride-potassium hexacyanoferrate (III) TS
Dissolve 0.1 g of potassium hexacyanoferrate (III) in 20 mL
of iron (III) chloride TS. Prepare before use.
Iron (III) chloride-pyridine TS, anhydrous Heat gradual-
ly 1.7 g of iron (III) chloride hexahydrate by direct applica-
tion of flame, melt, and solidify. After cooling, dissolve the
residue in 100 mL of chloroform, add 8 mL of pyridine, and
filter.
Iron (III) chloride TS Dissolve 9 g of iron (III) chloride
hexahydrate in water to make 100 mL (1/3 mol/L).
Iron (III) chloride TS, acidic To 60 mL of acetic acid
(100) add 5 mL of sulfuric acid and 1 mL of iron (III) chlo-
ride hexahydrate TS.
Iron (III) chloride TS, dilute Dilute 2 mL of iron (III)
chloride hexahydrate TS with water to make 100 mL. Prepare
before use.
Iron (III) nitrate enneahydrate Fe(N0 3 ) 3 .9H 2
[K 8559, Special class]
Iron (III) nitrate TS Dissolve 1 g of iron (III) nitrate en-
neahydrate in hydrochloric acid-potassium chloride buffer
solution (pH 2.0) to make 300 mL.
Iron (III) perchlorate-ethanol TS Dissolve 0.8 g of iron
(III) perchlorate hexahydrate in perchloric acid-ethanol TS to
make 100 mL.
Storage — Preserve in tight containers, in a cold place.
Iron (III) perchlorate hexahydrate Fe(C104) 3 .6H 2
Hygroscopic, light purple crystals, and a solution in ethanol
(99.5) (1 in 125) is clear and orange in color.
Iron (III) sulfate B-hydrate Fe 2 (S0 4 ) 3 .«H 2 [K
Special class]
SI,
Iron (III) sulfate TS Dissolve 50 g of iron (III) sulfate «-
hydrate in an excess of water, and add 200 mL of sulfuric
acid and water to make 1000 mL.
Iron (II) sulfate heptahydrate FeS0 4 .7H 2 [K 8978,
Special class]
Iron (II) sulfate TS Dissolve 8 g of iron (II) sulfate hepta-
hydrate in 100 mL of freshly boiled and cooled water. Pre-
pare before use.
Iron (II) sulfide
velopment]
FeS [K 8948, for hydrogen sulfide de-
Iron (II) tartrate TS Dissolve 1 g of iron (II) sulfate hep-
tahydrate, 2 g of potassium sodium tartrate tetrahydrate and
0.1 g of sodium hydrogen sulfite in water to make 100 mL.
Iron (II) thiocyanate TS Add 3 mL of dilute sulfuric acid
to 35 mL of water, and remove the dissolved oxygen by boil-
ing the solution. Dissolve 1 g of iron (II) sulfate heptahydrate
in this hot solution, cool, and then dissolve 0.5 g of potassi-
um thiocyanate. When the solution is pale red in color,
decolorize by adding reduced iron, separate the excess of
reduced iron by decanting, and preserve the solution with
protection from oxgen. Do not use a solution showing a pale
red color.
Iron (II) trisodium pentacyanoamine TS To 1.0 g of sodi-
um pentacyanonitrosylferrate (III) dihydrate add 3.2 mL of
ammonia water, shake, stopper closely, and allow to stand in
a refrigerator overnight. Add this solution to 10 mL of etha-
nol (99.5), filter a yellow colored precipitate by suction, wash
with ether (99.5), dry, and preserve in a desiccator. Before us-
ing, dissolve in water to make a solution of 1.0 mg/mL, and
store in a refrigerator. Use within 7 days after preparation.
Iron-phenol TS Dissolve 1.054 g of ammonium iron (II)
sulfate hexahydrate in 20 mL of water, add 1 mL of sulfuric
acid and 1 mL of hydrogen peroxide (30), heat until efferves-
cence ceases, and dilute with water to make 50 mL. To 3
volumes of this solution contained in a volumetric flask add
sulfuric acid, with cooling, to make 100 volumes, yielding the
iron-sulfuric acid solution. Purify phenol by distillation,
discarding the first 10% and the last 5%, and collect the dis-
tillate, with exclusion of moisture, in a dry, tared, glass-stop-
pered flask of about twice the volume of the phenol. Stopper
the flask, solidify the phenol in an ice bath, breaking the top
crust with a glass rod to ensure complete crystallization, and
after drying, weigh the flask. To the glass-stoppered flask add
1.13 times the mass of phenol of the iron sulfuric acid solu-
206
Reagents, Test Solutions / General Tests
JP XV
tion, insert the stopper in the flask, and allow to stand,
without cooling but with occasional shaking, until the phenol
is liquefied, then shake the mixture vigorously. Allow to
stand in a dark place for 16 to 24 hours. To the mixture add
diluted sulfuric acid (10 in 21) equivalent to 23.5% of its
mass, mix well, transfer to dry glass-stoppered bottles, and
preserve in a dark place, with protection from atmospheric
moisture. Use within 6 months.
Iron-phenol TS, dilute To 10 mL of iron-phenol TS add
4.5 mL of water. Prepare before use.
Iron powder Fe A lusterless, gray to grayish black pow-
der, being attracted by a magnet.
Identification — To 1 mL of a solution in hydrochloric acid
(1 in 50) add water to make 15 mL, and add 0. 1 mL of potas-
sium hexacyanoferrate (III) TS: a blue color appears.
Iron salicylate TS Dissolve 0.1 g of ammonium iron (III)
sulfate dodecahydrate in 50 mL of diluted sulfuric acid (1 in
250), and add water to make 100 mL. Measure 20 mL of this
solution, and add 10 mL of a solution of sodium salicylate
(23 in 2000), 4 mL of dilute acetic acid, 16 mL of sodium ace-
tate TS and water to make 100 mL. Prepare before use.
Isatin See 2,3-indolinedione.
Isoamyl acetate See 3-methylbutyl acetate.
Isoamyl alcohol See 3-methyl-l-butanol.
Isoamyl benzoate C 12 H 16 2
Specific gravity <2.56> d\ 5 : 0.993
Boiling point <2.57>: 260 - 262°C
Isoamyl parahydroxybenzoate C 12 H 16 3 White crystal-
line powder, having a faint characteristic odor.
It is very soluble in acetonitrile, in ethanol (95), in acetone
and in diethyl ether, and practically insoluble in water.
Melting point <2.60>: 62 - 64°C
Isobutanol See 2-methyl-l-propanol.
Isobutyl parahydroxybenzoate C u H 14 3 Colorless
crystals or white crystalline powder. Odorless. Freely soluble
in ethanol (95), in acetone and in diethyl ether, and practical-
ly insoluble in water.
Melting point <2.60>: 75 - 77°C
Residue on ignition <2.44>: not more than 0.1%.
Content: not less than 98.0%. Assay — Proceed as direct-
ed in the Assay under Ethyl Parahydroxybenzoate.
Each mL of 1 mol/L sodium hydroxide VS
= 194.2 mg of C u H 14 3
Isobutyl salicylate CnH 14 3 Colorless, clear liquid,
having a characteristic odor.
Refractive index <2.45> n™: 1.506-1.511
Specific gravity <2.56> df: 1.068 - 1.073
Boiling point <2.57>: 260 - 262°C
Purity — Perform the test with 1 /uL of isobutyl salicylate as
directed under Gas Chromatography <2.02> according to the
following conditions. Measure each peak area by the auto-
matic integration method, and calculate the amount of iso-
butyl salicylate by the area percentage method: It shows the
purity of not less than 97.0%.
Operating conditions
Detector: A thermal conductivity detector.
Column: A column about 3 mm in inside diameter and
about 2 m in length, packed with siliceous earth for gas chro-
matography, 180 to 250 ^m in particle diameter, coated with
polythylene glycol 20 M for gas chromatography at the ratio
of 10%.
Column temperature: A constant temperature of about 220
°C.
Carrier gas: Helium
Flow rate: About 20 mL per minute.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of isobutyl salicylate obtained from 1 fiL
of the sample solution is about 60% to 80% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of isobutyl salicylate beginning after the sol-
vent peak.
Isoelectric point markers for teceleukin Dissolve 0.02 to
0.05 mg of cytochrome C, trypsinogen, lentil-lectin basic
band, lentil-lectin middle band, lentil-lectin acidic band,
horse myoglobin basic band, horse myoglobin acidic band,
human carbonic anhydrase B, bovine carbonic anhydrase B,
and /Mactoglobulin A, in 0.1 mL of saccharose solution (3 in
10).
L-Isoleucine C 6 H 13 N0 2 [Same as the namesake mono-
graph]
Isoniazid for assay C 6 H 7 N 3 [Same as the monograph
Isoniazid. When dried, it contains not less than 99.0% of iso-
niazid (C 6 H 7 N 3 0).]
Isoniazid C 6 H 7 N 3 [Same as the namesake mono-
graph]
Isoniazid TS Dissolve 0.1 g of isoniazid for assay in a
mixture of 50 mL of methanol and 0.12 mL of hydrochloric
acid, and add methanol to make 200 mL.
Isonicotinic acid White, crystals or powder. Melting
point: about 315°C (decomposition).
Isonicotinic acid amide C 6 H 6 N 2 White, crystals or
crystalline powder.
Melting point <2.60>: 155 - 158°C
Purity Clarity of solution — Dissolve 1 .0 g of the sub-
stance to be tested in 20 mL of methanol: the solution is
clear.
Content: not less than 99.0%. Assay — Weigh accurately
about 0.3 g of isonicotinic acid amide, previously dried, and
dissolve in 20 mL of acetic acid (100) by heating. After cool-
ing, add 100 mL of benzene, and titrate <2.50> with 0.1 mol/
L perchloric acid VS until the color of the solution changes
from purple to blue-green (indicator: 3 drops of crystal violet
TS). Perform a blank determination and make any necessary
correction.
Each mL of 0.1 mol/L perchloric acid VS
= 11.21mg of C 6 H 6 N 2
Isooctane See octane, iso.
Isopromethazine hydrochloride for thin-layer chromato-
graphy Ci 7 H 20 N 2 S.HCl White, odorless, crystalline pow-
der. Freely soluble in water, in ethanol (95) and in chloro-
form, and practically insoluble in diethyl ether.
Melting point <2.60>: 193 - 197°C
Purity Related substances — Dissolve 5.0 mg of iso-
JPXV
General Tests / Reagents, Test Solutions
207
promethazine hydrochloride for thin-layer chromatography
in exactly 25 mL of ethanol, and perform the test with this so-
lution as directed in the Purity (3) under Promethazine Hy-
drochloride: any spot other than the principal spot at the Ri
value of about 0.65 does not appear.
Isopropanol See 2-propanol.
Isopropanol for liquid chromatography See 2-propanol
for liquid chromatography.
Isopropyl benzoate C 6 H 5 COOCH(CH 3 ) 2 A clear,
colorless liquid, having a characteristic odor.
Referactive index <2.45> n!§: 1.490 - 1.498
Specific gravity <2.56> df : 1.008 - 1.016
Isopropylamine See propylamine, iso.
Isopropylamine-ethanol TS To 20 mL of isopropylamine
add ethanol (99.5) to make 100 mL. Prepare before use.
Isopropylether See propylether, iso.
Isopropyl iodide for assay C 3 H 7 I Colorless, clear liq-
uid. On exposure to light it liberates iodine and becomes
brown. Miscible with ethanol (95), with diethyl ether and
with petroleum benzin, and not miscible with water. Use the
distillate obtained between 89.0°C and 89.5 °C.
Specific gravity <2.56> df: 1.700-1.710
Purity — Perform the test with 1 iiL of isopropyl iodide for
assay as directed under Gas Chromatography <2.02> accord-
ing to the operating conditions in the Assay under Hypromel-
lose. Measure each peak area by the automatic integration
method, and calculate the amount of isopropyl iodide by the
area percentage method: It shows the purity of not less than
99.8%. Adjust the detection sensitivity so that the peak
height of isopropyl iodide from 1 /uL of isopropyl iodide for
assay is about 80% of the full scale.
Content: not less than 98.0%. Assay — Transfer 10 mL of
ethanol (95) into a brown volumetric flask, weigh accurately,
add 1 mL of isopropyl iodide for assay, and weigh accurately
again. Add ethanol (95) to make exactly 100 mL, pipet 20 mL
of this solution into the second brown volumetric flask, add
exactly 50 mL of 0.1 mol/L silver nitrate VS and then 2 mL
of nitric acid, stopper, shake occasionally for 2 hours in a
dark place, and allow to stand overnight in a dark place.
Shake occasionally for 2 hours, add water to make exactly
100 mL, and filter through dry filter paper. Discard the first
20 mL of the filtrate, pipet the next 50 mL, and titrate <2.50>
the excess silver nitrate with 0.1 mol/L ammonium thio-
cyanate VS (indicator: 2 mL of ammonium iron (III) sulfate
TS). Perform a blank determination in the same manner.
Each mL of 0.1 mol/L silver nitrate VS
= 17.00 mg of C 3 H 7 I
Isopropyl myristate C 17 H 34 2 Colorless, clear, oily liq-
uid, and odorless. Congeals at about 5°C. Soluble in 90% al-
cohol, miscible with many organic solvents and with solid
oils, and insoluble in water, in glycerin and in propylene gly-
col.
Refractive index <2.45> n 2 °: 1.432-1.436
Specific gravity <2.56> df : 0.846 - 0.854
Saponification value <1.I3>: 202 - 212
Acid value <1.13>; not more than 1.
Iodine value <1J3>: not more than 1.
Residue on ignition <2.44>: not more than 0.1% (1 g).
Isopropyl myristate for sterility test C I7 H 34 2 Transfer
100 mL of isopropyl myristate into a centrifuge tube, add 100
mL of twice-distilled water, and shake vigorously for 10
minutes. Then centrifuge at a rate of 1800 revolutions per
minute for 20 minutes, separate the supernatant liquid
(isopropyl myristate layer), and determine the pH of the
residual water layer: not less than 5.5.
Treat isopropyl myristate which meets the requirements of
pH determination as follows: 500 mL of isopropyl myristate,
which has met the requirements of pH determination, is per-
colated through a 15-cm high layer of activated alumina filled
in a glass column 20 mm in diameter and 20 cm in length with
a slightly positive pressure in order to facilitate adequate
flow, and then sterilized by filtration.
Isopropyl p-aminobenzoate See isopropyl 4-aminobenzo-
ate.
Isopropyl 4-aminobenzoate
Pale brown crystals.
Melting point <2.60>: 83 - 86°C
NH 2 C 6 H 4 COOCH(CH 3 ) 2
Isopropyl p-hydroxybenzoate See isopropyl parahydrox-
ybenzoate.
Isopropyl parahydroxybenzoate C I0 H 12 O 3 Odorless
and colorless fine crystals, or white, crystalline powder. Free-
ly soluble in ethanol (95), in acetone and in diethyl ether, and
very slightly soluble in water.
Melting point <2.60>: 84 - 86°C
Residue on ignition <2.44>: not more than 0.1%.
Content: not less than 99.0%. Assay — Proceed as direct-
ed in the Assay under Ethyl Parahydroxybenzoate.
Each mL of 1 mol/L sodium hydroxide VS
= 180.2 mg of C 10 H 12 O 3
Isotonic sodium chloride solution [Same as the namesake
monograph]
Japanese acid clay Natural hydrous aluminum silicate,
grayish white powder, having a particle size of about 74 iim.
Loss on drying <2.41>: not more than 10% (1 g, 105°C, 4
hours).
Water adsorbing capacity: not less than 2.5%. Weigh ac-
curately about 10 g of Japanese acid clay in weighing bottle,
allow to stand for 24 hours with cover in a chamber, in which
humidity is maintained to 80% by means of sulfuric acid
(specific gravity 1.19), reweigh, and determine the increase of
mass of the sample.
Jesaconitine for purity C 35 H 49 NOi 2 A white powder.
Freely soluble in acetonitrile, in ethanol (99.5) and in diethyl
ether, and practically insoluble in water.
Identification — Determine the infrared absorption spec-
trum of jesaconitine for purity as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3500 cm" 1 , 1715 cm" 1 , 1607 cm" 1 , 1281cm" 1 , 1259 cm" 1 ,
1099 cm" 1 and 772 cm" 1 .
Absorbance <2.24> E\ a/ ° m (258 nm): 270 - 291 [5 mg dried
for not less than 12 hours in a desiccator (reduced pressure
not exceeding 0.67 kPa, phosphorus (V) oxide, 40°C),
ethanol (99.5), 200 mL].
Purity Related substances — (1) Dissolve 5.0 mg of
208
Reagents, Test Solutions / General Tests
JP XV
jesaconitine for purity in 2 mL of acetonitrile, and use as the
sample solution. Pipet 1 mL of the sample solution, add
acetonitrile to make exactly 50 mL, and use as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 /xL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography, and proceed the test as
directed in the Identification in Processed Aconite Root: the
spot other than the principal spot is not more intense than the
spot with the standard solution.
(2) Dissolve 5.0 mg of jesaconitine for purity in 5 mL of
acetonitrile, and use as the sample solution. Pipet 1 mL of the
sample solution, add acetonitrile to make exactly 50 mL, and
use as the standard solution. Perform the test with exactly 10
/xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peaks of jesaconitine and the solvent is not
larger than the peak area of jesaconitine with the standard so-
lution.
Operating conditions
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity under Proc-
essed Aconite Root.
Mobile phase: A mixture of phosphate buffer solution for
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust the flow rate so that the retention time of
jesaconitine is about 36 minutes.
Time span of measurement: About 3 times as long as the
retention time of jesaconitine.
System suitability
Test for required detectability: Pipet 1 mL of the standard
solution, and add acetonitrile to make exactly 20 mL.
Confirm that the peak area of jesaconitine obtained from
10 /xL of this solution is equivalent to 3.5 to 6.5% of that
obtained from 10 /xL of the standard solution.
System performance: Dissolve 5 mg each of aconitine for
purity, hypaconitine for purity and mesaconitine for purity,
and 1 mg of jesaconitine for purity in 200 mL of acetonitrile.
When the procedure is run with 10 /xL of this solution under
the above operating conditions, mesaconitine, hypaconitine,
aconitine and jesaconitine are eluted in this order, and each
resolution between these peaks is not less than 1.5, respec-
tively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
jesaconitine is not more than 1.5%.
Water <2.48>: not more than 1.0% [5 mg dried for not less
than 12 hours in a desiccator (reduced pressure not exceeding
0.67 kPa, phosphorus (V) oxide, 40°C), coulometric
titration].
Josamycin C4 2 H 69 NO I5 [Same as the namesake mono-
graph]
Josamycin propionate C 45 H 73 NO I6 [Same as the
namesake monograph]
Kainic acid QoH^NCvHjO [Same as the monograph
Kainic Acid Hydrate]
Kainic acid for assay [Same as the monograph Kainic
Acid Hydrate]
Kanamycin sulfate C 1 8H3 6 N 4 11 .xH2S04 [Same as the
namesake monograph]
Karl Fischer TS See Water Determination <2.48>.
Kerosene It is mainly a mixture of hydrocarbons in the
methane series, and a colorless, clear liquid, having not a dis-
agreeable, characteristic odor.
Specific gravity <2.56>: about 0.80
Distilling range <2.57>: 180 - 300°C
Kininogen Produced by purifying from bovine plasma.
Dissolve an appropriate amount of kininogen in 0.02 mol/L
phosphate buffer solution, pH 8.0 so that 10 mL of the solu-
tion contains 1 mg of kininogen, and use this solution as the
sample solution. Perform the following tests with the sample
solution: it meets the requirement of each test.
(i) Immediately after the sample solution is prepared, add
0. 1 mL of a solution of trichloroacetic acid (1 in 5) to 0.5 mL
of the sample solution, shake, and centrifuge. To 0.5 mL of
the supernatant liquid add 0.5 mL of gelatin-tris buffer solu-
tion, pH 8.0, and shake. To 0.1 mL of this solution add 1.9
mL of trichloroacetic acid-gelatin-tris buffer solution. Pro-
ceed with 0.1 mL of this solution as directed in the Purity (2)
under Kallidinogenase, and determine the amount of kinin:
kinin is not detected.
(ii) Warm 0.5 mL of the sample solution at 30 ± 0.5 °C
for 20 minutes, and proceed as directed in (i): kinin is not de-
tected.
(iii) Perform the test with 0.5 mL of the sample solution
as directed in the Purity (2) under Kallidinogenase: the
decomposition of bradykinin is not observed.
(iv) To 0.5 mL of the sample solution add 0.5 mL of 0.02
mol/L phosphate buffer solution, pH 8.0 containing 500 /xg
of crystal trypsin, previously warmed at 30 ± 0.5°C for 5
minutes, warm this solution at 30 ± 0.5°C for 5 minutes,
add 0.2 mL of a solution of trichloroacetic acid (1 in 5), and
shake. Then boil for 3 minutes, cool in ice immediately, and
centrifuge. To 0.5 mL of the supernatant liquid add 0.5 mL
of gelatin-tris buffer solution, pH 8.0, and
shake. To 0.1 mL of this solution add 0.9 mL of trichloro-
acetic acid-gelatin-tris buffer solution. To 0.1 mL of this solu-
tion add trichloroacetic acid-gelatin-tris buffer solution to
make 20 mL, then proceed as directed in (i), and determine
the amount, B K , of kinin per well. Calculate the kinin-releas-
ing activity per mg by the following equation: not less than 10
/xg bradykinin equivalent per mg.
Kinin-releasing activity per mg (jxg bradykinin
equivalent /mg) = B K x 0.0096
Kininogen TS Dissolve a sufficient quantity of kininogen
in 0.02 mol/L phosphate buffer solution, pH 8.0 to prepare a
solution having an ability in each mL to release kinin cor-
responding to not less than 1 /xg of bradykinin.
Lactic acid CH 3 CH(OH)COOH [K 8726, Special class]
Lactic acid TS Dissolve 12.0 g of lactic acid in water to
make 100 mL.
ce-Lactoalbumin White powder. Derived from milk.
Molecular weight of about 14,200.
Lactobionic acid Q2H22O12 Colorless crystals or white
crystalline powder, having no odor.
Melting point <2.60>: 113 - 118°C
JPXV
General Tests / Reagents, Test Solutions
209
Purity — Dissolve 0.10 g of lactobionic acid in 10 mL of a
mixture of methanol and water (3:2), and perform the test
with 10 fiL of this solution as directed in the Identification (2)
under Erythromycin Lactobionate: the spot other than the
principal spot is not found.
/?-LactoglobuIin Prepare from milk. White to light yel-
low powder.
Nitrogen content <1.08>: not less than 14% (calculated on
the dried basis).
Lactose See lactose monohydrate.
a-Lactose and /Mactose mixture (1:1) Use a mixture of
lactose monohydrate and anhydrous lactose (3:5).
Lactose broth After adding lactose monohydrate to ordi-
nary broth in the ratio of 0.5%, add about 12 mL of bromo-
thymol blue-sodium hydroxide TS to 1000 mL of the medi-
um. Then dispense portions of about 10 mL into tubes for
fermentation, and sterilize fractionally on each of three suc-
cessive days for 15 to 30 minutes at 100°C by using an au-
toclave, or sterilize by autoclaving for not more than 20
minutes at 121 °C, and cool quickly by immersing in cold
water.
Lactose broth, three times concentrated Add lactose
monohydrate to ordinary broth prepared by using 330 mL in
place of 1000 mL of water in the ratio of 1.5%, and prepare
according to the method of preparation under lactose broth,
with 25 mL portions in tubes for fermentation.
Lactose broth, twice concentrated Add lactose monohy-
drate to ordinary broth prepared by using 500 mL in place of
1000 mL of water in the ratio of 1 .0% and prepare according
to the method of preparation under lactose broth.
Lactose monohydrate C12H22O11.H2O [Same as the
monograph Lactose].
Lactose substrate TS Dissolve 6.0 g of lactose monohy-
drate in diluted disodium hydrogen phosphate-citric acid
buffer solution, pH 4.5 (1 in 10) to make 100 mL.
Lactose substrate TS for /?-galactosidase (penicillium)
Dissolve 6.0 g of lactose monohydrate in diluted disodium
hydrogen phosphate-citric acid buffer solution, pH 4.5 (1 in
10) to make 100 mL.
Lanthanum-alizarin complexone TS To 1 mL of ammo-
nia water (28) add 10 mL of water. To 4 mL of this solution
add 4 mL of a solution of ammonium acetate (1 in 5) and 192
mg of alizarin complexone, and label this solution as alizarin
complexone stock solution. Dissolve 41 g of sodium acetate
trihydrate in 400 mL of water, and add 24 mL of acetic acid
(100). To this solution add the total volume of the alizarin
complexone stock solution, add 400 mL of acetone, and label
this solution as alizarin complexone solution. To 10 mL of
diluted hydrochloric acid (1 in 6) add 163 mg of lanthanum
(III) oxide, heat to dissolve, and label this solution as lantha-
num solution. To the alizarin complexone solution add the
lanthanum solution, and mix. After cooling, adjust to pH 4.7
with acetic acid (100) or ammonia water (28), and add water
to make 1000 mL. Prepare before use.
Lanthanum (III) oxide La 2 3 White crystals.
Loss on ignition <2.43>: not more than 0.5% (1 g, 1000°C,
1 hour)
Lauromacrogol [Same as the namesake monograph]
Lead acetate See lead (II) acetate trihydrate.
Lead acetate TS See lead (II) acetate TS.
Lead (II) acetate TS To 9.5 g of lead (II) acetate trihy-
drate add freshly boiled and cooled water to make 100 mL.
Preserve in tightly stoppered bottles (0.25 mol/L).
Lead dioxide See lead (IV) oxide.
Lead (II) acetate trihydrate Pb(CH 3 COO) 2 .3H 2
[K 8374, Special class]
Lead (II) nitrate Pb(N0 3 ) 2 [K 8563, Special class]
Lead (II) oxide PbO [K 8090, Special class]
Lead (IV) oxide Pb0 2 A dark brown to black-brown,
powder or granules.
Identification — A supernatant liquid of a solution in dilute
acetic acid (1 in 100) responds to Quality Tests <1.09> (3) for
lead salt.
Lead monoxide See lead (II) oxide.
Lead nitrate See lead (II) nitrate.
Lead subacetate TS Place the yellowish mixture, ob-
tained by triturating 3 g of lead (II) acetate trihydrate and 1 g
of lead (II) oxide with 0.5 mL of water, in a beaker, and heat
on a water bath, covering with a watch glass, until it shows a
homogeneous, white to reddish white color. Then add 9.5 mL
of hot water in small portions, cover it again with a watch g-
lass, and set it aside. Decant the supernatant liquid, and ad-
just the specific gravity to 1.23 to 1.24 (15 °C) by adding
water. Preserve in tightly stoppered bottles.
Lead subacetate TS, dilute To 2 mL of lead subacetate
TS add freshly boiled and cooled water to make 100 mL. Pre-
pare before use.
L-Leucine C 6 H 13 N0 2 [Same as the namesake monograph]
Levallorphan tartrate for assay C 19 H 25 NO.C 6 H 6 6
[Same as the monograph Levallorphan Tartrate.
When dried, it contains not less than 99.0% of
C 19 H 25 NO.QH 6 6 .]
Levothyroxine sodium C^HnLNNaCvx^O [Same as
the monograph Levothyroxine Sodium Hydrate]
Levothyroxine sodium for thin-layer chromatography
[Same as the monograph Levothyroxine Sodium Hydrate.
Proceed the test as directed in the Identification (3) under
Levothyroxine Sodium Hydrate: any spot other than the
principal spot at the Ri value of about 0.26 does not appear.
Lidocaine for assay (C 14 H 22 N 2 0) [same as the mono-
graph Lidocaine]
Limonene C I0 H 16 Clear and colorless liquid, having a
specific perfume and a bitter taste.
Refractive index <2.45> n$: 1.427 - 1.474
Specific gravity <2.56> df : 0.841 -0.846
Melting point <2.60>: 176 - 177°C
Purity Related substances — Dissolve 0.1 g of limonene in
25 mL of hexane and use this solution as the sample solution.
Perform the test with 2//L of the sample solution as directed
210
Reagents, Test Solutions / General Tests
JP XV
under Gas Chromatography <2.02> according to the follow-
ing conditions. Measure each peak area by the automatic in-
tegration method and calculate the amount of limonene: it is
not less than 97.0%.
Operating conditions
Proceed the operating conditions in the Assay under Eu-
calyptus Oil except detection sensitivity and time span of
measurement.
Detection sensitivity: Measure 1 mL of limonene, add hex-
ane to make 100 mL, and adjust the detection sensitivity so
that the peak height of limonene obtained from 2 liL of this
solution is 40% to 60% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of limonene beginning after the solvent peak.
(Z)-Ligustilide for thin-layer chromatography Ci2H 14 2
A clear, yellow-grown liquid, having a characteristic odor.
Miscible with methanol and with ethanol (99.5), and practi-
cally insoluble in water.
Identification — Determine the absorption spectrum of a
solution in methanol (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 320 nm and 324 nm.
Purity Related substances — Dissolve 1 mg in 10 mL of
methanol. Proceed the test with 1 iiL of this solution as
directed in the Identification (5) under Hochuekkito Extract:
no spot other than the principle spot of around Rf 0.6 ap-
pears.
Liothyronine sodium Ci 5 H u I 3 NNa0 4
namesake monograph]
[Same as the
Liothyronine sodium for thin-layer chromatography
[Same as the monograph Liothyronine Sodium. Proceed as
directed for the Identification (1) under Liothyronine Sodium
Tablets: any spot other than the principal spot at the Rf value
of 0.3 to 0.4 does not appear.]
Liquid paraffin See paraffin, liquid.
Liquiritin for thin-layer chromatography C21H22O9.XH2O
White crystals or crystalline powder. Sparingly soluble in
methanol, slightly soluble in ethanol (99.5), and practically
insoluble in water. Melting point: about 210°C (with decom-
position).
Identification — Determine the absorption spectrum of a
solution in diluted methanol (1 in 2) (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits maxima between 215 nm and 219 nm, and between 275
nm and 279 nm.
Purity Related substances — Dissolve 1.0 mg in 1 mL of
methanol, and perform the test with 1 /uL of this solution as
directed in the Identification (5) under Kakkonto Extract: no
spot other than the principal spot (Rf value is about 0.4) ap-
pears.
Lisinopril C 2 iH 31 N 3 5 .2H 2 [Same as the monograph
Lisinopril Hydrate]
Lisinopril for assay C 2 iH 31 N 3 5 .2H 2 [Same as the
monograph Lisinopril Hydrate. It contains not less than 99.5
% of lisinopril (C 2 iH 31 N 3 5 : 405.49), calculated on the anhy-
drous basis.]
Lithium acetate dihydrate CH 3 COOLi.2H 2 Colorless
crystals.
Dilute acetic acid insoluble substances — To 40.0 g of lithi-
um acetate dihydrate add 45 mL of water, heat in a water
bath to dissolve, cool, then dissolve in dilute acetic acid, and
filter by suction. Wash the filter with water, dry the filter at
105 ± 2°C for 1 hour, and weigh the mass of the residue af-
ter cooling: not more than 0.0025%.
Content: not less than 97.0%. Assay — Weigh accurately
0.3 g of lithium acetate dihydrate, add exactly 50 mL of
acetic acid (100) and exactly 5 mL of acetic anhydride, dis-
solve by heating in a water bath, and titrate <2.50> with 0.1
mol/L perchloric acid VS after cooling (potentiometric titra-
tion). Perform a blank determination in the same manner,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 10.20 mg of CH 3 COOLi.2H 2
Lithium bromide LiBr White crystals or crystalline pow-
der. It is hygroscopic.
Purity (1) Chloride <1.03>: not more than 0.1%.
(2) Sulfate <1.14>: not more than 0.01%.
Lithium chloride LiCl White crystals or masses.
Identification — Perform the test as directed under Flame
Coloration Test (1) <1.04>: a persistent red color appears.
Lithium sulfate See lithium sulfate monohydrate.
Lithium sulfate monohydrate Li 2 S0 4 .H 2 [K 8994,
Special class]
Lithocholic acid for thin-layer chromatography
C 2 4H 40 O 3 White crystals or crystalline powder. Soluble in
ethanol (95), in acetic acid (100) and in acetone, slightly solu-
ble in chloroform, and practically insoluble in water. Melting
point: about 186°C.
Purity Related substances — Dissolve 25 mg of lithocholic
acid for thin-layer chromatography in a mixture of chloro-
form and ethanol (95) (9:1) to make exactly 25 mL. Dilute 1.0
mL of this solution with a mixture of chloroform and ethanol
(95) (9:1) to make exactly 100 mL. Perform the test with 10
jXL of this solution as directed in the Purity (7) under Ur-
sodeoxycholic Acid: any spot other than the principal spot
with the Rf value of about 0.7 does not appear.
Content: 98.0%. Assay — Weigh accurately about 0.5 g
of lithocholic acid for thin-layer chromatography, previously
dried at 80°C for 4 hours under reduced pressure (phospho-
rus (V) oxide), dissolve in 40 mL of neutralized ethanol and
20 mL of water. Add 2 drops of phenolphthalein TS, titrate
<2.50> with 0.1 mol/L sodium hydroxide VS, add 100 mL of
freshly boiled and cooled water near the end point, and con-
tinue the titration.
Each mL of 0.1 mol/L sodium hydroxide VS
= 37.66 mg of C 2 4H 40 H 3
Locke-Ringer's TS
Sodium chloride
Potassium chloride
Calcium chloride dihydrate
Magnesium chloride hexahydrate
Sodium hydrogen carbonate
Dextrose
Water, freshly distilled with
a hard-glass apparatus
9.0 g
0.42 g
0.24 g
0.2 g
0.5 g
0.5 g
a sufficient quantity
To make
1000 mL
Prepare before use. The constituents except dextrose and
JPXV
General Tests / Reagents, Test Solutions
211
sodium hydrogen carbonate can be made up in concentrated
stock solutions, stored in a dark place, and diluted before
use.
Loganin for thin-layer chromatography Ci 7 IT 26 Oio
White, crystals or crystalline powder. Soluble in water,
sparingly soluble in methanol, and very slightly soluble in
ethanol (99.5). Melting point: 221 -227°C.
Purity Related substances — Dissolve 1.0 mg of loganin
for thin-layer chromatography in 2 mL of methanol. Per-
form the test with 10 /uL of this solution as directed in the
Identification under Cornus Fruit: any spot other than the
principal spot at the Rf value of about 0.4 does not appear.
Low-molecular weight heparin for calculation of molecu-
lar mass.
It is a low-molecular weight heparin with a disaccharide
unit prepared, and display the molecular mass distribution
between 600 and more than 10,000. When the average of
molecular mass of Low-molecular weight heparin interna-
tional standard is determined as a reference with this, the
difference compared as a reference with the Low-molecular
weight heparin international standard is not less than 5%.
Luteolin for thin-layer chromatography C 15 H 10 O 6
Light yellow to yellow-brown crystalline powder. Slightly
soluble in methanol and in ethanol (99.5), and practically in-
soluble in water. Melting point: about 310°C (with decompo-
sition).
Purity Related substances — Dissolve 1.0 mg of luteolin
for thin-layer chromatography in 1 mL of methanol. Proceed
the test with 10 fiL of this solution as directed in the Identifi-
cation under Chrysanthemum Flower: any spot other than
the principal spot of Rf about 0.7 does not appear.
Lysate reagent A lyophilized product obtained from
amebocyte lysate of horseshoe crab (Limulus polyphemus or
Tachypleus tridentatus). Amebocyte lysate preparations
which do not react to /?-glucans are available: they are pre-
pared by removing the G factor reacting to /3-glucans from
amebocyte lysate or by inhibiting the G factor reacting sys-
tem of amebocyte lysate.
Lysate TS Dissolve a lysate reagent in water for bacterial
endotoxins test, or in a suitable buffer, by gentle stirring.
Lysil endopeptidase White powder or masses, An exotox-
in produced by Achromobacter. Molecular weight: 27,500.
L-Lysine hydrochloride C 6 H I4 N 2 02.HC1 [Same as the
namesake monograph]
Macrogol 600 HOCH 2 (CH 2 OCH 2 )nCH 2 OH, n=ll-13
Clear, colorless, viscous liquid or a white, petrolatum-like
solid, having a faint, characteristic odor. Very soluble in
water, in ethanol (95), in acetone and in macrogol 400, solu-
ble in diethyl ether, and practically insoluble in petroleum
benzine. Congealing point: 18-23°C
Average molecular weight: When perform the test as
directed in the Average molecular weight test under Macrogol
400, it is between 570 and 630.
4-(N-Maleimidylmethyl)-cyclohexane-l-carboxylate-N-
hydroxysuccinimide ester C 16 H 18 N 2 6 Colorless crystals.
Being decomposed by acid and alkaline treatment.
Magnesia TS Dissolve 5.5 g of magnesium chloride hexa-
hydrate and 7 g of ammonium chloride in 65 mL of water,
add 35 mL of ammonia TS, allow the mixture to stand for a
few days in tightly stoppered bottles, and filter. If the solu-
tion is not clear, filter before use.
Magnesium Mg [K 8875, Special class]
Magnesium chloride See magnesium chloride hexahy-
drate.
Magnesium chloride hexahydrate MgCl 2 .6H 2
[K 8159, Special class]
Magnesium nitrate See magnesium nitrate hexahydrate.
Magnesium nitrate hexahydrate Mg(N0 3 ) 2 .6H 2
[K 8567, Special class]
Magnesium oxide MgO [K 8432, Special class]
Magnesium powder Mg [K 8876, Special class]
Magnesium sulfate See magnesium sulfate heptahy-
drate.
Magnesium sulfate heptahydrate MgS0 4 .7H 2
[K 8995, Special class]
Magnesium sulfate TS Dissolve 12 g of magnesium sul-
fate hexahydrate in water to make 100 mL (0.5 mol/L).
Magneson [K 8879, Special class]
Magneson TS Dissolve 0.1 g of magneson in 100 mL of
A^TV-dimethylformamide.
Magnolol for component determination Ci 8 H 18 2
Odorless white, crystals or crystalline powder. Freely soluble
in methanol and in diethyl ether, and practically insoluble in
water. Melting point: about 102 C C.
Absorbance <2.24> E[ * (290 nm): 270 - 293 [0.01 g dried
for 1 hour or more a desiccator (silica gel), methanol, 500
mL].
Purity Related substances — (1) Dissolve 1.0 mg of
magnolol for component determination in exactly 1 mL of
methanol, and perform the test with this solution as directed
under Thin-layer Chromatography <2.03>. Spot 10 fiL of the
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography, then develop the plate with a
mixture of hexane, acetone and acetic acid (100) (20:15:1) to
a distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): any spot
other than the principal spot at the Rf value of about 0.5 does
not appear.
(2) Dissolve 5.0 mg of magnolol for component determi-
nation in 10 mL of the mobile phase, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the area of each
peak from these solutions by the automatic integration
method: the total area of peaks other than the peak of mag-
nolol from the sample solution is not larger than the peak
area of magnolol from the standard solution.
Operating conditions
Proceed the operating conditions under Magnolia Bark ex-
cept detection sensitivity and time span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution,
212
Reagents, Test Solutions / General Tests
JP XV
add the mobile phase to make exactly 20 mL. Adjust the sen-
sitivity so that the peak area of magnolol obtained with 10 /xL
of this solution can be measured, and is about 20% of full-
scale by the automatic integration method.
Time span of measurement: About 3 times as long as the
retention time of magnolol beginning after the solvent peak.
Malachite green See malachite green oxalate.
Malachite green oxalate C 52 H5 4 N 2 I2 [K 8878,
Malachite green (oxalate), Special class]
Maleic acid C 4 H 4 4 [K 8884, Special class]
Maltose See maltose monohydrate.
Maltose monohydrate C 12 H 22 0n.H 2 [Same as the
namesake monograph].
Manganese dioxide Mn0 2 Black to black-brown, mass-
es or powder.
Identification — To 0.5 g add 20 mL of water and 3 mL of
hydrochloric acid, and 3 mL of hydrogen peroxide (30).
Alkalinize the solution with ammonia solution (28) while
cooling, and add 25 mL of hydrogen sulfide TS: pale red
precipitates appear.
D-Mannitol
Mannitol]
C 6 H 14 6 [Same as the monograph D-
D-Mannose C 6 H 12 6 White crystal or crystalline pow-
der. It is very soluble in water. Melting point: about 132°C
(with decomposition).
Optical rotation <2.49> [a]™: + 13.7 - + 14.7° (4 g, diluted
ammonia TS (1 in 200), 20 mL, 100 mm).
Marker protein for celmoleukin molecular weight determi-
nation Add 10 /xL of cytochrome C prepared to a concen-
tration of 2 mg per mL to 10 /uL of a commercially available
marker protein with a known molecular weight (6 in-
gredients: phosphorylase b, bovine serum albumin, ovalbu-
min, carbonic dehydratase, soy trypsin inhibitor, and lyso-
zyme) and then dilute 10-fold with buffer solution for cel-
moleukin.
Identification: Use the solution to be examined as the sam-
ple solution. Separately, to an amount of cytochrom C add
distilled water for injection so that each mL contains 100 mg
of protein, and use this as the standard solution. When 20 /xL
each of the sample solution and standard solution are tested
using SDS polyacrylamide gel electrophoresis under the oper-
ating conditions outlined in the Identification (3) of Cel-
moleukin (Genetical Recombination), the sample solution ex-
hibits 7 major electrophoretic bands. Furthermore, the
degree of mobility of the sample solution cytochrome C is
consistent with that of the band obtained from the standard
solution.
Meat extract Concentrated extract of fresh meat of
bovine, equine or other animals. A yellow-brown to dark
brown paste-like mass, having a meat-like odor.
Medium for float culture Dissolve 6.000 g of sodium
chloride, 0.400 g of potassium chloride, 0.677 g of anhydrous
sodium dihydrogen phosphate (NaH 2 P0 4 ), 0.100 g of calci-
um nitrite tetrahydrate, 0.100 g of magnesium sulfate hy-
drate, 2.000 g of glucose, 0.164 g of sodium succinate hexa-
hydrate, 46 mg of succinic acid, 0.240 g of L-arginine
hydrochloride, 56.8 mg of L-asparagine monohydrate, 20 mg
of L-aspartic acid, 72.9 mg of L-cysteine hydrochloride
monohydrate, 20 mg of L-glutamic acid, 1 mg of glutathione,
10 mg of glycine, 20.3 mg of L-histidine hydrochloride mono-
hydrate, 20 mg of L-hydroxyproline, 50 mg of L-isoleucine,
40 mg of L-lysine hydrochloride, 15 mg of methionine, 20 mg
of L-threonine, 5 mg of L-tryptophan, 20 mg of L-valine, 50
mg of L-leucine, 15 mg of L-phenylalanine, 20 mg of L-pro-
line, 30 mg of L-serine, 20 mg of L-tyrosine, 0.2 mg of D-bio-
tin (crystals), 0.25 mg of calcium pantothenate, 3 mg of cho-
line chloride, 35 mg of /-inositol, 1 mg of 4-aminobenzoic
acid, 5 /ug of cyanocobalamin, 1 mg of folic acid, 1 mg of
nicotinamide, 0.2 mg of riboflavin, 1 mg of thiamine
hydrochloride, 1 mg of pyridoxine hydrochloride, and 5 mg
of phenol red in a suitable amount of water, add 1 mL of
kanamycin sulfate solution (3 in 50), add water to make 1000
mL, and then sterilize by autoclaving for 15 minutes at 121°
C. After cooling, add 10 mL of L-glutamine solution (3 in
100) and 20 mL of 7% sodium bicarbonate injection, and
then mix. Store at 4°C.
Mefruside for assay Ci3H 19 ClN 2 5 S 2 [Same as the
monograph Mefruside. When dried, it contains not less than
99.0% of mefruside (C 13 H 19 C1N 2 5 S 2 ).]
Meglumine C7IL7NO5 [same as the namesake mono-
graph]
Mentha oil [Same as the namesake monograph]
Menthol C 10 H 20 O [Same as the monograph tf/-Menthol
or /-Menthol]
/-Menthol for assay [Same as the monograph /-Menthol.
It contains not less than 99.0% of C 10 H 20 O and meets the fol-
lowing additional specifications.]
Optical rotation <2.49> [a]™: -48.0- -51.0° (2.5 g, eth-
anol (95), 25 mL, 100 mm).
Purity Related substances — Dissolve 0.10 g of /-menthol
for assay in 10 mL of dichloromethane, and use this solution
as the sample solution. Pipet 1 mL of this solution, add
dichloromethane to make exactly 100 mL, and use this solu-
tion as the standard solution (1). Perform the test with exact-
ly 5 juL each of the sample solution and standard solution (1)
as directed under Gas Chromatography <2.02> according to
the following conditions, measure each peak area of these so-
lutions by the automatic integration method: the total peak
area other than the peak area of /-menthol from the sample
solution is not larger than the peak area of /-menthol from
the standard solution (1).
Operatin conditions
Proceed the operating conditions in the Assay under Men-
tha Oil except detection sensitivity and time span of measure-
ment.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add dichloromethane to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of /-menthol obtained from 5
iXL of the standard solution (2) can be measured, and the
peak height of /-menthol from 5 fiL of the standard solution
(1) is about 20% of the full scale.
Time span of measurement: About twice as long as the
retention time of /-menthol beginning after the solvent peak.
Mepivacaine hydrochloride for assay
C 15 H 22 N 2 0.HC1 [Same as the monograph Mepivacaine
Hydrochloride. When dried, it contains not less than 99.0%
JPXV
General Tests / Reagents, Test Solutions
213
of mepivacaine hydrochloride (Ci 5 H 2 2N 2 O.HCl).]
Mercapto acetic acid HSCH 2 COOH [K 8630, Special
class] Place in an ampule, and preserve in a dark, cold
place. Do not use after storing for a long period.
2-Mercaptoethanol HSCH 2 CH 2 OH Clear and colorless
liquid.
Specific gravity <2.56> elf: 1.112 - 1.117
Content: not less than 97.0%. Assay — Perform the test
with 0.6 fiL of the substance to be examined as directed under
Gas Chromatography <2.02> according to the following con-
ditions, and determine the peak areas of each component by
the automatic integration method.
Content (%) = (the peak area of 2-mercaptoethanol/the total
of the peak areas of each component) x 100
Operating conditions
Detector: A hydrogen flame-ionization detector
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with siliceous earth for gas chro-
matography (177-250 fxva in particle diameter) coated in 20%
with 50% phenyl-methyl silicone polymer for gas chro-
matography.
Column temperature: A constant temperature of about 120
°C
Carrier gas: Helium
Flow rate: Adjust the flow rate of about 50 mL/min and so
that the retention time of 2-mercaptoethanol is 3-4 minutes.
Time span of measurement: About 7 times as long as the
retention time of 2-mercaptoethanol.
Mercaptopurine C 5 H 4 N 4 S.H 2 [Same as the mono-
graph Mercaptopurine Hydrate]
Mercuric acetate See mercury (II) acetate.
Mercuric acetate TS for nonaqueous titration See mercu-
ry (II) acetate TS for nonaqueous titration.
Mercuric chloride See mercury (II) chloride.
Mercury Hg [K 8572, Special class]
Mercury (II) acetate Hg(CH 3 COO) 2 [K 8369, Special
class]
Mercury (II) acetate TS for nonaqueous titration Dis-
solve 5 g of mercury (II) acetate in acetic acid (100) for non-
aqueous titration to make 100 mL.
Mercury (II) chloride HgCl 2 [K 8139, Special class]
Mercury (II) chloride TS Dissolve 5.4 g of mercury (II)
chloride in water to make 100 mL.
Mesaconitine for purity C33H45NO11 White, crystals or
crystalline powder. Slightly soluble in acetonitrile and in
ethanol (99.5), very slightly soluble in diethyl ether, and prac-
tically insoluble in water. Melting point: about 190°C (with
decomposition).
Identification — Determine the infrared absorption spec-
trum of mesaconitine for purity as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3510 cm" 1 , 1713 cm" 1 , 1277 cm" 1 , 1116 cm" 1 , 1098 cm" 1
and 717 cm" 1 .
Absorbance <2.24> E\°j m (230 nm): 211 - 247 [5 mg dried
for not less than 12 hours in a desiccator (reduced pressure
not exceeding 0.67 kPa, phosphorus (V) oxide, 40°C),
ethanol (99.5), 200 mL].
Purity Related substances — (1) Dissolve 5.0 mg of
mesaconitine for purity in 2 mL of acetonitrile, and use as the
sample solution. Pipet 1 mL of the sample solution, add
acetonitrile to make exactly 50 mL, and use as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography, and proceed the test as
directed in the Identification under Processed Aconite Root:
the spot other than the principal spot is not more intense than
the spot with the standard solution.
(2) Dissolve 5.0 mg of mesaconitine for purity in 5 mL of
acetonitrile, and use as the sample solution. Pipet 1 mL of the
sample solution, add acetonitrile to make exactly 50 mL, and
use as the standard solution. Perform the test with exactly 10
,uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peaks of mesaconitine and the solvent is not
larger than the peak area of mesaconitine with the standard
solution.
Operating conditions
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity under Proc-
essed Aconite Root.
Mobile phase: A mixture of phosphate buffer solution for
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust the flow rate so that the retention time of
mesaconitine is about 19 minutes.
Time span of measurement: About 3 times as long as the
retention time of mesaconitine.
System suitability
Test for required detectability: Pipet 1 mL of the standard
solution, and add acetonitrile to make exactly 20 mL.
Confirm that the peak area of mesaconitine obtained from 10
fiL of this solution is equivalent to 3.5 to 6.5% of that
obtained from 10 fiL of the standard solution.
System performance: Dissolve 1 mg each of aconitine for
purity, hypaconitine for purity and mesaconitine for purity,
and 8 mg of jesaconitine for purity in 200 mL of acetonitrile.
When the procedure is run with 10 fiL of this solution under
the above operating conditions, mesaconitine, hypaconitine,
aconitine and jesaconitine are eluted in this order, and each
resolution between these peaks is not less than 1.5, respec-
tively.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
mesaconitine is not more than 1.5%.
Water <2.48>: not more than 1.0% [5 mg dried for not less
than 12 hours in a desiccator (reduced pressure not exceeding
0.67 kPa, phosphorus (V) oxide, 40°C), coulometric titra-
tion].
Mesityl oxide CH 3 COCH = C(CH 3 ) 2 A colorless or pale
yellow, clear liquid, having a characteristic odor.
Specific gravity <2.56> df a : 0.850 - 0.860
Metacycline hydrochloride C 22 H 22 N 2 8 .HC1 Yellow to
dark yellow, crystals or crystalline powder.
Purity Related substances — Dissolve 20 mg of metacy-
214
Reagents, Test Solutions / General Tests
JP XV
cline hydrochloride in 25 mL of 0.01 mol/L hydrochloric
acid TS, and use this solution as the sample solution. Proceed
the test with 20 /uL of the sample solution as directed in the
Purity (2) under Doxycycline Hydrochloride Hydrate, deter-
mine each peak area by the automatic integration method,
and calculate the amounts of them by the area percentage
method: the total area of peaks other than metacycline is not
more than 10%.
Metallic sodium See sodium.
Metanil yellow C 18 H I4 N 3 Na03S Yellow-brown powder.
Sparingly soluble in water, and very slightly soluble in etha-
nol (95) and in 7V,iV-dimethylformamide.
Metanil yellow TS Dissolve 0.1 g of metanil yellow in 200
mL of 7V,7V-dimethylformamide.
Metaphosphoric acid HP0 3 [K 8890, Special class]
Metaphosphoric acid-acetic acid TS Dissolve 15 g of
metaphosphoric acid and 40 mL of acetic acid (100) in water
to make 500 mL. Preserve in a cold place, and use within 2
days.
Metenolone enanthate C27H42O3 [Same as the name-
sake monograph]
Metenolone enanthate for assay To 1 g of metenolone
enanthate add 30 mL of water, and add slowly 70 mL of
methanol with warming to dissolve. Filter while hot, and al-
low the filtrate to stand on a water bath for 30 minutes. Allow
to stand overnight in a cold place, collect the crystals thus
formed, and wash with a small amount of diluted methanol
(1 in 3). Recrystallize in the same manner, and dry the crys-
tals in a desiccator (in vacuum, phosphorus (V) oxide) for 4
hours. It is white, odorless crystals.
Absorbance <2.24> E\°f m (242 nm): 321 - 328 (1 mg, meth-
anol, 100 mL).
Optical rotation <2.49> [ a ]™: +40- +42° (0.2 g, chloro-
form, 10 mL, 100 mm).
Melting point <2.60>: 69 - 72°C
Purity Related substances — Dissolve 50 mg of meteno-
lone enanthate for assay in chloroform to make exactly 10
mL, and use this solution as the sample solution. Proceed
with 10 fiL of this solution as directed in the Purity (3) under
Metenolone Enanthate: any spot other than the principal
spot does not appear.
Metformin hydrochloride for assay C 4 H U N 5 .HC1
[Same as the monograph Metformin Hydrochloride. When
dried, it contains not less than 99.0% of metformin
hydrochloride (C 4 H„N 5 .HC1)]
Methanesulfonic acid CH3SO3H Clear, colorless liquid
or colorless or white, crystalline mass, having a characteristic
odor. Miscible with water, with ethanol (95) and with diethyl
ether.
Congealing point <2.42>: 15 -20 C C
Specific gravity <2.56> df : 1.483 - 1.488
Content: not less than 99.0%. Assay — Weigh accurately
about 2 g of methanesulfonic acid, dissolve in 40 mL of
water, and titrate <2.50> with 1 mol/L sodium hydroxide VS
(indicator: 2 drops of bromothymol blue TS).
Each mL of 1 mol/L sodium hydroxide VS
= 96.11 mg of CH3SO3H
Methanesulfonic acid TS To 35 mL of methanesulfonic
acid add 20 mL of acetic acid (100) and water to make 500
mL.
0.1 mol/L Methanesulfonic acid TS To 4.8 g of methane-
sulfonic acid add water to make 500 mL.
Methanol CH 3 OH [K 8891, Special class]
Methanol, anhydrous CH4O To 1000 mL of methanol
add 5 g of magnesium powder. If necessary, add 0.1 mL of
mercury (II) chloride TS to start the reaction. After the evolv-
ing of a gas is stopped, distillate the solution, and preserve
the distillate protecting from moisture. Water content per
mL is not more than 0.3 mg.
Methanol for Karl Fischer method See Water Determina-
tion <2.48>.
Methanol for liquid chromatography CH 3 OH A clear,
colorless liquid. Mixable with water.
Purity Ultraviolet-absorbing substances — Perform the
test as directed in Ultraviolet-visible Spectrophotometry
<2.24> using water as the blank: the absorbances at 210 nm,
at 220 nm, at 230 nm, at 240 nm and at 254 nm are not more
than 0.70, 0.30, 0.15, 0.07 and 0.02, respectively.
Methanol-free ethanol See ethanol (95), methanol-free.
Methanol-free ethanol (95) See ethanol (95), methanol-
free.
Methanol, purified Distil methanol before use.
Methionine C 5 H u N0 2 S [Same as the monograph l-
Methionine]
2-Methoxyethanol CH 3 OCH 2 CH 2 OH [K 8895, Special
class]
4-Methoxybenzaldehyde C 8 H 8 2 Clear, colorless to
light yellow liquid. Miscible with ethanol (95) and with
diethyl ether, and practically insoluble in water.
Specific gravity <2.56> df: 1.123 - 1.129
Content: not less than 97.0%. Assay— Weigh accurately
about 0.8 g of 4-methoxybenzaldehyde, add exactly 7.5 mL
of hydroxylamine TS, shake well, allow to stand for 30
minutes, and titrate <2.50> with 0.5 mol/L hydrochloric acid
VS (indicator: 3 drops of bromophenol blue TS) until the col-
or of the solution changes from blue through green to yellow-
green. Perform a blank determination.
Each mL of 0.5 mol/L hydrochloric acid VS
= 68.08 mg of C 8 H 8 2
4-Methoxybenzaldehyde-acetic acid TS To 0.5 mL of 4-
methoxybenzaldehyde add acetic acid (100) to make 100 mL.
4-Methoxybenzaldehyde-sulfuric acid TS To 9 mL of
ethanol (95) add 0.5 mL of 4-methoxybenzaldehyde and 0.5
mL of sulfuric acid, and mix thoroughly.
l-Methoxy-2-propanol C 4 H 10 O 2 A colorless, clear liq-
uid.
Clarity of solution — To 5 mL of l-methoxy-2-propanol
add 20 mL of water, and mix: the solution is clear.
Specific gravity <2.56> df: 0.920 - 0.925
Refractive index <2.45> ng: 1.402 - 1.405
Water <2.48>: not more than 0.5% (5 g).
JPXV
General Tests / Reagents, Test Solutions
215
Content: not less than 98.0%.
Assay — Proceed as directed under Gas Chromatography
<2.02> using the area percentage method according to the fol-
lowing conditions:
Operating conditions
Detector: Thermal conductivity detector
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with siliceous earth for gas
chromatography (150 to 180^<m) coated with polyethylene
glycol 20 M for gas chromatography in 20%.
Column temperature: A constant temperature of about
90°C.
Carrier gas: Helium
Flow rate: A constant flow rate of 20 mL per minute.
p-Methyl aminophenol sulfate See 4-methyl animo-
phenol sulfate.
4-MethyI aminophenol sulfate (HOC 6 H 4 NHCH 3 ) 2 .H 2 S04
White to pale yellow or very pale grayish white, crystals or
crystalline powder. Melting point: about 260°C (with decom-
position).
p-Methyl aminophenol sulfate TS See 4-methyl amino-
phenol sulfate TS.
4-MethyI aminophenol sulfate TS Dissolve 0.35 g of 4-
methyl aminophenol sulfate and 20 g of sodium hydrogen
sulfite in water to make 100 mL. Prepare before use.
2-MethyIaminopyridine C 6 H 8 N 2 A pale yellow liquid.
Specific gravity <2.56> df Q : 1.050 - 1.065
Boiling point <2.57>: 200 - 202°C
Water <2.48>: less than 0.1%.
2-Methylamino pyridine for Karl Fischer method See
Water Determination <2.48>.
Methyl behenate C 23 H 46 2 White, odorless and taste-
less, scaly crystals or powder. Dissolves in acetone, in diethyl
ether and in chloroform.
Melting point <2.60>: 54°C
Saponification value <1.13>: 155.5 - 158.5
Methyl benzoate C 6 H 5 COOCH 3 Clear, colorless liquid.
Refractive index <2.45> n™: 1.515 - 1.520
Specific gravity <2.56> df a ; 1.087 - 1.095
Purity — Dissolve 0.1 mL of methyl benzoate in the mobile
phase in Assay under Thiamine Hydrochloride to make 50
mL. Perform the test as directed under Liquid Chromatogra-
phy <2.01> with 10 iuL of this solution according to the Assay
under Thiamine Hydrochloride. Measure each peak area by
the automatic integration method in a range about twice the
retention time of methyl benzoate, and calculate the amount
of methyl benzoate by the area percentage method: it shows
the purity of not less than 99.0%.
Methyl benzoate for estriol test C 8 H 8 2 Clear, colorless
liquid, having a characterisic odor.
Refractive index <2.45> n™: 1.515 - 1.520
Specific gravity <2.56> df : 1.087 - 1.095
Acid value <1.13>: not more than 0.5.
D-( + )-a-Methylbenzylamine C 6 H 5 CH(CH 3 )NH 2 Color-
less or pale yellow clear liquid, having an amine like odor.
Miscible with ethanol (95) and with acetone, and slightly
soluble in water.
Refractive index <2.45> n™: 1.524-1.529
Optical rotation <2.49> [a]™'- + 37 - + 41 ° (50 mm)
Content: not less than 98.0%. Assay — Perform the test
with exact 0.6 /xL of D-( + )-a-methylbenzylamine as directed
under Gas Chromatography <2.02> according to the follow-
ing conditions. Measure each peak area by the automatic in-
tegration method, and calculate the amount of D-( + )-
a-methylbenzylamine .
Operating conditions
Detector: Hydrogen flame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with siliceous earth for gas
chromatography (180 to 250 /um in particle diameter) coated
with polyethylene glycol 20 M for gas chromatography and
potassium hydroxide at the ratio of 10% and 5%, respec-
tively.
Column temperature: A constant temperature of about 140
°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
D-( + )-a-methylbenzylamine is about 5 minutes.
Selection of column: To 5 mL of D-( + )-a-methylbenzyl-
amine add 1 mL of pyridine. Proceed with 0.6 fiL of this solu-
tion under the above operating conditions, and calculate the
resolution. Use a column giving elution of pyridine and
D-( + )-a-methylbenzylamine in this order with the resolution
between these peaks being not less than 3.
Time span of measurement: About 3 times as long as the
retention time of D-( + )-a-methylbenzylamine beginning af-
ter the solvent peak.
3-Methyl-l-butanol C 5 H 12 [K 8051, Special class]
3-Methylbutyl acetate CH 3 COOCH 2 CH 2 CH(CH 3 ) 2
[K 8358, Special class]
Methyl cellosolve See 2-methoxyethanol.
Methyl docosanate C 23 H 46 2 White, tabular crystals or
crystalline powder.
Melting point <2.60>: 51.0 - 56.0°C
Methyldopa C 10 H 13 NO 4 .l y H 2 [Same as the mono-
graph Methyldopa Hydrate]
Methyldopa for assay Ci H 13 NO 4 .l yH 2 [Same as the
monograph Methyldopa Hydrate. When dried, it contains
not less than 99.0% of methyldopa (C 10 H 13 NO 4 ).]
JV,./V'-MethylenebisacryIamide CH 2 (NHCOCHCH 2 ) 2
White crystalline powder.
Content: not less than 97.0%.
Methylene blue See methylene blue trihydrate.
Methylene blue-potassium perchlorate TS To 500 mL of
a solution of potassium perchlorate (1 in 1000) add dropwise,
with shaking, a solution of methylene blue (1 in 100) until a
slight, permanent turbidity results. Allow the solution to
stand, and filter the supernatant liquid.
Methylene blue-sulf uric acid-monobasic sodium phosphate
TS See methylene blue-sulfuric acid-sodium dihydrogen-
phosphate TS.
Methylene blue-sulfuric acid-sodium dihydrogenphosphate
TS To 30 mL of a solution of methylene blue (1 in 1000)
216
Reagents, Test Solutions / General Tests
JP XV
add 500 mL of water, 6.8 mL of sulfuric acid and 50 g of so-
dium dihydrogenphosphate dihydrate, dissolve, and add
water to make 1000 mL.
Methylene blue trihydrate
[K 8897, Special class]
C 16 H 18 C1N 3 S.3H 2
Methylene blue TS Dissolve 0.1 g of methylene blue tri-
hydrate in water to make 100 mL. Filter if necessary.
tf/-Methylephedrine hydrochloride
[Same as the namesake monograph]
C„H 17 NO.HCl
tf/-Methylephedrine hydrochloride for assay [Same as the
monograph (//-Methylephedrine Hydrochloride]
Methylergometrine maleate for assay
C20H25N3O2.C4H4O4 [Same as the monograph Methyler-
gometrine Maleate. When dried, it contains not less than
99.0% of methylergometrine maleate (C 20 H 25 N 3 O 2 .C 4 H 4 O 4 ).]
Methyl ethyl ketone See 2-butanone.
Methyl iodide See iodomethane.
Methyl iodide for assay CH 3 I Clear, colorless liquid.
On exposure to light, it liberates iodine and becomes brown.
Miscible with ethanol (95) and with diethyl ether, and spar-
ingly soluble in water. Use the distillate obtained between
42.2°C and 42.6°C.
Specific gravity <2.56> clf 5 : 2.27 - 2.28
Purity — Perform the test with 1 liL of methyl iodide for as-
say as directed under Gas Chromatography <2.02> according
to the operating conditions in the Assay under Hypromellose.
Measure each peak area by the automatic integration meth-
od, and calculate the amount of methyl iodide by the area
percentage method: it shows the purity of not less than
99.8%. Adjust the detection sensitivity so that the peak
height of methyl iodide from 1 iiL of methyl iodide for assay
is about 80% of the full scale.
Content: not less than 98.0%. Assay — Proceed as direct-
ed in the Assay under Isopropyl iodide for assay.
Each mL of 0.1 mol/L silver nitrate VS
= 14.19 mg of CH3I
Methyl isobutyl ketone See 4-methyl-2-pentanone.
3-O-Methylmethyldopa for thin-layer chromatography
C„H 15 N0 4
Purity Related substances — Dissolve 5 mg of 3-0-
methylmethyldopa for thin-layer chromatography in
methanol to make exactly 100 mL. Perform the test with 20
/iL of this solution as directed in the Purity (5) under Methyl-
dopa: any spot other than the principal spot at the Rf value
of about 0.7 does not appear.
2-Methyl-5-nitroimidazole for thin-layer chromato-
graphy C 4 H 5 N 3 2 White crystalline powder. Slightly
soluble in water and in acetone. Melting point: about 253 °C
(with decomposition).
Purity Related substances — Dissolve 40 mg of 2-methyl-
5-nitroimidazole for thin-layer chromatography in 8 mL of
acetone, and use as the sample solution. Pipet 2.5 mL of the
sample solution, add acetone to make exactly 100 mL, and
use as the standard solution. Perform the test as directed in
the Purity (3) under Metronidazole: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Methyl orange Ci 4 H 14 N 3 Na0 3 S [K 8893, Special
class]
Methyl orange-boric acid TS Add 0.5 g of methyl orange
and 5.2 g of boric acid in 500 mL of water, and dissolve by
warming on a water bath. After cooling, wash this solution
with three 50-mL portions of chloroform.
Methyl orange TS Dissolve 0. 1 g of methyl orange in 100
mL of water, and filter if necessary.
Methyl orange-xylenecyanol FF TS Dissolve 1 g of meth-
yl orange and 1 .4 g of xylene cyanol FF in 500 mL of dilute
ethanol.
Methyl parahydroxybenzoate HOC 6 H 4 COOCH 3
[Same as the namesake monograph]
4-Methylpentan-2-ol C 6 H 14 A clear and colorless,
volatile liquid.
Refractive index <2.45> ri$: about 1.411
Specific gravity <2.56> df : about 0.802
Boiling point <2.57>: about 132°C
4-Methyl-2-pentanone
[K 8903, Special class]
CH 3 COCH 2 CH(CH 3 ) 2
3-Methyl-l-phenyl-5-pyrazolone
Special class]
C10H10N7
[K 9548,
Methyl prednisolone
monograph]
C 22 H 30 O 3 [Same as the namesake
2-Methyl-l-propanol (CH 3 ) 2 CHCH 2 OH [K 8811, Spe-
cial class]
/V-Methylpyrrolidine C 5 H U N Colorless, clear liquid,
having a characteristic order.
Identification — Determine the spectrum of iV-methylpyr-
rolidine in a solution of deuterated chloroform for nuclear
magnetic resonance spectroscopy (4 in 50) as directed under
Nuclear Magnetic Resonance Spectroscopy <2.21> (*H): it ex-
hibits a big signal, at around 5 2.3 ppm.
Content: not less than 95%. Assay — Put 30 mL of water in
a beaker, weigh accurately the beaker, add dropwise about
0.15 g of /V-methylpyrrolidine, weigh accurately the beaker
again, and titrate <2.50> with 0.05 mol/L sulfuric acid VS
(potentiometric titration). Perform a blank determination in
the same manner and make any necessary correction.
Each mL of 0.05 mol/L sulfuric acid
= 8.515 mg of C 5 H U N
Methyl red C 15 H 15 N 3 2 [K 8896, Special class]
Methyl red-methylene blue TS Dissolve 0.1 g of methyl
red and 0.1 g of methylene blue in ethanol (95) to make 100
mL, and filter if necessary. Preserve in light-resistant contain-
ers.
Methyl red TS Dissolve 0.1 g of methyl red in 100 mL of
ethanol (95), and filter if necessary.
Methyl red TS , dilute Dissolve 25 mg of methyl red in 1 00
mL of ethanol (99.5), and filter if necessary. Prepare before
use.
Methyl red TS for acid or alkali test To 0.1 g of methyl
red add 7.4 mL of 0.05 mol/L sodium hydroxide VS or 3.7
JPXV
General Tests / Reagents, Test Solutions
217
mL of 0.1 mol/L sodium hydroxide VS, triturate to dissolve
in a mortar, and add freshly boiled and cooled water to make
200 mL. Preserve in light-resistant, glass-stoppered bottles.
Methylrosaniline chloride See crystal violet.
Methylrosaniline chloride TS See crystal violet TS.
Methyl salicylate C 8 H 8 3
graph]
[Same as the namesake mono-
Methylsilicone polymer for gas chromatography Pre-
pared for gas chromatography.
Methyltestosterone C 2 oH 30 2 [Same as the namesake
monograph]
l-MethyI-l//-tetrazoIe-5-thiol for liquid chromatography
C 2 H 4 N 4 S White, crystals or crystalline powder. Very solu-
ble in methanol, and freely soluble in water.
Melting point <2.60>: 123 - 127°C
Loss on drying <2.41>: not more than 1.0% (1 g, in vacu-
um, phosphorous (V) oxide, 2 hours).
Content: not less than 99.0%. Assay — Weigh accurately
about 0.2 g of l-methyl-l//-tetrazole-5-thiol, previously
dried, dissolve in 80 mL of TV, TV-dim ethylformamide, and ti-
trate <2.50> with 0.1 mol/L sodium methoxide VS (indicator:
3 drops of thymol blue-TV, TV-dimethylformamide TS). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L sodium methoxide VS
= 11.61 mg of C 2 H 4 N 4 S
Methylthymol blue C 37 H 43 N 2 Na0 13 S [K 9552]
Methylthymol blue-potassium nitrate indicator Mix 0.1 g
of methylthymol blue with 9.9 g of potassium nitrate, and tri-
turate until the mixture becomes homogeneous.
Sensitivity — When 0.02 g of methylthymol blue-potassium
nitrate indicator is dissolved in 100 mL of 0.02 mol/L sodium
hydroxide VS, the solution is slightly blue in color. On add-
ing 0.05 mL of 0.01 mol/L barium chloride VS to this solu-
tion, the solution shows a blue color, then on the subsequent
addition of 0.1 mL of 0.01 mol/L disodium ethylene-
diamineteraacetate VS, it becomes colorless.
Methylthymol blue-sodium chloride indicator Mix 0.25 g
of methylthymol blue and 10 g of sodium chloride, and grind
to homogenize.
l-MethyI-Lff-tetrazoIe-5-thiol C 2 H 4 N 4 S White, crystals
or crystalline powder.
Melting point <2.60>: 125 - 129 C C
Identification — (1) Determine the ultraviolet-visible ab-
sorption spectrum of a solution of l-methyl-l//-tetrazole-5-
thiol (1 in 200,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 222
nm and 226 nm.
(2) Determine the infrared absorption spectrum of 1-
methyl-l//-tetrazole-5-thiol according to the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3060 cm- 1 , 2920 cm- 1 , 2780 cm- 1 , 1500 cm- 1 , 1430 cm- 1
and 1410 cm- 1 .
Purity Related substances — Dissolve 0.10 g of 1-methyl-
l//-tetrazole-5-thiol in exactly 100 mL of water. Perform the
test with 1 /xL of this solution as directed in the Purity (4) un-
der Cefmetazole Sodium: any spot other than the principal
spot at the i?f value of about 0.77 does not appear.
Methyl yellow C 14 H 15 N 3 [K 8494, Special class]
Methyl yellow TS Dissolve 0.1 g of methyl yellow in 200
mL of ethanol (95).
Metoclopramide for assay C I4 H 22 C1N 3 2 [Same as the
monograph Metoclopramide. When dried, it contains not
less than 99.0% of metoclopramide (C 14 H 22 C1N 3 2 ).]
Metoprolol tartrate for assay (C 15 H 25 N0 3 ) 2 .C 4 H 6 6
[Same as the monograph Metoprolol Tartrate. When dried, it
contains not less than 99.5% of metoprolol tartrate ((Ci 5 H 25
N0 3 ) 2 .C 4 H 6 6 ).]
Metronidazole C 6 H 9 N 3 3
graph]
[Same as the namesake mono-
Metronidazole for assay C 6 H 9 N 3 3 [Same as the mono-
graph Metronidazole. It meets the following additional re-
quirement.]
Related substances— Weigh accurately about 25 mg of
metronidazole for assay, dissolve in a mixture of water and
methanol (4:1) to make exactly 100 mL, and use this solution
as the sample solution. Pipet 2 mL of the sample solution,
and add the mixture of water and methanol (4:1) to make ex-
actly 50 mL. Pipet 2.5 mL of this solution, add the mixture
of water and methanol (4:1) to make exactly 20 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the total area of
the peaks other than metronidazole is not more than the peak
area of metronidazole with the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay under Metronidazole Tablets.
Time span of measurement: About 4 times as long as the
retention time of metronidazole.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, add a mixture of water and methanol
(4:1) to make exactly 20 mL. Confirm that the peak area of
metronidazole obtained with 10 fiL of this solution is equiva-
lent to 7 to 13% of that with the standard solution.
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of metronidazole are not less than 3000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
metronidazole is not more than 2.0%.
Microplates Polystyrene plates with an inside diameter of
7 (upper edge) to 6.4 (lower edge) mm, and 11.3 mm thick-
ness. Have 96 flat-bottomed truncated cone-shaped wells.
Milk casein See casein, milk.
Milk of lime Place 10 g of calcium oxide in a mortar, and
add gradually 40 mL of water under grinding.
218
Reagents, Test Solutions / General Tests
JP XV
Mixture of petroleum hexamethyl tetracosane branching
hydrocarbons (L) for gas chromatography Prepared for gas
chromatography.
Molecular weight markers for teceleukin Dissolve 0.4 mg
each of lysozyme, soy trypsin inhibitor, carbonic anhydrase,
egg white albumin, bovine serum albumin, and phosphory-
lase b in 200 /uL of diluted glycerin (1 in 2).
Molybdenum (III) oxide M0O3 A white to yellowish
green powder.
Identification — Dissolve 0.5 g in 5 mL of ammonia solu-
tion (28), acidify 1 mL of this solution with a suitable amount
of nitric acid, add 5 mL of sodium phosphate TS, and warm:
yellow precipitates appear.
Molybdenum (III) oxide-citric acid TS To 54 g of molyb-
denum (III) oxide and 11 g of sodium hydroxide add 200 mL
of water, and dissolve by heating while stirring. Separately,
dissolve 60 g of citric acid monohydrate in 250 mL of water,
and add 140 mL of hydrochloric acid. Mix these solutions,
filter if necessary, add water to make 1000 mL, and add a so-
lution of potassium bromate (1 in 100) until a yellow-green
color appears.
Storage — Preserve in tightly stopered containers, protected
from light.
Molybdenum trioxide See molybdenum (III) oxide.
Molybdenum trioxide-citric acid TS See molybdenum
(III) oxide-citric acid TS.
Monobasic ammonium phosphate See ammonium di-
hydrogenphosphate.
Monobasic potassium phosphate See potassium di-
hydrogenphosphate.
Monobasic potassium phosphate for pH determination
See potassium dihydrogenphosphate for pH determination.
0.05 moI/L Monobasic potassium phosphate, pH 3.0 See
0.05 mol/L potassium dihydrogenphosphate, pH 3.0.
0.02 mol/L Monobasic potassium phosphate TS See 0.02
mol/L potassium dihydrogenphosphate TS.
0.05 mol/L Monobasic potassium phosphate TS See 0.05
mol/L potassium dihydrogenphosphate TS.
0.2 mol/L Monobasic potassium phosphate TS See 0.2
mol/L potassium dihydrogenphosphate TS.
0.2 mol/L Monobasic potassium phosphate TS for buffer
solution See 0.2 mol/L potassium dihydrogenphosphate TS
for buffer solution.
0.05 mol/L Monobasic potassium phosphate TS, pH 4.7
See 0.05 mol/L potassium dihydrogenphosphate TS, pH 4.7.
Monobasic sodium phosphate See sodium dihydrogen-
phosphate dihydrate.
0.05 mol/L Monobasic sodium phosphate TS See 0.05
mol/L sodium dihydrogenphosphate TS.
0.1 mol/L Monobasic sodium phosphate TS See 0.1 mol
/L sodium dihydrogenphosphate TS.
2 mol/L Monobasic sodium phosphate TS See 2 mol/L
sodium dihydrogenphosphate TS.
0.05 mol/L Monobasic sodium phosphate TS, pH 2.6
See 0.05 mol/L sodium dihydrogenphosphate TS, pH 2.6.
0.05 mol/L Monobasic sodium phosphate TS, pH 3.0
See 0.05 mol/L sodium dihydrogenphosphate TS, pH 3.0.
0.1 mol/L Monobasic sodium phosphate TS, pH 3.0 See
0.1 mol/L sodium dihydrogenphosphate TS, pH 3.0.
0.02 mol/L Monobasic ammonium phosphate TS See
0.02 mol/L ammonium dihydrogenphosphate TS.
Monoethanolamine See 2-Aminoethanol.
Morphine hydrochloride [Same as the monograph Mor-
phine Hydrochloride Hydrate]
Morphine hydrochloride for assay
CnHj9NO3.HCl.3H2O [Same as the monograph Morphine
Hydrochloride Hydrate. It contains not less than 99.0% of
morphine hydrochloride (C17H19NO3.HCI), calculated on the
anhydrous basis.]
3-(iV-Morpholino)propanesulfonic acid C7H15NO4S
White crystalline powder, freely soluble in water, and practi-
cally insoluble in ethanol (99.5).
Melting point <2.60>: 275 - 280°C
0.02 mol/L 3-(iV-Morpholino)propanesulfonic acid buffer
solution, pH 7.0 Dissolve 4.2 g of 3-(/V-mor-
pholino)propanesulfonic acid in 900 mL of water, adjust the
pH to 7.0 with dilute sodium hydroxide TS, and add water to
make 1000 mL.
0.02 mol/L 3-(iV-Morpholino)propanesulfonic acid buffer
solution, pH 8.0 Dissolve 4.2 g of 3-(/V-mor-
pholino)propanesulfonic acid in 700 mL of water, adjust the
pH to 8.0 with dilute sodium hydroxide TS, and add water to
make 1000 mL.
0.1 mol/L 3-(./V-Morphorino)propanesurfonic acid buffer
solution, pH 7.0 Dissolve 20.92 g of 3-(/V-mor-
pholino)propanesulfonic acid in 900 mL of water, adjust the
pH to 7.0 with sodium hydroxide TS, and add water to make
1000 mL.
MTT TS Dissolve 8 g of sodium chloride, 0.2 g of calci-
um chloride, 1.15 g of anhydrous sodium dihydrogen phos-
phate (NaH 2 P0 4 ) and 0.2 g of potassium dihydrogen phos-
phate (KH 2 P0 4 ) in water to make 1000 mL, and sterilize in an
autoclave for 15 minutes at 121 °C to make the PBS( — ) solu-
tion. Dissolve 0.3 g of 3-(4,5-dimethylthiazole- 2-yl)-2,5-
diphenyl-2//-tetrazolium bromide in this PBS( — ) solution to
make 100 mL. Sterilize by membrane filtration (pore size,
0.45 ^m), and store in a cool place shielded from light.
Murexide C 8 H 8 N 6 6 Red-purple powder. Practically
insoluble in water, in ethanol (95) and in diethyl ether.
Purity Clarity of solution — Dissolve 10 mg of murexide
in 100 mL of water: the solution is clear.
Residue on ignition <2.44>: not more than 0.1% (1 g).
Sensitivity — Dissolve 10 mg of murexide in 2 mL of ammo-
nia-ammonium chloride buffer solution, pH 10.0, and add
water to make 100 mL, and use this solution as the sample so-
lution. Separately, add 2 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.0, to 5 mL of diluted Standard
Calcium Solution (1 in 10), add water to make 25 mL, and
render the solution to pH 11.3 with sodium hydroxide TS.
JP XV
General Tests / Reagents, Test Solutions 219
Add 2 mL of the sample solution and water to this solution to
make 50 mL: a red-purple color develops.
Murexide-sodium chloride indicator Prepared by mixing
0.1 g of murexide and 10 g of sodium chloride and grinding
to get homogeneous.
Storage — Preserve in light-resistant containers.
Myoglobin A hemoprotein obtained from horse heart
muscle. White crystalline powder. It contains not less than 95
% of myoglobin in the total protein.
Nalidixic acid Ci 2 H 12 N 2 3 [Same as the namesake
monograph]
Naphazoline nitrate
namesake monograph]
C 14 H, 4 N 7 .HNO,
[Same as the
Naphazoline nitrate for assay [Same as the monograph
Naphazoline Nitrate. When dried, it contains not less than
99.0% of naphazoline nitrate (C 14 H 14 N 2 .NH0 3 ).]
Naphthalene C 10 H 8 Colorless flake-like or
stick-like crystals, having a characteristic odor.
Melting point <2.60>: 78 - 82°C
lustrous
1,3-Naphthalenediol C 10 H 8 O 2 Red-brown crystals or
gray-brown powder. Freely soluble in water, in methanol and
in ethanol (99.5). Melting point: about 124°C.
1,3-Naphthalenediol TS Dissolve 50 mg of 1,3-
naphthalenediol in 25 mL of ethanol (99.5), and add 2.5 mL
of phosphoric acid.
2-NaphthalenesuIfonic acid Ci H 8 O 3 S.H 2 O White to
pale yellowish white powder. Very soluble in water, in
methanol and in ethanol (95), and sparingly soluble in diethyl
ether and in chloroform.
Water <2.48>: 7.0-11.5% (0.5 g, volumetric titration,
direct titration).
Content: not less than 95.0%, calculated on the anhydrous
basis. Assay — Weigh accurately about 0.5 g of 2-
naphthalenesulfonic acid, dissolve in 30 mL of water, and ti-
trate <2.50> with 0.1 mol/L sodium hydroxide VS (indicator:
3 drops of bromothymol blue TS). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L soduim hydroxide VS
= 20.82 mg of C 10 H 8 O 3 S
1-Naphthol C 10 H 7 OH [K
in light-resistant containers.
Special class] Preserve
2-Naphthol C 10 H 7 OH [K 8699, Special class] Preserve
in light-resistant containers.
a-Naphthol See 1-naphthol.
/?-Naphthol See 2-naphthol.
p-Naphtholbenzein C 27 H 20 O 3 [K 8693, Special class]
a-Naphtholbenzein See /?-naphtholbenzein.
p-Naphtholbenzein TS Dissolve 0.2 g of jO-naphtholben-
zein in acetic acid (100) to make 100 mL.
Purity Clarity and color of solution — Dissolve 0.1 g of p-
naphtholbenzein in 100 mL of ethanol (95): the solution is
red in color and clear.
Sensitivity — Add 100 mL of freshly boiled and cooled
water to 0.2 mL of a solution of /?-naphtholbenzein in etha-
nol (95) (1 in 1000), and add 0.1 mL of 0.1 mol/L sodium hy-
droxide VS: a green color develops. Add subsequently 0.2
mL of 0.1 mol/L hydrochloric acid VS: the color of the solu-
tion changes to yellow-red.
a-Naphtholbenzein TS See p-naphtholbenzein TS.
1-NaphthoI-sulfuric acid TS Dissolve 1.5 g of 1-naphthol
in 50 mL of ethanol (95), add 3 mL of water and 7 mL of sul-
furic acid, and mix well. Prepare before use.
1-Naphthol TS Dissolve 6 g of sodium hydroxide and 16
g of anhydrous sodium carbonate in water to make 100 mL.
In this solution dissolve 1 g of 1-naphthol. Prepare before
use.
2-Naphthol TS Dissolve 1 g of 2-naphthol in sodium car-
bonate TS to make 100 mL. Prepare before use.
a-Naphthol TS See 1-naphthol TS.
/?-NaphthoI TS See 2-naphthol TS.
1-Naphthylamine C 10 H 7 NH 2 [K 8692, Special class]
Preserve in light-resistant containers.
a-Naphthylamine See 1-naphthylamine.
./V-U-NaphthyO-Af'-diethylethylenediamine oxalate See
N, ./V-diethyl-Af'-l-naphthylethylenediamine oxalate.
■/V-(1-Naphthyl)-Af' -die! h > lelhy lenediamine oxalate-ace-
tone TS See N, Af-diethyl-/V'-l-naphthylethylenediamine
oxalate-acetone TS.
./V-U-NaphthyO-Af'-diethylethylenediamine oxalate TS
See N, Af-diethyWV'-l-naphthylethylenediamine oxalate TS.
Naphthylethylenediamine TS Dissolve 0.1 g of JV-1-
naphthylethylenediamine dihydrochloride in water to make
100 mL. Prepare before use.
./V-1-Naphthylethylenediamine dihydrochloride
C 10 H 7 NHCH 2 CH 2 NH 2 .2HC1 [K8197, Special class]
Naringin for thin-layer chromatography
C 27 H 32 N 14 .2H 2 White crystals or crystalline powder. Free-
ly soluble in ethanol (95) and in acetone, and slightly soluble
in water. Melting point: about 170°C (with decomposition).
Optical rotation <2.49> [«]£?: ~ 87 - - 93° (0. 1 g, ethanol
(95), 10 mL, 100 mm).
Purity Related substances — Proceed with 10,uL of a so-
lution, prepared by dissolving 10 mg of naringin for thin-lay-
er chromatography in 10 mL of ethanol (95), as directed in
the Identificaiton under Bitter Orange Peel: any spot other
than the principal spot at the Rf value of about 0.4 does not
appear.
Neutral alumina containing 4% of water Take 50 g of
neutral alumina for column chromatography, previously d-
ried at 105 °C for 2 hours, in a tight container, add 2.0 mL of
water, shake well to make homogeneous, and allow to stand
for more than 2 hours.
Neutral detergent Synthetic detergent containing anionic
or non-ionic surfactant, and pH of its 0.25% solution is be-
tween 6.0 and 8.0. Dilute to a suitable concentration before
use.
Neutralized ethanol See ethanol, neutralized.
Neutral red Ci 5 H 17 N 4 Cl Slightly metallic, dark green
220
Reagents, Test Solutions / General Tests
JP XV
powder or masses.
Identification — Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3310 cm -1 , 3160 cm -1 , 1621
cm -1 , 1503 cm -1 , 1323 cm -1 , 1199 cm -1 and 732 cm -1 .
Neutral red TS Dissolve 0.1 g of neutral red in acetic acid
(100) to make 100 mL.
Nicardipine hydrochloride for assay C 26 H 29 N 3 06.]-[C1
[Same as the monograph Nicardipine Hydrochloride. When
dried, it contains not less than 99.0% of nicardipine
hydrochloride (C 26 H 29 N 3 6 .HC1).]
Nicergoline for assay C 24 H 26 BrN 3 3 [Same as the
monograph Nicergoline, or Nicergoline purified according to
the method below. When dried, it contains not less than 99.0
% of nicergoline (C 24 H 26 BrN 3 3 ), and when perform the test
of the Purity (2) under Nicergoline, the total area of the
peaks other than nicergoline from the sample solution is not
more than 2.5 times the peak area of nicergoline from the
standard solution.
Method of purification: Dissolve 1 g of Nicergoline in 20
mL of acetonitrile, allow to stand in a dark place for about 36
hours, filter, and dry the crystals so obtained at 60°C for 2
hours in vacuum.]
Nicomol for assay C 34 H 32 N 4 9 [Same as the mono-
graph Nicomol. When dried, it contains not less than 99.0%
of C 34 H 32 N 4 9 ].
Nicotinamide C 6 H 6 N 2 [Same as the namesake mono-
graph]
/?-Nicotinamide-adenine dinucleotide (jS-NAD)
C 21 H 27 N 7 14 P 2 [K 9802]
Content: not less than 94.5%. Assay — Weigh accurately
about 25 mg of /?-nicotinamide-adenine dinucleotide, oxi-
dized form, and dissolve in water to make exactly 25 mL.
Pipet 0.2 mL of this solution, add 0.1 mol/L phosphate
buffer solution, pH 7.0, to make exactly 10 mL, and use this
solution as the sample solution. Determine the absorbances,
A T and A B , of the sample solution and 0.1 mol/L phosphate
buffer solution, pH 7.0, at 260 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
blank.
Amount (mg) of C 21 H 27 N 7 14 P 2
0.6634X10
17.6x0.20
x (A T - A B ) x 25
Nicotinamide adenine dinucleotide TS See /?-
nicotinamide-adenine dinucleotide TS.
^-Nicotinamide adenine dinucleotide (/?-NAD) TS Dis-
solve 40 mg of /^-nicotinamide adenine dinucleotide, oxidized
form in 10 mL of water. Prepare before use.
Nifedipine C 17 H 18 N 2 6 [Same as the namesake mono-
graph]
Ninhydrin C 9 H 6 4 [K 8870, Special class]
Ninhydrin TS Dissolve 0.2 g of ninhydrin in water to
make 10 mL. Prepare before use.
Ninhydrin-acetic acid TS Dissolve 1.0 g of ninhydrin in
50 mL of ethanol (95), and add 10 mL of acetic acid (100).
Ninhydrin-L-ascorbic acid TS Dissolve 0.25 g of nin-
hydrin and 0.01 g of L-ascorbic acid in water to make 50 mL.
Prepare before use.
Ninhydrin-butanol TS Dissolve 0.3 g of ninhydrin in 100
mL of 1-butanol, and add 3 mL of acetic acid (100).
Ninhydrin-citric acid-acetic acid TS Dissolve 70 g of
citric acid monohydrate in 500 mL of water, add 58 mL of
acetic acid (100), 70 mL of a solution of sodium hydroxide
(21 in 50) and water to make 1000 mL. In 100 mL of this so-
lution dissolve 0.2 g of ninhydrin.
Ninhydrin-stannous chlorid TS See ninhydrin-tin (II)
chloride TS.
Ninhydrin-sulfuric acid TS Dissolve 0. 1 g of ninhydrin in
100 mL of sulfric acid. Prepare before use.
Ninhydrin-tin (II) chloride TS Dissolve 21.0 g of citric
acid in water to make 200 mL, adjust the pH to 5.6 ± 0.2 by
adding sodium hydroxide TS, add water to make 500 mL,
and dissolve 1.3 g of tin (II) chloride. To 50 mL of the solu-
tion, add 50 mL of a 2-methoxethanol solution of ninhydrin
(2 in 50). Prepare before use.
0.2% Ninhydrin-water saturated 1-butanol TS Dissolve 2
g of ninhydrin in 1-butanol saturated with water to make
1000 mL.
Nitrendipine for assay [Same as the monograph Nitrendi-
pine. It, when dried, contains not less than 99.0% of nitren-
dipine (C 18 H 20 N 2 O 6 ), and meets the following requirement.
When perform the test as directed in the Purity (2) under
Nitrendipine, the area of the peak of dimethyl ester, having
the relative retention time of about 0.8 with respect to nitren-
dipine is not larger than 1/2 times the peak area of nitrendi-
pine from the standard solution, the area of the peak other
than nitrendipine and the dimethyl ester is not larger than 1/5
times the peak area of nitrendipine from the standard solu-
tion, and the total area of the peak other than nitrendipine is
not larger than 1/2 times the peak area of nitrendipine from
the standard solution.]
Nitric acid HN0 3 [K 8541, Special class, Concentra-
tion: 69-70%, Density: about 1.42 g/mL]
Nitric acid, dilute Dilute 10.5 mL of nitric acid with
water to make 100 mL (10%).
Nitric acid, fuming [K 8739, Special class, Concentra-
tion: not less than 97%, Density: 1.52 g/mL]
Nitric acid TS, 2 mol/L Dilute 12.9 mL of nitric acid
with water to make 100 mL.
2,2',2"-Nitrilotrisethanol (CH 2 CH 2 OH) 3 N [K 8663,
Special class]
2,2',2"-Nitrilotrisethanol buffer solution, pH 7.8
Dissolve 149.2 g of 2,2',2"-nitrilotrisethanol in about 4500
mL of water, adjust to pH 7.8 with 4 mol/L hydrochloric
acid, and add water to make 5000 mL.
p-Nitroaniline See 4-nitroaniline.
4-Nitroaniline 2 NC 6 H 4 NH 2 [K 8708, p-Nitroaniline,
Special class]
/j-Nitroaniline-sodium nitrite TS See 4-nitroaniline-sodi-
JPXV
General Tests / Reagents, Test Solutions
221
um nitrite TS.
4-Nitroaniline-sodium nitrite TS To 90 mL of a solution
of 0.3 g of 4-nitroaniline in 100 mL of 10 mol/L hydrochloric
acid TS add 10 mL of a solution of sodium nitrite (1 in 20),
and mix well. Prepare before use.
o-Nitrobenzaldehyde See 2-nitrobenzaldehyde.
2-Nitrobenzaldehyde 2 NC 6 H 4 CHO Pale yellow crys-
tals or crystalline powder.
Melting point <2.60>: 42 - 44°C
Nitrobenzene C 6 H 5 N0 2 [K 8723, Special class]
p-Nitrobenzenediazonium chloride TS See 4-nitroben-
zenediazonium chloride TS.
4-Nitrobenzenediazonium chloride TS Dissolve 1.1 g of
4-nitroaniline in 1.5 mL of hydrochloric acid, add 1.5 mL of
water, and then add a solution prepared by dissolving 0.5 g of
sodium nitrite in 5 mL of water, while cooling in an ice bath.
Prepare before use.
p-Nitrobenzenediazonium chloride TS for spraying See
4-nitrobenzenediazonium chloride TS for spraying.
4-Nitrobenzenediazonium chloride TS for spraying Dis-
solve 0.4 g of 4-nitroaniline in 60 mL of 1 mol/L hydro-
chloric acid TS, and add, while cooling in an ice bath, sodium
nitrite TS until the mixture turns potassium iodide-starch
paper to blue in color. Prepare before use.
p-Nitrobenzenediazonium fluoroborate See 4-nitroben-
zenediazonium fluoroborate.
4-Nitrobenzenediazonium fluoroborate
O2NC6H4N2BF4 Pale yellowish white, almost odorless pow-
der. Freely soluble in dilute hydrochloric acid, slightly solu-
ble in water, and very slightly solute in ethanol (95) and in
chloroform. Melting point: about 148°C (with decomposi-
tion).
Identification — Add 1 mL each of a solution of phenol (1
in 1000) and sodium hydroxide TS to 10 mL of a solution of
4-nitrobenzenediazonium fluoroborate (1 in 1000): a red col-
or develops.
Loss on drying <2.41>: not more than 1.0% (1 g, silica gel,
2 hours).
p-Nitrobenzoyl chloride See 4-nitrobenzoyl chloride.
4-Nitrobenzoyl chloride 2 NC 6 H 4 C0C1 Light yellow
crystals.
Melting point <2.60>: 70 - 74°C
Content: not less than 98.0%. Assay — Weigh accurately
about 0.5 g of 4-nitrobenzoyl chloride, add an excess of silver
nitrate-ethanol TS, and boil under a reflux condenser for 1
hour. After cooling, filter the precipitate, wash with water,
dry at 105°C to constant mass, and weigh. The mass of
4-nitrobenzoyl chloride, multiplied by 1.107, represents the
mass of 4-nitrobenzoyl chloride (C 7 H 4 C1N0 3 ).
p-Nitrobenzyl chloride See 4-nitrobenzyl chloride.
4-Nitrobenzyl chloride 2 NC 6 H 4 CH 2 C1 Light yellow
crystals or crystalline powder. Soluble in ethanol (95).
Melting point <2.60>: 71 -73°C
Content: not less than 98.0%. Assay — Weigh accurately
about 0.5 g of 4-nitrobenzyl chloride, add 15 mL of a solu-
tion prepared by dissolving 4 g of silver nitrate in 10 mL of
water and adding ethanol (95) to make 100 mL, and heat on a
water bath under a reflex condensor for 1 hour. After cool-
ing, filter the precipitate with a glass filter, wash with water,
dry at 105°C to constant mass, and weigh. The mass of the
precipitate represents the amount of silver chloride (AgCl:
143.32).
Amount (mg) of 4-nitrobenzyl chloride
= amount (mg) of silver chloride (AgCl: 143.32) X 1.197
4-(4-Nitrobenzyl)pyridine Ci 2 H 10 N 2 O 2 Pale yellow, crystal-
line powder. Freely soluble in acetone, and soluble in ethanol
(95).
Melting point <2.60>: 69 - 71 °C
Nitrogen [Same as the namesake monograph]
Nitrogen monoxide NO A colorless gas. Prepare by
adding sodium nitrite TS to a solution of iron (II) sulfate hep-
tahydrate in dilute sulfuric acid. Nitrogen monoxide from a
metal cylinder may be used.
Nitromethane CH 3 N0 2 [K 9523, Special class]
3-Nitrophenol C 6 H 5 N0 3 A light yellow crystalline pow-
der.
Melting point <2.60>: 96 - 99°C
4-Nitrophenol C 6 H 5 N0 3 [K 8721, Special class]
o-Nitrophenol C 6 H 5 N0 3 [K 8719, Special class]
o-Nitrophenyl-/?-D-galactopyranoside See 2-nitrophenyl-
jS-D-galactopyranoside.
2-Nitrophenyl-jS-D-galactopyranoside C 12 H 15 N0 8
White crystalline powder. Odorless. It is sparingly soluble in
water, slightly soluble in ethanol (95), and practically insolu-
ble in diethyl ether.
Melting point <2.60>: 193 - 194°C
Purity Clarity and color of solution — A solution of 2-
nitrophenyl-/?-D-galactopyranoside (1 in 100) is clear and
colorless.
Loss on drying <2.41>: not more than 0.1% (0.5 g, 105 C C,
2 hours).
Content: not less than 98.0%. Assay — Weigh accurately
about 0.05 g of 2-nitrophenyl-/?-D-galactopyranoside, previ-
ously dried, dissolve in water to make exactly 100 mL. Pipet
20 mL of this solution, and add water to make exactly 50 mL.
Determine the absorbance, A, of this solution at 262 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of 2-nitrophenyl-/?-D-galactopyranoside
A
133
x 25,000
l-Nitroso-2-naphthol C 10 H 7 NO 2
class]
[K 8713, Special
l-Nitroso-2-naphthol TS Dissolve 0.06 g of l-nitroso-2-
naphthol in 80 mL of acetic acid (100), and add water to
make 100 mL.
a-Nitroso-jS-naphthol See l-nitroso-2-naphthol.
a-Nitroso-jS-naphthoI TS See l-nitroso-2-naphthol TS.
Nitrous oxide N 2 Colorless and odorless gas. Use ni-
trous oxide from a metal cylinder.
NK-7 cells Cells derived from mouse NK cells
222
Reagents, Test Solutions / General Tests
JP XV
NN Indicator Mix 0.5 g of 2-hydroxy-l-(2'-hydroxy-4'-
sulfo-l'-naphthylazo)-3-naphthoic acid with 50 g of anhy-
drous sodium sulfate, and triturate until the mixture becomes
homogeneous.
Nonylphenoxypoly(ethyleneoxy)ethanoI for gas
matography Prepared for gas chromatography.
chro-
Normal agar media for teceleukin Dissolve 5.0 g of meat
extract, 10.0 g of peptone, 5.0 g of sodium chloride, and 15.0
to 20.0 g of agar in water to make 1000 mL, and sterilize. Ad-
just the pH to 6.9 to 7.1.
Normal human plasma Dissolve the lyophilized normal
human plasma derived from 1 mL of normal human plasma
with 1 mL of water.
B-Octadecane C 18 H 38 Colorless or white solid at ordina-
ry temperature.
Purity Clarity of solution — A solution of w-octadecane in
chloroform (1 in 25) is clear.
Octadecylsilanized silica gel for pretreatment Prepared
for pretreatment.
M-Octane C 8 H 18
Specific gravity <2.56> df: 0.700 - 0.705
Purity — Perform the test with 2 iiL of n-octane as directed
under Gas Chromatography <2.02> according to the condi-
tions in the Assay under Hypromellose. Measure each peak
area by the automatic integration method, and calculate the
amount of M-octane by the area percentage method: not less
than 99.0%.
Octane, iso A colorless liquid. Practically insoluble in
water. Miscible with diethyl ether and with chloroform.
Purity — Determine the absorbances of isooctane at 230
nm, 250 nm and 280 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using water as the blank solution:
these values are not more than 0.050, 0.010 and 0.005, re-
spectively.
1-Octanol CH 3 (CH 2 ) 6 CH 2 OH [K 8213, Special class]
Octyl alcohol See 1-octanol.
M-Octylbenzen C 14 H 22 Clear and colorless liquid, hav-
ing a characteristic odor.
Specific gravity <2.56> df: 0.854 - 0.863
Distillation test <2.57>: 263 - 265 °C, not less than 95 vol%.
Ofloxacin demethyl substance (±)-9-Fluoro-2,3-dihydro-
3-methyl-7-oxo-7//-10-(l-piperazinyl)-pirido[l, 2, 3-de] [1,
4]benzoxazine-6-carboxylic acid C 17 H I8 FN 3 4 White
to light green-yellowish white, crystals or crystalline powder.
Identification — Determine the infrared absorption spec-
trum of ofloxacin demethyl substance as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3050 cm-', 2840 cm-', 1619 cm-', 1581
cm-', 1466 cm-', 1267 cm-', 1090 cm-', 1051 cm-' and 816
cm-'.
Olive oil [Same as the namesake monograph]
Orcine C 7 H 3 2 White to light red-brown crystals or
crystalline powder, having an unpleasant, sweet taste. It
turns to red in color when oxidized in air. Soluble in water, in
ethanol (95), and in diethyl ether.
Meting point <2.60>: 107 - 111°C
Orcine-ferric chloride TS See orcine-iron (III) chloride
TS.
Orcine-iron (III) chloride TS Dissolve 10 mg of orcine in
1 mL of a solution of iron (III) chloride hexahydrate in hy-
drochloric acid (1 in 1000). Prepare before use.
Ordinary agar medium Dissolve 25 to 30 g of agar in
1000 mL of ordinary broth with the aid of heat, add water to
make up for the loss, adjust the pH to between 6.4 and 7.0,
and filter. Dispense the filtrate, and sterilize by autoclaving.
When powdered agar is used, 15 to 20 g of it is dissolved.
Ordinary broth Dissolve 5 g of beef extract and 10 g of
peptone in 1000 mL of water by gentle heating. Adjust the
pH of the mixture between 6.4 and 7.0 after sterilization,
cool, add water to make up for the loss, and filter. Sterilize
the filtrate by autoclaving for 30 minutes at 121 °C.
Osthole for thin-layer chromatography Ci 5 H 16 3 A
white crystalline powder, having no odor. Freely soluble in
methanol and in ethyl acetate, soluble in ethanol (99.5), and
practically insoluble in water. Melting point: 83 - 84°C.
Purity Related substances — Dissolve 1.0 mg of osthole
for thin-layer chromatography in 1 mL of methanol. Per-
form the test with 10 iiL of this solution as directed in the
Identification under Cnidium Monnieri Fruit: on spot ap-
pears other than the principal spot at around Rf 0.3.
Oxalate pH standard solution See pH Determination
<2.54>.
Oxalic acid See oxalic acid dihydrate.
Oxalic acid dihydrate H 2 C 2 4 .2H 2 [K 8519, Special
class]
Oxalic acid TS Dissolve 6.3 g of oxalic acid dihydrate in
water to make 100 mL (0.5 mol/L).
Oxycodone hydrochloride for assay
C 18 H 21 N0 4 .HC1.3H 2 [Same as the monograph Oxyco-
done Hydrochloride Hydrate. It contains not less than 99.0%
of oxycodone hydrochloride (C 18 H 21 N0 4 .HC1), calculated on
the anhydrous basis.]
Oxygen 2 [K 1101]
8-Oxyquinoline See 8-quinolinol.
2-Oxy-l-(2'-oxy-4'-sulfo-l'-naphthylazo)-3-naphthoic
acid See 2-hydroxy-l-(2'-hydroxy-4'-sulfo-l'-naphthyl-
azo)-3-naphthoic acid.
Oxytocin C 43 H 66 N 12 12 S 2 [Same as the namesake
monograph]
Paeonifiorin for thin-layer chromatography
C 23 H 28 O u -«H 2 Colorless, odorless powder. Freely soluble
in water and in methanol, and not dissolves in diethyl ether.
Melting point: 123 - 125 °C (with decomposition).
Purity Related substances — Dissolve 1 .0 mg of paeoni-
fiorin for thin layer chromatography in exactly 1 mL of meth-
anol. Perform the test with 20 iiL of this solution as directed
in the Identification (2) under Peony Root: any spot other
than the principal spot at the Rf value of about 0.3 does not
JPXV
General Tests / Reagents, Test Solutions
223
appear.
Palladium chloride See palladium (II) chloride.
Palladium chloride TS See palladium (II) chloride TS.
Palladium (II) chloride PdCl 2 [K 8154, Special class]
Palladium (II) chloride TS Dissolve 0.2 g of palladium
(II) chloride in 500 mL of 0.25 mol/L sulfuric acid TS, by
heating if necessary, cool, and add 0.25 mol/L sulfuric acid
TS to make 1000 mL.
Palmatin chloride C21H22CINO4.XH2O A yellow-brown
crystalline powder.
Purity — Dissolve 1 mg of palmatin chloride in 10 mL of
methanol, and use this solution as the sample solution. Pro-
ceed with 20 /xL of the sample solution as directed in the As-
say under Phellodendron Bark: when measure the peak areas
2 times as long as the retention time of palmatin, the total
area of the peaks other than palmatin is not larger than 1/10
of the total area except the area of solvent peak.
Palmitic acid for gas chromatography C 16 H 32 02 [K
8756, Special class]
Papaverine hydrochloride C20H21NO4.HCI [Same as
the namesake monograph]
Papaverine hydrochloride for assay C20H21NO4.HCI
[Same as the monograph Papaverine Hydrochloride. When
dried, it contains not less than 99.0% of papaverine
hydrochloride (C20H21NO4.HCI).]
Paraffin [Same as the namesake monograph]
Paraffin, liquid [Same as the monograph Light Liquid
Paraffin]
Parahydroxybenzoic acid C 7 H 6 3 White crystals.
Melting point <2.60>: 212 - 216°C
Content: not less than 98.0 %. Assay — Weigh accurately
about 0.7 g of parahydroxybenzoic acid, dissolve in 50 mL of
acetone, add 100 mL of water, and titrate <2.50> with 0.5 mol
/L sodium hydroxide VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.5 mol/L sodium hydroxide VS
= 69.06 mg of C 7 H 6 3
PBS containing bovine serum To 100 mL of bovine se-
rum add 900 mL of phosphate-buffered sodium chloride TS
containing thimerosal (0.1 g) to make 1000 mL.
Storage — Store in a cool place shielded from light.
PBS containing bovine serum albumin Add PBS to 10 g
of bovine serum albumin and 0.1 g of thimerosal to make
1000 mL.
Storage — Store in a cool, dark place.
Peanut oil [Same as the namesake monograph]
Pentane CH 3 (CH 2 ) 3 CH 3 Clear and colorless liquid.
Specific gravity <2.56> df Q : 0.620 - 0.650
Distilling range <2.57>: 35.5 - 37°C, not less than 98 vol%.
Peonol for component determination Use peonol for
thin-layer chromatography meeting the following additional
specifications.
Absorbance <2.24> E\°^ m (274 nm): 853 - 934 [5 mg after
drying in a desiccator (calcium chloride for drying) for 1 hour
or more, methanol, 1000 mL].
Purity Related substances — Dissolve 5.0 mg of peonol
for component determination in 50 mL of the mobile phase,
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add the mobile phase to make exactly
100 mL, and use this solution as the standard solution (1).
Perform the test with exactly 10 /uL each of the sample solu-
tion and standard solution (1) as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and measure each peak area of these solutions by the auto-
matic integration method: the total area of the peaks other
than peonol from the sample solution is not larger than the
peak area of peonol from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Component deter-
mination under Moutan Bark except detection sensitivity and
time span of measurement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of peonol obtained from 10
iXL of the standard solution (2) can be measured, and the
peak height of peonol from 10 /xL of the standard solution (1)
is about 20% of the full scale.
Time span of measurement: About 3 times as long as the
retention time of peonol beginning after the solvent peak.
Peonol for thin-layer chromatography C 9 H 10 O 3
White, crystals or crystalline powder, having a specific odor.
Freely soluble in methanol and in diethyl ether, and slightly
soluble in water. Melting point: about 50°C
Purity Related substances — Dissolve 1.0 mg of peonol
for thin-layer chromatography in exactly 1 mL of methanol,
and perform with 10 /xL of this solution as directed in the
Identification under Moutan Bark: any spot other than the
principal spot at the Rf value of near 0.5 does not appear.
Peptone Prepared for microbial test.
Peptone, animal tissue Prepared for microbial test.
Peptone, casein Grayish yellow powder, having a charac-
teristic but not putrescent odor. It dissolves in water, but not
in ethanol (95) and in diethyl ether.
Loss on drying <2.41>: not more than 7% (0.5 g, 105°C,
constant mass).
Residue on ignition <2.44>: not more than 15% (0.5 g).
Degree of digestion — Dissolve 1 g of casein peptone in 10
mL of water, and perform the following test using this solu-
tion as the sample solution:
(1) Overlay 1 mL of the sample solution with 0.5 mL of a
mixture of 1 mL of acetic acid (100) and 10 mL of dilute etha-
nol: no ring or precipitate forms at the junction of the two
liquids, and on shaking, no turbidity results.
(2) Mix 1 mL of the sample solution with 4 mL of a satu-
rated solution of zinc sulfate heptahydrate: a small quantity
of precipitate is produced (proteoses).
(3) Filter the mixture of (2), and to 1 mL of the filtrate
add 3 mL of water and 4 drops of bromine TS: a red-purple
color is produced.
Nitrogen content <1.08>: not less than 10% (105°C, con-
stant mass, after drying).
Peptone, gelatin Prepared for microbial test.
224
Reagents, Test Solutions / General Tests
JP XV
Peptone, soybean Prepared for microbial test.
Perchloric acid HC10 4 [K 8223, Special class, Density:
about 1.67 g/mL. Concentration: 70.0-72.0%]
Perchloric acid-dehydrated ethanol TS See perchloric
acid-ethanol TS.
Perchloric acid-ethanol TS Add cautiously 25.5 mL of
perchloric acid to 50 mL of ethanol (99.5), cool, and add eth-
anol (99.5) to make 100 mL (3 mol/L).
Performic acid Mix 9 volumes of formic acid and 1
volume of hydrogen peroxide (30), and leave at room temper-
ature for 2 hours.
Storage — Store in a cool place.
Peroxidase Obtained from horse-radish. A red-brown
powder. It is freely soluble in water. It contains about 250 u-
nits per mg. One unit indicates an amount of the enzyme
which produces 1 mg of purpurogallin in 20 seconds at 20°C
and pH 6.0, from pyrogallol and hydrogen peroxide (30) used
as the substrate.
Peroxidase-labeled antibody stock solution 1 w/v% bo-
vine serum albumin-phosphate buffer-sodium chloride TS
containing antibody fragment (Fab') bound to peroxidase.
Peroxidase-labeled bradykinin A solution of horseradish
origin peroxidase-binding bradykinin in gelatin-phosphate
buffer solution, pH 7.0. A colorless to light brown clear solu-
tion.
Peroxidase-labeled bradykinin TS To 0.08 mL of perox-
idase-labeled bradykinin, 8 mg of sodium tetraborate deca-
hydrate, 8 mg of bovine serum albumin and 0.8 mL of
gelatin-phosphate buffer solution, pH 7.0 add water to make
8 mL, and lyophilize. Dissolve this in 8 mL of water. Prepare
before use.
Peroxidase-labeled rabbit anti-ECP antibody Fab' TS
Mix 1 volume of ECP standard substance (equivalent to
about 1 mg of protein) and 1 volume of Freund's complete
adjuvant, and then immunize rabbits subcutaneously in the
back region and intramuscularly in the hind leg muscle with
this solution 5 times at 2 week intervals. Harvest blood on the
10 th day after completing the immunization to obtain rabbit
antiserum. Rabbit anti-ECP antibody Fab' is obtained by
preparing an immobilized ECP column in which ECP stan-
dard substance is bound to agarose gel and then purifying by
affinity column chromatography to obtain rabbit anti-ECP
antibody which undergoes pepsin digestion to yield F(ab')2
which is reacted with 2-aminoethanethiol hydrochloride.
Horseradish peroxidase is reacted with maleimido reagent
[4-(maleimidomethyl) cyclohexyl carbonic acid-A^-hydrox-
ysuccinimide imidoester] to yield maleimido peroxidase. Per-
form a coupling reaction by mixing rabbit anti-ECP antibody
Fab' and maleimido peroxidase at 4°C to prepare perox-
idase-labeled rabbit anti-ECP antibody Fab'. Take a specific
amount of peroxidase-labeled rabbit anti-ECP antibody Fab'
and dilute using PBS containing bovine serum albumin. The
peroxidase-labeled rabbit anti-ECP antibody Fab' TS is a
diluted solution with a concentration that gives a good
calibration curve with assay characteristics.
Description: Clear and colorless solution
Identification: Pipet 100 juL of the TS to be examined into
fiat-bottomed microtest plates. When substrate buffer solu-
tion for celmoleukin is added to this, it immediately exhibits
a dark violet color, which changes to yellowish-red with time.
Perphenazine maleate for assay [Same as the monograph
Perphenazine Maleate. When dried, it contains not less than
99.0 % of perphenazine maleate
(C 21 H 26 ClN 3 OS.2C4H 4 04).]
Pethidine hydrochloride for assay C 15 H 2 iN0 2 .HCl
[Same as the monograph Pethidine Hydrochloride. When
dried, it contains not less than 99.0% of pethidine hydrochlo-
ride C 15 H 21 N0 2 .HC1.]
Petrolatum [Same as the monograph Yellow Petrolatum
or White Petrolatum]
Petroleum benzine [K 8594, Special class]
Petroleum ether [K 8593, Special class]
Phenacetin Ci H 13 NO 2 White crystals or crystalline
powder. Soluble in ethanol (95), and very slightly soluble in
water.
Melting point <2.60>: 134 - 137°C
Loss on drying <2.41>: not more than 0.5% (1 g, 105°C, 2
hours).
o-Phenanthroline See 1,10-phenanthrolinemonohydrate.
o-Phenanthroline hydrochloride See 1,10-phenanthroli-
um chloride monohydrate.
1,10-Phenanthroline monohydrate C 12 H 8 N 2 .H 2 [K 8789,
Special class]
o-Phenanthroline TS See 1,10-phenanthroline TS.
1,10-Phenanthroline TS Dissolve 0.15 g of 1,10-phenan-
throline monohydrate in 10 mL of a freshly prepared ferrous
sulfate heptahydrate solution (37 in 2500) and 1 mL of dilute
sulfuric acid. Preserve in tightly stoppered containers.
1,10-Phenanthrolinium chloride monohydrate
C 12 H 8 N 2 .HC1.H 2 [K 8202, Special class]
Phenethylamine hydrochloride C 6 H 5 CH 2 CH 2 NH 2 .HC1
White crystals or crystlline powder.
Melting point: <2.60> 220 - 225°C
Phenobarbital sodium C 12 H u N 2 Na0 3 [Same as the
namesake monograph]
Phenol C 6 H 5 OH [K 8798, Special class]
Phenol for assay C 6 H 5 OH [K 8798, Special class]
Phenol-hydrochloric acid TS Dissolve 0.2 g of phenol in
10 mL of 6 mol/L hydrochloric acid TS.
Phenolphthalein C 20 H 14 O 4 [K 8799, Special class]
Phenolphthalein-thymol blue TS Solution A: Dissolve
0.1 g of phenolphthalein in diluted ethanol (99.5) (4 in 5). So-
lution B: Dissolve 0.1 g of thymol blue in 50 mL of a mixture
of ethanol and dilute sodium hydroxide TS, add water to
make 100 mL. Mix 2 volumes of solution A and 3 volumes of
solution B before use.
Phenolphthalein TS Dissolve 1 g of phenolphthalein in
100 mL of ethanol (95).
Phenolphthalein TS, alkaline See Alcohol Number De-
termination <1.01>.
JPXV
General Tests / Reagents, Test Solutions
225
Phenol red C 19 H 14 5 S [K 8800, Special class]
Phenol red TS Dissolve 0.1 g of phenol red in 100 mL of
ethanol (95), and filter if necessary.
Phenol red TS, dilute To 235 mL of a solution of ammo-
nium nitrate (1 in 9400) add 105 mL of 2 mol/L sodium
hydroxide TS and 135 mL of a solution prepared by dissolv-
ing 24 g of acetic acid (100) in water to make 200 mL. To this
solution add 25 mL of a solution prepared by dissolving 33
mg of phenol red in 1 .5 mL of 2 mol/L sodium hydroxide TS
and adding water to make 100 mL. If necessary, adjust the
pH to 4.7.
Phenol-sodium nitroprusside TS See phenol-sodium pen-
tacyanonitrosylferrate (III) TS.
Phenol-sodium pentacyanonitrosylferrate (III) TS Dis-
solve 5 g of phenol and 25 mg of sodium pentacyanonitrosyl-
ferrate (III) dihydrate in sufficient water to make 500 mL.
Preserve in a dark, cold place.
Phenolsulfonphthalein for assay Ci 9 H I4 5 S [Same as
the monograph Phenolsulfonphthalein. When dried, it con-
tains not less than 99.0% of phenolsulfonphthalein
(CHmOjS).]
50% Phenyl-50% methylpolysiloxane for gas chromato-
graphy Prepared for gas chromatography.
Phenylalanine C 9 H u N0 2 [Same as the monograph l-
Phenylalanine]
Phenyl benzoate C 6 H 5 COOC 6 H5 White crystals or crys-
talline powder, having a slight, characteristic odor.
Melting point <2.60>: 68 - 70°C
Purity Clarity of solution — Dissolve 1 .0 g of phenyl ben-
zoate in 20 mL of methanol: the solution is clear.
o-Phenylenediamine H 2 NC 6 H 4 NH 2 White to dark
brown crystals or crystalline powder. Freely soluble in
ethanol (95) and in acetone, and soluble in water.
Content: not less than 95.0%. Assay — Accurately weigh
about 0.15 g of o-phenylenediamine, add 50 mL of acetic
acid for nonaqueous titration to dissolve, and then titrate
<2.50> with 0.1 mol/L of perchloric acid VS (potentiometric
titration). Correct by conducting a blank test using the same
method.
Each mL of 0.1 mol/L perchloric acid VS
= 10.81 mg of H 2 NC 6 H 4 NH 2
o-Phenylenediamine dihydrochloride
H 2 NC 6 H 4 NH 2 .2HC1 White to pale yellow or pale red crys-
tals or crystalline powder.
Clarity: a solution (1 in 20) is clear.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.15 g of o-phenylenediamine dihydrochloride, dis-
solve in 50 mL of water, and titrate <2.50> with 0. 1 mol/L so-
dium hydroxide VS (potentiometric titration).
Each mL of 0.1 mol/L sodium hydroxide VS
= 9.053 mg of H 2 NC 6 H 4 NH 2 .2HC1
Phenylfluorone Ci 9 H 12 5 [K 9547]
Phenylfluorone-ethanol TS Dissolve 50 mg of phenyl-
fluorone in 10 mL of a mixture of ethanol (95) and diluted hy-
drochloric acid (1 in 3), and add thanol (95) to make exactly
500 mL.
D-Phenylglycine C 8 H 9 N0 2 White, crystals or crystalline
powder. Slightly soluble in water.
Loss on drying <2.41>: not more than 0.5% (1 g, 105°C,
3 hours).
Content: not less than 98.5%. Assay — Weigh accurately
about 0.3 g of D-phenylglycine, previously dried, dissolve in 3
mL of formic acid, add 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 15.12 mg of C 8 H 9 N0 2
Phenylhydrazine C 6 H 5 NHNH 2 Colorless or light yellow,
clear liquid, having a faint aromatic odor.
Content: not less than 99.0%. Assay — Weigh accurately
about 1 g, add 30 mL of diluted hydrochloric acid (1 in 100)
and water to make exactly 100 mL. Put exactly 20 mL of this
solution in a glass-stoppered conical flask, and add 40 mL of
diluted hydrochloric acid (3 in 4). After cooling, add 5 mL of
chloroform, and titrate <2.50> with 0.05 mol/L potassium io-
date VS while shaking vigorously until the red color of the
chloroform layer disappears. Perform a blank determination
in the same manner, and make any necessary correction.
Each mL of 0.05 mol/L potassium iodate VS
= 5 .407 mg of C 6 H 5 NHNH 2
Phenylhydrazine hydrochloride See phenylhydrazinium
chloride.
Phenylhydrazine hydrochloride TS See phenylhydrazini-
um chloride TS.
Phenylhydrazinium chloride C 6 H 5 NHNH 2 .HC1
[K 8203, Special class]
Phenylhydrazinium chloride TS Dissolve 65 mg of phe-
nylhydrazinium chloride recrystallized from dilute ethanol,
in 100 mL of a solution previously prepared by adding cauti-
ously 170 mL of sulfuric acid to 80 mL of water.
35% Phenyl-methyl silicone polymer for gas chromato-
graphy Prepared for gas chromatography.
50% Phenyl-methyl silicone polymer for gas chromato-
graphy Prepared for gas chromatography.
65% Phenyl-methyl silicone polymer for gas chromato-
graphy Prepared for gas chromatography.
25% Phenyl-25% cyanopropyl-methylsilicone polymer for
gas chromatography Prepared for gas chromatography.
l-phenyl-3-methyl-5-pyrazolone See 3-methyl-l-phenyl-
5-pyrazolone.
Phenylpiperazine hydrochloride C 10 H 14 N 2 .HC1 A white
powder. Melting point: about 247°C (with decomposition).
Phloroglucin See phloroglucinol dihydrate.
Phloroglucin dihydrate See phloroglucinol dihydrate.
Phloroglucinol dihydrate C 6 H 3 (OH) 3 .2H 2 White to
pale yellow, crystals or crystalline powder.
Melting point <2.60>: 215 -219°C (after drying).
Loss on drying <2.41>: 18.0 - 24.0% (1 g, 105°C, 1 hour).
226
Reagents, Test Solutions / General Tests
JP XV
Phosphate buffer solution for component determination of
bupleurum root To 100 mL of 0.2 mol/L potassium di-
hydrogen phosphate TS add 59 mL of sodium hydrogen
phosphate TS.
Phosphate buffer solution for pancreatin Dissolve 3.3 g
of anhydrous disodium hydrogen phosphate, 1.4 g of potas-
sium dihydrogen phosphate and 0.33 g of sodium chloride in
water to make 100 mL.
Phosphate buffer solution, pH 3.0 Dissolve 136 g of
potassium dihydrogen phosphate in water to make 1000 mL,
and adjust to pH 3.0 with phosphoric acid.
0.02 mol/L Phosphate buffer solution, pH 3.0 Dissolve
3.1 g of sodium dihydrogen phosphate dihydrate in 1000 mL
of water, and adjust the pH to 3.0 with diluted phosphoric
acid (1 in 10).
Phosphate buffer solution, pH 3.1 Dissolve 136.1 g of
potassium dihydrogen phosphate in 500 mL of water, and
add 6.3 mL of phosphoric acid and water to make 1000 mL.
0.05 mol/L Phosphate buffer solution, pH 3.5 To 1000
mL of 0.05 mol/L potassium dihydrogen phosphate TS add a
suitable amount of a solution of phosphoric acid (49 in
10,000) to make a solution having pH 3.5.
0.02 mol/L Phosphate buffer solution, pH 3.5 Dissolve
3.1 g of sodium dihydrogen phosphate dihydrate in 1000 mL
of water, and adjust the pH to 3.5 with diluted phosphoric
acid (1 in 10).
0.1 mol/L Phosphate buffer solution, pH 4.5 Dissolve
13.61 g of potassium dihydrogen phosphate in 750 mL of
water, adjust to pH 4.5 with potassium hydroxide TS, and
add water to make 1000 mL.
0.1 mol/L Phosphate buffer solution, pH 5.3 Dissolve
0.44 g of disodium hydrogen phosphate dodecahydrate and
13.32 g of potassium dihydrogen phosphate in 750 mL of
water, adjust the pH to 5.3 with sodium hydroxide TS or
phosphoric acid, and add water to make 1000 mL.
1/15 mol/L Phosphate buffer solution, pH 5.6 Dissolve
9.07 g of potassium dihydrogen phosphate in about 750 mL
of water, adjust the pH to 5.6 with potassium hydroxide TS,
and add water to make 1000 mL.
Phosphate buffer solution, pH 5.9 Dissolve 6.8 g of
potassium dihydrogen phosphate in 800 mL of water, adjust
the pH to 5.9 with diluted potassium hydroxide TS (1 in 10),
and add water to make 1000 mL.
Phosphate buffer solution, pH 6.0 Dissolve 8.63 g of
potassium dihydrogen phosphate and 1.37 g of anhydrous
disodium hydrogen phosphate in 750 mL of water, adjust the
pH to 6.0 with sodium hydroxide TS or diluted phosphoric
acid (1 in 15), and add water to make 1000 mL.
0.05 mol/L Phosphate buffer solution, pH 6.0 To 50 mL
of 0.2 mol/L potassium dihydrogen phosphate TS for buffer
solution add 5.70 mL of 0.2 mol/L sodium hydroxide TS and
water to make 200 mL.
Phosphate buffer solution, pH 6.2 Dissolve 9.08 g of
potassium dihydrogen phosphate in 1000 mL of water (solu-
tion A). Dissolve 9.46 g of disodium hydrogen phosphate in
1000 mL of water (solution B). Mix 800 mL of the solution A
and 200 mL of the solution B, and adjust the pH to 6.2 with
the solution A or the solution B if necessary.
Phosphate buffer solution, pH 6.5 Mix 50 mL of 0.2 mol
/L potassium dihydrogen phosphate TS for buffer solution
and 15.20 mL of 0.2 mol/L sodium hydroxide VS, and add
water to make 200 mL.
Phosphate buffer solution, pH 6.8 Dissolve 3.40 g of
potassium dihydrogen phosphate and 3.55 g of disodium
hydrogen phosphate in water to make 1000 mL.
0.01 mol/L Phosphate buffer solution, pH 6.8 Dissolve
1.36 g of potassium dihydrogen phosphate in 900 mL of
water, adjust the pH to 6.8 with 0.2 mol/L sodium hydroxide
TS, and add water to make 1000 mL.
0.1 mol/L Phosphate buffer solution, pH 6.8 Dissolve
6.4 g of potassium dihydrogen phosphate and 18.9 g of diso-
dium hydrogen phosphate dodecahydrate in about 750 mL of
water, adjust the pH to 6.8 with sodium hydroxide TS if
necessary, and add water to make 1000 mL.
Phosphate buffer solution, pH 7.0 Mix 50 mL of 0.2 mol
/L potassium dihydrogen phosphate TS for buffer solution
and 29.54 mL of 0.2 mol/L sodium hydroxide VS, and add
water to make 200 mL.
0.05 mol/L Phosphate buffer solution, pH 7.0 Dissolve
4.83 g of dipotassium hydrogen phosphate and 3.02 g of
potassium dihydrogen phosphate in 1000 mL of water, and
adjust to pH 7.0 with phosphoric acid or potassium
hydroxide TS.
0.1 mol/L Phosphate buffer solution, pH 7.0 Dissolve
17.9 g of disodium hydrogen phosphate dodecahydrate in
water to make 500 mL (solution A). Dissolve 6.8 g of potassi-
um dihydrogen phosphate in water to make 500 mL (solution
B). To a volume of solution A add solution B until the mix-
ture is adjusted to pH 7.0 (about 2:1 by volume of solutions
A and B).
Phosphate buffer solution, pH 7.2 Mix 50 mL of 0.2 mol
/L potassium dihydrogen phosphate TS for buffer solution
and 34.7 mL of 0.2 mol/L sodium hydroxide VS, and add
water to make 200 mL.
Phosphate buffer solution, pH 7.4 Mix 50 mL of 0.2 mol
/L potassium dihydrogen phosphate TS for buffer solution
and 39.50 mL of 0.2 mol/L sodium hydroxide VS, and add
water to make 200 mL.
0.03 mol/L Phosphate buffer solution, pH 7.5 Dissolve
4.083 g of potassium dihydrogen phosphate in 800 mL of
water, adjust the pH to 7.5 with 0.2 mol/L sodium hydroxide
TS, and add water to make 1000 mL.
Phosphate buffer solution, pH 8.0 Mix 50 mL of 0.2 mol
/L potassium dihydrogen phosphate TS for buffer solution
and 46.1 mL of 0.2 mol/L sodium hydroxide VS, and add
water to make 200 mL.
0.1 mol/L Phosphate buffer solution for antibiotics, pH
8.0 Dissolve 16.73 g of dipotassium hydrogen phosphate
and 0.523 g of potassium dihydrogen phosphate in about 750
mL of water, adjust the pH to 8.0 with phosphoric acid, and
add water to make 1000 mL.
0.02 mol/L Phosphate buffer solution, pH 8.0 To 50 mL
JPXV
General Tests / Reagents, Test Solutions
227
of 0.2 mol/L potassium dihydrogen phosphate TS add 300
mL of water, adjust the pH to 8.0 with sodium hydroxide TS,
and add water to make 500 mL.
0.1 mol/L Phosphate buffer solution, pH 8.0 Dissolve
13.2 g of anhydrous disodium hydrogen phosphate and 0.91
g of potassium dihydrogen phosphate in about 750 mL of
water, adjust to pH 8.0 with phosphoric acid, and add water
to make 1000 mL.
0.2 mol/L Phosphate buffer solution, pH 10.5 Dissolve
34.8 g of dipotassium hydrogen phosphate in 750 mL of
water, adjust to pH 10.5 with 8 mol/L sodium hydroxide TS,
and add water to make 1000 mL.
Phosphate buffer solution, pH 12 To 5.44 g of disodium
hydrogen phosphate add 36.5 mL of sodium hydroxide TS
and about 40 mL of water, dissolve by shaking well, and add
water to make 100 mL.
Phosphate buffer solution for antibiotics, pH 6.5
Dissolve 10.5 g of disodium hydrogen phosphate dodecahy-
drate and 5.8 g of potassium dihydrogen phosphate in 750
mL of water, adjust the pH to 6.5 with sodium hydroxide TS,
and add water to make 1000 mL.
Phosphate buffer solution for microplate washing Dis-
solve 0.62 g of sodium dihydrogen phosphate dihydrate, 9.48
g of disodium hydrogen phosphate dodecahydrate, 52.6 g of
sodium chloride, 3.0 g of polysorbate 80 and 1.8 g of polyox-
yethylene (40) octylphenyl ether in water to make 600 mL.
Dilute this solution 10 times with water before use.
Phosphate buffer solution for processed aconite root Dis-
solve 19.3 g of disodium hydrogen phosphate dodecahydrate
in 3660 mL of water, and add 12.7 g of phosphoric acid.
Phosphate-buffered sodium chloride TS Dissolve 8.0 g of
sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodi-
um hydrogen phosphate dodecahydrate, and 0.2 g of potassi-
um dihydrogen phosphate in water to make 1000 mL.
0.01 mol/L Phosphate buffer-sodium chloride TS, pH 7.4
Dissolve 2.93 g of disodium hydrogen phosphate dodecahy-
drate (NaH 2 P0 4 12H 2 0), 0.25 g of potassium dihydrogen
phosphate (KH 2 P0 4 ), and 9.0 g of sodium chloride in water
to make 1000 mL.
Phosphinic acid H 3 P0 2 [K 8440, First class]
Phosphate TS Dissolve 2.0 g of dipotassium hydrogen
phosphate and 8.0 g of potassium dihydrogen phosphate in
water to make 1000 mL.
Phosphomolybdic acid See phosphomolybdic acid n-
hydrate.
Phosphomolybdic acid n-hydrate P 2 5 .24Mo0 3 .«H 2
Yellow crystals or crystalline powder.
Identification — (1) To 10 mL of a solution (1 in 10) add
0.5 mL of ammonia TS: yellow precipitates appear, which
disappear by the addition of 2 mL of ammonia TS, and yel-
low precipitates appear by further addition of 5 mL of dilut-
ed nitric acid (1 in 2).
(2) To 5 mL of a solution (1 in 10) add 1 mL of ammonia
TS and 1 mL of magnesia TS: white precipitates appear.
Phosphoric acid H 3 P0 4 [K 9005, Special class]
Phosphoric acid-sodium sulfate buffer solution, pH 2.3
Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of
water, and add 2.7 mL of phosphoric acid. If necessary, ad-
just to pH 2.3 with 2-aminoethanol.
Phosphoric acid-acetic acid-boric acid buffer solution, pH
2.0 Dissolve 6.77 mL of phosphoric acid, 5.72 mL of acetic
acid (100) and 6.18 g of boric acid in water to make 1000 mL.
Adjust the pH of this solution to 2.0 with 0.5 mol/L sodium
hydroxide VS.
Phosphorus pentoxide See phosphorus (V) oxide.
Phosphorus, red P An odorless dark red powder. Practi-
cally insoluble in carbon disulfide and in water.
Free phosphoric acid: Not more than 0.5%.
To 5 g add 10 mL of a solution of sodium chloride (1 in 5),
mix, then add 50 mL of the solution of sodium chloride (1 in
5), allow to stand for 1 hour, and filter. Wash the residue
with three 10-mL portions of the solution of sodium chloride
(1 in 5), combine the filtrate and the washings, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of thymol blue TS). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 4.90 mg of H3PO4
Phosphorus (V) oxide P 2 5 [K 8342, Special class]
Phosphotungstic acid
hydrate.
See phosphotungstic acid n-
Phosphotungstic acid w-hydrate P 2 5 .24W0 3 .«H 2
White to yellowish green, crystals or crystalline powder.
Identification — To 5 mL of a solution (1 in 10) add 1 mL of
acidic tin (II) chloride TS, and heat: blue precipitates appear.
Phosphotungstic acid TS Dissolve 1 g of phospho-
tungstic acid n-hydrate in water to make 100 mL.
o-Phthalaldehyde C 6 H 4 (CHO) 2 Light yellow to yellow
crystals.
Content: not less than 99%. Assay — Dissolve 1 g of o-
phthalaldehyde in 10 mL of ethanol (95). Proceed with 2,mL
of this solution as directed in Gas Chromatography <2.02> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method.
Content (%) = peak area of o-phthalaldehyed/total area
of all peaks x 100
Operating conditions —
Detector: A thermal conductivity detector
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with siliceous earth for gas chro-
matography treated with acid and silane (177 - 250 ,Mm),
coated with methyl silicon polymer for gas chromatography
in 10%.
Column temperature: A constant temperature of about 180
°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
o-phthalaldehyde is 3 - 4 minutes.
Time span of measurement: About 7 times as long as the
retention time of o-phthalaldehyde, beginning after the sol-
vent peak.
Phthalein purple C 32 H 32 N 2 Oi 2 .xH 2 Yellowish white
to brown power. Soluble in ethanol (95), and practically in-
soluble in water.
228
Reagents, Test Solutions / General Tests
JP XV
Sensitivity test — Dissolve 10 mg of phthalein purple in 1
mL of ammonia solution (28), and add water to make 100
mL. To 5 mL of this solution add 95 mL of water, 4 mL of
ammonia solution (28), 50 mL of ethanol (95) and 0.1 mL of
diluted barium chloride TS (1 in 5): the solution shows a blue-
purple color which disappears on the addition of 0.15 mL of
0.1 mol/L disodium dihydrogen ethylenediamine tetraacetate
TS.
Phthalic acid C 8 H 6 4 Colorless or white crystalline
powder. Soluble in methanol and in ethanol (95), sparingly
soluble in water, and practically insoluble in chloroform.
Melting point: about 200°C (with decomposition).
Content: not less than 98%. Assay — Weigh accurately
about 2.8 g of phthalic acid, add exactly 50 mL of 1 mol/L
sodium hydroxide VS and 25 mL of water, and dissolve by
heating on a hot plate. After cooling, add 5 drops of
phenolphthalein TS, and titrate the excess sodium hydroxide
with 0.5 mol/L sulfuric acid VS. Perform a blank determina-
tion, and make any necessary correction.
Each mL of 1 mol/L sodium hydroxide VS
= 83.07 mg of C 8 H 6 4
Phthalic anhydride C 8 H 4 3 [K 8887, Special class]
Phthalimide C 8 H 5 N0 2 White to pale brown crystals or
powder.
Melting point <2.60>: 232-237°C
Clarity — 1.0 g of phthalimide dissolves in 20 mL of sodium
hydroxide TS as a slight turbid solution.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.3 g of the substance to be tested, dissolve in 40 mL of
7V,7V-dimethylformamide, and titrate <2.50> with 0.1 mol/L
sodium meth oxide VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium methoxide VS
= 14.71 mg of C 8 H 5 N0 2
Phytonadione C 31 H 46 2 [Same as the namesake mono-
graph]
Picric acid See 2,4,6-trinitrophenol.
Picric acid-ethanol TS See 2,4,6-trinitrophenol-ethanol
TS.
Picric acid TS See 2,4,6-trinitrophenol TS.
Picric acid TS, alkaline See 2,4,6-trinitrophenol TS,
alkaline.
Piperidine hydrochloride C 5 H U N.HC1 A white crystal-
line powder. Dissolves in water and in methanol. The pH of
the aqueous solution (1 in 20) is between 3.0 and 5.0.
Melting point <2.60>: 247 - 252°C
Purity Clarity and color of solution — Dissolve 1.0 g of
piperidine hydrochloride in 20 mL of water: the solution is
clear and colorless.
Residue on ignition <2.44>: not more than 0.1% (1 g).
Content: not less than 99.0%. Assay — Dissolve about
0.25 g of piperidine hydrochloride, accurately weighed, in
50 mL of water, add 5 mL of diluted nitric acid (1 in 3), and
titrate <2.50> with 0.1 mol/L silver nitrate VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 12.16 mg of C 5 H U N.HC1
Polyalkylene glycol for gas chromatography Prepared
for gas chromatography.
Polyalkylene glycol monoether for gas chromatography
Prepared for gas chromatography.
Polyethylene glycol 15000-diepoxide for gas chromato-
graphy Prepared for gas chromatography.
Polyethylene glycol 20 M for gas chromatography
Prepared for gas chromatography.
Polyethylene glycol 400 for gas chromatography
Prepared for gas chromatography.
Polyethylene glycol 600 for gas chromatography Pre-
pared for gas chromatography.
Polyethylene glycol 1500 for gas chromatography Pre-
pared for gas chromatography.
Polyethylene glycol 6000 for gas chromatography
Prepared for gas chromatography.
Polyethylene glycol ester for gas chromatography Pre-
pared for gas chromatography.
Polyoxyethylene hydrogenated castor oil 60 A nonionic
surfactant prepared by addition polymerization of ethylene
oxide with hydrogenated castor oil. Average molar number
of ethylene oxide added is about 60. A white or pale yellow
petrolatum-like or waxy substance, having a faint, character-
istic odor and a slight bitter taste. Very soluble in ethyl ace-
tate and in chloroform, freely soluble in ethanol (95), slightly
soluble in water, and practically insoluble in diethyl ether.
Identification — (1) To 0.5 g of polyoxyethylene hydro-
genated castor oil 60 add 10 mL of water and 5 mL of ammo-
nium thiocyanate-cobalt (II) nitrate TS, and shake thor-
oughly. Add 5 mL of chloroform, shake, and allow to stand:
a blue color develops in the chloroform layer.
(2) To 0.2 g of polyoxyethylene hydrogenated castor oil
60 add 0.5 g of potassium bisulfate, and heat: an acrolein-
like, irritating odor is perceptible.
(3) To 0.5 g of polyoxyethylene hydrogenated castor oil
60 add 10 mL of water, shake, and add 5 drops of bromine
TS: the color of the test solution does not disappear.
Congealing point <2.42>: 30 - 34°C
pH <2.54> — To 1 .0 g of polyoxyethylene hydrogenated cas-
tor oil 60 add 20 mL of water, and dissolve by heating: the
pH of the solution is between 3.6 and 6.0.
Acid value <1.13>: not more than 1.0.
Saponification value <I.13>; 41 - 51
Hydroxy I value <1.13>: 39-49
Purity (1) Clarity and color of solution — Dissolve 1.0 g
of polyoxyethylene hydrogenated castor oil 60 in 20 mL of
ethanol: the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of polyoxy-
ethylene hydrogenated castor oil 60 according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of polyoxyethylene hydrogenated castor oil 60 according to
Method 3, and perform the test (not more than 2 ppm).
Water <2.48>: not more than 2.0% (1 g).
Residue on ignition <2.44>: not more than 0.1% (1 g).
Storage — Preserve in tight containers.
JPXV
General Tests / Reagents, Test Solutions
229
Polyoxyethylene (23) lauryl ether
C 12 H 25 (OCH 2 CH 2 )«OH White masses,
about 40°C
Melting point:
Polyoxyethylene (40) octylphenyl ether Obtained by the
addition polymerization with ethylene oxide to octylphenol.
A colorless or white to pale yellow, liquid, vaseline-like or
waxy, having slightly a characteristic odor.
Clarity: a solution (1 in 20) is clear.
Specific gravity <2.56> df: 1.10-1.11
pH <2.54>: 7.0 - 9.5 (5 w/v%, 25°C).
Polysorbate 20 Chiefly consists of addition polymer of
sorbitan monolaurate and ethylene oxide. Pale yellow to yel-
low liquid, having a faint, characteristic odor.
Identification— (I) To 0.5 g of polysorbate 20 add 10 mL
of water and 10 mL of sodium hydroxide TS, boil for 5
minutes, and acidify with dilute hydrochloric acid: an oily
fraction is separated.
(2) To 0.5 g of polysorbate 20 add 10 mL of water,
shake, and add 5 drops of bromine TS: the red color of the
test solution does not disappear.
(3) Saponify 5 g of polysorbate 20 as directed under
Saponification Value, and evaporate ethanol completely. Dis-
solve the residue in 50 mL of water, acidity with hydrochloric
acid (methyl orange), and extract with two 30 mL portions of
diethyl ether. Combine the diethyl ether layer, wash with 20
mL portions of water until the washings become neutral, and
evaporate the diethyl ether on a water bath: the acid value of
the residue is between 275 and 285. Use 50 mL of 0.5 mol/L
potassium hydroxide-ethanol VS for saponification.
Acid value <1.13>: not more than 4.0.
Saponification value <1.13>: 43 - 55
Loss on drying <2.41>: not more than 3.0% (5 g, 105°C, 1
hour).
Residue on ignition — Weigh accurately 3 g of polysorbate
20, heat gently at first, and ignite gradually (800 in 1200°C)
until the residue is completely incinerated. If any carbonized
substance remains, extract with hot water, filter through a
sheet of filter paper for quantitative analysis (5C), and ignite
the residue with the filter paper. Add the filtrate to it,
evaporate to dryness, and ignite carefully until the car-
bonized substance does not remain. If any carbonized sub-
stance still remains, add 15 mL of ethanol (95), crush the car-
bonized substance with a glass rod, burn the ethanol, and ig-
nite carefully. Cool in a desiccator (silica gel), and weigh the
residue accurately: not more than 1.0%.
Polysorbate 80 [Same as the namesake monograph].
Polyvinyl alcohol (-CH 2 CHOH-)„ [K 9550, Special
class]
Polyvinyl alcohol I Colorless to white, or pale yellow
granules or powder. It is odorless, or has a faint odor of
acetic acid. It is tasteless. Practically insoluble in ethanol (95)
or in diethyl ether. To polyvinyl alcohol I add water, and
heat: A clear, viscous solution is obtained. It is hygroscopic.
Viscosity <2.53>: 25.0 - 31.0 mm 2 /s. Weigh 4.000 g of
polyvinyl alcohol I, previously dried, add 95 mL of water, al-
low to stand for 30 minutes, and heat to dissolve on a water
bath under a reflux condenser for 2 hours while stirring. Af-
ter cooling, add water to make 100.0 g, and mix. Allow to
stand still to remove bubbles, and perform the test at 20 ±
0.1 °C as directed in Method 1.
pH <2.54> — The pH of a solution of polyvinyl alcohol I (1
in 25) is between 5.0 and 8.0.
Clarity and color of solution — To 1.0 g of polyvinyl alco-
hol I add 20 mL of water, disperse by well stirring, warm be-
tween 60°C and 80°C for 2 hours, and cool: the solution is
colorless and clear.
Saponification value: 98.0 - 99.0 mol%. Weigh accurately
about 3.0 g of polyvinyl alcohol I, previously dried, transfer
to a glass-stoppered conical flask, add 100 mL of water, and
dissolve by heating on a water bath. After cooling, add ex-
actly 25 mL of 0.1 mol/L sodium hydroxide VS, stopper
tightly, and allow to stand for 2 hours. Then add exactly 30
mL of 0.05 mol/L sulfuric acid VS, shake well, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion in the same manner, and make any necessary correction.
However, when the volume of 0.1 mol/L sodium hydroxide
VS consumed in the test is 25 mL or more, use about 2.0 g of
the sample.
Saponification value (mol%)
44.05,4
60.05 -0.42,4
100-
0.6005 x(g- b)f
amount (g) of the sample
a: Volume (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed in the test
b: Volume (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed in the blank test
/: Molarity factor of 0.1 mol/L sodium hydroxide VS
Polyvinyl alcohol II Colorless to white or pale yellow
granules or powder. It is odorless, or has a faint odor of acet-
ic acid. It is tasteless. Practically insoluble in ethanol (95) or
in diethyl ether. To polyvinyl alcohol II add water, and heat:
A clear, viscous solution is obtained. It is hygroscopic.
Viscosity <2.53>: 4.6 - 5.4 mm 2 /s. Weigh 4.000 g of poly-
vinyl alcohol II, previously dried, add 95 mL of water, allow
to stand for 30 minutes, and dissolve by stirring on a water
bath between 60°C and 80°C for 2 hours. After cooling, add
water to make 100.0 g, and mix. Allow to stand still to re-
move bubbles, and perform the test at 20 ± 0.1 °C as directed
in Method 1.
pH <2.54> — The pH of a solution of polyvinyl alcohol II (1
in 25) is between 5.0 and 8.0.
Clarity and color of solution — To 1.0 g of polyvinyl alco-
hol II add 20 mL of water, disperse by well stirring, heat on a
water bath for 2 hours, and cool: the solution is clear and
colorless.
Saponification value: 86.5 - 89.5 mol%. Weigh accurately
about 2.0 g of polyvinyl alcohol II, previously dried, transfer
to a glass-stoppered, conical flask, add 100 mL of water, and
warm while stirring for 2 hours. After cooling, add exactly 25
mL of 0.5 mol/L sodium hydroxide VS, stopper tightly, and
allow to stand for 2 hours. Then add exactly 30 mL of 0.25
mol/L sulfuric acid VS, shake well, and titrate <2.50> with
0.5 mol/L sodium hydroxide VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination in the
same manner, and make any necessary correction.
Saponification value (mol%)
44.05,4
60.05 -0.42,4
100-
230
Reagents, Test Solutions / General Tests
JP XV
A _ 3.0025 x(g-fr)/
amount (g) of the sample
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the test
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the blank test
/: Molarity factor of 0.5 mol/L sodium hydroxide VS
Polyvinyl alcohol TS Weigh exactly 0.50 g of polyvinyl
alcohol, and add water to make exactly 100 mL.
Potassium acetate CH 3 COO.K [K 8363, Special class]
Potassium acetate TS Dissolve 10 g of potassium acetate
in water to make 100 mL (1 mol/L).
Potassium aluminum sulfate See aluminum potassium
sulfate dodecahydrate.
Potassium bicarbonate See potassium hydrogen car-
bonate.
Potassium biphthalate See potassium hydrogen phtha-
late.
Potassium biphthalate buffer solution, pH 3.5 See potas-
sium hydrogen phthalate buffer solution, pH 3.5.
Potassium biphthalate buffer solution, pH 4.6 See potas-
sium hydrogen phthalate buffer solution, pH 4.6.
Potassium biphthalate buffer solution, pH 5.6 See potas-
sium hydrogen phthalate buffer solution, pH 5.6.
Potassium biphthalate for pH determination See potassi-
um hydrogen phthalate for pH determination.
Potassium biphthalate (standard reagent) See potassium
hydrogen phthalate (standard reagent).
0.2 mol/L Potassium biphthalate TS for buffer solution
See 0.2 mol/L potassium hydrogen phthalate TS for buffer
solution.
Potassium bisulfate See potassium hydrogen sulfate.
Potassium bromate KBr0 3 [K 8530, Special class]
Potassium bromide KBr [K 8506, Special class]
Potassium bromide for infrared spectrophotometry
Crush homocrystals of potassium bromide or potassium
bromide, collect a powder passed through a No. 200 (75 /am)
sieve, and dry at 120°C for 10 hours or at 500°C for 5 hours.
Prepare tablets with this powder, and determine the infrared
absorption spectrum <2.25>: any abnormal absorption does
not appear.
Potassium carbonate K 2 C0 3 [K 8615, Special class]
Potassium carbonate, anhydrous See potassium car-
bonate.
Potassium carbonate-sodium carbonate TS Dissolve 1.7
g of potassium carbonate and 1 .3 g of anhydrous sodium car-
bonate in water to make 100 mL.
Potassium chlorate KC10 3 [K 8207, Special class]
Potassium chloride KC1 [K 8121, Special class]
Potassium chloride for conductivity measurement
[K 8121, Potassium chloride for conductivity measurement]
Potassium chloride for infrared spectrophotometry
Crush homocrystals of potassium chloride or potassium chlo-
ride (Special class), collect the powder passed through a No.
200 (75 ,um) sieve, and dry at 120°C for 10 hours or at 500°C
for 5 hours. Prepare tablets with this powder, and determine
the infrared absorption spectrum <2.25>: any abnormal ab-
sorption does not appear.
Potassium chloride-hydrochloric acid buffer solution To
250 mL of a solution of potassium chloride (3 in 20) add 53
mL of 2 mol/L hydrochloric acid TS and water to make 1000
mL.
Potassium chloride TS, acidic Dissolve 250 g of potassi-
um chloride in water to make 1000 mL, and add 8.5 mL of
hydrochloric acid.
0.2 mol/L Potassium chloride TS Dissolve 14.9 g of
potassium chloride in water to make 1000 mL. Prepare before
use.
Potassium chromate K 2 Cr0 4 [K 8312, Special class]
Potassium chromate TS Dissolve 10 g of potassium chro-
mate in water to make 100 mL.
Potassium cyanide KCN [K 8443, Special class]
Potassium cyanide TS Dissolve 1 g of potassium cyanide
in water to make 10 mL. Prepare before use.
Potassium dichromate K 2 Cr 2 7 [K 8517, Special class]
Potassium dichromate (standard reagent) K 2 Cr 2 7
[K 8005, Standard reagent for volumetric analysis]
Potassium dichromate-sulfuric acid TS Dissolve 0.5 g of
potassium dichromate in diluted sulfuric acid (1 in 5) to make
100 mL.
Potassium dichromate TS Dissolve 7.5 g of potassium
dichromate in water to make 100 mL.
Potassium dihydrogen phosphate KH 2 P0 4 [K 9007,
Special class]
Potassium dihydrogen phosphate for pH determination
KH 2 P0 4 [K 9007, for pH determination]
0.02 mol/L Potassium dihydrogen phosphate TS
Dissolve 2.72 g of potassium dihydrogen phosphate in water
to make 1000 mL.
0.05 mol/L Potassium dihydrogen phosphate TS Dis-
solve 6.80 g of potassium dihydrogen phosphate in water to
make 1000 mL.
0.1 mol/L Potassium dihydrogen phosphate TS, pH 2.0
Dissolve 13.6 g of potassium dihydrogen phosphate in water
to make 1000 mL. Adjust the pH to 2.0 with phosphoric acid.
0.25 mol/L Potassium dihydrogen phosphate TS, pH 3.5
Dissolve 34 g of potassium dihydrogen phosphate in 900 mL
of water, adjust the pH to 3.5 with phosphoric acid, and add
water to make 1000 mL.
0.33 mol/L Potassium dihydrogen phosphate TS Dis-
solve 4.491 g of potassium dihydrogen phosphate in water to
make 100 mL.
0.05 mol/L Potassium dihydrogen phosphate, pH 3.0
Adjust the pH of 0.05 mol/L potassium dihydrogen
JPXV
General Tests / Reagents, Test Solutions
231
phosphate TS to 3.0 with phosphoric acid.
0.05 mol/L Potassium dihydrogen phosphate TS, pH 4.7
Dissolve 6.80 g of potassium dihydrogen phosphate in 900
mL of water, adjust the pH to exactly 4.7 with dilute sodium
hydrochloride TS, and add water to make 1000 mL.
0.1 mol/L Potassium dihydrogen phosphate TS Dissolve
13.61 g of potassium dihydrogen phosphate in water to make
1000 mL.
0.2 mol/L Potassium dihydrogen phosphate TS
Dissolve 27.22 g of potassium dihydrogen phosphate in water
to make 1000 mL.
0.2 mol/L Potassium dihydrogen phosphate TS for buffer
solution Dissolve 27.218 g of potassium dihydrogen
phosphate for pH determination in water to make 1000 mL.
Potassium disulfate K 2 S 2 7 [K 8783, Potassium Di-
sulfate, Special class]
Potassium ferricyanide See potassium hexacyanoferrate
(III).
Potassium ferricyanide TS See potassium hexacyanofer-
rate (III) TS.
Potassium ferricyanide TS, alkaline See potassium hex-
acyanoferrate (III) TS, alkaline.
Potassium ferrocyanide See potassium hexacyanoferrate
(II) trihydrate.
Potassium ferrocyanide TS See potassium hexacyanofer-
rate (II) TS.
Potassium guaiacolsulfonate C7H7KO5S [Same as the
namesake monograph]
Potassium hexacyanoferrate (II) trihydrate
K 4 Fe(CN) 6 .3H 2 [K 8802, Special class]
Potassium hexacyanoferrate (II) TS Dissolve 1 g of po-
tassium hexacyanoferrate (II) trihydrate in water to make 10
mL (1/4 mol/L). Prepare before use.
Potassium hexacyanoferrate (III) K 3 Fe(CN) 6 [K 8801,
Special class]
Potassium hexacyanoferrate (III) TS Dissolve 1 g of po-
tassium hexacyanoferrate (III) in water to make 10 mL
(i/ 3 mol/L). Prepare before use.
Potassium hexacyanoferrate (III) TS, alkaline Dissolve
1.65 g of potassium hexacyanoferrate (III) and 10.6 g of an-
hydrous sodium carbonate in water to make 100 mL.
Preserve in light-resistant containers.
Potassium hexahydroxoantimonate (V) K 2 H 2 Sb207.4H 2
White granules or crystalline powder.
Identification — To 1 g add 100 mL of water, and dissolve
by warming. To 20 mL of this solution add 0.2 mL of sodium
chloride TS: white precipitates appear. Rubbing the inside
wall of the vessel with a glass rod accelerates the forming of
the precipitates.
Potassium hexahydroxoantimonate (V) TS To 2 g of
potassium hexahydroxoantimonate (V) add 100 mL of water.
Boil the solution for about 5 minutes, cool quickly, add 10
mL of a solution of potassium hydroxide (3 in 20), allow to
stand for 1 day, and filter.
Potassiumhydrogencarbonate KHCO3 [K 8621, Special
class]
Potassium hydrogen phthalate C 6 H 4 (COOK)(COOH)
[K 8809, Special class]
Potassium hydrogen phthalate buffer solution, pH 3.5
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate TS
for buffer solution and 7.97 mL of 0.2 mol/L hydrochloric
acid VS with water to make 200 mL.
Potassium hydrogen phthalate buffer solution, pH 4.6
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate TS
for buffer solution and 12.0 mL of 0.2 mol/L sodium hy-
droxide VS with water to make 200 mL.
Potassium iodide TS, saturated Saturate 20 g of potassi-
um iodide in 10 mL of fleshly boiled and cooled water. Pre-
pare before use.
0.3 mol/L Potassium hydrogen phthalate buffer solution,
pH 4.6 Dissolve 61.26 g of potassium hydrogen phthalate
in about 800 mL of water, adjust the pH to 4.6 with sodium
hydroxide TS, and add water to make 1000 mL.
Potassium hydrogen phthalate buffer solution, pH 5.6
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate TS
for buffer solution and 39.7 mL of 0.2 mol/L sodium hy-
droxide VS with water to make 200 mL.
Potassium hydrogen phthalate for pH determination
C 6 H 4 (COOK)(COOH) [K 8809, For pH determination]
Potassium hydrogen phthalate (standard reagent)
C 6 H 4 (COOK)(COOH) [K 8005, Standard reagent for volu-
metric analysis]
0.2 mol/L Potassium hydrogen phthalate TS for buffer so-
lution Dissolve 40.843 g of potassium hydrogen phthalate
for pH determination in water to make 1000 mL.
Potassium hydrogen sulfate KHS0 4
class]
[K 8972, Special
Potassium hydroxide KOH [K 8574, Special class]
Potassium hydroxide-ethanol TS Dissolve 10 g of potas-
sium hydroxide in ethanol (95) to make 100 mL. Prepare be-
fore use.
0.1 mol/L Potassium hydroxide-ethanol TS To 1 mL of
dilute potassium hydroxide-ethanol TS add ethanol (95) to
make 5 mL. Prepare before use.
Potassium hydroxide-ethanol TS, dilute Dissolve 35 g of
potassium hydroxide in 20 mL of water, and add ethanol (95)
to make 1000 mL (0.5 mol/L). Preserve in tightly stoppered
bottles.
Potassium hydroxide TS Dissolve 6.5 g of potassium
hydroxide in water to make 100 mL (1 mol/L). Preserve in
polyethylene bottles.
0.02 mol/L Potassium hydroxide TS Dilute 2 mL of
potassium hydroxide TS with water to make 100 mL. Prepare
before use.
0.05 mol/L Potassium hydroxide TS Dilute 5 mL of
potassium hydroxide TS with water to make 100 mL. Prepare
before use.
8 mol/L Potassium hydroxide TS Dissolve 52 g of
232
Reagents, Test Solutions / General Tests
JP XV
potassium hydroxide in water to make 100 mL. Preserve in
polyethylene bottles.
Potassium iodate KI0 3 [K 8922, Special class]
Potassium iodate (standard reagent) KI0 3 [K 8005,
Standard reagent for volumetric analysis]
Potassium iodide KI [K 8913, Special class]
Potassium iodide for assay [Same as the monograph
Potassium Iodide]
Potassium iodide-starch TS Dissolve 0.5 g of potassium
iodide in 100 mL of freshly prepared starch TS. Prepare be-
fore use.
Potassium iodide TS Dissolve 16.5 g of potassium iodide
in water to make 100 mL. Preserve in light-resistant contain-
ers. Prepare before use (1 mol/L).
Potassium iodide TS, concentrated Dissolve 30 g of
potassium iodide in 70 mL of water. Prepare before use.
Storage — Preserve in light-resistant containers.
Potassium iodide-zinc sulfate TS Dissolve 5 g of potassi-
um iodide, 10 g of zinc sulfate, and 50 g of sodium chloride
in water to make 200 mL.
Potassium methanesulfonate CH 3 S0 3 K White crystals
or crystalline powder.
Purity Clarity and color of solution — Dissolve 1.0 g of
potassium methanesulfonate in 20 mL of water: the solution
is transparent and colorless.
Content: not less than 98.0%. Assay— Dissolve about 0.1
g of potassium methanesulfonate, accurately weighed, in 10
mL of acetic acid (100), add 20 mL of acetic anhydride, and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 13.42 mg of CH 3 S0 3 K
Potassium naphthoquinone sulfonate See potassium 1,2-
naphthoquinone-4-sulfonate.
Potassiuml,2-naphthoquinone-4-sulfonate CioH 5 2 S0 3 K
[K 8696, /3-Naphthoquinone-4-sulfonic acid potassium salt,
Special class]
Potassium l,2-naphthoquinone-4-sulfonate TS Dissolve
0.5 g of potassium l,2-naphthoquinone-4-sulfonate in water
to make 100 mL. Prepare before use.
Potassium nitrate KN0 3 [K 8548, Special class]
Potassium nitrite KN0 2 [K 8017, Special class]
Potassium perchlorate KC10 4 [K 8226, Special class]
Potassium periodate KI0 4 [K 8249, Special class]
Potassium periodate TS To 2.8 g of potassium per-
iodate add 200 mL of water, dissolve by adding dropwise 20
mL of sulfuric acid under shaking, cool, and add water to
make 1000 mL.
Potassium permanganate KMn0 4 [K 8247, Special
class]
Potassium permanganate TS Dissolve 3.3 g of potas-
sium permanganate in water to make 1000 mL (0.02
mol/L).
Potassium permanganate TS, acidic To 100 mL of potas-
sium permanganate TS add 0.3 mL of sulfuric acid.
Potassium peroxodisulfate K 2 S 2 8 [K 8253, Special
class]
Potassium persulfate See potassium peroxodisulfate.
Potassium pyroantimonate See potassium hexahydrox-
oantimonate (V).
Potassium pyroantimonate TS See potassium hexa-
hydroxoantimonate (V) TS.
Potassium pyrophosphate K 4 7 P 2 White, crystalline
powder, very soluble in water.
Melting point <2.60>: 1109°C
Potassium pyrosulfate See potassium disulfate.
Potassium sodium tartarate See potassium sodium tar-
tarate tetrahydrate.
Potassium sodium tartarate tetrahydrate KNaC 4 H 4 6 .4H 2
[K 8536, ( + )-Potassium sodium tartrate tetrahydrate, Spec-
ial class]
Potassium sulfate K 2 S0 4 [K 8962, Special class]
Potassium sulfate TS Dissolve 1 g of potassium sulfate in
water to make 100 mL.
Potassium tartrate 2C 4 H 4 K 2 6 .H 2 [K8535,
Potassium Tartrate-Water (2/1), Special class]
Potassium tellurite K 2 Te0 3 White powder or small
masses obtained by melting an equimolar mixture of telluri-
um dioxide and potassium carbonate in a stream of carbon
dioxide. Soluble in water.
Content: not less than 90.0%. Assay— Dissolve about
1 .0 g of potassium tellurite, accurately weighed, in 100 mL of
water, add 5 mL of diluted acetic acid (31) (1 in 3), and boil.
After cooling, filter by suction through a crucible glass filter
(1G4), previously dried at 105 ± 2°C for 1 hour to constant
mass (6(g)). Wash the filtrate with water, dry the glass filter at
110 C C for 3 hours, and measure the mass a (g).
Content (%) of potassium tellurite (K 2 Te0 3 )
_ (g - 6) x 1.5 902
~ S
X100
S: Mass (g) of potassium tellurite taken.
Potassium tetraoxalate for pH determination See potas-
sium trihydrogen dioxalate dihydrate for pH determination.
Potassium thiocyanate KSCN [K 9001, Special class]
Potassium thiocyanate TS Dissolve 1 g of potassium
thiocyanate in water to make 10 mL.
Potassium trihydrogen dioxalate dihydrate for pH deter-
mination KH 3 (C 2 4 ) 2 .2H 2 [K 8474]
Potato extract Prepared for microbial test.
Potato starch [Same as the namesake monograph]
Potato starch TS Prepare as directed under starch TS
with 1 g of potato starch.
JPXV
General Tests / Reagents, Test Solutions
233
Potato starch TS for amylolytic activity test Dry about 1
g of potato starch, accurately weighed, at 105°C for 2 hours,
and measure the loss. Weigh accurately an amount of potato
starch, equivalent to 1.000 g on the dried basis, place into a
conical flask, add 20 mL of water, and make it pasty by grad-
ually adding 5 mL of a solution of sodium hydroxide (2 in 25)
while shaking well. Heat in a water bath for 3 minutes while
shaking, add 25 mL of water, and cool. Neutralize exactly
with 2 mol/L hydrochloric acid TS, add 10 mL of 1 mol/L a-
cetic acid-sodium acetate buffer solution, pH 5.0, and add
water to make exactly 100 mL. Prepare before use.
Powdered tragacanth
graph]
[Same as the namesake mono-
Prazepam for assay Ci 9 H 17 ClN 2 [Same as the mono-
graph Prazepam. When dried, it contains not less than 99.0%
of C 19 H 17 C1N 2 0.]
Prednisolone
graph]
C21H28O5 [Same as the namesake mono-
Prednisone C 2 iH 2 60 5 White, crystalline powder. Slight-
ly soluble in methanol, in ethanol (95) and in chloroform,
and very slightly soluble in water.
Optical rotation <2.49> [a]™: +167- +175° (after
drying, 0.1 g, 1,4-dioxane, 10 mL, 100 mm).
Loss on drying <2.41>: not more than 1.0% (1 g, 105°C, 3
hours).
Content: 96.0 - 104.0%. Assay— Weigh accurately about
20 mg of prednisone, and dissolve in methanol to make ex-
actly 100 mL. Pipet 5 mL of this solution, dilute with metha-
nol to make exactly 100 mL. Perform the test with this solu-
tion as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and read the absorbance A at the wavelength of maxi-
mum absorption at about 238 nm.
Amount (mg) of C 2 iH 26 5
440
x 20,000
Prednisolone acetate C 23 H 30 O 6 [Same as the namesake
monograph]
Probenecid C13H19NO4S [Same as the namesake mono-
graph]
Procainamide hydrochloride Ci 3 H 21 N 3 O.HCl [Same as
the namesake monograph]
Procainamide hydrochloride for assay
C 13 H 21 N 3 O.HCl [Same as the monograph Procainamide
Hydrochloride. When dried, it contains not less than 99.0%
of procainamide hydrochloride (C 8 H 8 N.HC1).]
Procaine hydrochloride Ci 3 H 20 N 2 O 2 .HCl [Same as the
namesake monograph]
Procaine hydrochloride for assay [Same as the mono-
graph Procaine Hydrochloride]
Procaterol hydrochloride Ci 6 H 22 N 2 3 .HCl. yH 2
[Same as the monograph Procain Hydrochloride Hydrate]
Progesterone C 21 H 30 O 2 [Same as the namesake mono-
graph]
L-Proline C 5 H 9 N0 2 [K 9107, Special class]
n-Propanol See 1-propanol.
1-Propanol CH 3 CH 2 CH 2 OH [K 8838, Special class]
2-Propanol (CH 3 ) 2 CHOH [K 8839, Special class]
2-Propanol for vitamin A assay (CH 3 ) 2 CHOH [K 8839,
Special class] When the absorbances at 300 nm and between
320 nm and 350 nm are determined as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
control, they are not more than 0.05 and not more than 0.01,
respectively. If necessary, purify by distillation.
2-Propanol for liquid chromatography
(CH 3 ) 2 CHOH Clear , colorless and volatile liquid, having a
characteristic odor. Miscible with water, with ethanol (95)
and with diethyl ether. Boiling point: about 82°C.
Refractive index <2.45> n 2 £: 1.376 - 1.378
Specific gravity <2.56> df a : 0.785 - 0.788
Purity (1) Ultraviolet absorbing substances — Perform
the test with 2-propanol for liquid chromatography as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, using
water as the blank: the absorbance at 230 nm is not more
than 0.2; at 250 nm, not more than 0.03; and between 280 nm
and 400 nm, not more than 0.01.
(2) Peroxide — Mix 100 mL of water and 25 mL of dilute
sulfuric acid, and add 25 mL of a solution of potassium
iodide (1 in 10). Add this solution to 20 g of 2-propanol for
liquid chromatography. Stopper tightly, shake, allow to
stand for 15 minute in a dark place, and titrate <2.50> with
0.01 mol/L sodium thiosulfate VS (indicator: 1 mL of starch
TS). Perform a blank determination in the same manner, and
make any necessary correction (not more than 0.0005%).
Propanol, iso See 2-propanol.
Propranolol hydrochloride for assay Ci 6 H 2 iN0 2 .HCl
[Same as the monograph Propranolol Hydrochloride. When
dried, it contains not less than 99.5% of propranolol
hydrochloride (C 16 H 21 N0 2 .HC1).]
Propantheline bromide C 23 H 30 BrNO 3 [Same as the
namesake monograph]
Propionic acid CH 3 CH 2 COOH Colorless liquid.
Purity — Clarity and color of solution — Dissolve 1 g of
propionic acid in 20 mL of ethanol (95): the solution is clear
and colorless.
Specific gravity <2.56> df : 0.998 - 1.004
Distilling range <2.57>: 139 - 143°C, not less than 95 vol%.
Propylamine, iso (CH 3 ) 2 CHNH 2 Colorless liquid, hav-
ing a characteristic, amine-like odor. Miscible with water,
with ethanol (95) and with diethyl ether.
Refractive index <2.45> n™: 1-374 - 1.376
Specific gravity <2.56> d\°: 0.685 - 0.690
Distilling range <2.57>: 31 - 33°C, not less than 95 vol%.
Propyl benzoate C 6 H 5 COOC 3 H 7 Clear, colorless liq-
uid, having a characteristic odor.
Refractive index <2.45> n™: 1-498 - 1.503
Specific gravity <2.56> df : 1.022 - 1.027
Propylene carbonate C 4 H 6 3 Colorless liquid.
Boiling point <2.57>: 240 - 242°C
Water <2.48>: less than 0.1%
Propylene carbonate for water determination See Water
Determination <2.48>.
234
Reagents, Test Solutions / General Tests
JP XV
Propylene glycol CH 3 CH(OH)CH 2 OH [K 8837, Spec-
ial class]
Propylene glycol cefatrizine Ci 8 H 18 N 6 5 S 2 .C 3 H 8 2
[Same as the namesake monograph]
Propylether, iso (CH 3 ) 2 CHOCH(CH 3 ) 2 Clear, colorless
liquid, having a characteristic odor. Not miscible with water.
Refractive index <2.45> n™: 1.368 - 1.369
Specific gravity <2.56> df: 0.723 - 0.725
Propyl parahydroxybenzoate HOC 6 H 4 COOCH 2 CH 2 CH 3
[Same as the namesake monograph]
Propylthiouracil for assay C 7 H 10 N 2 OS [Same as the
monograph Propylthiouracil. When dried, it contains not
less than 99.0% of propylthiouracil (C 7 H 10 N 2 OS).]
Prostaglandin Ax C 20 H 32 O 4 White crystals or crystalline
powder. Very soluble in ethanol (95) and in ethyl acetate, and
very slightly soluble in water.
Purity Related substances — Dissolve 5 mg of prostaglan-
din A, in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 3 mL of the sample solution, add
ethanol (95) to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing operating conditions. Determine areas of all peaks of
both solutions by the automatic integration method: the total
area of the peaks other than the peak of prostaglandin A[
from the sample solution is not larger than the peak area of
prostaglandin A! from the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed the operating
conditions in the Assay of Alprostadil Alfadex.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of prostaglandin A t obtained from 10 /xL
of the standard solution is 5 to 10% of the full scale.
Time span of measurement: About twice as long as the
retention time of prostaglandin A! beginning after the solvent
peak.
Protein digestive enzyme TS A solution of lysyl endopep-
tidase in 0.05 mol/L tris buffer solution, pH 8.6 (1 in 5000).
Pseudoephedrine hydrochloride Ci H 15 NO.HCl White,
crystals or crystalline powder. Freely soluble in water, in
methanol and in acetic acid (100), soluble in ethanol (99.5),
and practically insoluble in acetic anhydride. Melting point:
182- 186°C
Purity Related substances — Dissolve 1 mg in 10 mL of
diluted methanol (1 in 2), and use this solution as the sample
solution. Perform the test with 10,mL of the sample solution
for twice as long as as the retention time of ephedrine as
directed in the Assay (1) under Kakkonto Extract: the total
area of the peaks other than pseudoephedrine and the solvent
is not larger than 1/10 times the total area of the peaks other
than the solvent.
Puerarin for thin-layer chromatography C 2I H 20 O 9
White crystalline powder. Freely soluble in methanol, and
practically insoluble in diethyl ether. Melting point: about
188°C (with decomposition).
Purity Related substances — Dissolve 1.0 mg of puerarin
for thin-layer chromatography in exactly 1 mL of methanol.
Perform the test with 2//L of this solution as directed in the
Identification under Pueraria Root: any spot other than the
principal spot at the Rf value of about 0.4 does not appear.
Pullulanase An enzyme obtained from Klebsiella pneu-
moniae. White crystals. It contains not less than 30 units per
mg. One unit is an enzymatic activity to produce 1 //ol of mal-
totriose from pullulan per minute at pH 5.0 and 30°C.
Pullulanase TS A solution of pullulanase containing 10
units per mL.
Purified hydrochloric acid See hydrochloric acid, puri-
fied.
Purified methanol See methanol, purified.
Purified sulfuric acid See sulfuric acid, purified.
Purified water [Same as the namesake monograph]
Purified water for ammonium limit test To 1500 mL of
purified water add cautiously 4.5 mL of sulfuric acid, distil
using a hard glass distiller, discard the first distillate, and use
the remaining distillate as ammonium-free purified water.
Purity — Mix 40 mL of purified water for ammonium limit
test with 6.0 mL of phenol-sodium pentacyanonitrosylferrate
(III) TS. Add 4.0 mL of sodium hypochlorite-sodium hy-
droxide TS, mix, and allow to stand for 60 minutes. Perform
the test with this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using water as the blank: absorb-
ance at the wavelength of 640 nm is not more than 0.010.
Pyrazole C 3 H 4 N 2 White to pale yellow crystals or crys-
talline powder.
Melting point <2.60>: 67 - 71 °C
Pyridine C 5 H 5 N [K 8777, Special class]
Pyridine-acetic acid TS Dilute 20 mL of pyridine with
sufficient diluted acetic acid (100) (1 in 25) to make 100 mL.
Prepare before use.
Pyridine, dehydrated C 5 H 5 N To 100 mL of pyridine
add 10 g of sodium hydroxide, and allow to stand for 24
hours. Decant the supernatant liquid, and distill.
Pyridine for Karl Fischer method See Water Determina-
tion <2.48>.
Pyridine-pyrazolone TS Dissolve, with thorough shak-
ing, 0.1 g of 3-methyl-l-phenyl-5-pyrazolone in 100 mL of
water by heating between 65°C and 70°C, and cool below
30°C. Mix this solution with a solution prepared by dissolv-
ing 0.02 g of bis-(l-phenyl-3-methyl-5-pyrazolone) in 20 mL
of pyridine. Prepare before use.
Pyridoxine hydrochloride C 8 H U N0 3 .HC1 [Same as the
namesake monograph]
l-(2-Pyridylazo)-2-naphthol C 15 H U N 3 Orange-
yellow or orange-red powder.
Absorbance — Dissolve 25 mg of l-(2-pyridylazo)-2-
naphthol in methanol to make exactly 100 mL. Pipet 2.0 mLof
this solution, and add methanol to make exactly 50 mL. Per-
form the test with this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using methanol as the
blank: absorbance at the wavelength of 470 nm is not less
than 0.55.
JPXV
General Tests / Reagents, Test Solutions
235
Melting point <2.60>: 137 - 140°C
Purity Clarity and color of solution — Dissolve 25 mg of
l-(2-pyridylazo)-2-naphthol in 100 mL of methanol: the solu-
tion is clear and orange-yellow.
Residue on ignition <2.44>: not more than 1.0%.
Sensitivity — On adding 50 mL of water, 30 mL of metha-
nol and 10 mL of acetic acid-sodium acetate buffer solution,
pH 5.5, to 0.2 mL of a solution of l-(2-pyridylazo)-2-
naphthol in methanol (1 in 4000), the solution is yellow in
color. Add 1 drop of a solution of copper (II) chloride dihy-
drate (1 in 600) to this solution: the solution is red-purple in
color. Add a subsequent 1 drop of diluted 0.1 mol/L disodi-
um dihydrogen ethylenediamine tetraacetate TS (1 in 10): the
color of the solution changes to yellow again.
l-(4-Pyridyl)pyridinium chloride hydrochloride
Ci H 9 ClN 2 .HCl White to yellowish white, crystalline pow-
der. Very soluble in water, very slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
Melting point <2.60>: 154- 156°C
Pyrogallol C 6 H 3 (OH) 3 [K 8780, Special class]
L-Pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochlo-
ride Ci 9 H 2 6N 8 6 .HCl White to light powder. Freely solu-
ble in water, in methanol and in acetic acid (100).
Absorbance <2.24> E\°^ (316 nm): 242-268 (2 mg,
water, 100 mL).
Optical rotation <2.49> [ a ]g: -51 - -56° [0.1 g, dilut-
ed acetic acid (100) (1 in 2), 10 mL, 100 mm].
Purity Related substances — Dissolve 0.05 g of L-pyro-
glutamylglycyl-L-arginine-p-nitroaniline hydrochloride in 10
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add methanol to make ex-
actly 50 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 20 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of 1-butanol, water, pyridine and
acetic acid (100) (15:12:10:3) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
L-Pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochlo-
ride TS Dissolve 25 mg of L-pyroglutamylglycyl-L-arginine-
/7-nitroaniline hydrochloride and 0.04 g of D-Mannitol in 2 to
3 mL of water, lyophilize, and add 16.7 mL of water to dis-
solve. To 1 volume of this solution add 9 volumes of water
before use.
Pyrole C 4 H 5 N Clear, colorless liquid, having a charac-
teristic oder. Soluble in ethanol (95) and in diethyl ether, and
practically insoluble in water.
Specific gravity <2.56> df Q : 0.965 - 0.975
Pyrophosphate buffer solution, pH 9.0 Dissolve 3.3 g of
potassium pyrophosphate, 15 mg of dithiothreitol and 40 mg
of disodium dihydrogen ethylenediamine tetraacetate dihy-
drate in 70 mL of water, adjust the pH with a solution of
citric acid monohydrate (21 in 100) to exactly 9.0, and add
water to make 100 mL.
0.05 mol/L Pyrophosphate buffer solution, pH 9.0
Dissolve 0.83 g of potassium pyrophosphate in 40 mL of water,
adjust the pH with 1 mol/L hydrochloric acid VS to 9.0, and
add water to make 50 mL. Adjust the temperature to 22 ±
2°C before use.
Quinhydrone C 6 H 4 (OH)2.C 6 H 4 02 Green crystals or
crystalline powder.
Melting point <2.60>: 169 - 172°C
Quinidine sulfate (C2oH 2 4N 2 2 )2-H 2 S04.2H 2 [Same as
the monograph Quinidine Sulfate Hydrate]
Quinine sulfate (C 20 H 21 N 2 O 2 ) 2 .H 2 SO 4 .2H 2 O [Same as
the namesake monograph]
Quinoline C 9 H 7 N [K 8279, Special class]
Quinoline TS Mix 50 mL of quinoline with 300 mL of
diluted hydrochloric acid (1 in 6), previously heated, cool,
and filter if necessary.
8-Quinolinol C 9 H 7 NO [K 8775, Special class]
Raney nickel catalyst Grayish black powder. An alloy
containing 40 to 50% of nickel and 50 to 60% of aluminum.
Ranitidinediamine (C 10 H 18 N 2 OS) 2 .C 4 H 4 O 4 White to
pale yellow crystalline powder.
Identification — Determine the infrared absorption spec-
trum of ranitidinediamine as directed in the paste method un-
der Infrared Spectrophotometry <2.25>: it exhibits absorption
at the wave numbers of about 2780 cm -1 , 1637 cm -1 , 1015
cm -1 and 788 cm -1 .
Content: not less than 95%. Assay — Weigh accurately
about 0.1 g of ranitidinediamine, dissolve in 50 mL of acetic
acid (100), and titrate <2.50> with 0.1 mol/L perchloric acid
VS until the color of the solution changes from purple to
green through blue (indicator: crystal violet TS). Perform the
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 13.62 mg of (C 10 H 18 N 2 OS) 2 .C 4 H 4 O 4
Reduced iron See iron powder.
Reference anti-interleukin-2 antibody for teceleukin
Monoclonal antibody obtained from a fusion cell strain from
mouse spleen cells sensitized to teceleukin and mouse mela-
noma cells, or alternately, rabbit antiserum towards human
interleukin-2, that is purified using affinity chromatography.
When determining the neutralizing activity, taking 1 neu-
tralizing unit as the titer that neutralizes one unit of activity
of teceleukin, contains at least 2000 neutralizing units per 1
mL.
Reinecke salt See reinecke salt monohydrate.
Reinecke salt monohydrate
NH 4 [Cr(NH 3 ) 2 (SCN) 4 ].H 2 [K 8926, Special class]
Reinecke salt TS To 20 mL of water add 0.5 g of Reine-
cke salt monohydrate, shake frequently for 1 hour, then
filter. Use within 48 hours.
Resazurin C I2 H 6 NNa0 4 Brownish purple powder. It
dissolves in water and the solution is purple in color.
Residue on ignition <2.44>: not less than 28.5% (1 g).
Resibufogenin for component determination
C 24 H 32 4 .xH 2 Odorless white crystalline powder.
236
Reagents, Test Solutions / General Tests
JP XV
Absorbance <2.24> EY° m (300 nm): 131-145 (0.01 g, meth-
anol, 250 mL), dried in a desiccator (silica gel) for 24 hours.
Purity Related substances — Weigh accurately 0.04 g of
resibufogenin for component determination and proceed as
directed in the Purity under bufalin for component determi-
nation.
Content: not less than 98.0%. Component deter-
mination — Weigh accurately about 10 mg of resibufogenin
for component determination, previously dried in a desicca-
tor (silica gel) for 24 hours, add methanol to make exactly 10
mL, and use this solution as the sample solution. Perform the
test with 20 /xL of this solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Measure each peak area by the automatic integration
method, and calculate the amount of resibufogenin by the
area percentage method.
Operating conditions
Detector: Ultraviolet absorption photometer (wavelength:
300 nm).
Column: A stainless steel column about 4 to 6 mm in inside
diameter and 15 to 30 cm in length, packed with octa-
decylsilanized silica gel for liquid chromatography (5 to 10
/urn in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (1:1).
Selection of column: Dissolve 0.01 g each of bufalin for
component determination, cinobufagin for component deter-
mination and resibufogenin for component determination in
methanol to make 200 mL. Perform the test with 20 /uh of
this solution according to the above operating conditions,
and calculate the resolution. Use a column giving elution of
bufalin, cinobufagin and resibufogenin in this odor, and
clearly dividing each peak.
Detection sensitivity: Pipet 1 mL of the sample solution,
add methanol to make exactly 100 mL, and use this solution
as the standard solution (1). Pipet 1 mL of the standard solu-
tion (1), add methanol to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of resibufogenin obtained
from 20 /xL of standard solution (2) can be measured by the
automatic integration method and the peak height of
resibufogenin from 20 /xh of the standard solution (1) is
about 20% of the full scale.
Time span of measurement: About twice as long as the
retention time of resibufogenin beginning after the peak of
solvent.
Resibufogenin for thin-layer chromatography
C24H320 4 .«H 2 White crystalline powder having no odor.
It is freely soluble in acetone and in methanol.
Purity Related substances — Dissolve 5.0 mg of the sub-
stance to be tested in exactly 5 mL of acetone. Perform the
test with 5 fiL of this solution as directed in the Identification
under Toad Venom: no other spots than the principal spot of
around Rf 0.4 appear.
Resolving gel for celmoleukin Prepare the resolving gel in
tris buffer solution, pH 8.8 using ammonium persulfate and
TEMED so the concentrations of acrylamide and sodium
lauryl sulfate are 13.5% and 0.1%, respectively.
Resorcin See resorcinol.
Resorcinol C 6 H 4 (OH) 2 [K 9032, Special class]
Resorcinol sulfuric acid TS Dissolve 0.1 g of resorcinol in
10 mL of diluted sulfuric acid (1 in 10).
Resorcinol TS Dissolve 0.1 g of resorcinol in 10 mL of
hydrochloric acid. Prepare before use.
Resorcin sulfuric acid TS See resorcinol sulfuric acid TS.
Resorcin TS See resorcinol TS.
L-Rhamnose monohydrate CglT^O.H^O White crystal-
line powder having sweet taste. Freely soluble in water, and
sparingly soluble in ethanol (95).
Optical rotation <2.49> [a]™: +7.8 - +8.3° (1 g, 20 mL of
water, 2 drops of ammonia TS, 100 mm).
Melting point <2.60>: 87-91°C
Purity Related substances — Dissolve 1.0 mg of L-rham-
nose monohydrate in 1 mL of water, and add methanol to
make exactly 10 mL. Proceed with 20 /uL of this solution as
directed in the Identification (2) under Acacia: any spot other
than the principal spot at the Rf value of about 0.5 does not
appear.
Rhein for thin-layer chromatography Ci 5 H 8 6 A yel-
low powder. Very slightly soluble in acetone, and practically
insoluble in water, in methanol, and in ethanol (99.5). Melt-
ing point: about 320°C (with decomposition).
Identification — Determine the absorption spectrum of a
solution in methanol (3 in 500,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 228 nm and 232 nm, between 255 nm and 259 nm,
and between 429 nm and 433 nm.
Purity Related substances — Dissolve 1.0 mg in 10 mL of
acetone, and perform the test with 2 /uL of this solution as
directed in the Identification (1) under Daiokanzoto Extract:
no spot other than the principal spot (Rf value is about 0.3)
appears.
Rhynchophylline for component determination
C22H28N2O4 White, crystals or crystalline powder. Sparing-
ly soluble in ethanol (99.5) and in acetone, and practically in-
soluble in water. Melting point: 205 - 209°C
Absorbance <2.24> £|% (245 nm): 473 - 502 (5 mg dried
in a desiccator (silica gel) for 24 hours, a mixture of methanol
and dilute acetic acid (7:3), 500 mL).
Purity Related substances —
(1) Dissolve 1.0 mg of rhynchophylline for component
determination in 1 mL of acetone, and perform the test with
this solution as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uh of the solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography, develop
with a mixture of 1-butanol, water and acetic acid (100)
(7:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
any spot other than the principal spot of Rf about 0.5 does
not appear.
(2) Dissolve 5 mg of rhynchophylline for component de-
termination in 100 mL of a mixture of methanol and dilute a-
cetic acid (7:3), and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add a mixture of
methanol and dilute acetic acid (7:3) to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 20 /uh each of the sample solution and stan-
dard solution as directed under Liquid Chromatography <
2.01> according to the following conditions. Determine each
JPXV
General Tests / Reagents, Test Solutions
237
peak area obtained from these solutions by the automatic in-
tegration method: the sum of the peak areas except the areas
of rhynchophylline and the solvent obtained from the sample
solution is not more than the peak area of rhynchophylline
from the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Component determination under Uncaria Thorn.
Time span of measurement: About 4 times as long as the
retention time of rhynchophylline beginning after the solvent
peak.
System suitability
Test for required detectability: Measure exactly 1 mL of
the standard solution, add a mixture of methanol and dilute
acetic acid (7:3) to make exactly 20 mL. Confirm that the
peak area of rhynchophylline obtained from 20 /xL of this so-
lution is equivalent to 3.5 to 6.5% of that from 20 /xL of the
standard solution.
System performance, and system repeatability: Proceed as
directed in the operating conditions in the Component deter-
mination under Uncaria Thorn.
Riboflavin C I7 H2oN 4 6 [Same as the namesake mono-
graph]
Riboflavin sodium phosphate C 17 H 2 oN 4 Na09P [Same
as the namesake monograph]
Ritodrine hydrochloride C 17 H 21 N0 3 .HC1 [Same as the
namesake monograph]
Rose Bengal See Microbial Limit Test for Crude Drugs
<5.02>.
Rose Bengal TS See Microbial Limit Test for Crude
Drugs <5.02>.
RPMI-1640 powdered medium Powder medium for cell
culture containing 6 g of sodium chloride, 400 mg of potassi-
um chloride, 800 mg of sodium dihydrogen phosphate, 100
mg of anhydrous calcium nitrate, 49 mg of anhydrous mag-
nesium sulfate, 2 g of dextrose, 200 mg of L-arginine, 1 mg of
glutathione, 50 mg of L-isoleucine, 15 mg of L-phenylalanine,
5 mg of L-tryptophan, 0.2 mg of biotin, 1 mg of
nicotinamide, 1 mg thiamine hydrochloride, 300 mg of L-
glutamine, 56.8 mg of L-asparagine, 10 mg of glycine, 50 mg
of L-leucine, 20 mg of L -proline, 20 mg of L-tyrosine, 0.25
mg of D-calcium pantothenate, 5 /xg of cyanocobalamin, 1
mg of aminobenzoic acid, 20 mg of L-aspartic acid, 15 mg of
L-histidine, 40 mg of L-lysine hydrochloride, 30 mg of L-ser-
ine, 20 mg of L-valine, 1 mg of folic acid, 1 mg of pyridoxine
hydrochloride, 20 mg of L-glutamic acid, 20 mg of L-hydrox-
yproline, 15 mg of L-methionine, 20 mg of L-threonine, 3 mg
of choline chloride, 35 mg of /-inositol, 0.2 mg of riboflavin,
59 mg of L-cystine, and 5 mg of phenol red.
Saccharated pepsin [Same as the namesake monograph]
Saikosaponin a for component determination Use sai-
kosaponin a for thin-layer chromatography meeting the fol-
lowing additional specifications.
Purity Related substances —
(1) Dissolve 2.0 mg of saikosaponin a for component de-
termination in 2 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of this solution, add methanol
to make exactly 100 mL, and use this as the standard solu-
tion. Proceed the test with exactly 10 /uL each of the sample
solution and standard solution as directed in the Purity (2)
under Bupleurum Root: the spot other than the principal spot
around Rf 0.4 is not larger and not more intense than the spot
obtained with the standard solution.
(2) Dissolve 10 mg of saikosaponin a for component de-
termination in 20 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of this solution, add
methanol to make exactly 100 mL, and use this as the stan-
dard solution. Perform the test with exactly 20 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine each peak area by the automatic in-
tegration method: the total area of the peaks other than sai-
kosaponin a and the solvent is not more than the peak area of
saikosaponin a obtained with the standard solution.
Operating conditions
Detector, and column: Proceed as directed in the operating
conditions in the Component determination under Bupleu-
rum Root.
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (13:7).
Flow rate: Adjust the flow rate so that the retention time of
saikosaponin a is about 16 minutes.
Time span of measurement: About 6 times as long as the
retention time of saikosaponin a beginning after the solvent
peak.
System suitability
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add methanol to make exactly 20
mL. Confirm that the peak area of saikosaponin a obtained
with 20 /xL of this solution is equivalent to 3.5 to 6.5% of that
with 20,mL of the standard solution.
System performance: Dissolve 6 mg each of saikosaponin a
for component determination and saikosaponin b 2 for com-
ponent determination in methanol to make 100 mL. When
the procedure is run with 20 /xL of the standard solution un-
der the above operating conditions, saikosaponin a and sai-
kosaponin b 2 are eluted in this order with the resolution be-
tween these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
saikosaponin a is not more than 1.0%.
Saikosaponin a for thin-layer chromatography A white,
crystalline powder or powder. Freely soluble in methanol and
in ethanol (99.5), and practically insoluble in water. Melting
point: 225 - 232°C (with decomposition).
Absorbance <2.24> E\ y ° m (206 nm): 60-68 (15 mg, metha-
nol, 200 mL). Previously dried in a desiccator (in vacuum, sil-
ica gel) for 24 hours.
Purity Related substances — Dissolve 1.0 mg of sai-
kosaponin a for thin-layer chromatography in exactly 1 mL
of methanol, and perform the test with 10 /xL of this solution
as directed in the Identification (2) under Bupleurum Root:
any spot other than the principal spot at the Rf value of
about 0.4 does not appear.
Salicylaldazine C 14 H 12 N 2 2 Dissolve 0.30 g of hydra-
zinium sulfate in 5 mL of water. To this solution add 1 mL of
acetic acid (100) and 2 mL of a freshly prepared solution of
salicylaldehyde in 2-propanol (1 in 5), shake well, and allow
238
Reagents, Test Solutions / General Tests
JP XV
to stand until a yellow precipitate is produced. Extract with
two 15 mL portions of dichloromethane, to the combined
dichloromethane extracts add 5 g of anhydrous sodium sul-
fate, shake, decant or filter, and evaporate the dichloro-
methane in the supernatant liquid or filtrate. Dissolve the
residue in a warmed mixture of toluene and methanol (3:2),
and cool. Filter the crystals produced, and dry in a desiccator
(in vacuum, silica gel) for 24 hours. It is a yellow, crystalline
powder.
Melting point <2.60>: 213 - 219°C
Purity — Dissolve 0.09 g of salicylaldazine in toluene to
make exactly 100 mL. Pipet 1 mL of this solution, add tol-
uene to make exactly 100 mL, and perform the test with this
solution as directed in the Purity (6) under Povidone: any
spot other than the principal spot does not appear.
Saikosaponin b 2 for component determination Saiko-
saponin b 2 for thin-layer chromatography. It meets the fol-
lowing requirements.
Purity Related substances — Dissolve 5 mg in 5 mL of the
mobile phase, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add the mobile phase to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with exactly 10 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the total area of the peaks other than the peaks
of saikosaponin b 2 and solvent is not larger than the peak
area of saikosaponin b 2 obtained with the standard solution.
Operating conditions
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay (1) under Saireito Extract.
Time span of measurement: About 6 times as long as the
retention time of saikosaponin b 2 .
System suitability
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of saikosaponin b 2 obtained with
10 /xL of this solution is equivalent to 3.5 to 6.5% of that with
10 /xL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay (1) under
Saireito Extract.
Saikosaponin b 2 for thin-layer chromatography
C 42 H 68 13 White crystals or crystalline powder. Freely solu-
ble in ethanol (99.5), soluble in methanol, and practically in-
soluble in water. Melting point: about 240°C
Absorbance <2.24> E\ 0/ ° m (252 nm): 352-424 (5 mg,
methanol, 250 mL). Previously dried in a desiccator (in vacu-
um, silica gel) for 24 hours.
Purity Related substances — Dissolve 2 mg in 2 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add methanol to make exactly
50 mL, and use this solution as the standard solution. Pro-
ceed the test with 10 /xL each of the sample solution and the
standard solution as directed in the Identification (1) under
Saireito Extract: the spot other than the principle spot of
around Rf 0.3 is not more intense than the spot obtained with
the standard solution.
Saikosaponin d for component determination A white,
crystalline powder or powder. Freely soluble in methanol and
in ethanol (99.5), and practically insoluble in water. Melting
point: about 240°C
Absorbance <2.24> E\ v ° m (206 nm): 63 - 71 (15 mg,
methanol, 200 mL). Previously dried in a desiccator (in vacu-
um, silica gel) for 24 hours.
Purity Related substances —
(1) Dissolve 2.0 mg in 2 mL of methanol, and use this so-
lution as the sample solution. Pipet 1 mL of this solution,
add methanol to make exactly 100 mL, and use this as the
standard solution. Proceed the test with 10 /xL each of the
sample solution and standard solution as directed in the Iden-
tification (2) under Bupleurum Root: the spot other than the
principal spot around Rf 0.4 is not larger and not more in-
tense than the spot obtained with the standard solution.
(2) Dissolve 10 mg in 20 mL of methanol, and use this so-
lution as the sample solution. Pipet 1 mL of this solution,
add methanol to make exactly 100 mL, and use this as the
standard solution. Perform the test with exactly 20 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the total area of the peaks other than
saikosaponin d and the solvent is not more than the peak area
of saikosaponin d obtained with the standard solution.
Operating conditions
Detector, and column: Proceed as directed in the operating
conditions in the Component determination under Bupleu-
rum Root.
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
saikosaponin d is about 13 minutes.
Time span of measurement: About 4 times as long as the
retention time of saikosaponin d beginning after the solvent
peak.
System suitability
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add methanol to make exactly 20
mL. Confirm that the peak area of saikosaponin d obtained
with 20 /xL of this solution is equivalent to 3.5 to 6.5% of that
with 20 jxL of the standard solution.
System performance: Dissolve 6 mg each of saikosaponin
d for component determination and saikosaponin a for com-
ponent determination in methanol to make 100 mL. When
the procedure is run with 20 /xL of the standard solution un-
der the above operating conditions, saikosaponin a and sai-
kosaponin d are eluted in this order with the resolution be-
tween these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
saikosaponin d is not more than 1.0%.
Salicylaldehyde HOC 6 H 4 CHO [K 8390, Special class]
Salicylamide C 7 H 7 N0 2 White crystals or crystalline
powder, and it is odorless and tasteless. Very soluble in N,N-
dimethylformamide, freely soluble in ethanol (95), soluble in
propylene glycol, sparingly soluble in diethyl ether, and
slightly soluble in water and in chloroform. It dissolves in so-
dium hydroxide TS.
Melting point <2.60>: 139 - 143°C
JPXV
General Tests / Reagents, Test Solutions
239
Purity Ammonium <1.02> — Shake 1.0 g of salicylamide
with 40 mL of water, and filter through filter paper previously
washed well with water. Discard the first 10 mL of the filtrate,
transfer the subsequent 20 mL to a Nessler tube, and add
water to make 30 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows:
transfer 2.5 mL of Standard Ammonium Solution to a Ness-
ler tube, and add water to make 30 mL.
Loss on drying <2.41>: not more than 0.5% (1 g, silica gel,
4 hours).
Residue on ignition <2.44>: not more than 0.1% (1 g).
Content: not less than 98.5%. Assay — Weigh accurately
about 0.2 g of salicylamide, previously dried, dissolve in 70
mL of A^A^-dimethylformamide, and titrate <2.50> with 0.1
mol/L tetramethylammonium hydroxide VS (potentiometric
titration). Separately, perform a blank determination with a
solution of 70 mL of AfJV-dimethylformamide in 15 mL of
water, and make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium hydroxide VS
= 13.71 mg of C 7 H 7 N0 2
Salicylic acid HOC 6 H 4 COOH [K 8392, Special class]
Salicylic acid for assay HOC 6 H 4 COOH [K 8392, Spec-
ial class]
Salicylic acid TS Dissolve 0.1 g of salicylic acid in 10 mL
of sulfuric acid. Prepare before use.
Santonin C 15 H 18 3 [Same as the namesake monograph]
Santonin for assay [Same as the monograph Santonin. It
contains not less than 99.0% of santonin (C 15 H 18 3 ).]
Schisandrin for thin-layer chromatography C24H32O7
White crystals for crystalline powder. Freely soluble in
methanol and diethyl ether, and practically insoluble in
water.
Melting point <2.60>: 130- 135°C
Purity Related substances — Dissolve 1.0 mg of schisan-
drin for thin-layer chromatography in exactly 1 mL of
methanol. Perform the test with 5 /iL of this solution as
directed in the Identification under Schisandra Fruit: any
spot other than the principal spot at the Rf value of about 0.4
does not appear.
Scopolamine hydrobromide CnH21NO4.HBr.3H2O
[Same as the monograph Scopolamine Hydrobromide Hy-
drate]
Scopolamine hydrobromide for thin-layer chromato-
graphy [Same as the monograph Scopolamine Hydro-
bromide Hydrate. Proceed as directed in the Identification (3)
under Opium Alkaloids and Atropine Injection: any spot
other than the principal spot at the Rf value of about 0.7 does
not appear.
Sea sand [K 8222, Special class]
2nd Fluid for disintegration test To 250 mL of 0.2 mol/L
potassium dihydrogen phosphate TS add 118 mL of 0.2
mol/L sodium hydroxide TS and water to make 1000 mL. It
is clear and colorless, and has a pH about 6.8.
2nd Fluid for dissolution test A mixture of phosphate
buffer solution, pH 6.8 and water (1:1).
Selenious acid H 2 Se0 3 [K 8035, Special class]
Selenious acid-sulfuric acid TS Dissolve 0.05 g of seleni-
ous acid in 10 mL of sulfuric acid.
Selenium Se [K 8598, Special class]
Selenium dioxide Se0 2 [K 8706, Special class]
Semicarbazide acetate TS Place 2.5 g of semicarbazide
hydrochloride, 2.5 g of anhydrous sodium acetate and 30 mL
of methanol in a flask, heat on a water bath for 2 hours, cool
to 20°C, and filter. To the filtrate add methanol to make 100
mL. Preserve in a cold place. Do not use the solution showing
a yellow color.
Semicarbazide hydrochloride H 2 NNHC0NH 2 .HC1 White
to light yellow crystals.
Identification — (1) To 10 mL of a solution (1 in 100) add
1 mL of silver nitrate TS: white precipitates appear.
(2) Determine the infrared absorption spectrum as direct-
ed in the potassium bromide disk method under Infrared
Spectrophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3420 cm" 1 , 3260 cm" 1 , 2670 cm" 1 , 1684
cm" 1 , 1582 cm" 1 , 1474 cm" 1 , 1386 cm" 1 , 1210 cm" 1 , 1181
cm" 1 , 770 cm" 1 and 719 cm" 1 .
Sennoside A for component determination Use sennoside
A for thin-layer chromatography meeting the following addi-
tional specifications.
Absorbance <2.24> E[ 0/ ° m (270 nm): 211 -226 [10 mg dried
in a desiccator (in vacuum at a pressure not exceeding 0.67
kPa, phosphorus (V) toxide) for not less than 12 hours, dilut-
ed sodium bicarbonate solution (1 in 100), 500 mL]
Purity Related substances — Dissolve 5.0 mg of sennoside
A for component determination in 50 mL of the mobile phase
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add the mobile phase to make exactly 25
mL, and use this solution as the standard solution (1). Per-
form the test with exactly 10 /uL each of the sample solution
and standard solution (1) as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and measure each peak area from these solutions by the auto-
matic integration method: the total peak area other than sen-
noside A obtained from the sample solution is not larger than
peak area of sennoside A from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Assay under Senna
Leaf except detection sensitivity and time span of measure-
ment.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of sennoside A obtained from
10 /iL of the standard solution (2) can be measured by the au-
tomatic integration method, and the peak height of sennoside
A from 10 /iL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement: About 3 times as long as the
retention time of sennoside A beginning after the peak of sol-
vent.
Sennoside A for thin-layer chromatography C 42 H 38 O20
Yellow crystalline powder. Insoluble in water, in chloroform
and in diethyl ether, and practically insoluble in methanol
and in acetone. Melting point: 200 - 240°C (with decomposi-
tion).
240
Reagents, Test Solutions / General Tests
JP XV
Purity Related substances — Dissolve 1.0 mg of sennoside
A for thin-layer chromatography in exactly 4 mL of a mix-
ture of tetrahydrofuran and water (7:3), and perform the test
with 80 /xL of this solution as directed in the identification un-
der Rhubarb: any spot other than the principal spot at the Rf
value of about 0.3 does not appear.
Sennoside B for component determination C42H 38 2 o
Yellow crystalline powder. Insoluble in water and in diethyl
ether, and practically insoluble in methanol and in acetone.
Melting point: 180- 186°C (with decomposition).
Absorbance <2.24> E\°f m (270 nm): 210 - 225 [10 mg dried
in a desiccator (in vacuum at a pressure not exceeding 0.67
kPa, phosphorus (V) toxide) for not less than 12 hours, dilut-
ed sodium bicarbonate solution (1 in 100), 500 mL]
Purity Related substances — (1) Dissolve 1.0 mg of Sen-
noside B for component determination in exactly 4 mL of a
mixture of tetrahydrofuran and water (7:3), and perform the
test as directed under Thin-layer Chromatography <2.03>
with this solution. Spot 80 /uL of this solution on a plate of
silica gel for thin-layer chromatography. Develop the plate
with a mixture of 1-propanol, ethyl acetate, water and formic
acid (7:7:4:2) to a distance of about 15 cm, and air-dry the
plate. Examine under the ultraviolet light (main wavelength:
365 nm): any spot other than the principal spot as the Rf
value of about 0.5 does not appear.
(2) Dissolve 5.0 mg of sennoside B for component deter-
mination in 50 mL of the mobile phase and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 25 mL, and use this so-
lution as the standard solution (1). Perform the test with ex-
actly 10 /uL each of the sample solution and standard solution
(1) as directed under Liquid Chromatography <2.01> accord-
ing to the following conditions, and measure each peak area
from these solutions by the automatic integration method:
the total peak area other than sennoside B obtained from the
sample solution is not larger than peak area of sennoside B
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Assay under Senna
Leaf except detection sensitivity and time span of measure-
ment.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of sennoside B obtained from
10 /xL of the standard solution (2) can be measured by the au-
tomatic integration method, and the peak height of sennoside
B from 10 /xL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement: About 4 times as long as the
retention time of sennoside B beginning after the peak of sol-
vent.
L-Serine C 3 H 7 N0 3 [K 9105, Special class]
Sesame oil [Same as the namesake monograph]
[6]-Shogaol for thin-layer chromatography C 17 H2 4 3 A
pale yellow oil. Miscible with methanol, ethanol (99.5) and
with diethyl ether, and practically insoluble in water.
Purity Related substances — Dissolve 1.0 mg of [6]-
shogaol for thin-layer chromatography in 2 mL of methanol,
and perform the test with this solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xL on a plate of
silica gel for thin-layer chromatography, develop the plate
with a mixture of ethyl acetate and hexane (1:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly 4-
dimethylaminobenzaldehyde TS on the plate, heat at 105°C
for 5 minutes, and allow to cool: no spot other than the
principal spot at around Rf 0.5 appears.
Silica gel An amorphous, partly hydrated silicic acid
occurring in glassy granules of various sizes. When used as a
desiccant, it is frequently coated with a substance that
changes color when the capacity to absorb water is exhaust-
ed. Such colored products may be regenerated by being heat-
ed at 110°C until the gel assumes the original color.
Loss on ignition <2.43>: not more than 6% (2 g, 950 ±
50°C).
Water absorption: not less than 31%. Weigh accurately
about 10 g of silica gel, and allow to stand for 24 hours in a
closed container in which the atmosphere is maintained at
80% relative humidity with sulfuric acid having a specific
gravity of 1.19. Weigh again, and calculate the increase in
mass.
Siliceous earth [K 8330, Diatomaceous earth, First class]
Silicone oil Colorless clear liquid, having no odor.
Viscosity <2.53>: 50 - 100 mm 2 /s.
Silicone resin Light gray, half-clear, viscous liquid or a
pasty material. It is almost odorless.
Viscosity and refractive index — Place 15 g of silicone resin
in a Soxhlet extractor, then extract with 150 mL of carbon
tetrachloride for 3 hours. The kinematic viscosity of the
residual liquid, obtained by evaporating carbon tetrachloride
from the extract on a water bath, is 100 to 1100 mm 2 /s
(25°C). Its refractive index is 1.400 to 1.410 (25°C).
Specific gravity <2.56> d: 0.98 - 1.02
Loss on drying <2.41>: 0.45 - 2.25 g with the extracted
residue obtained in the Viscosity and refractive index (100°C,
1 hour).
Silicotungstic acid 26-water Si0 2 .12W0 3 .26H 2
White to slightly yellowish, crystals. Deliquescent. Very solu-
ble in water and in ethanol (95).
Loss on ignition <2.43>: 14 - 15% (2 g, dry at 110°C for 2
hours then 700 - 750°C, constant mass).
Clarity and color of solution: a solution (1 in 20) is clear
and colorless.
Silver chromate-saturated potassium chromate TS Dis-
solve 5 g of potassium chromate in 50 mL of water, add silver
nitrate TS until a pale red precipitate is produced, and filter.
To the filtrate add water to make 100 mL.
Silver diethyldithiocarbamate See silver N, A^-diethyl-
dithiocarbamate.
Silver nitrate AgN0 3 [K 8550, Special class]
Silver nitrate-ammonia TS Dissolve 1 g of silver nitrate in
20 mL of water, and add ammonia TS dropwise with stirring
until the precipitate is almost entirely dissolved.
Storage — Preserve in tight, light-resistant containers.
Silver nitrate TS Dissolve 17.5 g of silver nitrate in water
to make 1000 mL (0. 1 mol/L). Preserve in light-resistant con-
tainers.
Silver Af,Af-diethyldithiocarbamate C 5 H 10 AgNS 2
JPXV
General Tests / Reagents, Test Solutions
241
[K 9512]
Soda lime [K 8603, First class]
Sodium acetate See sodium acetate trihydrate.
Sodium acetate-acetone TS Dissolve 8.15 g of sodium
acetate trihydrate and 42 g of sodium chloride in 100 mL of
water, and add 68 mL of 0.1 mol/L hydrochloric acid VS,
150 mL of acetone and water to make 500 mL.
Sodium acetate, anhydrous CH 3 COONa [K 8372,
Special class]
Sodium acetate trihydrate CH 3 COONa.3H 2
[K 8371, Special class]
Sodium acetate TS Dissolve 13.6 g of sodium acetate tri-
hydrate in water to make 100 mL (1 mol/L).
Sodium benzoate for assay [Same as the monograph
Sodium Benzoate]
Sodium bicarbonate See sodium hydrogen carbonate.
Sodium bicarbonate for pH determination See sodium
hydrogen carbonate for pH determination.
Sodium bicarbonate TS See sodium hydrogen carbonate
TS.
7% Sodium bicarbonate injection [Same as the mono-
graph Sodium Bicarbonate Injection. However, labeled
amount should be 7 w/v%].
Sodium bismuthate See bismuth sodium trioxide.
Sodium bisulfite See sodium hydrogen sulfite.
Sodium bisulfite TS See sodium hydrogen sulfite TS.
Sodium bitartrate See sodium hydrogen tartrate mono-
hydrate.
Sodium bitartrate TS See sodium hydrogen tartrate TS.
Sodium borate for pH determination See sodium
tetraborate for pH determination.
Sodium borate decahydrate See sodium tetraborate deca-
hydrate.
Sodium borohydride NaBH 4 White to grayish white,
crystals, powder or masses. Freely soluble in water.
Content: not less than 95%. Assay — Weigh accurately 0.25
g of sodium borohydride, dissolve in 20 mL of diluted
sodium hydroxide TS (3 in 10), and add water to make ex-
actly 500 mL. Pipet 20 mL of this solution, put in a glass-
stoppered iodine flask, and cool in ice. Add exactly 40 mL of
iodine TS, allow to stand at a dark place for 10 minutes, add
exactly 10 mL of diluted sulfuric acid (1 in 6), and titrate
<2.50> with 0.1 mol/L sodium thiosulfate VS (back titration)
(indicator: starch solution). Perform a blank determination
in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 0.4729 mg of NaBH 4
Sodium bromide NaBr [K 8514, Special class]
Sodium carbonate See sodium carbonate decahydrate.
Sodium carbonate, anhydrous Na 2 C0 3 [K 8625, Sodi-
um carbonate, Special class]
Sodium carbonate decahydrate Na 2 CO 3 .10H 2 O
[K 8624, Special class]
Sodium carbonate for pH determination Na 2 C0 3
[K 8625, for pH determination]
Sodium carbonate (standard reagent) Na 2 C0 3 [K 8005,
Standard reagent for volumetric analysis]
Sodium carbonate TS Dissolve 10.5 g of anhydrous sodi-
um carbonate in water to make 100 mL (1 mol/L).
0.55 mol/L Sodium carbonate TS Dissolve 5.83 g of an-
hydrous sodium carbonate in water to make 100 mL.
Sodium chloride NaCl [K 8150, Special class]
Sodium chloride (standard reagent) NaCl [K 8005,
Standard reagent for volumetric analysis]
Sodium chloride TS Dissolve 10 g of sodium chloride in
water to make 100 mL.
0.1 mol/L Sodium chloride TS Dissolve 6 g of sodium
chloride in water to make 1000 mL.
1 mol/L Sodium chloride TS Dissolve 29.22 g of sodium
chloride in water to make 500 mL.
Sodium citrate See trisodium citrate dihydrate.
Sodium cobaltinitrite See sodium hexanitrocobaltate
(III).
Sodium cobaltinitrite TS See sodium hexanitrocobaltate
(III) TS.
Sodium 1-decanesulfonate Ci H 2 iNaO 3 S A white pow-
der.
Purity Clarity and color of solution — Dissolve 1 .0 g in 20
mL of water: the solution is clear and colorless.
Loss on drying <2.41>: not more than 3.0% (1 g, 105°C,
3 hours).
Content: not less than 98.0%. Assay — Weigh accurately
about 0.45 g of sodium 1-decanesulfonate, dissolve in 50 mL
of water, and pass through a column, about 1.2 cm in inside
diameter and about 25 cm in length, packed with about
20 mL of strongly acidic ion-exchange resin (0.3 to 1.0 mm,
H type) at a flow rate of about 4 mL per minute. Wash with
150 mL of water at a flow rate of about 4 mL per minute.
Combine the washing and the elute, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS (potentiometric titration).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 24.43 mg of C 10 H 21 NaO 3 S
0.0375 mol/L Sodium 1-decanesulfonate TS Dissolve
3.665 g of sodium 1-decanesulfonate in 400 mL of water.
Sodium desoxycholate C 24 H 39 Na0 4 White, odorless,
crystalline powder.
Identification — Determine the infrared absorption spec-
trum of sodium desoxycholate, previously dried, according
to the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3400 cm -1 , 2940 cm -1 , 1562 cm -1 and
1408 cm- 1 .
Purity Related substances — Dissolve 0.10 g of sodium
242
Reagents, Test Solutions / General Tests
JP XV
desoxycholate in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of this solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, methanol and acetic acid
(100) (80:40:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly concentrated sulfuric acid on the plate,
and heat at 105°C for 10 minutes: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Sodium diethyldithiocarbamate See sodium N, A'-diethyl-
dithiocarbamate trihydrate.
Sodium iV,./V-diethyldithiocarbamate trihydrate
(C 2 H 5 ) 2 NCS 2 Na.3H 2 [K 8454, Special class]
Sodium dihydrogen phosphate NaH 2 P0 4 A white,
powder or crystalline powder. Freely soluble in water, and
very slightly soluble in ethanol (99.5). It has a hygroscopic
property.
A solution is acidic.
Sodium dihydrogen phosphate anhydrous See sodium di-
hydrogen phosphate.
2 mol/L Sodium dihydrogen phosphate TS Dissolve
312.02 g of sodium dihydrogen phosphate dihydrate in water
to make 1000 mL.
Sodium dihydrogen phosphate TS, pH 2.5 Dissolve 2.7 g
of sodium dihydrogen phosphate dihydrate in 1000 mL of
water, and adjust the pH to 2.5 with phosphoric acid.
Sodium dihydrogen phosphate dihydrate
NaH 2 P0 4 .2H 2 [K 9009, Special class]
0.05 mol/L Sodium dihydrogen phosphate TS Dissolve
7.80 g of sodium dihydrogen phosphate dihydrate in water to
make 1000 mL.
0.1 mol/L Sodium dihydrogen phosphate TS Dissolve
7.80 g of sodium dihydrogen phosphate dihydrate in 450 mL
of water, adjust to a pH of 5.8 exactly with sodium hydroxide
TS, and add water to make 500 mL.
0.05 mol/L Sodium dihydrogen phosphate TS, pH 2.6
Dissolve 7.80 g of sodium dihydrogen phosphate dihydrate in
900 mL of water, adjust the pH to exactly 2.6 and add water
to make 1000 mL.
0.05 mol/L Sodium dihydrogen phosphate TS, pH 3.0
Dissolve 3.45 g of sodium dihydrogen phosphate dihydrate in
500 mL of water (solution A). Dilute 2.45 g of phosphoric
acid with water to make 500 mL (solution B). To a volume of
solution A add solution B until the mixture is adjusted to pH
3.0.
0.1 mol/L Sodium dihydrogen phosphate TS, pH 3.0
Dissolve 15.60 g of sodium dihydrogen phosphate dihydrate
in 900 mL of water, adjust the pH to 3.0 with phosphoric
acid, and add water to make 1000 mL.
Sodium disunite Na 2 S 2 5 [K 8501, First class]
Sodium disulfite TS Dissolve 0.10 g of sodium disulfite in
10 mL of 1 mol/L hydrochloric acid TS, and add acetone to
make 100 mL.
Sodium dodecylbenzene sulfonate C 18 H 29 S0 3 Na White,
crystalline powder or mass.
pH <2.54> — The pH of a solution of 0.5 g of sodium dode-
cylbenzene sulfonate in 50 mL of freshly boiled and cooled
water is between 5.0 and 7.0. Measure the pH at 25°C passing
nitrogen with stirring.
Loss on drying <2.41>: not more than 0.5% (1 g, 105°C, 2
hours).
Content: not less than 99.0%. Assay — Weigh accurately
about 40 mg of sodium dodecylbenzene sulfonate, previously
dried, and perform the test as directed in (4) Sulfur in the
Procedure of determination under Oxygen Flask Combustion
Method <1.06>, using a mixture of 20 mL of water and 2 mL
of strong hydrogen peroxide water as absorbing solution.
Each mL of 0.01 mol/L barium perchlorate VS
= 1.742 mg of C 18 H 29 S0 3 Na
Sodium fluoride NaF [K 8821, Special class]
Sodium fluoride (standard reagent) NaF [K 8005, Stan-
dard reagent for volumetric analysis]
Sodium fluoride TS Dissolve 0.5 g of sodium fluoride in
100 mL of 0.1 mol/L hydrochloric acid TS. Prepare before
use.
Sodium 1-heptane sulfonate C 7 H 15 Na0 3 S White, crys-
tals or crystalline powder.
Purity Clarity and color of solution — Dissolve 1.0 g of
sodium 1-heptane sulfonate in 10 mL of water: the solution is
clear and colorless.
Loss on drying <2.41>: not more than 3.0% (1 g, 105 °C, 3
hours).
Content: not less than 99.0%. Assay — Dissolve about 0.4
g of sodium 1-heptane sulfonate, previously dried and
weighed accurately, in 50 mL of water, transfer to a chromat-
ographic column, prepared by packing a chromatographic
tube 9 mm in inside diameter and 160 mm in height with 10
mL of strongly acidic ion exchange resin for column chroma-
tography (425 to 600 /xm in particle diameter, H type), and
flow at a flow rate of about 4 mL per minute. Wash the
column at the same flow rate with 150 mL of water, combine
the washings with the effluent solution, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS (indicator: 10 drops of bro-
mothymol blue TS) until the color of the solution changes
from yellow to blue.
Each mL of 0.1 mol/L sodium hydroxide VS
= 20.23 mg of C 7 H 15 Na0 3 S
Sodium 1-hexane sulfonate C 6 H 13 Na0 3 S White, crys-
tals or crystalline powder.
Loss on drying <2.41>: not more than 3.0% (1 g, 105 °C, 2
hours).
Content: not less than 98.0%. Assay — Weigh accurately
about 0.4 g of sodium 1-hexane sulfonate, previously dried,
and dissolve in 25 mL of water. Transfer 15-20 mL of this so-
lution into a chromatographic column about 11 mm in di-
ameter and about 500 mm in height of strongly acidic ion ex-
change resin for column chromatography (246 /xm to 833 /um
in particle diameter, type H), and elute at the rate of about
5-10 mL per minute, then wash the column with five 50-mL
portions of water at the rate of about 5-10 mL per minute.
JPXV
General Tests / Reagents, Test Solutions
243
Combine the washings to the eluate, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS (indicator: 3 drops of
phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 18.82 mg of C 6 H 13 Na0 3 S
Sodium hexanitrocobaltate (III) Na 3 Co(N0 2 ) 6
[K 8347, Special class]
Sodium hexanitrocobaltate (III) TS Dissolve 10 g of sodi-
um hexanitrocobaltate (III) in water to make 50 mL, and
filter if necessary. Prepare before use.
Sodium hydrogen carbonate NaHC0 3 [K 8622, Special
class]
Sodium hydrogen carbonate for pH determination
NaHC0 3 [K 8622, Sodium hydrogen carbonate, for pH de-
termination]
Sodium hydrogen carbonate TS Dissolve 5.0 g of sodium
hydrogen carbonate in water to make 100 mL.
Sodium hydrogen sulfite [K 8059, Special class]
Sodium hydrogen sulfite TS Dissolve 10 g of sodium
hydrogen sulfite in water to make 30 mL. Prepare before use.
Sodium hydrogen tartrate monohydrate
NaHC 4 H 4 6 .H 2 [K 8538, Sodium hydrogentartrate mon-
ohydrate, Special class]
Sodium hydrogen tartrate TS Dissolve 1 g of sodium
bitartrate in water to make 10 mL (0.5 mol/L). Prepare be-
fore use.
Sodium hydrosulfite Na 2 S 2 4 [K 8737, Sodium dithio-
nite, Special class]
Sodium hydroxide NaOH [K 8576, Special class]
Sodium hydroxide-dioxane TS Dissolve 0.80 g of sodium
hydroxide in a mixture of 1,4-dioxane and water (3:1) to
make 100 mL.
Sodium hydroxide-methanol TS Dissolve by thorough
shaking 4 g of sodium hydroxide in methanol to make 100
mL. To the supernatant liquid obtained by centrifugation
add methanol to make 500 mL. Prepare before use.
Sodium hydroxide TS Dissolve 4.3 g of sodium hydrox-
ide in water to make 100 mL (1 mol/L). Preserve in poly-
ethylene bottles.
Sodium hydroxide TS, dilute Dissolve 4.3 g of sodium
hydroxide in freshly boiled and cooled water to make 1000
mL. Prepare before use (0.1 mol/L).
0.01 mol/L Sodium hydroxide TS Dilute 10 mL of sodi-
um hydroxide TS with water to make 1000 mL. Prepare be-
fore use.
0.05 mol/L Sodium hydroxide TS To 10 mL of 0.5 mol/
L sodium hydroxide TS add water to make 100 mL.
0.2 mol/L Sodium hydroxide TS Dissolve 8.0 g of sodi-
um hydroxide in freshly boiled and cooled water to make
1000 mL. Prepare before use.
0.5 mol/L Sodium hydroxide TS Dissolve 22 g of sodium
hydroxide in water to make 1000 mL. Preserve in poly-
ethylene bottles.
2 mol/L Sodium hydroxide TS Dissolve 86 g of sodium
hydroxide in water to make 1000 mL. Preserve in polyethy-
lene bottles.
4 mol/L Sodium hydroxide TS Dissolve 168 g of sodium
hydroxide in water to make 1000 mL. Preserve in poly-
ethylene bottles.
6 mol/L Sodium hydroxide TS Dissolve 252 g of sodium
hydroxide in water to make 1000 mL. Preserve in a polyethy-
lene bottle.
8 mol/L Sodium hydroxide TS Dissolve 336 g of sodium
hydroxide in water to make 1000 mL. Preserve in poly-
ethylene bottles.
Sodium hypobromite TS To 8 mL of bromine TS add 25
mL of water and 25 mL of sodium carbonate TS. Prepare be-
fore use.
Sodium hypochlorite-sodium hydroxide TS To a volume
of sodium hypochlorite TS for ammonium limit test, equiva-
lent to 1.05 g of sodium hypochlorite (NaCIO: 74.44), add 15
g of sodium hydroxide and water to make 1000 mL. Prepare
before use.
Sodium hypochlorite TS Prepare the solution by passing
chlorine into sodium hydroxide TS while cooling with ice, so
as to contain 5% of sodium hypochlorite (NaCIO: 74.44).
Prepare before use.
Sodium hypochlorite TS for ammonium limit test
Clear, colorless or pale green-yellow solution prepared by
passing chlorine into sodium hydroxide or sodium carbonate
solution, having the odor of chlorine.
Content: not less than 4.2 w/v% as sodium hypochlorite
(NaCIO: 74.44). Assay— Pipet 10 mL of sodium hypo-
chlorite TS for ammonium limit test, and add water to make
exactly 100 mL. Transfer exactly 10 mL of this solution to a
glass-stoppered flask, add 90 mL of water, then add 2 g of
potassium iodide and 6 mL of diluted acetic acid (1 in 2),
stopper tightly, shake well, and allow to stand for 5 minutes
in a dark place. Titrate <2.50> the liberated iodine with 0.1
mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 3.722 mg of NaCIO.
Sodium iopodate for assay Ci 2 H 12 I 3 N 2 Na0 2 [Same as
the monograph Sodium Iopodate. It contains not less than
99.0% of C 12 H 12 I 3 N 2 Na0 2 , calculated on the dried basis.
Sodium lauryl sulfate [Same as the namesake mono-
graph]
Sodium lauryl sulfate TS Dissolve 100 g of sodium lauryl
sulfate in 900 mL of water, add 10 mL of 1 mol/L
hydrochloric acid TS, and add water to make 1000 mL.
0.2% Sodium lauryl sulfate TS Dissolve 0.1 g of sodium
lauryl sulfate in 0.1 mol/L of pH 7.0 sodium phosphate
buffer to make 50 mL.
Sodium metabisulfite See sodium disulfite.
Sodium metabisulfite TS See sodium disulfite TS.
Sodium, metallic Na [K 8687, Sodium, Special class]
244
Reagents, Test Solutions / General Tests
JP XV
Sodium l-methyl-l//-tetrazole-5-thiolate
C2H 3 N4NaS.2H 2 White, crystals or crystalline powder.
Melting point <2.60>: 90 - 94°C
Purity Related substances — Dissolve 10 mg of sodium 1-
methyl-l//-tetrazole-5-thiolate in 10 mL of water, and use
this solution as the sample solution. Perform the test with
this solution as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL of the sample solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of ethyl acetate, acetone,
water and acetic acid (100) (10:2:1:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): any spot other than the principal
spot does not appear.
Sodium molybdate See sodium molybdate dihydrate.
Sodium molybdate dihydrate Na 2 Mo0 4 .2H 2
[K 8906, Special class]
Sodium Na [K 8687, special class]
Sodium 2-naphthalenesulfonate
brown, crystals or powder.
Content: not less than 98.0%.
Ci H 7 NaO 3 S Pale
Sodium naphthoquinone sulfonate TS Dissolve 0.25 g of
sodium ^-naphthoquinone sulfonate in methanol to make
100 mL.
Sodium ^-naphthoquinone sulfonate C 10 H 5 NaO 5 S
Yellow to orange-yellow, crystals or crystalline powder. Solu-
ble in water, and practically insoluble in ethanol (95).
Loss on drying <2.41>: Not more than 2.0% (1 g, in vacu-
um, 50°C).
Residue on ignition <2.44>: 26.5-28.0% (1 g, after
drying).
Sodium nitrate NaN0 3 [K 8562, Special class]
Sodium nitrite NaN0 2 [K 8019, Special class]
Sodium nitrite TS Dissolve 10 g of sodium nitrite in
water to make 100 mL. Prepare before use.
Sodium nitroprusside See sodium pentacyanonitrosylfer-
rate (III) dihydrate.
Sodium nitroprusside TS See sodium pentacyano-
nitrosylferrate (III) TS.
Sodium 1-octane sulfonate CH 3 (CH 2 ) 7 S0 3 Na White
crystals or powder.
Residue on ignition <2.44>: 32.2 - 33.0% (1.0 g).
Sodium oxalate (standard reagent) C 2 4 Na 2 [K 8005,
Standard reagent for volumetric analysis]
Sodium pentacyanoammine ferroate (II) n-hydrate
Na 3 [Fe(CN) 5 NH 3 ].«H 2 [K 8689, First class]
Sodium pentacyanonitrosylferrate (Ill)-potassium hexac-
yanoferrate (III) TS Mix an equal volume of a solution of
sodium pentacyanonitrosylferrate (III) dihydrate (1 in 10), a
solution of potassium hexacyanoferrate (III) (1 in 10) and a
solution of sodium hydroxide (1 in 10), and allow to stand for
30 minutes. Use after the color of the solution is changed
from a dark red to yellow. Prepare before use.
Sodium pentacyanonitrosylferrate (HI) dihydrate
Na 2 Fe(CN) 5 (NO).2H 2 [K 8722, Special class]
Sodium pentacyanonitrosylferrate (III) TS Dissolve 1 g
of sodium pentacyanonitrosylferrate (III) dihydrate in water
to make 20 mL. Prepare before use.
Sodium 1-pentane sulfonate C 5 H u Na0 3 S White, crys-
tals or crystalline powder. Freely soluble in water, and practi-
cally insoluble in acetonitrile.
Purity Clarity and color of solution — Dissolve 1.0 g of
sodium 1-pentane sulfonate in 10 mL of water: the solution is
colorless and clear.
Water <2.48>: not more than 3.0% (0.2 g).
Content: not less than 99.0%, calculated on the anhydrous
basis. Assay — Dissolve about 0.3 g of sodium 1-pentane
sulfonate, accurately weighed, in 50 mL of water. Transfer
this solution to a chromatographic column, prepared by
pouring 10 mL of strongly acidic ion-exchange resin (H type)
(424 - 600 /xm in particle diameter) into a chromatographic
tube, 9 mm in inside diameter and 160 mm in height, and e-
lute at the rate of about 4 mL per minute. Wash the chromat-
ographic column with 50 mL of water at the rate of about 4
mL per minute, and wash again with 100 mL of water in the
same manner. Combine the washings with the eluate, and ti-
trate <2.50> with 0.1 mol/L sodium hydroxide VS (indicator:
10 drops of bromothymol blue TS) until the yellow color of
the solution changes to blue.
Each mL of 0.1 mol/L sodium hydroxide VS
= 17.42 mg of C 5 H n Na0 3 S
Sodium perchlorate See sodium perchlorate monohy-
drate.
Sodium perchlorate monohydrate NaC10 4 .H 2
[K 8227, Special class]
Sodium periodate NaI0 4 [K 8256, Special class]
Sodium periodate TS Dissolve 60.0 g of sodium perio-
date in 120 mL of 0.05 mol/L sulfuric acid TS, and add water
to make 1000 mL. If the solution is not clear, filter this
through a glass-filter. Keep in a light-resistant vessel.
Sodium peroxide Na 2 2 [K 8231, Special class]
Sodium phosphate See trisodium phosphate dodecahy-
drate.
Sodium phosphate TS Dissolve 5.68 g of disodium
hydrogen phosphate and 6.24 g of sodium dihydrogen phos-
phate dihydrate in water to make 1000 mL.
Sodium p-phenol sulfonate C 6 H 5 4 NaS.2H 2 White
to light yellow, crystals or crystalline powder, having a spec-
ific odor.
Identification — (1) To 10 mL of a solution of sodium p-
phenol sulfonate (1 in 10) add 1 drop of iron (III) chloride
TS: a purple color develops.
(2) Determine the absorption spectrum of a solution of
sodium /?-phenol sulfonate (1 in 5000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 269 nm and 273 nm and between 276 nm and
280 nm.
Purity Clarity and color of solution — Dissolve 1.0 g of
sodium p-phenol sulfonate in 25 mL of water: the solution is
clear and colorless.
Content: not less than 90.0%. Assay— Dissolve about
JPXV
General Tests / Reagents, Test Solutions
245
0.5 g of sodium p-phenol sulfonate, accurately weighed, in 50
mL of water. Transfer the solution to a chromatographic
column, prepared by pouring strongly acidic ion exchange re-
sin (H type) for column chromatography (150 to 300 /um in
particle diameter) into a chromatographic tube about 1 cm in
inside diameter and about 30 cm in height, and allow to flow.
Wash the chromatographic column with water until the
washing is no longer acidic, combine the washings with the
above effluent solution, and titrate <2.50> with 0.1 mol/L so-
dium hydroxide VS (indicator: 5 drops of bromocresol green-
methyl red TS). Separately, dissolve 0.5 g of sodium p-
phenol sulfonate, previously weighed accurately, in 50 mL of
water and titrate with 0.1 mol/L sodium hydroxide VS, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 23.22 mg of C 6 H 5 4 NaS.2H 2
0.1 mol/L Sodium phosphate buffer solution, pH 7.0
Dissolve 17.9 g of disodium hydrogen phosphate dodecahy-
drate (NaH 2 P0 4 .12H 2 0) in water to make 500 mL. Add to
this solution to a 500 mL solution prepared by dissolving 7.8
g of disodium hydrogen phosphate dihydrate in water until
the pH becomes 7.0.
Sodium pyruvate Prepared for microbial test.
Sodium salicylate HOC 6 H 4 COONa [K 8397, Special c-
lass]
Sodium salicylate-sodium hydroxide TS Dissolve 1 g of
sodium salicylate in 0.01 mol/L sodium hydroxide VS to
make 100 mL.
Sodium selenite Na 2 Se0 3 [K 8036, Special class]
Sodium p-styrenesulfonate C 8 H 7 Na0 3 S White crystals
or crystalline powder. Freely soluble in water, slightly soluble
in ethanol (99.5), and practically insoluble in diethyl ether.
Recrystalize from diluted ethanol (1 in 2), and dry in vacu-
um.
Identification — Determine the infrared absorption spec-
trum of sodium /?-styrenesulfonate according to the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
1236 cm- 1 , 1192 cm- 1 , 1136 cm- 1 , 1052 cm- 1 , 844 cm- 1 and
688 cm- 1 .
Purity — Perform the test with 10 fiL of a solution of sodi-
um p-styrenesulfonate (1 in 1000) as directed in the Assay un-
der Panipenem: Any obstructive peaks for determination of
panipenem are not observed.
Sodium sulfate See sodium sulfate decahydrate.
Sodium sulfate, anhydrous Na 2 S0 4 [K 8987, Special c-
lass]
Sodium sulfate decahydrate Na 2 SO 4 .10H 2 O [K 8986,
Special class]
Sodium sulfide See sodium sulfide enneahydrate.
Sodium sulfide enneahydrate Na 2 S.9H 2 [K 8949,
Special class]
Sodium sulfide TS Dissolve 5 g of sodium sulfide ennea-
hydrate in a mixture of 10 mL of water and 30 mL of gly-
cerin. Or dissolve 5 g of sodium hydroxide in a mixture of 30
mL of water and 90 mL of glycerin, saturate a half volume of
this solution with hydrogen sulfide, while cooling, and mix
with the remaining half. Preserve in well-filled, light-resistant
bottles. Use within 3 months.
Sodium sulfite See sodium sulfite heptahydrate.
Sodium sulfite, anhydrous Na 2 S0 3 [K 8061, Sodium
sulfite, Special class]
Sodium sulfite heptahydrate Na 2 S0 3 .7H 2 [K 8060,
Special class]
1 mol/L Sodium sulfite TS Dissolve 1.26 g of anhydrous
sodium sulfite in water to make 10 mL.
Sodium tartrate See sodium tartrate dihydrate.
Sodium tartrate dihydrate C 4 H 4 Na 2 6 .2H 2
[K 8540, Special class]
Sodium tetraborate-calcium chloride buffer solution, pH
8.0 Dissolve 0.572 g of sodium tetraborate decahydrate and
2.94 g of calcium chloride dihydrate in 800 mL of freshly
boiled and cooled water, adjust the pH to 8.0 with 1 mol/L
hydrochloric acid TS, and add water to make 1000 mL.
Sodium tetraborate decahydrate Na 2 B 4 O 7 .10H 2 O
[K 8866, Special class]
Sodium tetraborate for pH determination
[K 8866, for pH standard solution]
Sodium tetraphenylborate (C 6 H 5 ) 4 BNa [K 9521]
Sodium thioglycolate HSCH 2 COONa A white powder,
having a characteristic odor.
Identification — (1) To a solution (1 in 10) add 1 drop
each of ammonia solution (28) and iron (III) chloride TS: a
dark red-purple color appears.
(2) Perform the test as directed under Flame Coloration
Test (1) <1.04>: a yellow color appears.
Purity Clarity and color of solution — Dissolve 1 g in 10
mL of water: the solution is clear and colorless.
Sodium thiosulfate See sodium thiosulfate pentahydrate.
Sodium thiosulfate pentahydrate Na 2 S 2 3 .5H 2
[K 8637, Special class]
Sodium thiosulfate TS Dissolve 26 g of sodium thio-
sulfate pentahydrate and 0.2 g of anhydrous sodium car-
bonate in freshly boiled and cooled water to make 1000 mL
(0.1 mol/L).
Sodium toluenesulfonchloramide trihydrate
C 7 H 7 ClNNa0 2 S.3H 2 [K 8318, Sodium p-toluenesulfon-
chloramide trihydrate, Special class]
Sodium toluenesulfonchloramide TS Dissolve 1 g of so-
dium toluensulfonchloramide trihydrate in water to make
100 mL. Prepare before use.
Sodium tridecanesulfonate C 13 H 27 S0 3 Na White, crys-
tals or powder.
Purity Absorbance — Dissolve 1.43 g of sodium tridecan-
sulfonate in 1000 mL of water, and perform the test as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: the
absorbances at 230 nm and 245 nm are not more than 0.05
and 0.01, respectively.
Sodium 3-trimethylsilylpropanesulfonate for nuclear mag-
netic resonance spectroscopy
246
Reagents, Test Solutions / General Tests
JP XV
(CH 3 ) 3 SiCH 2 CH 2 CH 2 S0 3 Na Prepared for nuclear magnet-
ic resonance spectroscopy.
Sodium 3-trimethyIsiIyIpropionate-d 4 for nuclear magnetic
resonance spectroscopy (CH 3 ) 3 SiCD 2 CD 2 COONa Pre-
pared for nuclear magnetic resonance spectroscopy.
Sodium tungstate See sodium tungstate (VI) dihydrate.
Sodium tungstate (VI) dihydrate Na 2 W0 4 .2H 2 [K
8612, Special class]
Solid plates Dilute anti-E. coli protein antibody stock so-
lution by adding 0.2 mol/L Tris hydrochloride buffer, pH
7.4, to a concentration of about 0.02mg/mL. Add exactly
0.1 mL of this solution to each well in the microplates, cover
with plate seal, and then shake gently. Centrifuge for 2
minutes if some solution sticks to the top of the microplate or
elsewhere. Dissolve 0.5 g of bovine serum albumin in 100 mL
of 0.01 mol/L phosphate buffer-sodium chloride TS (pH 7.4)
to make the wash solution. After leaving the microplates for
16 to 24 hours at a constant temperature of about 25 °C, re-
move the solution in each well by aspiration, add 0.25 mL of
the wash solution, shake gently, and then remove this solu-
tion by aspiration. Repeat this procedure 2 more times for
each well using 0.25 mL of the wash solution. Add 0.25 mL
of the block buffer solution to each well, gently shake, and let
stand for 16 to 24 hours at a constant temperature of about
25°C to make solid plates. When using, remove the solution
from the wells by aspiration, add 0.25 mL of the wash solu-
tion to each well, shake gently, and then remove this solution
by aspiration. Repeat this procedure 2 more times using 0.25
mL of the wash solution.
Soluble starch See starch, soluble.
Soluble starch TS Triturate 1 g of soluble starch in 10 mL
of cooled water, pour gradually into 90 mL of boiled water
while costantly stirring, boil gently for 3 minutes, and cool.
Prepare before use.
Solution of factor Xa 7 \nkat s _ 2 22i of Factor Xa in 10 mL
of water.
Sorbitan sesquioleate [Same as the namesake mono-
graph]
D-Sorbitol [Same as the namesake monograph]
D-Sorbitol for gas chromatography Prepared for gas
chromatography.
Soybean-casein digest medium See Sterility Test <4.06>.
Soybean oil [Same as the namesake monograph]
Soybean peptone See peptone, soybean.
Stacking gel for celmoleukin In 0.5 mol/L Tris buffer so-
lution, pH 6.8, prepare stacking the gel using ammonium
persulfate and TEMED so the acrylamide concentration is
5.2% and the sodium lauryl sulfate concentration is 0.1%.
Stannous chloride See tin (II) chloride dihydrate.
Stannous chloride-sulfuric acid TS See tin (II) chloride-
sulfuric acid TS.
Stannous chloride TS See tin (II) chloride TS.
Stannous chloride TS, acidic See tin (II) chloride TS,
acidic.
Starch [K 8658, Special class]
Starch-sodium chloride TS Saturate starch TS with sodi-
um chloride. Use within 5 to 6 days.
Starch, soluble [K 8659, Special class]
Starch TS Triturate 1 g of starch with 10 mL of cold
water, and pour the mixture slowly, with constant stirring,
into 200 mL of boiling water. Boil the mixture until a thin,
translucent fluid is obtained. Allow to settle, and use the
supernatant liquid. Prepare before use.
Stearic acid for gas chromatography C 18 H 36 2
[K 8585, Special class]
Stearyl alcohol [Same as the namesake monograph]
Strong ammonia water See ammonia solution (28).
Strong cupric acetate TS See copper (II) acetate TS,
strong.
Strong hydrogen peroxide water See hydrogen peroxide
(30).
Strongly acidic ion exchange resin Contains strong acid
ion exchange residues. Particle diameter is about 100 /im.
Strongly basic ion exchange resin Contains strong basic
ion exchange residues. Particle diameter is about 100 /um.
Strontium chloride SrCl 2 .6H 2 [K 8132, Special class]
Strychnine nitrate for assay C 2 iH 2 2N 2 2 .HN0 3 To 1 g
of strychnine nitrate add 14 mL of water and about 10 mg of
active carbon, heat in a water bath for 10 minutes, filter while
hot, cool the filtrate quickly to form crystals, and filter the
crystals. Add 8 mL of water to the crystals, dissolve by heat-
ing in a water bath, filter while hot, cool quickly, and filter
the crystals formed. Repeat this procedure with 8 mL of
water, and dry the crystals in a desiccator (in vacuum, silica
gel) for 24 hours. Colorless or white crystals or crystalline
powder. Sparingly soluble in water, in glycerin and in chloro-
form, slightly soluble in ethanol (95), and practically insolu-
ble in diethyl ether.
Purity Related substances — Dissolve 35 mg of strychnine
nitrate for assay in 100 mL of the mobile phase and use this
solution as the sample solution. Pipet 2 mL of the sample so-
lution, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution (1). Perform the test
with exactly 20 /xL each of the sample solution and standard
solution (1) as directed under Liquid Chromatography <2.01>
according to the following conditions. Measure each peak
area of these solutions by the automatic integration method:
the total area of the peaks other than strychnine from the
sample solution is not larger than the peak area of strychnine
from the standard solution (1).
Operating conditions
Proceed the operating conditions in the Assay under Nux
Vomica except detection sensitivity and time span of meas-
urement.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add the mobile phase to make exactly 40 mL, and use this
solution as the standard solution (2). Adjust the detection
sensitivity so that the peak area of strychnine obtained from
20 iuL of the standard solution (2) can be measured by the au-
tomatic integration method and the peak height of strychnine
JPXV
General Tests / Reagents, Test Solutions
247
from 20 iuL of the standard solution (1) is about 20% of the
full scale.
Time span of measurement: About 3 times as long as the
retention time of strychnine beginning after the solvent peak.
Loss on drying <2.41>: not more than 0.5% (0.2 g, 105°C,
3 hours).
Content: not less than 99.0% calculated on the dried basis.
Assay — Dissolve about 0.5 g of strychnine nitrate for
assay, accurately weighed, in 40 mL of a mixture of acetic an-
hydride and acetic acid (100) (4:1), heat if necessary, cool,
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.74 mg of C 21 H 22 N 2 2 .HN0 3
Styrene C 8 H 8 Colorless, clear liquid.
Specific gravity <2.56> d: 0.902 - 0.910
Purity — Perform the test with 1 /uL of styrene as directed
under Gas Chromatography <2.02> according to the follow-
ing conditions. Measure each peak area by the automatic in-
tegration method and calculate the amount of styrene by the
area percentage method: it shows the purity of not less than
99%.
Operating conditions
Detector: Thermal conductivity detector.
Column: A glass column, about 3 mm in inside diameter
and about 2 m in length, packed with siliceous earth (180 to
250 /urn in particle diameter) coated with polyethylene glycol
20 M at the ratio of 10%.
Column temperature: A constant temperature of about 100
°C.
Temperature of sample vaporization chamber: A constant
temperature of about 150°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
styrene is about 10 minutes.
Time span of measurement: About twice as long as the
retention time of styrene, beginning after the solvent peak.
Substrate buffer for celmoleukin Dissolve 32.4 g of
tripotassium citrate monohydrate in water to make 1000 mL,
and add 1 mol/L citric acid TS for buffer solution to adjust
the pH to 5.5. To 100 mL of this solution add and dissolve
0.44 g of o-phenylenediamine and then 60 /xh of hydrogen
peroxide (30). Prepare at the time of use.
Substrate solution for lysozyme hydrochloride To a suit-
able amount of dried cells of Micrococcus luteus add a suita-
ble amount of phosphate buffer solution, pH 6.2, gently
shake to make a suspension, and add the substrate cells or the
same buffer solution so that the absorbance of the suspension
at 640 nm is about 0.65. Prepare before use.
Substrate solution for peroxidase determination Dissolve
0.195 mL of hydrogen peroxidase (30), 8.38 g of disodium
hydrogen phosphate dodecahydrate and 1.41 g of citric acid
monohydrate in water to make 300 mL. To 15 mL of this so-
lution add 13 mg of o-phenylenediamine dihydrochloride be-
fore use.
Substrate TS for kallidinogenase assay (1) Dissolve an
appropriate amount of H-D-valyl-L-leucyl-L-arginine p-
nitroanilide dihydrochloride in 0.1 mol/L tris buffer solu-
tion, pH 8.0 to prepare a solution containing 1 mg of H-D-
valyl-L-leucyl-L-arginine jP-nitroanilide dihydrochloride in 5
mL.
Substrate TS for kallidinogenase assay (2) Dissolve 17.7
mg of A'-a-benzoyl-L-arginine ethyl ester hydrochloride in 0.1
mol/L tris buffer solution, pH 8.0 to make 100 mL.
Substrate TS for kallidinogenase assay (3) Suspend 0.6 g
of milk casein purified by the Hammerstein's method in 80
mL of 0.05 mol/L sodium hydrogen phosphate TS, and dis-
solve by warming at 65 °C for 20 minutes. After cooling, ad-
just to pH 8.0 with 1 mol/L hydrochloric acid TS or sodium
hydroxide TS, and add water to make exactly 100 mL. Pre-
pare before use.
Substrate TS for kallidinogenase assay (4)
Dissolve 25 mg of H-D-valyl-L-leucyl-L-arginine-4-nitro-
anilide dihydrochloride in 28.8 mL of water.
Succinic acid, anhydrous C 4 H 4 3 White or pale yellow-
ish white crystals or flakes. It is odorless. Soluble in water,
freely soluble in hot water, and sparingly soluble in ethanol
(95).
Purity (1) Chloride <1.03>: not more than 0.005%.
(2) Iron <7.70>: not more than 0.001%.
Residue on ignition <2.44>: not more than 0.1% (1 g).
Content: not less than 98.0%. Assay— Dissolve about 1 g
of anhydrous succinic acid, accurately weighed, in 50 mL of
water by warming, cool, and titrate <2.50> with 1 mol/L sodi-
um hydroxide VS (indicator: 2 drops of phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 50.04 mg of C4H4O3.
Sucrose C I2 H 22 O u [Same as the monograph Sucrose]
Sudan III C 22 H 16 N 4 Red-brown powder. It dissolves
in acetic acid (100) and in chloroform, and insoluble in water,
in ethanol (95), in acetone and in ether.
Melting point <2.60>: 170 - 190°C
Sudan III TS Dissolve 0.01 g of sudan III in 5 mL of eth-
anol (95), filter, and add 5 mL of glycerin to the filtrate. Pre-
pare before use.
Sulbactam sodium for sulbactam penicillamine
C 8 H 10 NNaO 5 S White to yellowish white crystalline powder.
Freely soluble in water, and slightly soluble in ethanol (95).
Identification — Determine the infrared absorption spec-
trum of sulbactam sodium for sulbactam penicillamine ac-
cording to the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits the absorption
at the wave numbers of about 1780 cm -1 , 1600 cm -1 , 1410
cm" 1 , 1400 cm- 1 , 1320 cm- 1 , 1300 cm- 1 , 1200 cm- 1 and
1130 cm- 1 .
Water <2.48>: not more than 1.0% (0.5 g).
Content: not less than 875 fig per mg, calculated on the an-
hydrous basis. Assay — Weigh accurately an amount of sul-
bactam sodium for sulbactam penicillamine and Sulbactam
Reference Standard, equivalent to about 0.10 g (potency),
dissolve each in a suitable volume of the mobile phase, add
exactly 10 mL of the internal standard solution and the mo-
bile phase to make 100 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 10 /iL each of these solutions as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the ratios, g T and Q s , of the peak
248
Reagents, Test Solutions / General Tests
JP XV
area of sulbactam to that of the internal standard.
Amount [/ug (potency)] of sulbactam (C 8 H u N0 5 S)
= Ws x ~^~ X1000
Qi_
Ss
W s : amount [mg (potency)] of Sulbactam Reference Stan-
dard
Internal standard solution A solution of ethyl parahydrox-
ybenzoate in the mobile phase (7 in 1000).
Operating conditions
Detector: Ultraviolet absorption photometer (wave-
length: 220 nm)
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 750 mL of 0.005 mol/L tetrabutylam-
monium hydroxide TS add 250 mL of acetonitrile for liquid
chromatography.
Flow rate: Adjust the flow rate so that the retention time of
sulbactam is about 6 minutes.
System suitability
System performance: When the procedure is run with 10
[iL of the standard solution according to the above operating
conditions, sulbactam and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution according to the above operat-
ing conditions, the relative standard deviation of the peak
areas of sulbactam is not more than 2.0%.
Sulfamic acid (standard reagent) See amido sulfuric acid
(standard reagent).
Sulfanilamide H 2 NC 6 H 4 S0 2 NH 2 [K 9066, Special
class]
Sulfanilamide for titration of diazotization H 2 NC 6 H 4 -
S0 2 NH 2 [K 9066, For titration of diazotization]
Sulfanilic acid H 2 NC 6 H 4 S0 3 H [K 8586, Special class]
Sulfathiazole C9H 9 N 3 3 S 2 White crystalline powder.
Melting point <2.60>: 200 - 204°C
Sulfosalicylic acid See 5-sulfosalicylic acid dihydrate.
5-Sulfosalicylic acid dihydrate C 7 H 6 6 S.2H 2
[K 8589, Special class]
Sulfosalicylic acid TS Dissolve 5 g of 5-sulfosalicylic acid
dihydrate in water to make 100 mL.
Sulfur S [K 8088, Special class]
Sulfur dioxide S0 2 Prepare by adding sulfuric acid
dropwise to a concentrated solution of sodium bisulfite. Col-
orless gas, having a characteristic odor.
Sulfuric acid H 2 S0 4 [K 8951, Special class]
Sulfuric acid, dilute Cautiously add 5.7 mL of sulfuric
acid to 10 mL of water, cool, and dilute with water to make
100 mL (10%).
Sulfuric acid-ethanol TS With stirring, add slowly 3 mL
of sulfuric acid to 1000 mL of ethanol (99.5), and cool.
Sulfuric acid for readily carbonizable substances To sul-
furic acid, the content of which has previously been deter-
mined by the following method, add water cautiously, and
adjust the final concentration to 94.5% to 95.5% of sulfuric
acid (H 2 S0 4 ). When the concentration is changed owing to
absorption of water during storage, prepare freshly.
Assay — Weigh accurately about 2 g of sulfuric acid in a
glass-stoppered flask rapidly, add 30 mL of water, cool, and
titrate <2.50> the solution with 1 mol/L sodium hydroxide VS
(indicator: 2 to 3 drops of bromothymol blue TS).
Each mL of 1 mol/L sodium hydroxide VS
= 49.04 mg of H 2 S0 4
Sulfuric acid, fuming H 2 S0 4 .«S0 3
class]
[K 8741, Special
Sulfuric acid-hexane-methanol TS To 230 mL of a mix-
ture of hexane and methanol (1:3) add cautiously 2 mL of
sulfuric acid.
Sulfuric acid-methanol TS Prepare carefully by adding
60 mL of sulfuric acid to 40 mL of methanol.
Sulfuric acid-methanol TS, 0.05 mol/L Add gradually 3
mL of sulfuric acid to 1000 mL of methanol, while stirring,
and allow to cool.
Sulfuric acid-monobasic sodium phosphate TS See sul-
furic acid-sodium dihydrogenphosphate TS.
Sulfuric acid, purified Place sulfuric acid in a beaker,
heat until white fumes are evolved, then heat for 3 minutes
cautiously and gently. Use after cooling.
Sulfuric acid-sodium dihydrogenphosphate TS Add 6.8
mL of sulfuric acid to 500 mL of water, then dissolve 50 g of
sodium dihydrogenphosphate dihydrate in this solution, and
add water to make 1000 mL.
Sulfuric acid-sodium hydroxide TS With stirring add
slowly 120 mL of sulfuric acid to 1000 mL of water, and cool
(solution A). Dissolve 88.0 g of sodium hydroxide in 1000
mL of freshly boiled and cooled water (solution B). Mix e-
qual volumes of solution A and solution B.
Sulfuric acid TS Cautiously add 1 volume of sulfuric acid
to 2 volumes of water, and while warming on a water bath
add dropwise potassium permanganate TS until a pale red
color of the solution remains.
0.05 mol/L Sulfuric acid TS Dilute 100 mL of 0.5 mol/L
sulfuric acid TS with water to make 1000 mL.
0.25 mol/L Sulfuric acid TS With stirring, add slowly 15
mL of sulfuric acid to 1000 mL of water, then cool.
0.5 mol/L Sulfurie acid TS With stirring, add slowly 30
mL of sulfuric acid to 1000 mL of water, then cool.
2 mol/L Sulfuric acid TS To 1000 mL of water add grad-
ually 120 mL of sulfuric acid with stirring, and cool.
Sulfurous acid See sulfurous acid solution.
Sulfurous acid solution H 2 S0 3 [K 8058, Special class]
Sulpiride for assay C 15 H 23 N 3 4 S [Same as the mono-
graph Sulpiride. When dried, it contains not less than 99.0%
of sulpiride (C 15 H 23 N 3 4 S).]
JPXV
General Tests / Reagents, Test Solutions
249
Sulpyrine C 13 H 16 N 3 Na0 4 S.H 2 [Same as the mono-
graph Sulpyrine Hydrate]
Sulpyrine for assay [Same as the monograph Sulpyrine
Hydrate. Calculated on the dried basis, it contains not less
than 99.0% of sulpyrine (Ci 3 H 16 N 3 Na0 4 S).]
Suxamethonium chloride for thin-layer chromatogra-
phy C I4 H 30 Cl 2 N 2 O 4 .2H 2 O [Same as the monograph Sux-
amethonium Chloride Hydrate]
Swertiamarin for thin-layer chromatography C 16 H 22 O 10
White, practically tasteless powder.
Melting point <2.60>: 113 - 114°C
Purity Related substances — Dissolve 2.0 mg of swer-
tiamarin for thin-layer chromatography in exactly 1 mL of
ethanol (95), and perform the test with 20 /uL of this solution
as directed in the Identification under Swertia Herb: any spot
other than the principal spot at the Rf value of about 0.5 does
not appear.
Syntheric zeolite for drying A mixture of
6(Na 2 0).6(Al 2 3 ).12(Si0 2 ) and 6(K 2 0).6(Al 2 3 ).12(Si0 2 )
prepared for drying. Usually, use the spherically molded
form, 2 mm in diameter, prepared by adding a binder. White
to grayish white, or color transition by adsorbing water.
Average fine pore diameter is about 0.3 nm, and the surface
area is 500 to 700 m 2 per g.
Loss on ignition <2.43>: not more than 2.0% [2 g, 550 -
600°C, 4 hours, allow to stand in a desiccator (phosphorus
(V) oxide)].
Talc [Same as the namesake monograph]
Tannic acid [Same as the namesake monograph]
Tannic acid TS Dissolve 1 g of tannic acid in 1 mL of eth-
anol (95), and add water to make 10 mL. Prepare before use.
Tartaric acid See L-tartaric acid.
L-Tartaric acid C 4 H 6 6
Special class].
[K 8532, l( + )-Tartaric acid,
Tartrate buffer solution, pH 3.0 Dissolve 1.5 g of L-tar-
taric acid and 2.3 g of sodium tartarate dihydrate in water to
make 1000 mL.
Taurine H 2 NCH 2 CH 2 S0 3 H White crystals or crystal-
line powder.
Contents: not less than 95.0%. Assay — Weigh accurately
about 0.2 g, dissolve in 50 mL of water, add 5 mL of formal-
dehyde solution, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.52 mg of C 2 H 7 N0 3 S
Terephthalic acid C 6 H 4 (COOH) 2 White crystals or
crystalline powder. Slightly soluble in ethanol (95), and prac-
tically insoluble in water and ether.
Residue on ignition <2.44>: not more than 0.3% (1 g).
Content: not less than 95.0%. Assay — Weigh accurately
about 2 g of terephthalic acid, dissolve in exactly 50 mL of 1
mol/L sodium hydroxide VS, and titrate <2.50> with 1 mol/L
hydrochloric acid VS (indicator: 3 drops of phenolphthalein
TS). Perform a blank determination in the same manner.
Each mL of 1 mol/L sodium hydroxide VS
= 83.07 mg of C 8 H 6 4
Terephthalic acid for gas chromatography
C 6 H 4 (COOH) 2 Terephthalic acid prepared for gas chroma-
tography.
Terphenyl C 18 H 14 White crystalline powder.
Melting point <2.60>: 208 - 213°C
Identification — Determine the absorption spectrum of a
solution of terphenyl in methanol (1 in 250,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 276 and 280 nm.
p-Terphenyl See terphenyl.
Test bacteria inoculation medium for teceleukin Dissolve
6.0 g of peptone, 3.0 g of yeast extract, 1.5 g of meat extract,
1.0 g of glucose, and 13.0 to 20.0 g of agar in 1000 mL of
water and sterilize. The pH is 6.5 to 6.6.
Test bacteria inoculation medium slant for teceleukin
Sterilized slant culture obtained by adding approximately 9
mL of bacteria inoculation medium for teceleukin to a test
tube with an inside diameter of 16 mm.
Testosterone propionate C 22 H 32 3 [Same as the name-
sake monograph]
Test solution for water determination See the Water De-
termination <2.48>.
Tetrabromophenolphthalein ethyl ester potassium salt
C 22 H 13 4 Br 4 K [K 9042, Special class]
Tetrabromophenolphthalein ethyl ester TS Dissolve
0.1 g of tetrabromophenolphthalein ethyl ester potassium
salt in acetic acid (100) to make 100 mL. Prepare before use.
Tetra B-butylammonium bromide [CH 3 (CH 2 ) 3 ] 4 NBr
White, crystals or crystalline powder, having a slight, charac-
teristic odor.
Melting point <2.60>: 101 - 105°C
Purity Clarity and color of solution — Dissolve 1.0 g of
tetra M-butylammonium bromide in 20 mL of water: the solu-
tion is clear and colorless.
Content: not less than 98.0%. Assay— Dissolve about
0.5 g of tetra M-butylammonium bromide, accurately weighed,
in 50 mL of water, add 5 mL of dilute nitric acid, and titrate
<2.50> with 0.1 mol/L silver nitrate VS while strongly shaking
(potentiometric titration). Perform a blank determination
and make any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 32.24 mg of C 16 H 36 NBr
Tetra B-butylammonium chloride C 16 H 36 C1N White
crystals, and it is deliquescent.
Water <2.48>: not more than 6.0% (0.1 g).
Content: not less than 95.0%, calculated on the anhydrous
basis. Assay — Weigh accurately about 0.25 g of
tetra«-butylammoniumchloride,dissolvein50 mLof water, and
titrate <2.50> with 0.1 mol/L silver nitrate VS (potentiometric
titration).
Each mL of 0.1 mol/L silver nitrate VS
= 27.97 mg of C 16 H 36 C1N
Tetrabutylammonium hydrogensulfate C 16 H 37 N0 4 S
250
Reagents, Test Solutions / General Tests
JP XV
White crystalline powder.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.7 g of tetrabutylammonium hydrogensulfate, dis-
solve in 100 mL of freshly boiled and cooled water, and ti-
trate <2.50> with 0.1 mol/L sodium hydroxide VS (indicator:
3 drops of bromocresol green-methyl red TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 33.95 mg of C 16 H 37 N0 4 S
40% Tetrabutylammonium hydroxide TS A solution
containing 40 g/dL of tetrabutylammonium hydroxide
[(C 4 H 9 ) 4 NOH: 259.47].
Content: 36 -44 g/dL. Assay— Pipet 10 mL of 40%
tetrabutylammonium hydroxide TS, and titrate <2.50> with 1
mol/L hydrochloric acid VS (indicator: 3 drops of methyl red
TS).
Each mL of 1 mol/L hydrochloric acid VS
= 259.5 mg of C 16 H 37 NO
0.005 mol/L Tetrabutylammonium hydroxide TS To 10
mL of tetrabutylammonium hydroxide TS add 700 mL of
water, adjust to pH 4.0 with diluted phosphoric acid (1 in
10), and add water to make 1000 mL.
Tetrabutylammonium hydroxide-methanol TS Methanol
solution containing 25 g/dL of tetrabutylammonium hydrox-
ide [(C 4 H 9 ) 4 NOH: 259.47]. Colorless to pale yellow solu-
tion, having an ammonium-like odor.
Content: 22.5 - 27.5 g/dL. Assay— Pipet 15 mL of tetra-
butylammonium hydroxide-methanol TS and titrate <2.50>
with 1 mol/L hydrochloric acid VS (indicator: 3 drops of
methyl red TS).
Each mL of 1 mol/L hydrochloric acid VS
= 259.5 mg of C 16 H 37 NO
10% Tetrabutylammonium hydroxide-methanol TS
A methanol solution containing 10 g/dL of tetrabutylammo-
nium hydroxide [(C 4 H 9 ) 4 NOH: 259.47].
Content: 9.0 - 11.0 g/dL. Assay— Pipet 2 mL of 10%
tetrabutylammonium hydroxide-methanol TS, transfer to a
glass-stoppered flask containing 20 mL of water, and titrate
<2.50> with 0.1 mol/L hydrochloric acid VS (indicator: 3
drops of methyl red TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 25.95 mg of C 16 H 37 NO
Tetrabutylammonium hydroxide TS A solution contain-
ing 13 g/dL of tetrabutylammonium hydroxide [(C 4 H 9 ) 4
NOH: 259.47].
Content: 11.7 - 14.3 g/dL. Assay — Pipet a quantity,
equivalent to about 0.3 g of tetrabutylammonium hydroxide
[(C 4 H 9 ) 4 NOH], transfer to a glass-stoppered flask containing
15 mL of water, accurately weighed, and titrate <2.50> with
0.1 mol/L hydrochloric acid VS (indicator: 3 drops of methyl
red TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 25.95 mg of C 16 H 37 NO
Tetrabutylammonium phosphate (C 4 H 9 ) 4 NH 2 P0 4
White powder. It is soluble in water.
Content: not less than 97.0%. Assay — Weigh accurately
1.5 g of tetrabutylammonium phosphate, dissolve in 80 mL
of water, and titrate <2.50> with 0.5 mol/L sodium hydroxide
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.5 mol/L sodium hydroxide VS
= 169.7 mg of (C 4 H 9 ) 4 NH 2 P0 4
Tetracycline C 22 H 24 N 2 8 Yellow to dark yellow, crys-
tals or crystalline powder. Sparingly soluble in ethanol, and
very slightly soluble in water.
Content: it contains not less than 870 fig (potency) per mg.
Assay — Proceed as directed in the Assay under Tetracycline
Hydrochloride. However, use the following formula.
Amount \pg (potency)] of tetracycline (C 22 H 24 N 2 8 )
= W s x(A T /A s )x\0Q0
W s : Amount [mg (potency)] of Tetracycline Hydrochlo-
ride Reference Standard
Tetracycline Hydrochloride C22H 24 N 2 8 .HC1 Yellow,
crystals or crystalline powder.
Purity Related substances — Dissolve 20 mg of tetracy-
cline hydrochloride in 25 mL of 0.01 mol/L hydrochloric
acid TS, and use this solution as the sample solution. Proceed
the test with 20 /xL of the sample solution as directed in the
Purity (2) under Oxytetracycline Hydrochloride, determine
each peak area by the automatic integration method, and cal-
culate the amounts of them by the area percentage method:
the total amount of the peaks other than tetracycline is not
more than 10%.
Tetraethylammonium hydroxide TS A solution contain-
ing 10% of tetraethylammonium hydroxide [(C 2 H 5 ) 4 NOH:
147.26]. A clear, colorless liquid, having a strong ammonia
odor. It is a strong basic and easily absorbs carbon dioxide
from the air.
Content: 10.0 - 11.0% Assay — Weigh accurately about 3 g
in a glass-stoppered flask containing 15 mL of water, and ti-
trate <2.50> with 0.1 mol/L hydrochloric acid VS (indicator:
3 drops of methyl red TS). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L hydrochloric acid VS
= 14.73 mg of C 8 H 2 iNO
Tetra-n-heptylammonium bromide [CH 3 (CH 2 )6] 4 NBr
White, crystals or crystalline powder, having a slight, charac-
teristic odor.
Melting point <2.60>: 87 - 89°C.
Content: not less than 98.0%. Assay — Dissolve about 0.5
g of tetra-M-heptylammonium bromide, accurately weighed,
in 50 mL of diluted acetonitrile (3 in 5), and 5 mL of dilute
nitric acid, and titrate <2.50> with 0.1 mol/L silver nitrate VS
while strongly shaking (potentiometric titration). Perform a
blank determination and make any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 49.07 mgC 28 H 60 NBr
Tetrahydrofuran CH 2 (CH 2 ) 2 CH 2 [K 9705, Special
class]
Tetrahydrofuran for gas chromatography Use tetra-
hydrofuran prepared by distilling with iron (II) sulfate hepta-
hydrate.
Storage — Preserve in containers, in which the air has been
displaced by nitrogen, in a dark, cold place.
Tetrahydrofuran for liquid chromatography C 4 H 8
JP XV
General Tests / Reagents, Test Solutions
251
Clear and colorless liquid.
Density <2.56> (20°C): 0.884 - 0.889 g/mL
Refractive index <2.45> n™: 1.406 - 1.409
Purity Ultraviolet absorbing substances — Determine the
absorption spectrum of tetrahydrofuran for liquid chro-
matography as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using water as the blank: the absor-
bences at 240 nm, 254 nm, 280 nm, 290 nm, and between 300
nm and 400 nm are not more than 0.35, 0.20, 0.05, 0.02 and
0.01, respectively.
Peroxide — Perform the test according to the method de-
scribed in JIS K 9705: not more than 0.01%.
Tetrahydroxyquinone C 6 H 4 6 Dark blue crystals. Its
color changes to yellow on exposure to light. Soluble in etha-
nol (95) and sparingly soluble in water.
Tetrahydroxyquinone indicator Mix 1 g of tetrahydro-
xyquinone with 100 g of sucrose homogeneously.
Tetrakishydroxypropylethylenediamine for gas chro-
matography Prepared for gas chromatography.
Tetramethylammonium hydroxide (CH 3 ) 4 NOH Ordi-
narily, available as an approximately 10% aqueous solution,
which is clear and colorless, and has a strong ammonia-like
odor. Tetramethylammonium hydroxide is a stronger base
than ammonia, and rapidly absorbs carbon dioxide from the
air. Use a 10% aqueous solution.
Purity Ammonia and other amines — Weigh accurately a
quantity of the solution, corresponding to about 0.3 g of
tetramethylammonium hydroxide [(CH 3 ) 4 NOH], in a weigh-
ing bottle already containing 5 mL of water. Add a slight ex-
cess of 1 mol/L hydrochloric acid TS (about 4 mL), and
evaporate on a water bath to dryness. The mass of the residue
(tetramethylammonium chloride), dried at 105 °C for 2 hours
and multiplied by 0.8317, represents the quantity of
tetramethylammonium hydroxide [(CH 3 ) 4 NOH], and cor-
responds to ±0.2% of that found in the Assay.
Residue on evaporation: not more than 0.02% (5 mL,
105°C, 1 hour).
Content: not less than 98% of the labeled amount. As-
say — Accurately weigh a glass-stoppered flask containing
about 15 mL of water. Add a quantity of the solution,
equivalent to about 0.2 g of tetramethylammonium
hydroxide [(CH 3 ) 4 NOH], weigh again, and titrate <2.50> with
0.1 mol/L hydrochloric acid VS (indicator: methyl red TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 9.115 mg of C 4 H 13 NO
Tetramethylammonium hydroxide-methanol TS A meth-
anol solution containing of 10 g/dL of tetramethylammoni-
um hydroxide [(CH 3 ) 4 NOH: 91.15]
Content: 9.0 - 11.0 g/dL. Assay— Pipet 2 mL of tetra-
methylammonium hydroxide-methanol TS, transfer to a
glass-stoppered flask containing 20 mL of water, and titrate
<2.50> with 0.1 mol/L hydrochloric acid VS (indicator: bro-
mocresol green-methyl red TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 9.115 mg of C 4 H 13 NO
Tetramethylammonium hydroxide TS Pipet 15 mL of
tetramethylammonium hydroxide, and add dehydrated etha-
nol (99.5) to make exactly 100 mL.
Tetramethylammonium hydroxide TS, pH 5.5 To 10 mL
of tetramethylammonium hydroxide add 990 mL of water,
and adjust the pH to 5.5 with diluted phosphoric acid (1 in
10).
3,3',5,5'-Tetramethylbenzidine dihydrochlorate dihydrate
Ci 6 H 22 Cl 2 N 2 .H 2 White to slightly reddish-white crystal-
line powder.
■/VjA^/V^/V-Tetramethylethylenediamine
(CH 3 ) 2 NCH 2 CH 2 N(CH 3 ) 2 Pale yellow clear liquid.
Specific gravity <2.56> df: 0.774 - 0.799
Content: not less than 99.0%.
Tetramethylsilane for nuclear magnetic resonance spectro-
scopy (CH 3 ) 4 Si Prepared for nuclear magnetic resonance
spectroscopy.
Tetra n-pentylammoniuin bromide [CH 3 (CH 2 ) 4 ] 4 NBr
White, crystals or crystalline powder. It is hygroscopic.
Melting point <2.60>: 100 - 101 °C
Tetraphenylboron potassium TS Add 1 mL of acetic acid
(31) to a solution of potassium biphthalate (1 in 500), then to
this solution add 20 mL of a solution of tetraphenylboron so-
dium (7 in 1000), shake well, and allow to stand for 1 hour.
Collect the produced precipitate on filter paper, and wash it
with water. To 1 /3 quantity of the precipitate add 100 mL of
water, warm, with shaking, at about 50°C for 5 minutes, cool
quickly, allow to stand for 2 hours with occasional shaking,
and filter, discarding the first 30 mL of the filtrate.
Tetraphenylboron sodium See sodium tetraphenyl-
borate.
Tetra-B-propylammonium bromide
[CH 3 CH 2 CH 2 ] 4 NBr White, crystals or crystalline powder.
Purity Clarity and color of solution — Dissolve 1.0 g of
tetra-M-propylammonium bromide in 20 mL of water: the so-
lution is clear and colorless.
Content: not less than 98.0%. Assay — Weigh accurately
about 0.4 g of tetra-M-propylammonium bromide, dissolve in
50 mL of water, add 5 mL of dilute nitric acid, and titrate
<2.50> with 0.1 mol/L silver nitrate VS while shaking strongly
(potentiometric titration).
Each mL of 0.1 mol/L silver nitrate VS
= 26.63 mg of C 12 H 28 NBr
Theophylline C 7 H 8 N 4 2 White powder. Slightly solu-
ble in water.
Melting point <2.60>: 269 - 274°C
Purity Caffeine, theobromine or paraxanthine — To 0.20
g of theophylline add 5 mL of potassium hydroxide TS or 5
mL of ammonia TS: each solution is clear.
Loss on drying <2.41>: not more than 0.5% (1 g, 105°C, 4
hours).
Content: not less than 99.0%. Assay — Weigh accurately
about 0.25 g of theophylline, previously dried, dissolve it in
40 mL of A^Af-dimethylformamide, and titrate <2.50> with
0.1 mol/L sodium methoxide VS (indicator: 3 drops of thy-
mol blue-/V,A r -dimethylformamide TS). Perform a blank de-
termination and make any necessary correction.
Each mL of 0.1 mol/L sodium methoxide VS
= 18.02 mg of C 7 H 8 N 4 2
Thiamine nitrate C 12 H 17 N 5 4 S [Same as the namesake
monograph]
252
Reagents, Test Solutions / General Tests
JP XV
Thianthol [Same as the monograph Thianthol. Proceed
as directed in the Identification (3) under Sulfur, Salicylic
Acid and Thianthol Ointment: any spot other than the prin-
cipal spot does not appear.]
3-Thienylethylpenicillin sodium Ci4H 15 N 2 Na04S2
White to pale yellowish white powder. Very soluble in water,
freely soluble in methanol, and sparingly soluble in ethanol
(95).
Optical rotation <2.49> [a]™: +265 - +290° (0.5 g calcu-
lated on the anhydrous bases, water, 50 mL, 100 mm).
Water <2.48>: Not more than 10.0% (0.2 g, volumetric
titration, direct titration).
Content: not less than 90% calculated on the anhydrous
basis. Assay — Weigh accurately about 0.1 g of 3-
thienylethylpenicillin sodium, dissolve in 35 mL of water,
add 0.75 mL of 0.1 mol/L hydrochloric acid TS, and adjust
to pH 8.5 with 0.1 mol/L sodium hydroxide TS. To this solu-
tion add 2 mL of a penicillinase solution prepared by dissolv-
ing penicillinase, equivalent to 513,000 Levy units, in 25 mL
of water and neutralizing with dilute sodium hydroxide TS
until a pale red color appears with 1 drop of a solution of
phenolphthalein in ethanol (95) (1 in 1000) as indicator, and
allow to stand at 25°C for 5 minutes. Titrate <2.50> this solu-
tion with 0.1 mol/L sodium hydroxide VS until the solution
reaches to pH 8.5 (potentiometric titration). Use the water
freshly boiled and cooled.
Each mL of 0.1 mol/L sodium hydroxide VS
= 36.24 mg of C 14 H 15 N 2 Na04S 2
Thimerosal C 9 H 9 HgNa0 2 S White or yellowish crystal-
line powder. Freely soluble in water.
Melting point <2.60>: 107 - 114°C.
Thioacetamide C 2 H 5 NS A white crystalline powder or
colorless crystals, having a characteristic odor. Freely soluble
in water and in ethanol (99.5). Melting point: 112 - 115°C
Thioacetamide-alkaline glycerin TS To 0.2 mL of thioa-
cetamide TS add 1 mL of alkaline glycerin TS, and heat for
20 minutes in a water bath. Prepare before use.
Thioacetamide TS To 0.2 mL of a solution of thioa-
cetamide (1 in 25) add 1 mL of a mixture of 15 mL of sodium
hydroxide TS, 5 mL of water and 20 mL of 85% glycerin,
and heat in a water bath for 20 seconds. Prepare before use.
Thiodiglycol S(CH 2 CH 2 OH) 2 [/?-Thiodiglycol for ami-
no acid autoanalysis] Colorless or pale yellow, clear liquid.
Specific gravity <2.56> df : 1.180 - 1.190
Water <2.48>: not more than 0.7%.
Thioglycolate medium I for sterility test See fluid
thioglycolate medium.
Thioglycolate medium II for sterility test See alternative
thioglycolate medium.
Thioglycolic acid See mercapto acetic acid.
Thionyl chloride SOCl 2 A colerless or light yellow,
clear liquid, having a pungent odor.
Specific gravity <2.56> df a : about 1.65 (Method 3)
Content: not less than 95.0%. Assay — Weigh accurately
0.1 g of thionyl chloride in a weighing bottle, put the bottle in
a glass-stoppered conical flask containing 50 mL of water
cooled to about 5°C, stopper immediately, dissolve the sam-
ple thoroughly, and transfer the solution to a 200-mL beaker.
Wash the conical flask and the weighing bottle in it with 30
mL of water, and combine the washings and the solution in
the beaker. Add 1 drop of an aqueous solution of polyvinyl
alcohol (100 g/L), and titrate <2.50> with 0.1 mol/L silver ni-
trate VS (potentiometric titration). Perform a blank determi-
nation in the same manner, and make any necessary correc-
tion.
Each mL of 0.1 mol/L silver nitrate VS
= 5.949 mg of SOCl 2
Thiopental for assay CnH 18 N 2 2 S Dissolve 10 g of
Thiopental Sodium in 300 mL of water. To this solution add
slowly 50 mL of dilute hydrochloric acid with stirring. Take
the produced crystals by filtration, wash with water until the
filtrate indicates no reaction to chloride, and air-dry. Add
diluted ethanol (99.5) (3 in 5), dissolve by heating in a water
bath, allow to stand, and take the produced crystals by filtra-
tion. Air-dry the crystals in air, and dry again at 105°C for 4
hours. White, odorless crystals.
Melting point <2.60>: 159 - 162°C
Purity (1) Clarity and color of solution — Dissolve 1.0 g
of thiopental for assay in dehydrated ethanol: the solution is
clear and light yellow.
(2) Related substances — Dissolve 0.05 g of thiopental for
assay in 15 mL of acetonitrile, add water to make 50 mL, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase in the Purity (4) under
Thiopental Sodium to make exactly 200 mL, and use this so-
lution as the standard solution. Proceed as directed in Purity
(4) under Thiopental Sodium.
Loss on drying <2.41>: not more than 0.20% (1 g, 105°C, 3
hours).
Content: not less than 99.0%. Assay — Weigh accurately
about 0.35 g of thiopental for assay, previously dried, dis-
solve in 5 mL of dehydrated ethanol and 50 mL of chloro-
form, and titrate <2.50> with 0.1 mol/L potassium hydrox-
ide-ethanol VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 24.23 mg of C„H I8 N 2 2 S
Thiosemicarbazide H 2 NCSNHNH 2 [K 8632, Special c-
lass]
Thiourea H 2 NCSNH 2 [K 8635, Special class]
Thiourea TS Dissolve 10 g of thiourea in water to make
100 mL.
L-Threonine C4H9NO3
graph]
[Same as the namesake mono-
Threoprocaterol hydrochloride Ci 6 H 22 N 2 3 .HCl To
procaterol hydrochloride add 10 volumes of 3 mol/L
hydrochloric acid TS, heat, and reflux for 3 hours. After
cooling, neutralize (pH 8.5) with sodium hydroxide TS, and
collect the crystals produced. Suspend the crystals in water,
dissolve by acidifying the solution at pH 1 to 2 with addition
of hydrochloric acid, neutralize (pH 8.5) by adding sodium
hydroxide TS, and separate the crystals produced. Suspend
the crystals in 2-propanol, and acidify the solution at pH 1 to
2 by adding hydrochloric acid. The crystals are dissolved and
reproduced. Collect the crystals, dry at about 60°C while
passing air. White to pale yellowish white, odorless crystals
JPXV
General Tests / Reagents, Test Solutions
253
or crystalline powder. Melting point: about 207 °C (with
decomposition).
Purity — Dissolve 0.10 g of threoprocaterol hydrochloride
in 100 mL of diluted methanol (1 in 2), and use this solution
as the sample solution. Perform the test with 2 /xh of the sam-
ple solution as directed under Liquid Chromatography
according to the operating conditions in the Purity (3) under
Procaterol Hydrochloride Hydrate. Measure each peak area
by the automatic integration method, and calculate the
amount of threoprocaterol by the area percentage method: it
shows the purity of not less than 95.0%. Adjust the detection
sensitivity so that the peak height of threoprocaterol obtained
from 2 /uL of the solution prepared by diluting 5.0 mL of the
sample solution with diluted methanol (1 in 2) to make 100
mL, is 5 to 10% of the full scale, and the time span of meas-
urement is about twice as long as the retention time of
threoprocaterol beginning after the peak of solvent.
Thrombin [Same as the namesake monograph]
Thymine C 5 H 6 N 2 2 : 126.11
Identification — Determine the infrared absorption spec-
trum of thymine, previously dried at 105°C for 3 hours, as
directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3030 cm -1 , 1734 cm -1 , 1676
cm -1 , 1446 cm - ' and 814 cm -1 .
Purity Related substances — Dissolve 50 mg of thymine in
100 mL of methanol. To 10 mL of this solution add the mo-
bile phase to make 100 mL, and use this solution as the sam-
ple solution. Proceed with 10 fiL of the sample solution as
directed in the Purity (3) under Aceglutamide Aluminum:
any peak does not appear at the retention time of
aceglutamide.
Thymol CH 3 C 6 H 3 (OH)CH(CH 3 ) 2 [Same as the name-
sake monograph]
Thymol blue C 27 H 30 O 5 S [K 8643, Special class]
Thymol bIue-iV,./V-dimethylformamide TS Dissolve 0.1 g
of thymol blue in 100 mL of A^iV-dimethylformamide.
Thymol blue-dioxane TS Dissolve 0.05 g of thymol blue
in 100 mL of 1,4-dioxane, and filter if necessary. Prepare be-
fore use.
Thymol blue TS Dissolve 0. 1 g of thymol blue in 100 mL
of ethanol (95), and filter if necessary.
Thymol blue TS, dilute Dissolve 0.05 g of thymol blue in
100 mL of ethanol (99.5), and filter if necessary. Prepare be-
fore use.
Thymol for assay [Same as the monograph Thymol. It
contains not less than 99.0% of thymol (C 10 H 14 O).]
Thymolphthalein C28H 30 O 4 [K 8642, Special class]
Thymolphthalein TS Dissolve 0.1 g of thymolphthalein
in 100 mL of ethanol (95), and filter if necessary.
Tiaramide hydrochloride for assay C 15 H I8 C1N 3 3 S-HC1
[Same as the monograph Tiaramide Hydrochloride. When
dried, it contains not less than 99.0% of tiaramide
hydrochloride (C 15 H 18 C1N 3 3 S.HC1).]
Tin Sn [K 8580, Special class]
Tin (II) chloride dihydrate SnCl 2 .2H 2 [K 8136, Spec-
ial class]
Tin (II) chloride-hydrochloric acid TS To 20 g of tin add
85 mL of hydrochloric acid, heat until hydrogen gas no lon-
ger are evolved, and allow to cool. Mix 1 volume of this solu-
tion and 10 volume of dilute hydrochloric acid. Prepare be-
fore use.
Tin (II) chloride-sulfuric acid TS Dissolve 10 g of tin (II)
chloride dihydrate in diluted sulfuric acid (3 in 200) to make
100 mL.
Tin (II) chloride TS Dissolve 1.5 g of Tin (II) chloride di-
hydrate in 10 mL of water containing a small amount of hy-
drochloric acid. Preserve in glass-stoppered bottles in which a
fragment of tin has been placed. Use within 1 month.
Tin (II) chloride TS, acidic Dissolve 8 g of Tin (II) chlo-
ride dihydrate in 500 mL of hydrochloric acid. Preserve in
glass-stoppered bottles. Use within 3 months.
Tipepidine hibenzate for assay Ci 5 H I7 NS 2 .C 14 H 10 O4
[Same as the monograph Tipepidine Hibenzate.
When dried, it contains not less than 99.0% of
C 15 H 17 NS 2 .C 14 H 10 O4.]
Titanium dioxide See titanium (IV) oxide.
Titanium dioxide TS See titanium (IV) oxide TS.
Titanium (III) chloride (20) TiCl 3 [K 8401, Titanium
(III) chloride solution, Special class] Store in light-resistant,
glass-stoppered containers.
Titanium (III) chloride-sulfuric acid TS Mix carefully 20
mL of titanium (III) chloride TS and 13 mL of sulfuric acid,
add carefully hydrogen peroxide (30) in small portions until a
yellow color develops, and heat until white fumes evolve. Af-
ter cooling, add water, heat again in the same manner, repeat
this procedure until the solution is colorless, and add water to
make 100 mL.
Titanium (III) chloride TS To titanium (III) chloride (20)
add dilute hydrochloric acid to obtain a solution containing
15g/dL of titanium (III) chloride (TiCl 3 ). Prepare before
use.
Content: 14.0 - 16.0 g/dL. Assay— To exactly 2 mL of
titanium (III) chloride add 200 mL of water and 5 mL of a
hydrochloric acid solution (2 in 3), and titrate <2.50> with 0.1
mol/L ferric ammonium sulfate VS under carbon dioxide un-
til a slight red color develops in the solution (indicator: 5 mL
of ammonium thiocyanate TS).
Each mL of 0.1 mol/L ferric ammonium sulfate VS
= 15.42 mg of TiCl 3
Titanium (IV) oxide Ti0 2 [K 8703, Special class]
Titanium (IV) oxide TS To 100 mL of sulfuric acid add
0.1 g of titanium (IV) oxide, and dissolve by gradually heat-
ing on a flame with occasional gentle shaking.
Titanium trichloride See titanium (III) chloride.
Titanium trichloride-sulfuric acid TS See titanium (III)
chloride-sulfuric acid TS.
Titanium trichloride TS See titanium (III) chloride TS.
Titanium yellow C 28 H 19 N 5 Na 2 6 S 4 [K 8639, Special
class]
254
Reagents, Test Solutions / General Tests
JP XV
Tocopherol C29H50O2 [Same as the namesake mono-
graph]
Tocopherol acetate C31H52O3 [Same as the namesake
monograph]
Tocopherol calcium succinate C 66 Hi 6CaOio [Same as
the namesake monograph]
Tocopherol succinate C33H54O5 Wet 0.5 g of tocoph-
erol calcium succinate with 5 mL of acetic acid (100), add 10
mL of toluene, and warm at 70°C for 30 minutes with oc-
casional shaking. After cooling, add 30 mL of water, shake
thoroughly, and allow to stand. Remove the water layer,
wash the toluene layer with several 30-mL portions of water
until the washings become neutral, and allow to stand. Shake
the toluene extract with 3 g of anhydrous sodium sulfate,
decant the toluene layer, distil the toluene under reduced
pressure, and obtain a light yellow, viscous liquid. When
preserved at room temperature for a long time, it becomes a
pale yellowish solid.
Absorbance <2.24> E\ v ° m (286 nm): 38.0-42.0 (10 mg,
chloroform, 100 mL).
Tolbutamide C 12 H 18 N 2 3 S [Same as the namesake
monograph]
Toluene C 6 H 5 CH 3 [K 8680, Special class]
o-Toluene sulfonamide C 7 H 9 N0 2 S Colorless crystals or
white crystalline powder. Soluble in ethanol (95), and spar-
ingly soluble in water.
Melting point <2.60>: 157 - 160°C
Purity /^-Toluene sulfonamide — Use a solution of o-
toluene sulfonamide in ethyl acetate (1 in 5000) as the sample
solution. Perform the test with 10,mL of the sample solution
as directed under Gas Chromatography <2.02> according to
the operating conditions in the Purity (6) under Saccharin So-
dium Hydrate: any peak other than the peak of o-toluene sul-
fonamide does not appear. Adjust the flow rate so that the
retention time of o-toluene sulfonamide is about 10 minutes,
and adjust the detection sensitivity so that the peak height of
o-toluene sulfonamide obtained from 10 /uL of the sample so-
lution is about 50% of the full scale. Time span of measure-
ment is about twice as long as the retention time of o-toluene
sulfonamide beginning after the solvent peak.
Water <2.48>: not more than 0.5% (4g, use 25 mL of
methanol for Karl Fischer method and 5 mL of pyridine for
Karl Fischer method).
Content: not less than 98.5%, calculated on the anhy-
drousbasis. Assay — Weighaccuratelyabout0.025 gofo-tol-
uene sulfonamide, and perform the test as directed under Ni-
trogen Determination <I.08>.
Each mL of 0.005 mol/L sulfuric acid VS
= 1.712 mg of C 7 H 9 N0 2 S
p-Toluene sulfonamide CH 3 C 6 H 4 S0 2 NH 2 White, crys-
tals or crystalline powder. Melting point: about 137°C
Purity Related substances — Dissolve 30 mg of p-toluene
sulfonamide in acetone to make exactly 200 mL. Proceed
with 10 /uL of this solution as directed in the Purity (3) under
Tolazamide: any spot other than the principal spot at the Rf
value of about 0.6 does not appear.
p-Toluene sulfonic acid See p-toluenesulfonic acid
monohydrate.
p-Toluenesulfonic acid monohydrate
CH 3 C 6 H 4 S03H.H 2 [K 8681, Special class]
o-Toluic acid C 8 H 8 2 White, crystals or crystalline
powder.
Melting point <2.60>: 102 - 105°C
Content: not less than 98.0%.
Toluidine blue See toluidine blue O
Toluidine blue O C 15 H I6 C1N 3 S Dark green powder,
soluble in water, and slightly soluble in ethanol (95).
Identification —
(1) A solution (1 in 100) shows a blue to purple color.
(2) A solution in ethanol (95) (1 in 200) shows a blue
color.
(3) A solution shows a maximum absorption at around
630 nm.
Triamcinolone acetonide C 24 H 31 F0 6 [Same as the
namesake monograph]
Trichloroacetic acid CCI3COOH [K 8667, Special
class]
Trichloroacetic acid-gelatin-tris buffer solution To 1
volume of a solution of trichloroacetic acid (1 in 5) add 6
volume of gelatin-tris buffer solution, pH 8.0 and 5 volume
of water.
Trichloroacetic acid TS Dissolve 1.80 g of trichloroacetic
acid, 2.99 g of sodium acetate trihydrate and 1.98 g of acetic
acid (31) in water to make 100 mL.
Trichloroacetic acid TS for serrapeptase Dissolve 1.80 g
of trichloroacetic acid and 1.80 g of anhydrous sodium
acetate in 5.5 mL of 6 mol/L acetic acid TS and water to
make 100 mL.
Trichlorofluoromethane CC1 3 F A colorless liquid or
gas.
Boiling point <2.57>: 23.7°C
Specific gravity <2.56> dl 1 - 2 : 1.494
l,l,2-Trichloro-l,2,2-trifluoroethane CFC1 2 .CF 2 C1
Colorless volatile liquid. Miscible with acetone and with
diethyl ether, and not with water.
Purity Related substances — Perform the test with 0.1 juL
of l,l,2-trichloro-l,2,2-trifluroethane as directed under Gas
Chromatography <2.02> according to the operating condi-
tions in the Purity (5) under Halothane: any peak other than
the peak of l,l,2-trichloro-l,2,2-trifluoroethane does not ap-
pear.
Tricine C 6 H 13 N0 5 White crystalline powder. Melting
point: 182 to 184°C (with decomposition).
Triethanolamine See 2,2',2"-nitrilotrisethanol.
Triethylamine (C 2 H 5 ) 3 N Clear colorless liquid, having a
strong amines odor. Miscible with methanol, with ethanol
(95) and with diethyl ether.
Melting point <2.60>: 89 - 90°C
Specific gravity <2.56> df: 0.722 - 0.730
Triethylamine buffer solution, pH 3.2 To 4 mL of
triethylamine add 2000 mL of water, and adjust the pH to 3.2
JPXV
General Tests / Reagents, Test Solutions
255
with phosphoric acid.
Triethylamine-phosphate buffer solution, pH 5.0 To 1.0
mL of triethylamine add 900 mL of water, adjust the pH to
5.0 with diluted phosphoric acid (1 in 10), and add water to
make 1000 mL.
Trifluoroacetic acid CF3COOH Colorless, clear liquid,
having a pungent odor. Miscible well with water.
Boiling point <2.57>: 72 - 73°C
Specific gravity <2.56> d 2 2 ° : 1.535
Trifluoroacetic acid for nuclear magnetic resonance spec-
troscopy CF3COOH Prepared for nuclear magnetic
resonance spectroscopy.
Trifluoroacetic acid TS To 1 mL of trifluoroacetic acid
add water to make 1000 mL.
Trifluoroacetic anhydride for gas chromatography
(CF 3 CO) 2 Colorless, clear liquid, having a pungent odor.
Boiling point <2.57>: 40 - 45°C
Trimetazidine hydrochloride for assay C14H22N2O3.2HCI
[Same as the monograph Trimetazidine Hydrochloride. It
contains not less than 99.0% of trimetazidine hydrochloride
(Q4H22N2O3.2HCI), calculated on the anhydrous basis.]
Trimethylsilyl imidazole C 6 H 12 N 2 Si Clear, colorless
to pale yellow liquid.
Refractive index <2.45> n™: 1.4744 - 1.4764
2,4,6-Trinitrobenzenesulfonic acid
C 6 H2(N02)3S03H.2H 2 Pale yellow to light yellow pow-
der.
Water <2.48>: 11 - 15% (0.1 g, volumetric titration, direct
titration).
Content: not less than 98%, calculated on the anhydrous
basis. Assay — Weigh accurately about 0.3 g of 2,4,6-
trinitrobenzenesulfonic acid, dissolve in 50 mL of a mixture
of water and ethanol (99.5) (1:1), and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (potentiometric titration). Per-
form a blank determination and make any necessary correc-
tion.
Each mL of 0.1 mol/L sodium hydroxide VS
= 29.32 mg of QH^NC^SOjH
2,4,6-Trinitrophenol HOC 6 H 2 (N0 2 ) 3 Light yellow to
yellow, moist crystals. It is added 15 to 25% of water for the
sake of safety, because it might explode by heating, mechani-
cal shocking and friction when it is dried.
Identification — To 0.1 g add 10 mL of water, dissolve by
warming, and add 12 mL of a mixture of 1% copper (II) sul-
fate solution and ammonia TS (5:1): green precipitates ap-
pear.
Content: not less than 99.5%. Assay — Weigh accurately
about 0.25 g, previously dried in a desiccator (silica gel) for
24 hours, dissolve in 50 mL of water by warming, and titrate
<2.50> with 0.1 mL sodium hydroxide VS (indicator: 3 drops
of phenolphthalein TS). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 22.91 mg of HOC 6 H 2 (N0 2 )3
2,4,6-Trinitrophenol-ethanol TS Dissolve 1.8 g of 2,4,6-
trinitrophenol in 50 mL of diluted ethanol (99.5) (9 in 10) and
30 mL of water, and add water to make 100 mL.
2,4,6-Trinitrophenol TS Dissolve 1 g of 2,4,6-trinitro-
phenol in 100 mL of hot water, cool, and filter if necessary.
2,4,6-Trinitrophenol TS, alkaline Mix 20 mL of 2,4,6-
trinitrophenol TS with 10 mL of a solution of sodium hy-
droxide (1 in 20), and add water to make 100 mL. Use within
2 days.
Triphenylchloromethane (Cgl-L^CCl White to grayish
or yellowish white, crystals or crystalline powder.
Melting point <2.60>: 107 - 115°C
Triphenyltetrazolium chloride See 2,3,5-triphenyl-2//-
tetrazolium Chloride.
Triphenyltetrazolium chloride TS See 2,3,5-triphenyl-
2//-tetrazolium chloride TS.
2,3,5-TriphenyI-2//-tetrazolium chloride Ci9H 15 ClN 4
[K 8214, Special class]
2,3,5-TriphenyI-2//-tetrazolium chloride TS Dissolve
0.25 g of 2,3,5-triphenyl-2//-tetrazolium chloride in ethanol
(99.5) to make 100 mL. Prepare before use.
Tripotassium citrate monohydrate C6H5K3O7.H2O
White crystals or crystalline powder. Very soluble in water,
and practically insoluble in ethanol (95).
Content: 99.0% or more Assay — Accurately weigh about
0.2 g of tripotassium citrate monohydrate, add 50 mL of
acetic acid for nonaqueous titration, dissolve by warming on
a water bath, cool, and then titrate <2.50> with 0.1 mol/L of
perchloric acid VS (potentiometric titration). Correct by con-
ducting a blank test using the same method.
Each mL of 0.1 mol/L perchloric acid VS
= 32.44 mg of C 6 H 5 K30 7 .H 2
Tris-acetic acid buffer solution, pH 6.5 Dissolve 13.57 g
of 2-amino-2-hydroxymethyl-l,3-propanediol and 6.73 g of
acetic acid (100) in water to make 1000 mL.
0.5 mol/L Tris buffer solution, pH 6.8 Dissolve 6 g of 2-
amino-2-hydroxymethyl-l,3-propanediol in 50 mL of water,
add 2 mol/L hydrochloric acid TS to adjust the pH to 6.8,
and then add water to make 100 mL. Filter if necessary.
Tris buffer solution, pH 7.0 Dissolve 24.3 g of 2-amino-
2-hydroxymethyl-l,3-propanediol in 1000 mL of water,
and adjust the pH to 7.0 with 0.1 mol/L hydrochloric acid
TS.
0.05 mol/L Tris buffer solution, pH 7.0 Dissolve 6.06 g
of 2-amino-2-hydroxymethyl-l,3-propanediol in about 750
mL of water, adjust to pH 7.0 with 1 mol/L hydrochloric
acid TS, and add water to make 1000 mL.
0.1 mol/L Tris buffer solution, pH 8.0 Dissolve 2.42 g of
2-amino-2-hydroxymethyl-l,3-propanediol in 100 mL of
water, adjust the pH to 8.0 with 0.2 mol/L hydrochloric acid
TS, and add water to make 200 mL.
Tris buffer solution, pH 8.2 Dissolve 24.2 g of 2-amino-
2-hydroxymethyl-l,3-propanediol and 0.5 g of polysorbate
20 in 800 mL of water, adjust to pH 8.2 with 1 mol/L
hydrochloric acid TS, and add water to make 1000 mL.
Tris buffer solution, pH 8.4 Dissolve 6.1 g of 2-amino-2-
hydroxymethyl-l,3-propanediol and 10.2 g of sodium chlo-
256
Reagents, Test Solutions / General Tests
JP XV
ride in 800 mL of water, adjust to pH 8.4 with 1 mol/L
hydrochloride TS, and add water to make 1000 mL.
0.05 mol/L Tris buffer solution, pH 8.6 Dissolve 6.1 g of
2-amino-2-hydroxymethyl-l,3-propanediol in 950 mL of
water, add 2 mol/L hydrochloric acid TS to adjust the pH to
8.6, and then add water to make 1000 mL.
Tris buffer solution, pH 8.8 Dissolve 18.2 g of 2-amino-
2-hydroxymethyl-l,3-propanediol in 75 mL of water, add 5
mol/L hydrochloric acid TS to adjust the pH to 8.8, and then
add water to make 100 mL. Filter if necessary.
Tris buffer solution, pH 9.5 Dissolve 36.3 g of 2-amino-
2-hydroxymethyl-l,3-propanediol in 1000 mL of water,
and adjust the pH to 9.5 by adding 1 mol/L hydrochloric
acid TS.
Tris buffer solution for bacterial endotoxins test Dissolve
18.2 g of 2-amino-2-hydroxymethyl-l,3-propanediol in 800
mL of water for bacterial endotoxins test, add 100 mL of 0. 1
mol/L hydrochloric acid TS and water for bacterial endotox-
ins test to make 1000 mL, and sterilize by heating in an au-
toclave at 121 °C for 90 minutes.
0.2 mol/L Tris-hydrochloride buffer solution, pH 7.4
Dissolve 6.61 g of 2-amino-2-hydroxymethyl-l,3-
propanediol hydrochloride and 0.97 g of 2-amino-2-hydrox-
ymethyl-l,3-propanediol in water to make 250 mL.
0.05 mol/L Tris-hydrochloride buffer solution, pH 7.5
Dissolve 6.35 g of 2-amino-2-hydroxymethyl-l,3-
propanediol hydrochloride and 1.18 g of 2-amino-2-hydrox-
ymethyl-l,3-propanediol in water to make 1000 mL.
Trishydroxymethylaminomethane
xymethyl-1 ,3-propanediol.
See 2-amino-2-hydro-
Trisodium citrate dihydrate C 6 H 5 Na 3 7 .2H 2 [K 8288,
or same as the monograph Sodium Citrate Hydrate]
Trisodium ferrous pentacyanoamine TS To 1.0 g of sodi-
um pentacyanonitrosylferrate (III) dihydrate add 3.2 mL of
ammonia TS, shake, and allow to stand in a tightly stoppered
bottle for a night in a refrigerator. Add this solution to 10 mL
of ethanol (99.5), filter to collect the yellow precipitate yield-
ed with suction. Wash the precipitate with dehydrated diethyl
ether, dry the precipitate, and keep in a desiccator. Dissolve
the precipitate in water to make a 1.0 mg/mL solution before
use, and keep in a refrigerator. Use within a week after
preparation.
Trisodium phosphate dodecahydrate Na 3 P0 4 .12H 2
[K 9012, Special class]
Trypsin for liquid chromatography An enzyme obtained
from the bovine pancreas. This one part digests 250 parts of
casein in the following reaction system.
Casein solution — To 0.1 g of milk casein add 30 mL of
water, disperse the casein well, add 1.0 mL of diluted sodium
hydroxide TS (1 in 10) to dissolve, and add water to make 50
mL. Prepare before use.
Sample solution — Dissolve 0.01 g of trypsin for liquid
chromatography in 500 mL of water.
Procedure — To 5 mL of the casein solution add 2 mL of
the sample solution and 3 mL of water, mix, then allow to
stand at 40°C for 1 hour, and add 3 drops of a mixture of
ethanol (95), water and acetic acid (100) (10:9:1): no
precipitate appears.
Trypsin inhibitor Produced by purifying soybean. Each
mg of trypsin inhibitor inhibits 10,000 to 30,000 BAEE Units
of trypsin. One BAEE Unit means a trypsin activity to indi-
cate an absorbance difference of 0.001 at 253 nm when 3.2
mL of the solution is reacted at 25 °C and pH 7.6, using N-a-
benzoyl-L-arginine ethyl ester as substrate.
Trypsin inhibitor TS Dissolve 5 mg of trypsin inhibitor in
0.05 mol/L phosphate buffer solution, pH 7.0 to make 10
mL.
Trypsin TS for test of elcatonin Dissolve 5 mg of trypsin
for liquid chromatography in 20 mL of a solution of ammo-
nium hydrogen carbonate (1 in 100). Prepare before use.
Trypsin TS for test of ulinastatin Dissolve crystal trypsin
for ulinastatin assay in ice-cooled 1 mmol/L hydrochloric
acid TS containing 1 mmol/L calcium chloride dihydrate so
that each mL of the solution contains 180 /ug of trypsin. Pre-
pare before use, and preserve in an ice-cooled water bath.
L-Tryptophan CnH 12 N 2 2 [Same as the namesake
monograph]
Turpentine oil [Same as the namesake monograph]
L-Tyrosine C 9 H u N0 3 White, crystals or crystalline
powder. Odorless and tasteless. Freely soluble in formic acid,
very slightly soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether. It dissolves in dilute
hydrochloric acid and in dilute nitric acid.
Optical rotation <2.49> [a]$: -10.5- -12.5° (after
drying, 2.5 g, 1 mol/L hydrochloric acid TS, 50 mL, 100
mm).
Loss on drying <2.41>: not more than 0.30% (1 g, 105°C,
3 hours).
Content: not less than 99.0%. Assay — Weigh accurately
about 0.3 g of L-tyrosine, previously dried, dissolve in 6 mL
of formic acid, add 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.12 mg of C 9 H„N0 3
Ubiquinone-9 Yellow to orange, crystalline powder.
Odorless and no taste.
Melting point <2.60>: about 44°C
Absorbance <2.24> £}* (275 nm): 163-190 (ethanol
(99.5))
Uracil C4H4N2O2 Needle crystals. Freely soluble in hot
water, and slightly soluble in cold water.
Melting point <2.60>: 335°C
Uranyl acetate See uranyl acetate dihydrate.
Uranyl acetate dihydrate U0 2 (CH 3 COO) 2 .2H 2 [K
8360: 1961, Special class]
Uranyl acetate TS Dissolve 1 g of uranyl acetate dihy-
drate in water to make 20 mL, and filter if necessary.
Uranyl acetate-zinc TS Dissolve 10 g of uranyl acetate di-
hydrate in 5 mL of acetic acid (31) and 50 mL of water by
heating. Separately, dissolve 30 g of zinc acetate dihydrate in
3 mL of acetic acid (31) and 30 mL of water by heating.
JPXV
General Tests / Reagents, Test Solutions
257
While the two solutions are still warm, mix them, cool, and
filter.
Urea H 2 NCONH 2 [K 8731, Special class]
Urethane See ethyl carbamate.
Ursodeoxycholic acid C24H40O4 [Same as the namesake
monograph]
M-Valerianic acid CH 3 (CH 2 ) 3 COOH Clear, colorless to
pale yellow liquid, having a characteristic odor. Miscible with
ethanol (95) and with diethyl ether, and soluble in water.
Distilling range <2.57>: 186 - 188°C, not less than 98 vol%.
Specific gravity <2.56> df: 0.936 - 0.942
L-Valine C 5 H u N0 2 [Same as the namesake mono-
graph]
H-D-Valyl-L-leucyl-L-arginine /j-nitroanilide dihydrochlo-
ride C 23 H 38 N 8 5 .2HC1 White to pale yellow, powder or
masses. Sparingly soluble in water.
Absorbance <2.24> E l °^ (316 nm): 214 - 236 (0.01 g, water,
500 mL).
Vanadium pentoxide See vanadium (V) oxide.
Vanadium pentoxide TS See vanadium (V) oxide TS.
Vanadium pentoxide TS, dilute See vanadium (V) oxide
TS, dilute.
Vanadium (V) oxide V 2 5 Orangish yellow to yellow-
brown powder.
Identification — Dissolve 0.3 g in 10 mL of ammonia TS
and 15 mL of water. To 2 mL of this solution add 20 mL of
water, mix, and add gently 1 mL of copper (II) sulfate TS:
yellow precipitates appear.
Vanadium (V) oxide TS Add vanadium (V) oxide to
phosphoric acid, saturate with vanadium (V) oxide by shak-
ing vigorously for 2 hours, and filter through a glass filter.
Vanadium (V) oxide TS, dilute Dilute 10 mL of vana-
dium (V) oxide TS with water to make 100 mL. Prepare be-
fore use.
Vanillin C 6 H 3 CHO(OCH 3 )(OH) [K 9544]
Vanillin-hydrochloric acid TS Dissolve 5 mg of vanillin
in 0.5 mL of ethanol (95), and to this solution add 0.5 mL of
water and 3 mL of hydrochloric acid. Prepare before use.
Vanillin-sulfuric acid-ethanol TS Dissolve 3 g of vanillin
in ethanol (99.5) to make 100 mL, and add 0.5 mL of sulfuric
acid.
Vanillin-sulfuric acid TS Add cautiously 75 mL of sulfu-
ric acid to 25 mL of ice-cold ethanol (95). After cooling, add
1 g of vanillin to dissolve. Prepare before use.
Vasopressin C 46 H 6 5N 15 1 2S2 A white powder.
Constituent amino acids — Perform the test as directed in
the Constituent amino acids under Oxytocin, and calculate
the respective molar ratios with respect to glycine: 0.9 - 1.1
for aspartic acid, 0.9-1.1 for glutamic acid, 0.9-1.1 for
proline, 0.8-1.1 for tyrosine, 0.9-1.1 for phenylalanine,
0.9 - 1.1 for arginine and 0.8 - 1.1 for cystine, and not more
than 0.03 for other amino acids.
Vegetable oil Vegetative oils specified in monographs.
Verapamil hydrochloride for assay C27H 38 N 2 4 .HC1
[Same as the monograph Verapamil Hydrochloride. When d-
ried, it contains not less than 99.0% of verapamil hydrochlo-
ride (C 27 H 38 N 2 4 .HC1).]
Vinblastine sulfate C 46 H5 8 N 4 09.H 2 S04 [Same as the
namesake monograph]
Vincristine sulfate C 46 H 56 N 4 O 10 .H 2 SO 4 [Same as the
namesake monograph]
Vinyl acetate C 4 H 6 2 Clear, colorless liquid.
Specific gravity <2.56>: 0.932 - 0.936
Water <2.48>: not more than 0.2%
Vinyl chloride C 2 H 3 C1 Colorless gas.
Boiling point <2.57>: - 14°C
Melting point <2.60>: -160°C
2-Vinylpyridine C 7 H 7 N A clear, colorless or dark
brown liquid.
Refractive index <2.45> n™: 1.546 - 1.552
Sepcific gravity <2.56> df a : 0.975 - 0.982
l-Vinyl-2-pyrrolidone C 6 H 9 NO Clear liquid.
Purity — Perform the test with 0.5 juL of l-vinyl-2-pyrroli-
done as directed under Gas Chromatography <2.02> accord-
ing to the following conditions. Determine each peak area of
the solutions by the automatic integration method, and calcu-
late the amount of l-vinyl-2-pyrrolidone by the area percen-
tage method: it is not less than 99.0%.
Operating conditions
Detector: A hydrogen flame-ionization detector.
Column: A hollow, capillary glass column about 0.53 mm
in inside diameter and about 30 m in length, having an about
1.0-yum layer of polyethylene glycol 20 M for gas chro-
matography on the inner side.
Column temperature: Maintain the temperature at 80°C
for 1 minute, then raise at the rate of 10°C per minute to
190°C, and hold constant to the temperature for 20 minutes.
Temperature of sample vaporization chamber: A constant
temperature of about 190°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
l-vinyl-2-pyrrolidone is about 15 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of l-vinyl-2-pyrrolidone from 0.5 //L of
l-vinyl-2-pyrrolidone is about 70% of the full scale.
Time span of measurement: About twice as long as the
retention time of l-vinyl-2-pyrrolidone beginning after the
solvent peak.
Water <2.48> — Take 50 mL of methanol for Karl Fischer
method and 10 mL of butyrolactone in a dry titration flask,
and titrate with Karl Fischer TS until end point. Weigh ac-
curately about 2.5 g of l-vinyl-2-pyrrolidone, transfer imme-
diately to a titration flask, and perform the test: water is not
more than 0.1%.
V8 protease A protease obtained from Staphylococus
aureus strain. When an amount of the enzyme hydrolyzes 1
fimo\ of /V-Z-butoxycarbonyl-L-glutamic acid-a-phenyl ester
in 1 minute at pH 7.8 and 37°C is defined as 1 unit, it con-
tains 500 - 1000 units per mg.
V8 protease TS Dissolve V8 protease in water to make a
solution of 1 mg/mL. Keep at a cold place and use within 6
258
Reagents, Test Solutions / General Tests
JP XV
days after preparation.
Voglibose for assay C 10 H 2 iNO 7 [Same as the mono-
graph Voglibose].
Warfarin potassium for assay [Same as the monograph
Warfarin Potassium. When dried, it contains not less than
99.0% of warfarin potassium (C 19 H 15 K0 4 ).]
25% Water containing benzoyl peroxide See Benzoyl
peroxide, 25% water containing.
Water for bacterial endotoxins test [Same as the mono-
graph Water for Injection or water produced by other proce-
dures that shows no reaction with the lysate reagent em-
ployed, at the detection limit of the reagent.]
Water for injection [Same as the namesake monograph]
Water, sterile purified [Same as the namesake mono-
graph]
Weakly acidic CM-bridged cellulose cation exchanger
(H type) Weakly acidic cation exchanger, intensified by
crosslinking porous spherical cellulose, into which carbox-
ymethyl growps have been introduced.
Wijs' TS Transfer 7.9 g of iodine trichloride and 8.9 g of
iodine to separate flasks, dissolve each with acetic acid (100),
mix both solutions, and add acetic acid (100) to make 1000
mL. Preserve in light-resistant, glass containers.
Wogonin for thin-layer chromatography C 16 H 12 5 Yel-
low crystals or crystalline powder. Slightly soluble in
methanol and in ethanol (99.5), and practically insoluble in
water. Melting point: 204 - 208°C
Identification — Determine the absorption spectrum of a
solution in methanol (1 in 200,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 207 nm and 211 nm, and between 273 nm and 277
nm.
Purity Related substances — Dissolve 1 mg in 1 mL of
methanol, and perform the test with 10 /xL of this solution as
directed in the Identification (3) under Saireito Extract: no
spot other than the principal spot (Rf value is about 0.4) ap-
pears.
Xanthene C 13 H I0 O White to light yellow crystals or
crystalline powder, having a slight, characteristic odor.
Melting point <2.60>: 98 - 102°C
Water <2.48>: not more than 0.5% (0.15 g).
Xanthene-9-carboxylic acid C 14 H 10 O3 Dissolve 0.25 g
of propantheline bromide in 5 mL of water and 10 mL of so-
dium hydroxide TS, heat the mixture to boiling, then con-
tinue to heat for 2 minutes. Cool to 60°C, add 5 mL of dilute
sulfuric acid, cool, filter the precipitate, and wash thoroughly
with water. Recrystallize the residue from dilute ethanol, and
dry for 3 hours in a desiccator (in vacuum, silica gel).
Melting point <2.60>: 217 - 222°C
Xanthone Ci 3 H 8 2 Light yellow powder. Freely soluble
in chloroform, and slightly soluble in hot water and in diethyl
ether.
Melting point <2.60>: 174- 176°C
Purity Related substances — Dissolve 0.050 g of xanthone
in chloroform to make exactly 10 mL. Perform the test with 5
luL of this solution as directed in the Purity under Propanthe-
line Bromide: any spot other than the principal spot at the Rf
value of about 0.7 does not appear.
Xanthydrol Ci 3 H 10 O 2 White to pale yellow powder.
Dissolves in ethanol (95), in diethyl ether, in chloroform, and
in acetic acid (100), and is practically insoluble in water.
Melting point <2.60>: 121 - 124°C
Residue on ignition <2.44>: not more than 2.0% (0.5 g).
Xylene C 6 H 4 (CH 3 ) 2 [K 8271, First class]
o-Xylene C 6 H 4 (CH 3 ) 2 Colorless, clear liquid.
Distilling range <2.57>: 143 - 146°C, not less than 95 vol%.
Refractive index <2.45> n 2 £: 1.501 - 1.506
Specific gravity <2.56> df: 0.875 - 0.885
Xylene cyanol FF C 25 H 27 N 2 Na0 7 S 2 [K 8272, Special c-
lass]
Xylenol orange C 31 H 30 N 2 Na 2 O 13 S [K 9563, Special
class]
Xylenol orange TS Dissolve 0.1 g of xylenol orange in
water to make 100 mL.
Xylitol C 5 H 12 5 [Same as the namesake monograph]
Xylose See D-xylose.
D-Xylose C 5 H 10 O 5 [Same as the monograph D-Xylose
of the Japanese Standards of Food Additives]
Yeast extract A peptone-like substance which represents
all the soluble product of yeast cells (Saccharomyces) pre-
pared under optimum conditions, clarified, and dried by
evaporating to a powder. Yeast extract (1 g) represents not
less than 7.5 g of yeast. A reddish yellow to brown powder,
having a characteristic but not putrescent odor. Soluble in
water, forming a yellow to brown solution, having a slight
acidic reaction. It contains no added carbohydrate.
Purity (1) Chloride <1.03> (calculated as NaCl): not
more than 5%.
(2) Coagulable protein — On heating a solution of yeast
extract (1 in 20) to boiling, no precipitate is produced.
Loss on drying <2.41>: not more than 5% (105°C, constant
mass).
Residue on ignition <2.44>: not more than 15% (0.5 g).
Nitrogen content <1.08>: 7.2-9.5% (105°C, constant
mass, after drying).
Yellow beeswax [Same as the namesake monograph]
Zaltoprofen C 17 H 14 3 S [Same as the namesake mono-
graph]
Zaltoprofen for assay C 17 H 14 3 S [Same as the mono-
graph Zaltoprofen. When dried, it contains not less than 99.5
% of zaltoprofen (C 17 H 14 3 S)].
Zinc Zn [K 8012, Special class]
Zinc acetate See zinc acetate dihydrate.
0.25 mol/L Zinc acetate buffer solution, pH 6.4 Dissolve
54.9 g of zinc acetate dihydrate in 150 mL of acetic acid (100)
and 600 mL of water, add 150 mL of ammonia water (28),
gently mix, and allow to cool to a room temperature. Adjust
to pH 6.4 with ammonia water (28), and add water to make
1000 mL.
Zinc acetate dihydrate Zn(CH 3 COO) 2 .2H 2 [K 8356,
JP XV
General Tests / Solid Supports/Column Packings for Chromatography
259
Special class]
Zinc, arsenic-free See zinc for arsenic analysis.
Zinc chloride ZnCl 2 [K 8111, Special class]
Zinc chloride TS Dissolve 10 g of zinc chloride and 10 g
of potassium hydrogen phthalate in 900 mL of water, adjust
the pH to 4.0 with sodium hydroxide TS, and add water to
make 1000 mL.
0.04 mol/L Zinc chloride TS Dissolve 5.453 g of zinc
chloride in water to make 1000 mL.
Zinc diethyldithiocarbamate
tic Containers <7.02>.
Zinc dibutyldithiocarbamate
tic Containers <7.02>
See Test Methods for Plas-
See Test Methods for Plas-
Zinc disodium ethylenediamine tetraacetate See zinc dis-
odium ethylenediamine tetraacetate tetrahydrate.
Zinc disodium ethylenediamine tetraacetate tetrahydrate
C 10 H 12 N 2 Na 2 O 8 Zn.4H 2 O White powder. The pH of a solu-
tion of zinc disodium ethylenediamine tetraacetate (1 in 100)
is between 6.0 and 9.0.
Purity Clarity and color of solution — Dissolve 0.10 g of
zinc disodium ethylenediamine tetraacetate tetrahydrate in 10
mL of freshly boiled and cooled water: the solution is clear
and colorless.
Content: not less than 98.0%. Assay— Dissolve about 0.5 g
of zinc disodium ethylenediamine tetraacetate tetrahydrate,
accurately weighed, in water to make exactly 100 mL. Pipet
10 mL of this solution, adjust the pH to about 2 with 80 mL
of water and dilute nitric acid, and titrate <2.50> with 0.01
mol/L bismuth nitrate VS until the color of the solution
changes from yellow to red (indicator: 2 drops of xylenol
orange TS).
Each mL of 0.01 mol/L bismuth nitrate VS
= 4.717 mg of Ci H 12 N 2 Na 2 O 8 Zn.4H 2 O
Zinc dust See zinc powder.
Zinc for arsenic analysis
about 800 /urn.
Zn [K 8012] Use granules of
Zinc iodide-starch TS To 100 mL of boiling water add a
solution of 0.75 g of potassium iodide in 5 mL of water, a so-
lution of 2 g of zinc chloride in 10 mL of water and a smooth
suspension of 5 g of starch in 30 mL of water, with stirring.
Continue to boil for 2 minutes, then cool.
Sensitivity — Dip a glass rod into a mixture of 1 mL of 0.1
mol/L sodium nitrite VS, 500 mL of water and 10 mL of hy-
drochloric acid, and touch on zinc iodide-starch paste TS: an
apparently blue color appeas.
Storage — Preserve in tightly stoppered bottles, in a cold
place.
Zincon
[K 9517, Special class]
Zincon TS Dissolve 0.1 g of zincon in 2 mL of 1 mol/L
sodium hydroxide VS, and add water to make 100 mL.
Zinc powder Zn [K 8013, Special class]
Zinc (standard reagent) Zn [K 8005, Standard reagent
for volumetric analysis]
Zinc sulfate See zinc sulfate heptahydrate.
Zinc sulfate for volumetric analysis See zinc sulfate hep-
tahydrate.
Zinc sulfate heptahydrate ZnS0 4 .7H 2 [K 8953, Spe-
cial class]
Zinc sulfate TS Dissolve 10 g of zinc sulfate heptahydrate
in water to make 100 mL.
Zirconyl-alizarin red S TS Dissolve 0.2 g of zirconyl ni-
trate in 5 mL of dilute hydrochloric acid, add 10 mL of aliza-
rin red S TS, and then add water to make 30 mL.
Zirconyl-alizarin S TS See zirconyl-alizarin red S TS.
Zirconyl nitrate See zirconyl nitrate dihydrate.
Zirconyl nitrate dihydrate ZrO(N0 3 ) 2 .2H 2 A white
crystalline powder. Freely soluble in water.
Identification — (1) To 5 mL of a solution (1 in 20) add 5
mL of sodium hydroxide TS: a white, milky precipitate is
formed.
(2) To 10 mL of a solution (1 in 20) add 10 mL of sulfuric
acid, cool, and superimpose 2 mL of iron (II) sulfate TS: a
brown ring is produced at the zone of contact.
9.42 Solid Supports/Column
Packings for Chromatography
Aminopropylsilanized silica gel for liquid chromato-
graphy Prepared for liquid chromatography.
Cellulose for thin-layer chromatography Use a high-
grade cellulose prepared for thin-layer chromatography.
Cellulose with fluorescent indicator for thin-layer chroma-
tography Use cellulose for thin-layer chromatography con-
taining a suitable fluorescent substance.
Cyanopropylsilanized silica gel for liquid chromatography
Prepared for liquid chromatography.
Diethylaminoethyl cellulose for column chromato-
graphy Prepared for column chromatography.
Diethylaminoethyl group bound to synthetic polymer for
liquid chromatography Produced by binding
diethylaminoethyl group to a hydrophilic synthetic polymer,
for liquid chromatography. Exchange volume is about 0.1
mg equivalents/cm 3 .
Dimethylaminopropylsilanized silica gel for liquid chroma-
tography Prepared for liquid chromatography.
Dimethylsilanized silica gel with fluorescent indicator for
thin-layer chromatography Dimethylsilanized silica gel for
thin-layer chromatography to which a fluorescent indicator is
added.
Divinylbenzene-methacrylate co-polymer for liquid chro-
matography Prepared for liquid chromatography.
Fluorosilanized silica gel for liquid chromatography
Prepared for liquid chromatography.
Gel-type strong acid cation-exchange resin for liquid chro-
matography (degree of cross-linkage: 8 %) Prepared for
260
Solid Supports/Column Packings for Chromatography / General Tests
JP XV
liquid chromatography.
Gel type strong acid ion-exchange resin for liquid chro-
matography (degree of cross-linkage: 6 %) Prepared for
liquid chromatography.
Glycol etherifized silica gel for liquid chromatography
Glycol group is bound to silica gel for liquid chro-
matography.
Graphite carbon for gas chromatography Prepared for
gas chromatography.
Hexasilanized silica gel for liquid chromatography Pre-
pared for liquid chromatography.
Hydrophilic silica gel for liquid chromatography Dio-
lized porous silica gel prepared for liquid chromatography
(5-10 //m in particle diameter).
Hydroxypropylsilanized silca gel for liquid chromato-
graphy Prepared for liquid chromatography.
Neutral alumina for chromatography Prepared for chro-
matography (75 - 180//m in particle diameter).
Neutral alumina for column chromatography Prepared
for column chromatography.
Octadecylsilanized polyvinyl alcohol gel polymer for liq-
uid chromatography Prepared for liquid chromato-
graphy.
Octadecylsilanized silica gel for liquid chromatography
Prepared for liquid chromatography.
Octadecylsilanized silica gel for thin-layer chromatography
Octadecylsilanized silica gel prepared for thin-layer chro-
matography.
Octadecylsilanized silica gel with fluorescent indicator for
thin-layer chromatography Octadecylsilanized silica gel for
thin-layer chromatography containing fluorescent indicator.
Octadecylsilanized silicone polymer coated silica gel for
liquid chromatography Prepared for liquid chro-
matography.
Octylsilanized silica gel for liquid chromatography Pre-
pared for liquid chromatography.
Pentaethylenehexaaminated polyvinyl alcohol polymer
bead for liquid chromatography Prepared for liquid chro-
matography.
Phenylated silica gel for liquid chromatography Pre-
pared for liquid chromatography.
Phenylsilanized silica gel for liquid chromatography Pre-
pared for liquid chromatography.
Polyamide for column chromatography Prepared for
column chromatography.
Polyamide for thin-layer chromatography Prepared for
thin-layer chromatography.
Polyamide with fluorescent indicator for thin-layer chro-
matography Add a fluorescent indicator to polyamide for
thin-layer chromatography.
Porous acrylonitrile-divinylbenzene copolymer for gas
chromatography (pore diameter: 0.06 - 0.08 fim, 100-200
m 2 /g) A porous acrylonitrile-divinylbenzene copolymer
prepared for gas chromatography.
Porous ethylvinylbenzene-divinylbenzene copolymer for
gas chromatography (average pore diameter: 0.0075 fim, 500
-600m 2 /g) A porous ethylvinylbenzene-divinylbenzene
copolymer prepared for gas chromatography. The average
pore diameter is 0.0075 /xm, and surface area is 500 to
600 m 2 per g.
Porous polymer beads for gas chromatography Prepared
for gas chromatography.
Porous silica gel for liquid chromatography A porous sil-
ica gel prepared for liquid chromatography.
Porous styrene-divinylbenzene copolymer for gas chro-
matography (average pore diameter: 0.0085 fim, 300 - 400
m 2 /g) A porous styrene-divinylbenzene copolymer prepared
for gas chromatography. The average pore diameter is 0.0085
/urn, and surface area is 300 to 400 m 2 /g.
Silica gel for gas chromatography A silica gel prepared
for gas chromatography.
Silica gel for liquid chromatography A silica gel prepared
for liquid chromatography.
Silica gel for thin-layer chromatography A silica gel pre-
pared for thin-layer chromatography.
Silica gel for thin -layer chlomatography (particle size 5~7
fim, with fluorescent indication) Prepared for high-perfor-
mance thin-layer chromatography.
Silica gel with complex fluorescent indicator for thin-layer
chromatography A silica gel for thin-layer chromatography
containing suitable complex fluorescent indicators.
Silica gel with fluorescent indiator for thin-layer chroma-
tography A silica gel for thin-layer chromatography con-
taining a suitable fluorescent indicator.
Siliceous earth for chromatography A siliceous earth pre-
pared for chromatography.
Siliceous earth for gas chromatography A siliceous earth
prepared for gas chromatography.
Strongly acidic ion exchange resin for column chromato-
graphy Prepared for column chromatography.
Strongly acidic ion exchange resin for liquid chromatogra-
phy Prepared for liquid chromatography.
Strongly acidic ion-exchange silica gel for liquid chro-
matography Prepared for liquid chromatography.
Styrene-divinylbenzene copolymer for liquid chromato-
graphy Prepared for liquid chromatography.
Synthetic magnesium silicate for column chromato-
graphy Prepared for column chromatography (150-250
fim in particle diameter).
Teflon for gas chromatography See Tetrafluoroethylene
polymer for gas chromatography.
Tetrafluoroethylene polymer for gas chromatography
Prepared for gas chromatography.
Trimethylsilanized silica gel for liquid chromatography
Prepared for liquid chromatography.
JPXV
General Tests / Standard Particles, etc.
261
Weakly acidic ion exchange resin for liquid chro-
matography Prepared for liquid chromatography.
Zeolite for gas chromatography (0.5 nm in pore diameter)
Zeolite prepared for gas chromatography.
9.43 Filter Papers, Filters for
filtration, Test Papers,
Crucibles, etc.
Filter paper [R 3801, Filter paper (for chemical analysis),
Filter paper for qualitative analysis]
No.l: For bulky gelatinous precipitate
No. 2: For moderate-sized precipitate
No. 3: For fine precipitate
No. 4: Hardened filter paper for fine precipitate
Filter paper for quantitative analysis [R 3801, Filter
paper (for chemical analysis), Filter paper for quantitative
analysis]
No. 5A: For bulky gelatinous precipitate
No. 5B: For moderate-sized precipitate
No. 5C: For fine precipitate
No. 6: Thin filter paper for fine precipitate
Porcelain crucible
cal analysis]
[R 1301, Porcelain crucible for chemi-
Sintered glass filter [R 3503, Glass appliance for chemical
analysis, Buchner funnel glass filter]
G3: 20-30 //m in pore size
G4: 5-10 //m in pore size
Blue litmus paper See litmus paper, blue.
Congo red paper Immerse filter paper in congo red TS,
and air-dry.
Glass fiber See glass wool.
Glass wool [K 8251, Special class]
Lead acetate paper See lead (II) acetate paper.
Lead (II) acetate paper Usually, immerse strips of filter
paper, 6 cm x 8 cm in size, in lead (II) acetate TS, drain off
the excess liquid, and dry the paper at 100°C, avoiding con-
tact with metals.
Litmus paper
paper]
Litmus paper.
paper]
, blue [K 9071, Litmus paper, Blue litmus
red [K 9071, Litmus paper, Red litmus
Phosgene test paper Dissolve 5 g of 4-dimethylamino-
benzaldehyde and 5 g of diphenylamine in 100 mL of ethanol
(99.5). Immerse a filter paper 5 cm in width in this solution,
and allow to dry spontaneously while the paper is suspended
in a dark place under clear air. Then cut off the 5-cm portions
from the upper side and lower side of the paper, and cut the
remaining paper to a length of 7.5 cm.
Preserve in tight, light-resistant containers. Do not use the
paper, which has changed to a yellow color.
Potassium iodate-starch paper Impregnate filter paper
with a mixture of equivalent volumes of a solution of potassi-
um iodate (1 in 20) and freshly prepared starch TS, and dry in
a clean room.
Storage — Preserve in a glass-stoppered bottle, protected
from light and moisture.
Potassium iodide-starch paper Impregnate filter paper
with freshly prepared potassium iodide-starch TS, and dry in
a clean room. Store in a glass-stoppered bottle, protected
from light and moisture.
Red litmus paper See litmus paper, red.
Turmeric paper Macerate 20 g of powdered dried
rhizome of Curcuma longa Linne with four 100 mL-portions
of cold water, decant the supernatant liquid each time, and
discard it. Dry the residue at a temperature not over 100°C.
Macerate the dried residue with 100 mL of ethanol (95) for
several days, and filter. Immerse filter paper in this ethanol
decoction, and allow the ethanol (95) to evaporate spontane-
ously in clean air.
Sensitivity — Dip a strip of turmeric paper, about 1.5 cm
length, in a solution of 1 mg of boric acid in a mixture of
1 mL of hydrochloric acid and 4 mL of water, after 1 minute
remove the paper from the liquid, and allow it to dry spon-
taneouly: the yellow color changes to brown. When the strip
is moistened with ammonia TS, the color of the strip changes
to greenish black.
Zinc iodide-starch paper Impregnate the filter paper for
volumetric analysis with freshly prepared zinc iodide-starch
TS, and dry in the clean room. Preserve in a glass-stoppered
bottle, protected from light and moisture.
9.44 Standard Particles, etc.
a-Alumina for specific surface area determination
a-Al 2 3 Prepared for specific surface area determination.
a-Alumina for thermal analysis a-Al 2 3 Prepared for
thermal analysis.
Calibration ball for particle density measurement
Calibration ball with a known volume prepared for measure-
ment of particle density. The volume of the calibration ball
must be accurately determined to the nearest 0.001 cm 3 .
Indium for thermal analysis In Prepared for thermal
analysis.
Content: not less than 99.99%.
Nickel for thermal analysis [K 9062 (Nickel), Special
class. Content: not less than 99.99%]
Standard particles for calibrating light-shielded automatic
fine particle counter Use plastic spherical particles of
known size and number.
Tin for thermal analysis Sn [K 8580 (Tin). Content: not
less than 99.99%]
Measuring Instruments and
Appliances, Thermometers, etc.
262
Optical Filters for Wavelength and Transmission Rate Calibration / General Tests
JP XV
graduation line olf.) ml
9.61 Optical Filters for
Wavelength and Transmission
Rate Calibration
Use optical filters for wavelength calibration and those for
transmission rate calibration shown in Table 9.61-1 and Ta-
ble 9.61-2, respectively. The optical filters for transmission
rate calibration are also used for the calibration of absor-
bances.
Table 9.61-1. Optical Filters for Wavelength Calibration
Type of filter
Range of
wavelength
calibration
(nm)
Product name
Neodymium optical filter for
wavelength calibration
Holmium optical filter for
wavelength calibration
400 - 750
250 - 600
JCRM 001
JCRM 002
Table 9.61-2.
Calibration
Optical Filters for Transmission Rate
Type of filter
Transmission rate
for calibration (%)
Product name
Optical filter for calibration
within the visible
wavelength range
1
10
20
30
40
50
JCRM 101
JCRM 110
JCRM 120
JCRM 130
JCRM 140
JCRM 150
Optical filter for calibration
within the ultraviolet
wavelength range
10
30
50
JCRM 210 A
JCRM 230 A
JCRM 250 A
Optical filter for calibration
within the near-ultraviolet
wavelength range
10
30
50
JCRM 310
JCRM 330
JCRM 350
20-22
:::
mL
50-
TTic figures arc in mm.
The figures are in mm.
Fig. 9.62-1 Fig. 9.62-2
The figures are in inm.
A: Gas buret (capacity of 100 mL, about 13.7 mm in
inside diameter, graduated in 0,2 mL divisions,
and graduated in 0. 1 mL divisions al the lower,
narrow part).
B: Gas buret (capacity of 1(X) mL, about 4.2 mm in
inside diameter at the upper stem with gradua-
tion in 0.02-mL division, about 28,5 mm in inside
diameter at the lower stem with graduation in 1-
mL divisions).
C: (Ct, Cj, Csand GO: Three-way stopcock.
D: Inlet of sample (bent forward at 20 mm in length).
E: Outlet of mixed gas (bent forward at 20 mm in-
lenglh).
F: Jacket (about 770 mm in length, about 40 mm in
outside diameter, almost completely filled with
water at room temperature) .
G: Rubber pressure tubing, about 4 mm in inside di-
ameter (Gi: about 80 cm in length; Ga and G3:
about 120 em in length).
H: Heavy-wall capillary tube (about 1 mm in inside
diameter),
K: Receiver,
L: leveling bulb (I-t: filled with about 50 nil, of mer-
cury; La and L3: filled with about 150 mL of mer-
cury).
Fig. 9.62-3
Measuring Instruments,
Appliances
9.62
Measuring Instruments are the instruments or machines
used for measuring mass or volume in the JP tests, and Ap-
pliances are the instruments specified in order to make test
conditions as consistent as possible in those tests.
Balances and weights (1) Chemical balances — Use
JPXV
General Tests / Thermometers
263
Table 9.62 Specification of Sieves
Nominal
size
(lira)
Specification of sieves
Sieve
Sieve opening (mm)
Wire (
mm)
number
Size
(mm)
Permissible variation
Diameter
Permissible
Average
Maximum
variation
3.5
5600
5.60
±0.14
0.42
1.66
±0.040
4
4750
4.75
±0.118
0.41
1.60
±0.040
4.7
4000
4.00
±0.100
0.37
1.40
±0.040
5.5
3350
3.35
±0.100
0.32
1.27
±0.030
6.5
2800
2.80
±0.084
0.28
1.11
±0.030
7.5
2360
2.36
±0.070
0.24
1.03
±0.030
8.6
2000
2.00
±0.060
0.20
0.953
±0.030
10
1700
1.70
±0.051
0.17
0.840
±0.025
12
1400
1.40
±0.042
0.14
0.717
±0.025
14
1180
1.18
±0.035
0.14
0.634
±0.025
16
1000
1.00
±0.030
0.14
0.588
±0.025
18
850
0.850
±0.034
0.127
0.523
±0.025
22
710
0.710
±0.028
0.112
0.450
±0.025
26
600
0.600
±0.024
0.101
0.390
±0.020
30
500
0.500
±0.020
0.089
0.340
±0.020
36
425
0.425
±0.017
0.081
0.290
±0.020
42
355
0.355
±0.013
0.072
0.250
±0.020
50
300
0.300
±0.012
0.065
0.208
±0.015
60
250
0.250
±0.0099
0.058
0.173
±0.015
70
212
0.212
±0.0087
0.052
0.151
±0.015
83
180
0.180
±0.0076
0.047
0.126
±0.015
100
150
0.150
±0.0066
0.043
0.104
±0.015
119
125
0.125
±0.0058
0.038
0.088
±0.015
140
106
0.106
±0.0052
0.035
0.075
±0.010
166
90
0.090
±0.0046
0.032
0.063
±0.010
200
75
0.075
±0.0041
0.029
0.052
±0.010
235
63
0.063
±0.0037
0.026
0.045
±0.005
282
53
0.053
±0.0034
0.024
0.037
±0.005
330
45
0.045
±0.0034
0.022
0.032
±0.005
391
38
0.038
±0.0026
0.018
0.027
±0.005
balances readable to the extent of 0.1 mg.
(2) Semimicrobalances — Use balances readable to the ex-
tent of 0.01 mg.
(3) Microbalances — Use balances readable to the extent
of 0.001 mg.
(4) Weights — Use calibrated weights.
Cassia flask Use glass-stoppered flasks, shown in Fig.
9.62-1, made of hard glass and having graduation lines of
volume on the neck.
Gas mixer Use the apparatus, shown in Fig. 9.62-3, made
of hard glass.
Nessler tube Use colorless, glass-stoppered cylinders
1.0 to 1.5 mm in thickness, shown in Fig. 9.62-2, made of hard
glass. The difference of the height of the graduation line of 50
mL from the bottom among cylinders does not exceed 2 mm.
Sieves Sieves conform to the specifications in Table 9.62.
Use the sieve number of nominal size as the designation.
Volumetric measures Use volumetric flasks, transfer
pipets, push-button micropipets, burets and measuring cylin-
ders conforming to the Japanese Industrial Standard.
9.63 Thermometers
Thermometers Ordinarily, use calibrated thermometers
with an immersion line (rod) or calibrated total immersion
mercury-filled thermometers according to the Japanese In-
dustrial Standards. Use the thermometers with the immersion
line (rod), shown in Table 9.63, for the tests in Congealing
Point, Melting Point (Method 1), Boiling Point and Distilling
Range.
264
Thermometers / General Tests
JP XV
Table 9.63 Thermometers with Immersion Line
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
Liquid
Mercury
Mercury
Mercury
Mercury
Mercury
Mercury
Gas filled above liquid
Nitrogen or
Nitrogen or
Nitrogen or
Nitrogen or
Nitrogen or
Nitrogen or
Argon
Argon
Argon
Argon
Argon
Argon
Temperature range
-17-50°C
40 - 100°C
90-150°C
140-200°C
190~250°C
240 - 320°C
Minimum graduation
0.2°C
0.2°C
0.2°C
0.2°C
0.2°C
0.2°C
Longer graduation lines at
each 1°C
each 1 °C
each 1°C
each 1°C
each 1°C
1°C
Graduation numbered at
each 2°C
each 2°C
each 2°C
each 2°C
each 2°C
2°C
Total length (mm)
280 - 300
280 - 300
280 - 300
280 - 300
280-300
280-300
Stem diameter (mm)
6.0±0.3
6.0±0.3
6.0±0.3
6.0±0.3
6.0±0.3
6.0±0.3
Bulb length (mm)
12-18
12- 18
12- 18
12- 18
12- 18
12- 18
Distance from bottom of
bulb to graduation at the
75-90
75-90
75-90
75-90
75-90
75-90
lowest temperature (mm)
Distance from top of
termometer to graduation
at the highest temperature
35-65
35-65
35-65
35-65
35-65
35-65
(mm)
Distance from bottom of
bulb to immersion
58-62
58-62
58-62
58-62
58-62
58-62
line(mm)
From of top of termometer
loop
loop
loop
loop
loop
loop
Test temperature
-15°C, 15°C,
45°C, 70°C,
95°C, 120°C,
145°C,
195°C,
245°C,
45 °C
95 °C
145°C
170°C, 195°C
220°C, 245°C
280°C, 315°C
Maximum scale error at
0.2°C
0.2°C
0.2°C
0.2°C
0.3°C (0.2°C,
0.4°C (0.5°C,
any point
at 195°C of
test temperature)
at 315°C of
test temperature)
Official Monographs
Absorptive Ointment
Method of preparation
White Petrolatum
Cetanol
White Beeswax
Sorbitan Sesquioleate
Lauromacrogol
Ethyl Parahydroxybenzoate or Methyl
Parahydroxybenzoate
Butyl Parahydroxybenzoate or Propyl
Parahydroxybenzoate
400 g
100 g
50 g
50 g
5g
lg
lg
Purified Water
a sufficient quantity
To make 1000 g
Melt White Petroleum, Cetanol, White Beeswax, Sorbitan
Sesquioleate and Lauromacrogol by heating on a water bath,
mix and maintain at about 75 °C. Add Methyl Parahydrox-
ybenzoate or Ethyl Parahydroxybenzoate and Propyl Para-
hydroxybenzoate or Butyl Parahydroxybenzoate to Purified
Water, dissolve by warming at 80°C. Combine both solu-
tions, mix to make emulsion, cool, and stir thoroughly until
it congeals.
Description Absorptive Ointment is white in color and is
lustrous. It has a slightly characteristic odor.
Containers and storage Containers — Tight containers.
Acebutolol Hydrochloride
7t7>P-Jlii^
H 3
-A
■ HCI
arxJ enanliomer
C 18 H 28 N 2 4 .HC1: 372.89
/V-{3-Acetyl-4-[(2flS)-2-hydroxy-
3-(l-methylethyl)aminopropyloxy]phenyl}butanamide
monohydrochloride [34381-68-5]
Acebutolol Hydrochloride, when dried, contains not
less than 98.0% and not more than 102.0% of
C 18 H 28 N 2 4 .HC1.
Description Acebutolol Hydrochloride occurs as white to
pale yellowish white crystals or crystalline powder.
It is freely soluble in water, in methanol, in ethanol (95)
and in acetic acid (100), and practically insoluble in diethyl
ether.
A solution of Acebutolol Hydrochloride (1 in 20) shows no
optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Acebutolol Hydrochloride in 0.01 mol/L
hydrochloric acid TS (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Acebutolol Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
(3) A solution of Acebutolol Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 141 - 145 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Acebutolol Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Acebutolol Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 40 mg of Acebutolol
Hydrochloride in 2 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 25 mL, and pipet 1 mL of this
solution, add methanol to make exactly 20 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 nL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with the upper layer of a
mixture of water, 1-butanol and acetic acid (100) (5:4:1) to a
distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 365 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 1.0% (0.5 g, 105 °C,
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.25 g of Acebutolol
Hydrochloride, previously dried, dissolve in 20 mL of acetic
acid (100), add 80 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 37.29 mg of C 18 H 28 N 2 4 .HC1
Containers and storage Containers — Well-closed contain-
ers.
265
266
Aceglutamide Aluminum / Official Monographs
JP XV
Aceglutamide Aluminum
H 2 N
C0 2 '
H NH
CH 3
Alj(OH) 4
C35H5 9 Al3N 10 O 2 4: 1084.84
Pentakis[(2S)-2-acetylamino-4-
carbamoylbutanoato]tetrahydroxotrialuminium
[12607-92-0]
Aceglutamide Aluminum contains not less than
85.4% and not more than 87.6% of aceglutamide
(C 7 H 12 N 2 4 : 188.18), and not less than 7.0% and not
more than 8.0% of aluminum (Al: 26.98), calculated
on the dried basis.
Description Aceglutamide Aluminum occurs as a white
powder, having astringent bitter taste.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
It dissolves in dilute hydrochloric acid.
It is hygroscopic.
Identification (1) Dissolve 0.03 g each of Aceglutamide
Aluminum and Aceglutamide Reference Standard in 5 mL of
water, and use these solutions as the sample solution and
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solution on a plate
of cellulose for thin-layer chromatography. Develop the plate
with a mixture of 1-propanol, water and acetic acid (100)
(16:8:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly a solution of bromocresol green in ethanol (95)
(1 in 1000), then spray evenly diluted ammonia solution (28)
(1 in 100): the spots from the sample solution and the stan-
dard solution show a light yellow and have the same Rf value.
(2) A solution of Aceglutamide Aluminum in dilute
hydrochloric acid (1 in 20) responds to the Qualitative Tests
<1.09> for aluminum salt.
Optical rotation <2.49> [ a \ 2 °: - 5.5 - - 7.5° (2 g calculated
on the dried basis, water, 50 mL, 100 mm).
Purity (1) Heavy metals < 7.07 > —Put 1.0 g of
Aceglutamide Aluminum in a porcelain crucible, cover the
crucible loosely, and heat gently to carbonize. After cooling,
add 2 mL of nitric acid and 1 mL of sulfuric acid, heat gently
until the white fumes no more evolve, and heat to incinerate
at 500 to 600°C. If the incineration is not accomplished, add
2 mL of nitric acid and 1 mL of sulfuric acid, heat in the
same manner as above, then ignite at 500 to 600°C to inciner-
ate. After cooling, add 2 mL of hydrochloric acid, proceed
with this solution according to Method 2, and perform the
test. Prepare the control solution as follows: proceed in the
same manner as the preparation of the test solution with the
same amount of the reagents, and add 2.0 mL of Standard
Lead Solution and water to make 50 mL (not more than 20
ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Aceglutamide Aluminum according to Method 1, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Aceglutamide
Aluminum in the mobile phase to make exactly 100 mL, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 100
mL, and use this solution as the standard solution (1).
Separately, dissolve 10 mg of 2-acetamidoglutarimide in the
mobile phase to make exactly 100 mL. Pipet 3 mL of this
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution (2). Perform the test
with exactly 20 /xL each of the sample solution and standard
solutions (1) and (2) as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the peak area of 2-acetamidoglutarimide from the
sample solution is not more than that from the standard solu-
tion (2), the peak areas other than aceglutamide and 2-
acetamidoglutarimide from the sample solution are not more
than 3/10 times the peak area of aceglutamide from the
standard solution (1), and the total of the peak areas other
than aceglutamide and 2-acetamidoglutarimide from the
sample solution is not more than the peak area of
aceglutamide from the standard solution (1).
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of aceglutamide.
System suitability —
Test for required detection: To exactly 5 mL of the
standard solution (1) add the mobile phase to make exactly 50
mL. Confirm that the peak area of aceglutamide obtained
from 20 [iL of this solution is equivalent to 7 to 13% of that
of aceglutamide obtained from 20 /xL of the standard solu-
tion.
System performance: Proceed as directed in the system
suitability in the Assay (1).
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution (1) under the above operating
conditions, the relative standard deviation of the peak area of
aceglutamide is not more than 2.0%.
Loss on drying <2.41> Not more than 5.0% (1 g, 130 C C,
5 hours).
Assay (1) Aceglutamide — Weigh accurately about 50 mg
of Aceglutamide Aluminum, dissolve in a suitable amount of
the mobile phase, add exactly 10 mL of the internal standard
solution and the mobile phase to make 50 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 45 mg of Aceglutamide Reference Standard, dissolve
in a suitable amount of the mobile phase, add exactly 10 mL
of the internal standard solution and the mobile phase to
make 50 mL, and use this solution as the standard solution.
Perform the test with 10 fxh each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of aceglutamide to
that of the internal standard.
Amount (mg) of aceglutamide (C7LL2N2O4)
JPXV
Official Monographs / Acetaminophen
267
= W s X (Qr/Qs)
W s : Amount (mg) of Aceglutamide Reference Standard
Internal standard solution — A solution of thymine in
methanol (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 210 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted perchloric acid (1 in
1000) and methanol (99:1).
Flow rate: Adjust the flow rate so that the retention time of
aceglutamide is about 5 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, aceglutamide and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 11,
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of aceglutamide to that of the internal standard is
not more than 1.0%.
(2) Aluminum — Weigh accurately about 3.0 g of
Aceglutamide Aluminum, add 20 mL of dilute hydrochloric
acid, and heat on a water bath for 60 minutes. After cooling,
add water to make exactly 200 mL. Pipet 20 mL of this solu-
tion, add exactly 25 mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS and 20 mL of acetic acid-
ammonium acetate buffer solution, pH4.8, and boil for
5 minutes. After cooling, add 50 mL of ethanol (95), and
titrate <2.50> with 0.05 mol/L zinc acetate VS until the color
of the solution changes from light dark green to light red (in-
dicator: 2 mL of dithizone TS). Perform a blank determina-
tion in the same manner.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS = 1.349 mg of Al
Containers and storage Containers — Tight containers.
Acetaminophen
Paracetamol
7th7J/7i>
C 8 H 9 N0 2 : 151.16
7V-(4-Hydroxyphenyl)acetamide [103-90-2]
Acetaminophen, when dried, contains not less than
98.0% of QH 9 N0 2 .
Description Acetaminophen occurs as white crystals or
crystalline powder.
It is freely soluble in methanol and in ethanol (95), sparing-
ly soluble in water, and very slightly, soluble in diethyl ether.
It dissolves in sodium hydroxide TS.
Identification Determine the infrared absorption spectra of
Acetaminophen, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of dried Acetaminophen Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Melting point <2.60> 169 - 172°C
Purity (1) Chloride < 1.03 > —Dissolve 4.0 g of
Acetaminophen in 100 mL of water by heating, cool with
shaking in ice water, allow to stand until ordinary tempera-
ture is attained, add water to make 100 mL, and filter. To 25
mL of the filtrate add 6 mL of dilute nitric acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution with 0.40 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.014%).
(2) Sulfate <1.14>— To 25 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test
solution. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.019%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of
Acetaminophen according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Acetaminophen according to Method 3, and perform the
test (not more than 2 ppm).
(5) Related substances — Dissolve 50 mg of
Acetaminophen in 1 mL of methanol, add the mobile phase-
to make 50 mL, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add the mobile phase to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with exactly 10 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of both solutions by the
automatic integration method: the total area of all peaks
other than the peak area of acetaminophen from the sample
solution is not larger than the peak area of acetaminophen
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column about 4 mm in inside
diameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogenphosphate, pH 4.7 and methanol (4:1)
Flow rate: Adjust the flow rate so that the retention time of
acetaminophen is about 5 minutes.
Selection of column: Dissolve 0.01 g each of
Acetaminophen and p-aminophenol in 1 mL of methanol,
add the mobile phase to make 50 mL, to 1 mL of this solution
268
Acetazolamide / Official Monographs
JP XV
add the mobile phase to make 10 mL. Proceed with 10 iuL of
this solution under the above operating conditions, and
calculate the resolution. Use a column giving elution of p-
aminophenol and acetaminophen in this order with the
resolution between these peaks being not less than 7.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of acetaminophen obtained from 10 /xL
of the standard solution is about 15% of the full scale.
Time span of measurement: About 6 times as long as the
retention time of acetaminophen beginning after the solvent
peak.
Loss on drying <2.41>
2 hours).
Not more than 0.3% (0.5 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 20 mg each of
Acetaminophen and Acetaminophen Reference Standard,
previously dried, dissolve in 2 mL of methanol, and add
water to make exactly 100 mL. Pipet 3 mL each of these
solutions, add water to make exactly 100 mL, and use these
solutions as the sample solution and standard solution,
respectively. Determine the absorbances, A T and A s , of the
sample solution and standard solution at the wavelength of
maximum absorption at about 244 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using water as
the blank.
Amount (mg) of C 8 H 9 N0 2 = W s x (A T /A S )
W s : Amount (mg) of Acetaminophen Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Acetazolamide
7t5"/7J K
H 3 C
O-'ri *
C 4 H 6 N 4 3 S 2 : 222.25
A/-(5-Sulfamoyl-l,3,4-thiadiazol-2-yl)acetamide [59-66-5]
Acetazolamide contains not less than 98.0% and not
more than 102.0% of C 4 H 6 N 4 3 S 2 , calculated on the
dried basis.
Description Acetazolamide occurs as a white to pale
yellowish white, crystalline powder. It is odorless, and has a
slight bitter taste.
It is slightly soluble in ethanol (95), very slightly soluble in
water, and practically insoluble in diethyl ether.
Melting point: about 255 °C (with decomposition).
Identification (1) To 0.1 g of Acetazolamide add 5 mL of
sodium hydroxide TS, then add 5 mL of a solution of 0.1 g of
hydroxylammonium chloride and 0.05 g of copper (II)
sulfate pentahydrate in 10 mL of water: a light yellow color
develops. Then heat this solution for 5 minutes: a deep yellow
color is produced gradually.
(2) To 0.02 g of Acetazolamide add 2 mL of dilute
hydrochloric acid, boil for 10 minutes, cool, and add 8 mL of
water: this solution responds to the Qualitative Tests <1.09>
for primary aromatic amines.
(3) To 0.2 g of Acetazolamide add 0.5 g of granulated
zinc and 5 mL of diluted hydrochloric acid (1 in 2): the gas
evolved darkens moistened lead (II) acetate paper.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Acetazolamide in 10 mL of sodium hydroxide TS: the
solution is clear and colorless to pale yellow.
(2) Chloride <1.03>— To 1.5 g of Acetazolamide add 75
mL of water, and warm at 70°C for 20 minutes with
occasional shaking. After cooling, filter, and to 25 mL of
the filtrate add 6 mL of dilute nitric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.20 mL of 0.01 mol/
L hydrochloric acid VS (not more than 0.014%).
(3) Sulfate <1.14>— To 25 mL of the filtrate obtained in
(2) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.40 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.038%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Acetazolamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(5) Silver-reducing substances — Wet 5 g of
Acetazolamide with 5 mL of aldehyde-free ethanol, and add
125 mL of water, 10 mL of nitric acid and exactly 5 mL of
0.1 mol/L silver nitrate VS. Stir for 30 minutes by protecting
from light, filter through a glass filter (G3), and wash the
residue on the glass filter with two 10-mL portions of water.
Combine the filtrate with the washings, to the solution add
5 mL of ferric ammonium sulface TS, and titrate <2.50> with
0.1 mol/L ammonium thiocyanate VS: not less than 4.8 mL
of 0.1 mol/L ammonium thiocyanate VS is consumed.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.15 g of Acetazolamide,
and dissolve in 400 mL of water in a water bath by heating.
After cooling, add water to make exactly 1000 mL. Pipet
5 mL of the solution, add 10 mL of 1 mol/L hydrochloric
acid TS, and then add water to make exactly 100 mL. Deter-
mine the absorbance A of this solution at the wavelength of
maximum absorption at about 265 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 4 H 6 N 4 3 S 2 = (A/414) x 200,000
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Acetic Acid
mm
Acetic Acid contains not less than 30.0 w/v% and
JP XV
Official Monographs / Acetohexamide 269
not more than 32.0 w/v% of C 2 H 4 2 : 60.05.
Description Acetic Acid is a clear, colorless liquid. It has a
pungent, characteristic odor and an acid taste.
It is miscible with water, with ethanol (95) and with
glycerin.
Specific gravity dl° : about 1.04
Identification Acetic Acid changes blue litmus paper to red,
and responds to the Qualitative Tests <1.09> for acetate.
Purity (1) Chloride— To 20 mL of Acetic Acid add 40 mL
of water, and use this solution as the sample solution. To 10
mL of the sample solution add 5 drops of silver nitrate TS: no
opalescence is produced.
(2) Sulfate — To 10 mL of the sample solution obtained in
(1) add 1 mL of barium chloride TS: no turbidity is
produced.
(3) Heavy metals <1.07> — Evaporate 10 mL of Acetic
Acid on a water bath to dryness, and to the residue add 2 mL
of dilute acetic acid and water to make 50 mL. Perform the
test with this solution as the test solution. Prepare the control
solution with 3.0 mL of Standard Lead Solution by adding 2
mL of dilute acetic acid and water to make 50 mL (not more
than 3 ppm).
(4) Potassium permanganate-reducing substances — To
20 mL of the sample solution obtained in (1) add 0.02 mol/L
potassium permanganate VS: the red color does not disap-
pear within 30 minutes.
(5) Non-volatile residue — Evaporate 30 mL of Acetic
Acid on a water bath to dryness, and dry at 105 C C for 1 hour:
the mass of the residue is not more than 1.0 mg.
Assay Measure exactly 5 mL of Acetic Acid, add 30 mL of
water, and titrate <2.50> with 1 mol/L sodium hydroxide VS
(indicator: 2 drops of phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 60.05 mg of C 2 H 4 2
Containers and storage Containers — Tight containers.
Glacial Acetic Acid
C 2 H 4 2 : 60.05
Acetic acid [64-19-7]
Glacial Acetic Acid contains not less than 99.0% of
C 2 H 4 2 .
Description Glacial Acetic Acid is a clear, colorless, volatile
liquid, or colorless or white, crystalline masses. It has a
pungent, characteristic odor.
It is miscible with water, with ethanol (95) and with diethyl
ether.
Boiling point: about 118°C
Specific gravity df : about 1.049
Identification A solution of Glacial Acetic Acid (1 in 3)
changes blue litmus paper to red, and responds to the
Qualitative Tests <1.09> for acetate.
Congealing point <2.42> Not below 14.5°C.
Purity (1) Chloride— To 10 mL of Glacial Acetic Acid
add water to make 100 mL, and use this solution as the sam-
ple solution. To 10 mL of the sample solution add 5 drops of
silver nitrate TS: no opalescence is produced.
(2) Sulfate — To 10 mL of the sample solution obtained in
(1) add 1 mL of barium chloride TS: no turbidity is
produced.
(3) Heavy metals <1.07> — Evaporate 2.0 mL of Glacial
Acetic Acid on a water bath to dryness. Dissolve the residue
in 2 mL of dilute acetic acid and water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion by adding 2.0 mL of dilute acetic acid and water to make
50 mL (not more than 10 ppm).
(4) Potassium permanganate-reducing substances — To
20 mL of the sample solution obtained in (1) add 0.10 mL of
0.1 mol/L potassium permanganate VS: the red color does
not disappear within 30 minutes.
(5) Non-volatile residue — Evaporate 10 mL of Glacial
Acetic Acid on a water bath to dryness, and dry at 105 C C for
1 hour: the mass of the residue is not more than 1.0 mg.
Assay Place 10 mL of water in a glass-stoppered flask, and
weigh accurately. Add about 1.5 g of Glacial Acetic Acid,
weigh accurately again, then add 30 mL of water, and titrate
<2.50> with 1 mol/L sodium hydroxide VS (indicator: 2 drops
of phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 60.05 mg of C 2 H 4 2
Containers and storage Containers — Tight containers.
Acetohexamide
y-tlr-'N+^S K
1-3C
o °
X
,jO
C 15 H 20 N 2 O 4 S: 324.40
4-Acetyl-/V-(cyclohexylcarbamoyl)benzenesulfonamide
[968-81-0]
Acetohexamide, when dried, contains not less than
98.0% and not more than 101.0% of C 15 H 20 N 2 O 4 S.
Description Acetohexamide occurs as a white to yellowish
white powder.
It is freely soluble in Af,Af-dimethylformamide, sparingly
soluble in acetone, slightly soluble in methanol and in ethanol
(99.5), and practically insoluble in water.
Melting point: about 185°C (with decomposition).
Identification (1) Dissolve 0.10 g of Acetohexamide in 100
mL of methanol. To 5 mL of the solution add 20 mL of 0.5
mol/L hydrochloric acid TS and 75 mL of methanol, and use
the solution as the sample solution (1). Determine the absorp-
tion spectrum of the sample solution (1) as directed under
Ultraviolet-visible Spectrophotometry < 2.24 > , using
methanol as the blank, and compare the spectrum with the
270
Ace toll exam ide / Official Monographs
JP XV
Reference Spectrum 1: both spectra exhibit similar intensities
of absorption at the same wavelengths. Separately, to exactly
10 mL of the sample solution (1) add methanol to make ex-
actly 50 mL, and use the solution as the sample solution (2).
Determine the absorption spectrum of the sample solution (2)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using methanol as the blank, and compare the spec-
trum with the Reference Spectrum 2: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Acetohexamide, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Chloride <I.03>— Dissolve 1.5 g of Acetohex-
amide in 40 mL of iV,./V-dimethylformamide, add 6 mL of di-
lute nitric acid and 7V,iV-dimethylformamide to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution as follows: to 0.45 mL of 0.01 mol/
L hydrochloric acid VS add 6 mL of dilute nitric acid and
A^TV-dimethylformamide to make 50 mL (not more than
0.011%).
(2) Sulfate <1.14> — Dissolve 2.0 g of Acetohexamide in
40 mL of iV,7V-dimethylformamide, and add 1 mL of dilute
hydrochloric acid and 7V,iV-dimethylformamide to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution as follows: to 0.40 mL of 0.005
mol/L sulfuric acid VS add 1 mL of dilute hydrochloric
acid and A^A^dimethylformamide to make 50 mL (not more
than 0.010%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Aceto-
hexamide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Related substances (i) Cyclohexylamine — Dissolve ex-
actly 1.0 g of Acetohexamide in exactly 30 mL of 0.5 mol/L
sodium hydroxide TS, add exactly 5 mL of hexane, shake
vigorously for 60 minutes, allow to stand for 5 minutes, and
use the upper layer as the sample solution. Separately, dis-
solve exactly 50 mg of cyclohexylamine in 0.5 mol/L sodium
hydroxide TS to make exactly 50 mL. Pipet 2 mL of this solu-
tion, and add 0.5 mol/L sodium hydroxide TS to make ex-
actly 300 mL. Pipet 30 mL of this solution, add exactly 5 mL
of hexane, shake vigorously for 60 minutes, allow to stand
for 5 minutes, and use the upper layer as the standard solu-
tion. Perform the test with exactly 2//L each of the sample
solution and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and determine the peak area of cyclohexylamine by the auto-
matic integration method: the peak area of cyclohexylamine
is not more than that with the standard solution.
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A fused-silica column 0.53 mm in inside diameter
and 30 m in length, coated the inner surface with methylsili-
cone polymer for gas chromatography 1.5 /urn in thickness.
Column temperature: A constant temperature of about
90°C.
Injection port temperature: A constant temperature of
about 150°C.
Detector temperature: A constant temperature of about
210°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
cyclohexylamine is about 4 minutes.
Split ratio: 1:1
System suitability —
System performance: When the procedure is run with 2 ftL
of the standard solution under the above operating
conditions, the number of theoretical plates of the peak of
cyclohexylamine is not less than 8000.
System repeatability: When the test is repeated 6 times with
2 /xL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of cy-
clohexylamine is not more than 5%.
(ii) Dicyclohexylurea — Dissolve exactly 1.0 g of Aceto-
hexamide in exactly 10 mL of 0.5 mol/L sodium hydroxide
TS, add exactly 20 mL of methanol, shake, then add exactly 5
mL of diluted hydrochloric acid (1 in 10), shake vigorously
for 15 minutes, and centrifuge. Filter 10 mL or more of the
supernatant liquid through a membrane filter with pore size
of not larger than 0.5 /xm. Discard the first 5 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, dissolve exactly 50 mg of dicyclohexylurea in
methanol to make exactly 100 mL. Pipet 2 mL of this solu-
tion, and add methanol to make exactly 100 mL. Pipet 20 mL
of this solution, add exactly 10 mL of 0.5 mol/L sodium
hydroxide TS, shake, then add exactly 5 mL of diluted
hydrochloric acid (1 in 10), shake, and use this solution as the
standard solution. Perform the test with exactly 50 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak area of dicyclohex-
ylurea by the automatic integration method: the peak area of
dicyclohexylurea is not more than that with the standard so-
lution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.5 g of sodium hydroxide in 1000
mL of 0.05 mol/L sodium dihydrogen phosphate TS, and
adjust the pH to 6.5 with 0.5 mol/L sodium hydroxide TS.
To 500 mL of this solution add 500 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
dicyclohexylurea is about 10 minutes.
System suitability —
System performance: When the procedure is run with
50 /uL of the standard solution under the above operating
conditions, the number of theoretical plates of the peak of
dicyclohexylurea is not less than 10,000.
System repeatability: When the test is repeated 6 times with
50 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
dicyclohexylurea is not more than 2.0%.
(iii) Other related substances — Dissolve 0.10 g of Aceto-
hexamide in 10 mL of acetone, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
acetone to make exactly 20 mL. Pipet two 1 mL portions of
JPXV
Official Monographs / Acetylcholine Chloride for Injection
271
this solution, add acetone to make exactly 10 mL and 25 mL,
respectively, and use these solutions as the standard solution
(1) and the standard solution (2). Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 [iL each of the sample solution and standard
solutions (1) and (2) on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, methanol, ammonia solution
(28) and cyclohexane (6:2:1:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than spot from
the standard solution (1), and the number of them which are
more intense than the spot from the standard solution (2) is
not more than 4.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Acetohexamide,
previously dried, dissolve in 30 mL of A^/V-dimethylfor-
mamide, add 10 mL of water, and titrate <2.50> with 0.1 mol
/L sodium hydroxide VS (potentiometric titration). Perform
a blank determination using a solution prepared by adding 19
mL of water to 30 mL of /V,A r -dimethylformamide, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 32.44 mg of C 15 H 20 N 2 O 4 S
Containers and storage Containers — Well-closed contain-
ers.
Acetylcholine Chloride for
Injection
aWfflT-tz^unio^fl:*
PigLr t-'Hri
CH,
C 7 H 16 C1N0 2 : 181.66
2-Acetoxy-7V,A r ,iV-trimethylethylaminium chloride
[60-31-1]
Acetylcholine Chloride for Injection is a preparation
for injection which is dissolved before use.
It contains not less than 98.0% and not more than
102.0% of acetylcholine chloride (C 7 H 16 C1N0 2 ), and
not less than 19.3% and not more than 19.8% of chlo-
rine (CI: 35.45), calculated on the dried basis.
It contains not less than 93% and not more than 107
% of the labeled amount of acetylcholine chloride
(C 7 H 16 C1N0 2 ).
Method of preparation Prepare as directed under Injec-
tions.
Description Acetylcholine Chloride for Injection occurs as
white crystals or crystalline powder.
It is very soluble in water, and freely soluble in ethanol
(95).
It is extremely hygroscopic.
Identification (1) Determine the infrared absorption
spectrum of Acetylcholine Chloride for Injection, previously
dried, as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(2) A solution of Acetylcholine Chloride for Injection
(1 in 10) responds to the Qualitative Tests <1.09> (2) for chlo-
ride.
Melting point <2.60> 149 - 152°C. Seal Acetylcholine
Chloride for Injection in a capillary tube for melting point
immediately after drying both of the sample and the tube at
105 C C for 3 hours, and determine the melting point.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Acetylcholine Chloride for Injection in 10 mL of water: the
solution is clear and colorless.
(2) Acidity — Dissolve 0.10 g of Acetylcholine Chloride
for Injection in 10 mL of freshly boiled and cooled water,
and add 1 drop of bromothymol blue TS, and 0.30 mL of
0.01 mol/L sodium hydroxide VS: the solution is blue in
color.
(3) Heavy metals <1.07> — Proceed with 2.0 g of Acetyl-
choline Chloride for Injection according to Method 1, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
Loss on drying <2.41>
hours).
Not more than 1.0% (1 g, 105 °C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Acetylcholine chloride — Weigh accurately the
contents of not less than 10 Acetylcholine Chloride for Injec-
tions. Weigh accurately about 0.5 g of the contents, dissolve
in 15 mL of water, then add exactly 40 mL of 0.1 mol/L sodi-
um hydroxide VS, stopper loosely, and heat on a water bath
for 30 minutes. Cool quickly, and titrate <2.50> the excess so-
dium hydroxide with 0.05 mol/L sulfuric acid VS (indicator:
3 drops of phenolphthalein TS). Perform a blank determina-
tion.
Each mL of 0.1 mol/L sodium hydroxide VS
= 18.17 mg of C 7 H 16 C1N0 2
(2) Chlorine — Titrate <2.50> the solution, which has been
titrated in (1), with 0.1 mol/L silver nitrate VS (indicator: 3
drops of fluorescein sodium TS).
Each mL of 0.1 mol/L silver nitrate VS
= 3.545 mg of CI
Containers and storage Containers — Hermetic containers.
272 Aclarubicin Hydrochloride / Official Monographs
JP XV
Aclarubicin Hydrochloride
TV^lltfi
'£K£
CH 3
OH • HC(
A °i I OH
°<u
C42H53NOU.HCI: 848.33
Methyl (li?,2i?,4S)-4-{2,6-dideoxy-4-0-[(2jR,6S)-6-
methyl-5-oxo-3,4,5,6-tetrahydro-2//-pyran-2-yl]-
a-L-/)>.ro-hexopyranosyl-(l->4)-2,3,6-trideoxy-3-
dimethylamino-a-L-/vxo-hexopyranosyloxy}-2-ethyl-2,5,7-
trihydroxy-6,ll-dioxo-l,2,3,4-tetrahydrotetracene-
1-carboxylate monohydrochloride [75443-99-0]
Aclarubicin Hydrochloride is the hydrochloride of
an anthracycline substance having antitumor activity
produced by the growth of Streptomyces galilaeus.
It contains not less than 920 /ug (potency) and not
more than 975 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Aclarubicin
Hydrochloride is expressed as mass (potency) of
aclarubicin (C42H53NOU: 811.87).
Description Aclarubicin Hydrochloride occurs as a yellow
to pale orange-yellow powder.
It is very soluble in chloroform and in methanol, freely
soluble in water, and slightly soluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Aclarubicin Hydrochloride in diluted methanol
(4 in 5) (3 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Aclarubicin Hydrochloride as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) A solution of Aclarubicin Hydrochloride in methanol
(1 in 200) responds to the Qualitative Tests <1.09> (2) for
chloride.
Optical rotation <2.49> [a£°: - 146 - - 162° (50 mg calculat-
ed on the anhydrous basis, water, 10 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving
0.05 g of Aclarubicin Hydrochloride in 10 mL of water is
between 5.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Aclarubicin Hydrochloride in 10 mL of water: the solution
is clear and yellow to pale orange-yellow.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Aclarubi-
cin Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 10 mg of Aclarubicin
Hydrochloride in 10 mL of the mobile phase, and use this
solution as the sample solution. Perform the test with 20 fiL
of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method. Calculate the amount of the related substances by
the area percentage method: the amount of aklavinone
having the relative retention time of about 0.6 to aclarubicin
is not more than 0.2%, aclacinomycin LI having the relative
retention time of about 0.75 to aclarubicin is not more than
0.5%, 1-deoxypyrromycin having the relative retention time
of about 1.7 to aclarubicin is not more than 1.5% and
aclacinomycin SI having the relative retention time of about
2.3 to aclarubicin is not more than 0.5%, and the total
amount of the peaks other than aclarubicin and the peaks
mentioned above is not more than 1.0% of the peak area of
aclarubicin.
Operating conditions —
Detector: A visible absorption photometer (wavelength:
436 nm).
Column: A stainless steel column 3.9 mm in inside
diameter and 30 cm in length, packed with silica gel for liquid
chromatography (10 ftm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of chloroform, methanol, acetic
acid (100), water and triethylamine (6800:2000:1000:200:1).
Flow rate: Adjust the flow rate so that the retention time of
aclarubicin is about 5 minutes.
Time span of measurement: As long as about 4 times of the
retention time of aclarubicin beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the solution for system suitability test.
Pipet 1 mL of the solution for system suitability test, and add
the mobile phase to make exactly 10 mL. Confirm that the
peak area of aclarubicin obtained from 20 fiL of this solution
is equivalent to 7 to 13% of that from 20 fiL of the solution
for system suitability test.
System performance: Dissolve 5 mg of Aclarubicin
Hydrochloride in 10 mL of 0.1 mol/L hydrochloric acid TS,
and allow to stand for 60 minutes. To 1 .0 mL of this solution
add 1.0 mL of 0.2 mol/L sodium hydroxide TS, 1.0 mL of
phosphate buffer solution, pH 8.0 and 1.0 mL of chlo-
roform, shake vigorously, and take the chloroform layer.
When the procedure is run with 20 fiL of the chloroform
under the above operating conditions, aclarubicin and 1-
deoxypyrromycin are eluted in this order with the resolution
between these peaks being not less than 3.0.
System repeatability: When the test is repeated 5 times with
20 [iL of the sample solution under the above operating
conditions, the relative standard deviation of the peak area of
JPXV
Official Monographs / Compound Acrinol and Zinc Oxide Oil
273
aclarubicin is not more than 2.0%.
Water <2.48> Not more than 3.5% (0.1 g, volumetric
titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Aclarubicin
Hydrochloride, equivalent to about 20 mg (potency), and dis-
solve in diluted methanol (4 in 5) to make exactly 100 mL.
Pipet 15 mL of this solution, add diluted methanol (4 in 5) to
make exactly 100 mL, and use this solution as the sample
solution. Separately, weigh accurately an amount of
Aclarubicin Reference Standard, equivalent to about 20 mg
(potency), add 0.6 mL of diluted hydrochloric acid (1 in 250)
and diluted methanol (4 in 5) to make exactly 100 mL. Pipet
15 mL of this solution, add diluted methanol (4 in 5) to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with the sample solution and standard
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and determine the absorbances, A T and A s , at
433 nm.
Amount L"g (potency)] of aclarubicin (C 4 2H53N0 15 )
= W s x (Aj/As) x 1000
W s : Amount [mg (potency)] of Aclarubicin Reference
Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant and at 5°C or below.
Acrinol and Zinc Oxide Oil
7^ l J/-Ji^-f>'?;i
Method of preparation
Acrinol, very finely powdered
Zinc Oxide Oil
10;
990;
To make 1000 g
Prepare by mixing the above ingredients.
Description Acrinol and Zinc Oxide Oil is a yellowish
white, slimy substance. Separation of a part of its ingredients
occurs on prolonged standing.
Identification (1) Shake well 1 g of Acrinol and Zinc
Oxide Oil with 10 mL of diethyl ether, 2 mL of acetic acid
(100) and 10 mL of water, and separate the water layer.
Shake the layer with 5 mL of hydrochloric acid and 2 to 3
drops of sodium nitrite TS, and allow to stand: a dark red
color is produced (acrinol).
(2) Place 1 g of Acrinol and Zinc Oxide Oil in a crucible,
melt by warming, heat, gradually raising the temperature
until the mass is thoroughly charred, and then ignite strongly:
a yellow color is produced, and disappears on cooling. To the
residue add 10 mL of water and 5 mL of dilute hydrochloric
acid, filter after thorough shaking, and to the filtrate add 2 to
3 drops of potassium hexacyanoferrate (II) TS: a white
precipitate is formed (zinc oxide).
(3) Shake well 0.2 g of Acrinol and Zinc Oxide Oil with
20 mL of ethanol (95) and 1 mL of acetic acid (100), cen-
trifuge, filter, and use the filtrate as the sample solution.
Separately, dissolve 5 mg of acrinol in 50 mL of ethanol (95)
and 2.5 mL of acetic acid (100), and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of 2-propanol and acetic acid (100) (9:1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 365 nm): the spots
from the sample solution and standard solution exhibit a blue
fluorescence and show the same Rf value.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Compound Acrinol and Zinc Oxide
Oil
a*7^U/-JL--5 1 >^»
Method of preparation
Acrinol, very finely powdered
Zinc Oxide Oil
Ethyl Aminobenzoate, finely powdered
White Beeswax
Hydrophilic Petrolatum
10 g
650 g
50 g
20 g
270 g
To make 1000 g
Prepare by mixing the above ingredients.
Description Compound Acrinol and Zinc Oxide Oil is light
yellow to yellow in color.
Identification (1) Shake well 1 g of Compound Acrinol
and Zinc Oxide Oil with 10 mL of diethyl ether, 2 mL of
acetic acid (100) and 10 mL of water, and separate the water
layer. Shake the layer with 5 mL of hydrochloric acid and 2
to 3 drops of sodium nitrite TS, and allow to stand: a dark
red color is produced (acrinol).
(2) Place 1 g of Compound Acrinol and Zinc Oxide Oil in
a crucible, melt by warming, heat, gradually raising the tem-
perature until the mass is thoroughly charred, and then ignite
strongly: a yellow color is produced, and disappears on
cooling. To the residue add 10 mL of water and 5 mL of
dilute hydrochloric acid, shake well, and filter. To the filtrate
add 2 to 3 drops of potassium hexacyanoferrate (II) TS: a
white precipitate is produced (zinc oxide).
(3) Shake well 0.2 g of Compound Acrinol and Zinc
Oxide Oil with 20 mL of ethanol (95) and 1 mL of acetic acid
(100), centrifuge, filter, and use the filtrate as the sample
solution. Separately, dissolve 5 mg of acrinol and 25 mg of
ethyl aminobenzoate in 50 mL of ethanol (95) and in 2.5 mL
of acetic acid (100), respectively, and use both solutions as
the standard solutions (1) and (2). Perform the test with these
solutions as directed under Thin-layer Chromatography <2.03
>. Spot 5 /uL each of the sample solution and standard solu-
tions on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of 2-propanol and acetic acid (100) (9:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 365 nm): the spots from the sample
solution and standard solution (1) exhibit a blue fluorescence,
and show the same Rf value. Also examine under ultraviolet
274
Acrinol and Zinc Oxide Ointment / Official Monographs
JP XV
light (main wavelength: 254 nm): the spots from the sample
solution and standard solution (2) exhibit a purple color, and
show the same Ri value.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Acrinol and Zinc Oxide Ointment
7?u/-;u-
&}»&?.»
Method of preparation
Acrinol, very finely powdered
Zinc Oxide Ointment
10 i
990;
To make 1000 g
Prepare as directed under Ointments, with the above
ingredients.
Description
color.
Acrinol and Zinc oxide Ointment is yellow in
Identification (1) Shake 0.5 g of Acrinol and Zinc Oxide
Ointment with 5 mL of diethyl ether, 5 mL of dilute
hydrochloric acid and 2 to 3 drops of sodium nitrite TS, and
allow to stand: a dark red color develops in the water layer
(acrinol).
(2) Ignite 0.5 g of Acrinol and Zinc Oxide Ointment to
char, and dissolve the residue in 5 mL of dilute hydrochloric
acid: the solution responds to the Qualitative Tests <1.09> for
zinc salt.
(3) Shake 0.5 g of Acrinol and Zinc Oxide Ointment with
5 mL of diethyl ether, 1 mL of acetic acid (100) and 5 mL of
water, separate the water layer, and use the water layer as the
sample solution. Dissolve 5 mg of acrinol in 1 mL of acetic
acid (100) and 5 mL of water, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of diethyl ether, ethanol (95) and acetic acid
(100) (40:10:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): the spots from the sample solution and the standard so-
lution exhibit a blue fluorescence and show the same Rf
value.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Acrinol Hydrate
Ethacridine Lactate
HjN
H 3 C COjH * H 2°
2-Ethoxy-6,9-diaminoacridine monolactate monohydrate
[1837-57-6]
Acrinol Hydrate contains not less than 98.5% and
not more than 101.0% of acrinol (QjH^NjO.CsHfA:
343.38), calculated on the anhydrous basis.
Description Acrinol Hydrate occurs as a yellow, crystalline
powder.
It is sparingly soluble in water, in methanol and in ethanol
(99.5).
Melting point: about 245°C (with decomposition).
The pH of a solution of Acrinol Hydrate (1 in 100) is be-
tween 5.5 and 7.0.
Identification (1) Determine the absorption spectrum of a
solution of Acrinol Hydrate (3 in 250,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Acrinol Hydrate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) To 5 mL of a solution of Acrinol Hydrate (1 in 100)
add 5 mL of dilute sulfuric acid, shake well, allow to stand
for about 10 minutes at room temperature, and filter: the
filtrate responds to the Qualitative Tests <1.09> for lactate.
Purity (1) Chloride <1.03>— Dissolve 1 .0 g of Acrinol Hy-
drate in 80 mL of water by warming on a water bath, cool,
and add 10 mL of sodium hydroxide TS and water to make
100 mL. Shake well, allow to stand for 30 minutes, filter, to
40 mL of the filtrate add 7 mL of dilute nitric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare 50 mL of the control solution with
4 mL of sodium hydroxide TS, 7 mL of dilute nitric acid,
0.30 mL of 0.01 mol/L hydrochloric acid VS and sufficient
water (not more than 0.026%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Acrinol
Hydrate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Volatile fatty acids — Dissolve 0.5 g of Acrinol Hy-
drate in a mixture of 20 mL of water and 5 mL of dilute sul-
furic acid, shake well, filter, and heat the filtrate: no odor of
volatile fatty acids is perceptible.
(4) Related substances — Dissolve 10 mg of Acrinol Hy-
JPXV
drate in 25 mL of the mobile phase, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution (1). Pipet 1 mL of the standard
solution (1), add the mobile phase to make exactly 10 mL,
and use this solution as the standard solution (2). Perform
the test with exactly 10 /uL each of the sample solution and
standard solutions (1) and (2) as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the area of the peak other than acrinol is not larger
than 3 times the peak area of acrinol obtained with the stan-
dard solution (2), and the total area of the peaks other than
acrinol is not larger than the peak area of acrinol with the
standard solution (1).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 7.8 g of sodium dihydrogen phos-
phate in 900 mL of water, adjust to pH 2.8 with phosphoric
acid, and add water to make 1000 mL. To 700 mL of this
solution add 300 mL of acetonitrile for liquid chro-
matography, and add 1.0 g of sodium 1-octanesulfonate to
dissolve.
Flow rate: Adjust the flow rate so that the retention time of
acrinol is about 15 minutes.
Time span of measurement: About 3 times as long as the
retention time of acrinol beginning after the solvent peak.
System suitability —
Test for required detectability: Confirm that the peak area
of acrinol obtained with 10 /xL of the standard solution (2) is
equivalent to 7 to 13% of that with 10 /xL of the standard
solution (1).
System performance: When the procedure is run with 10
fiL of the standard solution (1) under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of acrinol are not less than 5000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution (1) under the above operating
conditions, the relative standard deviation of the peak area of
acrinol is not more than 1.5%.
Water <2.48> 4.5 - 5.5% (0.2 g, volumetric titration, direct
titration)
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.27 g of Acrinol Hydrate,
dissolve in 5 mL of formic acid, add 60 mL of a mixture of a-
cetic anhydride and acetic acid (100) (1:1), and titrate <2.50>
immediately with 0.1 mol/L perchloric acid VS (potentiomet-
ric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.34 mg of acrinol (dsH^O^HsO,)
Containers and storage Containers — Tight containers.
Official Monographs / Actinomycin D 275
Storage — Light-resistant.
Actinomycin D
N !)— Th r-D-Val-Pro-MeQ ly-MeVal
H
-Th r-D- Val-Pro-MeGly-Ma Val '
MeGly =
MeVal =
W-Methylglycine
W-Methylvaline
C 62 H 86 N 12 16 : 1255.42
[50-76-0]
Actinomycin D is a peptide substance having an-
titumor activity produced by the growth of Strep-
tomyces parvulus.
It, when dried, contains not less than 950 tig
(potency) and not more than 1030 Lig (potency) per mg.
The potency of Actinomycin D is expressed as mass
(potency) of actinomycin D (C 62 H g6 N 12 16 ).
Description Actinomycin D occurs as an orange-red to red
crystalline powder.
It is freely soluble in acetone, sparingly soluble in acetoni-
trile and in methanol, slightly soluble in ethanol (99.5), and
very slightly soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Actinomycin D in methanol (3 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Actinomycin D Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Dissolve 0.1 g each of Actinomycin D and Actinomy-
cin D Reference Standard in 10 mL of acetone, and use these
solutions as the sample solution and standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 [iL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of 1-butanol, water and methanol
(4:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the Rf value of the principal spot from the sample solution is
the same as that from the standard solution.
Optical rotation <2.49> [ce]l
■292- -317° (after drying,
10 mg, methanol, 10 mL, 100 mm).
Loss on drying <2.41> Not more than 5.0% (1 g, in vacuum,
60°C, 3 hours).
Assay Weigh accurately an amount of Actinomycin D
and Actinomycin D Reference Standard, previously dried,
equivalent to about 60 mg (potency), dissolve each in the
mobile phase to make exactly 50 mL, and use these solutions
as the sample solution and standard solution. Perform the
276 Adrenaline / Official Monographs
JP XV
test with exactly 25 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak area of actinomycin D, A T and A s , of both solu-
tions.
Amount Lug (potency)] of C 62 H 86 N 12 16
= W s x (Aj/As) x 1000
W s : Amount [mg (potency)] of Actinomycin D Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.02 mol/L acetic acid-sodium
acetate TS and acetonitrile (25:23).
Flow rate: Adjust the flow rate so that the retention time of
actinomycin D is about 23 minutes.
System suitability —
System performance: When the procedure is run with
25 /xL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of actinomycin D are not less than 2000
steps and not more than 1.5, respectively.
System repeatability: When the test is repeated 5 times with
25 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
actinomycin D is not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Adrenaline
Epinephrine
CH 3
C 9 H 13 N0 3 : 183.20
4-[(li?)-l-Hydroxy-2-(methylamino)ethyl]benzene-l,2-diol
[51-43-4]
Adrenaline, when dried, contains not less than
98.0% of C 9 H 13 N0 3 .
Description Adrenaline occurs as a white to grayish white,
crystalline powder. It has no odor.
It is freely soluble in acetic acid (100), very slightly soluble
in water, and practically insoluble in methanol, in ethanol
(95) and in diethyl ether.
It dissolves in dilute hydrochloric acid.
It gradually changes to brown in color by air and by light.
Identification (1) Dissolve 0.01 g of Adrenaline in 10 mL
of diluted acetic acid (31) (1 in 500), and use this solution as
the sample solution. To 1 mL of the sample solution add 4
mL of water and 1 drop of iron (III) chloride TS: a deep
green color is produced, and it gradually changes to red.
(2) Place 1 mL each of the sample solution obtained in (1)
in test tubes A and B. Add 10 mL of potassium hydrogen
phthalate buffer solution, pH 3.5, to A, and add 10 mL of
phosphate buffer solution, pH 6.5, to B. To each of the test
tubes add 1 mL of iodine TS, allow to stand for 5 minutes,
and add 2 mL each of sodium thiosulfate TS: a red color de-
velops in test tube A, and a deep red color develops in test
tube B.
Optical rotation <2.49> [a]™: -50.0- -53.5° (after
drying, 1 g, 1 mol/L hydrochloric acid TS, 25 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Adrenaline in 10 mL of dilute hydrochloric acid: the solu-
tion is clear, and is not more colored than Matching Fluid A.
(2) Adrenalone — Dissolve 50 mg of Adrenaline in 0.05
mol/L hydrochloric acid TS to make exactly 25 mL, and de-
termine the absorbance of this solution at 310 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it is not
more than 0.40.
(3) Noradrenaline — Dissolve 10.0 mg of Adrenaline in
2.0 mL of a L-tartaric acid solution (1 in 200). Pipet 1 mL of
the solution, add 3.0 mL of pyridine, then add 1.0 mL of
freshly prepared sodium naphthoquinone sulfonate TS, and
allow to stand in a dark place for 30 minutes. To this solution
add 5.0 mL of pyridine containing 0.05 g of L-ascorbic acid:
the solution is not more colored than the following control
solution.
Control solution: Dissolve 2.0 mg of Noradrenaline Bitar-
trate Reference Standard and 90 mg of Adrenaline Bitartrate
Reference Standard in methanol to make exactly 10 mL.
Pipet 1 mL of this solution, and proceed in the same manner.
Loss on drying <2.41> Not more than 1.0% (2 g, in vacuum,
silica gel, 18 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Adrenaline, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 2 drops
of crystal violet TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.32 mg of C 9 H 13 N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant, under nitrogen atmosphere, and
in a cold place.
Adrenaline Injection
Epinephrine Injection
Adrenaline Injection is aqueous solution for injec-
tion.
It contains not less than 0.085 w/v% and not more
JPXV
Official Monographs / Afloqualone 277
than 0.115 w/v% of adrenaline (C 9 H 13 N0 3 : 183.20).
Method of preparation Dissolve Adrenaline in diluted
Hydrochloric Acid (9 in 10,000), and prepare as directed un-
der Injections.
Description Adrenaline Injection is a colorless, clear liquid.
It changes gradually to pale red and then to brown on
exposure to air and light.
pH: 2.3-5.0
Identification (1) To 1 mL of Adrenaline Injection add 4
mL of water and 1 drop of iron (III) chloride TS: a deep
green color is produced, and it gradually changes to red.
(2) Place 1 mL each of Adrenaline Injection in test tubes
A and B, and proceed as directed in the Identification (2)
under Adrenaline.
Extractable volume <6.05> It meets the requirement.
Assay Pipet 30 mL of Adrenaline Injection into a separa-
tor, add 25 mL of carbon tetrachloride, shake vigorously for
1 minute, allow the liquids to separate, and discard the car-
bon tetrachloride. Repeat this procedure three times. Rinse
the stopper and mouth of the separator with a small amount
of water. Add 0.2 mL of starch TS, then while swirling the
separator add iodine TS dropwise until a persistent blue color
develops, and immediately add sodium thiosulfate TS to dis-
charge the blue color. Add 2.1 g of sodium hydrogen car-
bonate to the liquid in the separator, preventing it from com-
ing in contact with the mouth of the separator, and shake
until most of the sodium hydrogen carbonate dissolves.
Rapidly inject 1.0 mL of acetic anhydride into the contents of
the separator. Immediately stopper the separator loosely, and
allow to stand until the evolution of gas ceases. Shake
vigorously, allow to stand for 5 minutes, extract with six
25-mL portions of chloroform, and filter each chloroform
extract through a pledget of absorbent cotton. Evaporate the
combined chloroform extracts on a water bath in a current of
air to 3 mL, completely transfer this residue by means of
small portions of chloroform to a tared beaker, and heat
again to evaporate to dryness. Dry the residue at 105 °C for
30 minutes, cool in a desiccator (silica gel), and accurately
measure the mass W (mg) of the dried residue. Dissolve in
chloroform to make exactly 5 mL, and determine the optical
rotation <2.49> [«]£> using a 100-mm cell.
Amount (mg) of adrenaline (C 9 H 13 N0 3 )
= H^x{0.5 + (0.5 x |[a]^ |)/93}x 0.5923
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
•Hermetic containers,
Adrenaline Solution
Epinephrine Solution
Adrenaline Solution contains not less than 0.085
w/v% and not more than 0.115 w/v% of adrenaline
(C 9 H 13 N0 3 : 183.20)
Method of preparation
Adrenaline 1 g
Sodium Chloride 8.5 g
Diluted Hydrochloric Acid (9 in 100) 10 mL
Stabilizer a suitable quantity
Preservative a suitable quantity
Purified Water a sufficient quantity
To make
Prepare by mixing the above ingredients.
1000 mL
Description Adrenaline Solution is clear, colorless or slight-
ly reddish liquid.
It changes gradually to pale red and then to brown on
exposure to air and light.
pH: 2.3-5.0
Identification Proceed as directed in the Identification
under Adrenaline Injection.
Assay Proceed as directed in the Assay under Adrenaline
Injection.
Amount (mg) of adrenaline (C 9 H 13 N0 3 )
= W*{0.5 + (0.5 x |[«]^|)/93}x 0.5923
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Afloqualone
77P^7n>
C 16 H 14 FN 3 0: 283.30
6-Amino-2-fluoromethyl-3-(2-tolyl)-3//-quinazolin-4-one
[56287-74-2]
Afloqualone, when dried, contains not less than
98.5% of C 16 H 14 FN 3 0.
Description Afloqualone occurs as white to light yellow
crystals or crystalline powder.
It is soluble in acetonitrile, sparingly soluble in ethanol
(99.5), and practically insoluble in water.
It is gradually colored by light.
Melting point: about 197°C (with decomposition).
278
Ajmaline / Official Monographs
JP XV
Identification (1) Conduct this procedure without ex-
posure to light, using light-resistant containers. Determine
the absorption spectrum of a solution of Afloqualone in
ethanol (99.5) (1 in 150,000) as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Afloqualone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Purity (1) Acidity or alkalinity — Take 1.0 g of Afloqua-
lone in a light-resistant vessel, add 20 mL of freshly boiled
and cooled water, shake well, and filter. To 10 mL of the
filtrate add 2 drops of bromothymol blue TS: a yellow color
develops. The color changes to blue by adding 0.20 mL of
0.01 mol/L sodium hydroxide TS.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Afloqua-
lone in a platinum crucible according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve 10 mg
of Afloqualone in 25 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 3 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL. Pipet 2
mL of this solution, add the mobile phase to make exactly 20
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the areas of each peak by the automatic integration method:
the total of the peak areas other than the peak area of aflo-
qualone from the sample solution is not more than the peak
area of afloqualone from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 7.2 g of disodium hydrogen
phosphate dodecahydrate in 1000 mL of water, adjust to pH
5.5 with diluted phosphoric acid (1 in 10). To 600 mL of this
solution add 400 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
afloqualone is about 5.5 minutes.
Time span of measurement: About 4 times as long as the
retention time of afloqualone beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 5 mL of the standard
solution, add the mobile phase to make exactly 25 mL, and
confirm that the peak area of afloqualone obtained from 20
/uL of this solution is equivalent to 15 to 25% of that of
afloqualone obtained from 20 iiL of the standard solution.
System performance: Dissolve 0.01 g of Afloqualone in a
suitable amount of the mobile phase, add 5 mL of a solution
of propyl parahydroxybenzoate in the mobile phase (1 in
2000) and the mobile phase to make 100 mL. When the
procedure is run with 20 fiL of this solution under the above
operating conditions, afloqualone and propyl parahydrox-
ybenzoate are eluted in this order with the resolution between
these peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of afloqualone is not more than 5%.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, 2 hours).
Residue on ignition <2.44> Not more than 0.1% (1.0 g,
platinum crucible).
Assay Weigh accurately about 0.4 g of Afloqualone, previ-
ously dried, dissolve in 10 mL of hydrochloric acid and 40
mL of water, and add 10 mL of a solution of potassium
bromide (3 in 10). After cooling at 15°C or below, titrate
<2.50> with 0.1 mol/L sodium nitrite VS according to the
potentiometric titration or amperometric titration under the
Electrometric Titration method.
Each mL of 0.1 mol/L sodium nitrite
= 28.33 mg of C, 6 H 14 FN 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ajmaline
7yvy
C 20 H 26 N 2 O 2 : 326.43
(17i?,21i?)-Ajmalan-17,21-diol [4360-12-7]
Ajmaline, when dried, contains not less than 96.0%
of C 20 H 26 N 2 O 2 .
Description Ajmaline occurs as a white to pale yellow,
crystalline powder. It is odorless, and has a bitter taste.
It is freely soluble in acetic anhydride and in chloroform,
sparingly soluble in methanol, in ethanol (95), in acetone and
in diethyl ether, and very slightly soluble in water.
It dissolves in dilute hydrochloric acid.
Melting point: about 195°C (with decomposition).
Identification (1) Dissolve 0.05 g of Ajmaline in 5 mL of
methanol, and use this solution as the sample solution. Add 3
mL of nitric acid to 1 mL of the sample solution: a deep red
color develops.
(2) Spot the sample solution of (1) on filter paper, and
spray Dragendorff's TS: an orange color develops.
Absorbance <2.24> E\ a/ ° m (249 nm): 257 - 271 (after drying,
2mg, ethanol (95), 100 mL).
JPXV
Official Monographs / Alacepril 279
EY° m (292 nm): 85-95 (after drying, 2 mg, ethanol (95),
100 mL).
Optical rotation <2.49> [a]™ : + 136 - + 151° (after drying,
0.5 g, chloroform, 50 mL, 100 mm).
Purity Related substances — Dissolve 0.10 g of Ajmaline in
10 mL of chloroform, and use this solution as the sample so-
lution. Pipet 1 mL of this solution, add chloroform to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform, acetone and
diethylamine (5:4:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 1.0% (0.6 g, in vacu-
um, 80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 0.3 g of Ajmaline, previous-
ly dried, dissolve in 50 mL of acetic anhydride and 50 mL of
acetone for nonaqueous titration, and titrate <2.50> with 0.05
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 16.32 mg of C 20 H 26 N 2 O 2
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Ajmaline Tablets
7-/vU>i£
Ajmaline Tablets contain not less than 90% and not
more than 110% of the labeled amount of ajmaline
(C 20 H 26 N 2 O 2 : 326.43).
Method of preparation Prepare as directed under Tablets,
with Ajmaline.
Identification (1) Shake a quantity of powdered Ajmaline
Tablets, equivalent to 0.1 g of Ajmaline according to the
labeled amount, with 30 mL of chloroform, and filter.
Evaporate the filtrate on a water bath to dryness. With the
residue, proceed as directed in the Identification under Ajma-
line.
(2) Dissolve 0.01 g of the residue of (1) in 100 mL of
ethanol (95). To 10 mL of this solution add ethanol (95) to
make 50 mL, and determine the absorption spectrum of the
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits maxima between 247 nm and 251 nm
and between 291 nm and 294 nm, and a minimum between
269 nm and 273 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Ajmaline Tablets at 100
revolutions per minute according to the Paddle method using
900 mL of 2 nd fluid for dissolution test as the test solution.
Take 20 mL or more of the dissolved solution 60 minutes af-
ter start of the test, and filter through a membrane filter with
a pore size not exceeding 0.8 /xm. Discard the first 10 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. Separately, weigh accurately about 28 mg of ajmaline
for assay, previously dried in vacuum at 80°C for 3 hours,
dissolve in 2 nd fluid for dissolution test to make exactly 500
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution at 288 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate of Ajmaline
Tablets in 60 minutes is not less than 75%.
Dissolution rate (%) with respect to the
labeled amount of ajmaline (C 20 H 26 N 2 O 2 )
= W s x (Aj/As) x (l/C) x 180
W s : Amount (mg) of ajmaline for assay.
C: Labeled amount (mg) of ajmaline (C 20 H 26 N 2 O 2 ) in 1
tablet.
Assay Weigh accurately and powder not less than 20 Ajma-
line Tablets. Weigh accurately a portion of the powder,
equivalent to about 0.3 g of ajmaline (C 20 H 26 N 2 O 2 ), add 15
mL of ammonia solution (28), and extract with four 25-mL
portions of chloroform. Combine the chloroform extracts,
wash with 10 mL of water, add 5 g of anhydrous sodium
sulfate, shake well, and filter. Wash the container and the
residue with two 10-mL portions of chloroform, and filter.
Evaporate the combined filtrate on a water bath to dryness,
dissolve the residue in 50 mL of acetic anhydride and 50 mL
of acetone for nonaqueous titration, and titrate <2.50> with
0.05 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.05 mol/L perchloric acid VS
= 16.32 mg of C 20 H 26 N 2 O 2
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Alacepril
77-te^'JJU
H CH 3
/ N v A ,
Oh « C03H
C 20 H 26 N 2 O 5 S: 406.50
(2S)-2-{(2S)-l-[(2S)-3-(Acetylsulfanyl)-
2-methylpropanoyl]pyrrolidine-2-carbonyl}amino-
3-phenylpropanoic acid
[74258-86-9]
Alacepril, when dried, contains not less than 98.5%
and not more than 101.0% of C 20 H 26 N 2 O 5 S.
280
Alacepril Tablets / Official Monographs
JP XV
Description Alacepril occurs as white crystals or crystalline
powder.
It is freely soluble in methanol, soluble in ethanol (95), and
slightly soluble in water.
It dissolves in sodium hydroxide TS.
Identification (1) To 20 mg of Alacepril add 0.1 g of sodi-
um hydroxide, and heat gradually to melt: the gas evolved
changes the color of a moisten red litmus paper to blue. After
cooling, to the melted substance add 2 mL of water, shake,
and add 1 mL of lead (II) acetate TS: a brown to black
precipitate is formed.
(2) Determine the infrared absorption spectrum of
Alacepril, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]™: -81-
0.25 g, ethanol (95), 25 mL, 100 mm).
Melting point <2.60> 153 - 157°C
■85° (after drying,
Purity (1) Chloride <1.03> — Dissolve 0.5 g of Alacepril in
30 mL of methanol, add 6 mL of dilute nitric acid and water
to make 50 mL, and perform the test with this solution as the
test solution. Prepare the control solution as follows: to 0.30
mL of 0.01 mol/L hydrochloric acid VS add 30 mL of
methanol, 6 mL of dilute nitric acid and water to make 50
mL (not more than 0.021%).
(2) Sulfate <1.14>— Dissolve 0.5 g of Alacepril in 30 mL
of methanol, add 1 mL of dilute hydrochloric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.50 mL of 0.005 mol/L sulfuric acid VS add 30 mL of
methanol, 1 mL of dilute hydrochloric acid and water to
make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Alacepril
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Related substances — Dissolve 50 mg of Alacepril in 5
mL of ethanol (95), and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add ethanol (95) to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with exactly 10 fiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the area of the peak other than alacepril from
the sample solution is not larger than 2/5 times the peak area
of alacepril from the standard solution, and the total area of
the peaks other than the peak of alacepril from the sample so-
lution is not larger than the peak area of alacepril from the
standard solution. For this calculation, use the areas of the
peaks, having the relative retention time of about 2.3 and
about 2.6 with respect to alacepril, after multiplying by their
sensitivity factors, 1.5 and 1.9, respectively.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fun in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (100) (1 in
100), acetonitrile, methanol and tetrahydrofuran (6:2:1:1).
Flow rate: Adjust the flow rate so that the retention time of
alacepril is about 5 minutes.
Time span of measurement: About 3 times as long as the
retention time of alacepril beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 4 mL of the
standard solution add ethanol (95) to make exactly 10 mL.
Confirm that the peak area of alacepril obtained with 10 fiL
of this solution is equivalent to 30 to 50% of that with 10 /uL
of the standard solution.
System performance: Dissolve 20 mg of Alacepril in 50 mL
of a solution of propyl parahydroxybenzoate in ethanol (95)
(1 in 80,000). When the procedure is run with lO^L of this
solution under the above operating conditions, alacepril and
propyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 7.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
alacepril is not more than 2.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Alacepril, previous-
ly dried, dissolve in 75 mL of a mixture of methanol and
water (2:1), and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (potentiometric titration). Perform a blank de-
termination in the same manner, and make any necessary
correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.65 mg of C 20 H 26 N 2 O 5 S
Containers and storage Containers — Tight containers.
Alacepril Tablets
Alacepril Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of
alacepril (C 20 H 26 N 2 O 5 S: 406.50).
Method of preparation Prepare as directed under Tablets,
with Alacepril.
Identification Shake well a quantity of powdered Alacepril
Tablets, equivalent to 0.1 g of Alacepril according to the la-
beled amount, with 10 mL of ethanol (95), filter, and use the
filtrate as the sample solution. Separately, dissolve 10 mg of
alacepril in 1 mL of ethanol (95), and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography, develop the plate with a mixture of ethanol
JPXV
Official Monographs / Alacepril Tablets
281
(99.5) and hexane (2:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the principal spot from the sample so-
lution and the spot from the standard solution show the same
color tone and Ri value.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Alacepril Tablets add 2 mL of water, dis-
perse the particle with the aid of ultrasonic wave, and add ex-
actly 2 mL of the internal standard solution every 10 mg of
alacepril (C20H26N2O5S) according to the labeled amount. To
this solution add a suitable amount of methanol, extract for
15 minutes with the aid of ultrasonic wave while occasional
shaking, and shake more 15 minutes. Add methanol to make
exactly FmL so that each mL of the solution contains about
0.5 mg of alacepril (C20H26N2O5S), centrifuge, and use the su-
pernatant liquid as the sample solution. Separately, weigh ac-
curately about 25 mg of alacepril for assay, previously dried
at 105 °C for 3 hours, add exactly 5 mL of the internal stan-
dard solution and methanol to make 50 mL, and use this so-
lution as the standard solution. Perform the test with 10 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Q T and Q s , of
the peak area of alacepril to that of the internal standard.
Amount (mg) of alacepril ^oH^NjOsS)
= W s x (Qj/Q s ) x (K/50)
W s : Amount (mg) of alacepril for assay
Internal standard solution — A solution of propyl para-
hydroxybenzoate in methanol (3 in 20,000).
Operating conditions —
Proceed as directed in the operating conditions in the As-
say.
System suitability—
Proceed as directed in the system suitability in the Assay.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Alacepril Tablets at 50
revolutions per minute according to the Paddle method using
900 mL of water as the dissolution medium. Withdraw not
less than 20 mL of the dissolution medium 30 minutes after
start of the test, and filter through a membrane filter with a
pore size not exceeding 0.45 11m. Discard the first 10 mL of
the filtrate, pipet KmL of the subsequent filtrate, add water
to make exactly V mL so that each mL contains about 14 11%
of alacepril (C20H26N2O5S) according to the labeled amount,
and use this solution as the sample solution. Separately,
weigh accurately about 14 mg of alacepril for assay, previ-
ously dried at 105°C for 3 hours, dissolve in 2 mL of
methanol, and add water to make exactly 100 mL. Pipet 5
mL of this solution, add water to make exactly 50 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A Tl and A sl , at 230 nm, and A T2 and A S2 , at 300
nm, of the sample solution and standard solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>. The dis-
solution rates for a 12.5-mg tablet and a 25-mg tablet in 30
minutes are not less than 75%, respectively, and that for a
50-mg tablet in 30 minutes is not less than 70%.
alacepril (C 2 oH 2 6N20 5 S)
= W s x ^ T '~^ T2 x ^ x I x 90
^ S1 -^ S2 V C
W s : Amount (mg) of alacepril for assay
C: Labeled amount (mg) of alacepril (C2oH 26 N 2 05S) in 1
tablet
Assay Weigh accurately, and powder not less than 20
Alacepril Tablets. Weigh accurately a portion of the powder,
equivalent to about 50 mg of alacepril (C20H26N2O5S),
moisten with 2 mL of water, add exactly 3 mL of the internal
standard solution and 40 mL of methanol, extract for 15
minutes with the aid of ultrasonic wave, cool, and add
methanol to make 50 mL. Centrifuge this solution, and use
the supernatant liquid as the sample solution. Separately,
weigh accurately about 50 mg of alacepril for assay, previ-
ously dried at 105 °C for 3 hours, add exactly 3 mL of the in-
ternal standard solution, dissolve with methanol to make 50
mL, and use this solution as the standard solution. Perform
the test with 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qt and Q s , of the peak area of alacepril to that of the internal
standard.
Amount (mg) of alacepril (C 20 H2 6 N 2 O 5 S) = W s x (Q T /Q S )
W s : Amount (mg) of alacepril for assay
Internal standard solution — A solution of propyl para-
hydroxybenzoate in methanol (3 in 20,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (100) (1 in
100), acetonitrile, methanol and tetrahydrofuran (13:5:1:1).
Flow rate: Adjust the flow rate so that the retention time of
alacepril is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, alacepril and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of alacepril to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Dissolution rate (%) with respect to the labeled amount of
282
Albumin Tannate / Official Monographs
JP XV
Albumin Tannate
Albumin Tannate is a compound of tannic acid and
a protein.
The label states the origin of the protein of Albumin
Tannate.
Description Albumin Tannate occurs as a light brown pow-
der. It is odorless, or has a faint, characteristic odor.
It is practically insoluble in water and in ethanol (95).
It dissolves in sodium hydroxide TS with turbidity.
Identification (1) To 0.1 g of Albumin Tannate add 10
mL of ethanol (95), and heat in a water bath for 3 minutes
with shaking. After cooling, filter, and to 5 mL of the filtrate
add 1 drop of iron (III) chloride TS: a blue-purple to bluish
black color is produced. On standing, a bluish black
precipitate is produced.
(2) To 0.1 g of Albumin Tannate add 5 mL of nitric acid:
an orange-yellow color develops.
Purity (1) Acidity — Shake 1 .0 g of Albumin Tannate with
50 mL of water for 5 minutes, and filter. To 25 mL of the
filtrate add 1 .0 mL of 0. 1 mol/L sodium hydroxide VS and 2
drops of phenolphthalein TS: a red color develops.
(2) Fats— To 2.0 g of Albumin Tannate add 20 mL of
petroleum benzine, shake vigorously for 15 minutes, and
filter. Evaporate 10 mL of the filtrate on a water bath: the
mass of the residue is not more than 50 mg.
Loss on drying <2.41> Not more than 6.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 1.0% (0.5 g).
Digestion test To 1.00 g of Albumin Tannate add 0.25 g of
saccharated pepsin and 100 mL of water, shake well, and
allow to stand for 20 minutes at 40 ± 1°C in a water bath.
Add 1.0 mL of dilute hydrochloric acid, shake, and allow to
stand for 3 hours at 40 ± 1°C. Cool rapidly to ordinary
temperature, and filter. Wash the residue with three 10-mL
portions of water, dry in a desiccator (silica gel) for 18 hours,
and dry at 105°C for 5 hours: the mass of the residue is 0.50
to 0.58 g.
Aldioxa
Containers and storage Containers-
Storage — Light-resistant.
-Tight containers.
>*
H;N
C 4 H 7 A1N 4 5 : 218.10
Dihydroxo(5-oxo-4-ureido-4,5-dihydro-l//-imidazol-
2-yl)oxoaluminium [5579-81-7]
Aldioxa is a condensation product of allantoin and
aluminum hydroxide.
When dried, it contains not less than 65.3% and not
more than 74.3% of allantoin (C 4 H 6 N 4 3 : 158.12), and
not less than 11.1% and not more than 13.0% of alu-
minum (Al: 26.98).
Description Aldioxa occurs as a white powder. It is odorless
and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute nitric
acid.
Melting point: about 230°C (with decomposition).
Identification (1) To 0.2 g of Aldioxa add 10 mL of dilute
hydrochloric acid, boil for 5 minutes, and add 10 mL of a
solution of phenylhydrazinium chloride (1 in 100). After
cooling, mix well with 0.5 mL of potassium hexacyanoferrate
(III) TS, and shake with 1 mL of hydrochloric acid: a red
color develops.
(2) To 0.2 g of Aldioxa add 10 mL of dilute hydrochloric
acid, dissolve by warming, and cool: the solution responds to
the Qualitative Tests <1.09> for aluminum salt.
Purity (1) Chloride <I.03>— To 0.10 g of Aldioxa add 6
mL of dilute nitric acid, boil to dissolve with shaking for 5
minutes, cool, and add water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.40 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.142%).
(2) Sulfate <I.I4>— To 0.20 g of Aldioxa add 6 mL of
dilute hydrochloric acid, boil to dissolve with shaking for 5
minutes, cool, and add water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the
control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS
(not more than 0.240%).
(3) Nitrate— To 0.10 g of Aldioxa add carefully 5 mL of
water and 5 mL of sulfuric acid, dissolve by shaking, cool,
and superimpose 2 mL of iron (II) sulfate TS: no brown ring
is produced at the zone of contact.
(4) Heavy metals <1.07> — To 1.0 g of Aldioxa add 3 mL
of hydrochloric acid and 3 mL of water, heat gently to boil
with shaking, and evaporate on a water bath to dryness. To
the residue add 30 mL of water, shake under warming, cool,
filter, and to the filtrate add 2 mL of dilute acetic acid (31)
and water to make 50 mL. Perform the test using this
solution as the test solution. Prepare the control solution as
follows: to 3 mL of hydrochloric acid add 3 mL of water,
JPXV
Official Monographs / Alimemazine Tartrate
283
evaporate on a water bath to dryness, and add 2.0 mL of
Standard Lead Solution, 2 mL of dilute acetic acid (31) and
water to make 50 mL (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Aldioxa according to Method 2, and perform the test (not
more than 2 ppm).
Loss on drying <2.41>
2 hours).
Not more than 4.0% (1 g, 105°C,
Assay (1) Allantoin — Weigh accurately about 0.1 g of
Aldioxa, previously dried, dissolve in 50 mL of dilute sulfuric
acid by heating, cool, and add water to make exactly 100 mL.
Pipet 10 mL of this solution, and perform the test as directed
under Nitrogen Determination <1.08>.
Each mL of 0.005 mol/L sulfuric acid VS
= 0.3953 mg of C 4 H 6 N 4 3
(2) Aluminum — Weigh accurately about 0.2 g of Aldiox-
a, previously dried, dissolve carefully in 50 mL of dilute
hydrochloric acid by heating, cool, and add dilute
hydrochloric acid to make exactly 100 mL. Pipet 4 mL of this
solution, add water to make exactly 25 mL, and use this
solution as the sample solution. Separately, pipet a suitable
quantity of Standard Aluminum Stock Solution, dilute with
water so that each mL of the solution contains not less than
16.0 fig and not more than 64.0 /ug of aluminum (Al: 26.98),
and use this solution as the standard solution. Perform the
test with the sample solution and standard solution as direct-
ed under Atomic Absorption Spectrophotometry <2.23> ac-
cording to the following conditions, and calculate the
aluminum content of the sample solution from the calibra-
tion curve obtained from the absorbance of the standard
solution.
Gas: Combustible gas — Acetylene
Supporting gas — Nitrous oxide
Lamp: An aluminum hollow cathode lamp
Wavelength: 309.2 nm
Containers and storage Containers — Well-closed contain-
ers.
Alimemazine Tartrate
7^7y>i5i^
CH 3 CH3
H0 2 C
COjH
OH
(C 18 H 22 N 2 S) 2 .C 4 H 6 6 : 746.98
A r ,7V,2-Trimethyl-3-(10//-phenothiazin-10-
yl)propylamine hemitartrate [41375-66-0]
Alimemazine Tartrate, when dried, contains not less
than 98.0% of (C 18 H 22 N 2 S) 2 .C 4 H 6 6 .
Description Alimemazine Tartrate occurs as a white
powder. It is odorless, and has a bitter taste.
It is freely soluble in water and in acetic acid (100), sparing-
ly soluble in ethanol (95), and practically insoluble in diethyl
ether.
The pH of a solution of Alimemazine Tartrate (1 in 50) is
between 5.0 and 6.5.
It is gradually colored by light.
Identification (1) To 2 mL of a solution of Alimemazine
Tartrate (1 in 100) add 1 drop of iron (III) chloride TS: a red-
brown color is produced, and immediately a yellow
precipitate is formed.
(2) Dissolve 1 g of Alimemazine Tartrate in 5 mL of
water, add 3 mL of sodium hydroxide TS, extract with two
10-mL portions of diethyl ether [use the aqueous layer ob-
tained in the Identification (4)]. Shake the combined diethyl
ether extracts with 3 g of anhydrous sodium sulfate, filter,
and evaporate the diethyl ether with the aid of a current of
air. Dry the residue in a desiccator (in vacuum, phosphorus
(V) oxide) for 16 hours: it melts <2.60> between 66°C and
70°C.
(3) Determine the absorption spectrum of a solution of
Alimemazine Tartrate (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) The aqueous layer, obtained in the identification (2),
when neutralized with dilute acetic acid, responds to the
Qualitative Tests <1.09> (1) and (2) for tartrate.
Melting point <2.60> 159 - 163 C C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Alimemazine Tartrate in 20 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Alimemazine Tartrate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Alimemazine Tartrate according to Method 3, and per-
form the test. Use a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 5) (not more than 2 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Alimemazine
Tartrate, previously dried, dissolve in 50 mL of acetic acid
(100), and titrate <2.50> with 0.1 mol/L perchloric acid VS
until the color of the solution changes from red through
brown to green-brown (indicator: 2 mL of p-naphtholben-
zein TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 37.35 mg of (C 18 H 22 N 2 S) 2 .C 4 H 6 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
284
Allopurinol / Official Monographs
JP XV
Allopurinol
n.
OH
C5H4N4O: 136.11
l//-Pyrazolo[3,4-(/]pyrimidin-4-ol
[575-50-0]
Allopurinol, when
98.0% of CH4N4O.
dried, contains not less than
Description Allopurinol occurs as white to pale yellowish
white crystals or crystalline powder. It is odorless.
It is slightly soluble in 7V,iV-dimethylformamide, very
slightly soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
It dissolves in sodium hydroxide TS and in ammonia TS.
Melting point: not lower than 320°C (with decomposition).
Identification (1) Dissolve 0.1 g of Allopurinol in 50 mL
of water by warming. To 5 mL of this solution add 1 mL of
ammonia TS and 1 mL of silver nitrate TS: a white
precipitate is produced.
(2) Dissolve 0.1 g of Allopurinol in 50 mL of water by
warming. To 5 mL of this solution add 0.5 mL of copper (II)
sulfate TS: a blue precipitate is produced.
(3) Determine the absorption spectrum of a solution of
Allopurinol (1 in 200,000) as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Allopurinol in 10 mL of sodium hydroxide TS: the solution is
clear, and has no more color than Matching Fluid D.
(2) Sulfate <1.14>— To 2.0 g of Allopurinol add 100 mL
of water, and boil for 5 minutes. Cool, add water to make
100 mL, and filter. To 25 mL of the filtrate add 1 mL of
dilute hydrochloric acid and water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 0.40 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.038%).
(3) Heavy metals <1.07>— Proceed with 1.0 g of Al-
lopurinol according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Allopurinol according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.05 g of Allopurinol in
10 mL of ammonia TS, and use this solution as the sample
solution. Pipet 1 mL of this solution, add ammonia TS to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 5 [iL each of
the sample solution and standard solution on a plate of cellu-
lose with fluorescent indicator for thin-layer chro-
matography. Develop the plate with ammonia TS-saturated
1-butanol to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0. 16 g of Allopurinol, previ-
ously dried, dissolve in 70 mL of A r ,A L dimethylformamide by
warming. Cool, and titrate <2.50> with 0.1 mol/L
tetramethylammonium hydroxide VS (potentiometric titra-
tion). To 70 mL of A^A^-dimethylformamide add 12 mL of
water, perform a blank determination, and make any neces-
sary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 13.61 mg of C 5 H 4 N 4
Containers and storage Containers — Tight containers.
Alprazolam
C 17 H 13 C1N 4 : 308.76
8-Chloro- 1 -methyl-6-phenyl-477-
[l,2,4]triazolo[4,3-a][l,4]benzodiazepine
[28981-97-7]
Alprazolam, when
98.5% of C 17 H 13 C1N 4 .
dried, contains not less than
Description Alprazolam occurs as white crystals or crystal-
line powder.
It is freely soluble in chloroform, soluble in methanol and
in ethanol (95), sparingly soluble in acetic anhydride, and
practically insoluble in water.
It dissolves in dilute nitric acid.
Identification (1) Determine the absorption spectrum of a
solution of Alprazolam in ethanol (95) (1 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Dissolve 0.05 g of Alprazolam in 0.7 mL of deu-
terochloroform for nuclear magnetic resonance spec-
troscopy, and determine the spectrum of this solution using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound, as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
OH): it exhibits a single signal A at around 5 2.6 ppm,
JPXV
Official Monographs / Alprenolol Hydrochloride
285
doublet signals B and C at around 6 4.0 ppm and 6 5.4 ppm,
and a broad signal D between 5 7.1 ppm and 7.9 ppm. The
ratio of integrated intensity of each signal, A:B:C:D, is about
3:1:1:8.
(3) Perform the test with Alprazolam as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 228 - 232°C
Purity (1) Chloride <1.03> — Dissolve 0.5 g of Alprazolam
in 10 mL of dilute nitric acid, and add water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.20 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.014%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Alprazol-
am according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(3) Related substances - Dissolve 50 mg of Alprazolam in
10 mL of methanol, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL, then pipet 1 mL of this solution, add
methanol to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
[iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop with a mixture of acetone, hexane,
ethyl acetate and ethanol (95) (4:2:2:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Alprazolam, previ-
ously dried, dissolve in 100 mL of acetic anhydride, and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 15.44 mg of C 17 H 13 C1N 4
Containers and storage Containers — Well-closed contain-
ers.
Alprenolol Hydrochloride
7JU7U/n-;U£@g±£
CH :
Y
CH 3
• HCI
and enantiomer
C 15 H 23 N0 2 .HC1: 285.81
(2RS)- 1 -(2-Allylphenoxy)-3-
[(1 -methylethyl)amino]propan-2-ol monohydrochloride
[13707-88-5]
less than 99.0% of C 15 H 23 N0 2 .HC1.
Description Alprenolol Hydrochloride occurs as white
crystals or crystalline powder.
It is freely soluble in water, in ethanol (95) and in acetic
acid (100), slightly soluble in acetic anhydride, and practical-
ly insoluble in diethyl ether.
Identification (1) To 2 mL of a solution of Alprenolol
Hydrochloride (1 in 100) add 0.05 mL of copper (II) sulfate
TS and 2 mL of sodium hydroxide TS: a blue-purple color
develops. To this solution add 1 mL of diethyl ether, shake
well, and allow to stand: a red-purple color develops in the
diethyl ether layer.
(2) Dissolve 0.05 g of Alprenolol Hydrochloride in 5 mL
of water, add 1 to 2 drops of bromine TS, and shake: the
color of the test solution disappears.
(3) Determine the absorption spectrum of a solution of
Alprenolol Hydrochloride in ethanol (95) (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of
Alprenolol Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(5) A solution of Alprenolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1 .0 g of Alprenolol Hydrochloride in 10
mL of water: the pH of this solution is between 4.5 and 6.0.
Melting point <2.60> 108-112°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Alprenolol Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1. 07>— Proceed with 2.0 g of Al-
prenolol Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Alprenolol Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Alprenolol
Hydrochloride in 10 mL of ethanol (95), and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
and add ethanol (95) to make exactly 100 mL. Pipet 2.5 mL
of this solution, add ethanol (95) to make exactly 10 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of
dichloromethane, acetone, acetic acid (100) and water
(60:42:5:3) to a distance of about 10 cm, air-dry the plate,
and then dry at 80 °C for 30 minutes. After cooling, allow the
plate to stand in iodine vapor for 30 minutes: the spots other
than the principal spot and the spot on the starting point
from the sample solution are not more intense than the spot
from the standard solution.
Alprenolol Hydrochloride, when dried, contains not Loss on dr ying <2 - 41> Not more than °- 5 % ( l S> in vacuum,
286
Alprostadil / Official Monographs
JP XV
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Alprenolol
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.58 mg of C 15 H 23 N0 2 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Alprostadil
Prostaglandin JL t
C20H34O5: 354.48
7-{(li?,2i?,3i?)-3-Hydroxy-2-[(l£',35)-3-
hydroxyoct- 1 -en- 1 -yl] -5-oxocyclopentyl} heptanoic acid
[745-65-3]
Alprostadil, when dried, contains not less than
97.0% and not more than 103.0% of C20H34O5.
Description Alprostadil occurs as white crystals or crystal-
line powder.
It is freely soluble in ethanol (99.5) and in tetrahydrofuran,
slightly soluble in acetonitrile, and practically insoluble in
water.
Identification (1) The absorption spectrum of a solution
of Alprostadil in ethanol (99.5) (1 in 100,000) determined as
directed under Ultraviolet-visible Spectrophotometry <2.24>
shows no absorption between 210 nm and 350 nm. Separate-
ly, to 10 mL of this solution add 1 mL of potassium hydrox-
ide-ethanol TS, allow to stand for 15 minutes, and determine
the absorption spectrum in the same way. Compare the
spectrum so obtained with the Reference Spectrum or the
spectrum of a solution of Alprostadil Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Alprostadil, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Alprostadil Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Optical rotation <2.49> [a]^,°: - 53 - - 61 ° (after drying, 25
mg, tetrahydrofuran, 5 mL, 100 mm).
Melting point <2. 60> 1 1 4 - 1 1 8 °C
Purity Related substances — Dissolve 4 mg of Alprostadil in
2 mL of a mixture of acetonitrile for liquid chromatography
and water (9:1), and use this solution as the sample solution.
Pipet 0.5 mL of the sample solution, and add the mixture of
acetonitrile for liquid chromatography and water (9:1) to
make exactly 10 mL. Pipet 2 mL of this solution, add the
mixture of acetonitrile for liquid chromatography and water
(9:1) to make exactly 10 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 5 /uL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine each peak area by the automatic in-
tegration method: the area of the peaks, having the relative
retention time of about 0.70 and 1.26 with respect to al-
prostadil, is not larger than 1/2 times the peak area of al-
prostadil with the standard solution, the area of the peaks,
having the relative retention time of about 0.88 and 1.18 with
respect to alprostadil, is not larger than the peak area of al-
prostadil with the standard solution, the area of the peaks
other than alprostadil and the peaks mentioned above is not
larger than 1/10 times the peak area of alprostadil with the
standard solution and the total area of the peaks other than
alprostadil is not larger than 2 times the peak area of al-
prostadil with the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 5 times as long as the
retention time of alprostadil beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, add the mixture of acetonitrile for
liquid chromatography and water (9:1) to make exactly
20 mL. Confirm that the peak area of alprostadil obtained
with 5 fiL of this solution is equivalent to 7 to 13% of that
with 5 /uL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 ,uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of al-
prostadil is not more than 1.5%.
Loss on drying <2.41> Not more than 1.0% (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Assay Weigh accurately about 5 mg each of Alprostadil
and Alprostadil Reference Standard, previously dried,
dissolve in exactly 5 mL of the internal standard solution,
add a mixture of acetonitrile for liquid chromatography and
water (9:1) to make 50 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 5 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qt and gs, of the peak area of alprostadil to that of the inter-
nal standard.
Amount (mg) of C 20 H 34 O 5 = W s x (Q T /Q S )
W s : Amount (mg) of Alprostadil Reference Standard
Internal standard solution — A solution of dimethyl phthalate
in the mixture of acetonitrile for liquid chromatography and
JPXV
Official Monographs / Alprostadil Alfadex
287
water (9:1) (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 196 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 9.07 g of potassium dihydrogen
phosphate in water to make 1000 mL. Adjust the pH to 6.3
with a solution prepared by dissolving 9.46 g of disodium
hydrogen phosphate in water to make 1000 mL, and dilute to
10 times its volume with water. To 360 mL of this solution
add 110 mL of acetonitrile for liquid chromatography and 30
mL of methanol for liquid chromatography.
Flow rate: Adjust the flow rate so that the retention time of
alprostadil is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, alprostadil and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 9.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of alprostadil to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and at a temperature not
exceeding 5°C.
Alprostadil Alfadex
Prostaglandin Ei a-Cyclodextrin Clathrate
Compound
-OH
H OH^
C2oH3405.^C36H 60 03o
7-{(lfl,2fl,3fl)-3-Hydroxy-2-[(l£',3S')-3-hydroxyoct-l-
en-l-yl]-5-oxocyclopentyl}heptanoic acid — a-cyclodextrin
[55648-20-9]
Alprostadil Alfadex is a ce-cyclodextrin clathrate
compound of alprostadil.
It contains not less than 2.8% and not more than 3.2
% of alprostadil (C 2 oH3 4 5 : 354.48), calculated on the
anhydrous basis.
Description Alprostadil Alfadex occurs as a white powder.
It is freely soluble in water, and practically insoluble in
ethanol (95), in ethyl acetate and in diethyl ether.
It is hygroscopic.
Identification (1) Dissolve 0.02 g of Alprostadil Alfadex
in 5 mL of water, add 5 mL of ethyl acetate, shake, and
centrifuge. Use the supernatant liquid as the sample solution
(1). Separately, to 0.02 g of Alprostadil Alfadex add 5 mL of
ethyl acetate, shake, and centrifuge. Use the supernatant
liquid as the sample solution (2). Evaporate the solvent from
these solutions under reduced pressure, add 2 mL of sulfuric
acid to the residue, and shake for 5 minutes: the liquid
obtained from the sample solution (1) shows an orange-yel-
low color, while the liquid obtained from the sample solution
(2) does not show that color.
(2) Dissolve 0.02 g of Alprostadil Alfadex in 5 mL of
water, add 5 mL of ethyl acetate, shake, centrifuge, and
evaporate the solvent from the supernatant liquid under
reduced pressure. Dissolve the residue in 2 mL of ethanol
(95), add 5 mL of 1,3-dinitrobenzene TS, then add 5 mL of a
solution of potassium hydroxide in ethanol (95) (17 in 100)
under ice-cooling, and allow to stand for 20 minutes in a dark
place under ice-cooling: a purple color develops.
(3) Dissolve 0.05 g of Alprostadil Alfadex in 1 mL of
iodine TS, by heating on a water bath, and allow to stand: a
dark blue precipitate is formed.
(4) Determine the absorption spectrum of a solution of
Alprostadil Alfadex in dilute ethanol (3 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits no absorption between 220 nm and 400 nm. Separate-
ly, to 10 mL of the solution add 1 mL of potassium hydrox-
ide-ethanol TS, allow to stand for 15 minutes, and determine
the absorption spectrum as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
Optical rotation <2.49> [ a ]^: +126- +138° (0.1 g calcu-
lated on the anhydrous basis, dilute ethanol, 20 mL, 100
mm).
pH <2.54> Dissolve 0.10 g of Alprostadil Alfadex in 20 mL
of water: the pH of this solution is between 4.0 and 5.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Alprostadil Alfadex in 10 mL of water: the solution is
colorless. Perform the test with this solution as directed un-
der Ultraviolet-visible Spectrophotometry within 30 minutes
after preparation of the solution: the absorbance at 450 nm is
not larger than 0.10.
(2) ProstaglandinA! — DissolveO.10 g of Alprostadil Alfa-
dex in 5 mL of water, add exactly 5 mL of the internal
standard solution and ethanol (95) to make 15 mL, and use
this solution as the sample solution. Separately, dissolve 1.5
mg of prostaglandin A t in ethanol (95) to make exactly 100
mL. Pipet 3 mL of this solution, add exactly 5 mL of the
internal standard solution, 2 mL of ethanol (95) and water to
make 15 mL, and use this solution as the standard solution.
Perform the test with 10 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the operating conditions described in the
Assay, and calculate the ratios, Q T and Q s , of the peak area
of prostaglandin A t to that of the internal standard: Q T is not
larger than Q s .
Internal standard solution — A solution of propyl para-
hydroxybenzoate in dilute ethanol (1 in 15,000).
(3) Related substances — Dissolve 0.10 g of Alprostadil
288
Dried Aluminum Hydroxide Gel / Official Monographs
JP XV
Alfadex in 3 mL of water, add exactly 3 mL of ethyl acetate,
shake, centrifuge, and use the supernatant liquid obtained as
the sample solution. Separately, dissolve 1.0 mg of
Prostaglandin A! in ethyl acetate to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, hexane and acetic acid (100) (10:2:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly a solution of
phosphomolybdic acid in ethanol (95) (1 in 4) on the plate,
and heat at 100°C for 5 minutes: the spots other than the
principal spot from the sample solution, and the spots other
than the spot corresponding to the spot from the standard
solution are all not more intense than the spot from the stan-
dard solution.
Water <2.4S> Not more than 6.0% (0.2 g, direct titration).
Assay Weigh accurately about 0.1 g of Alprostadil Alfa-
dex, dissolve in 5 mL of water, add exactly 5 mL of the
internal standard solution and water to make 15 mL, and use
this solution as the sample solution. Separately, weigh
accurately about 3 mg of Alprostadil Reference Standard,
dissolve in 5 mL of ethanol (95), add exactly 5 mL of the
internal standard solution and water to make 15 mL, and use
this solution as the standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Q T and Q s ,
of the peak area of alprostadil to that of the internal
standard.
Amount (mg) of alprostadil (C20H34O5)
= W s x (Q T /Q S )
W s : Amount (mg) of Alprostadil Reference Standard
Internal standard solution— A solution of propyl para-
hydroxybenzoate in dilute ethanol (1 in 15,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 205 nm).
Column: A stainless steel column about 5 mm in inside
diameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.02 mol/L potassium di-
hydrogenphosphate and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
alprostadil is about 6 minutes.
Selection of column: Dissolve about 0.1 g of Alprostadil
Alfadex in 5 mL of water, add 5 mL of a solution of
prostaglandin A, in ethanol (95) (3 in 200,000) and 5 mL of
the internal standard solution. Proceed with 10 ^L of this
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of alprostadil, the
internal standard and prostaglandin A! in this order and
complete separation of these peaks.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at a temperature not exceeding
5°C.
Expiration date 24 months after preparation.
Dried Aluminum Hydroxide Gel
Dried Aluminum Hydroxide Gel contains not less
than 50.0% of aluminum oxide (A1 2 3 : 101.96).
Description Dried Aluminum Hydroxide Gel occurs as a
white, amorphous powder. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Most of it dissolves in dilute hydrochloric acid and in
sodium hydroxide TS.
Identification To 0.2 g of Dried Aluminum Hydroxide Gel
add 20 mL of dilute hydrochloric acid, warm, and centrifuge:
the supernatant liquid responds to the Qualitative Tests
<1.09> for aluminum salt.
Purity (1) Acidity or alkalinity — To 1.0 g of Dried Alumi-
num Hydroxide Gel add 25 mL of water, shake well, and
centrifuge: the supernatant liquid is neutral.
(2) Chloride <1.03>— To 1.0 g of Dried Aluminum
Hydroxide Gel add 30 mL of dilute nitric acid, heat gently to
boil while shaking, cool, add water to make 100 mL, and
centrifuge. To 5 mL of the supernatant liquid add 6 mL of
dilute nitric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control
solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS
(not more than 0.284%).
(3) Sulfate <U4>— To 1.0 g of Dried Aluminum
Hydroxide Gel add 15 mL of dilute hydrochloric acid, heat
gently to boil while shaking, cool, add water to make 250
mL, and centrifuge. To 25 mL of the supernatant liquid add
1 mL of dilute hydrochloric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 1 .0 mL of 0.005 mol/L sulfuric
acid VS (not more than 0.480%).
(4) Nitrate — To 0.10g of Dried Aluminum Hydroxide
Gel add 5 mL of water, then carefully add 5 mL of sulfuric
acid, shake well to dissolve, and cool. Superimpose the
solution on 2 mL of iron (II) sulfate TS: no brown-colored
ring is produced at the zone of contact.
(5) Heavy metals <1.07> — Dissolve 2.0 g of Dried Alumi-
num Hydroxide Gel in 10 mL of dilute hydrochloric acid by
heating, filter if necessary, and add water to make 50 mL.
Perform the test with this solution as the test solution.
Prepare the control solution as follows: evaporate 10 mL of
dilute hydrochloric acid to dryness, and add 2.0 mL of
Standard Lead Solution, 2 mL of dilute acetic acid and water
to make 50 mL (not more than 10 ppm).
(6) Arsenic <1.11> — To 0.8 g of Dried Aluminum
Hydroxide Gel add 10 mL of dilute sulfuric acid, heat gently
to boil while shaking, cool, and filter. Take 5 mL of the
filtrate, use this solution as the test solution, and perform the
test (not more than 5 ppm).
Acid-consuming capacity Weigh accurately about 0.2 g of
Dried Aluminum Hydroxide Gel, and transfer to a glass-
stoppered flask. Add exactly 100 mL of 0.1 mol/L
JPXV
Official Monographs / Natural Aluminum Silicate
289
hydrochloric acid VS, stopper the flask, shake at 37 ± 2°C
for 1 hour, and filter. Measure exactly 50 mL of the filtrate,
and titrate <2.50> while thoroughly stirring, the excess
hydrochloric acid with 0.1 mol/L sodium hydroxide VS until
the pH of the solution becomes to 3.5. The volume of 0.1
mol/L hydrochloric acid VS consumed is not less than 250
mL per g of Dried Aluminum Hydroxide Gel.
Assay Weigh accurately about 2 g of Dried Aluminum
Hydroxide Gel, add 15 mL of hydrochloric acid, heat on a
water bath with shaking for 30 minutes, cool, and add water
to make exactly 500 mL. Pipet 20 mL of this solution, add
exactly 30 mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS and 20 mL of acetic acid
(31)-ammonium acetate buffer solution, pH 4.8, boil for 5
minutes, and cool. Add 55 mL of ethanol (95), and titrate
<2.50> with 0.05 mol/L zinc acetate VS until the color of the
solution changes from light dark green to light red. (indica-
tor: 2 mL of dithizone TS). Perform a blank determination,
and make any necessary correction.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.549 mg of A1 2 3
Containers and storage Containers — Tight containers.
Dried Aluminum Hydroxide Gel
Fine Granules
fcjfcMMfcyjus -^A^juwi
Dried Aluminum Hydroxide Gel Fine Granules
contain not less than 47.0% of aluminum oxide (A1 2 3 :
101.96).
Method of preparation Prepare with Dried Aluminum
Hydroxide Gel as directed under Powders.
Identification To 0.2 g of Dried Aluminum Hydroxide Gel
Fine Granules add 20 mL of dilute hydrochloric acid, warm
and centrifuge: the supernatant liquid responds to the
Qualitative Tests <1.09> for aluminum salt.
Acid-consuming capacity Proceed as directed for Acid-
consuming capacity under Dried Aluminum Hydroxide Gel:
the volume of 0.1 mol/L hydrochloric acid VS consumed is
not less than 235 mL per g of Dried Aluminum Hydroxide
Gel Fine Granules.
Assay Proceed as directed in the Assay under Dried Alumi-
num Hydroxide Gel.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.549 mg of A1 2 3
Containers and storage Containers — Tight containers.
Natural Aluminum Silicate
Description Natural Aluminum Silicate occurs as a white or
slightly colored powder. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Natural Aluminum Silicate (1 g) dissolves when heated in
20 mL of a solution of sodium hydroxide (1 in 5), with some
decomposition, leaving a large amount of insoluble sub-
stance.
Identification (1) To 0.5 g of Natural Aluminum Silicate
add 3 mL of diluted sulfuric acid (1 in 3), heat until white
fumes evolve, cool, add 20 mL of water, and filter. Render
the filtrate slightly acid with ammonia TS: the solution
responds to the Qualitative Tests <1.09> for aluminum salt.
(2) Prepare a bead by fusing ammonium sodium
hydrogenphosphate tetrahydrate on a platinum loop. Place
the bead in contact with Natural Aluminum Silicate, and fuse
again: an infusible material appears in the bead, producing,
upon cooling, an opaque bead with a web-like structure.
Purity (1) Acidity or alkalinity — Shake 5.0 g of Natural
Aluminum Silicate with 100 mL of water, and centrifuge: the
supernatant liquid so obtained is neutral.
(2) Chloride <1.03>— To 5.0 g of Natural Aluminum Sili-
cate add 100 mL of water, boil gently for 15 minutes while
shaking, then cool, add water to restore the original volume,
and centrifuge. To 10 mL of the supernatant liquid add 6 mL
of dilute nitric acid, dilute to 50 mL with water, and perform
the test using this solution as the test solution. Prepare the
control solution with 0.30 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.021%).
(3) Sulfate <1.14> — To the residue obtained in (6) add 3
mL of dilute hydrochloric acid, heat on a water bath for 10
minutes, dilute to 50 mL with water, and filter. To 2.0 mL of
the filtrate add 1 mL of dilute hydrochloric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 1.0 mL of 0.005
mol/L sulfuric acid VS (not more than 0.480%).
(4) Heavy metals <1.07> — To 1 .5 g of Natural Aluminum
Silicate add 50 mL of water and 5 mL of hydrochloric acid,
boil gently for 20 minutes while shaking, then cool, cen-
trifuge, remove the supernatant liquid, wash the residue with
two 10-mL portions of water, centrifuging each time, com-
bine these washings with the filtrate, and add ammonia solu-
tion (28) dropwise, until a precipitate just appears. Add drop-
wise dilute hydrochloric acid with vigorous shaking and
redissolve the precipitate. Heat the mixture with 0.45 g of
hydroxylammonium chloride, cool, and add 0.45 g of sodi-
um acetate trihydrate, 6 mL of dilute acetic acid and water to
make 150 mL. Perform the test, using 50 mL of this solution
as the test solution. Prepare the control solution with 2.0 mL
of Standard Lead Solution, 0.15 g of hydroxylammonium
chloride, 0.15 g of sodium acetate trihydrate, 2 mL of dilute
acetic acid and water to make 50 mL (not more than 40 ppm).
(5) Arsenic <1.11> — To 1.0 g of Natural Aluminum
Silicate, add 5 mL of dilute hydrochloric acid, heat gently to
boil while shaking well, cool rapidly, and centrifuge. Mix the
290
Synthetic Aluminum Silicate / Official Monographs
JP XV
The figures are in mm.
A: Distilling flask of about 300-mL capacity.
R: Steam generator of about 1000- ml- capacity, contain-
ing a few boiling tips to prevent bumping
C: Condenser
D: Receiver: 200-mL volumetric flask
E: Steam-introducing tube having an internal diameter
of about 8 mm
K G: Rubber tube with a clamp
H: Thermometer
fluoride under Oxygen Flask Combustion Method <1.06>. No
corrective solution is used in this procedure.
Amount (mg) of fluoride (F: 19.00) in the test solution
= amount (mg) of fluoride in 5 mL of
the standard solution
x (A T /A S ) x (200/ *0
The content of fluoride (F) is not more than 0.01%.
Loss on drying <2.41> Not more than 20.0% (1 g, 105°C,
3 hours).
Adsorptive power To 0.10 g of Natural Aluminum Silicate
add 20 mL of a solution of methylene blue trihydrate (3 in
2000), shake for 15 minutes, allow to stand for 5 hours at
37 ± 2°C, and centrifuge. Dilute 1.0 mL of the supernatant
liquid with water to 200 mL. Place 50 mL of the solution in a
Nessler tube and observe horizontally or vertically against a
white background: the color of the solution is not deeper
than that of the following control solution.
Control solution: Dilute 1 .0 mL of a solution of methylene
blue trihydrate (3 in 2000) with water to 400 mL, and use 50
mL of this solution.
Containers and storage Containers — Well-closed contain-
ers.
Synthetic Aluminum Silicate
residue with 5 mL of dilute hydrochloric acid with shaking,
centrifuge, then add 10 mL of water to the residue, and
repeat the extraction in the same manner. Concentrate the
combined extracts on a water bath to 5 mL. Use this solution
as the test solution, and perform the test (not more than 2
ppm).
(6) Soluble salts — Evaporate 50 mL of the supernatant
liquid obtained in (1) on a water bath to dryness, and ignite
the residue at 700°C for 2 hours: the mass of the ignited
residue is not more than 40 mg.
(7) Fluoride — (i) Apparatus: Use a hard glass apparatus
as illustrated in the figure. Ground-glass joints may be used.
(ii) Procedure: Transfer 5.0 g of Natural Aluminum Sili-
cate to the distilling flask A with the aid of 20 mL of water,
add about 1 g of glass wool and 50 mL of diluted purified
sulfuric acid (1 in 2), and connect A to the distillation
apparatus, previously washed with steam streamed through
the steam introducing tube E. Connect the condenser C with
the receiver D containing 10 mL of 0.01 mol/L sodium
hydroxide VS and 10 mL of water so that the lower end of C
is immersed in the solution. Heat A gradually until the tem-
perature of the solution in A reaches 130°C, then open the
rubber tube F, close the rubber tube G, boil water in the
steam generator B vigorously, and introduce the generated
steam into F. Simultaneously, heat A, and maintain the
temperature of the solution in A between 135 C C and 145°C.
Adjust the distilling rate to about 10 mL per minute. Collect
about 170 mL of the distillate, then stop the distillation, wash
C with a small quantity of water, combine the washings with
the distillate, add water to make exactly 200 mL, and use this
solution as the test solution. Perform the test with the test
solution as directed in the procedure of determination for
Description Synthetic Aluminum Silicate occurs as a white
powder. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Synthetic Aluminum Silicate (1 g) dissolves when heated in
20 mL of a solution of sodium hydroxide (1 in 5), leaving a
small amount of insoluble substance .
Identification (1) To 0.5 g of Synthetic Aluminum Silicate
add 3 mL of diluted sulfuric acid (1 in 3), heat until white
fumes evolve, cool, add 20 mL of water, and filter. Render
the filtrate slightly acid with ammonia TS: the solution
responds to the Qualitative Tests <1.09> for aluminum salt.
(2) Prepare a bead by fusing ammonium sodium
hydrogenphosphate tetrahydrate on a platinum loop. Place
the bead in contact with Synthetic Aluminum Silicate, and
fuse again: an infusible material appears in the bead, produc-
ing, upon cooling, an opaque bead with a web-like structure.
Purity (1) Acidity or alkalinity — Shake 1.0 g of Synthetic
Aluminum Silicate with 20 mL of water, and centrifuge: the
supernatant liquid so obtained is neutral.
(2) Chloride <1.03>— To 5.0 g of Synthetic Aluminum
Silicate add 100 mL of water, boil gently for 15 minutes while
shaking, then cool, add water to restore the original volume,
and centrifuge. To 10 mL of the supernatant liquid add 6 mL
of dilute nitric acid and water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution with 0.30 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.021%).
(3) Sulfate <1.14> — To 2.0 mL of the supernatant liquid
obtained in (2) add 1 mL of dilute hydrochloric acid and
JPXV
Official Monographs / Aluminum Monostearate
291
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 1.0 mL of
0.005 mol/L sulfuric acid VS (not more than 0.480%).
(4) Heavy metals <1.07> — To 3.0 g of Synthetic Alumi-
num Silicate add 50 mL of water and 5 mL of hydrochloric
acid, boil gently for 20 minutes while shaking, then after
cooling, centrifuge, remove the supernatant liquid, wash the
precipitate with two 10-mL portions of water, centrifuging
each time, combine these washings with the filtrate, and add
ammonia solution (28) dropwise until a precipitate just
appears. Add dropwise dilute hydrochloric acid with
vigorous shaking to redissolve the precipitate. Heat the
solution with 0.45 g of hydroxylammonium chloride, and
after cooling, add 0.45 g of sodium acetate trihydrate, 6 mL
of dilute acetic acid and water to make 150 mL. Perform the
test with 50 mL of this solution as the test solution. Prepare
the control solution with 3.0 mL of Standard Lead Solution,
0.15 g of hydroxylammonium chloride, 0.15 g of sodium
acetate trihydrate, 2 mL of dilute acetic acid and water to
make 50 mL (not more than 30 ppm).
(5) Arsenic <1.11> — To 1.0 g of Synthetic Aluminum
Silicate add 10 mL of dilute hydrochloric acid, heat gently to
boiling while shaking well, cool rapidly, and centrifuge. Mix
the residue with 5 mL of dilute hydrochloric acid with shak-
ing, centrifuge, then add 10 mL of water to the residue, and
repeat the extraction in the same manner. Concentrate the
combined extracts on a water bath to 5 mL. Use this solution
as the test solution, and perform the test (not more than 2
ppm).
Loss on drying <2.41> Not more than 20.0% (1 g, 105°C,
3 hours).
Acid-consuming capacity <6.04> Weigh accurately about 1
g of Synthetic Aluminum Silicate, transfer to a glass-stop-
pered flask, add 200 mL of 0.1 mol/L hydrochloric acid VS,
exactly measured, stopper the flask, and shake at 37 ± 2°C
for 1 hour. Filter, pipet 50 mL of the filtrate, and titrate
<2.50> by stirring well the excess hydrochloric acid with 0.1
mol/L sodium hydroxide VS until the pH of the solution
changes to 3.5. The volume of 0.1 mol/L hydrochloric acid
VS consumed is not less than 50.0 mL per g of Synthetic Alu-
minum Silicate.
Containers and storage Containers — Well-closed contain-
ers.
Aluminum Monostearate
Aluminum Monostearate is mainly aluminum com-
pounds of stearic acid (Ci 8 H 36 2 : 284.48) and palmitic
acid (C 16 H 32 2 : 256.42).
Aluminum Monostearate, when dried, contains not
less than 7.2% and not more than 8.9% of aluminum
(Al: 26.98).
Description Aluminum Monostearate occurs as a white to
yellowish white powder. It is odorless or has a faint, charac-
teristic odor.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Identification (1) Heat 3 g of Aluminum Monostearate
with 30 mL of hydrochloric acid in a water bath with oc-
casional shaking for 10 minutes. After cooling, shake the
mixture vigorously with 50 mL of water and 30 mL of diethyl
ether for 3 minutes, and allow to stand. To the separated
aqueous layer add sodium hydroxide TS until the solution
becomes slightly turbid, and filter: the filtrate responds to the
Qualitative Tests <1.09> for aluminum salt.
(2) Wash the diethyl ether layer separated in (1) with two
20-mL portions of water, and evaporate the diethyl ether
layer on a water bath: the residue melts <1.13> at above 54°C.
Acid value for fatty acid <1.13> 193 - 210. Weigh accurately
about 1 g of fatty acid obtained in the Identification (2),
transfer a 250-mL glass-stoppered flask, add 100 mL of a
mixture of diethyl ether and ethanol (95) (2:1), warm to dis-
solve, add several drops of phenolphthalein TS, and proceed
as directed under Acid Value.
Purity (1) Free fatty acid — Mix 1.0 g of Aluminum
Monostearate with about 50 mL of a mixture of neutralized
ethanol and diethyl ether (1:1), filter through dry filter paper,
wash the vessel and the filter paper with a small amount of a
mixture of neutralized ethanol and diethyl ether (1:1),
combine the filtrate and the washings, and add 2.1 mL of 0.1
mol/L potassium hydroxide VS: a red color develops.
(2) Water-soluble salts — Heat 2.0 g of Aluminum
Monostearate with 80 mL of water in a loosely stoppered
conical flask on a water bath for 30 minutes with occasional
shaking. After cooling, filter through dry filter paper, wash
the residue with a small amount of water, combine the
washings with the filtrate, add water to make 100 mL,
evaporate 50 mL of this solution on a water bath, and heat
strongly at 600°C: the mass of the residue is not more than
10.0 mg.
(3) Heavy metals <1.07> — Heat 1.0 g of Aluminum Mono-
stearate over a small flame with caution at the beginning, and
continue the heating, gradually raising the temperature, to
ash. After cooling, add 10 mL of diluted hydrochloric acid (1
in 2), evaporate on a water bath, and boil the residue with 20
mL of water for 1 minute. Cool, filter, wash the residue with
water, combine the filtrate and the washings, and add 2 mL
of dilute acetic acid and water to make 50 mL. Perform the
test using this solution as the test solution. Evaporate 10 mL
of diluted hydrochloric acid (1 in 2) on a water bath to
dryness, add 2 mL of dilute acetic acid and 5.0 mL of
Standard Lead Solution, dilute with water to make 50 mL,
and use this solution as the control solution (not more than
50 ppm).
(4) Arsenic < 1.11 > — Mix 1.0 g of Aluminum
Monostearate with 2 g of magnesium nitrate hexahydrate,
ignite over a small flame, moisten the residue after cooling
with 0.5 mL of nitric acid, and heat. Heat again the residue
with 10 mL of dilute sulfuric acid until white fumes evolve,
add water to make 5 mL, and perform the test with this
solution as the test solution (not more than 2 ppm).
Loss on drying <2.4I> Not more than 3.0% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 1 g of Aluminum
Monostearate, previously dried, ignite gently to ash, and
cool. Add dropwise 0.5 mL of nitric acid, evaporate on a
water bath by heating, and then heat strongly between 900°C
292
Aluminum Potassium Sulfate Hydrate / Official Monographs
JP XV
and 1100°C to a constant mass. After cooling, weigh rapidly
the ignited residue, and designate the mass as aluminum
oxide (A1 2 3 : 101.96).
Amount (mg) of aluminum (Al)
= amount (mg) of aluminum oxide (A1 2 3 ) X 0.5293
Containers and storage Containers — Well-closed contain-
ers.
Aluminum Potassium Sulfate
Hydrate
Alum
A1K(S0 4 ) 2 .12H 2 0: 474.39
Aluminum Potassium Sulfate Hydrate contains not
less than 99.5% of A1K(S0 4 ) 2 .12H 2 0.
Description Aluminum Potassium Sulfate Hydrate occurs
as colorless or white crystals or powder. It is odorless. It has a
slightly sweet, strongly astringent taste.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
A solution of Aluminum Potassium Sulfate Hydrate (1 in
20) is acid.
Identification A solution of Aluminum Potassium Sulfate
Hydrate (1 in 10) responds to the Qualitative Tests <1.09> for
aluminum salt, to the Qualitative Tests <1.09> (1), (3) and (4)
for potassium salt, and to the Qualitative Tests <1.09> (1) and
(3) for sulfate.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Aluminum Potassium Sulfate Hydrate according to Method
1, and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(2) Iron <1.10> — Prepare the test solution with 1.0 g of
Aluminum Potassium Sulfate Hydrate according to Method
1, and perform the test according to Method A. Prepare the
control solution with 2.0 mL of Standard Iron Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.6 g
of Aluminum Potassium Sulfate Hydrate, according to
Method 1, and perform the test (not more than 3.3 ppm).
Assay Weigh accurately about 4.5 g of Aluminum Potassi-
um Sulfate Hydrate, and dissolve in water to make exactly
200 mL. Take exactly 20 mL of this solution, and add exactly
30 mL of 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS and 20 mL of acetic acid-ammonium acetate
buffer solution, pH 4.8, boil for 5 minutes, and cool. Add 55
mL of ethanol (95), and titrate <2.50> with 0.05 mol/L zinc
acetate VS (indicator: 2 mL of dithizone TS), until the color
of the solution changes from light dark green to light red.
Perform a blank determination.
Each mL of 0.05 mol/L disodium dihydorgen
ethylenediamine tetetraacetate VS
= 23.72 mg of A1K(S0 4 ) 2 .12H 2
Containers and storage Containers — Tight containers.
Dried Aluminum Potassium Sulfate
Burnt Alum
A1K(S0 4 ) 2 : 258.21
Dried Aluminum Potassium Sulfate, when dried,
contains not less than 98.0% of A1K(S0 4 ) 2 .
Description Dried Aluminum Potassium Sulfate occurs as
white masses or white powder. It is odorless. It has a slightly
sweet, astringent taste.
It is freely soluble in hot water and practically insoluble in
ethanol (95).
It dissolves slowly in water.
Identification A solution of Dried Aluminum Potassium
Sulfate (1 in 20) responds to the Qualitative Tests <1.09> for
aluminum salt, to the Qualitative Tests <1.09> (1), (3) and (4)
for potassium salt, and to the Qualitative Tests <1.09> (1) and
(3) for sulfate.
Purity (1) Water-insoluble substances — To 2.0 g of Dried
Aluminum Potassium Sulfate add 40 mL of water, shake fre-
quently, and allow to stand for 48 hours. Collect the insolu-
ble residue on a glass filter (G4), wash with 50 mL of water,
and dry at 105°C for 2 hours: the mass of the residue is not
more than 50 mg.
(2) Heavy metals <1.07> — Dissolve 0.5 g of Dried Alumi-
num Potassium Sulfate in 45 mL of water, and filter, if neces-
sary. Add 2 mL of dilute acetic acid and water to make 50
mL, and perform the test using this solution as the test solu-
tion. Prepare the control solution with 2.0 mL of Standard
Lead Solution, 2 mL of dilute acetic acid and water to make
50 mL (not more than 40 ppm).
(3) Iron <1.10> — Prepare the test solution with 0.54 g of
Dried Aluminum Potassium Sulfate according to Method 1,
and perform the test according to Method A. Prepare the
control solution with 2.0 mL of Standard Iron Solution (not
more than 37 ppm).
(4) Arsenic </.//> — Prepare the test solution with 0.40 g
of Dried Aluminum Potassium Sulfate, according to Method
1, and perform the test (not more than 5 ppm).
Loss on drying <2.41> Not more than 15.0% (2 g, 200°C,
4 hours).
Assay Weigh accurately about 1.2 g of Dried Aluminum
Potassium Sulfate, previously dried, add 80 mL of water,
and heat on a water bath with occasional shaking for 20
minutes. Cool, add water to make exactly 100 mL, and filter,
if necessary. Discard the first 30 mL of the filtrate, take exact-
ly the subsequent 20 mL of the filtrate, and add exactly 30
mL of 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS and 20 mL of acetic acid-ammonium acetate
buffer solution, pH 4.8, boil for 5 minutes, and cool. Add 55
mL of ethanol (95), and titrate <2.50> with 0.05 mol/L zinc
acetate VS (indicator: 2 mL of dithizone TS), until the color
of the solution changes from light dark green to light red.
Perform a blank determination.
JPXV
Official Monographs / Amantadine Hydrochloride
293
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 12.91 mg of A1K (S0 4 ) 2
Containers and storage Containers — Tight containers.
Amantadine Hydrochloride
77>^y>iii
Alum Solution
■ HCI
Alum Solution contains not less than 0.27 w/v% and
not more than 0.33 w/v% of aluminum potassium
sulfate Hydrate [A1K(S0 4 ) 2 .12H 2 0: 474.39].
Method of preparation
Aluminum Potassium Sulfate Hydrate 3 g
Mentha Water 50 mL
Water or Purified Water a sufficient quantity
To make 1000 mL
Dissolve and mix the above ingredients.
Description Alum Solution is a clear, colorless liquid. It has
the odor of the mentha oil and an astringent taste.
Identification (1) To 5 mL of Alum Solution add 3 mL of
ammonium chloride TS and 1 mL of ammonia TS: a white,
gelatinous precipitate is produced, which changes to red upon
the addition of 5 drops of alizarin red S TS (aluminum sul-
fate).
(2) Place 100 mL of Alum Solution in an evaporating
dish, evaporate on a water bath to dryness, and dissolve the
residue in 5 mL of water: the solution responds to the
Qualitative Tests <1.09> for potassium salt.
(3) Alum Solution responds to the Qualitative Tests
<1.09> (1) and (2) for sulfate.
Assay Pipet 50 mL of Alum Solution, add exactly 30 mL of
0.02 mol/L disodium dihydrogen ethylenediamine tetraa-
cetate VS, and further add 20 mL of acetic acid-ammonium
acetate buffer solution, pH 4.8. Boil for 5 minutes, cool, add
55 mL of ethanol (95), and titrate <2.50> with 0.02 mol/L
zinc acetate VS (indicator: 2 mL of dithizone TS), until the
color of the solution changes from light dark green to light
red. Perform a blank determination.
Each mL of 0.02 mol/L disodium dihydrogen ethylene-
diamine tetraacetate VS
= 9.488 mg of A1K(S0 4 ) 2 -12H 2
Containers and storage Containers — Tight containers.
C 10 H 17 N.HC1: 187.71
Tricyclo [3.3.1. I 3 ' 7 ] dec- 1 -ylamine monohydrochloride
[665-66-7]
Amantadine Hydrochloride, when dried, contains
not less than 99.0% of C 10 H 17 N.HC1.
Description Amantadine Hydrochloride occurs as a white,
crystalline powder. It is odorless, and has a bitter taste.
It is very soluble in formic acid, freely soluble in water, in
methanol and in ethanol (95), and practically insoluble in
diethyl ether.
Identification (1) To 0.1 g of Amantadine Hydrochloride
add 1 mL of pyridine and 0.1 mL of acetic anhydride,
dissolve by boiling for 1 minute, add 10 mL of dilute
hydrochloric acid, and cool in ice water. Filter the crystals
separated, wash with water, and dry at 105°C for 1 hour: the
residue melts <2.60> between 147°C and 151°C.
(2) Determine the infrared absorption spectrum of
Amantadine Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
(3) A solution of Amantadine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Amantadine Hydrochloride in
5 mL of water: the pH of this solution is between 4.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Amantadine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Amanta-
dine Hydrochloride according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than lOppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Amantadine Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.50 g of Amantadine
Hydrochloride in 10 mL of water, add 10 mL of sodium
hydroxide TS and 10 mL of chloroform, and shake. Filter the
chloroform layer through absorbent cotton with 3 g of
anhydrous sodium sulfate on a funnel, and use the filtrate as
the sample solution. Pipet 1 mL of the sample solution, add
chloroform to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 2 /uL each
of the sample solution and standard solution as directed un-
der Gas Chromatography <2.02> according to the following
conditions. Determine each peak area of these solutions by
the automatic integration method: each peak area other than
that of amantadine from the sample solution is not larger
294
Ambenonium Chloride / Official Monographs
JP XV
than 1/3 of the peak area of amantadine from the standard
solution, and the total area of each peak is not larger than the
peak area of amantadine from the standard solution.
Operating conditions—
Detector: A hydrogen fiame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with siliceous earth for gas
chromatography (150 to 180 /um in particle diameter) coated
with a mixture (L) of branched hydrocarbon of petroleum
hexamethyltetracosane group for gas chromatography and
potassium hydroxide at the ratios of 2% and 1%, respective-
ly.
Column temperature: Inject at a constant temperature of
about 125°C, maintain the temperature for 5 minutes, raise
at the rate of 5°C per minute to 150°C, and maintain at a
constant temperature of about 150°C for 15 minutes.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
amantadine is about 11 minutes.
Selection of column: Dissolve 0.15 g of naphthalene in 5
mL of the sample solution, and add chloroform to make 100
mL. Proceed with 2//L of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of naphthalene and amantadine in this
order with the resolution between these peaks being not less
than 2.5.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of amantadine obtained from 2 /xL of the
standard solution composes about 10% of the full scale.
Time span of measurement: About twice as long as the
retention time of amantadine beginning after the solvent
peak.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.2 g of Amantadine
Hydrochloride, previously dried, dissolve in 2 mL of formic
acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS, and
heat on a water bath for 30 minutes. After cooling, add acetic
acid (100) to make 70 mL, and titrate <2.50> the excess per-
chloric acid with 0.1 mol/L sodium acetate VS (potentiomet-
ric titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.77 mg of C 10 Hi 7 N.HCl
Containers and storage Containers — Well-closed contain-
ers.
Ambenonium Chloride
U
r^«y^7r6
2CI
C 28 H 42 Cl4N 4 2 : 608.47
2,2 ' - [( 1 ,2-Dioxoethane- 1 ,2-diyl)diimino]bis [N-
(2-chlorobenzyl)-A f ,A r -diethylethylaminium] dichloride
[115-79-7]
Ambenonium Chloride contains not less than 98.5%
of C28H42CI4N4O2, calculated on the dried basis.
Description Ambenonium Chloride occurs as a white
powder.
It is freely soluble in water, in methanol and in acetic acid
(100), soluble in ethanol (95), and slightly soluble in acetic
anhydride.
It is hygroscopic.
Melting point: about 205°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ambenonium Chloride in methanol (1 in 5000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Ambenonium Chloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Ambenonium Chloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ambenonium Chloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Ambeno-
nium Chloride according to Method 4, and perform the test.
Use a solution of magnesium nitrate in ethanol (95) (1 in 5).
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Ambenonium
Chloride in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
methanol to make exactly 20 mL . Pipet 1 mL of this solution,
add methanol to make exactly 10 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
5 /uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, formic acid and water
(12:6:5) to a distance of about 10 cm, and air-dry the plate.
Allow the plate to stand in iodine vapor: the spots other than
the principal spot from the sample solution are not more
JPXV
Official Monographs / Amidotrizoic Acid
295
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 11.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Ambenonium Chlo-
ride, and dissolve in 50 mL of a mixture of acetic anhydride
and acetic acid (100) (7:3). Titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.42 mg of C 28 H 42 Cl4N 4 2
Containers and storage Containers — Tight containers.
Amidotrizoic Acid
7 5 K h U 7K
H 3 C
COsH
C„H 9 I 3 N 2 4 : 613.91
3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid
[117-96-4]
Amidotrizoic Acid, calculated on the dried basis,
contains not less than 98.0% of CnHgL^CV
Description Amidotrizoic Acid occurs as a white, crystal-
line powder. It is odorless.
It is slightly soluble in ethanol (95), very slightly soluble in
water, and practically insoluble in diethyl ether.
It dissolves in sodium hydroxide TS.
Identification (1) Heat 0.1 g of Amidotrizoic Acid over a
flame: a purple gas is evolved.
(2) Determine the infrared absorption spectrum of
Amidotrizoic Acid as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Amidotrizoic Acid in 10 mL of 0.2 mol/L sodium hydroxide
TS: the solution is clear and colorless.
(2) Primary aromatic amines — Dissolve 0.20 g of
Amidotrizoic Acid in 5 mL of water and 1 mL of sodium
hydroxide TS, add 4 mL of a solution of sodium nitrite (1 in
100) and 10 mL of 1 mol/L hydrochloric acid TS, shake, and
allow to stand for 2 minutes. Add 5 mL of ammonium
amidosulfate TS, shake well, allow to stand for 1 minute,
and add 0.4 mL of a solution of 1-naphthol in ethanol (95) (1
in 10), 15 mL of sodium hydroxide TS and water to make
exactly 50 mL. Determine the absorbance of this solution at
485 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> using a solution, prepared in the same manner,
as the blank: the absorbance is not more than 0.15.
(3) Soluble halides — Dissolve 2.5 g of Amidotrizoic Acid
in 20 mL of water and 2.5 mL of ammonia TS, add 20 mL of
dilute nitric acid and water to make 100 mL, allow to stand
for 15 minutes with occasional shaking, and filter. Discard
the first 10 mL of the filtrate, transfer the subsequent 25 mL
of the filtrate to a Nessler tube, and add ethanol (95) to make
50 mL. Proceed as directed under Chloride Limit Test <1.03>
using this solution as the test solution. Prepare the control so-
lution as follows: to 0.10 mL of 0.01 mol/L hydrochloric
acid VS, add 6 mL of dilute nitric acid and water to make 25
mL, then ethanol (95) to make 50 mL.
(4) Iodine — Dissolve 0.20 g of Amidotrizoic Acid in 2.0
mL of sodium hydroxide TS, add 2.5 mL of 0.5 mol/L sul-
furic acid TS, allow to stand for 10 minutes with occasional
shaking, add 5 mL of chloroform, shake well, and allow to
stand: the solution is colorless in the chloroform layer.
(5) Heavy metals <1.07> — Proceed with 2.0 g of
Amidotrizoic Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 0.6 g
of Amidotrizoic Acid according to Method 3, and perform
the test (not more than 3.3 ppm).
Loss on drying <2.41> Not more than 7.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Transfer about 0.5 g of Amidotrizoic Acid, accurate-
ly weighed, to a saponification flask, dissolve in 40 mL of
sodium hydroxide TS, add 1 g of zinc powder, connect to a
reflux condenser, boil for 30 minutes, cool, and filter. Wash
the flask and the filter paper with 50 mL of water, and com-
bine the washings and the filtrate. Add 5 mL of acetic acid
(100) to this solution, and titrate <2.50> with 0.1 mol/L silver
nitrate VS until the color of the precipitate changes from yel-
low to green (indicator: 1 mL of tetrabromophenolphthalein
ethyl ester TS).
Each mL of 0.1 mol/L silver nitrate VS
= 20.46 mg of CnH 9 I 3 N 2 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
296
Amikacin Sulfate / Official Monographs
JP XV
Amikacin Sulfate
7 5fi->>filEK£
" F N
2H 2 SQ 4
C 22 H 4 3N 5 I3 .2H 2 S04: 781.76
3-Amino-3-deoxy-a-D-glucopyranosyl-(l -> 6)-
[6-amino-6-deoxy-a-D-glucopyranosyl-(l->4)]-l-Af-
[(2<S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine
disulfate [39831-55-5]
Amikacin Sulfate is the sulfate of a derivative of
kanamycin.
It contains not less than 691 fig (potency) and not
more than 791 fig (potency) per mg, calculated on the
dried basis. The potency of Amikacin Sulfate is ex-
pressed as mass (potency) of amikacin (C 2 2H43N50 13 :
585.60).
Description Amikacin Sulfate occurs as a white to yellowish
white powder.
It is very soluble in water, and practically insoluble in
ethanol (95).
Identification (1) Determine the infrared absorption spec-
trum of Amikacin Sulfate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Amikacin Sulfate
Reference Standard previously dried: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(2) Dissolve 0.1 g each of Amikacin Sulfate and Amika-
cin Sulfate Reference Standard in 4 mL of water, and use
these solutions as the sample solution and standard solution.
Perform the test with these solutions as directed under Thin-
layer chromatography <2.03>. Spot 2 /uL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of water, ammonia water (28), methanol and tetrahydrofu-
ran (1:1:1:1) to a distance of about 10cm, and air-dry the
plate. Spray evenly ninhydrin-citric acid-acetic acid TS on the
plate, and heat at 100°C for 10 minutes: the principal spots
from the sample solution and the standard solution exhibit a
red-purple color and show the same Rf value.
(3) A solution of Amikacin Sulfate (1 in 100) responds to
the Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [a]™: +76 - +84° (1 g, water, 100
mL, 100 mm).
pH <2.54> Dissolve 1 .0 g of Amikacin Sulfate in 100 mL of
water: the pH of the solution is between 6.0 and 7.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Amikacin Sulfate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.10 g of Amikacin
Sulfate in 4 mL of a water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer chromatography <2.03>. Spot 2 fiL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of water, ammonia water (28), methanol and tetra-
hydrofuran (1:1:1:1) to a distance of about 10 cm, and air-
dry the plate. Spray evenly ninhydrin-citric acid-acetic acid
TS on the plate, and heat at 100°C for 10 minutes: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 4.0% (1 g, in vacuum,
60°C, 3 hours).
Assay Weigh accurately an amount of Amikacin Sulfate
and Amikacin Sulfate Reference Standard, equivalent to
about 50 mg (potency), dissolve each in water to make exactly
50 mL. Pipet 200 //L each of these solutions in the test tube
with glass stopper, add exactly 3 mL of pyridine and exactly 2
mL of a solution of 2,4,6-trinitrobenzenesulfonic acid (1 in
100), stopper tightly, and heat in a water bath at 70°C for 30
minutes. After cooling, add exactly 2 mL each of acetic aid
(100), and use these solutions as the sample solution and stan-
dard solution, respectively. Perform the test with exactly 20
fiL each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the heights, H T and H s , of the peak of amika-
cin derivative.
Amount [fig (potency)] of amikacin (C 2 2H43N 5 13 )
= W s x (H T /H S ) x 1000
W s : Amount [mg (potency)] of Amikacin Sulfate Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 340 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 2.72 g of potassium dihydrogen-
phosphate in 800 mL of water, adjust to pH 6.5 with a solu-
tion of potassium hydroxide (1 in 40), and add water to make
1000 mL. To 280 mL of this solution add 720 mL of
methanol, and mix.
Flow rate: Adjust the flow rate so that the retention time of
amikacin derivative is about 9 minutes.
System suitability —
System performance: Dissolve about 5 mg (potency) of
Amikacin Sulfate and about 5 mg (potency) of Kanamycin
JPXV
Official Monographs / Aminophylline Injection
297
Sulfate in 5 mL of water. Transfer 200 /xL of this solution in
a glass-stopperd test tube, add 3 mL of pyridine and 2 mL of
a solution of 2,4,6-trinitrobenzenesulfonic acid (1 in 100),
stopper tightly, heat in a water bath at 70°C for 30 minutes.
After cooling, add 2 mL of acetic acid (100). When the proce-
dure is run with 20 liL of this solution under the above oper-
ating conditions, amikacin derivative and kanamycin deriva-
tive are eluted in this order with the resolution between these
peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of amikacin derivative is not more than 2.0%.
Containers and storage Containers — Hermetic containers.
Aminophylline Hydrate
aw
HjN..
■xHiO
"NH ?
C 14 H 16 N 8 4 .C 2 H 8 N 2 .xH 2
l,3-Dimethyl-l//-purine-2,6 (3i/,7W)-dione
hemi(ethylenediamine) hydrate [5877-66-5 , dihydrate]
Aminophylline Hydrate contains not less than 84.0
% and not more than 86.0% of theophylline
(C 7 H 8 N 4 2 : 180.16), and not less than 14.0% and not
more than 15.0% of ethylenediamine (C 2 H 8 N 2 : 60.10),
calculated on the anhydrous basis.
Description Aminophylline Hydrate occurs as white to pale
yellow granules or powder. It is odorless or slightly am-
monia-like odor, and has a bitter taste.
It is soluble in water, slightly soluble in methanol, and
practically insoluble in ethanol (95) and in diethyl ether.
To 1 g of Aminophylline Hydrate add 5 mL of water, and
shake: it dissolves almost completely. Separation of crystals
begins in 2 to 3 minutes, and these crystals dissolve on the ad-
dition of a small amount of ethylenediamine.
It is gradually affected by light, and gradually loses
ethylenediamine in air.
Identification (1) Dissolve 0.75 g of Aminophylline Hy-
drate in 30 mL of water, and use this solution as the sample
solution. To 20 mL of the sample solution add 1 mL of dilute
hydrochloric acid: a precipitate is gradually formed. Filter
the precipitate, recrystallize from water, and dry at 105 °C for
1 hour: the crystals so obtained melt <2.60> between 271 °C
and 275 °C.
(2) Dissolve 0.1 g of the crystals obtained in (1) in 50 mL
of water, and to 2 mL of this solution add tannic acid TS
dropwise: a white precipitate is produced, and this precipitate
dissolves upon dropwise addition of tannic acid TS.
(3) To 0.01 g of the crystals obtained in (1) add 10 drops
of hydrogen peroxide TS and 1 drop of hydrochloric acid,
and evaporate on a water bath to dryness: the residue shows a
yellow-red color. Invert the dish containing the residue over a
vessel containing 2 to 3 drops of ammonia TS: the color of
the residue changes to red-purple, which is destroyed on the
addition of 2 to 3 drops of sodium hydroxide TS.
(4) Dissolve 0.01 g of the crystals obtained in (1) in 5 mL
of water, add 3 mL of ammonia-ammonium chloride buffer
solution, pH 8.0, and 1 mL of copper (II) sulfate-pyridine
TS, and mix. Add 5 mL of chloroform to the mixture, and
shake: the chloroform layer develops a green color.
(5) To 5 mL of the sample solution obtained in (1) add 2
drops of copper (II) sulfate TS: a purple color develops. Add
1 mL of copper (II) sulfate TS: the color changes to blue, and
green precipitates are formed on standing.
pH <2.54> Dissolve 1.0 g of Aminophylline Hydrate in 25
mL of water: the pH of the solution is between 8.0 and 9.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Aminophylline Hydrate in 10 mL of hot water: the solution is
clear and colorless to pale yellow.
(2) Heavy metals <1.07>— Proceed with 1.0 g of
Aminophylline Hydrate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
Water <2.48> Not more than 7.9% (0.3 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Theophylline — Weigh accurately about 0.25 g
of Aminophylline Hydrate, and dissolve in 50 mL of water
and 8 mL of ammonia TS by gentle warming on a water bath.
Add exactly 20 mL of 0. 1 mol/L silver nitrate VS, warm on a
water bath for 15 minutes, allow to stand between 5°C and
10°C for 20 minutes, collect the precipitate by suction, and
wash with three 10-mL portions of water. Combine the
filtrate and washings, and add dilute nitric acid to make
neutral. Add 3 mL of dilute nitric acid, and titrate <2.50> the
excess silver nitrate with 0.1 mol/L ammonium thiocyanate
VS (indicator: 2 mL of ammonium iron (III) sulfate TS). Per-
form a blank determination.
Each mL of 0.1 mol/L silver nitrate VS
= 18.02 mg of theophylline (C 7 H 8 N 4 2 )
(2) Ethylenediamine — Weigh accurately about 0.5 g of
Aminophylline Hydrate, dissolve in 30 mL of water, and ti-
trate <2.50> with 0.1 mol/L hydrochloric acid VS (indicator:
3 drops of bromophenol blue TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 3.005 mg of ethylenediamine (C 2 H 8 N 2 )
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Aminophylline Injection
7 5/7-f y >a*f*
Aminophylline Injection is an aqueous solution for
injection.
It contains not less than 75% and not more than 86
% of the labeled amount of theophylline
(C 7 H 8 N 4 2 : 180.16), and not less than 13% and not
more than 20% of ethylenediamine (C 2 H 8 N 2 : 60.10).
298
Amitriptyline Hydrochloride / Official Monographs
JP XV
The concentration of Aminophylline Injection is
expressed as the quantity of aminophylline dihydrate
(C 16 H 24 N 10 O 4 .2H 2 O: 456.46).
Method of preparation Prepare as directed under Injec-
tions, with Aminophylline Hydrate. It may be prepared with
Theophylline and its equivalent Ethylenediamine, instead of
Aminophylline Hydrate.
It may contain not more than 60 mg of Ethylenediamine as
a stabilizer for each g of Aminophylline Hydrate.
Description Aminophylline Injection is a clear and colorless
liquid. It has a slightly bitter taste.
It gradually changes in color by light.
pH: 8.0-10.0
Identification To a volume of Aminophylline Injection,
equivalent to 0.75 g of Aminophylline Hydrate according to
the labeled amount, add water to make 30 mL. Proceed with
this solution as directed in the Identification under
Aminophylline Hydrate.
Extractable volume <6.0J> It meets the requirement.
Assay (1) Theophylline — To an accurately measured
volume of Aminophylline Injection, equivalent to about
0.2 g of theophylline (C 7 H 8 N 4 2 ) (about 0.25 g of
Aminophylline Hydrate), add 15 mL of water, 8 mL of am-
monia TS and 20 mL of silver nitrate TS, and warm on a
water bath for 15 minutes. Cool to between 5°C and 10°C for
20 minutes, filter the precipitate through a glass filter (G4),
and wash with three 10-mL portions of water. Dissolve the
precipitate in 5 mL of nitric acid, and wash the filter with
three 10-mL portions of water. Combine the nitric acid solu-
tion and washings, and titrate <2.50> with 0.1 mol/L ammo-
nium thiocyanate VS (indicator: 2 mL of ammonium iron
(III) sulfate TS).
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 18.02 mg of theophilline (C 7 H 8 N 4 2 )
(2) Ethylenediamine — To an accurately measured volume
of Aminophylline Injection, equivalent to about 30 mg of
ethylenediamine (C 2 H 8 N 2 ) (about 0.2 g of Aminophylline
Hydrate), add water to make 30 mL, and titrate <2.50> with
0.1 mol/L hydrochloric acid VS (indicator: 2 to 3 drops of
bromophenol blue TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 3.005 mg of ethylenediamine (C 2 H 8 N 2 )
Containers and storage Containers-
Storage — Light-resistant.
■Hermetic containers.
Amitriptyline Hydrochloride
7 5 r- U -i^ U >WI£ik
ylidene)-/V,A f -dimethylpropylamine monohydrochloride
[549-18-8]
Amitriptyline Hydrochloride, when dried, contains
not less than 99.0% of C 20 H 23 N.HC1.
Description Amitriptyline Hydrochloride occurs as color-
less crystals or a white to pale yellow crystalline powder. It
has a bitter taste and a numbing effect.
It is freely soluble in water, in ethanol (95) and in acetic
acid (100), soluble in acetic anhydride, and practically insolu-
ble in diethyl ether.
The pH of a solution of Amitriptyline Hydrochloride (1 in
20) is between 4.0 and 5.0.
Identification (1) Dissolve 5 mg of Amitriptyline
Hydrochloride in 3 mL of sulfuric acid: a red color develops.
Add 5 drops of potassium dichromate TS to this solution: it
turns dark brown.
(2) Acidify 1 mL of a solution of Amitriptyline
Hydrochloride (1 in 500) with 0.5 mL of dilute nitric acid,
and add 1 drop of silver nitrate TS: a white, opalescent
precipitate is produced.
(3) Determine the absorption spectrum of a solution of
Amitriptyline Hydrochloride (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Amitriptyline Hydrochloride Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
Melting point <2.60> 195 - 198°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Amitriptyline Hydrochloride in 20 mL of water: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Amitrip-
tyline Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Amitriptyline
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.39 mg of C 20 H 23 N.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
„CH 3
C 20 H 23 N.HC1: 313.86
3-(10,ll-Dihydro-5//-dibenzo[a,rf]cyclohepten-5-
JPXV
Official Monographs / Ammonia Water
299
Amitriptyline Hydrochloride
Tablets
Amitriptyline Hydrochloride Tablets contain not
less than 90% and not more than 110% of the
labeled amount of amitriptyline hydrochloride
(C 2 oH 23 N.HCl: 313.86).
Method of preparation Prepare as directed under Tablets,
with Amitriptyline Hydrochloride.
Identification (1) Weigh a quantity of powdered Amitrip-
tyline Hydrochloride Tablets, equivalent to 0.1 g of Amitrip-
tyline Hydrochloride according to the labeled amount. Add
10 mL of chloroform, shake thoroughly, and filter.
Evaporate the filtrate on a water bath to about 2mL, add
diethyl ether until turbidity is produced, and allow to stand.
Filter the crystals formed through a glass filter (G4), and
proceed as directed in the Identification (1) and (2) under
Amitriptyline Hydrochloride.
(2) Determine the absorption spectrum of a solution of
the crystals obtained in (1) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 238 nm and 240 nm, and a minimum be-
tween 228 nm and 230 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Amitriptyline Hydrochlo-
ride Tablets at 50 revolutions per minute according to the
Paddle method using 900 mL of 2 nd fluid for dissolution test
as the test solution. Take 20 mL or more of the dissolved so-
lution 60 minutes after starting the test, and filter through a
membrane filter with a pore size not exceeding 0.8 fim. Dis-
card the first 10 mL of the filtrate, pipet the subsequent KmL
of the filtrate, add 2 nd fluid for dissolution test to make ex-
actly V mL so that each mL contains about 1 1 fig of amitrip-
tyline hydrochloride (C20H23N.HCI) according to the labeled
amount, and use this solution as the sample solution.
Separately, weigh accurately about 55 mg of Amitriptyline
Hydrochloride Reference Standard, previously dried at
105 °C for 2 hours, and dissolve in 2 nd fluid for dissolution
test to make exactly 250 mL. Pipet 5 mL of this solution, add
2 nd fluid for dissolution test to make exactly 100 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A T and A s , of the sample solution and standard
solution at 239 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
The dissolution rate of Amitriptyline Hydrochloride
Tablets in 60 minutes should be not less than 70%.
Dissolution rate (%) with respect to the labeled
amount of amitriptyline hydrochloride (C20H23N.HCI)
= W s x (A T /A S ) x (V'/V) x (1/C) x 18
W s : Amount (mg) of Amitriptyline Hydrochloride
Reference Standard.
C: Labeled amount (mg) of amitriptyline hydrochloride
(C20H23N.HCI) in 1 tablet.
Assay Weigh accurately and powder not less than 20
Amitriptyline Hydrochloride Tablets. Weigh accurately a
portion of the powder, equivalent to about 20 mg of amitrip-
tyline hydrochloride (C20H23N.HCI), and add 75 mL of
diluted methanol (1 in 2). After shaking for 30 minutes, add
diluted methanol (1 in 2) to make exactly 100 mL, and filter.
Discard the first 20-mL portion of the filtrate, measure
exactly the subsequent 5-mL portion, add methanol to make
exactly 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 20 mg of Amitriptyline
Hydrochloride Reference Standard, previously dried at
105 °C for 2 hours, and dissolve in diluted methanol (1 in 2)
to make exactly 100 mL. Measure exactly 5 mL of this
solution, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 239 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, respectively.
Amount (mg) of amitriptyline hydrochloride
(C20H23N.HCI)
= W s x (A T /A S )
Ws' Amount (mg) of Amitriptyline Hydrochloride Refer-
ence Standard
Containers and storage Containers — Tight containers.
Ammonia Water
7>T-77k
Ammonia Water contains not less than 9.5 w/v%
and not more than 10.5 w/v% of ammonia (NH 3 :
17.03).
Description Ammonia Water occurs as a clear, colorless
liquid, having a very pungent, characteristic odor.
It is alkaline.
Specific gravity df : 0.95 - 0.96
Identification (1) Hold a glass rod moistened with
hydrochloric acid near the surface of Ammonia Water: dense
white fumes are produced.
(2) Hold moistened red litmus paper near the surface of
Ammonia Water: it turns blue.
Purity (1) Residue on evaporation — Evaporate 10.0 mL
of Ammonia Water to dryness, and dry the residue at 105 °C
for 1 hour: the mass of the residue is not more than 2.0 mg.
(2) Heavy metals <1.07> — Evaporate 5.0 mL of Ammo-
nia Water to dryness on a water bath, add 1 mL of dilute
hydrochloric acid to the residue, and evaporate to dryness.
Dissolve the residue in 2 mL of dilute acetic acid, add water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution with 2.5 mL of
Standard Lead Solution, 2 mL of dilute acetic acid and water
to make 50 mL (not more than 5 ppm).
(3) Potassium permanganate-reducing substances — To
10.0 mL of Ammonia Water add 40 mL of dilute sulfuric
acid while cooling, and add 0.10 mL of 0.02 mol/L potassi-
um permanganate VS: the red color of the potassium per-
manganate does not disappear within 10 minutes.
300
Amobarbital / Official Monographs
JP XV
Assay Measure exactly 5 mL of Ammonia Water, add 25
mL of water, and titrate <2.50> with 0.5 mol/L sulfuric acid
VS (indicator: 2 drops of methyl red TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 17.03 mg of NH 3
Containers and storage Containers-
Storage — Not exceeding 30°C.
Amobarbital
T=EJi)l>¥9-)\,
-Tight containers.
"'°Kyy
O
C„H 18 N 2 3 : 226.27
5-Ethyl-5-(3-methylbutyi)pyrimidine-
2,4,6(l//,3//,5//)-trione [57-43-2]
Amobarbital, when dried,
99.0% of C„H 18 N 2 3 .
contains not less than
Description Amobarbital occurs as white crystals or crystal-
line powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in ethanol (95), in acetone and in diethyl
ether, sparingly soluble in chloroform, and practically insolu-
ble in water.
It dissolves in sodium hydroxide TS and in sodium car-
bonate TS.
The pH of a saturated solution of Amobarbital is between
5.0 and 5.6.
Identification (1) Boil 0.2 g of Amobarbital with 10 mL of
sodium hydroxide TS: the gas evolved changes moistened red
litmus paper to blue.
(2) Dissolve 0.05 g of Amobarbital in 2 to 3 drops of
ammonia-ammonium chloride buffer solution, pH 10.7, and
5 mL of diluted pyridine (1 in 10). Add 5 mL of chloroform
and 0.3 mL of copper (II) sulfate TS to the solution: a red-
purple precipitate is produced in the aqueous layer. Shake the
mixture: a red-purple color is produced in the chloroform
layer.
(3) To 0.4 g of Amobarbital add 0.1 g of anhydrous
sodium carbonate and 4 mL of water, shake, and add a
solution of 0.3 g of 4-nitrobenzyl chloride in 7 mL of ethanol
(95). Heat the mixture on a water bath for 30 minutes under a
reflux condenser, and allow to stand for 1 hour. Filter the
crystals produced, wash with 7 mL of sodium hydroxide TS
and a small portion of water, recrystallize from ethanol, and
dry at 105°C for 30 minutes: the crystals so obtained melt
<2.60> between 168°C and 173°C or between 150°C and 154°
C.
Melting point <2.60> 157 - 160°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Amobarbital in 5 mL of sodium hydroxide TS: the solution is
clear and colorless.
(2) Chloride <1.03>— Dissolve 0.30 g of Amobarbital in
20 mL of acetone, and add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: take
0.30 mL of 0.01 mol/L hydrochloric acid VS, 20 mL of
acetone and 6 mL of dilute nitric acid, and add water to make
50 mL (not more than 0.035%).
(3) Sulfate <1.14>— Dissolve 0.40 g of Amobarbital in 20
mL of acetone, and add 1 mL of dilute hydrochloric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: take
0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of acetone,
and 1 mL of dilute hydrochloric acid, and add water to make
50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1 .0 g of Amobar-
bital according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(5) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Amobarbital. The solution is not more
colored than Matching Fluid A.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Amobarbital, previ-
ously dried, and dissolve in 5 mL of ethanol (95) and 50 mL
of chloroform. Titrate <2.50> with 0.1 mol/L potassium
hydroxide-ethanol VS until the color of the solution changes
from yellow through light blue to purple (indicator: 1 mL of
alizarin yellow GG-thymolphthalein TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 22.63 mg of C u H I8 N 2 3
Containers and storage Containers — Well-closed contain-
ers.
Amobarbital Sodium for Injection
aatfflT^M'JU^-JUt- r- U ^7 A
H 3 C
„ONa
C u H 17 N 2 Na0 3 : 248.25
Monosodium 5-ethyl-5-(3-methylbutyl)-4,6-
dioxo-l,4,5,6-tetrahydropyrimidin-2-olate [64-43-7]
Amobarbital Sodium for Injection is a preparation
for injection which is dissolved before use.
When dried, it contains not less than 98.5% of
amobarbital sodium (C 11 H 17 N 2 Na0 3 ), and not less
than 92.5% and not more than 107.5% of the labeled
amount of amobarbital sodium (C 11 H 17 N 2 Na0 3 ).
Method of preparation
tions.
Prepare as directed under Injec-
Description Amobarbital Sodium for Injection occurs as
white crystals or a crystalline powder. It is odorless, and has a
bitter taste.
JPXV
Official Monographs / Amoxapine
301
It is freely soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether and in chloroform.
The pH of its solution (1 in 10) is between 10.0 and 11.0.
It is hygroscopic.
Identification (1) Dissolve 1.5 g of Amobarbital Sodium
for Injection in 20 mL of water, and add 10 mL of dilute
hydrochloric acid with stirring: a white precipitate is
produced. Collect the precipitate, wash with four 10-mL por-
tions of water, and dry at 105 C C for 3 hours: it melts <2.60>
between 157°C and 160°C. With this precipitate, proceed as
directed in the Identification under Amobarbital.
(2) Ignite 0.5 g of Amobarbital Sodium for Injection,
cool, and dissolve the residue in 10 mL of water: the solution
responds to the Qualitative Tests <1.09> (1) for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Amobarbital Sodium for Injection in 10 mL of freshly boiled
and cooled water: the solution is clear and colorless.
(2) Chloride <1.03>— Dissolve 1.0 g of Amobarbital
Sodium for Injection in 49 mL of water, add 1 mL of acetic
acid (100), shake, and filter. Discard the first 10 mL of the
filtrate, and to the subsequent 30 mL of the filtrate add 6 mL
of dilute nitric acid and water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the
control solution as follows: to 0.30 mL of 0.01 mol/L
hydrochloric acid VS add 0.5 mL of acetic acid (100), 6 mL
of dilute nitric acid and water to make 50 mL (not more than
0.018%).
(3) Sulfate <1.14>— Dissolve 2.0 g of Amobarbital
Sodium for Injection in 49 mL of water, add 1 mL of acetic
acid (100), shake, and filter. Discard the first 10 mL of the
filtrate, and to the subsequent 25 mL of the filtrate add 2.5
mL of dilute hydrochloric acid and water to make 50 mL.
Perform the test using this solution as the test solution.
Prepare the control solution as follows: to 0.40 mL of 0.005
mol/L sulfuric acid VS add 0.5 mL of acetic acid (100), 1 mL
of dilute hydrochloric acid and water to make 50 mL (not
more than 0.019%).
(4) Heavy metals <1.07> — Dissolve 2.0 g of Amobarbital
Sodium for Injection in 45 mL of water, add 5 mL of dilute
hydrochloric acid, shake vigorously, and warm on a water
bath for 2 minutes with occasional shaking. Cool, add 30 mL
of water, shake, and filter. Discard the first 10 mL of the
filtrate, add 1 drop of phenolphthalein TS to the subsequent
40 mL of the filtrate, add ammonia TS until a slight red color
develops, and add 2.5 mL of dilute hydrochloric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
2.5 mL of dilute hydrochloric acid add 1 drop of
phenolphthalein TS, add ammonia TS until a pale red color
develops, and add 2.5 mL of dilute acetic acid, 2.0 mL of
Standard Lead Solution and water to make 50 mL (not more
than 20 ppm).
(5) Neutral or basic substances — Dissolve about 1 g of
Amobarbital Sodium for Injection, accurately weighed, in 10
mL of water and 5 mL of sodium hydroxide TS, then add 40
mL of chloroform, and shake well. Separate the chloroform
layer, wash with two 5-mL portions of water, and filter.
Evaporate the filtrate on a water bath to dryness, and dry the
residue at 105°C for 1 hour: the mass of the residue is not
more than 0.30%.
(6) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Amobarbital Sodium for Injection: the so-
lution is not more colored than Matching Fluid A.
Loss on drying <2.4I> Not more than 1.0% (1 g, 105°C,
4 hours).
Assay Weigh accurately the contents of not less than 10
samples of Amobarbital Sodium for Injection. Weigh
accurately about 0.5 g of the contents, previously dried,
transfer to a separator, dissolve in 20 mL of water, add 5 mL
of ethanol (95) and 10 mL of dilute hydrochloric acid, and
extract with 50 mL of chloroform, then with three 25-mL
portions of chloroform. Combine the chloroform extracts,
wash with two 5-mL portions of water, and extract the
washings with two 10-mL portions of chloroform. Filter the
combined chloroform extracts into a conical flask, and wash
the filter paper with three 5-mL portions of chloroform.
Combine the filtrate and the washings, and add 10 mL of
ethanol (95). Titrate <2.50> with 0.1 mol/L potassium
hydroxide-ethanol VS until the color of the solution changes
from yellow through light blue to purple (indicator: 2 mL of
alizarin yellow GG-thymolphthalein TS). Perform a blank
determination with a mixture of 160 mL of chloroform and
30 mL of ethanol (95), and make any necessary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 24.83 mg of C„H 17 N 2 Na0 3
Containers and storage Containers — Hermetic containers.
Amoxapine
C 17 H 16 C1N 3 0: 313.78
2-Chloro- 1 1 -(piperazin- 1 -yl)dibenzo [b,f] [1,4] oxazepine
[14028-44-5]
Amoxapine, when dried, contains not less than
98.5% of C 17 H 16 C1N 3 0.
Description Amoxapine occurs as white to light yellowish
white crystals or crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
ethanol (95) and in diethyl ether, and practically insoluble in
water.
Identification (1) Determine the absorption spectrum of a
solution of Amoxapine in 0.1 mol/L hydrochloric acid TS
(1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption as the same wavelengths.
(2) Determine the infrared absorption spectrum of
Amoxapine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
302
Amoxicillin Hydrate / Official Monographs
JP XV
(3) Perform the test with Amoxapine as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 178 - 182°C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Amoxapine according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 15 ppm).
(2) Related substances — Dissolve 0.5 g of Amoxapine in
10 mL of a mixture of ethanol (95) and acetic acid (100) (9:1),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, and add a mixture of ethanol (95) and
acetic acid (100) (9:1) to make exactly 10 mL. Pipet 1 mL of
this solution, add a mixture of ethanol (95) and acetic acid
(100) (9:1) to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of ethanol (95)
and acetic acid (100) (9:1) to a distance of about 10 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.4% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Amoxapine, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to greenish
blue (indicator: 2 drops of crystal violet TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 15.69 mg of C 17 H 16 C1N 3
Containers and storage Containers — Tight containers.
Amoxicillin Hydrate
•3HjO
C 16 H 19 N 3 5 S.3H 2 0: 419.45
(2S,5i?,6i?)-6-[(2i?)-2-Amino-2-(4-hydroxyphenyl)-
acetylamino] -3 , 3-dimethyl-7-oxo-4-thia- 1 -
azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate
[61336-70-7]
Amoxicillin Hydrate contains not less than 950 fig
(potency) and not more than 1010 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Amoxicillin Hydrate is expressed as mass (potency) of
amoxicillin (C 16 H 19 N 3 O s S: 365.40).
Description Amoxicillin Hydrate occurs as white to light
yellowish white, crystals or crystalline powder.
It is slightly soluble in water and in methanol, and very
slightly soluble in ethanol (95).
Identification Determine the infrared absorption spectrum
of Amoxicillin Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Amoxicillin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™'- +290 - +315° (0.1 g calculated
on the anhydrous basis, water, 100 mL, 100 mm).
Purity (1) Heavy metals <1.07> — To 1.0 g of Amoxicillin
Hydrate add 2 mL of a solution of magnesium sulfate hepta-
hydrate (1 in 4), mix, and heat on a water bath to dryness.
Carbonize the residue by gently heating. After cooling, add 1
mL of sulfuric acid, heat carefully, then heat at 500 - 600°C
to incinerate. After cooling, add 1 mL of hydrochloric acid to
the residue, and heat on a water bath to dryness. Then add 10
mL of water to the residue, and heat on a water bath to dis-
solve. After cooling, add ammonia TS to adjust the pH to 3 -
4, and add 2 mL of dilute acetic acid. If necessary, filter,
wash the residue on the filter with 10 mL of water, transfer
the filtrate and washings into a Nessler tube, add water to
make 50 mL, and use this solution as the test solution. Pre-
pare the control solution as follows: To 2.0 mL of Standard
Lead Solution add 2 mL of a solution of magnesium sulfate
heptahydrate (1 in 4), then proceed in the same manner as for
preparation of the test solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Amoxicillin Hydrate according to Method 4, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Amoxicillin
Hydrate in 50 mL of a solution of sodium tetraborate deca-
hydrate (1 in 200), and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add a solution of so-
dium tetraborate decahydrate (1 in 200) to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 10 fiL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
each peak area by the automatic integration method: the area
of the peak other than amoxicillin obtained from the sample
solution is not more than the peak area of amoxicillin from
the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.361 g of sodium acetate trihy-
drate in 750 mL of water, adjust the pH to 4.5 with acetic
acid (31), and add water to make 1000 mL. To 950 mL of this
solution add 50 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
amoxicillin is about 8 minutes.
Time span of measurement: About 4 times as long as the
JPXV
Official Monographs / Amphotericin B
303
retention time of amoxicillin.
System suitability —
Test for required detection: To exactly 1 mL of the
standard solution add a solution of sodium tetraborate
decahydrate (1 in 200) to make exactly 10 mL. Confirm that
the peak area of amoxicillin obtained from 10 fiL of this solu-
tion is equivalent to 7 to 13% of that of amoxicillin obtained
from 10 fiL of the standard solution.
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the number of theoretical plates of the peak of
amoxicillin is not less than 2500.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
amoxicillin is not more than 1.0%.
Water <2.48> Not less than 11.0% and not more than
15.0% (0.1 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Amoxicillin Hydrate
and Amoxicillin Reference Standard, equivalent to about 30
mg (potency), dissolve each in a solution of boric acid (1 in
200) to make exactly 100 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
exactly 10 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the peak
areas, A T and A s , of amoxicillin of each solution.
Amount \jug (potency)] of amoxicillin (C I6 H 19 N 3 05S)
= W s x (Aj/A s ) x 1000
W s : Amount [mg (potency)] of Amoxicillin Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.361 g of sodium acetate trihy-
drate in 750 mL of water, adjust the pH to 4.5 with acetic
acid (31), and add water to make 1000 mL. To 950 mL of this
solution add 50 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
amoxicillin is about 8 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the number of theoretical plates of the peak of
amoxicillin is not less than 2500.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
amoxicillin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Amphotericin B
H OH
H CH 3
C 47 H 73 N0 17 : 924.08
(IR,3S,5R,6R,9R, IIR, 15S, 16R, 17R, 18S, 19£, 21E,
23£,25£,27£',29£',31£,33fl,35S',36S,37S')-33-(3-
Amino-3,6-dideoxy-/?-D-mannopyranosyloxy)-
l,3,5,6,9,ll,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-
14,39-dioxabicyclo[33.3.1]nonatriaconta-
19,21,23,25,27,29,31-heptaene-36-carboxylic acid
[1397-89-3]
Amphotericin B is a polyene macrolide substance
having antifungal activity produced by the growth of
Streptomyces nodosus.
It contains not less than 840 tig (potency) per mg,
calculated on the dried basis. The potency of Am-
photericin B is expressed as mass (potency) of am-
photericin B (C 47 H 73 N0 17 ).
Description Amphotericin B occurs as a yellow to orange
powder.
It is freely soluble in dimethylsulfoxide and practically
insoluble in water and in ethanol (95).
Identification (1) Dissolve 5 mg of Amphotericin B in 10
mL of dimethylsulfoxide. To 1 mL of this solution add 5 mL
of phosphoric acid: a blue color develops between the two
layers, and the solution becomes blue by shaking. After
addition of 15 mL of water it becomes yellow to light yellow-
brown by shaking.
(2) Dissolve 25 mg of Amphotericin B in 5 mL of
dimethylsulfoxide, and add methanol to make 50 mL. To 1
mL of this solution add methanol to make 50 mL. Determine
the absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
Amphotericin B Reference Standard: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Purity Amphotericin A — Weigh accurately about 50 mg
each of Amphotericin B and Amphotericin B Reference
Standard, add exactly 10 mL each of dimethylsulfoxide to
dissolve, and add methanol to make exactly 50 mL. Pipet
4 mL each of these solutions, add methanol to make exactly
50 mL, and use these solutions as the sample solution and
standard solution (1), respectively. Separately, weigh ac-
curately about 20 mg of Nystatin Reference Standard, add
exactly 40 mL of dimethylsulfoxide to dissolve, then add
methanol to make exactly 200 mL. Pipet 4 mL of this solu-
304
Amphotericin B for Injection / Official Monographs
JP XV
tion, add methanol to make exactly 50 mL, and use this solu-
tion as the standard solution (2). Perform the test with these
solutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> using a solution obtained in the same manner as
the sample solution as the blank, and determine the absor-
bances at 282 nm and at 304 nm. Calculate the amount of
amphotericin A by the following equation: not more than 5%
for Amphotericin B used for injections, and not more than
15% for Amphotericin B not used for injections.
Amount (%) of amphotericin A
= W s x {Q4 Sal x A T2 ) - l4 Sa2 x A T1 )} x 25
W T x {(,4 Sal x A sb2 ) - (A Sa2 x A sbl )}
W s : Amount (mg) of Nystatin Reference Standard
W T : Amount (mg) of the sample
A S!il : Absorbance at 282 nm of the standard solution (1)
^4 sbl : Absorbance at 282 nm of the standard solution (2)
A S z 2 '- Absorbance at 304 nm of the standard solution (1)
A sb2 : Absorbance at 304 nm of the standard solution (2)
A Tl : Absorbance at 282 nm of the sample solution
A T2 '- Absorbance at 304 nm of the sample solution
Loss on drying <2.41> Not more than 5.0% (0.1 g, in vacu-
um, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Saccharomyces cerevisiae ATCC 9763
(ii) Culture medium — Use the medium 2) under (1) Agar
media for seed and base layer.
(iii) Preparation of cylinder-agar plate — Proceed as
directed in 5 under the Cylinder plate method, using Petri
dish plates not dispensing the agar medium for base layer and
dispensing 8.0 mL of the seeded agar medium.
(iv) Standard solution — Use light-resistant vessels. Weigh
accurately an amount of Amphotericin B Reference Standard
equivalent to about 20 mg (potency), dissolve in dimethylsul-
f oxide to make exactly 20 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
5 C C or below and use within 24 hours. Take exactly a suitable
amount of the standard stock solution before use, and add
dimethylsulfoxide to make solutions so that each mL con-
tains 200 ^g (potency) and 50 fig (potency). Pipet 1 mL each
of these solutions, add 0.2 mol/L phosphate buffer solution,
pH 10.5 to make exactly 20 mL, and use these solutions as the
high concentration standard solution and low concentration
standard solution, respectively.
(v) Sample solution — Use light-resistant vessels. Weigh
accurately an amount of Amphotericin B equivalent to about
20 mg (potency), dissolve in dimethylsulfoxide to make
exactly 20 mL, and use this solution as the sample stock
solution. Take exactly a suitable amount of the sample stock
solution, add dimethylsulfoxide to make solutions so that
each mL contains 200 fig (potency) and 50 fig (potency).
Pipet 1 mL each of these solutions, add 0.2 mol/L phosphate
buffer solution, pH 10.5 to make exactly 20 mL, and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Amphotericin B for Injection
al7ffl7A*xU->>B
Amphotericin B for Injection is a preparation for
injection which is dissolved before use.
It contains not less than 90.0% and not more than
120.0% of the labeled amount of amphotericin B
(C 47 H 73 N0 17 : 924.08).
Method of preparation Prepare as directed under Injec-
tions, with Amphotericin B.
Description Amphotericin B for Injection occurs as yellow
to orange, powder or masses.
Identification To an amount of Amphotericin B for Injec-
tion, equivalent to 25 mg (potency) of Amphotericin B ac-
cording to the labeled amount, add 5 mL of dimethylsul-
foxide and 45 mL of methanol, and shake. To 1 mL of this
solution add methanol to make 50 mL, and filter if necessary.
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits maxima between 361 nm and 365 nm, between 380 nm
and 384 nm and between 403 nm and 407 nm.
pH <2.54> Dissolve an amount of Amphotericin B for Injec-
tion, equivalent to 50 mg (potency) of Amphotericin B ac-
cording to the labeled amount, in 10 mL of water. To 1 mL
of this solution add water to make 50 mL: 7.2 - 8.0.
Purity Clarity and color of solution — Dissolve an amount
of Amphotericin B for Injection, equivalent to 50 mg (poten-
cy) of Amphotericin B according to the labeled amount, in 10
mL of water: the solution is clear and yellow to orange.
Loss on drying <2.41> Not more than 8.0% (0.3 g, in vacu-
um, 60°C, 3 hours).
Bacterial endotoxins <4.01> Less than 3.0EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test. However, use the average of the
limits specified in the potency definition for T.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, preparation of cylin-
der-agar plate and standard solutions — Proceed as directed in
the Assay under Amphotericin B.
(ii) Sample solutions — Prepare using light-resistant con-
tainers. Weigh accurately an amount of Amphotericin B for
Injection, equivalent to about 50 mg (potency) according to
the labeled amount, dissolve in dimethylsulfoxide to make
exactly 50 mL, and use this solution as the sample stock solu-
JPXV
Official Monographs / Amphotericin B Tablets
305
tion. Measure exactly a suitable quantity of the sample stock
solution, add dimethylsulfoxide to make solutions so that
each mL contains about 200 /ug (potency) and 50 /ug (poten-
cy). Pipet 1 mL each of these solutions, add 0.2 mol/L phos-
phate buffer solution, pH 10.5 to make exactly 20 mL, and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Hermetic containers.
Storage — Store in a cold place.
Amphotericin B Syrup
Amphotericin B Syrup contain not less than 90.0%
and not more than 115.0% of the labeled amount of
amphotericin B (C 47 H 73 N0 17 : 924.08).
Method of preparation
with Amphotericin B.
Prepare as directed under Syrup,
Identification To an amount of Amphotericin B Syrup,
equivalent to 25 mg (potency) of Amphotericin B according
to the labeled amount, add 5 mL of dimethylsulfoxide and 45
mL of methanol, and shake. To 1 mL of this solution add
methanol to make 50 mL, and filter, if necessary. Determine
the absorption spectrum of the solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 361 nm and 365 nm, between 380 nm and 384
nm and between 403 nm and 407 nm.
pH <2.54> 5.0-7.0.
Microbial limits <4.05> Perform the test as directed under
Microbial Limit Test: the total microbial count is not more
than 100 per mL, and the total fungus and yeast count is not
more than 50 per mL.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, preparation of cylin-
der-agar plate and standard solutions — Proceed as directed in
the Assay under Amphotericin B.
(ii) Sample solutions — Prepare using light-resistant con-
tainers. Weigh accurately an amount of Amphotericin B
Syrup, equivalent to about 0.1 g (potency) according to the
labeled amount, add about 70 mL of dimethylsulfoxide,
shake, then add dimethylsulfoxide to make exactly 100 mL,
and use this solution as the sample stock solution. Measure
exactly a suitable amount of the sample stock solution, add
dimethylsulfoxide to make solutions so that each mL con-
tains about 200 /ug (potency) and 50 /xg (potency). Pipet 1 mL
each of these solutions, add 0.2 mol/L phosphate buffer solu-
tion, pH 10.5 to make exactly 20 mL, and use these solutions
as the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Amphotericin B Tablets
7A*x'J->>Bffi
Amphotericin B Tablets contain not less than 90.0%
and not more than 120.0% of the labeled amount of
amphotericin B (C 47 H73N0 17 : 924.08).
Method of preparation
with Amphotericin B.
Prepare as directed under Tablets,
Identification To an amount of pulverized Amphotericin B
Tablets, equivalent to 25 mg (potency) of Amphotericin B ac-
cording to the labeled amount, add 5 mL of dimethylsul-
foxide and 45 mL of methanol, and shake. To 1 mL of this
solution add methanol to make 50 mL, and filter, if necessa-
ry. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 361 nm and 365 nm, between 380
nm and 384 nm and between 403 nm and 407 nm.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test. However, use the average of the
limits specified in the potency definition for T.
Loss on drying <2.41>
urn, 60°C, 3 hours).
Not more than 5.0% (0.3 g, in vacu-
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, preparation of cylin-
der-agar plate and standard solutions — Proceed as directed in
the Assay under Amphotericin B.
(ii) Sample solutions — Prepare using light-resistant con-
tainers. Weigh accurately and powder not less than 20 tablets
of Amphotericin B Tablets. Weigh accurately a part of the
powder, equivalent to about 0.1 g (potency) according to the
labeled amount, add about 70 mL of dimethylsulfoxide,
shake, then add dimethylsulfoxide to make exactly 100 mL,
centrifuge, and use the supernatant liquid as the sample stock
solution. Measure exactly a suitable amount of the sample
stock solution, add dimethylsulfoxide to make solutions so
that each mL contains 200 /xg (potency) and 50 /xg (potency).
Pipet 1 mL each of these solutions, add 0.2 mol/L phosphate
buffer solution, pH 10.5 to make exactly 20 mL, and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Well-closed contain-
ers.
306
Anhydrous Ampicillin / Official Monographs
JP XV
Anhydrous Ampicillin
Anhydrous Aminobenzylpenicillin
&7k7>fcfvU >
"V ^
- COeH
H H
C 16 H 19 N 3 4 S: 349.40
(2S,5/?,6/?)-6-[(2/?)-2-Amino-2-phenylacetylamino]-
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-
carboxylic acid [69-53-4]
Anhydrous Ampicillin contains not less than 960 fig
(potency) and not more than 1005 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Anhydrous Ampicillin is expressed as mass (potency)
of ampicillin (C 16 H 19 N 3 4 S).
Description Anhydrous Ampicillin occurs as white to light
yellowish white, crystals or crystalline powder.
It is sparingly soluble in water, slightly soluble in
methanol, very slightly soluble in ethanol (95), and practical-
ly insoluble in acetonitrile.
Identification Determine the infrared absorption spectrum
of Anhydrous Ampicillin as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Optical rotation <2.49> [a]^: +280- +305° (0.5 g calcu-
lated on the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Anhydrous Ampicillin in 100 mL of water is between 4.0
and 5.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Anhydrous Ampicillin according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Anhydrous Ampicillin according to Method 3, and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.05 g of Anhydrous
Ampicillin in the mobile phase to make 50 mL, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the area of
each peak other than ampicillin from the sample solution is
not more than the peak area of ampicillin from the standard
solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: As long as about 10 times of
the retention time of ampicillin.
System suitability —
Test for required detectability: To exactly 1 mL of the
standard solution add the mobile phase to make exactly
10 mL. Confirm that the peak area of ampicillin obtained
from 10 ^L of this solution is equivalent to 7 to 13% of that
from 10 fiL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Water <2.48> Not more than 2.0% (2.5 g, volumetric
titration, direct titration).
Assay Weigh accurately an amount of Anhydrous Ampicil-
lin and Ampicillin Reference Standard, equivalent to about
50 mg (potency), add exactly 5 mL each of the internal
standard solution and the mobile phase to make 50 mL, and
use these solutions as the sample solution and standard solu-
tion. Perform the test with 10 fiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, g T and Q s , of the peak area of am-
picillin to that of the internal standard.
Amount [fig (potency)] of ampicillin (Q6LL9N3O4S)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Ampicillin Reference
Standard
Internal standard solution — A solution of guaifenesin in the
mobile phase (1 in 200).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.94 g of diammonium hydrogen
phosphate in 850 mL of water, add 100 mL of acetonitrile,
adjust the pH to 5.0 with phosphoric acid, and add water to
make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
ampicillin is about 6 minutes.
System suitability —
System performance: When the procedure is run with
10 [iL of the standard solution under the above operating
conditions, ampicillin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 40.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ampicillin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Ampicillin Hydrate
307
Ampicillin Hydrate
Aminobenzylpenicillin Hydrate
H £ N H
" y CO,H
■ 3H s O
C 16 H 19 N 3 4 S.3H 2 0: 403.45
(2S,5/?,6/?)-6-[(2/?)-2-Amino-2-phenylacetylamino]-
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-
carboxylic acid trihydrate [7177-48-2]
Ampicillin Hydrate contains not less than 960 fig
(potency) and not more than 1005 fig (potency) per mg,
calculated on the anhydrous basis. The potency of Am-
picillin Hydrate is expressed as mass (potency) of am-
picillin (C 16 H 19 N 3 4 S: 349.40).
Description Ampicillin Hydrate occurs as a white to light
yellowish white, crystals or crystalline powder.
It is sparingly soluble in water, slightly soluble in
methanol, very slightly soluble in ethanol (95), and practical-
ly insoluble in acetonitrile.
Identification Determine the infrared absorption spectrum
of Ampicillin Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Ampicillin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49>[ a ] 2 £: +280- +305° (0.5 g calculated
on the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Ampicillin Hydrate in 400 mL of water is between 3.5 and
5.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Ampicillin Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Ampicillin hydrate according to Method 3, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Ampicillin hy-
drate in the mobile phase to make 50 mL, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 fiL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: each peak area
other than that of ampicillin obtained from the sample solu-
tion is not more than the peak area of ampicillin from the
standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 10 times as long as the
retention time of ampicillin.
System suitability —
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of ampicillin
obtained from 1 fiL of this solution is equivalent to 7 to 1 3 %
of that from 10 fiL of the standard solution.
(4) TV.A'-Dimethylaniline — Weigh accurately about 1 g of
Ampicillin Hydrate, dissolve in 5 mL of sodium hydroxide
TS, add exactly 1 mL of the internal standard solution, shake
vigorously for 1 minute, and use the upper layer liquid
obtained after allowing it to stand as the sample solution.
Separately, weigh accurately about 50 mg of N,N-
dimethylaniline, dissolve in 2 mL of hydrochloric acid and 20
mL of water, add water to make exactly 50 mL, and use this
solution as the standard stock solution. Pipet 5 mL of the
standard stock solution, and add water to make exactly 250
mL. Pipet 1 mL of this solution, add 5 mL of sodium
hydroxide TS and exactly 1 mL of the internal standard
solution, shake vigorously for 1 minute, and use the upper
layer liquid obtained after allowing it to stand as the standard
solution. Perform the test with 1 fiL each of the sample
solution and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
determine the ratios, Q T and Q s , of the peak area of N,N-
dimethylaniline to that of the internal standard, and calculate
the amount of A^Af-dimethylaniline by the following
equation: not more than 20 ppm.
Amount (ppm) of A^Af-dimethylaniline
= (W S /W T ) x (Qj/Qs) x 400
W s : Amount (g) of TV.Af-dimethylaniline
W T : Amount (g) of the sample
Internal standard solution — A solution of naphthalene in
cyclohexane (1 in 20,000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 2.6 mm in inside diameter and
2 m in length, packed with siliceous earth for gas chro-
matography (180 - 250 fan in particle diameter) coated with
50% phenyl-50% methyl polysiloxane for gas chro-
matography at the ratio of 3%.
Column temperature: A constant temperature of about
120°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
7V,7V-dimethylaniline is about 5 minutes.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard stock solution, and add water to make exactly
250 mL. Pipet 1 mL of this solution, add 5 mL of sodium
hydroxide TS and exactly 1 mL of the internal standard
solution, shake vigorously for 1 minute, and use the upper
layer liquid obtained after allowing it to stand for the test.
Confirm that when the procedure is run with 1 fiL of the
upper layer liquid under the above operating conditions, the
308
Ampicillin Sodium / Official Monographs
JP XV
ratio of the peak area of A^/V-dimethylaniline to that of the
internal standard is equivalent to 15 - 25% of the ratio of the
peak area of iV./V-dimethylaniline to that of the internal
standard obtained from the standard solution.
System performance: Dissolve 50 mg of 7V,/V-dimethylani-
line in cyclohexane to make 50 mL. To 1 mL of this solution
add the internal standard solution to make 50 mL, and use
this solution as the solution for system suitability test. When
the procedure is run with 1 /XL of the solution for system
suitability test under the above operating conditions, N,N-
dimethylaniline and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
1 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the ratios of the peak area of A^/V-dimethylaniline to that
of the internal standard is not more than 2.0%.
Water <2.48> 12.0-15.0% (0.1 g, volumetric titration,
direct titration).
Assay Weigh accurately an amount of Ampicillin Hydrate
and Ampicillin Reference Standard, equivalent to about 50
mg (potency), dissolve in a suitable volume of the mobile
phase, add exactly 5 mL each of the internal standard solu-
tion and the mobile phase to make 50 mL, and use these solu-
tions as the sample solution and standard solution. Perform
the test with 10 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine the
ratios, Q T and Q s , of the peak area of ampicillin to that of the
internal standard.
Amount \p.g (potency)] of ampicillin C 16 H 19 N 3 4 S
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Ampicillin Reference
Standard
Internal standard solution— A solution of guaifenesin in the
mobile phase (1 in 200).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.94 g of diammonium hydrogen
phosphate in 850 mL of water, add 100 mL of acetonitrile,
adjust the pH to 5.0 with phosphoric acid, and add water to
make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
ampicillin is about 6 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, ampicillin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 40.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ampicillin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Ampicillin Sodium
Aminobenzylpenicillin Sodium
H H
C 16 H 18 N 3 Na0 4 S: 371.39
Monosodium (2S,5i?,6i?)-6-[(2i?)-2-amino-2-
phenylacetylamino]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate [69-52-3]
Ampicillin Sodium contains not less than 850 (xg
(potency) and not more than 950 [xg (potency) per mg,
calculated on the anhydrous basis. The potency of
Ampicillin Sodium is expressed as mass (potency) of
ampicillin (C 16 H 19 N 3 4 S: 349.40).
Description Ampicillin Sodium occurs as white to light
yellowish white, crystals or crystalline powder.
It is very soluble in water, and sparingly soluble in ethanol
(99.5).
Identification (1) Determine the infrared absorption spec-
trum of Ampicillin Sodium, previously dried in a desiccator
(reduced pressure not exceeding 0.67 kPa, 60°C) for 3 hours,
as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(2) Ampicillin Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [a]™: +246- +272° (1 g calculated
on the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Ampicillin Sodium in 10 mL of water is between 8.0 and
10.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ampicillin Sodium in 10 mL of water: the solution is clear
and colorless or pale yellow.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Ampicil-
lin Sodium according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ampicillin Sodium according to Method 1 , and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Ampicillin
Sodium in 50 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
JPXV
Official Monographs / Amyl Nitrite
309
solution as the standard solution. Perform the test with
exactly 10 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the peak area
other than ampicillin obtained from the sample solution is
not more than the peak area of ampicillin from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ion in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.94 g of diammonium hydrogen
phosphate in 850 mL of water, add 100 mL of acetonitrile,
adjust the pH to 5.0 with phosphoric acid, and add water to
make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
ampicillin is about 6 minutes.
Time span of measurement: About 10 times as long as the
retention time of ampicillin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of ampicillin
obtained from 10 /uL of this solutionis equivalent to 7 to 13%
of that of ampicillin obtained from 10 liL of the standard
solution.
System performance: Dissolve 50 mg of Ampicillin
Reference Standard in a suitable amount of the mobile phase,
add 5 mL of a solution of guaifenesin in the mobile phase (1
in 200) and the mobile phase to make 50 mL, and use this
solution as the solution for system suitability test. When the
procedure is run with 10 fiL of the solution for system
suitability test under the above operating conditions,
ampicillin and guaifenesin are eluted in this order with the
resolution between these peaks being not less than 35.
System repeatability: When the test is repeated 6 times with
10 iiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the ratios of the peak area of ampicillin to that of
guaifenesin is not more than 1.0%.
Water <2.48> Not more than 2.0% (0.2 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer, having pH 6.5 to 6.6 af-
ter sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Ampicillin Reference Standard, equivalent to about 25 mg
(potency), and dissolve in phosphate buffer solution, pH 6.0
to make exactly 50 mL. Take exactly a suitable amount of
this solution, add phosphate buffer solution, pH 6.0 to make
solutions so that each mL contains 5 /ug (potency) and 1 .25 /ug
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Ampicillin Sodium, equivalent to about 25 mg (potency), and
dissolve in phosphate buffer solution, pH 6.0 to make exactly
50 mL. Take exactly a suitable amount of this solution, add
phosphate buffer solution, pH 6.0 to make solutions so that
each mL contains 5 iig (potency) and 1.25 tig (potency), and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Amyl Nitrite
117.15
Amyl Nitrite is the nitrous acid ester of 3 -m ethyl -
butanol-1 and contains a small quantity of 2-methyl-
butanol-1 and the nitrous acid esters of other homo-
logues.
It contains not less than 90.0 % of
C 5 H„N0 2 .
Description Amyl Nitrite is a clear, light yellowish liquid,
and has a characteristic, fruity odor.
It is miscible with ethanol (95), and with diethyl ether.
It is practically insoluble in water.
It is affected by light and by heat.
It is volatile at ordinary temperature and flammable even at
a low temperature.
Boiling point: about 97°C
Identification Determine the infrared spectrum of Amyl
Nitrite as directed in the liquid film method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
Specific gravity <2.56> df : 0.871 - 0.880
Purity (1) Acidity — To 5 mL of Amyl Nitrite add a mix-
ture of 1.0 mL of 1 mol/L sodium hydroxide VS, 10 mL of
water and 1 drop of phenolphthalein TS, shake, and allow to
stand for 1 minute: the light red color of the water layer does
not disappear.
(2) Water — Allow 2.0 mL of Amyl Nitrite to stand in ice
water: no turbidity is produced.
(3) Aldehyde — To 3 mL of a mixture of equal volumes of
silver nitrate TS and aldehyde free-ethanol add ammonia TS
dropwise until the precipitate first formed is redissolved. Add
1.0 mL of Amyl Nitrite, and warm between 60°C and 70°C
for 1 minute: a brown to black color is not produced.
(4) Residue on evaporation — Evaporate 10.0 mL of
Amyl Nitrite on a water bath in a draft, carefully protecting
from flame, and dry the residue at 105 °C for 1 hour: the mass
of the residue is not more than 1.0 mg.
Assay Weigh accurately a volumetric flask containing 10
mL of ethanol (95), add about 0.5 g of Amyl Nitrite, and
weigh accurately again. Add exactly 25 mL of 0.1 mol/L sil-
310
Dental Antiformin / Official Monographs
JP XV
ver nitrate VS, then add 15 mL of potassium chlorate solu-
tion (1 in 20) and 10 mL of dilute nitric acid, stopper the flask
immediately, and shake it vigorously for 5 minutes. Dilute
with water to make exactly 100 mL, shake, and filter through
dry filter paper. Discard the first 20 mL of the filtrate,
measure exactly 50 mL of the subsequent filtrate, and titrate
<2.50> the excess silver nitrate with 0.1 mol/L ammonium
thiocyanate VS (indicator: 2 mL of ammonium iron (III) sul-
fate TS). Perform a blank determination.
Each mL of 0.1 mol/L silver nitrate VS
= 35.14 mg of C 5 H„N0 2
Containers and storage Containers — Hermetic containers
not exceeding 10-ml capacity.
Storage — Light-resistant, in a cold place, and remote from
fire.
Dental Antiformin
Dental Sodium Hypochlorite Solution
Dental Antiformin contains not less than 3.0 w/v%
and not more than 6.0w/v% of sodium hypochlorite
(NaCIO: 74.44).
Description Dental Antiformin is a slightly light yellow-
green, clear liquid. It has a slight odor of chlorine.
It gradually changes by light.
Identification (1) Dental Antiformin changes red litmus
paper to blue, and then decolorizes it.
(2) To Dental Antiformin add dilute hydrochloric acid: it
evolves the odor of chlorine, and the gas changes potassium
iodide starch paper moistened with water to blue.
(3) Dental Antiformin responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Assay Measure exactly 3 mL of Dental Antiformin in a
glass-stoppered flask, add 50 mL of water, 2 g of potassium
iodide and 10 mL of acetic acid (31), and titrate <2.50> the
liberated iodine with 0.1 mol/L sodium thiosulfate VS (indi-
cator: 3 mL of starch TS).
Each mL of 0.1 mol/L sodium thiosulfate VS
= 3.722 mg of NaCIO
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 10°C.
Antipyrine
Phenazone
r„JD
H;C
C„H 12 N 2 0: 188.23
l,5-Dimethyl-2-phenyl-l,2-dihydro-3//-pyrazol-3-one
[60-80-0]
Antipyrine, when dried, contains not less than
99.0% of C„H 12 N 2 0.
Description Antipyrine occurs as colorless or white crystals,
or a white, crystalline powder. It is odorless, and has a slight-
ly bitter taste.
It is very soluble in water, freely soluble in ethanol (95),
and sparingly soluble in diethyl ether.
A solution of Antipyrine (1 in 10) is neutral.
Identification (1) To 5 mL of a solution of Antipyrine (1
in 100) add 2 drops of sodium nitrite TS and 1 mL of dilute
sulfuric acid: a deep green color develops.
(2) To 2 mL of a solution of Antipyrine (1 in 100) add 4
drops of dilute iron (III) chloride TS: a yellow-red color de-
velops. Then add 10 drops of dilute sulfuric acid: the color
changes to light yellow.
(3) To 5 mL of a solution of Antipyrine (1 in 100) add 2
to 3 drops of tannic acid TS: a white precipitate is produced.
(4) To 0.1 g of Antipyrine add 0.1 g of vanillin, 5 mL of
water and 2 mL of sulfuric acid, boil the mixture, and cool: a
yellow-red precipitate is produced.
Melting point <2.60> 111 - 113°C
Purity (1) Chloride <7.0?>— Perform the test with 1.0 g of
Antipyrine. Prepare the control solution with 0.40 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.014%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Antipy-
rine according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Readily carbonizable substances<A/5> — Perform the
test with 0.5 g of Antipyrine: the solution remains colorless.
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 0.2 g of Antipyrine, previously dried
and accurately weighed, in 20 mL of sodium acetate TS, add
exactly 30 mL of 0.05 mol/L iodine VS, and allow to stand
for 20 minutes with occasional shaking. Dissolve the
precipitate in 10 mL of chloroform, and titrate <2.50> the ex-
cess iodine with 0.1 mol/L sodium thiosulfate VS (indicator:
3 mL of starch TS). Perform a blank determination.
Each mL of 0.05 mol/L iodine VS
= 9.411 mg of C H H 12 N 2
JPXV
Official Monographs / Arbekacin Sulfate 311
Containers and storage Containers — Well-closed contain-
ers.
Arbekacin Sulfate
C 22 H 4 4N 6 O 10 .xH 2 SO4 (x = 2 - 2V 2 )
3-Amino-3-deoxy-a-D-glucopyranosyl-(l -> 6)-
[2,6-diamino-2,3,4,6-tetradeoxy-a-D-eo';/!ro-
hexopyranosyl-(1^4)]-l-N-[(25')-4-amino-2-
hydroxybutanoyl]-2-deoxy-D-streptamine sulfate
[51025-85-5, Arbekacin]
Arbekacin Sulfate is the sulfate of a derivative of
dibekacin.
It contains not less than 670 /ug (potency) and not
more than 750 fig (potency) per mg, calculated on the
dried basis. The potency of Arbekacin Sulfate is ex-
pressed as mass (potency) of arbekacin (C22H 44 N 6 O 10 :
552.62).
Description Arbekacin Sulfate occurs as a white powder.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 10 mg each of Arbekacin
Sulfate and Arbekacin Sulfate Reference Standard in 1 mL of
water, and use these solutions as the sample solution and
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of ammonia solution (28), methanol, chlo-
roform and ethanol (95) (7:6:4:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly 0.2% ninhydrin-water
saturated 1-butanol TS on the plate, and heat at 100°C for 10
minutes: the principal spot obtained from the sample solu-
tion and the spot from the standard solution are purple-
brown in color and their Rf values are the same.
(2) A solution of Arbekacin Sulfate (1 in 50) responds to
the Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [a]™: +69- +79° (0.25 g after
drying, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.75
g of Arbekacin Sulfate in 10 mL of water is between 6.0 and
8.0.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 1 .0 g of Arbekacin Sulfate in 5 mL of
water is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Arbeka-
cin Sulfate according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Dibekacin — Weigh accurately about 20 mg of
Arbekacin Sulfate, add exactly 10 mL of the internal stan-
dard solution to dissolve, add water to make 20 mL, and use
this solution as the sample solution. Separately, weigh
accurately an amount of Dibekacin Sulfate Reference
Standard, equivalent to about 10 mg (potency), and dissolve
in water to make exactly 50 mL. Pipet 5 mL of this solution,
add exactly 10 mL of the internal standard solution and
water to make 20 mL, and use this solution as the standard
solution. Perform the test with 5 fiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, g T and Q s , of the peak area
of dibekacin to that of the internal standard. Calculate the
amount of dibekacin by the following equation: not more
than 2.0%.
Amount (%) of dibekacin
= (W S /W T ) x (Q T /Qs) x (1/10) x 1000
W s : Amount [mg (potency)] of Dibekacin Sulfate Refer-
ence Standard
W T : Amount (mg) of the sample
Internal standard solution — A solution of bekanamycin
sulfate (1 in 2000).
Operating conditions —
Detector: Fluorometric detector (excitation wavelength:
340 nm, detection wavelength: 460 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Reaction coil: A column about 0.3 mm in inside diameter
and about 3 m in length.
Reaction coil temperature: A constant temperature of
about 50°C.
Mobile phase: Dissolve 8.70 g of sodium 1-pentane sul-
fonate and 8.52 g of anhydrous sodium sulfate in 980 mL of
water, adjust the pH to 4.0 with acetic acid (100), and add
water to make 1000 mL. To 230 mL of this solution add
20 mL of methanol.
Reagent: Dissolve 12.36 g of boric acid in 960 mL of water,
add 10 mL of a solution of o-phthalaldehyde in ethanol
(99.5) (1 in 25), adjust the pH to 10.5 with 8 mol/L potassium
hydroxide TS, and add water to make 1000 mL. To this
solution add 1 mL of 2-mercaptoethanol.
Reaction temperature: A constant temperature of about
50°C.
Flow rate of the mobile phase: 0.5 mL per minute.
Flow rate of the reagent: 1 mL per minute.
System suitability —
System performance: Dissolve 20 mg each of Arbekacin
Sulfate, becanamycin sulfate and dibekacin sulfate in 200 mL
312 Arbekacin Sulfate Injection / Official Monographs
JP XV
of water. When the procedure is run with 5 /uh of this
solution under the above operating conditions, becanamycin,
arbekacin and dibekacin are eluted in this order, and the
resolution between the peaks, becanamycin and arbekacin is
not less than 5 and arbekacin and dibekacin is not less than
1.5, respectively.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of dibekacin to that of the internal standard is not
more than 2.0%.
(4) Related substances — Dissolve 20 mg of Arbekacin
Sulfate in 20 mL of water, and use this solution as the sample
solution. Pipet 3 mL of the sample solution, add water to
make exactly 250 mL, and use this solution as the standard
solution. Perform the test with exactly 5 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the area of each peak by the automatic
integration method: the total area of the peaks other than ar-
bekacin and dibekacin obtained from the sample solution is
not more than the peak area of arbekacin from the standard
solution.
Operating conditions —
Detector, column, column temperature, reaction coil, reac-
tion coil temperature, mobile phase, reagent, reaction tem-
perature, flow rate of mobile phase, and flow rate of reagent:
Proceed as directed in the operating conditions in the Purity
(3).
Time span of measurement: About 1.5 times as long as the
retention time of arbekacin.
System suitability —
System performance: Dissolve 10 mg each of Arbekacin
Sulfate and dibekacin sulfate in 200 mL of water. When the
procedure is run with 5 /uL of this solution under the above
operating conditions, arbekacin and dibekacin are eluted in
this order with the resolution between these peaks being not
less than 1.5.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
arbekacin is not more than 5.0%.
Loss on drying <2.41> Not more than 5.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer, having pH 7.8 - 8.0 after
sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Arbekacin Sulfate Reference Standard, previously dried,
equivalent to about 20 mg (potency), dissolve in diluted phos-
phate buffer solution, pH 6.0 (1 in 2) to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5 to 15°C and use within 30 days.
Take exactly a suitable amount of the standard stock solution
before use, add 0.1 mol/L phosphate buffer solution, pH 8.0
to make solutions so that each mL contains 20 /ug (potency)
and 5 /xg (potency), and use these solutions as the high con-
centration standard solution and low concentration standard
solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Arbekacin Sulfate, equivalent to about 20 mg (potency), and
dissolve in water to make exactly 50 mL. Take exactly a suita-
ble amount of this solution, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
20 /ug (potency) and 5 /xg (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Arbekacin Sulfate Injection
Arbekacin Sulfate Injection is an aqueous injection.
It contains not less than 90.0% and not more than
110.0% of the labeled amount of arbekacin sulfate
(C 22 H44N 6 O 10 : 552.62).
Method of preparation Prepare as directed under Injec-
tions, with Arbekacin Sulfate.
Description Arbekacin Sulfate Injection occurs as a clear
and colorless liquid.
Identification To 0.2 mL of Arbekacin Sulfate Injection
add 1 mL of water, and use this solution as the sample solu-
tion. Separately, dissolve 10 mg of Arbekacin Sulfate Refer-
ence Standard in 1 mL of water, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop with a
mixture of ammonia solution (28), methanol, chloroform
and ethanol (95) (7:6:4:1) to a distance of about 12 cm, and
air-dry the plate. Spray evenly 0.2% ninhydrin-water saturat-
ed 1-butanol TS on the plate, and heat at 80°C for 10
minutes: the principal spot with the sample solution and the
spot with the standard solution show a purple-brown color
and the same Rf value.
Osmotic pressure ratio <2.47> 0.8 - 1.2 (for the preparation
intended for intramuscular use).
pH <2.54> 6.0-8.0
Bacterial endotoxins <4.01> Less than 0.50 EU/mg (poten-
cy).
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to the Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, Culture medium and Standard solu-
tions: Proceed as directed in the Assay under Arbekacin Sul-
JPXV
Official Monographs / L-Arginine Hydrochloride 313
fate.
(ii) Sample solutions — Take exactly a volume of Arbeka-
cin Sulfate Injection, equivalent to about 20 mg (potency),
and add water to make exactly 50 mL. Take exactly a suitable
amount of this solution, add 0.1 mol/L phosphate buffer so-
lution, pH 8.0 to make solutions so that each mL contains 20
H% (potency) and 5 n% (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Hermetic containers.
L-Arginine
l-t;u^->
C 6 H 14 N 4 2 : 174.20
(25)-2-Amino-5-guanidinopentanoic acid
[74-79-3]
L-Arginine, when dried, contains not less than
98.5% and not more than 101.0% of C 6 H 14 N 4 2 .
Description L-Arginine occurs as white crystals or crystal-
line powder. It has a characteristic odor.
It is freely soluble in water and in formic acid, and practi-
cally insoluble in ethanol (99.5).
It dissolves in dilute hydrochloric acid.
It is hygroscopic.
Identification Determine the infrared absorption spectrum
of previously dried L-Arginine as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [«]*>: +26.9- +27.9° (after
drying, 2 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of L-Arginine in 10 mL of water is between 10.5 and 12.0.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 1.0 g of L-Arginine in 10 mL of water is
clear and colorless.
(2) Chloride <1.03>— Perform the test with 0.5 g of
L-Arginine. Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1. 14>— Perform the test with 0.6 g of L-Argi-
nine. Prepare the control solution with 0.35 mL of 0.005
mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> Perform the test with 0.25 g of L-
Arginine, using the distillation under reduced pressure. Pre-
pare the control solution with 5.0 mL of Standard Ammoni-
um Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Dissolve 2.0 g of L-Arginine in
30 mL of water, add 1 drop of phenolphthalein TS, neutral-
ize with dilute hydrochloric acid, add 2 mL of dilute acetic
acid and water to make 50 mL, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(6) Iron <1.10> — Prepare the test solution with 1 .0 g of L-
Arginine according to Method 1, and perform the test using
Method A. Prepare the control solution with 1.0 mL of Stan-
dard Iron Solution (not more than 10 ppm).
(7) Related substances — Dissolve 0.10 g of L-Arginine in
10 mL of water, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add water to make ex-
actly 50 mL. Pipet 2 mL of this solution, add water to make
exactly 20 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /iL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of 2-propanol and ammonia solution (28) (7:3) to a distance
of about 10 cm, and dry the plate at 80°C for 30 minutes.
Spray evenly ninhydrin-butanol TS on the plate, and heat at
80°C for 10 minutes: the spot other than the principal spot
with the sample solution is not more intense than the spot
with the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 80 mg of L-Arginine, previ-
ously dried, dissolve in 3 mL of formic acid, add 50 mL of
acetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation in the same manner, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 8.710 mg of C 6 H I4 N 4 2
Containers and storage Containers — Tight containers.
L-Arginine Hydrochloride
X.
^COjH
H NHj
•HCI
C 6 H 14 N 4 2 .HC1: 210.66
(2S)-2-Amino-5-guanidinopentanoic acid
monohydrochloride [1119-34-2]
L-Arginine Hydrochloride, when dried, contains not
less than 98.5% of C 6 H 14 N 4 2 .HC1.
Description L-Arginine Hydrochloride occurs as white
crystals or crystalline powder. It is odorless, and has a slight,
characteristic taste.
It is freely soluble in water and in formic acid, and very
slightly soluble in ethanol (95).
Identification (1) Determine the infrared absorption spec-
trum of L-Arginine Hydrochloride, previously dried, as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
314 L-Arginine Hydrochloride Injection / Official Monographs
JP XV
(2) A solution of L-Arginine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [ a ]o- +21.5 -+23.5° (after
drying, 2 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of L-Arginine Hydrochloride in 10
mL of water: the pH of this solution is between 4.7 and 6.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
L-Arginine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1. 14>— Perform the test with 0.6 g of L-Argi-
nine Hydrochloride. Prepare the control solution with 0.35
mL of 0.005 mol/L sulfuric acid VS (not more than 0.028%).
(3) Ammonium <1.02> — Perform the test with 0.25 g of
L-Arginine Hydrochloride, using the distillation under
reduced pressure. Prepare the control solution with 5.0 mL
of Standard Ammonium Solution (not more than 0.02%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of L-Argi-
nine Hydrochloride according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(5) Arsenic </.//> — Prepare the test solution with 1.0 g
of L-Arginine Hydrochloride according to Method 1, and
perform the test (not more than 2 ppm).
(6) Related substances — Dissolve 0.20 g of L-Arginine
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
water to make exactly 10 mL. Pipet 1 mL of this solution,
add water to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of ethanol (99.5), water, 1-butanol and am-
monia water (28) (2:1:1:1) to a distance of about 10 cm, and
dry the plate at 100°C for 30 minutes. Spray evenly a solution
of ninhydrin in acetone (1 in 50) on the plate, and heat at
80°C for 5 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of L-Arginine
Hydrochloride, previously dried, dissolve in 2 mL of formic
acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS, and
heat on a water bath for 30 minutes. After cooling, add 45
mL of acetic acid (100), and titrate <2.50> the excess perchlor-
ic acid with 0.1 mol/L sodium acetate VS (potentiometric
titration). Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 10.53 mg of C 6 H I4 N 4 2 HC1
Containers and storage Containers — Tight containers.
L-Arginine Hydrochloride Injection
L-7;u^->^K^a*t«
L-Arginine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 9.5 w/v% and not more
than 10.5 w/v% of L-arginine hydrochloride (C 6 H 14 N 4
2 .HC1: 210.66).
Method of preparation
L-Arginine Hydrochloride
Water for Injection
100 g
a sufficient quantity
To make
1000 mL
Prepare as directed under Injections, with the above
ingredients.
No preservative is added.
Description L-Arginine Hydrochloride Injection is a clear,
colorless liquid.
Identification (1) To 5 mL of a solution of L-Arginine
Hydrochloride Injection (1 in 100) add 1 mL of ninhydrin
TS, and heat for 3 minutes: a blue-purple color develops.
(2) To 5 mL of a solution of L-Arginine Hydrochloride
Injection (1 in 10) add 2 mL of sodium hydroxide TS and 1 to
2 drops of a solution of 1-naphthol in ethanol (95) (1 in
1000), allow to stand for 5 minutes, and add 1 to 2 drops of
sodium hypochlorite TS: a red-orange color develops.
pH <2.54> 5.0-6.0
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Perform the test with L-Arginine
Hydrochloride Injection stored in a container in a volume ex-
ceeding 10 mL: it meets the requirement.
Assay Pipet 20 mL of L-Arginine Hydrochloride Injection,
add 7.5 mol/L hydrochloric acid TS to make exactly 100 mL,
and determine the optical rotation a D as directed under
Optical Rotation Determination <2.49> at 20 ± 1°C in a
100-mm cell.
Amount (mg) of L-arginine hydrochloride (C 6 H 14 N 4 2 .HC1)
= a D x 4444
Containers and storage Containers — Hermetic containers.
Arotinolol Hydrochloride
X
NH Z . HCi
and enantiomer
C 15 H 2I N 3 2 S 3 .HC1: 408.00
5-{2-[(2i?S)-3-(l,l-Dimethylethyl)amino-
2-hy droxypropylsulf anyl] - 1 , 3-thiazol-4-yl} thiophene-
2-carboxamide monohydrochloride [68377-91-3]
JPXV
Official Monographs / Arsenical Paste 315
Arotinolol Hydrochloride, when dried, contains not
less than 99.0% of C 15 H 21 N 3 2 S3.HC1.
Description Arotinolol Hydrochloride occurs as a white to
light yellow crystalline powder.
It is freely soluble in dimethylsulfoxide, slightly soluble in
methanol and in water, very slightly soluble in ethanol (99.5),
and practically insoluble in diethyl ether.
A solution of Arotinolol Hydrochloride in methanol (1 in
125) does not show optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Arotinolol Hydrochloride in methanol (1 in
75,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Arotinolol Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Arotinolol Hydrochloride (1 in 200)
responds to the Qualitative Tests <1.09> (2) for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Arotinolol Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances - Dissolve 0.05 g of Arotinolol
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL. Pipet 1 mL of this
solution, add methanol to make exactly 10 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 40 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol, acetone and ammonia solution
(28) (30:10:10:1) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.20% (1 g, in vacu-
um, 105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1.5 g of Arotinolol
Hydrochloride, previously dried, dissolve in dimethylsul-
foxide to make exactly 25 mL. Pipet 5 mL of this solution,
add 100 mL of water and 5 mL of sodium hydroxide TS, and
extract with three 50-mL portions of dichloromethane. Filter
each dichloromethane extract through a pledget of absorbent
cotton with anhydrous sodium sulfate on it. Evaporate
combined filtrate to dryness in vacuum. Dissolve the residue
in 70 mL of acetic acid (100), and titrate <2.50> with 0.05 mol
/L perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 20.40 mg of C 15 H 21 N 3 2 S3.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Arsenical Paste
Arsenical Paste contains not less than 36.0% and not
more than 44.0% of arsenic (III) trioxide (As 2 3 :
197.84).
Method of preparation
Arsenic Trioxide, finely powdered
Procaine Hydrochloride, finely
powdered
Hydrophilic Ointment
Clove Oil
Medicinal Carbon
40 g
10 g
30 g
a suitable quantity
a suitable quantity
To make 100 g
Mix Arsenic Trioxide and Procaine Hydrochloride with
Hydrophilic Ointment, and add Clove Oil to make a suitably
viscous liquid, followed by Medicinal Carbon for coloring.
Description Arsenical Paste is grayish black and has the
odor of clove oil.
Identification (1) Place 0.1 g of Arsenical Paste in a small
flask, add 5 mL of fuming nitric acid and 5 mL of sulfuric
acid, and heat over a flame until the reacting liquid becomes
colorless and white fumes begin to evolve. After cooling, add
the reacting liquid to 20 mL of water cautiously, and add
10 mL of hydrogen sulfide TS while warming: a yellow
precipitate is produced (arsenic (III) trioxide).
(2) Shake thoroughly 0.5 g of Arsenical Paste with 25 mL
of diethyl ether, 5 mL of dilute hydrochloric acid and 20 mL
of water, separate the water layer, and filter: 5 mL of the
filtrate responds to the Qualitative Tests <1.09> for primary
aromatic amines (procaine hydrochloride).
(3) Shake thoroughly 0.5 g of Arsenical Paste with 25 mL
of diethyl ether and 25 mL of water, separate the water layer,
filter, and use the filtrate as the sample solution. Dissolve
0.01 g of procaine hydrochloride in 5 mL of water, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 fiL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, ethanol (99.5) and ammonia solution (28)
(50:5:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 mm):
the spots from the sample solution and standard solution ex-
hibit the same Rf value.
Assay Weigh accurately about 0.3 g of Arsenical Paste into
a 1 50-mL Kjeldahl flask, add 5 mL of fuming nitric acid and
10 mL of sulfuric acid, and shake thoroughly. Heat cautious-
ly the mixture, gently at first, and then continue strong heat-
ing, until red fumes of nitrogen oxide are sparingly evolved.
After cooling, add 5 mL of fuming nitric acid, heat again
until red fumes of nitrogen oxide are no longer evolved and
316
Arsenic Trioxide / Official Monographs
JP XV
the reacting liquid becomes clear, and cool. Add 30 mL of a
saturated solution of ammonium oxalate monohydrate, heat
again until white fumes of sulfuric acid are evolved, and
continue the heating for 10 minutes. Decompose completely
oxalic acid, cool, transfer cautiously the colorless reacting
liquid to a glass-stoppered flask, containing 40 mL of water.
Wash thoroughly the Kjeldahl flask with 60 mL of water, add
the washings to the content of the glass-stoppered flask, and
cool. Dissolve 3 g of potassium iodide in this solution, allow
to stand in a dark place at room temperature for 45 minutes,
and titrate <2.50> the liberated iodine with 0.1 mol/L sodium
thiosulfate VS (indicator: 5 mL of starch TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 4.946 mg of As 2 3
Containers and storage Containers — Tight containers.
Arsenic Trioxide
Arsenous Acid
As 2 3 : 197.84
Arsenic Trioxide, when dried, contains not less than
99.5% of As 2 3 .
Description Arsenic Trioxide occurs as a white powder.
It is odorless. It is practically insoluble in water, in ethanol
(95) and in diethyl ether.
It dissolves in sodium hydroxide TS.
Identification Dissolve 0.2 g of Arsenic Trioxide in 40 mL
of water by heating on a water bath: the solution responds to
the Qualitative Tests <1.09> for arsenite.
Purity Clarity of solution — To 1.0 g of Arsenic Trioxide
add 10 mL of ammonia TS, and heat gently: the solution is
clear.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 0.15 g of Arsenic Trioxide,
previously dried, dissolve in 20 mL of a solution of sodium
hydroxide (1 in 25), by warming, if necessary. Add 40 mL of
water and 2 drops of methyl orange TS, then add dilute
hydrochloric acid until the color of the solution becomes light
red. Add 2 g of sodium hydrogen carbonate and 50 mL of
water to this solution, and titrate <2.50> with 0.05 mol/L io-
dine VS (indicator: 3 mL of starch TS).
Each mL of 0.05 mol/L iodine VS = 4.946 mg of As 2 3
Containers and storage Containers — Tight containers.
Ascorbic Acid
Vitamin C
H OH
L-threo-Hex-2-eaono- 1 ,4-lactone [50-81-7]
Ascorbic Acid, when dried, contains not less than
99.0% of L-ascorbic acid (C 6 H 8 6 ).
Description Ascorbic Acid occurs as white crystals or a
white, crystalline powder. It is odorless, and has an acid
taste.
It is freely soluble in water, sparingly soluble in ethanol
(95), and practically insoluble in diethyl ether.
Melting point: about 190°C (with decomposition).
Identification (1) To 5 mL each of a solution of Ascorbic
Acid (1 in 50) add 1 drop of potassium permanganate TS or 1
to 2 drops of 2,6-dichloroindophenol sodium TS: the color of
the solution is discharged immediately in each case.
(2) Dissolve 0.1 g of Ascorbic Acid in 100 mL of a solu-
tion of metaphosphoric acid (1 in 50). To 5 mL of the solu-
tion add iodine TS until the color of the solution becomes
light yellow. Then add 1 drop of a solution of copper (II)
sulfate pentahydrate (1 in 1000) and 1 drop of pyrrole, and
warm the mixture at 50°C for 5 minutes: a blue color de-
velops.
Optical rotation <2.49> [«]£," : + 20.5 - + 21.5 c
water, 25 mL, 100 mm).
(2.5 g,
pH <2.54> Dissolve 1.0 g of Ascorbic Acid in 20 mL of
water: the pH of this solution is between 2.2 and 2.5.
Purity (1) Clarity and color of solution — Dissolve 1.0 of
Ascorbic Acid in 20 mL of water: the solution is clear and
colorless.
(2) Heavy metals <1.07>— Perform the test with 1.0 g of
Ascorbic Acid according to Method 1. Prepare the control
solution with 2.0 mL of Standard Lead Solution (not more
than 20 ppm).
Loss on drying <2.41> Not more than 0.20% (1 g, silica gel,
24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Ascorbic Acid,
previously dried, dissolve in 50 mL of a solution of
metaphosphoric acid (1 in 50), and titrate <2.50> with 0.05
mol/L iodine VS (indicator: 1 mL of starch TS).
Each mL of 0.05 mol/L iodine VS
= 8.806 mg of L-ascorbic acid (C 6 H 8 6 )
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Ascorbic Acid Powder 317
Ascorbic Acid Injection
Vitamin C Injection
conds. Perform a blank determination, and make any neces-
sary correction. Calculate the quantity (A mg) of L-ascorbic
acid (C 6 H 8 6 ) equivalent to 1 mL of this test solution.
Containers and storage Containers — Hermetic containers.
Storage — Under nitrogen atmosphere.
Ascorbic Acid Injection is an aqueous solution for
injection.
It contains not less than 95% and not more than 115
% of the labeled amount of L-ascorbic acid (C 6 H 8 6 :
176.12).
Method of preparation Prepare as directed under Injec-
tions, with the sodium salt of Ascorbic Acid.
Description Ascorbic Acid Injection occurs as a clear,
colorless liquid.
Identification (1) Measure a volume of Ascorbic Acid
Injection, equivalent to 0.5 g of Ascorbic Acid according to
the labeled amount, and add water to make 25 mL. Proceed
with 5 mL each of the solution as directed in the Identifica-
tion (1) under Ascorbic Acid.
(2) Measure a volume of Ascorbic Acid Injection,
equivalent to 5 mg of Ascorbic Acid according to the labeled
amount. Add a solution of metaphosphoric acid (1 in 50) to
make 5 mL, and proceed with this solution as directed in the
Identification (2) under Ascorbic Acid.
(3) Ascorbic Acid Injection responds to the Qualitative
Tests (1) for sodium salt.
pH <2.54> 5.6-7 .4
Extractable volume <6.05> It meets requirement.
Assay Measure exactly a volume of Ascorbic Acid Injec-
tion, equivalent to about 0.1 g of L-ascorbic acid (C 6 H 8 6 ),
previously diluted with metaphosphoric acid-acetic acid TS,
if necessary, and add metaphosphoric acid-acetic acid TS to
make exactly 200 mL. Measure exactly 2 mL of the solution,
and shake with 8 mL of metaphosphoric acid-acetic acid TS
and 2 mL of hydrogen peroxide TS. Titrate <2.50> with 2,6-
dichloroindophenol sodium TS for titration until a light red
color persists for 5 seconds. Perform a blank determination,
and make any necessary correction.
Each mL of 2, 6-dichlorophenol-indophenol
sodium TS for titration
= A mg of L-ascorbic acid (C 6 H 8 6 )
A is decided by the following standardization of 2,6-
dichloroindophenol sodium TS for titration.
2,6-Dichlorophenol-indophenol sodium TS for titration:
Preparation — Dissolve 42 mg of sodium hydrogen car-
bonate in 50 mL of water, add 0.5 g of 2,6-dichloroin-
dophenol sodium dihydrate and water to make 200 mL, and
filter. Prepare before use.
Standardization — Weigh accurately about 50 mg of
Ascorbic Acid Reference Standard, previously dried in a
desiccator (silica gel) for 24 hours, and dissolve in
metaphosphoric acid-acetic acid TS to make exactly 100 mL.
Pipet 2 mL of this solution, shake with 8 mL of
metaphosphoric acid-acetic acid TS and 2 mL of hydrogen
peroxide TS, and titrate <2.50> with 2,6-dichloroindophenol
sodium TS for titration until a light red color persists for 5 se-
Ascorbic Acid Powder
Vitamin C Powder
7X=UL-tr>gtlft
Ascorbic Acid Powder contains not less than 95%
and not more than 120% of the labeled amount of
L-ascorbic acid (C 6 H g 6 : 176.12).
Method of preparation
with Ascorbic Acid.
Prepare as directed under Powders,
Identification (1) Weigh a portion of Ascorbic Acid Pow-
der, equivalent to 0.5 g of Ascorbic Acid according to the
labeled amount, add 30 mL of water, shake for 1 minute, and
filter. Proceed with 5 mL each of the filtrate as directed in the
Identification (1) under Ascorbic Acid.
(2) Weigh a portion of Ascorbic Acid Powder, equivalent
to about 0.01 g of Ascorbic Acid according to the labeled
amount, add 10 mL of a solution of metaphosphoric acid (1
in 50), shake for 1 minute, and filter. Proceed with 5 mL of
the filtrate as directed in the Identification (2) under Ascorbic
Acid.
Purity Rancidity — Ascorbic Acid Powder is free from any
unpleasant or rancid odor and taste.
Assay Weigh accurately a portion of Ascorbic Acid Pow-
der, equivalent to about 0.1 g of L-ascorbic acid (C 6 H 8 6 )
according to the labeled amount, extract with several succes-
sive portions of metaphosphoric acid-acetic acid TS, combine
the extracts, and filter. Wash the residue with metaphosphor-
ic acid-acetic acid TS. Combine the filtrates and washings,
and add metaphosphoric acid-acetic acid to make exactly 200
mL. Pipet 2 mL of the solution, and shake with 8 mL of
metaphosphoric acid-acetic acid TS and 2 mL of hydrogen
peroxide TS. Titrate <2.50> with 2,6-dichloroindophenol so-
dium TS for titration until a light red color persists for 5 se-
conds. Perform a blank determination, and make any neces-
sary correction.
Each mL of 2, 6-dichlorophenol-indophenol
sodium TS for titration
= A mg of C 6 H 8 6
A is decided by the following standardization of 2,6-
dichloroindophenol sodium TS for titration.
2,6-Dichlorophenol-indophenol sodium TS for titration:
Preparation — Dissolve 42 mg of sodium hydrogen car-
bonate in 50 mL of water, add 0.05 g of 2,6-dichloroin-
dophenol sodium dihydrate and water to make 200 mL, and
filter. Prepare before use.
Standardization — Weigh accurately about 50 mg of
Ascorbic Acid Reference Standard, previously dried in a
desiccator (silica gel) for 24 hours, and dissolve in
metaphosphoric acid-acetic acid TS to make exactly 100 mL.
318
L-Aspartic Acid / Official Monographs
JP XV
Pipet 2 mL of this solution, shake with 8 mL of
metaphosphoric acid-acetic acid TS and 2 mL of hydrogen
peroxide TS, and titrate <2.50> with 2,6-dichloroindophenol
sodium TS for titration until a light red color persists for 5 se-
conds. Perform a blank determination, and make any neces-
sary correction. Calculate the quantity (A mg) of L-ascorbic
acid (C 6 H 8 6 ) equivalent to 1 mL of this test solution.
Containers and storage Containers — Tight containers.
L-Aspartic Acid
CO z H
H NH 2
C 4 H 7 N0 4 : 133.10
(2S)-2-Aminobutanedioic acid
[56-84-8]
L-Aspartic Acid, when dried, contains not less than
98.5% and not more than 101.0% of C 4 H 7 N0 4 .
Description L-Aspartic Acid occurs as white, crystals or
crystalline powder.
It is sparingly soluble in water, and practically insoluble in
ethanol (99.5).
It dissolves in dilute hydrochloric acid and in 0.2 mol/L so-
dium hydroxide TS.
Identification Determine the infrared absorption spectrum
of L-Aspartic Acid as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [ a \^: +24.0- +26.0° (2 g, after
drying, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 0.4 g of L-Aspartic Acid in 100 mL of
water by warming, and allow to cool: between 2.5 and 3.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
L-Aspartic Acid in 20 mL of 1 mol/L hydrochloric acid TS:
the solution is clear and colorless.
(2) Chloride <1.03> — Dissolve 0.5 g of L-Aspartic Acid in
6 mL of dilute nitric acid and 20 mL of water, add water to
make 50 mL, and perform the test with this solution as the
test solution. Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1. 14>— Dissolve 0.6 g of L-Aspartic Acid in 5
mL of dilute hydrochloric acid and 30 mL of water, add
water to make 45 mL, and add 5 mL of barium chloride TS.
Perform the test with this solution as the test solution. Pre-
pare the control solution with 0.35 mL of 0.005 mol/L sul-
furic acid VS, add 5 mL of dilute hydrochloric acid and water
to make 45 mL, and add 5 mL of barium chloride (not more
than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Aspartic Acid. Prepare the control solution with 5.0 mL of
Standard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of L-Aspar-
tic Acid according to Method 4, and perform the test. Pre-
pare the control solution with 1.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(6) Iron <1.10> — Prepare the test solution with 1 .0 g of L-
Aspartic Acid according to Method 1, and perform the test
according to Method A. Prepare the control solution with 1 .0
mL of Standard Iron Solution (not more than 10 ppm).
(7) Related substances — Dissolve 0.20 g of L-Aspartic
Acid in 10 mL of 0.2 mol/L sodium hydroxide TS, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add water to make exactly 10 mL. Pipet 1 mL of
this solution, add water to make exactly 50 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography, develop with a mixture of 1-butanol, water and
acetic acid (100) (3:1:1) to a distance of about 10 cm, and dry
the plate at 80°C for 30 minutes. Spray evenly a solution of
ninhydrin in a mixture of methanol and acetic acid (100)
(97:3) (1 in 100), and heat at 80°C for 10 minutes: the spot
other than the principal spot is not more intense than the spot
obtained with the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.15 g of L-Aspartic Acid,
previously dried, dissolve in 50 mL of water by warming. Af-
ter cooling, titrate <2.50> with 0.1 mo/L sodium hydroxide
VS (potentiometric titration). Perform a blank determination
in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 13.31 mg of C 4 H 7 N0 4
Containers and storage Containers — Tight containers.
Aspirin
Acetylsalicylic Acid
77.t°U>
CO;H
O
CH,
C 9 H 8 4 : 180.16
2-Acetoxybenzoic acid [50-78-2]
Aspirin, when dried, contains not less than 99.5% of
C 9 H 8 04.
Description Aspirin occurs as white crystals, granules or
powder. It is odorless, and has a slight acid taste.
It is freely soluble in ethanol (95) and in acetone, soluble in
diethyl ether, and slightly soluble in water.
It dissolves in sodium hydroxide TS and in sodium
carbonate TS.
In moist air, it gradually hydrolyzes to salicylic acid and
acetic acid.
Melting point: about 136°C (bath fluid is heated at 130°C
previously).
JPXV
Official Monographs / Aspirin Aluminum 319
Identification (1) Boil 0.1 g of Aspirin in 5 mL of water
for 5 to 6 minutes, cool, and add 1 to 2 drops of iron (III)
chloride TS: a red-purple color is produced.
(2) Boil 0.5 g of Aspirin in 10 mL of sodium carbonate
TS for 5 minutes, and add 10 mL of dilute sulfuric acid: the
odor of acetic acid is perceptible, and a white precipitate is
produced. Filter the precipitate, add 3 mL of ethanol (95) and
3 mL of sulfuric acid to the filtrate, and heat: the odor of
ethyl acetate is perceptible.
Purity (1) Clarity of solution — Dissolve 0.5 g of Aspirin
in 10 mL of warm sodium carbonate TS: the solution is clear.
(2) Salicylic acid — Dissolve 2.5 g of Aspirin in 25 mL of
ethanol (95), and add 1.0 mL of this solution to a solution
which is prepared by transferring 1 mL of a freshly prepared
dilute ammonium iron (III) sulfate TS to a Nessler tube and
diluting with water to 50 mL. Allow to stand for 30 seconds:
the solution has no more color than the following control
solution.
Control solution: Dissolve 0.100 g of salicylic acid in
water, and add 1 mL of acetic acid (100) and water to make
1000 mL. Add 1.0 mL of this solution to a solution which is
prepared by transferring 1 mL of freshly prepared dilute
ammonium iron (III) sulfate TS and 1 mL of ethanol (95) to a
Nessler tube and diluting with water to 50 mL. Allow to stand
for 30 seconds.
(3) Chloride <1.03>— Boil 1.8 g of Aspirin in 75 mL of
water for 5 minutes, cool, add water to make 75 mL, and
filter. To 25 mL of the filtrate add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this
solution as the test solution. Prepare the control solution
with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.015%).
(4) Sulfate <1.14>— To 25 mL of the filtrate obtained in
(3) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.50 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.040%).
(5) Heavy metals <1.07> — Dissolve 2.5 g of Aspirin in 30
mL of acetone, add 2 mL of dilute acetic acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution with 2.5 mL of
Standard Lead Solution, 30 mL of acetone, 2 mL of dilute
acetic acid and water to make 50 mL (not more than 10 ppm).
(6) Readily carbonizable substances <1J5> — Weigh 0.5 g
of Aspirin, and perform the test. The solution has no more
color than Matching Fluid Q.
Loss on drying <2.41> Not more than 0.5% (3 g, silica gel,
5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1.5 g of Aspirin, previously
dried, add exactly 50 mL of 0.5 mol/L sodium hydroxide VS,
and boil gently for 10 minutes under a reflux condenser with a
carbon dioxide-absorbing tube (soda lime). Cool, and titrate
<2.50> immediately the excess sodium hydroxide with 0.25
mol/L sulfuric acid VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination.
Each mL of 0.5 mol/L sodium hydroxide VS
= 45.04 mg of C 9 H 8 4
Containers and storage Containers — Well-closed contain-
Aspirin Tablets
Acetylsalicylic Acid Tablets
7XtfU>§£
Aspirin Tablets contain not less than 95% and not
more than 105% of the labeled amount of aspirin
(C 9 H 8 4 : 180.16).
Method of preparation Prepare as directed under Tablets,
with Aspirin.
Identification (1) Weigh a quantity of powdered Aspirin
Tablets, equivalent to 0.1 g of Aspirin according to the
labeled amount, add 10 mL of water, and boil for 5 to 6
minutes. After cooling, filter, and add 1 to 2 drops of iron
(III) chloride TS to the filtrate: a red-violet color develops.
(2) Weigh a portion of powdered Aspirin Tablets,
equivalent to 0.5 g of Aspirin according to the labeled
amount, extract with two 10-mL portions of warm ethanol
(95), and filter the combined extracts. Evaporate the filtrate
to dryness, and boil the residue with 10 mL of sodium car-
bonate TS for 5 minutes. Proceed as directed in the Identifi-
cation (2) under Aspirin.
Purity Salicylic acid — Take a portion of the powdered
Aspirin Tablets, equivalent to 1.0 g of Aspirin according to
the labeled amount, shake with 15 mL of ethanol (95) for 5
minutes, filter, discard the first 5 mL of the filtrate, and add
1.0 mL of the subsequent filtrate to a solution which is pre-
pared by transferring 1 mL of freshly prepared dilute ammo-
nium iron (III) sulfate TS to a Nessler tube and diluting with
water to make 50 mL. Proceed as directed in the Purity (2)
under Aspirin.
Assay Weigh accurately and powder not less than 20
Aspirin Tablets. Weigh accurately a portion of the powder,
equivalent to about 1.5 g of aspirin (C 9 H 8 4 ), add exactly 50
mL of 0.5 mol/L sodium hydroxide VS, and proceed as
directed in the Assay under Aspirin.
Each mL of 0.5 mol/L sodium hydroxide VS
= 45.04 mg of aspirin (C 9 H 8 4 )
Containers and storage Containers — Well-closed contain-
ers.
Aspirin Aluminum
Aluminum Acetylsalicylate
[AI(OH)f +
C 18 H 15 A10 9 : 402.29
Bis(2-acetoxybenzoato)hydroxoaluminium [23413-80-1 ]
Aspirin Aluminum contains not less than 83.0% and
320
Aspoxicillin Hydrate / Official Monographs
JP XV
not more than 90.0% of aspirin (C 9 H 8 4 : 180.16), and
not less than 6.0% and not more than 7.0% of alumi-
num (Al: 26.98), calculated on the anhydrous basis.
Description Aspirin Aluminum occurs as a white, crystal-
line powder. It is odorless or has a slight, acetic odor.
It is practically insoluble in water, in methanol, in ethanol
(95) and in diethyl ether.
It dissolves, with decomposition, in sodium hydroxide TS
and in sodium carbonate TS.
Identification (1) Dissolve 0.1 g of Aspirin Aluminum in
10 mL of sodium hydroxide TS by heating, if necessary. Neu-
tralize 2 mL of the solution with hydrochloric acid, and add 1
to 2 drops of iron (III) chloride TS: a red-purple color de-
velops.
(2) Determine the absorption spectrum of the sample
solution obtained in the Assay (1) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 277 nm and 279 nm.
(3) Place 2 g of Aspirin Aluminum in a platinum cruci-
ble, and ignite until charred. To the residue add 1 g of anhy-
drous sodium carbonate, and ignite for 20 minutes. After
cooling, to the residue add 15 mL of dilute hydrochloric acid,
shake, and filter: the filtrate responds to the Qualitative Tests
<1.09> for aluminum salt.
Purity (1) Salicylate — Using A T2 and A S2 obtained in the
Assay (1), calculate the amount of salicylate [as salicylic acid
(C 7 H 6 3 : 138.12)] by the following equation: salicylate
content is not more than 7.5%, calculated on the anhydrous
basis.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= W s x (A T2 /A S2 ) x ( 1 /4)
W s : Amount (mg) of salicylic acid for assay
(2) Heavy metals <1.07> — Place 2.0 g of Aspirin Alumi-
num in a porcelain crucible, cover the crucible loosely, and
ignite at a low temperature until charred. After cooling, add
2 mL of nitric acid and 1 mL of sulfuric acid to the content of
the crucible, heat gently the crucible until white fumes are
evolved, and continue the heating until white fumes are no
longer evolved, then ignite between 500°C and 600°C until
the carbon is incinerated. When the incineration is not com-
pleted, add 2 mL of nitric acid and 1 mL of sulfuric acid, and
heat gently in the same manner, then ignite between 500°C
and 600°C to incinerate completely. After cooling, add 2 mL
of hydrochloric acid, and proceed as directed in Method 2,
and perform the test. Prepare the control solution by using
the same quantities of the same reagents as directed for the
preparation of the test solution, and add 2.0 mL of Standard
Lead Solution and water to make 50 mL (not more than 10
ppm).
(3) Arsenic <1.11> — Dissolve 1.0 g of Aspirin Aluminum
in 15 mL of sodium hydroxide TS. To this solution add 1
drop of phenolphthalein TS, and with stirring, add dropwise
hydrochloric acid until the red color of the solution disap-
pears. Then add 2 mL of hydrochloric acid, cool with oc-
casional shaking for 10 minutes, and filter with a glass filter
(G3). Wash the residue with two 5 mL portions of 1 mol/L
hydrochloric acid TS, and combine the filtrate and the
washings. Use this solution as the test solution, and perform
the test (not more than 2 ppm).
Water <2.48> Not more than 4.0% (0.15 g, direct titration).
Assay (1) Aspirin — Weigh accurately about 0.1 g of Aspi-
rin Aluminum, add 40 mL of sodium fluoride TS, and shake
for 5 minutes. Allow the solution to stand for 10 minutes
with frequent shaking. Extract the solution with six 20-mL
portions of chloroform. Combine all chloroform extracts,
and add chloroform to make exactly 200 mL. Measure exact-
ly 10 mL of this solution, add chloroform to make exactly
100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.09 g of salicylic acid for
assay, previously dried in a desiccator (silica gel) for 3 hours,
and dissolve in chloroform to make exactly 200 mL. Measure
exactly 5 mL of this solution, add chloroform to make exact-
ly 200 mL, and use this solution as the standard solution (1).
Then weigh accurately about 0.09 g of aspirin for assay,
previously dried in a desiccator (silica gel) for 5 hours, and
dissolve in chloroform to make exactly 200 mL. Measure
exactly 10 mL of this solution, add chloroform to make
exactly 100 mL, and use this solution as the standard solution
(2). Perform the test with these solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>. Determine the
absorbances, A Tl and A sl , of the sample solution and stan-
dard solution (1) at 278 nm, and absorbances, A T2 and A S2 ,
of these solution, at 308 nm, respectively. Then determine the
absorbance A S3 of the standard solution (2) at 278 nm.
Amount (mg) of aspirin (C 9 H 8 4 )
A„ x A.
W, x
A,. -
W s : Amount (mg) of aspirin for assay
(2) Aluminum — Weigh accurately about 0.4 g of Aspirin
Aluminum, and dissolve in 10 mL of sodium hydroxide TS.
Add dropwise 1 mol/L hydrochloric acid TS to adjust the
solution to a pH of about 1 , add 20 mL of acetic acid-ammo-
nium acetate buffer solution, pH 3.0, and 0.5 mL of Cu-PAN
TS, and heat. While boiling, titrate <2.50> with 0.05 mol/L
disodium dihydrogen ethylenediamine tetraacetate VS until
the color of the solution changes from red to yellow and per-
sists for 1 minute. Perform a blank determination, and make
any necessary correction.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 1.349 mg of Al
Containers and storage Containers — Well-closed contain-
ers.
Aspoxicillin Hydrate
7XtK + :
') >*»*
H NH 2
H 3 C^
•SHjjO
H H
C 2 iH 27 N 5 7 S.3H 2 0: 547.58
(2S,5i?,6i?)-6-[(2i?)-2-[(2i?)-2-Amino-3-
methylcarbamoylpropanoylamino]-
JPXV
Official Monographs / Aspoxicillin Hydrate 321
2-(4-hydroxyphenyl)acetylamino]-3,3-dimethyl-7-oxo-
4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid
trihydrate [63358-49-6, anhydride]
Aspoxicillin Hydrate contains not less than 950 fig
(potency) and not more than 1020 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Aspoxicillin Hydrate is expressed as mass (potency) of
aspoxicillin (C 21 H 27 N 5 7 S: 493.53).
Description Aspoxicillin Hydrate occurs as a white, crystals
or crystalline powder.
It is freely soluble in 7V,./V-dimethylformamide, sparingly
soluble in water, and practically insoluble in acetonitrile, in
methanol and in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Aspoxicillin Hydrate (1 in 4000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Aspoxicillin Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Aspoxicillin Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or
spectrum of Aspoxicillin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™: +170- +185° (0.2 g calcu-
lated on the anhydrous bases, water, 20 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Aspoxicillin Hydrate in 50 mL
of water: the pH of the solution is between 4.2 and 5.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Aspoxicillin Hydrate in 50 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Aspox-
icillin Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Aspoxicillin Hydrate according to Method 5, and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve 0.05 g of Aspoxicillin
Hydrate in 10 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10,mL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the areas of each peak by the automatic
integration method: the area of each peak other than aspox-
icillin from the sample solution is not more than 3/10 of the
peak area of aspoxicillin from the standard solution, and the
total of peak areas other than aspoxicillin from the sample
solution is not more than the peak area of aspoxicillin from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 6 times as long as the
retention time of aspoxicillin.
System suitability —
Test for required detection: Pipet 2 mL of the standard
solution, add the mobile phase to make exactly 10 mL. Con-
firm that the peak area of aspoxicillin obtained from 10 fiL of
this solution is equivalent to 15 to 25% of that of aspoxicillin
obtained from 10 fiL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of aspoxicillin is not more than 5%.
Water <2.48> Not less than 9.5% and not more than 13.0%
(0.2 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Aspoxicillin Hydrate
and Aspoxicillin Reference Standard, equivalent to about 0.1
g (potency), dissolve each in a suitable amount of water, add
exactly 10 mL of the internal standard solution, 6.5 mL of
acetonitrile and water to make 50 mL, and use these solutions
as the sample solution and standard solution, respectively.
Perform the test with 10 ftL each of these solutions as direct-
ed under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of aspoxicillin to that of the internal standard of
each solution.
Amount [fig (potency)] of aspoxicillin (C21H27N5O7S)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Aspoxicillin Reference
Standard
Internal standard solution — A solution of iV-(3-hydrox-
yphenyl)acetamide (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 130 mL of acetonitrile add potassium
dihydrogenphosphate TS, pH 3.0 to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
aspoxicillin is about 3 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, aspoxicillin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of aspoxicillin to that of the internal standard is
not more than 0.8%.
Containers and storage Containers — Tight containers.
322 Astromicin Sulfate / Official Monographs
JP XV
Astromicin Sulfate
7Xr-nv-f ->>fi?tl£±M
2H2SO,
C 17 H35N 5 6 .2H 2 S04: 601.65
2,6-Diamino-2,3,4,6,7-pentadeoxy- > 5-L-/)'xo-
heptopyranosyl-(l— ►3)-4-amino-l-(2-aminoacetyl-/V-
methylamino)-l,4-dideoxy-6-0-methyl-lL-c/»>o-
inositol disulfate
[72275-67-3]
Astromicin Sulfate is the sulfate of an aminoglyco-
side substance having antibacterial activity produced
by the growth of Micromonospora olivasterospora.
It contains not less than 610 /ug (potency) and not
more than 680 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Astromicin Sulfate
is expressed as mass (potency) of astromicin (Q7H35N5
6 : 405.49).
Description Astromicin Sulfate occurs as a white to light
yellowish white, powder or masses.
It is very soluble in water, sparingly soluble in ethylene
glycol, and practically insoluble in methanol and in ethanol
(99.5).
It is hygroscopic.
Identification (1) Dissolve 10 mg each of Astromicin Sul-
fate and Astromicin Sulfate Reference Standard in 10 mL of
water. To 5 mL each of these solutions add water to make
100 mL, and use these solutions as the sample solution and
standard solution. Perform the test with 10 fiL each of these
solutions as directed under Liquid Chromatography <2.01>
according to the following conditions: the retention time of
astromicin obtained from the sample solution is the same
with that from the standard solution.
Operating conditions —
Detector, column, column temperature, reaction coil, tem-
perature of reaction coil, mobile phase, reaction reagent,
reaction temperature, flow rate of mobile phase, and flow
rate of reaction reagent: Proceed as directed in the operating
conditions in the Purity (3).
(2) To 2 mL of a solution of Astromicin Sulfate (1 in 100)
add 2 to 3 drops of barium chloride TS: a white precipitate is
formed, and it does not dissolve by addition of dilute nitric
acid.
Optical rotation <2.49> [«]£>: +90 - +110° (0.25 g calculated
on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Astromicin Sulfate in 10 mL of water is between 4.5 and
6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Astromicin Sulfate in 10 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Astromi-
cin Sulfate according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Astromicin
Sulfate in 100 mL of water, and use this solution as the
sample solution. Pipet 2 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 10 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the peak areas of the related substance
III, having the relative retention time of about 0.1, and the
related substance I, having the relative retention time of
about 1.2 with respect to the peak of astromicin, from the
sample solution are not more than the peak area of astromi-
cin from the standard solution, the peak area of the related
substance II, having the related retention time of about 0.8, is
not more than 2.0 times the peak area of astromicin from the
standard solution, and the total area of the peaks other than
astromicin from the sample solution is not more than 3.5
times the peak area of astromicin from the standard solution.
Operating conditions —
Detector: A fluorophotometer (excitation wavelength:
340 nm; detection wavelength: 430 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Reaction coil: A stainless steel tube 0.25 mm in inside
diameter and 150 cm in length.
Temperature of reaction coil: 50°C
Mobile phase: To 800 mL of a solution of anhydrous sodi-
um sulfate (71 in 2000) add 25 mL of a solution of sodium 1-
heptanesulfonate (1 in 1000) and 1 mL of acetic acid (100),
and add water to make 1000 mL.
Reaction reagent: Dissolve 1 1 .2 g of potassium hydroxide,
0.458 g of polyoxyethylene (23) lauryl ether, 0.300 g of o-
phthalaldehyde and 1 mL of 2-mercaptoethanol in 400 mL of
a solution of boric acid (31 in 1000), and add water to make
500 mL.
Reaction temperature: 50°C
Flow rate of mobile phase: 0.7 mL per minute
Flow rate of reaction reagent: 0.2 mL per minute
Time span of measurement: About 2 times as long as the
retention time of astromicin.
System suitability —
Test for required detectability: To 5 mL of the sample
solution add water to make 100 mL, and use this solution as
the solution for system suitability test. Pipet 2 mL of the
solution for system suitability test, and add water to make
exactly 100 mL. Confirm that the peak area of astromicin
obtained from 10 fiL of this solution is equivalent to 1.5 to
2.5% of that from 10 fiL of the solution for system suitability
test.
System performance: To 100 mL of water add 5 mL of
the sample solution and 2 mL of a solution of L-valine (1 in
5000). When the procedure is run with 10 fiL of this solution
under the above operating conditions, L-valine and astromi-
JPXV
Official Monographs / Atenolol 323
cin are eluted in this order with the resolution between these
peaks being not less than 1.5, and when the procedure is run
with 10 /xL of the solution for system suitability test under the
above operating conditions, the symmetry factor of the peak
of astromicin is not more than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of astromicin is not more than 2.0%.
Water <2.48> Not more than 8.0% (0.2 g, volumetric titra-
tion, back titration). Use a mixture of methanol for water
determination and ethylene glycol for water determination
(1:1) instead of methanol for water determination.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount of
Astromicin Sulfate Reference Standard, equivalent to about
25 mg (potency), dissolve in diluted hydrochloric acid (1 in
1000) to make exactly 25 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
5 - 15°C, and use within 30 days. Take exactly a suitable
amount of the standard stock solution before use, add
0.1 mol/L phosphate buffer solution for antibiotics, pH 8.0
to make solutions so that each mL contains 4 /ug (potency)
and 1 /xg (potency), and use these solutions as the high con-
centration standard solution and low concentration standard
solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Astromicin Sulfate, equivalent to about 25 mg (potency), and
dissolve in 0.1 mol/L phosphate buffer solution for
antibiotics, pH 8.0 to make exactly 25 mL. Take exactly a
suitable amount of this solution, add 0.1 mol/L phosphate
buffer solution for antibiotics, pH 8.0 to make solutions so
that each mL contains 4 /xg (potency) and 1 /xg (potency), and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Atenolol
7x/P-JU
HO H
and enantiomer
C 14 H 22 N 2 3 : 266.34
2-(4- { (2i?S)-2-Hydroxy-3-
[(l-methylethyl)amino]propyloxy}phenyl)acetamide
[29122-68-7]
Atenolol, when dried, contains not less than 99.0%
and not more than 101.0% of C 14 H 2 2N 2 3 .
Description Atenolol occurs as a white to pale yellow crys-
talline powder.
It is freely soluble in methanol and in acetic acid (100),
soluble in ethanol (99.5), and slightly soluble in water.
A solution of Atenolol in methanol (1 in 25) shows no opti-
cal rotation.
Identification (1) Determine the absorption spectrum of a
solution of Atenolol in methanol (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Atenolol as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Melting Pint <2.60> 152 - 156°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Atenolol according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 50 mg of Atenolol in 25
mL of the mobile phase, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add the mobile
phase to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the area of the peak other than
atenolol is not larger than 1/2 times the peak area of atenolol
obtained with the standard solution, and the total area of the
peaks other than atenolol is not larger than the peak area of
atenolol with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 226 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen
phosphate in 1000 mL of water, and adjust to pH 3.0 with
phosphoric acid. To 40 volume of this solution add 9 volume
of methanol and 1 volume of tetrahydrofuran. Dissolve 1 g
of sodium 1-octanesulfonate and 0.4 g of tetrabutylammoni-
um hydrogensulfate in 1000 mL of this solution.
Flow rate: Adjust the flow rate so that the retention time of
atenolol is about 8 minutes.
Time span of measurement: About 4 times as long as the
retention time of atenolol.
System suitability —
Test for required detectability: To exactly 10 mL of the
standard solution add the mobile phase to make exactly 50
mL. Confirm that the peak area of atenolol obtained with 10
/xL of this solution is equivalent to 14 to 26% of that obtained
with 10 fiL of the standard solution.
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
324 Atropine Sulfate Hydrate / Official Monographs
JP XV
tor of the peak of atenolol are not less than 5000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
atenolol is not more than 1.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Atenolol, previously
dried, dissolve in 100 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 26.63 mg of C 14 H 22 N 2 3
Containers and storage Containers — Tight containers.
Atropine Sulfate Hydrate
7hnt?>filEK£*sfQ*i
• H^CX, ■ HjO
(C 17 H 23 N0 3 ) 2 .H 2 S0 4 .H 2 0: 694.83
(li?,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl [(2RS)-
3-hydroxy-2-phenyl]propanoate hemisulfate hemihydrate
[5908-99-6]
Atropine Sulfate Hydrate, when dried, contains not
less than 98.0% of atropine sulfate [(C 17 H 23 N0 3 ) 2 .H 2
S0 4 : 676.82].
Description Atropine Sulfate Hydrate occurs as colorless
crystals or a white, crystalline powder. It is odorless.
It is very soluble in water and in acetic acid (100), freely
soluble in ethanol (95), and practically insoluble in diethyl
ether.
Melting point: 188 - 194°C (with decomposition). In-
troduce a capillary tube charged with dried sample into a
bath previously heated to 180°C, and continue to heat at a
rate of rise of about 3°C per minute.
It is affected by light.
Identification (1) To 1 mg of Atropine Sulfate Hydrate
add 3 drops of fuming nitric acid, and evaporate the mixture
on a water bath to dryness. Dissolve the residue in 1 mL of
A^N-dimethylformamide, and add 5 to 6 drops of
tetraethylammonium hydroxide TS: a red-purple color de-
velops.
(2) To 2 mL of a solution of Atropine Sulfate Hydrate (1
in 50) add 4 to 5 drops of hydrogen tetrachloroaurate (III)
TS: a lusterless, yellowish white precipitate is formed.
(3) To 5 mL of a solution of Atropine Sulfate Hydrate (1
in 25) add 2 mL of ammonia TS, and allow to stand for 2 to 3
minutes. Collect the precipitate, wash with water, and dry in
a desiccator (in vacuum, silica gel) for 4 hours: it melts <2.60>
between 115°C and 118°C.
(4) A solution of Atropine Sulfate Hydrate (1 in 20)
responds to the Qualitative Tests <1.09> for sulfate.
Purity (1) Clarity and color of solution — Dissolve 0.5 g
of Atropine Sulfate Hydrate in 10 mL of water: the solution
is clear and colorless.
(2) Acidity — Dissolve 1.0 g of Atropine Sulfate Hydrate
in 20 mL of water, and add 0.30 mL of 0.02 mol/L sodium
hydroxide VS and 1 drop of methyl red-methylene blue TS: a
green color develops.
(3) Related substances — Dissolve 0.25 g of Atropine Sul-
fate Hydrate in 1 mL of diluted hydrochloric acid (1 in 10),
add water to make 15 mL, and use this solution as the sample
solution.
(i) To 5 mL of the sample solution add 2 to 3 drops of
hydrogen hexachloroplatinate (IV) TS: no precipitate is
formed.
(ii) To 5 mL of the sample solution add 2 mL of ammo-
nia TS, and shake vigorously: the turbidity of the solution is
not greater than that of the following control solution.
Control solution: To 0.30 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 50
mL. To this solution add 1 mL of silver nitrate TS, and allow
7 mL of the mixture to stand for 5 minutes.
(4) Hyoscyamine — Weigh accurately about 1 g of Atro-
pine Sulfate Hydrate, previously dried, and dissolve in water
to make exactly 10 mL: the specific optical rotation [a]p
<2.49> of this solution in a 100-mm cell is between —0.60°
and +0.10°.
(5) Readily carbonizable substances <1.15> — Take 0.20 g
of Atropine Sulfate Hydrate, and perform the test: the solu-
tion has no more color than Matching Fluid A.
Loss on drying <2.41> Not more than 4.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 110°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 0.25 g of Atropine Sulfate Hydrate,
previously dried and accurately weighed, in 30 mL of acetic
acid (100). If necessary, dissolve it by warming, and cool. Ti-
trate <2.50> with 0.05 mol/L perchloric acid VS until the
color of the solution changes from purple through blue to
blue-green (indicator: 3 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 33.84 mg of atropine sulfate [(C 17 H 23 N0 3 ) 2 .H 2 S0 4 ]
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Atropine Sulfate Injection
7hPtf>StK^att*
Atropine Sulfate Injection is an aqueous solution for
injection.
It contains not less than 93.0% and not more than
107.0% of the labeled amount of atropine sulfate hy-
drate [(C 17 H 23 N0 3 ) 2 .H 2 S0 4 .H 2 0: 694.83].
Method of preparation Prepare as directed under Injec-
JPXV
Official Monographs / Azathioprine 325
tions, with Atropine Sulfate Hydrate.
Description Atropine Sulfate Injection is a clear, colorless
liquid.
pH: 4.0-6.0
Identification (1) Evaporate a volume of Atropine Sulfate
Injection, equivalent to 1 mg of Atropine Sulfate Hydrate ac-
cording to the labeled amount, on a water bath to dryness.
Proceed with the residue as directed in the Identification (1)
under Atropine Sulfate Hydrate.
(2) Evaporate an exactly measured volume of Atropine
Sulfate Injection, equivalent to 5 mg of Atropine Sulfate Hy-
drate according to the labeled amount, on a water bath to
dryness. After cooling, dissolve the residue in 1 mL of
ethanol (95), and use this solution as the sample solution. If
insoluble substance remains, crush it, allow to stand, and use
the supernatant liquid as the sample solution. Separately,
dissolve 10 mg of Atropine Sulfate Reference Standard in
2 mL of ethanol (95), and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of acetone, water and ammonia water (28) (90:7:3) to a
distance of about 10 cm, and dry the plate at 80°C for 10
minutes. After cooling, spray evenly Dragendorff's TS for
spraying on the plate: the spots obtained from the sample so-
lution and the standard solution show an orange color and
the same Rf value.
(3) Atropine Sulfate Injection responds to the Qualitative
Tests <].09> for sulfate.
Bacterial endotoxins <4.01> Less than 75 EU/mg.
Extractable volume <6.05> It meets the requirements.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test: it meets the requirement.
Assay To an exactly measured volume of Atropine Sulfate
Injection, equivalent to about 5 mg of atropine sulfate Hy-
drate [(C 17 H 2 3N0 3 )2.H 2 S04.H 2 0], add exactly 3 mL of the in-
ternal standard solution and water to make 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Atropine Sulfate Reference Stan-
dard, separately determine its loss on drying <2.41> in the
same conditions as for Atropine Sulfate Hydrate, and dis-
solve in water to make exactly 50 mL. Pipet 10 mL of this so-
lution, add exactly 3 mL of the internal standard solution
and water to make 50 mL, and use this solution as the stan-
dard solution. Perform the test with 20 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Q T and Q s , of the peak area
of atropine to that of the internal standard.
Amount (mg) of atropine sulfate Hydrate
[(C 17 H 23 N0 3 ) 2 .H 2 S0 4 .H 2 0]
= Ws x (Qt/Qs) x (1/5) x 1.0266
W s : Amount (mg) of Atropine Reference Standard, calcu-
lated based on the dried basis
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 0.4 g of sodium lauryl sulfate add
500 mL of diluted phosphoric acid (1 in 1000) to dissolve, and
adjust the pH to 3.0 with sodium hydroxide TS. To 240 mL
of this solution add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
atropine is about 16 minutes.
System suitability —
System performance: When the procedure is run with 20
iiL of the standard solution under the above operating
conditions, the internal standard and atropine are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of atropine to that of the internal standard is not
more than 1.5%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Azathioprine
h;C
L,
o
C 9 H 7 N 7 2 S: 277.26
6-(l-Methyl-4-nitro-l//-imidazol-5-ylthio)purine
[446-86-6]
Azathioprine, when
98.5% of C 9 H 7 N 7 2 S.
dried, contains not less than
Description Azathioprine is light yellow crystals or crystal-
line powder. It is odorless.
It is sparingly soluble in A^A^-dimethylformamide and in
pyridine, very slightly soluble in water and in ethanol (99.5),
and practically insoluble in chloroform and in diethyl ether.
It dissolves in sodium hydroxide TS and in ammonia TS.
It is gradually colored by light.
Melting point: about 240°C (with decomposition).
Identification (1) Dissolve 0.01 g of Azathioprine in 50
mL of water by warming. To 5 mL of this solution add 1 mL
of dilute hydrochloric acid and 0.01 g of zinc powder, and
allow to stand for 5 minutes: a yellow color is produced.
326
Azathioprine Tablets / Official Monographs
JP XV
Filter this solution: the filtrate responds to the Qualitative
Tests <1.09> for primary aromatic amines, and a red color is
produced.
(2) Dissolve 0.01 g of Azathioprine in 50 mL of water by
warming. To 1 mL of this solution add 0.5 mL of phos-
photungstic acid TS and 0.5 mL of dilute hydrochloric acid: a
white precipitate is formed.
(3) Prepare the test solution by proceeding with 0.03 g of
Azathioprine according to the Oxygen Flask Combustion
Method <1.06>, using 20 mL of water as the absorbing liquid:
the test solution responds to the Qualitative Tests <1.09> (1)
for sulfate.
(4) Dissolve 0.01 g of Azathioprine in 2 mol/L
hydrochloric acid TS to make 100 mL. Dilute 5 mL of the
solution with water to make 50 mL. Determine the absorp-
tion spectrum of the solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum or the spectrum of a solution of
Azathioprine Reference Standard prepared in the same
manner as the sample solution: both spectra exhibit similar
intensities of absorption at the same wavelengths.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Azathioprine in 50 mL of A^A^-dimethylformamide: the
solution is clear and shows a light yellow color.
(2) Acidity or alkalinity — Add 100 mL of water to 2.0 g
of Azathioprine, shake well for 15 minutes, centrifuge for 5
minutes at 10,000 revolutions per minute, and filter. Discard
the first 20 mL of the filtrate, add 2 drops of methyl red TS to
40 mL of the subsequent filtrate, and use this solution as the
sample solution
(i) Add 0.10 mL of 0.02 mol/L hydrochloric acid VS to
20 mL of the sample solution: a red color develops.
(ii) Add 0. 10 mL of 0.02 mol/L sodium hydroxide VS to
20 mL of the sample solution: a yellow color develops.
(3) Sulfate <1.14>— To 25 mL of the filtrate obtained in
(2) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test
solution. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.038%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of
Azathioprine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Azathioprine, according to Method 3, and perform the
test (not more than 2 ppm).
(6) Related substances — Dissolve 10 mg of Azathioprine
in 80 mL of the mobile phase by warming, cool, add the
mobile phase to make 100 mL, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 /uL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area of both solutions
by the automatic integration method: the total area of the
peaks other than that of azathioprine from the sample solu-
tion is not larger than 1/2 of the peak area of azathioprine
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 296 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 Lira in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust to pH 2.5 of a solution of 0.05 mol/
L potassium dihydrogenphosphate TS (1 in 2) with diluted
phosphoric acid (3 in 2000). To 800 mL of this solution add
200 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
azathioprine is about 8 minutes.
Time span of measurement: About three times as long as
the retention time of azathioprine beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add water to make exactly 50 mL. Confirm that
the peak area of azathioprine obtained from 20 /xL of this
solution is equivalent to 8 to 12% of that of azathioprine
obtained from 20 /xL of the standard solution.
System performance: Dissolve 10 mg of Azathioprine in 80
mL of water by warming, cool, and add water to make 100
mL. To 2 mL of this solution add 2 mL of a solution,
separately prepared by dissolving 0.06 g of benzoic acid in 3
mL of methanol and diluting with water to make 10 mL, and
add the mobile phase to make 25 mL. When the procedure is
run with 20 LiL of this solution under the above operating
conditions, azathioprine and benzoic acid are eluted in this
order with the resolution between these peaks being not less
than 9.
System repeatability: When the test is repeated 6 times with
20 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of azathioprine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Azathioprine, previ-
ously dried, add 80 mL of A r .,A L dimethylformamide, and
warm to dissolve. After cooling, titrate <2.50> with 0.1 mol/L
tetramethylammonium hydroxide VS until the color of the
solution changes from yellow through yellow-green to blue-
green (indicator: 1 mL of thymol blue-dimethylformamide
TS). Perform a blank determination, and make any necessary
correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 27.73 mg of C 9 H 7 7 2 S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Azathioprine Tablets
Azathioprine Tablets contain not less than 95.0%
JPXV
Official Monographs / Azithromycin Hydrate 327
and not more than 105.0% of the labeled amount of
azathioprine (C 9 H 7 N 7 2 S: 277.26).
Method of preparation
with Azathioprine.
Prepare as directed under Tablets,
Azithromycin Hydrate
Identification (1) Weigh a quantity of powdered
Azathioprine Tablets, equivalent to 0.01 g of Azathioprine
according to the labeled amount. Add 50 mL of water, shake
well while warming, and filter. Proceed with 5 mL of the
filtrate as directed in the Identification (1) under
Azathioprine.
(2) Proceed with 1 mL of the filtrate obtained in (1) as
directed in the Identification (2) under Azathioprine.
(3) Determine the absorption spectrum of the sample
solution in the Assay as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
278 nm and 282 nm.
(4) Weigh a quantity of powdered Azathioprine Tablets,
equivalent to 0.1 g of Azathioprine to the labeled amount.
Add 10 mL of a solution of ammonia solution (28) in
methanol (1 in 10), shake well, filter, and use the filtrate
as the sample solution. Separately, dissolve 0.1 g of
Azathioprine Reference Standard in 10 mL of a solution of
ammonia solution (28) in methanol (1 in 10), and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, a solution of ammonia solution (28) in
methanol (1 in 10), M-butyl formate and 1,2-dichloroethane
(15:10:5:2) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots from the sample solution and the standard so-
lution show the same Ri value.
Assay Weigh accurately and powder not less than 20
Azathioprine Tablets. Weigh accurately a portion of the
powder, equivalent to about 0.1 g of azathioprine
(C 9 H 7 N 7 2 S), add 20 mL of dimethylsulfoxide for ultrav-
iolet-visible spectrophotometry, shake well, add 0.1 mol/L
hydrochloric acid TS to make exactly 500 mL, and filter. Dis-
card the first 20 mL of the filtrate, measure exactly 3 mL of
the subsequent filtrate, add 0.1 mol/L hydrochloric acid TS
to make exactly 100 mL, and use this solution as the sample
solution. Separately, weigh accurately about 0.1 g of
Azathioprine Reference Standard, previously dried at 105°C
for 5 hours, dissolve in 20 mL of dimethylsulfoxide for
ultraviolet-visible spectrophotometry, and add 0.1 mol/L
hydrochloric acid TS to make exactly 500 mL. Measure
exactly 3 mL of this solution, add 0.1 mol/L hydrochloric
acid TS to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and A s , of
the sample solution and standard solution at 280 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
respectively.
Amount (mg) of azathioprine (C 9 H 7 N 7 2 S)
= W s x (A T /A S )
W s : Amount (mg) of Azathioprine Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
C 38 H 72 N 2 12 .2H 2 0: 785.02
(2R,3SAS,5R,6R,SR,llR,12R,13S,14R)-5-
(3,4,6-Trideoxy-3-dimethylamino-/?-D-xy/o-
hexopyranosyloxy)-3-(2,6-dideoxy-3-
C-methyl-3-O-methyl-a-L-ribo-hexopyranosyloxy)-
10-aza-6,12,13-trihydroxy-2,4,6,8,10,ll,13-
heptamethylhexadecan-14-olide dihydrate
[117772-70-0]
Azithromycin Hydrate is the derivative of
erythromycin.
It contains not less than 945 fig (potency) and not
more than 1030 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Azithromycin Hy-
drate is expressed as mass (potency) of azithromycin
(C 38 H 72 N 2 12 : 748.98).
Description Azithromycin Hydrate occurs as a white
crystalline powder.
It is freely soluble in methanol and in ethanol (99.5), and
practically insoluble in water.
Identification Determine the infrared absorption spectrum
of Azithromycin Hydrate as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Azithromycin Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Optical rotation <2.49> [a]g>: -45- -49° (0.4 g calculated
on the anhydrous basis, ethanol (99.5), 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Azithromycin Hydrate according to Method 2, and perform
the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Being specified separately.
(3) Residual solvent — Being specified separately.
Water <2.48> Not less than 4.0% and not more than 5.0%
(0.4 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Azithromycin
Hydrate and Azithromycin Reference Standard, equivalent
to about 50 mg (potency), dissolve each in an adequate
amount of a mixture of acetonitrile and water (3:2), add
328
Aztreonam / Official Monographs
JP XV
exactly 2 mL of the internal standard solution and the
mixture of acetonitrile and water (3:2) to make 50 mL, and
use these solutions as the sample solution and standard solu-
tion. Perform the test with 5 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, g T and Q s , of the peak area of
azithromycin to that of the internal standard.
Amount [/<g (potency)] of azithromycin (C38H72N2O12)
= W s x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Azithromycin Reference
Standard
Internal standard solution — A solution of 4,4'-
bis(diethylamino)benzophenone in acetonitrile (3 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
polyvinyl alcohol gel polymer for liquid chromatography (5
/um in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 6.97 g of dipotassium hydrogen
phosphate in about 750 mL of water, adjust the pH to 11.0
with potassium hydroxide TS, and add water to make 1000
mL. To 400 mL of this solution add 600 mL of acetonitrile
for liquid chromatography.
Flow rate: Adjust the flow rate so that the retention time of
azithromycin is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 /xL
of the standard solution under the above operating
conditions, azithromycin and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 nL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of azithromycin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Aztreonam
7XH/3TtA
H 3 C
CO5H
H;N-
i O ^SOsH
N >— N
H CHg
Ci 3 H 17 N s O,S 2 : 435.43
2-{(Z)-(2-Aminothiazol-4-yl)-[(2S,3S)-2-methyl-
4-oxo- 1-sulf oazetidin-3-ylcarbamoyl]methyleneaminooxy}-
2-methyl-l-propanoic acid [78110-38-0]
Aztreonam contains not less than 920 tig (potency)
and not more than 1030 tig (potency) per mg, calculat-
ed on the anhydrous basis. The potency of Aztreonam
is expressed as mass (potency) of aztreonam (C 13 H 17 N 5
o 8 s 2 ).
Description Aztreonam occurs as a white to yellowish white
crystalline powder.
It is freely soluble in dimethylsulfoxide, slightly soluble in
water and in methanol, and very slightly soluble in ethanol
(95).
Identification (1) Determine the absorption spectrum of a
solution of Aztreonam (3 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
Aztreonam Reference Standard: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(2) Determine the spectrum of a solution of Aztreonam
in deuterated dimethylsulfoxide for nuclear magnetic
resonance spectroscopy (1 in 10), using a light hydrogen sub-
stance existing in deuterated dimethylsulfoxide for nuclear
magnetic resonance spectroscopy as an internal reference
compound and 2.50 ppm for its chemical shift, as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
OH): it exhibits a multiple signal at around <5 1.5 ppm, and a
single signal at around 5 7.0 ppm. The ratio of integrated
intensity of each signal is 9:1.
Optical rotation <2.49> [ a ]™: -26 - -32° (0.25 g calculat-
ed on the anhydrous bases, water, 50 mL, 100 mm).
pH <2.54> Dissolve 0.05 g of Aztreonam in 10 mL of water:
the pH of this solution is between 2.2 and 2.8.
Purity (1) Clarity and color of solution — Dissolve 0.1 g of
Aztreonam in 20 mL of water: the solution is clear and
colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Aztreo-
nam according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Aztreonam according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.04 g of Aztreonam in
100 mL of water, and use this solution as the sample solution.
Pipet 2 mL of the sample solution, add water to make exactly
100 mL, and use this solution as the standard solution.
Perform the test with exactly 25 /xL each of these solutions as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the areas of each peak
by the automatic integration method: the area of each peak is
not more than the peak area of aztreonam from the standard
solution, and the total area of peaks other than aztreonam
from the sample solution is not more than 2.5 times of the
peak area of aztreonam from the standard solution.
Operating conditions —
Column, column temperature, mobile phase, and flow
rate: Proceed as directed in the operating conditions in the
Assay.
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Time span of measurement: About 4 times as long as the
retention time of aztreonam beginning after the solvent peak.
System suitability —
JPXV
Official Monographs / Bacampicillin Hydrochloride
329
Test for required detection: Pipet 5 mL of the standard
solution, add water to make exactly 10 mL, and use this
solution as the solution for the test for required detection.
Pipet 1 mL of the solution, and add water to make exactly 10
mL. Confirm that the peak area of aztreonam obtained from
25 /xL of this solution is equivalent to 7 to 13% of that
obtained from 25 /xL of the solution for the test for required
detection.
System performance: When the procedure is run under the
above operating conditions with 25 /xL of the standard solu-
tion obtained in the Assay, the internal standard and aztreo-
nam are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
25 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of aztreonam is not more than 2.0%.
Water <2.48> Not more than 2.0% (0.5 g, volumetric
titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Aztreonam and
Aztreonam Reference Standard, equivalent to about 20 mg
(potency), dissolve each in 70 mL of water, add exactly 10
mL of the internal standard solution and water to make 100
mL, and use these solutions as the sample solution and stan-
dard solution, respectively. Perform the test with 25 /xL each
of these solutions as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of aztreonam to that
of the internal standard of each solution.
Amount \ii% (potency)] of aztreonam (Ci3H 17 N 5 8 S2)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Aztreonam Reference Stan-
dard
Internal standard solution — A solution of 4-aminobenzoic
acid (1 in 6250).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.7 g of tetrabutylammonium
hydrogensulfate in 300 mL of water, adjust to pH 3.0 with
0.5 mol/L disodium hydrogenphosphate TS, and add water
to make 1000 mL. To 650 mL of this solution add 350 mL of
methanol.
Flow rate: Adjust the flow rate so that the retention time of
aztreonam is about 8 minutes.
System suitability —
System performance: When the procedure is run with 25
jxL of the standard solution under the above operating condi-
tions, the internal standard and aztreonam are eluted in this
order with the resolution between these peaks being not less
than 4.
System repeatability: When the test is repeated 6 times with
25 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of aztreonam to that of the internal standard is not
more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Bacampicillin Hydrochloride
Ampicillin Ethoxycarbonyloxyethyl
Hydrochloride
CH 3 O
H IU
C 21 H 27 N 3 7 S.HC1: 501.98
1-Ethoxycarbonyloxyethyl (2 S,5R,6R )-6-[(2R )-2-amino-
2-phenylacetylamino]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate monohydrochloride
[37661-08-8]
Bacampicillin Hydrochloride is a hydrochloride of
ampicilline ethoxycarbonyloxyethyl ester.
It contains not less than 626 /ug (potency) per mg,
calculated on the anhydrous basis. The potency of
Bacampicillin Hydrochloride is expressed as mass
(potency) of ampicillin (Ci 6 H 19 N 3 4 S: 349.40).
Description Bacampicillin Hydrochloride occurs as a white
to pale yellow crystalline powder. It has a characteristic odor.
It is freely soluble in methanol and in ethanol (95), and
soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Bacampicillin Hydrochloride in methanol (1 in
1000) as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of Bacampicillin Hydrochloride
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Bacampicillin Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference
Spectrum or the spectrum of Bacampicillin Hydrochloride
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Bacampicillin Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [a]™: + 140 - + 170° (0.1 g calculated
on the anhydrous basis, ethanol (95), 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bacampicillin Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
330
Bacitracin / Official Monographs
JP XV
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bacampicillin Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(3) Free ampicillin — Weigh accurately about 0.1 g of
Bacampicillin Hydrochloride, transfer into a 100-mL
separater, add exactly 15 mL of ice-cold water to dissolve,
add and mix with exactly 10 mL of ice-cold 0.05 mol/L phos-
phate buffer solution, pH 7.0, then add 25 mL of ice-cold
chloroform, shake, and abandon the chloroform layer.
Repeat the procedure twice with two 25-mL portions of ice-
cold chloroform. Centrifuge the water layer, filter the super-
natant, and use the filtrate as the sample solution. Separately,
weigh accurately an amount of Ampicillin Reference
Standard, equivalent to about 20 mg, and dissolve in water to
make exactly 100 mL. Pipet 5 mL of this solution, add 10 mL
of 0.05 mol/L phosphate buffer solution, pH 7.0 and water
to make exactly 25 mL, and use this solution as the standard
solution. To exactly 10 mL each of the sample solution and
standard solution add exactly 2 mL of sodium hydroxide TS,
allow to stand for exactly 15 minutes, add exactly 2 mL of 1
mol/L hydrochloric acid TS, exactly 10 mL of 0.3 mol/L
potassium hydrogen phthalate buffer solution, pH 4.6, and
exactly 10 mL of 0.005 mol/L iodine VS, allow to stand for
exactly 20 minutes without exposure to light. Titrate <2.50>
these solutions with 0.01 mol/L sodium thiosulfate VS until
the color of the solution changes to colorless. Separately, to
exactly 10 mL each of the sample solution and the standard
solution add exactly 10 mL of 0.3 mol/L potassium hydrogen
phthalate buffer solution, pH 4.6 and exactly 10 mL of 0.005
mol/L iodine VS, and perform a blank determination with
the same manner. Determine the consumed amounts (mL) of
0.005 mol/L iodine VS, V T and V s , of the sample solution
and the standard solution: the amount of ampicillin is not
more than 1.0%.
Amount (mg) of ampicillin (C^H^^C^S)
= W s x (V T /V S ) x (1/20)
W s : Amount (mg) of Ampicillin Reference Standard
Water <2.48> Not more than 1.0% (0.5 g, volumetric
titration, direct titration).
Residue on ignition <2.44> Not more than 1.5% (1 g).
Assay Weigh accurately an amount of Bacampicillin
Hydrochloride and Bacampicillin Hydrochloride Reference
Standard, equivalent to about 40 mg (potency), dissolve each
in water to make exactly 100 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with exactly 20 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the peak
areas, A T and A s , of bacampicillin of these solutions.
Amount [fig (potency)] of ampicillin (C 16 H 19 N 3 04S)
= W s x (A T /A S ) x 1000
fV s : Amount [mg (potency)] of Bacampicillin Hydrochlo-
ride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 500 mL of diluted 2 mol/L sodium
dihydrogen phosphate TS (1 in 100), add diluted 0.05 mol/L
disodium hydrogen phosphate TS (2 in 5) to adjust the pH to
6.8. To 500 mL of this solution add 500 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
bacampicillin is about 6.5 minutes.
System suitability —
System performance: When the procedure is run with
20 fiL of this solution under the above operating conditions,
the number of theoretical plates and the symmetry factor of
the peak of bacampicillin are not less than 10,000 and not
more than 2, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of peak areas of
bacampicillin is not more than 2.0%.
Containers and storage Containers — Tight containers.
Bacitracin
[1405-87-4]
Bacitracin is a mixture of peptide substances having
antibacterial activity including bacitracin A as the main
component produced by the growth of Bacillus subtilis
or Bacillus licheniformis .
It contains not less than 60 Units per mg. The poten-
cy of Bacitracin is expressed as unit calculated from the
amount of bacitracin A (C 66 H 103 N 17 O 16 S: 1422.69).
One unit of Bacitracin is equivalent to 23.8 fig of
bacitracin A (C 66 H 103 N 17 O 16 S).
Description Bacitracin occurs as a white to light brown
powder.
It is freely soluble in water, and slightly soluble in ethanol
(99.5).
Identification (1) To 3 mL of a solution of Bacitracin (1 in
100) add 3 mL of 4-dimethylaminobenzaldehyde TS, shake
until red-rosy to red-purple color appears, then add several
drops of a solution of sodium nitrite (1 in 100), and shake: a
green to dark green color is produced.
(2) Dissolve 60 mg each of Bacitracin and Bacitracin
Reference Standard in 10 mL of water, and use these
solutions as the sample solution and standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 1 fiL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatograph. Develop the plate with a mixture of 1-
butanol, acetic acid (100), water, pyridine and ethanol (99.5)
(30:15:10:6:5) to a distance of about 10 cm, and air-dry the
plate. Spray evenly ninhydrin TS on the plate, and heat at 110
°C for 5 minutes: the spots obtained from the sample
solution and standard solution show the same Rf value.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bacitracin according to Method 2, and perform the test. Pre-
JPXV
Official Monographs / Baclofen 331
pare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.15 g of Bacitracin in
0.05 mol/L sulfuric acid TS to make 100 mL. To 2 mL of this
solution add 0.05 mol/L sulfuric acid TS to make 10 mL, and
determine the absorbances of this solution, A\ and A 2 , at 252
nm and 290 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: A 2 /At is not more than 0.20.
Loss on drying <2.41> Not more than 5.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Micrococcus luteus ATCC 10240.
(ii) Culture medium — Use the medium iii in 3) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount of
Bacitracin Reference Standard, equivalent to about 400
units, dissolve in phosphate buffer solution, pH 6.0 to make
exactly 20 mL, and use this solution as the standard stock
solution. Keep the standard stock solution at not exceeding
10°C and use within 2 days. Take exactly a suitable amount
of the standard stock solution before use, add phosphate
buffer solution, pH 6.0 to make solutions so that each mL
contains 2 units and 0.5 units, and use these solutions as the
high concentration standard solution and low concentration
standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Bacitracin, equivalent about 400 units, dissolve in phosphate
buffer solution, pH 6.0 to make exactly 20 mL. Take exactly
a suitable amount of this solution, add phosphate buffer
solution, pH 6.0 to make solutions so that each mL contains
2 units and 0.5 units, and use these solutions as the high con-
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Baclofen
/Wn7i >
C 10 H 12 ClNO 2 : 213.66
(3/?5')-4-Amino-3-(4-chlorophenyl)butanoic acid
[1134-47-0]
Baclofen contains not less than 98.5% of
C 10 H 12 ClNO 2 , calculated on the anhydrous basis.
Description Baclofen occurs as a white to pale yellowish
white, crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
water, very slightly soluble in methanol and in ethanol (95),
and practically insoluble in diethyl ether.
It dissolves in dilute hydrochloric acid.
Identification (1) To 5 mL of a solution of Baclofen (1 in
1000) add 1 mL of ninhydrin TS, and heat on a water bath
for 3 minutes: a blue-purple color develops.
(2) Determine the absorption spectrum of a solution of
Baclofen in 0.1 mol/L hydrochloric acid TS (1 in 2000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Baclofen Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Perform the test with Baclofen as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Purity (1) Chloride <1.03>— Dissolve 0.5 g of Baclofen in
50 mL of acetic acid (100), and add water to make 100 mL.
To 10 mL of this solution add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.30 mL of 0.01 mol/L hydrochloric acid VS add 5 mL of
acetic acid (100), 6 mL of dilute nitric acid and water to make
50 mL (not more than 0.21%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Baclofen
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Baclofen according to Method 3, and perform the test (not
more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Baclofen in 50
mL of the mobile phase, and use this solution as the sample
solution. Pipet 1 .0 mL and 1 .5 mL of the sample solution, to
each add the mobile phase to make exactly 100 mL, and use
these solutions as the standard solutions (1) and (2), respec-
tively. Perform the test with exactly 25 /xL each of the sample
solution and standard solutions (1) and (2) as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine each peak height of these solutions:
each height of the peaks other than the peak of baclofen from
the sample solution is not larger than the peak height of
baclofen from the standard solution (1), and the total height
of these peaks is not larger than the peak height of baclofen
from the standard solution (2).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 [im in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted acetic
acid (100) (1 in 900) (3:2).
Flow rate: Adjust the flow rate so that the retention time of
baclofen is about 4 minutes.
Time span of measurement: About 3 times as long as the
retention time of baclofen beginning after the solvent peak.
System suitability —
Test for required detection: Adjust the sensitivity so that
the peak height of baclofen obtained from 25 fiL of the
standard solution (1) is between 5 and 10 mm.
System performance: Dissolve 0.40 g of Baclofen and 5 mg
332 Baclofen Tablets / Official Monographs
JP XV
of methyl parahydroxybenzoate in 200 mL of the mobile
phase. To 10 mL of this solution add the mobile phase to
make 100 mL. When the procedure is run with 25 fiL of this
solution under the above operating conditions, baclofen and
methyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
25 /xL of the standard solution (1) under the above operating
conditions, the relative standard deviation of the peak height-
s of baclofen is not more than 3.0%.
Water <2.48> Not more than 1.0% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.5 g of Baclofen, dissolve in
80 mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L
perchloric acid VS until the color of the solution changes
from purple through blue to greenish blue (indicator: 2 drops
of crystal violet TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 21.37 mg of C 10 H 12 ClNO 2
Containers and storage Containers — Well-closed contain-
ers.
Baclofen Tablets
Baclofen Tablets contain not less than 93% and not
more than 107% of the labeled amount of baclofen
(C 10 H 12 ClNO 2 : 213.66).
Method of preparation Prepare as directed under Tablets,
with Baclofen.
Identification (1) To a portion of powdered Baclofen
Tablets, equivalent to 0.01 g of Baclofen according to the
labeled amount, add 10 mL of water, shake well, and filter.
To 5 mL of the filtrate add 1 mL of ninhydrin TS, and pro-
ceed as directed in the Identification (1) under Baclofen.
(2) To a portion of powdered Baclofen Tablets, equiva-
lent to 25 mg of Baclofen according to the labeled amount,
add 50 mL of 0.1 mol/L hydrochloric acid TS, shake for 15
minutes, and filter. Determine the absorption spectrum of the
filtrate as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits maxima between 257 nm and 261
nm, between 264 nm and 268 nm, and between 272 nm and
276 nm.
(3) To a portion of powdered Baclofen Tablets, equiva-
lent to 0.01 g of Baclofen according to the labeled amount,
add 2 mL of a mixture of methanol and acetic acid (100)
(4:1), shake well, centrifuge, and use the supernatant liquid
as the sample solution. Separately, dissolve 0.01 g of
Baclofen Reference Standard in 2 mL of a mixture of
methanol and acetic acid (100) (4:1), and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
20 /xL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (4:1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spot from the sample
solution and that from the standard solution show the same
Rf value.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Baclofen Tablets at 50
revolutions per minute according to the Paddle method using
500 mL of water as the dissolution medium. Take 20 mL or
more of the dissolved solution 45 minutes after starting the
test, and filter through a membrane filter with pore size of not
more than 0.8 ^m. Discard the first 10 mL of the filtrate,
pipet FmL of the subsequent, add water to make exactly V
mL so that each mL contains about 10 //g of baclofen (Ci H 12
C1N0 2 ) according to the labeled amount, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 10 mg of Baclofen Reference Standard (separately de-
termine the water content <2.48> in the same manner as
Baclofen), dissolve in water to make exactly 100 mL, then
pipet 10 mL of this solution, add water to make exactly 100
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and the
standard solution at 220 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>.
The dissolution rate (%) of Baclofen Tablets in 45 minutes
is not less than 70%.
Dissolution rate (%) with respect to the labeled amount
of baclofen (C 10 H 12 ClNO 2 )
= W s x (A T /A S ) x (V'/V) x (1/C) x 50
W s : Amount (mg) of Baclofen Reference Standard.
C: Labeled amount (mg) of baclofen (C 10 H 12 ClNO 2 ) in 1
tablet.
Assay Weigh accurately and powder not less than 20
Baclofen Tablets. Weigh accurately a portion of the powder,
equivalent to about 50 mg of baclofen (Ci H 12 ClNO 2 ), add
130 mL of 0.1 mol/L hydrochloric acid TS, shake for 10
minutes, add 0.1 mol/L hydrochloric acid TS to make exact-
ly 200 mL, and centrifuge. Pipet 10 mL of the supernatant
liquid, add 2 drops of phenolphthalein TS, neutralize with
dilute sodium hydroxide TS, add water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 0.25 g of Baclofen Reference
Standard (separately determine the water content <2.4S> in
the same manner as Baclofen), and dissolve in 0.1 mol/L
hydrochloric acid TS to make exactly 100 mL. Pipet 10 mL
of this solution, and add 0.1 mol/L hydrochloric acid TS to
make exactly 100 mL. Pipet 10 mL of this solution, add 2
drops of phenolphthalein TS, neutralize with dilute sodium
hydroxide TS, add water to make exactly 50 mL, and use this
solution as the standard solution. Pipet 2 mL each of the
sample solution and the standard solution, to each add 4 mL
of ninhydrin-stannous chloride TS, shake, heat on a water
bath for 20 minutes, and shake at once vigorously for 2
minutes. After cooling, to each solution add a mixture of
water and 1-propanol (1:1) to make exactly 25 mL. Deter-
mine the absorbances, A T and A s , of these solutions at 570
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using a blank prepared with 2 mL of water in the
same manner.
Amount (mg) of baclofen (C 10 H 12 ClNO 2 )
JPXV
Official Monographs / Barbital 333
= W s x (Aj/As) x (1/5)
W s ; Amount (mg) of Baclofen Reference Standard, calcu-
lated on the anhydrous basis
Containers and storage Containers — Well-closed contain-
ers.
Bamethan Sulfate
H OH
■ H?SO„
and enantiomer
(C 12 H, 9 N0 2 ) 2 .H 2 S0 4 : 516.65
(li?S)-2-Butylamino-l-
(4-hydroxyphenyl)ethanol hemisulfate [5716-20-1]
Bamethan Sulfate, when dried, contains not less
than 99.0% of (C 12 H 19 N0 2 ) 2 .H 2 S0 4 .
Description Bamethan Sulfate occurs as white crystals or
crystalline powder. It is odorless, and has a bitter taste.
It is freely soluble in water and in acetic acid (100), soluble
in methanol, slightly soluble in ethanol (95), and practically
insoluble in diethyl ether.
Melting point: about 169°C (with decomposition).
Identification (1) To 1 mL of a solution of Bamethan
Sulfate (1 in 1000) add 5 mL of a solution of 4-nitroben-
zenediazonium fluoroborate (1 in 2000) and 10 mL of boric
acid-potassium chloride-sodium hydroxide buffer solution,
pH 9.2: an orange-red color develops.
(2) Determine the absorption spectrum of a solution of
Bamethan Sulfate in 0.01 mol/L hydrochloric acid TS (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Bamethan Sulfate, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>: it exhabits absorption at the wave numbers of
about 1618, 1597, 1518, 1118 and 833 cm" 1 .
(4) A solution of Bamethan Sulfate (1 in 100) responds to
the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 1.0 g of Bamethan Sulfate in 10 mL of
water: the pH of this solution is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Bamethan Sulfate in 20 mL of water: the solution is clear,
and has no more color than the following control solution.
Control solution: To 1.5 mL of Matching Fluid O add
diluted hydrochloric acid (1 in 40) to make 200 mL.
(2) Chloride <1.03>— Perform the test with 3.5 g of
Bamethan Sulfate. Prepare the control solution with 0.25 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.002%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Bame-
than Sulfate according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bamethan Sulfate according to Method 3, and perform the
test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Bamethan
Sulfate in 2 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform and methanol (7:2) in a
developing vessel saturated with ammonia vapor to a distance
of about 12 cm, and air-dry the plate. Spray evenly Dragen-
dorff's TS for spraying on the plate, air-dry for 15 minutes,
spray Dragendorff's TS for spraying again, then, after 1
minute, spray evenly a solution of sodium nitrite (1 in 20),
and immediately put a glass plate on the plate. Examine the
plate after 30 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.75 g of Bamethan Sulfate,
previously dried, dissolve in 80 mL of acetic acid (100), and
titrate with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 51.67 mg of (C 12 H I9 N0 2 ) 2 .H 2 S0 4
Containers and storage Containers — Tight containers.
Barbital
i$)l£9-)l
HaC-^I
O
C 8 H 12 N 2 3 : 184.19
5,5-Diethylpyrimidine-2,4,6(l//,3//,5//)-trione
[57-44-3]
Barbital, when dried, contains not less than 99.0%
of QH 12 N 2 3 .
Description Barbital occurs as colorless or white crystals or
a white, crystalline powder. It is odorless, and has a slightly
bitter taste.
It is freely soluble in acetone and in pyridine, soluble in
ethanol (95), sparingly soluble in diethyl ether, and slightly
soluble in water and in chloroform.
It dissolves in sodium hydroxide TS and in ammonia TS.
The pH of its saturated solution is between 5.0 and 6.0.
Identification (1) Boil 0.2 g of Barbital with 10 mL of
sodium hydroxide TS: the gas evolved changes moistened red
334 Barium Sulfate / Official Monographs
JP XV
litmus paper to blue.
(2) Dissolve 0.05 g of Barbital in 5 mL of diluted pyridine
(1 in 10), add 0.3 mL of copper (II) sulfate TS, shake, and
allow to stand for 5 minutes: a red-purple precipitate is
formed. Shake the mixture with 5 mL of chloroform: a red-
purple color develops in the chloroform layer. Separately,
dissolve 0.05 g of Barbital in 2 to 3 drops of ammonia-ammo-
nium chloride buffer solution, pH 10.7, and 5 mL of diluted
pyridine (1 in 10). Add 5 mL of chloroform and 0.3 mL of
copper (II) sulfate TS to the solution: a red-purple precipitate
is produced in the aqueous layer. The red-purple precipitate
is not dissolved in the chloroform by shaking.
(3) To 0.4 g of Barbital add 0.1 g of anhydrous sodium
carbonate and 4 mL of water, shake, and add a solution of
0.3 g of 4-nitrobenzyl chloride in 7 mL of ethanol (95). Heat
the mixture on a water bath under a reflux condenser for 30
minutes, and allow to stand for 1 hour. Collect the separated
crystals, wash with 7 mL of sodium hydroxide TS and a small
amount of water, recrystallize from a mixture of ethanol (95)
and chloroform (1:1), and dry at 105°C for 30 minutes: the
crystals melt <2.60> between 192°C and 196°C.
Melting point <2.60> 189 - 192°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Barbital in 5 mL of sodium hydroxide TS: the solution is
clear and colorless.
(2) Chloride <1.03>— Dissolve 0.30 g of Barbital in 20 mL
of acetone, and add 6 mL of dilute nitric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution as follows: take 0.30
mL of 0.01 mol/L hydrochloric acid VS, 20 mL of acetone
and 6 mL of dilute nitric acid, and add water to make 50 mL
(not more than 0.035%).
(3) Sulfate <1.14>— Dissolve 0.40 g of Barbital in 20 mL
of acetone, and add 1 mL of dilute hydrochloric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: take
0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of acetone,
and 1 mL of dilute hydrochloric acid, and add water to make
50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Barbital
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead solution (not
more than 20 ppm).
(5) Readily carbonizable substances <1J5> — Perform the
test with 0.5 g of Barbital. The solution is not more colored
than Matching Fluid A.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Barbital, previously
dried, and dissolve in 5 mL of ethanol (95) and 50 mL of
chloroform. Titrate <2.50> with 0.1 mol/L potassium
hydroxide-ethanol VS until the color of the solution changes
from yellow through light blue to purple (indicator: 1 mL of
alizarin yellow GG-thymolphthalein TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 18.42 mg of C 8 H 12 N 2 3
Containers and storage Containers — Well-closed contain-
ers.
Barium Sulfate
tB^U^A
BaSCv 233.39
Description Barium Sulfate occurs as a white powder. It is
odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It does not dissolve in hydrochloric acid, in nitric acid and
in sodium hydroxide TS.
Identification (1) Mix 0.5 g of Barium Sulfate with 2 g
each of anhydrous sodium carbonate and potassium car-
bonate in a crucible, heat the mixture until fusion is com-
plete, treat the cooled mass with hot water, and filter. The
filtrate, acidified with hydrochloric acid, responds to the
Qualitative Tests <1.09> for sulfate.
(2) Wash the hot water-insoluble residue obtained in (1)
with water, dissolve in 2 mL of acetic acid (31), and filter, if
necessary: the solution responds to the Qualitative Tests
<1.09> for barium salt.
Purity (1) Acidity or alkalinity — Agitate 1.0 g of Barium
Sulfate with 20 mL of water for 5 minutes: the solution is
neutral.
(2) Phosphate— Boil 1 .0 g of Barium Sulfate with 3 mL
of nitric acid and 5 mL of water for 5 minutes, cool, and add
water to restore the original volume. Filter through a filter
paper, previously washed with dilute nitric acid, to the filtrate
add an equal volume of hexaammonium heptamolybdate TS,
and allow to stand between 50°C and 60°C for 1 hour: no
yellow precipitate is produced.
(3) Sulfide— Place 10 g of Barium Sulfate in a 250-mL
conical flask, add 10 mL of dilute hydrochloric acid and
water to make 100 mL, and boil for 10 minutes: the gas
evolved does not darken moistened lead (II) acetate paper.
(4) Heavy metals <1.07>— Boil 5.0 g of Barium Sulfate
with 2.5 mL of acetic acid (100) and 50 mL of water for 10
minutes, cool, add 0.5 mL of ammonia TS and water to make
100 mL, and filter. Perform the test with a 50-mL portion of
this filtrate. Prepare the control solution with 2.5 mL of
Standard Lead Solution, 1.25 mL of acetic acid (100), 0.25
mL of ammonia TS and water to make 50 mL (not more than
10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Barium Sulfate according to Method 1, and perform the
test (not more than 1 ppm).
(6) Hydrochloric acid-soluble substances and soluble
barium salts — Cool the solution obtained in (3), add water to
make 100 mL, and filter. Evaporate 50 mL of the filtrate on a
water bath to dryness, add 2 drops of hydrochloric acid and
10 mL of warm water, filter through filter paper for assay,
and wash with 10 mL of warm water. Evaporate the com-
bined filtrate and washings on a water bath to dryness, and
dry the residue at 105 °C for 1 hour: the residue weighs not
more than 15 mg. Shake the residue, if any, with 10 mL of
water, and filter. To the filtrate add 0.5 mL of dilute sulfuric
acid, and allow to stand for 30 minutes: no turbidity is
JPXV
Official Monographs / Beclometasone Dipropionate
335
produced.
Containers and storage Containers — Well-closed contain-
ers.
Freeze-dried BCG Vaccine
(for Percutaneous Use)
fclt BCG 7 ?^>
Freeze-dried BCG Vaccine (for Percutaneous Use) is
a preparation for injection which is dissolved before
use.
It contains live bacteria derived from a culture of the
bacillus of Calmette and Guerin.
It conforms to the requirements of Freeze-dried
BCG Vaccine (for Percutaneous Use) in the Minimum
Requirements for Biological Products.
Description Freeze-dried BCG Vaccine (for Percutaneous
Use) becomes a white to light yellow, turbid liquid on addi-
tion of solvent.
Beclometasone Dipropionate
C 28 H 37 C10 7 : 521.04
9-Chloro- 1 1/?, 1 7 ,21 -trihydroxy- 1 6/?-methylpregna- 1 ,4-
diene-3,20-dione 17,21-dipropanoate [5534-09-8]
Beclometasone Dipropionate, when dried, contains
not less than 97.0% and not more than 103.0% of
C28H37CIO7.
Description Beclometasone Dipropionate occurs as a white
to pale yellow powder. It is odorless.
It is freely soluble in chloroform, soluble in methanol,
sparingly soluble in ethanol (95) and in 1,4-dioxane, slightly
soluble in diethyl ether, and practically insoluble in water.
Melting point: about 208°C (with decomposition).
Identification (1) Dissolve 2 mg of Beclometasone
Dipropionate in 2 mL of sulfuric acid: initially a yellowish
color develops, and gradually changes through orange to
dark red-brown. To this solution add carefully 10 mL of
water: the color changes to bluish green, and a flocculent
precipitate is formed.
(2) Dissolve 0.01 g of Beclometasone Dipropionate in 1
mL of methanol, add 1 mL of Fehling's TS, and heat: a red
to red-brown precipitate is formed.
(3) Prepare the test solution with 0.02 g of Beclometa-
sone Dipropionate as directed under Oxygen Flask Combus-
tion Method <1.06>, using a mixture of 1 mL of sodium
hydroxide TS and 20 mL of water as an absorbing liquid: the
test solution responds to the Qualitative Tests <1.09> for chlo-
ride.
(4) Determine the infrared absorption spectrum of
Beclometasone Dipropionate, previously dried, as directed in
the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum or the spectrum of previously dried
Beclometasone Dipropionate Reference Standard: both spec-
tra exhibit similar intensities of absorption at the same wave
numbers. If any difference appears between the spectra, dis-
solve Beclometasone Dipropionate and Beclometasone
Dipropionate Reference Standard in ethanol (95), respective-
ly, then evaporate the ethanol to dryness, and repeat the test
on the residues.
Optical rotation <2.49> [a]™: +i
g, 1,4-dioxane, 10 mL, 100 mm).
+ 94° (after drying, 0.1
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Beclometasone Dipropionate according to Method 2, and
perform the test. Prepare the control solution with 1.5 mL of
Standard Lead Solution (not more than 30 ppm).
(2) Related substances — Dissolve 20 mg of Beclometa-
sone Dipropionate in 5 mL of a mixture of chloroform and
methanol (9:1), and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add a mixture of chlo-
roform and methanol (9:1) to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of 1,2-
dichloroethane, methanol and water (475:25:1) to a distance
of about 15 cm, and air-dry the plate. Spray evenly alkaline
blue tetrazolium TS on the plate: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 20 mg each of Beclometa-
sone Dipropionate and Beclometasone Dipropionate Refer-
ence Standard, previously dried, and dissolve each in
methanol to make exactly 50 mL. Pipet 10 mL each of these
solutions, add exactly 10 mL of the internal standard solu-
tion and methanol to make 50 mL, and use these solutions as
the sample solution and standard solution, respectively. Per-
form the test with 20 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of beclometasone
dipropionate to that of the internal standard, respectively.
Amount (mg) of C 2 8H 37 C107
= W s x (Q T /Q S )
W s : Amount (mg) of Beclometasone Dipropionate Refer-
ence Standard
Internal standard solution — A solution of testosterone
propionate in methanol (1 in 4000).
336 Beef Tallow / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetonitrile and water (3:2).
Flow rate: Adjust the flow rate so that the retention time of
beclometasone dipropionate is about 6 minutes.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, beclometasone dipropionate and the internal standard
are eluted in this order with the resolution between these
peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of beclometasone dipropionate to that of the inter-
nal standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Beef Tallow
Sevum Bovinum
Beef Tallow is a purified fat obtained by wet steam
rendering from the fresh fatty tissues of Bos taurus
Linne var. domesticus Gmelin (Bovidae).
Description Beef Tallow occurs as a white, uniform mass.
It has a characteristic odor and a mild taste.
It is freely soluble in diethyl ether and in petroleum ether,
very slightly soluble in ethanol (95), and practically insoluble
in water.
It is breakable at a low temperature, but softens above
30°C.
Melting point: 42 - 50°C
Acid value <1.13> Not more than 2.0.
Saponification value <7.73> 193 - 200
Iodine value <1.I3> 33-50 (When the sample is insoluble in
20 mL of cyclohexane, dissolve it by shaking a glass-stop-
pered flask in warm water. Then, if insoluble, increase the
volume of solvent.)
Purity (1) Moisture and coloration — Beef Tallow (5.0 g),
melted by heating on a water bath, forms a clear liquid, from
which no water separates. In a 10-mm thick layer of the liq-
uid, it is colorless or slightly yellow.
(2) Alkalinity— To 2.0 g of Beef Tallow add 10 mL of
water, melt by heating on a water bath, and shake vigorously.
After cooling, add 1 drop of phenolphthalein TS to the sepa-
rated water layer: no color develops.
(3) Chloride— To 1.5 g of Beef Tallow add 30 mL of
ethanol (95), boil for 10 minutes under a reflux condenser,
and filter after cooling. To 20 mL of the filtrate add 5 drops
of a solution of silver nitrate in ethanol (95) (1 in 50): the
turbidity of the mixture does not exceed that of the following
control solution.
Control solution: To 1.0 mL of 0.01 mol/L hydrochloric
acid VS add ethanol (95) to make 20 mL, then add 5 drops of
an ethanolic solution of silver nitrate (1 in 50).
Containers and storage Containers — Well-closed contain-
ers.
White Beeswax
Cera Alba
White Beeswax is bleached Yellow Beeswax.
Description White Beeswax occurs as white to yellowish
white masses. It has a characteristic odor. It is comparatively
brittle when cooled, and the fractured surface is granular,
and non-crystalline.
It is slightly soluble in diethyl ether, and practically insolu-
ble in water and in ethanol (99.5).
Acid value <1.13> 5 - 9 or 17 - 22 Weigh accurately about
6 g of White Beeswax, place in a glass-stoppered 250-mL
flask, and add 50 mL of ethanol (99.5). Warm the mixture to
dissolve the wax, add 1 mL of phenolphthalein TS, and pro-
ceed as directed in the Acid value. Perform a blank determi-
nation using solvent which is not previously neutralized, and
make any necessary correction.
Saponification value <1.13> 80-100 Weigh accurately
about 3 g of White Beeswax, place in a glass-stoppered
250-mL flask, and add exactly 25 mL of 0.5 mol/L potassium
hydroxide-ethanol VS and 50 mL of ethanol (95), heat for 4
hours on a water bath under a reflux condenser, and proceed
as directed in the Saponification value.
Melting point <7.75> 60 - 67°C.
Purity Paraffin, fat, Japan wax or resin — Melt White
Beeswax at the lowest possible temperature, drip the liquid
into a vessel containing ethanol (95) to form granules, and
allow them to stand in air for 24 hours. Drop the granules
into two mixtures of ethanol (95) and water, one adjusted so
as to have a specific gravity of 0.95 and the other 0.97: the
granules sink or are suspended in the mixture with the specific
gravity of 0.95, and float or are suspended in the other mix-
ture.
Containers and storage Containers — Well-closed contain-
ers.
Yellow Beeswax
Cera Flava
Yellow Beeswax is the purified wax obtained from
honeycombs such as those of Apis indica
JPXV
Official Monographs / Bekanamycin Sulfate 337
Radoszkowski or Apis mellifera Linne (Apidae).
Description Yellow Beeswax occurs as light yellow to
brownish yellow masses. It has a characteristic odor, which is
not rancid. It is comparatively brittle when cooled, and the
fractured surface is granular, and non-crystalline.
Acid value <1.13> 5 - 9 or 17 - 22 Weigh accurately about
6 g of Yellow Beeswax, place in a glass-stoppered 250-mL
flask, and add 50 mL of ethanol (99.5). Warm the mixture to
dissolve the wax, add 1 mL of phenolphthalein TS, and pro-
ceed as directed in the Acid value. Perform a blank determi-
nation using solvent which is not previously neutralized, and
make any necessary correction.
Saponification value <1.13> 80 - 100 Weigh accurately
about 3 g of Yellow Beeswax, place in a 250-mL glass-stop-
pered flask, and add 25 mL of 0.5 mol/L potassium hydrox-
ide-ethanol and 50 mL of ethanol (95), insert a reflux con-
denser, heat for 4 hours on a water bath, and proceed as
directed in the Saponification value.
Melting point <7.75> 60 - 67°C.
Purity Paraffin, fat, Japan wax or resin — Melt Yellow
Beeswax at the lowest possible temperature, drip the liquid
into a glass vessel containing ethanol (95) to form granules,
and allow them to stand in air for 24 hours. Drop the gran-
ules into two mixtures of ethanol (95) and water, one adjust-
ed so as to have a specific gravity of 0.95 and the other 0.97:
the granules sink or are suspended in the mixture with the
specific gravity of 0.95, and float or are suspended in the
other mixture.
Containers and storage Containers — Well-closed contain-
ers.
Bekanamycin Sulfate
H 2 N
• JtHjSOj
C 18 H 37 N 5 O 10 .xH 2 SO4
3-Amino-3-deoxy-a-D-glucopyranosyl-(l->6)-[2,6-
diamino-2,6-dideoxy-a-D-glucopyranosyl-(l->4)]-2-deoxy-
D-streptamine sulfate [70550-99-1]
Bekanamycin Sulfate is the sulfate of an
aminoglycoside substance having antibacterial activity
produced by the growth of the mutant of Streptomyces
kanamyceticus.
It contains not less than 680 /ug (potency) and not
more than 770 ,ug (potency) per mg, calculated on the
dried basis. The potency of Bekanamycin Sulfate is
expressed as mass (potency) of bekanamycin (C 18 H 37 N 5
O 10 : 483.51).
Description Bekanamycin Sulfate occurs as a white pow-
der.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 20 mg of Bekanamycin Sulfate
in 2 mL of 1/15 mol/L phosphate buffer solution, pH 5.6,
add 1 mL of ninhydrin TS, and boil: a blue-purple color
develops.
(2) Dissolve 30 mg each of Bekanamycin Sulfate and
Bekanamycin Sulfate Reference Standard in 5 mL of water,
and use these solutions as the sample solution and standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
solution of potassium dihydrogen phosphate (3 in 40) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
0.2% ninhydrin-water saturated 1-butanol TS, and heat at
100°C for 10 minutes: the principal spots obtained from the
sample solution and the standard solution show a purple-
brown color and the same Ri value.
(3) To a solution of Bekanamycin Sulfate (1 in 5) add 1
drop of barium chloride TS: a white turbidity is produced.
Optical rotation <2.49> [a]™: +102- +116° (after drying,
0.25 g, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.50
g of Bekanamycin Sulfate in 10 mL of water is between 6.0
and 8.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Bekanamycin Sulfate in 5 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Be-
kanamycin Sulfate according to Method 4, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 30 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Bekanamycin Sulfate according to Method 1, and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve 60 mg of Bekanamycin
Sulfate in 10 mL of water, and use this solution as the sample
solution. Pipet 3 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 5 /xL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
solution of potassium dihydrogen phosphate (3 in 40) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
0.2% ninhydrin-water saturated 1-butanol TS on the plate,
and heat at 100°C for 10 minutes: the spot other than the
principal spot obtained from the sample solution is not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 5.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
338
Benidipine Hydrochloride / Official Monographs
JP XV
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer having pH <2.54> 7.8 to
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Bekanamycin Sulfate Reference Standard, previously dried,
equivalent to about 20 mg (potency), dissolve in diluted phos-
phate buffer solution, pH 6.0 (1 in 2) to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5 to 15°C and use within 30 days.
Take exactly a suitable amount of the standard stock solution
before use, add 0.1 mol/L phosphate buffer solution, pH 8.0
to make solutions so that each mL contains 10 /ug (potency)
and 2.5 /xg (potency), and use these solutions as the high
concentration standard solution and low concentration stan-
dard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Bekanamycin Sulfate, equivalent to about 20 mg (potency),
and dissolve in water to make exactly 50 mL. Take exactly a
suitable amount of this solution, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 10,ag (potency) and 2.5 /xg (potency), and use these
solutions as the high concentration sample solution and low
concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Benidipine Hydrochloride
^— vt: >^ig^
• HCI
Yl
l^ll and enantiomer
C 28 H 31 N 3 6 .HC1: 542.02
3-[(3i?S)-l-Benzylpiperidin-3-yl] 5-methyl (4RS)-
2,6-dimethyl-4-(3-nitrophenyi)-l,4-dihydropyridine-3,5-
dicarboxylate monohydrochloride [91599-74-5]
Benidipine Hydrochloride, when dried, contains not
less than 99.0% and not more than 101.0% of
C 28 H 31 N 3 6 .HC1.
Description Benidipine Hydrochloride occurs as a yellow
crystalline powder.
It is very soluble in formic acid, soluble in methanol,
sparingly soluble in ethanol (99.5), and practically insoluble
in water.
A solution of Benidipine Hydrochloride in methanol (1 in
100) shows no optical rotation.
Melting point: about 200°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Benidipine Hydrochloride in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Benidipine Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) To 5 mL of a solution of Benidipine Hydrochloride (1
in 10) add 5 mL of ammonia TS, heat on a water bath for 5
minutes, cool, and filter. The filtrate, which is acidified with
dilute nitric acid, responds to the Qualitative Tests <1.09> (2)
for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Benidipine Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 20 mg of Benidipine
Hydrochloride in 100 mL of a mixture of water and methanol
(1:1), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add the mixture of water and
methanol (1:1) to make exactly 500 mL, and use this solution
as the standard solution. Perform the test with exactly 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the peak areas of bisbenzyl-
piperidyl ester having the relative retention time of about
0.35 with respect to benidipine, dehydro derivative having the
relative retention time of about 0.75 and other related sub-
stances are not larger than 1/2 times the peak area of benidi-
pine with the standard solution, and the total area of the
peaks other than benidipine is not larger than the peak area
of benidipine with the standard solution. For this calculation,
use the peak areas of bisbenzylpiperidyl ester and dehydro
derivative after multiplying by their response factors, 1.6, re-
spectively.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 237 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0, methanol and tetrahydrofu-
ran (65:27:8).
Flow rate: Adjust the flow rate so that the retention time of
benidipine is about 20 minutes.
Time span of measurement: About 2 times as long as the
retention time of benidipine beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add the mixture of water and
methanol (1:1) to make exactly 20 mL. Confirm that the peak
area of benidipine obtained with 10 ^L of this solution is e-
quivalent to 18 to 32% of that with 10 /uL of the standard so-
lution.
JPXV
Official Monographs / Benidipine Hydrochloride Tablets
339
System performance: Dissolve 6 mg of Benidipine
Hydrochloride and 5 mg of benzoin in 200 mL of the mixture
of water and methanol (1:1). When the procedure is run with
10 /xL of this solution under the above operating conditions,
benzoin and benidipine are eluted in this order with the reso-
lution between these peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
benidipine is not more than 3.5%.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Benidipine
Hydrochloride, previously dried, dissolve in 10 mL of formic
acid, add 70 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 54.20 mg of C 28 H 31 N 3 6 .HC1
Containers and storage Containers — Tight containers.
Benidipine Hydrochloride Tablets
Benidipine Hydrochloride Tablets contain not
less than 95.0% and not more than 105.0% of the
labeled amount of benidipine hydrochloride
(C 28 H 31 N 3 6 .HC1: 542.02).
Method of preparation Prepare as directed under Tablets,
with Benidipine Hydrochloride.
Identification Shake well a quantity of powdered Benidi-
pine Hydrochloride Tablets, equivalent to 10 mg of Benidi-
pine Hydrochloride according to the labeled amount, with
100 mL of methanol, and centrifuge. To 10 mL of the super-
natant liquid add methanol to make 100 mL, and use this so-
lution as the sample solution. Determine the absorption spec-
trum of the sample solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits maxima between
235 nm and 239 nm, and between 350 nm and 360 nm.
Purity Dehydro derivative — Powder Benidipine Hydro-
chloride Tablets in an agate mortar. To an amount of the
powder, equivalent to 20 mg of Benidipine Hydrochloride,
add about 80 mL of a mixture of diluted phosphoric acid (1
in 500) and methanol (1:1), shake well, and add the mixture
of diluted phosphoric acid (1 in 500) and methanol (1:1) to
make exactly 100 mL. Filter through a membrane filter with
pore size of 0.45 /um, and use the filtrate as the sample solu-
tion. Separately, dissolve 20 mg of benidipine hydrochloride
for assay in the mixture of diluted phosphoric acid (1 in 500)
and methanol (1:1) to make exactly 100 mL. Pipet 1 mL of
this solution, add the mixture of diluted phosphoric acid (1 in
500) and methanol (1:1) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area by
the automatic integration method: the peak area of dehydro
derivative having the relative retention time of about 0.75
with respect to benidipine is not larger than 1/2 times the
peak area of benidipine with the standard solution. For this
calculation, use the peak area of dehydro derivative after
multiplying by the relative response factor, 1.6.
Operating conditions —
Perform as directed in the operating conditions in the
Assay.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mixture of diluted phos-
phoric acid (1 in 500) and methanol (1:1) to make exactly
20 mL. Confirm that the peak area of benidipine obtained
with 10 /xL of this solution is equivalent to 7 to 13% of that
with 10 iuL of the standard solution.
System performance: Dissolve 6 mg of benidipine
hydrochloride and 5 mg of benzoin in 200 mL of a mixture of
water and methanol (1:1). When the procedure is run with 10
/xL of this solution under the above operating conditions,
benzoin and benidipine are eluted in this order with the
resolution between these peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
benidipine is not more than 2.0%.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Benidipine Hydrochloride Tablets add 40
mL of a mixture of diluted phosphoric acid (1 in 500) and
methanol (1:1), shake to disintegrate, and add a suitable
amount of the mixture of diluted phosphoric acid (1 in 500)
and methanol (1:1) to make exactly KmL of a solution, con-
taining 40 fig of benidipine hydrochloride (C 28 H 31 N 3 CvHCl)
per mL. Centrifuge the solution, pipet 20 mL of the super-
natant liquid, add exactly 10 mL of the internal standard so-
lution and the mixture of diluted phosphoric acid (1 in 500)
and methanol (1:1) to make 50 mL, and use this solution as
the sample solution. Proceed as directed in the Assay.
Amount (mg) of benidipine hydrochloride (C 2 8H 31 N 3 6 .HC1)
= ^sX(Gt/Gs)x(F/1000)
W s : Amount (mg) of benidipine hydrochloride for assay
Internal standard solution — A solution of benzoin in a mix-
ture of water and methanol (1:1) (13 in 200,000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Benidipine Hydrochloride
Tablets at 50 revolutions per minute according to the Paddle
method using the sinker, using 900 mL of 1st fluid for disso-
lution test as the dissolution medium. Withdraw 20 mL or
more of the dissolution medium 30 minutes after starting the
test for 2-mg and 4-mg tablts and 45 minutes after starting
the test for 8-mg tablet, and filter through a membrane filter
with a pore size not exceeding 0.45 fim. Discard the first 10
mL of the filtrate pipet the subsequent KmL, and add 1st
fluid for dissolution test to make exactly K'mL so that each
mL contains about 2.2 /ug of benidipine hydrochloride (C 28
340
Benserazide Hydrochloride / Official Monographs
JP XV
H 31 N 3 6 .HC1) according to the labeled amount. Pipet 5 mL
of this solution, add exactly 5 mL of the mobile phase, and
use this solution as the sample solution. Separately, weigh ac-
curately about 22 mg of benidipine hydrochloride for assay,
previously dried at 105°C for 2 hours, and dissolve in the mo-
bile phase to make exactly 100 mL. Pipet 2 mL of this solu-
tion, and add the mobile phase to make exactly 50 mL. Pipet
5 mL of this solution, and add the mobile phase to make ex-
actly 20 mL. Pipet 5 mL of this solution, add exactly 5 mL of
1st fluid for dissolution test, and use this solution as the stan-
dard solution. Perform the test with exactly 50 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas, A T and A s , of benidi-
pine: the dissolution rates for 2-mg or 4-mg tablet in 30
minutes and for 8-mg tablet in 45 minutes are not less than 80
% and not less than 85%, respectively.
Dissolution rate (%) of benidipine hydrochloride
(C 2 8H 31 N 3 6 .HCr) with respect to the labeled amount
= W s x (A T /A S ) x (V'/V) x (I/O x 9
W s : Amount (mg) of benidipine hydrochloride for assay
C: Labeled amount (mg) of benidipine hydrochloride
(C 28 H 31 N 3 6 .HC1) in 1 tablet.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 237 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0 and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
benidipine is about 5 minutes.
System suitability —
System performance: When the procedure is run with 50
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of benidipine are not less than 3000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
50 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
benidipine is not more than 1.5%.
Assay Weigh accurately the mass of not less than 20 Benidi-
pine Hydrochloride Tablets, and power using an agate mor-
tar. Weigh accurately a part of the powder, equivalent to
about 8 mg of benidipine hydrochloride (C 28 H 31 N 3 6 .HC1),
add about 150 mL of a mixture of diluted phosphoric acid (1
in 500) and methanol (1:1), shake, then add the mixture of
diluted phosphoric acid (1 in 500) and methanol (1 : 1) to make
exactly 200 mL, and centrifuge. Pipet 20 mL of the super-
natant liquid, add exactly 10 mL of the internal standard so-
lution and the mixture of diluted phosphoric acid (1 in 500)
and methanol (1:1) to make 50 mL, and use this solution as
the sample solution. Separately, weigh accurately about 40
mg of benidipine hydrochloride for assay, previously dried at
105 °C for 2 hours, and dissolve in the mixture of diluted
phosphoric acid (1 in 500) and methanol (1:1) to make exactly
100 mL. Pipet 2 mL of this solution, add exactly 10 mL of
the internal standard solution and the mixture of diluted
phosphoric acid (1 in 500) and methanol (1:1) to make 50
mL, and use this solution as the standard solution. Perform
the test with 10 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Q T and Qs> of the peak area of benidipine to that of the inter-
nal standard.
Amount (mg) of benidipine hydrochloride (C 2 8H 31 N 3 6 .HC1)
= ^ S x(Gt/Gs)x(1/5)
W s : Amount (mg) of benidipine hydrochloride for assay
Internal standard solution — A solution of benzoin in a mix-
ture of water and methanol (1:1) (13 in 200,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 237 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0, methanol and tetrahydrofu-
ran (65:27:8).
Flow rate: Adjust the flow rate so that the retention time of
benidipine is about 20 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, the internal standard and benidipine are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of benidipine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Benserazide Hydrochloride
"o-tzv-x h*we
HO
H NH 2h 0H
■ HCI
OH and enantiomer
C 10 H 15 N 3 O 5 .HCl: 293.70
(2i?5')-2-Amino-3-hydroxy-
Af'-(2,3,4-trihydroxybenzyl)propanoyihydrazide
monohydrochloride [14919-77-8]
Benserazide Hydrochloride contains not less than
98.0% of C 10 H 15 N 3 O5.HCl, calculated on the anhy-
drous basis.
Description Benserazide Hydrochloride occurs as a white to
JPXV
Official Monographs / Bentonite 341
grayish white, crystalline powder.
It is freely soluble in water and in formic acid, soluble in
methanol, very slightly soluble in ethanol (95), and practical-
ly insoluble in diethyl ether.
The pH of a solution of Benserazide Hydrochloride (1 in
100) is between 4.0 and 5.0.
It is hygroscopic.
It is gradually colored by light.
A solution of Benserazide Hydrochloride (1 in 100) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Benserazide Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 10,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Benserazide Hydrochloride as directed in the potassium
bromide disk method under Infrared Spectrophotometry <
2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) To 10 mL of a solution of Benserazide Hydrochloride
(1 in 30) add silver nitrate TS: a white precipitate is formed.
To a portion of this precipitate add dilute nitric acid: the
precipitation does not dissolve.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Benserazide Hydrochloride in 10 mL of water, and perform
the test with this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: the absorbance of this solution at
430 nm is not more than 0.10.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Benser-
azide Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.25 g of Benserazide Hydrochloride in 10 mL of methanol,
and use this solution as the sample solution. Pipet 1 mL and 3
mL of the sample solution, add methanol to make exactly 200
mL, and use these solutions as the standard solution (1) and
(2), respectively. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2 juL
each of the sample solution and standard solutions (1) and (2)
on a plate of cellulose for thin-layer chromatography.
Develop the plate with a solution of formic acid in sodium
chloride TS (1 in 1000) to a distance of about 10 cm, and air-
dry the plate. Spray evenly sodium carbonate TS, air-dry,
and then spray evenly Folin's TS on the plate: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution (2), and the
number of the spots which intense more than the spot from
the standard solution (1) are not more than 2.
Water <2.48> Not more than 2.5% (0.5 g, direct titration).
Use a solution of salicylic acid in methanol for Karl Fischer
method (3 in 20) instead of methanol for Karl Fischer
method.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Benserazide
Hydrochloride, dissolve in 5 mL of formic acid, add 50 mL
of acetic acid (100), and titrate <2.50> immediately with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.37 mg of CjoHuNjOj.HCI
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Bentonite
^> h-M h-
Bentonite is a natural, colloidal, hydrated aluminum
silicate.
Description Bentonite occurs as a very fine, white to light
yellow-brown powder. It is odorless. It has a slightly earthy
taste.
It is practically insoluble in water and in diethyl ether.
It swells in water.
Identification (1) Add 0.5 g of Bentonite to 3 mL of dilut-
ed sulfuric acid (1 in 3), and heat until white fumes are
evolved. Cool, add 20 mL of water, and filter. To 5 mL of the
filtrate add 3 mL of ammonia TS: a white, gelatinous
precipitate is produced, which turns red on the addition of 5
drops of alizarin red S TS.
(2) Wash the residue obtained in (1) with water, add 2 mL
of a solution of methylene blue trihydrate (1 in 10,000), and
wash again with water: the residue is blue in color.
pH <2.54> To 1.0 g of Bentonite add 50 mL of water, and
shake: the pH of the suspension is between 9.0 and 10.5.
Purity (1) Heavy metals <l.07> — To 1.5 g of Bentonite
add 80 mL of water and 5 mL of hydrochloric acid, and boil
gently for 20 minutes with thorough stirring. Cool, cen-
trifuge, collect the supernatant liquid, wash the residue with
two 10-mL portions of water, and centrifuge each. Combine
the supernatant liquid and the washings, and add dropwise
ammonia solution (28). When a precipitate is produced, add
dropwise dilute hydrochloric acid with vigorous stirring, and
dissolve. To the solution add 0.45 g of hydroxylammonium
chloride, and heat. Cool, and add 0.45 g of sodium acetate
trihydrate, 6 mL of dilute acetic acid and water to make 150
mL. Pipet 50 mL of the solution, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: mix 2.5 mL of Standard Lead Solution, 0.15 g of
hydroxylammonium chloride, 0.15 g of sodium acetate trihy-
drate, and 2 mL of dilute acetic acid, and add water to make
50 mL (not more than 50 ppm).
(2) Arsenic <1. 11>— To 1 .0 g of Bentonite add 5 mL of di-
lute hydrochloric acid, and gently heat to boil while stirring
well. Cool immediately, and centrifuge. To the residue add 5
mL of dilute hydrochloric acid, shake well, and centrifuge.
To the residue add 10 mL of water, and perform the same
operations. Combine all the extracts, and heat on a water
bath to concentrate to 5 mL. Perform the test with this solu-
tion as the test solution (not more than 2 ppm).
(3) Foreign matter — Place 2.0 g of Bentonite in a mortar,
add 20 mL of water to swell, disperse evenly with a pestle,
and dilute with water to 100 mL. Pour the suspension
342
Benzalkonium Chloride / Official Monographs
JP XV
through a No. 200 (74 /um) sieve, and wash the sieve
thoroughly with water. No grit is felt when the fingers are
rubbed over the wire mesh of the sieve.
Loss on drying <2.4I> 5.0 - 10.0% (2 g, 105°C, 2 hours).
Gel formation Mix 6.0 g of Bentonite with 0.30 g of mag-
nesium oxide. Add the mixture, in several portions, to 200
mL of water contained in a glass-stoppered 500-mL cylinder.
Agitate for 1 hour, transfer 100 mL of the suspension to a
100-mL graduated cylinder, and allow to stand for 24 hours:
not more than 2 mL of supernatant appears on the surface.
Swelling power To 100 mL of water in a glass-stoppered
100-mL cylinder add 2.0 g of Bentonite in ten portions,
allowing each portion to settle before adding the next, and
allow to stand for 24 hours: the apparent volume of the
sediment at the bottom is not less than 20 mL.
Containers and storage Containers — Well-closed contain-
ers.
Benzalkonium Chloride
Benzalkonium Chloride is represented by the for
mula [C 6 H 5 CH 2 N(CH 3 ) 2 R]C1, in which R extends from
C 8 H 17 to C 18 H 37 , with C 12 H 25 and C 14 H 29 comprising
the major portion.
It contains not less than 95.0% and not more than
105.0% of benzalkonium chloride (as C 22 H 40 C1N:
354.01), calculated on the anhydrous basis.
Description Benzalkonium Chloride occurs as a white to
yellowish white powder, colorless to light yellow, gelatinous
pieces, or jelly-like fluid or mass. It has a characteristic odor.
It is very soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
A solution of Benzalkonium Chloride foams strongly when
shaken.
Identification (1) Dissolve 0.2 g of Benzalkonium Chlo-
ride in 1 mL of sulfuric acid, add 0.1 g of sodium nitrate, and
heat for 5 minutes on a water bath. After cooling, add 10 mL
of water and 0.5 g of zinc powder, heat for 5 minutes, cool,
and filter: the filtrate responds to the Qualitative Tests <1.09>
for primary aromatic amines. The color of the solution is red.
(2) To 2 mL of a solution of Benzalkonium Chloride (1 in
1000) add a mixture of 0.2 mL of a solution of bromophenol
blue (1 in 2000) and 0.5 mL of sodium hydroxide TS: a blue
color develops. Add 4 mL of chloroform to this solution, and
shake vigorously: the blue color shifts to the chloroform
layer. Collect the chloroform layer, and add dropwise, with
stirring, a solution of sodium lauryl sulfate (1 in 1000): the
chloroform layer turns colorless.
(3) Determine the absorption spectrum of a solution of
Benzalkonium Chloride in 0.1 mol/L hydrochloric acid TS (1
in 2000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(4) To 1 mL of a solution of Benzalkonium Chloride (1 in
100) add 2 mL of ethanol (95), 0.5 mL of dilute nitric acid
and 1 mL of silver nitrate TS: a white precipitate is produced.
This precipitate does not dissolve on the addition of dilute
nitric acid, but dissolves on the addition of ammonia TS.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Benzalkonium Chloride in 10 mL of water: the solution is
clear and colorless to light yellow.
(2) Petroleum ether-soluble substances — To 3.0 g of
Benzalkonium Chloride add water to make 50 mL, then add
50 mL of ethanol (99.5) and 5 mL of 0.5 mol/L sodium
hydroxide TS, and extract with three 50-mL portions of
petroleum ether. Combine the petroleum ether extracts, and
wash with three 50-mL portions of dilute ethanol. After
shaking well with 10 g of anhydrous sodium sulfate, filter
through a dry filter paper, and wash the filter paper with two
10-mL portions of petroleum ether. Evaporate the petroleum
ether on a water bath by heating, and dry the residue at
105°C for 1 hour: the residue is not more than 1.0%.
Water <2.48> Not more than 15.0%.
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.15 g of Benzalkonium
Chloride, and dissolve in 75 mL of water. Adjust the pH
between 2.6 and 3.4 by adding dropwise diluted dilute
hydrochloric acid (1 in 2), add 1 drop of methyl orange TS,
and titrate <2.50> with 0.02 mol/L sodium tetraphenylboron
VS until the color of the solution becomes red.
Each mL of 0.02 mol/L sodium tetraphenylboron VS
= 7.080 mg of C 22 H 40 C1N
Containers and storage Containers — Tight containers.
Benzalkonium Chloride Solution
Benzalkonium Chloride Solution is an aqueous solu-
tion containing not more than 50.0 w/v% of benzalko-
nium chloride.
It contains not less than 93% and not more than
107% of the labeled amount of benzalkonium chloride
(C 22 H 40 C1N: 354.01).
Method of preparation Dissolve Benzalkonium Chloride in
Water or Purified Water. It is also prepared by diluting
Concentrated Benzalkonium Chloride Solution 50 with
Water or Purified Water.
Description Benzalkonium Chloride Solution is a clear,
colorless to light yellow liquid, having a characteristic odor.
It foams strongly on shaking.
Identification (1) Evaporate a volume of Benzalkonium
Chloride Solution, equivalent to 0.2 g of Benzalkonium
Chloride according to the labeled amount, on a water bath to
dryness, and proceed with the residue as directed in the
Identification (1) under Benzalkonium Chloride.
(2) To a volume of Benzalkonium Chloride Solution,
equivalent to 0.01 g of Benzalkonium Chloride according to
the labeled amount, add water to make 10 mL. Proceed with
2 mL of this solution as directed in the Identification (2)
under Benzalkonium Chloride.
JPXV
Official Monographs / Benzbromarone
343
(3) To a volume of Benzalkonium Chloride Solution,
equivalent to 1 g of Benzalkonium Chloride according to the
labeled amount, add water or concentrate on a water bath, if
necessary, to make 10 mL. To 1 mL of this solution add 0.1
mol/L hydrochloric acid VS to make 200 mL, and proceed as
directed in the Identification (3) under Benzalkonium Chlo-
ride.
(4) To a volume of Benzalkonium Chloride Solution,
equivalent to 0.1 g of Benzalkonium Chloride according to
the labeled amount, add water or concentrate on a water
bath, if necessary, to make 10 mL. Proceed with 1 mL of this
solution as directed in the Identification (4) under Benzalko-
nium Chloride.
Assay Pipet a volume of Benzalkonium Chloride Solution,
equivalent to about 0.15 g of benzalkonium chloride (C22H40
C1N), dilute with water to make 75 mL, if necessary, and pro-
ceed as directed in the Assay under Benzalkonium Chloride.
Each mL of 0.02 mol/L sodium tetraphenylboron VS
= 7.080 mg of benzalkonium chloride (C 2 2H 40 C1N)
Containers and storage Containers — Tight containers.
Benzalkonium Chloride
Concentrated Solution 50
SO tf )l =1 - O ^{tWte. 50
Benzalkonium Chloride Concentrated Solution
50 is an aqueous solution, presented as
[C 6 H 5 CH 2 N(CH 3 ) 2 R]C1, where R ranges from C 8 H 17 to
C lg H 37 , and mainly consisting of C 12 H 25 and C 14 H 29 .
It contains more than 50.0 w/v% and not more than
55.0 w/v% of benzalkonium chloride (C 22 H 40 C1N:
354.01).
Description Benzalkonium Chloride Concentrated Solution
50 is a colorless to light yellow liquid or jelly-like fluid, and
has a characteristic odor.
It is very soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
A solution prepared by adding water to it vigorously foams
when shaken.
Identification (1) Dissolve 0.4 g of Benzalkonium Chlo-
ride Concentrated Solution 50 in 1 mL of sulfuric acid, add
0.1 g of sodium nitrate, and heat for 5 minutes on a water
bath. After cooling, add 10 mL of water and 0.5 g of zinc
powder, heat for 5 minutes, cool, and filter: the filtrate
responds to the Qualitative Tests <1.09> for primary aromatic
amines. The color of the solution is red.
(2) To 2 mL of a solution of Benzalkonium Chloride
Concentrated Solution 50 (1 in 500) add a mixture of 0.2 mL
of a solution of bromophenol blue (1 in 2000) and 0.5 mL of
sodium hydroxide TS: a blue color develops. Add 4 mL of
chloroform to this solution, and shake vigorously: the blue
color shifts to the chloroform layer. Collect the chloroform
layer, and add dropwise, with stirring, a solution of sodium
lauryl sulfate (1 in 1000): the chloroform layer turns color-
less.
(3) Determine the absorption spectrum of a solution of
Benzalkonium Chloride Concentrated Solution 50 in 0.1 mol
/L hydrochloric acid TS (1 in 1000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) To 1 mL of a solution of Benzalkonium Chloride
Concentrated Solution 50 (1 in 50) add 2 mL of ethanol (95),
0.5 mL of dilute nitric acid and 1 mL of silver nitrate TS: a
white precipitate is produced. This precipitate does not dis-
solve on the addition of dilute nitric acid, but dissolves on the
addition of ammonia TS.
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
Benzalkonium Chloride Concentrated Solution 50 in 10 mL
of water: the solution is clear and colorless to light yellow.
(2) Petroleum ether-soluble substances — To 6.0 g of
Benzalkonium Chloride Concentrated Solution 50 add water
to make 50 mL, then add 50 mL of ethanol (99.5) and 5 mL
of 0.5 mol/L sodium hydroxide TS, and extract with three
50-mL portions of petroleum ether. Combine the petroleum
ether extracts, and wash with three 50-mL portions of dilute
ethanol. After shaking well with 10 g of anhydrous sodium
sulfate, filter through a dry filter paper, and wash the filter
paper with two 10-mL portions of petroleum ether.
Evaporate the petroleum ether on a water bath by heating,
and dry the residue at 105°C for 1 hour: the residue is not
more than 1.0%.
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Benzalkonium
Chloride Concentrated Solution 50, and dissolve in 75 mL of
water. Adjust the pH to between 2.6 and 3.4 by adding
dropwise diluted dilute hydrochloric acid (1 in 2), add 1 drop
of methyl orange TS, and titrate <2.50> with 0.02 mol/L sodi-
um tetraphenylboron VS until the color of the solution
becomes red.
Each mL of 0.02 mol/L sodium tetraphenylborate VS
= 7.080 mg of benzalkonium chloride (C 22 H 40 C1N)
Containers and storage Containers — Tight containers.
Benzbromarone
^>X7P7P >
C 17 H 12 Br 2 3 : 424.08
3,5-Dibromo-4-hydroxyphenyl 2-ethylbenzo[6]furan-3-yl
ketone [3562-84-3]
Benzbromarone, when dried, contains not less than
98.5% and not more than 101.0% of C 17 H 12 Br 2 3 .
Description Benzbromarone occurs as a white to light yel-
low, crystalline powder.
It is very soluble in N, /V-dimethylformamide, freely solu-
ble in acetone, sparingly soluble in ethanol (99.5), and practi-
cally insoluble in water.
344 Benzethonium Chloride / Official Monographs
JP XV
It dissolves in dilute sodium hydroxide TS.
Identification (1) Determine the absorption spectrum of a
solution of Benzbromarone in 0.01 mol/L sodium hydroxide
TS (1 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Benzbromarone, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 149 - 153°C
Purity (1) Sulfate <1.14> — Dissolve 1.0 g of Benzbroma-
rone in 40 mL of acetone, and add 1 mL of dilute
hydrochloric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control
solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid
VS add 40 mL of acetone, 1 mL of dilute hydrochloric acid
and water to make 50 mL (not more than 0.019%).
(2) Soluble halides — Dissolve 0.5 g of Benzbromarone in
40 mL of acetone, and add 6 mL of dilute nitric acid and
water to make 50 mL. Proceed with this solution as directed
under Chloride Limit Test <1.03>. Prepare the control solu-
tion as follows: to 0.25 mL of 0.01 mol/L hydrochloric acid
VS add 40 mL of acetone, 6 mL of dilute nitric acid and
water to make 50 mL.
(3) Heavy metals <1.07> — Proceed with 2.0 g of Ben-
zbromarone according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Iron <1.10> — Prepare the test solution with 1.0 g of
Benzbromarone according to Method 3, and perform the test
according to Method A. Prepare the control solution with 2.0
mL of Standard Iron Solution (not more than 20 ppm).
(5) Related substances — Dissolve 0.10 g of Benzbroma-
rone in 10 mL of acetone, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add acetone to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10,uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of cyclohexane, 4-methyl-2-
pentanone, ethanol (99.5) and acetic acid (100) (100:20:2:1)
to a distance of about 15 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, phosphorus (V) oxide,
50°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Benzbromarone,
previously dried, dissolve in 30 mL of AfJV-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L tetramethylam-
monium hydroxide VS (indicator: 5 drops of thymol blue-
dimethylformamide TS). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 42.41 mg of C 17 H 12 Br 2 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Benzethonium Chloride
h 3 C
HjCHjC CH 3
C 27 H 42 C1N0 2 : 448.08
7V-Benzyl-AgV-dimethyl-2-{2-[4-(l,l,3,3-
tetramethylbutyl)phenoxy]ethoxy}ethylaminium
chloride [121-54-0]
Benzethonium Chloride, when dried, contains not
less than 97.0% of C 27 H 42 C1N0 2 .
Description Benzethonium Chloride occurs as colorless or
white crystals. It is odorless.
It is very soluble in ethanol (95), freely soluble in water,
and practically insoluble in diethyl ether.
A solution of Benzethonium Chloride foams strongly when
shaken.
Identification (1) Dissolve 0.2 g of Benzethonium Chlo-
ride in 1 mL of sulfuric acid, add 0.1 g of sodium nitrate, and
heat for 5 minutes on a water bath. After cooling, add 10 mL
of water and 0.5 g of zinc powder, heat for 5 minutes, cool,
and filter: the filtrate responds to the Qualitative Tests <1.09>
for primary aromatic amines, developing a red color.
(2) To 2 mL of a solution of Benzethonium Chloride (1 in
1000) add a mixture of 0.2 mL of a solution of bromophenol
blue (1 in 2000) and 0.5 mL of sodium hydroxide TS: a blue
color develops. Add 4 mL of chloroform to this solution, and
shake vigorously: the blue color shifts to the chloroform
layer. Collect the chloroform layer, and add dropwise a
solution of sodium lauryl sulfate (1 in 1000) with stirring: the
chloroform layer turns colorless.
(3) Determine the absorption spectrum of a solution of
Benzethonium Chloride in 0.1 mol/L hydrochloric acid TS (1
in 5000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(4) To 1 mL of a solution of Benzethonium Chloride (1 in
100) add 2 mL of ethanol (95), 0.5 mL of dilute nitric acid
and 1 mL of silver nitrate TS: a white precipitate is produced.
This precipitate does not dissolve on addition of dilute nitric
acid, but dissolves on addition of ammonia TS.
Melting point <2.60> 158 - 164°C (after drying).
Purity Ammonium — Dissolve 0.10 g of Benzethonium
Chloride in 5 mL of water, and boil with 3 mL of sodium
hydroxide TS: the evolving gas dose not change moistened
red litmus paper to blue.
JPXV
Official Monographs / Benzoic Acid
345
Loss on drying <2.41>
4 hours).
Not more than 5.0% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Benzethonium
Chloride, previously dried, dissolve in 75 mL of water, add
diluted dilute hydrochloric acid (1 in 2) dropwise to adjust the
pH to 2.6-3.4, then add 1 drop of methyl orange TS, and
titrate <2.50> with 0.02 mol/L tetraphenylboron VS until the
solution develops a red.
Each mL of 0.02 mol/L sodium tetraphenylboron VS
= 8.962 mg of C 27 H 42 C1N0 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Benzethonium Chloride Solution
Benzethonium Chloride Solution contains not less
than 93% and not more than 107% of the labeled
amount of benzethonium chloride (C27H42CINO2:
448.08).
Method of preparation Dissolve Benzethonium Chloride in
Water or Purified Water.
Description Benzethonium Chloride Solution is a clear,
colorless liquid. It is odorless.
It foams strongly when shaken.
Identification (1) Evaporate a volume of Benzethonium
Chloride Solution, equivalent to 0.2 g of Benzethonium
Chloride according to the labeled amount, on a water bath to
dryness, and proceed with the residue as directed in the
Identification (1) under Benzethonium Chloride.
(2) To a volume of Benzethonium Chloride Solution,
equivalent to 0.01 g of Benzethonium Chloride according to
the labeled amount, add water to make 10 mL, proceed with
2 mL of this solution as directed in the Identification (2)
under Benzethonium Chloride.
(3) To a volume of Benzethonium Chloride Solution,
equivalent to 1 g of Benzethonium Chloride according to the
labeled amount, and add water or concentrate on a water
bath to make 10 mL. To 1 mL of this solution add 0.1 mol/L
hydrochloric acid TS to make 500 mL, and determine the
abosprition spectrum as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 262
nm and 264 nm, between 268 nm and 270 nm, and between
274 nm and 276 nm.
(4) To a volume of Benzethonium Chloride Solution,
equivalent to 0.1 g of Benzethonium Chloride according to
the labeled amount, add water, or concentrate on a water
bath, if necessary, to make 10 mL, and proceed with 1 mL of
this solution as directed in the Identification (4) under Ben-
zethonium Chloride.
Purity (1) Nitrite — Add 1.0 mL of Benzethonium Chlo-
ride Solution to a mixture of 1 mL of a solution of glycine (1
in 10) and 0.5 mL of acetic acid (31): no gas is evolved.
(2) Oxidizing substances — To 5 mL of Benzethonium
Chloride Solution add 0.5 mL of potassium iodide TS and 2
to 3 drops of dilute hydrochloric acid: no yellow color is
produced.
Assay Pipet a volume of Benzethonium Chloride Solution,
equivalent to about 0.2 g of benzethonium chloride
(C27H42CINO2), dilute with water to make 75 mL, if necessa-
ry, and proceed as directed in the Assay under Benzethonium
Chloride.
Each mL of 0.02 mol/L sodium tetraphenylboron VS
= 8.962 mg of benzethonium chloride (C27H42CINO2)
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Benzoic Acid
^X0 2 H
u
C 7 H 6 2 : 122.12
Benzoic acid [65-85-0]
Benzoic Acid, when dried, contains not less than
99.5% of C 7 H 6 2 .
Description Benzoic Acid occurs as white crystals or crys-
talline powder. It is odorless, or has a faint, benzaldehyde-
like odor.
It is freely soluble in ethanol (95), in acetone and in diethyl
ether, soluble in hot water, and slightly soluble in water.
Identification Dissolve 1 g of Benzoic Acid in 8 mL of sodi-
um hydroxide TS, and add water to make 100 mL. This solu-
tion responds to the Qualitative Tests <1.09> (2) for benzoate.
Melting point <2.60> 121 - 124°C
Purity (1) Heavy metals <1.07> — Dissolve 1.0 g of Benzoic
Acid in 25 mL of acetone, add 2 mL of dilute acetic acid and
water to make 50 mL, and perform the test using this solution
as the test solution. Prepare the control solution as follows:
to 2.0 mL of Standard Lead Solution add 2 mL of dilute
acetic acid, 25 mL of acetone and water to make 50 mL (not
more than 20 ppm).
(2) Chlorinated compounds — Take 0.5 g of Benzoic Acid
and 0.7 g of calcium carbonate in a crucible, mix with a small
amount of water, and dry. Ignite it at about 600°C, dissolve
in 20 mL of dilute nitric acid, and filter. Wash the residue
with 15 mL of water, combine the filtrate and the washing,
add water to make 50 mL, and add 0.5 mL of silver nitrate
TS: this solution has not more turbid than the following con-
trol solution.
Control solution: Dissolve 0.7 g of calcium carbonate in 20
mL of dilute nitric acid, and filter. Wash the residue with 15
mL of water, combine the filtrate and the washings, add 1.2
mL of 0.01 mol/L hydrochloric acid VS and water to make
50 mL, and add 0.5 mL of silver nitrate TS.
(3) Potassium permanganate-reducing substances — Add
0.02 mol/L potassium permanganate VS dropwise to a boil-
ing mixture of 100 mL of water and 1.5 mL of sulfuric acid,
until a red color persists for 30 seconds. Dissolve 1.0 g of
346 Benzyl Alcohol / Official Monographs
JP XV
Benzoic Acid in this boiling solution, and add 0.50 mL of
0.02 mol/L potassium permanganate VS: a red color persists
for at least 15 seconds.
(4) Phthalic acid — To 0.10 g of Benzoic Acid add 1 mL
of water and 1 mL of resorcinol-sulfuric acid TS, and heat
the mixture in an oil bath heated at a temperature between
120°C and 125 °C. After evaporating the water, heat the
residue for 90 minutes, cool, and dissolve in 5 mL of water.
To 1 mL of the solution add 10 mL of a solution of sodium
hydroxide (43 in 500), shake, then examine under light at a
wavelength between 470 nm and 490 nm: the green fluores-
cence of the solution is not more intense than that of the fol-
lowing control solution.
Control solution: Dissolve 61 mg of potassium hydrogen
phthalate in water to make exactly 1000 mL. Measure exactly
1 mL of the solution, add 1 mL of resorcinol-sulfuric acid
TS, and proceed as directed above.
(5) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Benzoic Acid. The solution is not more
colored than Matching Fluid Q.
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
3 hours).
Residue on ignition <2.44> Not more than 0.05% (1 g).
Assay Weigh accurately about 0.5 g of Benzoic Acid, previ-
ously dried, dissolve in 25 mL of neutralized ethanol and 25
mL of water, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.21 mg of C 7 H 6 2
Containers and storage Containers — Well-closed contain-
ers.
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers. ♦
Benzyl Alcohol
^>vJU7JU=l-JU
OH
C 7 H 8 0: 108.14
Benzyl alcohol [100-51-6]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Benzyl Alcohol contains not less than 98.0% and not
more than 100.5% of C 7 H 8 0.
♦The label states, where applicable, that it is suitable
for use in the manufacture of injection forms. ♦
♦Description Benzyl Alcohol is a clear, colorless oily liq-
uid.
It is miscible with ethanol (95), with fatty oils and with es-
sential oils.
It is soluble in water.
Specific gravity df : 1.043 - 1.049*
♦Identification Determine the infrared absorption spec-
trum of Benzyl Alcohol as directed in the liquid film method
Refractive index <2.45>
n- a -
"D •
1.538- 1.541
Purity (1) Clarity and color of solution — Dissolve 2.0 mL
of Benzyl Alcohol in 60 mL of water: the solution is clear and
colorless.
(2) Acidity— To 10 mL of Benzyl Alcohol add 10 mL of
neutralized ethanol, 2 drops of phenolphthalein TS and 1.0
mLofO.l mol/L sodium hydroxide VS: a red color develops.
(3) Benzaldehyde and other related substances — Use Ben-
zyl Alcohol as the sample solution. Separately, weigh exactly
0.750 g of benzaldehyde and 0.500 g of cyclohexylmethanol,
and add Benzyl Alcohol to make exactly 25 mL. Pipet 1 mL
of this solution, add exactly 2mL of ethylbenzene internal
standard solution and exactly 3 mL of dicyclohexyl internal
standard solution, then add Benzyl Alcohol to make exactly
20 mL, and use this solution as the standard solution (1). Per-
form the test with 0.1 /xL each of the sample solution and
standard solution (1) as directed under Gas Chromatography
<2.02> according to the following conditions: no peaks of
ethylbenzene and dicyclohexyl appear on the chromatogram
obtained with the sample solution. When 0.1 /xh of the stan-
dard solution (1) is injected, adjust the sensitivity of the de-
tector so that the peak height of ethylbenzene is not more
than 30% of the full scale of the recorder. The peak area of
benzaldehyde obtained with the sample solution is not more
than the deference between the peak areas of benzaldehyde of
the sample solution and the standard solution (1) (0.15%),
and the peak area of cyclohexylmethanol with the sample so-
lution is not more than the deference between the peak areas
of cyclohexylmethanol of the sample solution and the stan-
dard solution (1) (0.10%). The total area of the peaks having
smaller retention time than benzyl alcohol and other than
benzaldehyde and cyclohexylmethanol obtained with the
sample solution is not more than 4 times the peak area of
ethylbenzene with the standard solution (1) (0.04%). The
total area of the peaks having larger retention time than ben-
zyl alcohol obtained with the sample solution is not more
than the peak area of dicyclohexyl with the standard solution
(1) (0.3%). For these calculations the peak areas less than
1/100 times the peak area of ethylbenzene with the standard
solution (1) are excluded.
Benzyl Alcohol labeled that it is suitable for use in the
manufacture of injection forms meets the following require-
ments.
Use Benzyl Alcohol as the sample solution. Separately,
weigh exactly 0.250 g of benzaldehyde and 0.500 g of cyclo-
hexylmethanol, and add Benzyl Alcohol to make exactly 25
mL. Pipet 1 mL of this solution, add exactly 2 mL of the
ethylbenzene internal standard solution and exactly 2 mL of
the dicyclohexyl internal standard solution, then add Benzyl
Alcohol to make exactly 20 mL, and use this solution as the
standard solution (2). Perform the test with 0.1 /xL each of
the sample solution and standard solution (2) as directed un-
der Gas Chromatography <2.02> according to the following
conditions: no peaks of ethylbenzene and dicyclohexyl ap-
pear on the chromatogram obtained with the sample solu-
tion. When 0.1 /uh of the standard solution (2) is injected, ad-
just the sensitivity of the detector so that the peak height of
ethylbenzene is not more than 30% of the full scale of the
recorder. The peak area of benzaldehyde of obtained with the
JPXV
Official Monographs / Benzyl Benzoate 347
sample solution is not more than the difference between the
peak areas of benzaldehyde of the sample solution and the
standard solution (2) (0.05%), and the peak area of cyclohex-
ylmethanol with the sample solution is not more than the
deference between the peak areas of cyclohexylmethanol of
the sample solution and the standard solution (2) (0.10%).
The total area of the peaks having smaller retention time than
benzyl alcohol and other than benzaldehyde and cyclohex-
ylmethanol obtained with the sample solution is not more
than 2 times the peak area of ethylbenzene with the standard
solution (2) (0.02%). The total area of the peaks having larg-
er retention time than benzyl alcohol obtained with the sam-
ple solution is not more than the peak area of dicyclohexyl
with the standard solution (2) (0.2%). For these calculation
the peak areas less than 1/100 times the peak area of ethyl-
benzene with the standard solution (2) are excluded.
Ethylbenzene internal standard solution: Dissolve exactly
0.100 g of ethylbenzene in Benzyl Alcohol to make exactly 10
mL. Pipet 2 mL of this solution, and add Benzyl Alcohol to
make exactly 20 mL.
Dicyclohexyl internal standard solution: Dissolve exactly
2.000 g of dicyclohexyl in Benzyl Alcohol to make exactly 10
mL. Pipet 2 mL of this solution, and add Benzyl Alcohol to
make exactly 20 mL.
Operating conditions —
Detector: A hydrogen flame - ionization detector
Column: A fused silica column 0.32 mm in inside diameter
and 30 m in length, coated inside with polyethylene glycol 20
M for gas chromatography in 0.5 //m thickness.
Column temperature: Raise the temperature at a rate of
5°C per minutes from 50°C to 220°C, and maintain at 220°C
for 35 minutes.
Temperature of injection port: A constant temperature of
about 200°C.
Temperature of detector: A constant temperature of about
310°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
benzyl alcohol is between 24 and 28 minutes.
Split ratio: Splitless
System suitability —
System performance: When the procedure is run with the
standard solution (1) under the above operating conditions,
the relative retention times of ethylbenzene, dicyclohexyl,
benzaldehyde and cyclohexylmethanol with respect to benzyl
alcohol are about 0.28, about 0.59, about 0.68 and about
0.71, respectively, and the resolution between the peaks of
benzaldehyde and cyclohexylmethanol is not less than 3.0. In
the case of Benzyl Alcohol labeled to use for injection, pro-
ceed with the standard solution (2) instead of the standard
solution (1).
(4) Peroxide value — Dissolve 5 g of Benzyl Alcohol in
30 mL of a mixture of acetic acid (100) and chloroform (3:2)
in a 250-mL glass-stoppered conical flask. Add 0.5 mL of
potassium iodide saturated solution, shake exactly for 1
minute, add 30 mL of water, and titrate <2.50> with 0.01
mol/L sodium thiosulfate VS until the blue color of the solu-
tion disappears after addition of 10 mL of starch TS near the
end point where the solution is a pale yellow color. Perform a
blank determination in the same manner. Calculate the
amount of peroxide by the following formula: not more than
5.
V\. Volume (mL) of 0.01 mol/L sodium thiosulfate VS
consumed in the test
V : Volume (mL) of 0.01 mol/L sodium thiosulfate VS
consumed in the blank
W: Amount (g) of the sample
(5) Residue on evaporation — Perform the test after con-
formation that the sample meets the requirement of the
peroxide value. Transfer 10.0 g of Benzyl Alcohol to a por-
celain or quartz crucible or platinum dish, previously
weighed accurately, and heat on a hot-plate at not exceeding
200°C, taking care to avoid boiling, to evaporate to dryness.
Dry the residue on the hot-plate for 1 hour, and allow to cool
in a desiccator: not more than 5 mg.
Assay Weigh accurately about 0.9 g of Benzyl Alcohol, add
exactly 15.0 mL of a mixture of pyridine and acetic anhydride
(7:1), and heat on a water bath under a reflux condenser for
30 minutes. Cool, add 25 mL of water, and titrate <2.50> the
excess acetic acid with 1 mol/L sodium hydroxide VS (indica-
tor: 2 drops of phenolphthalein TS). Perform a blank deter-
mination.
Each mL of 1 mol/L sodium hydroxide VS
= 108.1 mg of C 7 H g O
♦Containers and storage Containers — Tight containers.
Storage — Light-resistant. »
Benzyl Benzoate
tr ■
C 14 H 12 2 : 212.24
Benzyl benzoate [120-51-4]
Benzyl Benzoate contains not less than 99.0% of
C 14 H 12 2 .
Description Benzyl Benzoate is a colorless, clear, viscous
liquid. It has a faint, aromatic odor and a pungent, burning
taste.
It is miscible with ethanol (95) and with diethyl ether.
It is practically insoluble in water.
Congealing point: about 17°C
Specific gravity d^: about 1.123
Boiling point: about 323 °C
Identification (1) Heat gently 1 mL of Benzyl Benzoate
with 5 mL of sodium carbonate TS and 2 mL of potassium
permanganate TS: the odor of benzaldehyde is perceptible.
(2) Warm the titrated mixture obtained in the Assay on a
water bath to remove ethanol, and add 0.5 mL of iron (III)
chloride TS: a light yellow-red precipitate is produced, which
turns white on the addition of dilute hydrochloric acid.
Refractive index <2.45> n™: 1.568 - 1.570
Purity Acidity — Dissolve 5.0 mL of Benzyl Benzoate in 25
mL of neutralized ethanol, and add 0.50 mL of 0. 1 mol/L so-
Amount (mEq/kg) of peroxide = {10x(Ki~ V a )}/W
348
Benzylpenicillin Benzathine Hydrate / Official Monographs
JP XV
dium hydroxide VS: a red color develops.
Residue on ignition <2.44> Not more than 0.05% (2 g).
Assay Weigh accurately about 2 g of Benzyl Benzoate, add
exactly 50 mL of 0.5 mol/L potassium hydroxide-ethanol
VS, and boil gently for 1 hour under a reflux condenser with a
carbon dioxide-absorbing tube (soda lime). Cool, and titrate
<2.50> the excess potassium hydroxide with 0.5 mol/L
hydrochloric acid VS (indicator: 2 drops of phenolphthalein
TS). Perform a blank determination.
Each mL of 0.5 mol/L potassium hydroxide-ethanol VS
= 106.1 mg of Ci 4 H 12 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Benzylpenicillin Benzathine Hydrate
onr
H COjH
jUO
(C 16 H I8 N 2 O 4 S) 2 .C 16 H 20 N 2 .4H 2 O: 981.18
(2S,5R,6R )-3 , 3-Dimethyl-7-oxo-6- [(phenylacetyl)amino] -
4-thia-l-azabicyclo[3.2.0]heptane-2-
carboxylic acid hemi(7V,./V'-dibenzylethylenediamine)
dihydrate [41372-02-5]
Benzylpenicillin Benzathine Hydrate is the N,N'-
dibenzylethylenediamine salt of a penicillin compound
having antibacterial activity produced by the growth of
Penicillium species.
It contains not less than 1213 Units and not more
than 1333 Units per mg, calculated on the anhydrous
basis. The potency of Benzylpenicillin Benzathine Hy-
drate is expressed as unit calculated from the amount
of benzylpenicillin sodium (C 16 H 17 N2Na0 4 S: 356.37).
1 Unit of Benzylpenicillin Benzathine Hydrate is
equivalent to 0.6 fig of benzylpenicillin sodium
(Ci 6 H 17 N 2 Na0 4 S). It contains not less than 24.0% and
not more than 27.0% of ./V.TV'-dibenzylethylenedia-
mine (C 16 H 2 oN 2 : 240.34), calculated on the anhydrous
basis.
Description Benzylpenicillin Benzathine Hydrate occurs as
a white crystalline powder.
It is slightly soluble in methanol and in ethanol (99.5), and
practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Benzylpenicillin Benzathine Hydrate in methanol
(1 in 2000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Benzylpenicillin Benzathine Hydrate as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Optical rotation <2.49> [a]™: +217 - +233° (0.1 g calculated
on the anhydrous basis, methanol, 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Benzylpenicillin Benzathine Hydrate according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Benzylpenicillin Benzathine Hydrate according to Method
3, and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 70 mg of Benzylpenicil-
lin Benzathine Hydrate in 25 mL of methanol, add a solution
prepared by dissolving 1.02 g of disodium hydrogen phos-
phate and 6.80 g of potassium dihydrogen phosphate in
water to make 1000 mL to make 50 mL, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase A to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area by
the automatic integration method: the area of the peak hav-
ing the relative retention time of about 2.4 with respect to
benzylpenicillin is not more than 2 times the total area of the
peaks of benzylpenicillin and 7V,Af'-dibenzylethylenediamine
obtained from the standard solution, and the area of the peak
other than benzylpenicillin, TV.Af'-dibenzylethylenediamine
and the peak having the relative retention time of about 2.4 is
not more than the total area of the peaks of benzylpenicillin
and A^Af'-dibenzylethylenediamine obtained from the stan-
dard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.0 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase A: A mixture of water, methanol and
0.25 mol/L potassium dihydrogen phosphate TS, pH 3.5
(6:3:1).
Mobile phase B: A mixture of methanol, water and
0.25 mol/L potassium dihydrogen phosphate TS, pH 3.5
(6:3:1).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-10
10-20
20-55
75
75 ->
25
25 -> 100
100
Flow rate: l.OmL/min
Time span of measurement: About 3 times as long as the
retention time of benzylpenicillin beginning after the solvent
peak.
System suitability —
JPXV
Official Monographs / Benzylpenicillin Potassium
349
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase A to make exactly 20 mL.
Confirm that the peak area of benzylpenicillin obtained from
20 fiL of this solution is equivalent to 3.5 to 6.5% of that
from the standard solution.
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, 7V,iV'-dibenzylethylenediamine and benzyl-
penicillin are eluted in this order with the resolution between
these peaks being not less than 25.
System repeatability: When the test is repeated 3 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of benzylpenicillin is not more than 2.0%.
Water <2.48> 5.0-8.0% (1 g, volumetric titration, direct
titration).
Assay (1) Benzylpenicillin — Weigh accurately an amount
of Benzylpenicillin Benzathine Hydrate, equivalent to about
85,000 Units, dissolve in 25 mL of methanol, and add a solu-
tion containing 1.02 g of disodium hydrogen phosphate and
6.80 g of potassium dihydrogen phosphate in 1000 mL of
water to make exactly 50 mL. Pipet 5 mL of this solution,
add a mixture of the solution containing 1.02 g of disodium
hydrogen phosphate and 6.80 g of potassium dihydrogen
phosphate in 1000 mL of water and methanol (1:1) to make
exactly 20 mL, and use this solution as the sample solution.
Separately, weigh accurately an amount of Benzylpenicillin
Potassium Reference Standard, equivalent to about 85,000
Units, and about 25 mg of /V.A^-dibenzylethylenediamine di-
acetate, dissolve in 25 mL of methanol, and add the solution
containing 1.02 g of disodium hydrogen phosphate and 6.80
g of potassium dihydrogen phosphate in 1000 mL of water to
make exactly 50 mL. Pipet 5 mL of this solution, add a mix-
ture of the solution containing 1.02 g of disodium hydrogen
phosphate and 6.80 g of potassium dihydrogen phosphate in
1000 mL of water and methanol (1:1) to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with exactly 20 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T ands A s , of benzylpenicillin.
Amount (unit) of benzylpenicillin sodium (Ci6H 17 N 2 Na0 4 S)
= W s x (A T /A S )
W s : Amount (unit) of Benzylpenicillin Potassium Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, methanol and 0.25
mol/L potassium dihydrogen phosphate TS, pH 3.5 (11:7:2).
Flow rate: Adjust the flow rate so that the retention time of
benzylpenicillin is about 18 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, A^/V'-dibenzylethylenediamine and benzyl-
penicillin are eluted in this order with the resolution between
these peaks being not less than 20.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviations of the peak areas
of JV,JV'-dibenzylethylenediamine and benzylpenicillin are
not more than 2.0%, respectively.
(2) A r ,./V'-Dibenzylethylenediamine — Determine the
areas, A T and A s , of the peak corresponding to 7V,/V-diben-
zylethylenediamine on the chromatograms obtained in (1)
with the sample solution and standard solution.
Amount (%) of 7V,/V'-dibenzylethylenediamine (C 16 H 20 N 2 )
= {W S /W T ) x (Aj/As) x 100 x 0.667
W s : Amount (mg) of /V,7V'-dibenzylethylenediamine
diacetate
W T \ Amount (mg) of the sample
0.667: Conversion factor for the molecular mass
of /V.A^-dibenzylethylenediamine diacetate
(C 16 H 20 N 2 -2CH 3 COOH) to that of N,N'-diben-
zylethylenediamine (benzathine, C 16 H 20 N 2 )
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Benzylpenicillin Potassium
Penicillin G Potassium
4P<
CH 3
CH :!
C 16 H 17 KN 2 4 S: 372.48
Monopotassium (2S',5/?,6/?)-3,3-dimethyl-7-oxo-6-
[(phenylacetyl)amino] -4-thia-
l-azabicyclo[3.2.0]heptane-
2-carboxylate [113-98-4]
Benzylpenicillin Potassium is the potassium salt of a
penicillin substance having antibacterial activity pro-
duced by the growth of Penicillium species.
It contains not less than 1430 units and not more
than 1630 units per mg, calculated on the dried basis.
The potency of Benzylpenicillin Potassium is expressed
as mass unit of benzylpenicillin potassium (C 16 H 17 KN 2
4 S). One unit of Benzylpenicillin Potassium is equiva-
lent to 0.57 Lig of benzylpenicillin potassium.
Description Benzylpenicillin Potassium occurs as white,
crystals or crystalline powder.
It is very soluble in water, and slightly soluble in ethanol
(99.5).
Identification (1) Determine the absorption spectrum of a
solution of Benzylpenicillin Potassium (1 in 1000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Benzylpenicillin Potassium Refer-
350
Benzylpenicillin Potassium / Official Monographs
JP XV
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(2) Determine the infrared absorption spectrum of Ben-
zylpenicillin Potassium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Benzylpenicillin Potassium Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) Benzylpenicillin Potassium responds to the Qualita-
tive Tests <1.09> (1) for potassium salt.
Optical rotation <2.49> [«]£>: +270- +300° (1.0 g calculated
on the dried basis, water, 50 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Benzylpenicillin Potassium in 100 mL of water is between
5.0 and 7.5.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 1 g of Benzylpenicillin Potassium in
10 mL of water is clear, and colorless or light yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Benzyl-
penicillin Potassium according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution by inciner-
ating 1 .0 g of Benzylpenicillin Potassium according to
Method 4, and perform the test. In the incineration, use a
crucible of porcelain, and after addition of 10 mL of a solu-
tion of magnesium nitrate hexahydrate in ethanol (95) (1 in
10) add 1 mL of hydrogen peroxide (30), then burn the
ethanol (not more than 2 ppm).
(4) Related substances — Dissolve 40 mg of Benzylpenicil-
lin Potassium in 20 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 fiL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
benzylpenicillin obtained from the sample solution is not
more than the peak area of benzylpenicillin from the stan-
dard solution, and the total area of the peaks other than ben-
zylpenicillin from the sample solution is not more than 3
times the peak area of benzylpenicillin from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of diammonium
hydrogen phosphate (33 in 5000) and acetonitrile (19:6),
adjusted the pH to 8.0 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
benzylpenicillin is about 7.5 minutes.
Time span of measurement: About 5 times as long as the
retention time of benzylpenicillin.
System suitability —
Test for required detection: Pipet 10 mL of the standard
solution, and add water to make exactly 100 mL. Confirm
that the peak area of benzylpenicillin obtained from 20 /xh of
this solution is equivalent to 7 to 13% of that from 20,mL of
the standard solution.
System performance: Dissolve 40 mg of Benzylpenicillin
Potassium in 20 mL of water. Separately, dissolve 10 mg of
methyl parahydroxybenzoate in 20 mL of acetonitrile. To
1 mL of this solution add water to make 20 mL. Mix 1 mL
each of these solutions, and add water to make 100 mL.
When the procedure is run with 20 fiL of this solution under
the above operating conditions, benzylpenicillin and methyl
parahydroxybenzoate are eluted in this order with the resolu-
tion between these peaks being not less than 8.
System repeatability: When the test is repeated 5 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
benzylpenicillin is not more than 2.0%.
Loss on drying <2.41> Not more than 1.0% (3 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium iii in 3) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount of
Benzylpenicillin Potassium Reference Standard, equivalent
to about 40,000 units, dissolve in phosphate buffer solution,
pH 6.0 to make exactly 100 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
not exceeding 5°C and use within 2 days. Take exactly a suit-
able amount of the standard stock solution before use, add
phosphate buffer solution, pH 6.0 to make solutions so that
each mL contains 2 units and 0.5 units, and use these solu-
tions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Benzylpenicillin Potassium, equivalent to about 40,000 units,
and dissolve in phosphate buffer solution, pH 6.0 to make
exactly 100 mL. Take exactly a suitable amount of this solu-
tion, add phosphate buffer solution, pH 6.0 to make
solutions so that each mL contains 2 units and 0.5 units, and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Berberine Chloride Hydrate 351
Berberine Chloride Hydrate
<ju^ u >ttfl:»*5|to*
CI- xHjO
C 20 H 18 ClNO 4 .xH 2 O
9,10-Dimethoxy-5,6-
dihydro[l,3]dioxolo[4,5-g]isoquino[3,2-a]isoquinolin-
7-ium chloride hydrate [633-65-8, anhydride]
Berberine Chloride Hydrate contains not less than
95.0% and not more than 102.0% of berberine chlo-
ride (C 2 oH 18 ClN0 4 : 371.81), calculated on the anhy-
drous basis.
Description Berberine Chloride Hydrate occurs as yellow
crystals or crystalline powder. It is odorless or has a faint,
characteristic odor. It has a very bitter taste.
It is sparingly soluble in methanol, slightly soluble in
ethanol (95), and very slightly soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Berberine Chloride Hydrate (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Berberine Chloride Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Ber-
berine Chloride Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Berberine Chloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Dissolve 0.1 g of Berberine Chloride Hydrate in 20
mL of water by warming, add 0.5 mL of nitric acid, cool, and
filter after allowing to stand for 10 minutes. To 3 mL of the
filtrate add 1 mL of silver nitrate TS, and collect the
produced precipitate: the precipitate does not dissolve in di-
lute nitric acid, but it dissolves in an excess amount of ammo-
nia TS.
Purity (1) Acidity — Shake thoroughly 0.10 g of Berberine
Chloride Hydrate with 30 mL of water, and filter. To the
filtrate add 2 drops of phenolphthalein TS and 0.10 mL of
0.1 mol/L sodium hydroxide VS: the yellow color changes to
an orange to red color.
(2) Sulfate <1.14>— Shake 1.0 g of Berberine Chloride
Hydrate with 48 mL of water and 2 mL of dilute hydrochlor-
ic acid for 1 minute, and filter. Discard the first 5 mL of the
filtrate, take the subsequent 25 mL of the filtrate, add water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution with 0.50 mL
of 0.005 mol/L sulfuric acid VS, 1 mL of dilute hydrochloric
acid, 5 to 10 drops of bromophenol blue TS and water to
make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Berber-
ine Chloride Hydrate according to Method 2, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 30ppm).
(4) Related substances — Dissolve 10 mg of Berberine
Chloride Hydrate in 100 mL of the mobile phase, and use this
solution as the sample solution. Pipet 4 mL of the sample so-
lution, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of both solutions by the automatic integration method:
the total of the peak of the areas other than berberine of the
sample solution is not larger than the peak area of berberine
of the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed as directed in the
operating conditions in the Assay.
Time span of measurement: About 2 times as long as the
retention time of berberine beginning after the solvent peak.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of berberine obtained from 10 /xL of the
standard solution is about 10% of the full scale.
Water <2.48> 8 - 12% (0.1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately 10 mg of Berberine Chloride Hy-
drate, dissolve in the mobile phase to make exactly 100 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of Berberine Chloride Refer-
ence Standard (separately, determine the water content <2.48>
in the same manner as Berberine Chloride Hydrate), and dis-
solve in the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the peak
areas, A T and A s of berberine in each solution.
Amount (mg) of berberine chloride (C 2 oH 18 ClN0 4 )
= W s x (A T /A S )
W s : Amount (mg) of Berberine Chloride Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 345 nm).
Column: A stainless steel column about 4 mm in inside
diameter and about 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /am in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 3.4 g of monobasic potassium
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
Selection of column: Dissolve each 1 mg of berberine
352 Berberine Tannate / Official Monographs
JP XV
chloride and palmatin chloride in the mobile phase to make
10 mL. Proceed with 10 ^L of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of palmatin and berberine in this order
with the resolution between these peaks being not less than
1.5.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak areas of berberine
is not more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Berberine Tannate
9>->^-Ol-<U >
Berberine Tannate is a compound of berberine and
tannic acid.
It contains not less than 27.0% and not more than
33.0%> of berberine (C 20 H 19 NO 5 : 353.37), calculated on
the anhydrous basis.
Description Berberine Tannate occurs as a yellow to light
yellow-brown powder. It is odorless or has a faint, charac-
teristic odor, and is tasteless.
It is practically insoluble in water, in acetonitrile, in
methanol and in ethanol (95).
Identification (1) To 0.1 g of Berberine Tannate add 10
mL of ethanol (95), and heat in a water bath for 3 minutes
with shaking. Cool, filter, and to 5 mL of the filtrate add 1
drop of iron (III) chloride TS: a blue-green color is produced,
and on allowing to stand, a bluish black precipitate is
formed.
(2) Dissolve 0.01 g of Berberine Tannate in 10 mL of
methanol and 0.4 mL of 1 mol/L hydrochloric acid TS, and
add water to make 200 mL. To 8 mL of the solution add
water to make 25 mL. Determine the absorption spectrum of
the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Ber-
berine Tannate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Berberine Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Purity (1) Acidity — To 0.10 g of Berberine Tannate add
30 mL of water, and filter after shaking well. To the filtrate
add 2 drops of phenolphthalein TS and 0.10 mL of 0.1 mol/L
sodium hydroxide VS: the color of the solution changes from
yellow to orange to red.
(2) Chloride <1.03>— Shake 1.0 g of Berberine Tannate
with 38 mL of water and 12 mL of dilute nitric acid for 5
minutes, and filter. Discard the first 5 mL of the filtrate, to 25
mL of the subsequent filtrate add water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 0.50 mL of 0.01 mol/L
hydrochloric acid VS by adding 6 mL of dilute nitric acid, 10
to 15 drops of bromophenol blue TS and water to make 50
mL (not more than 0.035%).
(3) Sulfate <1.14>— Shake 1.0 g of Berberine Tannate
with 48 mL of water and 2 mL of dilute hydrochloric acid for
1 minute, and filter. Discard the first 5 mL of the filtrate, take
the subsequent 25 mL of the filtrate, add water to make 50
mL, and perform the test using this solution as the test
solution. Prepare the control solution with 0.50 mL of 0.005
mol/L sulfuric acid VS, 1 mL of dilute hydrochloric acid, 5
to 10 drops of bromophenol blue TS and water to make 50
mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Berber-
ine Tannate according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
(5) Related substances — Dissolve 10 mg of Berberine
Tannate in 100 mL of the mobile phase, and use this solution
as the sample solution. Pipet 4 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine each peak area of
both solutions by the automatic integration method: the total
of the peak areas other than berberine of the sample solution
is not larger than the peak area of berberine of the standard
solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of berberine beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of berberine obtained with 10 /uL
of this solution is equivalent to 7 to 13% of that with 10 /xL of
the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
berberine is not more than 3.0%.
Water <2.48> Not more than 6.0% (0.7 g, direct titration).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Weigh accurately about 30 mg of Berberine Tannate,
dissolve in the mobile phase to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh
accurately about 10 mg of Berberine Chloride Reference
Standard (separately, determine the water content <2.48> in
the same manner as Berberine Chloride Hydrate), dissolve in
the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine the peak areas, A T
and A s , of berberine in each solution.
Amount (mg) of berberine (C 2 oH 19 N0 5 )
= W s x (A T /A S ) x 0.9504
JPXV
Official Monographs / Betahistine Mesilate
353
W s : Amount (mg) of Berberine Chloride Reference Stan-
dard, calculated on the dehydrated basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 345 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
System suitability —
System performance: Dissolve 1 mg each of berberine chlo-
ride and palmatin chloride in the mobile phase to make 10
mL. When the procedure is run with 10 /xL of this solution
under the above operating conditions, palmatin and berber-
ine are eluted in this order with the resolution between these
peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
berberine is not more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Betahistine Mesilate
CHs »2 HaC-SQjH
C 8 H 12 N 2 .2CH 4 3 S: 328.41
A r -Methyl-2-pyridin-2-ylethylamine
dimethanesulfonate [5638-76-6, Betahistine]
Betahistine Mesilate, when dried, contains not less
than 98.0% and not more than 101.0% of
C 8 H 12 N 2 .2CH 4 3 S.
Description Betahistine Mesilate occurs as white crystals or
crystalline powder.
It is very soluble in water, freely soluble in acetic acid
(100), and sparingly soluble in ethanol (99.5).
It dissolves in dilute hydrochloric acid.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Betahistine Mesilate in 0.1 mol/L hydrochloric
acid (1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Beta-
histine Mesilate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) To 30 mg of Betahistine Mesilate add 0. 1 g of sodium
nitrate and 0.1 g of anhydrous sodium carbonate, mix well,
and heat gradually. After cooling, dissolve the residue in 2
mL of dilute hydrochloric acid and 10 mL of water, filter if
necessary, and to the filtrate add 1 mL of barium chloride TS:
a white precipitate is formed.
Melting point <2.60> 110 - 114°C (after drying).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Betahistine Mesilate according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 50 mg of Betahistine
Mesilate in 10 mL of a mixture of water and acetonitrile
(63:37), and use this solution as the sample solution. Pipet 1
mL of the sample solution, add the mixture of water and
acetonitrile (63:37) to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with exactly
20 [iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of the peak other
than betahistine with the sample solution is not larger than
1/10 times the peak area of betahistine with the standard so-
lution, and the total area of the peaks other than the peak of
betahistine with the sample solution is not larger than 1/2
times the peak area of betahistine with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 261 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 5 mL of diethyl amine and 20 mL of
acetic acid (100) add water to make 1000 mL. Dissolve 2.3 g
of sodium lauryl sulfate in 630 mL of this solution, and add
370 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
betahistine is about 5 minutes.
Time span of measurement: About 3 times as long as the
retention time of betahistine beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add the mixture of water and acetonitrile
(63:37) to make exactly 50 mL. Confirm that the peak area of
betahistine obtained with 20 /xL of this solution is equivalent
to 7 to 13% of that with 20 /xL of the standard solution.
System performance: Dissolve 10 mg of betahistine mesi-
late and 10 mg of 2-vinylpyridine in 50 mL of the mixture of
water and acetonitrile (63:37). To 2 mL of this solution add
the mixture of water and acetonitrile (63:37) to make 50 mL.
When the procedure is run with 20 /iL of this solution under
the above operating conditions, 2-vinylpyridine and beta-
histine are eluted in this order with the resolution between
these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
354 Betahistine Mesilate Tablets / Official Monographs
JP XV
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
betahistine is not more than 1.0%
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
phosphorus (V) oxide, 70°C, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Betahistine Mesi-
late, previously dried, dissolve in 1 mL of acetic acid (100),
add 50 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.42 mg of C 8 H, 2 N 2 .2CH 4 03S
Containers and storage Containers — Tight containers.
Betahistine Mesilate Tablets
Betahistine Mesilate Tablets contain not less than
93.0% and not more than 107.0% of the labeled
amount of betahistine mesilate (C 8 H 12 N2.2CH40 3 S:
328.41).
Method of preparation Prepare as directed under Tablets,
with Betahistine Mesilate.
Identification To 5 mL of the sample solution obtained in
the Assay add 0.1 mol/L hydrochloric acid TS to make 100
mL, and determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 259 nm and 263 nm.
Purity Related substances — Powder not less than 20 Beta-
histine Mesilate Tablets. To a portion of the powder, equiva-
lent to about 50 mg of Betahistine Mesilate, add 10 mL of a
mixture of water and acetonitrile (63:37), agitate for 10
minutes with the aid of ultrasonic waves, centrifuge, and use
the supernatant liquid as the sample solution. Pipet 1 mL of
this solution, add the mixture of water and acetonitrile
(63:37) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the area of the peak, having the rela-
tive retention time of about 1.9 with respect to betahistine, is
not more than 3/5 times the peak area of betahistine obtained
from the standard solution, and the total area of the peaks
other than betahistine is not more than the peak area of beta-
histine from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the Assay.
Time span of measurement: About 8 times as long as the
retention time of betahistine beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add the mixture of water and acetonitrile
(63:37) to make exactly 50 mL. Confirm that the peak area of
betahistine obtained with 20 /uL of this solution is equivalent
to 7 to 13% of that with 20 /uL of the standard solution.
System performance: Dissolve 10 mg of betahistine mesi-
late and 10 mg of 2-vinylpyridine in 50 mL of the mixture of
water and acetonitrile (63:37). To 2 mL of this solution add
the mixture of water and acetonitrile (63:37) to make 50 mL.
When the procedure is run with 20 fiL of this solution under
the above operating conditions, 2-vinylpyridine and beta-
histine are eluted in this order with the resolution between
these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
betahistine is not more than 1.0%.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Betahistine Mesilate Tablets add exactly V
mL of 0.1 mol/L hydrochloric acid TS so that each mL
contains about 0.4 mg of betahistine mesilate (C 8 H 12 N 2 .2CH 4
3 S), agitate for about 10 minutes with the aid of ultrasonic
waves to disintegrate the tablet, then centrifuge, and use the
supernatant liquid as the sample solution. Proceed as direct-
ed in the Assay
Amount (mg) of betahistine mesilate (C 8 H 12 N2.2CH 4 03S)
= W s x (A T /A S ) x (F/250)
W s : Amount (mg) of betahistine mesilate for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Betahistine Mesilate
Tablets at 50 revolutions per minute according to the Paddle
method using 900 mL of water as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 15
minutes after starting the test, and filter through a membrane
filter with a pore size not exceeding 0.45 fim. Discard the first
10 mL of the filtrate, pipet KmL of the subsequent filtrate,
add water to make exactly V mL so that each mL contains
about 6.7 /xg of betahistine mesilate (C 8 H 12 N2.2CH 4 03S) ac-
cording to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 17 mg of
betahistine mesilate for assay, previously dried under reduced
pressure with phosphorous (V) oxide at 70°C for 24 hours,
and dissolve in water to make exactly 100 mL. Pipet 4 mL of
this solution, add water to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the peak areas, A T
and A s , of betahistine. The dissolution rate in 15 minutes is
not less than 85%.
Dissolution rate (%) with respect to the labeled amount of
betahistine mesilate (C 8 H 12 N 2 .2CH 4 3 S)
= W s x (A T /A S ) x (V'/V) x (l/Q x 36
W s : Amount (mg) of betahistine mesilate for assay
C: Labeled amount (mg) of betahistine mesilate
(C 8 H 12 N 2 .2CH 4 3 S) in 1 tablet
Operating conditions —
Proceed as directed in the Assay.
System suitability —
JPXV
Official Monographs / Betamethasone 355
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of betahistine are not less than 2000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
betahistine is not more than 2.0%.
Assay Weigh accurately the mass of not less than 20 Beta-
histine Mesilate Tablets, and powder. Weigh accurately a
portion of the powder, equivalent to about 20 mg of beta-
histine mesilate (C 8 H 12 N 2 .2CH 4 3 S), add 40 mL of 0.1
mol/L hydrochloric acid TS, agitate for 10 minutes with the
aid of ultrasonic waves, and add 0.1 mol/L hydrochloric acid
TS to make exactly 50 mL. Centrifuge, and use the super-
natant liquid as the sample solution. Separately, weigh ac-
curately about 0.1 g of betahistine mesilate for assay, previ-
ously dried under reduced pressure with phosphorous (V)
oxide at 70°C for 24 hours, and dissolve in 0.1 mol/L
hydrochloric acid TS to make exactly 50 mL. Pipet 10 mL of
this solution, add 0.1 mol/L hydrochloric acid TS to make
exactly 50 mL, and use this solution as the standard solution.
Perform the test with exactly 5 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01>, according to the following conditions,
and determine the peak areas, A T and A s , of betahistine.
Amount (mg) of betahistine mesilate (C8LL2N2.2CH4O3S)
= W s x (A T /A S ) x (1/5)
W s : Amount (mg) of betahistine mesilate for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 261 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 5 mL of diethylamine and 20 mL of acet-
ic acid (100) add water to make 1000 mL. In 630 mL of this
solution dissolve 2.3 g of sodium lauryl sulfate, and add 370
mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
betahistine is about 5 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of betahistine are not less than 2000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of be-
tahistine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Betamethasone
^.9 / 9*/>
9-Fluoro- 1 1/3,17,21 -trihy droxy- 1 6/?-methylpregna-
l,4-diene-3,20-dione [378-44-9]
Betamethasone, when dried, contains not less than
96.0% and not more than 103.0%, of C 22 H 29 F0 5 .
Description Betamethasone occurs as a white to pale yel-
lowish white, crystalline powder.
It is sparingly soluble in methanol, in ethanol (95) and in
acetone, and practically insoluble in water.
Melting point: about 240°C (with decomposition).
Identification (1) Proceed 10 mg of Betamethasone as
directed under Oxygen Flask Combustion Method <1.06>, us-
ing a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS
and 20 mL of water as an absorbing liquid, and prepare the
test solution: the test solution so obtained responds to the
Qualitative Tests <1.09> for fluoride.
(2) Dissolve 1.0 mg of Betamethasone in 10 mL of
ethanol (95). Mix 2.0 mL of the solution with 10 mL of
phenylhydrazinium hydrochloride TS, heat in a water bath at
60°C for 20 minutes, and cool the solution. Determine the
absorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using as the blank the
solution prepared with 2.0 mL of ethanol (95) in the same
manner as the former solution, and compare the spectrum
with the Reference Spectrum or the spectrum of a solution of
Betamethasone Reference Standard prepared in the same
manner as the sample solution: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Be-
tamethasone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Betamethasone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve Betamethasone and Be-
tamethasone Reference Standard in acetone, respectively,
then evaporate the acetone to dryness, and repeat the test on
the residues.
Optical rotation <2.49> [«]£>: +118
0.1 g, methanol, 20 mL, 100 mm).
126° (after drying,
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Betamethasone according to Method 2, and perform the test.
Prepare the control solution with 1.5 mL of Standard Lead
Solution (not more than 30 ppm).
(2) Related substances — Dissolve 10 mg of Betametha-
sone in 5 mL of a mixture of chloroform and methanol (9:1),
356 Betamethasone Tablets / Official Monographs
JP XV
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add a mixture of chloroform and
methanol (9:1) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
dichloromethane, diethyl ether, methanol and water
(385:75:40:6) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.5% (0.1 g,
platinum crucible).
Assay Dissolve about 20 mg each of Betamethasone and
Betamethasone Reference Standard, previously dried and ac-
curately weighed, in methanol to make exactly 50 mL. Pipet 5
mL each of these solutions, add exactly 5 mL each of the in-
ternal standard solution, then add methanol to make 50 mL,
and use these solutions as the sample solution and standard
solution, respectively. Perform the test with lO^L each of
these solutions as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and g s , of the peak area of betamethasone to
that of the internal standard, respectively.
Amount (mg) of C22H29FO5
= Ws x (St/Qs)
W s : Amount (mg) of Betamethasone Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (2 in 3500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column about 4.0 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone is about 4 minutes.
System suitability —
System performance: When proceed the test with 10 jxL of
the standard solution under the above operating conditions,
betamethasone and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of betamethasone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Betamethasone Tablets
Betamethasone Tablets contain not less than 90.0%
and not more than 107.0% of the labeled amount of
betamethasone (C22H29FO5: 392.46).
Method of preparation Prepare as directed under Tablets,
with Betamethasone.
Identification Pulverize Betamethasone Tablets. To a por-
tion of the powder, equivalent to 2 mg of Betamethasone ac-
ceding to the labeled amount, add 20 mL of methanol, shake
for 5 minutes, and filter. Evaporate the filtrate on a water
bath to dryness, dissolve the residue after cooling in 2 mL of
methanol, filter if necessary, and use this as the sample solu-
tion. Separately, dissolve 2 mg of Betamethasone Reference
Standard in 2 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography, develop with a mixture of 1-butanol, water and
acetic anhydride (3:1:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the principal spot obtained with the
sample solution and the spot with the standard solution show
the same Rf value.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Betamethasone Tablets add KmL of water
so that each mL contains about 50 /xg of betamethasone (C22
H 29 F0 5 ) according to the labeled amount, add exactly an
amount of the internal standard solution equivalent to 2 mL
per 50 /xg of betamethasone, shake vigorously for 10 minutes,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 20 mg of Be-
tamethasone Reference Standard, previously dried in a desic-
cator (in vacuum, phosphorus (V) oxide) for 4 hours, and dis-
solve in acetonitrile to make exactly 200 mL. Pipet 5 mL of
this solution, add exactly 20 mL of the internal standard so-
lution, and use this solution as the standard solution. Per-
form the test with 50 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, g T and Q s , of the peak area of betamethasone to
that of the internal standard.
Amount (mg) of betamethasone (C22H29FO5)
= W s x (Q T /Q S ) x (K/400)
W s : Amount (mg) of Betamethasone Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 40,000)
Operating conditions —
Proceed as directed in the Assay
System suitability —
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
JPXV
Official Monographs / Betamethasone Tablets 357
tions, betamethasone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of betamethasone to that of the internal standard is
not more than 1.0%.
Dissolution <6.10> Perform the test according the following
method: it meets the requirement.
Perform the test with 1 tablet of Betamethasone Tablets at
50 revolutions per minute according to the Paddle method us-
ing 900 mL of water as the dissolution medium. Withdraw 20
mL or more of the dissolution medium 30 minutes after start-
ing the test, and filter through a membrane filter with a pore
size not exceeding 0.45 //m. Discard the first 10 mL of the
filtrate, pipet the subsequent FmL of the filtrate, add water
to make exactly V mL so that each mL contains about 0.56
H% of betamethasone (C22H29FO5) according to the labeled
amount, and use this solution as the sample solution.
Separately, weigh accurately about 28 mg of Betamethasone
Reference Standard, previously dried in a desiccator (in vacu-
um, phosphorus (V) oxide) for 4 hours, dissolve in methanol
to make exactly 100 mL. Pipet 5 mL of this solution, and add
water to make exactly 100 mL. Pipet 4 mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 100 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of betamethasone. The dissolution rate in 30 minutes is not
less than 85%.
Dissolution rate (%) with respect to the labeled amount of
betamethasone (C22H29FO5)
= W s x (Aj/As) x (V'/V) x (l/Q x (9/5)
W s : Amount (mg) of Betamethasone Reference Standard
C: Labeled amount (mg) of betamethasone (C22H29FO5) in
1 tablet
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 241 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (3:2).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone is about 7 minutes.
System suitability —
System performance: When the procedure is run with 100
[iL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of betamethasone are not less than 3000 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
100 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
betamethasone is not more than 2.0%.
tamethasone Tablets, and powder. Weigh accurately a por-
tion of the powder, equivalent to about 5 mg of betametha-
sone (C22H29FO5), add 25 mL of water, then add exactly 50
mL of the internal standard solution, and shake vigorously
for 10 minutes. Filter through a membrane filter with pore
size of not more than 0.5 pm, discard the first 5 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 20 mg of Betamethasone
Reference Standard, previously dried in a desiccator (in vacu-
um, phosphorus (V) oxide) for 4 hours, and dissolve in
acetonitrile to make exactly 50 mL. Pipet 5 mL of this solu-
tion, add exactly 20 mL of the internal standard solution and
5 mL of water, and use this solution as the standard solution.
Perform the test with 20 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Qj and Q s , of the peak area of betamethasone to
that of the internal standard.
Amount (mg) of betamethasone (C22H29FO5)
= W s x (Q T /Q S ) x (1/4)
W s : Amount (mg) of Betamethasone Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone is about 4 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, betamethasone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of betamethasone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Assay Weigh accurately the mass of not less than 20 Be-
358
Betamethasone Dipropionate / Official Monographs
JP XV
Betamethasone Dipropionate
A^c Hj
C 28 H 37 F0 7 : 504.59
9-Fluoro- 1 1/6, 1 7 ,2 1 -trihydroxy- 1 6/?-methylpregna- 1,4-
diene-3,20-dione 17,21-dipropanoate [5593-20-4]
Betamethasone Dipropionate, when dried, contains
not less than 97.0% and not more than 103.0% of
C28H37FO7, and not less than 3.4% and not more than
4.1% of fluorine (F:19.00).
Description Betamethasone Dipropionate occurs as a white
to pale yellowish white, crystalline powder. It is odorless.
It is freely soluble in acetone, in 1,4-dioxane and in chlo-
roform, soluble in methanol, sparingly soluble in ethanol
(95), slightly soluble in diethyl ether, and practically insoluble
in water and in hexane.
It is affected gradually by light.
Identification (1) To 1 mL of a solution of Betamethasone
Dipropionate in methanol (1 in 10,000) add 4 mL of isoniazid
TS, and heat on a water bath for 2 minutes: a yellow color
develops.
(2) Proceed with 0.01 g of Betamethasone Dipropionate
as directed under Oxygen Flask Combustion Method <1.06>,
using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide
TS and 20 mL of water as the absorbing liquid: the test solu-
tion so obtained responds to the Qualitative Tests <1.09> for
fluoride.
(3) Determine the absorption spectrum of a solution of
Betamethasone Dipropionate in methanol (3 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of
Betamethasone Dipropionate, previously dried, as directed in
the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
Melting point <2.60> 176 - 180°C
Optical rotation <2.49> [a]™'- +63 - +70° (after drying, 50
mg, 1,4-dioxane, 10 mL, 100 mm).
Purity (1) Fluoride — To 0.10 g of Betamethasone
Dipropionate add 10.0 mL of diluted 0.01 mol/L sodium
hydroxide TS (1 in 20), shake for 10 minutes, and filter
through a membrane filter (0.4-^m pore size). Place 5.0 mL
of the filtrate in a 20-mL volumetric flask, and add 10 mL of
a mixture of alizalin complexone TS, acetic acid-potassium
acetate buffer solution, pH 4.3, and cerium (III) nitrate TS
(1:1:1), add water to make 20 mL, allow to stand for 1 hour,
and use this solution as the sample solution. Separately, place
1.0 mL of Standard Fluorine Solution in a 20-mL volumetric
flask, add 5.0 mL of diluted 0.01 mol/L sodium hydroxide
TS (1 in 20), then 10 mL of a mixture of alizalin complexone
TS, acetic acid-potassium acetate buffer solution, pH 4.3,
and cerium (III) nitrate TS (1:1:1), proceed in the same
manner as the preparation of the sample solution, and use
this solution as the standard solution. Place 5.0 mL of
diluted 0.01 mol/L sodium hydroxide TS (1 in 20) in a 20-mL
volumetric flask, and proceed in the same manner as the
preparation of the sample solution. Using this solution as the
blank, determine the absorbances of the sample solution and
standard solution at 600 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>: the absorbance of the sam-
ple solution is not greater than that of the standard solution
(not more than 0.012%).
(2) Heavy metals <1.07>— Proceed with 1.0 g of Be-
tamethasone Dipropionate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.010 g of Betamethasone Dipropionate in 10 mL of chlo-
roform, and use this solution as the sample solution. Pipet 3
mL of the sample solution, add chloroform to make exactly
100 mL, and use this solution as the standard solution.
Perform the test as directed under Thin-layer Chro-
matography <2.03> with these solutions. Spot 20 /xL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and acetone
(7:1) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g,
platinum crucible).
Assay (1) Betamethasone dipropionate — Weigh accurate-
ly about 15 mg of Betamethasone Dipropionate, previously
dried, and dissolve in methanol to make exactly 100 mL.
Pipet 5 mL of this solution, and dilute with methanol to
exactly 50 mL. Determine the absorbance A of this solution
at the wavelength of maximum absorption at about 239 nm
as directed under Ultraviolet-visible Spectrophotometry
<2.24>.
Amount (mg) of C 2 8H 37 F07
= (.4/312) x 10,000
(2) Fluorine — Weigh accurately about 10 mg of Be-
tamethasone Dipropionate, previously dried, and proceed as
directed in the procedure of determination for fluorine under
Oxygen Flask Combustion Method <1.06>, using a mixture of
0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of
water as the absorbing liquid.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Betamethasone Sodium Phosphate
359
Storage — Light-resistant.
Betamethasone Sodium Phosphate
^<9 / 9 >/> V >m±*T )li- h U^A
.PO^fl;.
C 22 H 28 FNa 2 8 P: 516.40
Disodium 9-fluoro-ll/?, 17,21 -trihydroxy-1 6/?-m ethylpregna-
l,4-diene-3,20-dione 21-phosphate [151-73-5]
Betamethasone Sodium Phosphate contains not less
than 97.0% and not more than 103.0% of
C 2 2H28FNa 2 O g P, calculated on the anhydrous basis.
Description Betamethasone Sodium Phosphate occurs as
white to pale yellowish white, crystalline powder or masses. It
is odorless.
It is freely soluble in water, sparingly soluble in methanol,
slightly soluble in ethanol (95), and practically insoluble in
diethyl ether.
It is hygroscopic.
Melting point: about 213°C (with decomposition).
Identification (1) Dissolve 2 mg of Betamethasone Sodi-
um Phosphate in 2 mL of sulfuric acid: a brown color de-
velops, and gradually changes to blackish brown.
(2) Prepare the test solution with 0.01 g of Betametha-
sone Sodium Phosphate as directed under Oxygen Flask
Combustion Method <1.06>, using a mixture of 0.5 mL of
0.01 mol/L sodium hydroxide TS and 20 mL of water as an
absorbing liquid: the test solution responds to the Qualitative
Tests <1.09> (2) for fluoride.
(3) Take 0.04 g of Betamethasone Sodium Phosphate in a
platinum crucible, and carbonize by heating. After cooling,
add 5 drops of nitric acid, and incinerate by heating. To the
residue add 10 mL of diluted nitric acid (1 in 50), and boil for
several minutes. After cooling, neutralize the solution with
ammonia TS, filter if necessary, and use this solution as the
sample solution. The sample solution responds to the
Qualitative Tests <1.09> for sodium salt and for phosphate.
(4) Determine the infrared absorption spectrum of Be-
tamethasone Sodium Phosphate, previously dried, as direct-
ed in the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum or the spectrum of previously dried
Betamethasone Sodium Phosphate Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Optical rotation <2.49> [a]™: +99 - + 105° (0.1 g, calculat-
ed on the anhydrous basis, water, 10 mL, 100 mm).
pH <2.54> Dissolve 0.10 g of Betamethasone Sodium Phos-
phate in 20 mL of water: the pH of this solution is between
7.5 and 9.0.
Purity (1) Clarity and color of solution — Dissolve 0.25 g
of Betamethasone Sodium Phosphate in 10 mL of water: the
solution is clear and colorless.
(2) Free phosphoric acid — Weigh accurately about 20 mg
of Betamethasone Sodium Phosphate, dissolve in 20 mL of
water, and use this solution as the sample solution. Separate-
ly, pipet 4 mL of Standard Phosphoric Acid Solution, add 20
mL of water, and use this solution as the standard solution.
To each of the sample solution and the standard solution add
exactly 7 mL of dilute sulfuric acid, exactly 2 mL of hexaam-
monium heptamolybdate-sulfuric acid TS and exactly 2 mL
of p-methylaminophenol sulfate TS, shake well, and allow to
stand at 20 ± 1°C for 15 minutes. To each add water to
make exactly 50 mL, and allow to stand at 20 ± 1°C for 15
minutes. Perform the test with these solutions as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using a
solution prepared with 20 mL of water in the same manner as
the blank. Determine the absorbances, A T and A s , of each so-
lution from the sample solution and standard solution at 730
nm: the amount of free phosphoric acid is not more than 0.5
%.
Amount (%) of free phosphoric acid (H 3 P0 4 )
= (A T /A S ) x (\/W) x 10.32
W: Amount (mg) of Betamethasone Sodium Phosphate,
calculated on the anhydrous basis.
(3) Betamethasone — Dissolve 20 mg of Betamethasone
Sodium Phosphate in exactly 2 mL of methanol, and use this
solution as the sample solution. Separately, dissolve 20 mg of
Betamethasone Reference Standard in exactly 10 mL of
methanol. Pipet 1 mL of this solution, add methanol to make
exactly 20 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a freshly prepared mixture of 1-butanol, water
and acetic anhydride (3 : 1 : 1) to a distance of about 1 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spot from the sample solution cor-
responding to the spot from the standard solution is not more
intense than the spot from the standard solution.
Water <2.48> Not more than 10.0% (0.2 g, back titration).
Assay Weigh accurately about 20 mg each of Betametha-
sone Sodium Phosphate and Betamethasone Sodium Phos-
phate Reference Standard (determine its water content <2.48>
before using in the same manner as Betamethasone Sodium
Phosphate), and dissolve each in methanol to make exactly
20 mL. Pipet 5 mL each of these solutions, and exactly 5 mL
of the internal standard solution, then add methanol to make
50 mL, and use these solutions as the sample solution and
standard solution, respectively. Perform the test with 10 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the
following conditions, and calculate the ratios, Q T and Q s , of
the peak area of betamethasone phosphate to that of the
internal standard, respectively.
Amount (mg) of C 22 H 28 FNa 2 8 P = W s x (g T /Q s )
W s : Amount (mg) of Betamethasone Sodium Phosphate
Reference Standard, calculated on the anhydrous
basis
Internal standard solution — A solution of butyl parahydrox-
360
Betamethasone Valerate / Official Monographs
JP XV
ybenzoate in methanol (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.6 g of tetra-M-butylammonium
bromide, 3.2 g of disodium hydrogen phosphate dodecahy-
drate and 6.9 g of potassium dihydrogen phosphate in 1000
mL of water, and add 1500 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
betamethasone phosphate is about 5 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, betamethasone phosphate and the internal stan-
dard are eluted in this order with the resolution between these
peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of betamethasone phosphate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Betamethasone Valerate
■<9 / 9 -/^lUiXxJU
C 27 H 37 F0 6 : 476.58
9-Fluoro- 1 1/3, 1 7 ,2 1 -trihydroxy- 1 6/?-methylpregna- 1 ,4-
diene-3,20-dione 17-pentanoate [2152-44-5]
Betamethasone Valerate, when dried, contains not
less than 97.0% and not more than 103.0% of
C 2 7H 37 F0 6 .
Description Betamethasone Valerate occurs as a white,
crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in ethanol (95),
sparingly soluble in methanol, slightly soluble in diethyl
ether, and practically insoluble in water.
Melting point: about 190°C (with decomposition).
Identification (1) Proceed with 0.01 g of Betamethasone
Valerate as directed under Oxygen Flask Combustion
Method <1.06>, using a mixture of 0.5 mL of 0.01 mol/L so-
dium hydroxide TS and 20 mL of water as the absorbing liq-
uid, and prepare the test solution: the test solution so ob-
tained responds to the Qualitative Tests <1.09> for fluoride.
(2) Determine the infrared absorption spectrum of
Betamethasone Valerate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of dried Betamethasone
Valerate Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Optical rotation <2.49> [ a ]™\ + 11 - +83° (after drying,
0.1 g, methanol, 20 mL, 100 mm).
Purity Related substances — Conduct this procedure
without exposure to daylight. Dissolve 0.02 g of Betametha-
sone Valerate in 5 mL of a mixture of chloroform and
methanol (9:1), and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add a mixture of chlo-
roform and methanol (9:1) to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chloroform
and methanol (9:1) to a distance of about 12 cm, and air-dry
the plate. Spray evenly alkaline blue tetrazolium TS on the
plate: the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g,
platinum crucible).
Assay Dissolve about 10 mg each of Betamethasone Valer-
ate and Betamethasone Valerate Reference Standard, previ-
ously dried and accurately weighed, in methanol to make
exactly 100 mL. Pipet 10 mL each of these solutions, add
10 mL each of the internal standard solution, and use these
solutions as the sample solution and standard solution, re-
spectively. Perform the test with 10 /xL each of the sample so-
lution and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, Q T and g s , of the peak area of be-
tamethasone valerate to that of the internal standard, respec-
tively.
Amount (mg) of C 27 H 37 F0 6 = W s x (Q T /Q S )
W s : Amount (mg) of Betamethasone Valerate Reference
Standard
Internal standard solution — A solution of isoamyl benzoate
in methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (7:3).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone valerate is about 10 minutes.
System suitability —
System performance: When the procedure is run with
JP XV
Official Monographs / Betamethasone Valerate and Gentamicin Sulfate Cream
361
10 fih of the standard solution under the above operating
conditions, betamethasone valerate and the internal standard
are eluted in this order with the resolution between these
peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of betamethasone valerate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Betamethasone Valerate and
Gentamicin Sulfate Cream
U-A
Betamethasone Valerate and Gentamicin Sulfate
Cream contains not less than 90.0% and not more than
110.0% of the labeled amount of betamethasone valer-
ate (C 27 H 37 F0 6 : 476.58) and not less than 90.0% and
not more than 115.0% of the labeled amount of gen-
tamicin C 1 (C 21 H4 3 N 5 7 : 477.60).
Method of preparation Prepare as directed under Cream,
with Betamethasone Valerate and Gentamicin Sulfate.
Identification (1) To a quantity of Betamethasone Valer-
ate and Gentamicin Sulfate Cream, equivalent to about 1.2
mg of Betamethasone Valerate according to the labeled
amount, add 20 mL of methanol and 20 mL of hexane, shake
vigorously for 10 minutes, and allow to stand. Take 15 mL of
the lower layer, evaporate the layer to dryness on a water
bath under a current of nitrogen. To the residue add 1 mL of
ethyl acetate, mix, and use as the sample solution. Separate-
ly, dissolve about 18 mg of Betamethasone Valerte Reference
Standard in 20 mL of ethyl acetate, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography, develop the
plate with ethyl acetate to a distance of about 10 cm, and air-
dry the plate. Spray evenly alkaline blue tetrazolium TS on
the plate, and heat at 100°C: the principal spot with the sam-
ple solution and the spot with the standard solution are pur-
ple in color, and their Rf values are the same.
(2) To a quantity of Betamethasone Valerate and Gen-
tamicin Sulfate Cream, equivalent to about 2 mg (potency) of
Gentamicin Sulfate according to the labeled amount, add 20
mL of ethyl acetate and 10 mL of water, shake vigorously for
10 minutes, and centrifuge. To 3 mL of the lower layer add 1
mL of dilute sodium hydroxide TS and 2 mL of ninhydrin
TS, and heat in a water bath at 90 - 95°C for 10 minutes: a
purple to dark purple color develops.
pH <2.54> To a quantity of Betamethasone Valerate and
Gentamicin Sulfate Cream, equivalent to 6 mg of Betametha-
sone Valerate according to the labeled amount, add 15 mL of
water, and mix while warming on a water bath to make a
milky liquid: the pH of the cooled liquid is between 4.0 and
6.0.
Purity Related substances — Weigh accurately an amount of
Betamethasone Valerate and Gentamicin Sulfate Cream,
equivalent to about 1 mg of Betamethasone Valerate accord-
ing to the labeled amount, and add 10 mL of a mixture of
methanol and water (7:3). Warm in a water bath at 60°C for
5 minutes, and shake vigorously for 20 minutes. Repeat this
procedure 2 times. After cooling for 15 minutes with ice, cen-
trifuge for 5 minutes, take away the bubbles from the upper
surface, and filter the remaining liquid. Discard first 2 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. Perform the test with 150 /xL of the sample solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, determine each peak area by the au-
tomatic integration method, and calculate these amounts by
the area percentage method: the amount of the substance
other than betamethasone valerate is not more than 3.5%,
and the total amount of them is not more than 7.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
45°C.
Mobile phase: A mixture of water, acetonitrile and
methanol (12:7:1)
Flow rate: Adjust the flow rate so that the retention time of
betamethasone valerate is about 16 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of betamethasone valerate beginning after the
solvent peak. The peaks of the compounding ingredients are
not determined.
System suitability —
Test for required detectability: Dissolve 20 mg of Be-
tamethasone Valerate in 100 mL of a mixture of methanol
and water (7:3). To exactly 1 mL of this solution add the mix-
ture of methanol and water (7:3) to make exactly 100 mL,
and use this solution as the solution for system suitability
test. To exactly 2.5 mL of the solution add the mixture of
methanol and water (7:3) to make exactly 50 mL. Confirm
that the peak area of betamethasone valerate obtained with
150 /xL of this solution is equivalent to 3.5 to 6.5% of that
with 150 /xL of the solution for system suitability test.
System performance: When the procedure is run with 150
/xL of the solution for system suitability test under the above
operating conditions, the number of theoretical plates and
the symmetry factor of the peak of betamethasone valerate
are not less than 4000 and 0.8 to 1.3, respectively.
System repeatability: When the test is repeated 6 times with
150 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of betamethasone valerate is not more than
2.0%.
Assay (1) Betamethasone valerate — Weigh accurately an
amount of Betamethasone Valerate and Gentamicin Sulfate
Cream, equivqlent to about 1 mg of betamethasone valerate
(C 27 H 37 F0 6 ), add 10 mL of a mixture of methanol and water
(7:3), and add exactly 10 mL of the internal standard solu-
tion. After warming in a water bath at 60°C for 5 minutes,
shake vigorously for 20 minutes. Repeat this procedure twice,
362
Betamethasone Valerate and Gentamicin Sulfate Ointment / Official Monographs
JP XV
cool with ice for 15 minutes, centrifuge for 5 minutes, then
filter the supernatant liquid, discard the first 5 mL of filtrate,
and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 25 mg of Betamethasone
Valerate Reference Standard, previously dried at 105 °C for 3
hours, and dissolve in methanol to make exactly 25 mL.
Pipet 5 mL of this solution, and add the mixture of methanol
and water (7:3) to make exactly 50 mL. Pipet 10 mL of this
solution, add exactly 10 mL of the internal standard solution,
mix, and use this solution as the standard solution. Perform
the test with 3 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Q T and Q s , of the peak area of betamethasone valerate to
that of the internal standard.
Amount (mg) of betamethasone valerate (C 2 7H 37 F0 6 )
= W s x (Q T /Q S ) x (1/25)
W s : Amount (mg) of Betamethasone Valerate Reference
Standard
Internal standard solution — Dissolve 20 mg of beclometa-
sone dipropionate in 10 mL of methanol, and add the mix-
ture of methanol and water (7:3) to make 200 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 2.1 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3.5 ^m in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (13:7).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone valerate is about 16 minutes.
System suitability —
System performance: When the procedure is run with 3 fiL
of the standard solution under the above operating condi-
tions, betamethasone valerate and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 4.
System repeatability: When the test is repeated 6 times with
3 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of betamethasone valerate to that of the internal
standard is not more than 1.0%.
(2) Gentamicin sulfate — Perform the test according to
the Cylinder-plate method as directed under Microbial Assay
for Antibiotics <4.02> according to the following conditions,
(i) Test organism, agar media for base layer and seed
layer, agar medium for transferring test organisms, and
standard solutions — Proceed as directed in the Assay un-
der Gentamicin Sulfate.
(ii) Sample solutions — Weigh accurately an amount of
Betamethasone Valerate and Gentamicin Sulfate Cream,
equivalent to about 1 mg (potency) of Gentamicin Sulfate,
add 100 mL of 0.1 mol/L phosphate buffer solution, pH
8.0, previously warmed to about 85°C, and shake well to
dissolve. After cooling, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make exactly 250 mL to make the high
concentration sample solution, which contains 4 fig (poten-
cy) per mL. Pipet a suitable amount of the high concentra-
tion sample solution, add 0.1 mol/L phosphate buffer so-
lution, pH 8.0 so that each mL contains 1 fig (potency),
and use this solution as the low concentration sample solu-
tion.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Betamethasone Valerate and
Gentamicin Sulfate Ointment
Betamethasone Valerate and Gentamicin Sulfate
Ointment contains not less than 95.0% and not more
than 110.0% of the labeled amount of betamethasone
valerate (C 27 H 37 F0 6 : 476.58) and not less than 90.0%,
and not more than 115.0% of the labeled potency of
gentamicin C, (C 21 H 43 N 5 7 : 477.60).
Method of preparation Prepare as directed under Oint-
ment, with Betamethasone Valerate and Gentamicin Sulfate.
Identification (1) To a quantity of Betamethasone Valer-
ate and Gentamicin Sulfate Ointment, equivalent to 1.2 mg
of Betamethasone Valerate according to the labeled amount,
add 20 mL of methanol and 20 mL of hexane, and disperse
the ointment with the aid of ultrasonic. Shake vigorously for
5 minutes, centrifuge for 5 minutes, cool for 15 minutes with
ice, and take 15 mL of the lower layer. Evaporate the layer to
dryness on a water bath under a current of nitrogen. To the
residue add 1 mL of ethyl acetate, apply ultrasonic waves,
filter, if necessary, and use the filtrate as the sample solution.
Separately, dissolve 18 mg of Betamethasone Valerte Refer-
ence Standard in 20 mL of ethyl acetate, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, de-
velop the plate with ethyl acetate to a distance of about 10
cm, and air-dry the plate. Spray evenly alkaline blue tetrazo-
lium TS on the plate, and heat at 100°C: the principal spot
from the sample solution and the spot from the standard so-
lution are purple in color, and their Rf values are the same.
(2) To a quantity of Betamethasone Valerate and Gen-
tamicin Sulfate Ointment, equivalent to 2 mg (potency) of
Gentamicin Sulfate according to the labeled amount, add 20
mL of hexane and 10 mL of water, shake vigorously for 10
minutes, and centrifuge. To 3 mL of the lower layer add 1
mL of dilute sodium hydroxide TS and 2 mL of ninhydrin
TS, and heat in a water bath at 90 - 95 °C for 10 minutes: a
red-brown color develops.
pH <2.54> To a quantity of Betamethasone Valerate and
Gentamicin Sulfate Ointment, equivalent to 6 mg of Be-
tamethasone Valerate according to the labeled amount, add
15 mL of water, and warm on a water bath to dissolve. After
cooling, separate the water layer: the pH of the layer is be-
tween 4.0 and 7.0.
Assay (1) Betamethasone valerate — Weigh accurately an
amount of Betamethasone Valerate and Gentamicin Sulfate
Ointment, equivalent to about 1 mg of betamethasone valer-
ate (C2 7 H 37 F0 6 ), add 10 mL of a mixture of methanol and
JPXV
Official Monographs / Bethanechol Chloride
363
water (7:3), and add exactly 10 mL of the internal standard
solution. After warming in a water bath at 75 °C for 5
minutes, shake vigorously for 10 minutes. Repeat this proce-
dure once more, cool with ice for 15 minutes, filter, discard
the first 5 mL of filtrate, and use the subsequent filtrate as the
sample solution. Separately, weigh accurately about 25 mg of
Betamethasone Valerate Reference Standard, previously
dried at 105 C C for 3 hours, and dissolve in methanol to make
exactly 25 mL. Pipet 5 mL of this solution, and add the mix-
ture of methanol and water (7:3) to make exactly 50 mL.
Pipet 10 mL of this solution, add exactly 10 mL of the inter-
nal standard solution, mix, and use this solution as the stan-
dard solution. Perform the test with 3 fiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, g T and Q s , of the peak area
of betamethasone valerate to that of the internal standard.
Amount (mg) of betamethasone valerate (C 27 H 37 F0 6 )
= W s x (Q T /Q S ) x (1/25)
W s : Amount (mg) of Betamethasone Valerate Reference
Standard
Internal standard solution — Dissolve 20 mg of beclometa-
sone dipropionate in 10 mL of methanol, and add the mix-
ture of methanol and water (7:3) to make 200 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 2.1 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3.5 ^m in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (13:7).
Flow rate: Adjust the flow rate so that the retention time of
betamethasone valerate is about 16 minutes.
System suitability—
System performance: When the procedure is run with 3 /uL
of the standard solution under the above operating condi-
tions, betamethasone valerate and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 4.
System repeatability: When the test is repeated 6 times with
3 jXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of betamethasone valerate to that of the internal
standard is not more than 1.0%.
(2) Gentamicin sulfate — Perform the test according to
the Cylinder-plate method as directed under Microbial Assay
for Antibiotics <4.02> according to the following conditions.
(i) Test organism, agar media for base layer and seed lay-
er, agar medium for transferring test organisms, and stan-
dard solutions — Proceed as directed in the Assay under Gen-
tamicin Sulfate.
(ii) Sample solutions — Weigh accurately an amount of
Betamethasone Valerate and Gentamicin Sulfate Ointment,
equivalent to about 1 mg (potency) of Gentamicin Sulfate,
transfer to a separator, add 50 mL of petroleum ether and ex-
actly 100 mL of 0.1 mol/L phosphate buffer solution, pH
8.0, shake for 10 minutes, and allow to stand. Pipet a suitable
amount of the water layer, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
4//g (potency) and 1 /ug (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Bethanechol Chloride
<?*=i-JH&ffc$l
hjN
O
A
Oh 3 c ch 3
H 3 (T ^ XH,
and enantiomer
C 7 H 17 C1N 2 2 : 196.68
(2i?5')-2-Carbamoyloxy-/V,/Y,A r -
trimethylpropylaminium chloride [590-63-6]
Bethanechol Chloride, when dried, contains not
less than 98.0% and not more than 101.0% of
C 7 H 17 C1N 2 2 .
Description Bethanechol Chloride occurs as colorless or
white crystals or a white, crystalline powder.
It is very soluble in water, freely soluble in acetic acid
(100), and sparingly soluble in ethanol (99.5).
A solution of Bethanechol Chloride (1 in 10) shows no op-
tical rotation.
It is hygroscopic.
Identification (1) To 2 mL of a solution of Bethanechol
Chloride (1 in 40) add 0.1 mL of a solution of cobalt (II)
chloride hexahydrate (1 in 100), then add 0.1 mL of potassi-
um hexacyanoferrate (II) TS: A green color is produced, and
almost entirely fades within 10 minutes.
(2) To 1 mL of a solution of Bethanechol Chloride (1 in
100) add 0.1 mL of iodine TS: a brown precipitate is pro-
duced, and the solution shows a greenish brown color.
(3) Determine the infrared absorption spectrum of
Bethanechol Chloride as directed in the paste method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(4) A solution of Bethanechol Chloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 217 - 221 °C (after drying).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bethanechol Chloride according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
(2) Related substances — Dissolve 1.0 g of Bethanechol
Chloride in 2.5 mL of water, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 1 /iL each of
the sample solution and standard solution on a plate of cellu-
lose for thin-layer chromatography. Develop the plate with a
mixture of a solution of ammonium acetate (1 in 100), ace-
tone, 1-butanol and formic acid (20:20:20:1) to a distance of
364
Bezafibrate / Official Monographs
JP XV
about 10 cm, and dry the plate at 105°C for 15 minutes.
Spray evenly hydrogen hexachloroplatinate (IV)-potassium
iodide TS on the plate, and allow to stand for 30 minutes: the
spot other than the principal spot from the sample solution is
not more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Bethanechol Chlo-
ride, previously dried, dissolve in 2 mL of acetic acid (100),
add 40 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.67 mg of C 7 H I7 C1N 2 2
Containers and storage Containers — Tight containers.
Bezafibrate
X
C 19 H 20 ClNO 4 : 361.82
2-(4-{2-[(4-Chlorobenzoyl)amino]ethyl}phenoxy)-2-
methylpropanoic acid
[41859-67-0]
Bezafibrate, when dried, contains not less than
98.5%> and not more than 101.0% of C 19 H 20 ClNO 4 .
Description Bezafibrate occurs as a white crystalline pow-
der.
It is freely soluble in /V,/V-dimethylformamide, soluble in
methanol, slightly soluble in ethanol (99.5), and practically
insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Bezafibrate in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Bezafibrate as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(3) Perform the test with Bezafibrate as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 181 - 186°C
Purity (1) Chloride <1.03>— Dissolve 3.0 g of Bezafibrate
in 15 mL of TV./V-dimethylformamide, add water to make 60
mL, shake well, allow to stand for more than 12 hours, and
filter. To 40 mL of the filtrate add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: To 0.70 mL of 0.01 mol/L hydrochloric acid VS add 10
mL of A^TV-dimethylformamide, 6 mL of dilute nitric acid
and water to make 50 mL (not more than 0.012%).
(2) Heavy metals <1.07>— Proceed with 2.0 g of
Bezafibrate according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Dissolve 0.10 g of Bezafibrate in
35 mL of methanol, add diluted 0.5 mol/L ammonium
acetate TS (1 in 50) to make 50 mL, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
70 mL of methanol and diluted 0.5 mol/L ammonium
acetate TS (1 in 50) to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with exactly
5 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the areas of the peaks hav-
ing the relative retention times of about 0.65 and 1.86 with
respect to bezafibrate obtained from the sample solution are
not larger than 1/2 times the peak area of bezafibrate from
the standard solution, the area of the peak other than those
and other than bezafibrate from the sample solution is not
larger than 1/5 times the peak area of bezafibrate from the
standard solution, and the total area of the peaks other than
the peak of bezafibrate from the sample solution is not larger
than 3/4 times the peak area of bezafibrate from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted acetic
acid (100) (1 in 100) (9:4).
Flow rate: Adjust the flow rate so that the retention time of
bezafibrate is about 6 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of bezafibrate beginning after the solvent
peak.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add a mixture of methanol and
diluted 0.5 mol/L ammonium acetate TS (1 in 50) (7:3) to
make exactly 50 mL. Confirm that the peak area of
bezafibrate obtained with 5 [iL of this solution is equivalent
to 7 to 13% of that with 5 fiL of the standard solution.
System performance: Dissolve 20 mg of Bezafibrate and 10
mg of 4-chlorobenzoate in 70 mL of methanol, and add dilut-
ed 0.5 mol/L ammonium acetate TS (1 in 50) to make 100
mL. When the procedure is run with 5 fiL of this solution un-
der the above operating conditions, 4-chlorobenzoate and
bezafibrate are eluted in this order with the resolution be-
tween these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
bezafibrate is not more than 2.0%.
JPXV
Official Monographs / Bezafibrate Sustained Release Tablets
365
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Bezafibrate, previ-
ously dried, dissolve in 50 mL of ethanol (99.5), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 36.18 mg of C 19 H 20 ClNO 4
Containers and storage Containers — Tight containers.
Bezafibrate Sustained Release
Tablets
Bezafibrate Sustained Release Tablets contain not
less than 95.0% and not more than 105. 0% of the la-
beled amount of bezafibrate (Ci 9 H 2 oClN0 4 : 361.82).
Method of preparation Prepare as directed under Tablets,
with Bezafibrate.
Identification Mix well an amount of powdered Bezafibrate
Sustained Release Tablets, equivalent to 0.1 g of Bezafibrate
according to the labeled amount, with 100 mL of methanol,
and filter. To 1 mL of the filtrate and add methanol to make
100 mL. Determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 227 nm and 231 nm.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Bezafibrate Sustained
Release Tablets at 50 revolutions per minute according to the
Paddle method using 900 mL of disodium hydrogen phos-
phate-citric acid buffer solution, pH 7.2 as the dissolution
medium. Withdraw exactly 20 mL of the dissolution medium
1.5 hours, 2.5 hours and 8 hours after starting the test, and
immediately fill up the dissolution medium each time with ex-
actly 20 mL of disodium hydrogen phosphate-citric acid
buffer solution, pH 7.2, previously warmed to 37±0.5°C.
Filter these media through a membrane filter with a pore size
not exceeding 0.45 fim. Discard the first 10 mL of the filtrate,
pipet the subsequent FmL, add disodium hydrogen phos-
phate-citric acid buffer solution, pH 7.2 to make exactly V
mL so that each mL contains about 13 fig of bezafibrate (Ci 9
H 20 ClNO 4 ) according to the labeled amount, and use these
solutions as the sample solutions. Separately, weigh accurate-
ly about 66 mg of bezafibrate for assay, previously dried at
105 °C for 3 hours, and dissolve in methanol to make exactly
50 mL. Pipet 2 mL of this solution, add disodium hydrogen
phosphate-citric acid buffer solution, pH 7.2 to make exactly
200 mL, and use this solution as the standard solution. Deter-
mine the absorbances, ^4t<ii) (n= 1,2,3) and A s , of the sample
solutions and standard solution at 228 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>: the dissolution
rates of a 100-mg tablet in 1.5 hours, in 2.5 hours and in 8
hours are 15 - 45%, 35 - 65% and not less than 80%, respec-
tively, and those of a 200-mg tablet in 1.5 hours, in 2.5 hours
and in 8 hours are 15 - 45%, 30 - 60% and not less than 75
%, respectively.
Dissolution rate (%) in each case of n with respect to the la-
beled amount of bezafibrate (C 19 H 2 oClN0 4 )
~ A n ~ 1 l A 1
W s x
^S l=1
'T(i)
A,
'45
V 1 ,c
x-x-xi8
W s : Amount (mg) of bezafibrate for assay
C: Labeled amount (mg) of bezafibrate (Ci 9 H 2 oClN0 4 ) in 1
tablet
Assay Weigh accurately, and powder not less than 20
Bezafibrate Sustained Release Tablets. Weigh accurately a
portion of the powder, equivalent to about 20 mg of
bezafibrate (Ci 9 H 20 ClNO 4 ), add 60 mL of methanol and ex-
actly 10 mL of the internal standard solution, and shake for
20 minutes. Add diluted 0.5 mol/L ammonium acetate TS (1
in 50) to make 100 mL, filter, and use the filtrate as the sam-
ple solution. Separately, weigh accurately about 20 mg of
bezafibrate for assay, previously dried at 105°C for 3 hours,
dissolve in 60 mL of methanol, add exactly 10 mL of the in-
ternal standard solution and diluted 0.5 mol/L ammonium
acetate TS (1 in 50) to make 100 mL, and use this solution as
the standard solution. Perform the test with 2 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak area
of bezafibrate to that of the internal standard.
Amount (mg) of bezafibrate (C 19 H 20 ClNO 4 ) = W s x (Q T /Q S )
W s : Amount (mg) of bezafibrate for assay
Internal standard solution — A solution of 4-nitrophenol in
methanol (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted acetic
acid (100) (1 in 100) (9:4).
Flow rate: Adjust the flow rate so that the retention time of
bezafibrate is about 6 minutes.
System suitability —
System performance: When the procedure is run with 2 fiL
of the standard solution under the above operating condi-
tions, the internal standard and bezafibrate are eluted in this
order with the resolution between these peaks being not less
than 4.
System repeatability: When the test is repeated 6 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of bezafibrate to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
366
Bifonazole / Official Monographs
JP XV
Bifonazole
tr*^-v-;u
and enanlbmer
C 22 H 18 N 2 : 310.39
l-[(i?S)-(Biphenyl-4-yl)(phenyl)methyl]-l//-imidazole
[60628-96-8]
mL each, and use these solutions as the standard solutions (1)
and (2), respectively. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL each of the sample solution and standard solutions (1) and
(2) on a plate of silica gel with fluorescent indicator for thin-
layer chromatography. Develop the plate with a mixture of
ethyl acetate and ammonia solution (28) (49:1) to a distance
of about 10 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot with Rf value
of about 0.20 from the sample solution is not more intense
than the spot from the standard solution (1). And the spots
other than the spot mentioned above and the principal spot
from the sample solution are not more intense than the spot
from the standard solution (2).
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 2 hours).
Bifonazole, when
98.5% of C 22 H 18 N 2 .
dried, contains not less than Residue on ignition «.«> Not more than 0.1% (1 g).
Description Bifonazole occurs as a white to pale yellow
powder. It is odorless and tasteless.
It is freely soluble in dichloromethane, soluble in
methanol, sparingly soluble in ethanol (95), slightly soluble in
diethyl ether, and practically insoluble in water.
A solution of Bifonazole in methanol (1 in 100) does not
show optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Bifonazole in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Bi-
fonazole, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2. 60> 1 47 - 1 5 1 °C
Purity (1) Chloride <7.0?>— To 2.0 g of Bifonazole add 40
mL of water, warm for 5 minutes, and after cooling, filter.
To 10 mL of the filtrate add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 0.30 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.021%).
(2) Sulfate <1.14>— To 10 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.50 mL of 0.005 mol
/L sulfuric acid VS (0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Bifona-
zole according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(4) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Bifonazole in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 3 mL of the sample solu-
tion, and add methanol to make exactly 100 mL. Pipet 25 mL
and 5 mL of this solution, add methanol to make exactly 50
Assay Weigh accurately about 0.15 g of Bifonazole, previ-
ously dried, and dissolve in dichloromethane to make exactly
50 mL. Pipet 5 mL of this solution in a glass-stoppered coni-
cal flask, add 10 mL of water, 5 mL of dilute sulfuric acid
and 25 mL of dichloromethane, and add 2 to 3 drops of a
solution of methyl yellow in dichloromethane (1 in 500) as in-
dicator, and titrate <2.50>, while shaking vigorously, with
0.01 mol/L sodium lauryl sulfate VS by a buret with 0.02-mL
minimum graduation. The end point is reached when the
color of the dichloromethane layer changes from yellow to
orange-red after dropwise addition of 0.01 mol/L sodium
lauryl sulfate VS, strong shaking, and standing for a while.
Each mL of 0.01 mol/L sodium lauryl sulfate VS
= 3. 104 mg of C 22 H I8 N 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Biperiden Hydrochloride
txijf>^i^
il ° h
C 21 H 29 N0.HC1: 347.92
l-(Bicyclo [2.2.1 ]hept-5-en-2-yl)-l-phenyl-3-(piperidin-l-
yl)propan-l-ol monohydrochloride [1235-82-1]
Biperiden Hydrochloride, when dried, contains not
less than 99.0% of C 21 H 29 NO.HCl.
Description Biperiden Hydrochloride occurs as a white to
brownish and yellowish white, crystalline powder.
It is freely soluble in formic acid, slightly soluble in water,
in methanol and in ethanol (95), and practically insoluble in
diethyl ether.
Melting point: about 270°C (with decomposition).
Identification (1) Dissolve 0.02 g of Biperiden Hydrochlo-
ride in 5 mL of phosphoric acid: a green color develops.
JPXV
Official Monographs / Bisacodyl 367
(2) Dissolve 0.01 g of Biperiden Hydrochloride in 5 mL
of water by heating, cool, and add 5 to 6 drops of bromine
TS: a yellow precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Biperiden Hydrochloride (1 in 2000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectrum of
Biperiden Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(5) Dissolve 0.02 g of Biperiden Hydrochloride in 10 mL
of water by heating, and cool: the solution responds to the
Qualitative Tests <1.09> for chloride.
Purity (1) Acidity or alkalinity — To 1.0 g of Biperiden
Hydrochloride add 50 mL of water, shake vigorously, filter,
and to 20 mL of the filtrate add 1 drop of methyl red TS: no
red to yellow color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Biperiden Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Biperiden Hydrochloride according to Method 3, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Biperiden
Hydrochloride in 20 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
50 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol and ammonia
solution (28) (80:15:2) to a distance of about 15 cm, and air-
dry the plate. Spray evenly Dragendorff's TS for spraying on
the plate: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Biperiden
Hydrochloride, previously dried, dissolve in 5 mL of formic
acid, add 60 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 34.79 mg of C 2 iH 29 NO.HCl
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Bisacodyl
tr-y-=ivju
C 22 H 19 N0 4 : 361.39
4,4'-(Pyridin-2-ylmethylene)bis(phenyl acetate) [603-50-9]
Bisacodyl, when dried, contains not less than 98.5%
of C 22 H 19 N0 4 .
Description Bisacodyl occurs as a white, crystalline pow-
der.
It is freely soluble in acetic acid (100), soluble in acetone,
slightly soluble in ethanol (95) and in diethyl ether, and prac-
tically insoluble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Determine the absorption spectrum of a
solution of Bisacodyl in ethanol (95) (3 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Bisacodyl Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Bi-
sacodyl, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of dried Bisacodyl Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Melting point <2.60> 132 - 136°C
Purity (1) Chloride <1.03> — Dissolve 1.0 g of Bisacodyl in
30 mL of acetone, and add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.35 mL of 0.01 mol/L hydrochloric acid VS add 30 mL of
acetone, 6 mL of dilute nitric acid and water to make 50 mL
(not more than 0.012%).
(2) Sulfate <1.14>— Dissolve 1.0 g of Bisacodyl in 2 mL of
dilute hydrochloric acid, and add water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 0.35 mL of 0.005 mol/L
sulfuric acid VS add 2 mL of dilute hydrochloric acid and
water to make 50 mL (not more than 0.017%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Bi-
sacodyl according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(4) Related substances — Dissolve 0.20 g of Bisacodyl in
10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add acetone to make
exactly 200 mL, and use this solution as the standard solu-
368
Bisacodyl Suppositories / Official Monographs
JP XV
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of 2-butanone, chloroform
and xylene (1:1:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Bisacodyl, previous-
ly dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from orange-yellow to green (indicator:
0.5 mL of /?-naphtholbenzein TS). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 36.14 mg of C 22 Hi 9 N0 4
Containers and storage Containers — Well-closed contain-
ers.
Bisacodyl Suppositories
tr-y-n^ju^sj
Bisacodyl Suppositories contain not less than 90%
and not more than 110% of the labeled amount of
bisacodyl (C 2 2H 19 N0 4 : 361.39).
Method of preparation Prepare as directed under Supposi-
tories, with Bisacodyl.
Identification (1) To a quantity of Bisacodyl Supposito-
ries, equivalent to 6 mg of Bisacodyl according to the labeled
amount, add 20 mL of ethanol (95), warm on a water bath
for 10 minutes, shake vigorously for 10 minutes, and allow to
stand in ice water for 1 hour. Centrifuge the solution, filter
the supernatant liquid, and to 2 mL of the filtrate add ethanol
(95) to make 20 mL. Determine the absorption spectrum of
the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 261
nm and 265 nm.
(2) Use the filtrate obtained in (1) as the sample solution.
Separately, dissolve 6 mg of Bisacodyl Reference Standard in
20 mL of ethanol (95), and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 20 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 2-butanone,
chloroform and xylene (1:1:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spot from the sample solution and
that from the standard solution show the same Rf value.
Assay Weigh accurately not less than 20 Bisacodyl Supposi-
tories, make them fine fragments carefully, and mix
uniformly. Weigh accurately a portion of the fragments,
equivalent to about 10 mg of bisacodyl (C 2 2H 19 N0 4 ), add
40 mL of tetrahydrofuran, warm to 40°C, dissolve by shak-
ing, cool, and add tetrahydrofuran to make exactly 50 mL.
Pipet 5 mL of this solution, add exactly 5 mL of the internal
standard solution, and add the mobile phase to make
100 mL. Cool this solution in ice for 30 minutes, centrifuge,
filter the supernatant liquid through a membrane filter with
pore size of 0.5 /urn, discard the first 10 mL of the filtrate, and
use the subsequent filtrate as the sample solution. Separately,
weigh accurately about 10 mg of Bisacodyl Reference Stan-
dard, previously dried at 105°C for 2 hours, and dissolve in
tetrahydrofuran to make exactly 50 mL. Pipet 5 mL of this
solution, proceed in the same manner as the sample solution,
and use this solution as the standard solution. Perform the
test with 20 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qt and Q s , of the peak area of bisacodyl to that of the inter-
nal standard, respectively.
Amount (mg) of bisacodyl (C 2 2H 19 N0 4 ) = W s x (Q T /Q S )
W s : Amount (mg) of Bisacodyl Reference Standard
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in acetonitrile (3 in 100,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 [im in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.01 mol/L citric acid TS,
acetonitrile and methanol (2:1:1).
Flow rate: Adjust the flow rate so that the retention time of
bisacodyl is about 8 minutes.
System suitability —
System performance: When the procedure is run with
20 /uL of the standard solution under the above operating
conditions, the internal standard and bisacodyl are eluted in
this order with the resolution between these peaks being not
less than 2.0.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of bisacodyl to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Bismuth Subgallate
Dermatol
Bismuth Subgallate, when dried, contains not less
than 47.0% and not more than 51.0% of bismuth (Bi:
208.98).
Description Bismuth Subgallate occurs as a yellow powder.
JPXV
Official Monographs / Bismuth Subnitrate
369
It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in dilute hydrochloric acid, in dilute nitric acid
and in dilute sulfuric acid on warming. It dissolves in sodium
hydroxide TS, forming a clear, yellow solution, which turns
red immediately.
It is affected by light.
Identification (1) Ignite 0.5 g of Bismuth Subgallate: it
chars at first, and leaves finally a yellow residue. The residue
responds to the Qualitative Tests <1.09> for bismuth salt.
(2) To 0.5 g of Bismuth Subgallate add 25 mL of water
and 20 mL of hydrogen sulfide TS, and shake well. Filter off
the blackish brown precipitate, and add 1 drop of iron (III)
chloride TS to the filtrate: a blue-black color is produced.
Purity (1) Clarity of solution — Dissolve 1.0 g of Bismuth
Subgallate in 40 mL of diluted sodium hydroxide TS (1 in 8):
the solution is clear.
(2) Sulfate — Ignite 3.0 g of Bismuth Subgallate in a por-
celain crucible, and cautiously dissolve the residue in 2.5 mL
of nitric acid by warming. Pour the solution into 100 mL of
water, shake, and filter. Evaporate 50 mL of the filtrate on a
water bath to 15 mL. Add water to make 20 mL, filter again,
and use the filtrate as the sample solution. To 5 mL of the
sample solution add 2 to 3 drops of barium nitrate TS: no
turbidity is produced.
(3) Nitrate— To 0.5 g of Bismuth Subgallate add 5 mL of
dilute sulfuric acid and 25 mL of iron (II) sulfate TS, shake
well, and filter. Superimpose carefully 5 mL of the filtrate on
sulfuric acid: no red-brown color develops at the zone of con-
tact.
(4) Ammonium — Dissolve 1 .0 g of Bismuth Subgallate in
5 mL of sodium hydroxide TS, and heat: the gas evolved does
not change moistened red litmus paper to blue.
(5) Copper — To 5 mL of the sample solution obtained in
(2) add 1 mL of ammonia TS, and filter: no blue color de-
velops in the filtrate.
(6) Lead — Ignite 1.0 g of Bismuth Subgallate at about
500°C in a porcelain crucible, dissolve the residue in a
smallest possible amount of nitric acid added dropwise,
evaporate over a low flame to dryness , and cool. Add 5 mL
of a solution of potassium hydroxide (1 in 6) to the residue,
boil carefully for 2 minutes, cool, and centrifuge. Take the
supernatant liquid in a test tube, add 10 drops of potassium
chromate TS, and acidify the solution by adding acetic acid
(100) dropwise: neither turbidity nor a yellow precipitate is
produced.
(7) Silver — To 5 mL of the sample solution obtained in
(2) add 0.5 mL of nitric acid and 2 to 3 drops of dilute
hydrochloric acid: no turbidity is produced.
(8) Alkaline earth metals and alkali metals — Boil 1.0 g of
Bismuth Subgallate with 40 mL of diluted acetic acid (31) (1
in 2) for 2 minutes, cool, add water to make 40 mL, and
filter. To 20 mL of the filtrate add 2 mL of dilute hydrochlor-
ic acid, boil, immediately pass hydrogen sulfide thoroughly
through the solution, filter the precipitate produced, and
wash with water. Combine the filtrate and the washings, add
5 drops of sulfuric acid, and evaporate to dryness. Ignite as
directed under Residue on Ignition <2.44>: the mass of the
residue does not more than 5.0 mg.
(9) Arsenic <1.11>— Mix well 0.20 g of Bismuth Subgal-
late with 0.20 g of calcium hydroxide, and ignite the mixture.
Dissolve the residue in 5 mL of dilute hydrochloric acid, use
this solution as the test solution, and perform the test (not
more than 10 ppm).
(10) Gallic acid— To 1.0 g of Bismuth Subgallate add 20
mL of ethanol (95), shake for 1 minute, and filter. Evaporate
the filtrate on a water bath to dryness: the mass of the residue
does not more than 5.0 mg.
Loss on drying <2.4I> Not more than 6.0% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 0.5 g of Bismuth Subgallate,
previously dried, ignite at about 500°C for 30 minutes, and
cool. Dissolve the residue in 5 mL of diluted nitric acid (2 in
5) by warming, and add water to make exactly 100 mL. Meas-
ure exactly 30 mL of this solution, add 200 mL of water, and
titrate <2.50> with 0.02mol/L disodium dihydrogen
ethylenediamine tetraacetate VS until the color of the solu-
tion changes from red-purple to yellow (indicator: 2 to 3
drops of xylenol orange TS).
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.180 mg of Bi
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Bismuth Subnitrate
Bismuth Subnitrate, when dried, contains not less
than 71.5% and not more than 74.5% of bismuth (Bi:
208.98).
Description Bismuth Subnitrate occurs as a white powder.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It readily dissolves in hydrochloric acid and in nitric acid
without effervescence.
It is slightly hygroscopic, and changes moistened blue lit-
mus paper to red.
Identification Bismuth Subnitrate responds to the Qualita-
tive Tests <1.09> for bismuth salt and nitrate.
Purity (1) Chloride <1.03>— Dissolve 0.7 g of Bismuth
Subnitrate in 2 mL of water and 2 mL of nitric acid, and add
6 mL of dilute nitric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: evaporate 2 mL of nitric acid on
a water bath to dryness, add 0.70 mL of 0.01 mol/L
hydrochloric acid VS, 6 mL of dilute nitric acid and water to
make 50 mL (not more than 0.035%).
(2) Sulfate— Dissolve 3.0 g of Bismuth Subnitrate in 3.0
mL of warmed nitric acid, pour this solution into 100 mL of
water, shake, and filter. Concentrate the filtrate on a water
bath to 30 mL, filter, and use this filtrate as the sample solu-
tion. To 5 mL of the sample solution add 2 to 3 drops of bari-
um nitrate TS: no turbidity is produced.
(3) Ammonium — Boil 0.10 g of bismuth Subnitrate with
5 mL of sodium hydroxide TS: the gas evolved does not
370
Bleomycin Hydrochloride / Official Monographs
JP XV
change moistened red litmus paper to blue.
(4) Copper — To 5 mL of the sample solution obtained in
(2) add 2 mL of ammonia TS, and filter: no blue color de-
velops.
(5) Lead — To 1.0 g of Bismuth Subnitrate add 5 mL of a
solution of sodium hydroxide (1 in 6), boil carefully for 2
minutes, cool and centrifuge. Transfer the supernatant liquid
to a test tube, add 10 drops of potassium chromate TS, and
add dropwise acetic acid (31) to render the solution acid: no
turbidity or yellow precipitate is produced.
(6) Silver — To 5 mL of the sample solution obtained in
(2) add 0.5 mL of nitric acid and 2 to 3 drops of dilute
hydrochloric acid: no turbidity is produced.
(7) Alkaline earth metals and alkali metals — Boil 2.0 g of
Bismuth Subnitrate with 40 mL of diluted acetic acid (31) (1
in 2) for 2 minutes, cool, add water to make 40 mL, and
filter. To 20 mL of the filtrate add 2 mL of dilute hydrochlor-
ic acid, boil, immediately pass hydrogen sulfide thoroughly
through the solution, filter, and wash the residue with water.
Combine the filtrate and the washings, add 5 drops of sulfur-
ic acid, evaporate to dryness, and ignite as directed under
Residue on Ignition <2.44>: the residue does not exceed 5.0
mg
(8) Arsenic <1.11>— To 0.20 g of Bismuth Subnitrate add
2 mL of sulfuric acid, heat until white fumes evolve, dilute
cautiously with water to 5 mL, use this solution as the test
solution, and perform the test (not more than 10 ppm).
Loss on drying <2.41>
2 hours).
Not more than 3.0% (2 g, 105°C,
Assay Weigh accurately about 0.4 g of Bismuth Subnitrate,
previously dried, dissolve in 5 mL of diluted nitric acid (2 in
5) by warming, and add water to make exactly 100 mL. Pipet
25 mL of the solution, add 200 mL of water and titrate <2.50>
with 0.02 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS until the color of the solution changes from
red-purple to yellow (indicator: 5 drops of xylenol orange TS)
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.180 mg of Bi
Containers and storage Containers — Well-closed contain-
ers.
Bleomycin Hydrochloride
H NH?
Bioornycmotc acid
Bleomycin A,
Bleomycin Deme1nyl-A z ; fl e
Bleomycin Aj : R =
Bleomycin A^ : R =
Bleomycin Az.b : H =
Bleomycin Ai : R =
Bleomycin Si' . R
Bleomycin Bj : R =
Bleomycinoic Acid
1-Bleomycinoic acid hydrochloride
Bleomycin A[
A^ 1 -[3-(Methylsulfinyl)propyl]bleomycinamide
hydrochloride
Bleomycin Demethyl-A 2
A rl -[3-(Methylsulfanyl)propyl]bleomycinamide
hydrochloride
Bleomycin A 2
Af 1 -[3-(Dimethylsulfonio)propyl]bleomycinamide
hydrochloride
Bleomycin A 2 ,_ a
Af 1 -(4-Aminobutyl)bleomycinamide hydrochloride
Bleomycin A 2 ._ b
A rl -(3-Aminopropyl)bleomycinamide hydrochloride
Bleomycin A 5
A rl -{3-[(4-Aminobutyl)amino]propyl}bleomycinamide
hydrochloride
Bleomycin B,-
Bleomycinamide hydrochloride
Bleomycin B 2
A^ I -(4-Guanidinobutyl)bleomycinamide
hydrochloride
Bleomycin B 4
Af'-{4-[3-(4-Guanidinobutyl)guanidino]butyl}-
bleomycinamide hydrochloride
[11056-06-7, Bleomycin]
Bleomycin Hydrochloride is the hydrochloride of a
JPXV
Official Monographs / Bleomycin Hydrochloride 371
mixture of substances having antitumor activity pro-
duced by the growth of Streptomyces verticillus.
It contains not less than 1400 fig (potency) and not
more than 2000 fig (potency) per mg, calculated on the
dried basis. The potency of Bleomycin Hydrochloride
is expressed as mass (potency) of bleomycin A 2 (C 55 H 84
C1N 17 21 S 3 : 1451.00).
Description Bleomycin Hydrochloride occurs as a white to
yellowish white powder.
It is freely soluble in water, and slightly soluble in ethanol
(95).
It is hygroscopic.
Identification (1) To 4 mg of Bleomycin Hydrochloride
add 5 fiL of copper (II) sulfate TS, and dissolve in water to
make 100 mL. Determine the absorption spectrum of this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Bleomycin Hydrochloride as directed in the potassium
bromide disk method under Infrared Spectrophotometry <
2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Bleomycin Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Bleomycin Hydrochloride in 20 mL of water is between
4.5 and 6.0.
Content ratio of the active principle Dissolve 10 mg of
Bleomycin Hydrochloride in 20 mL of water, and use this
solution as the sample solution. Perform the test with 20 fiL
of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the peak area of bleomycin A 2 (the first principal
peak) is between 55% and 70%, that of bleomycin B 2 (the
second principal peak) is between 25% and 32%, the total
peak area of bleomycin A 2 and bleomycin B 2 is not less than
85%, the peak area of demethylbleomycin A 2 (a peak having
the relative retention time of 1.5 - 2.5 to bleomycin A 2 ) is not
more than 5.5%, and the total area of the rest peaks is not
more than 9.5%.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 ftm in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase stock solution: Dissolve 0.96 g of sodium
1-pentanesulfonate and 1.86 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in 1000 mL of water
and 5 mL of acetic acid (100), and adjust the pH to 4.3 with
ammonia TS.
Mobile phase A: A mixture of the mobile phase stock
solution and methanol (9:1).
Mobile phase B: A mixture of the mobile phase stock solu-
tion and methanol (3:2).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-60
60-75
100^0
0^ 100
100
Flow rate: About 1.2 mL per minute.
Time span of measurement: 20 minutes after elution of the
peak of demethylbreomycin A 2 beginning after the solvent
peak.
System suitability —
System performance: When the procedure is run with
20 fiL of the sample solution under the above operating con-
ditions, bleomycin A 2 and bleomycin B 2 are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
20 fiL of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of
bleomycin A 2 is not more than 2.0%.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 80 mg of Bleomycin Hydrochloride in
4 mL of water is clear and colorless.
(2) Copper — Dissolve exactly 75 mg of Bleomycin
Hydrochloride in exactly 10 mL of diluted nitric acid (1 in
100), and use this solution as the sample solution. Separately,
to exactly 15 mL of Standard Copper Solution add diluted
nitric acid (1 in 100) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with the
sample solution and standard solution as directed under A-
tomic Absorption Spectrophotometry <2.23> according to the
following conditions: the absorbance of the sample solution
is not more than that of the standard solution (not more than
200 ppm).
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Copper hollow-cathode lamp
Wavelength: 324.8 nm
Loss on drying <2.41> Not more than 5.0% (60 mg, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours). Take the sample
to be tested while avoiding moisture absorption.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Mycobacterium smegmatis ATCC
607
(ii) Agar medium for seed, base layer and transferring the
test organism
Glycerin 10.0 g
Peptone 10.0 g
Meat extract 10.0 g
Sodium chloride 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components, and sterilize. Adjust the pH after
sterilization to 6.9 - 7.1 with sodium hydroxide TS.
(iii) Liquid media for suspending the test organism
372 Bleomycin Sulfate / Official Monographs
JP XV
Glycerin
10.0 g
Peptone
10.0 g
Meat extract
10.0 g
Sodium chloride
3.0g
Water
1000 mL
Bleomycin Sulfate
Mix all the components, and sterilize. Adjust the pH after
sterilization to 6.9-7.1 with sodium hydroxide TS.
(iv) Preparation of seeded agar layer — Cultivate the test
organism on the slant of the agar medium for transferring the
test organism at 27°C for 40 to 48 hours, then inoculate the
test organism thus obtained in 100 mL of the liquid media for
suspending the test organism, cultivate with shaking at be-
tween 25 °C and 27 °C for 5 days, and use this as the suspen-
sion of test organism. Store the suspension of test organism
at a temperature not exceeding 5°C, and use within 14 days.
Add 0.5 mL of the suspension of test organism in 100 mL of
the agar medium for seed previously kept at 48°C, mix thor-
oughly, and use as the seeded agar layer.
(v) Preparation of cylinder-agar plate — Proceed as
directed in the Preparation of cylinder-agar plate under the
Microbial Assay for Antibiotics, dispensing 5.0 mL of agar
medium for base layer and 8.0 mL of the agar medium for
seed into the Petri dish.
(vi) Standard solutions — Weigh accurately an amount of
Bleomycin A 2 Hydrochloride Reference Standard, previously
dried under reduced pressure not exceeding 0.67 kPa at an or-
dinary temperature for 3 hours, equivalent to about 15 mg
(potency), dissolve in 0.1 mol/L phosphate buffer solution,
pH 6.8 to make exactly 100 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
5°C or below, and use within 30 days. Take exactly a suitable
amount of the standard stock solution before use, add 0.1
mol/L phosphate buffer solution, pH 6.8 to make solutions
so that each mL contains 30 /xg (potency) and 15 ^g (poten-
cy), and use these solutions as the high concentration stan-
dard solution and low concentration standard solution, re-
spectively.
(vii) Sample solutions — Weigh accurately an amount of
Bleomycin Hydrochloride, equivalent to about 15 mg (poten-
cy), and dissolve in 0.1 mol/L phosphate buffer solution, pH
6.8 to make exactly 100 mL. Take exactly a suitable amount
of this solution, add 0.1 mol/L phosphate buffer solution,
pH 6.8 to make solutions so that each mL contains 30 /ug
(potency) and 15 /xg (potency), and use these solutions as the
high concentration sample solution and low concentration
sample solution, respectively.
Containers and storage Containers — Tight containers.
"/U3"7>f
<wmm
Bleomycinoic ackj
E n^Tiycin A-
Bleomycin Darmeuiyl-A 2 ; R
Bleomycin A? ; R
Bleomycin A^ ^
Bleomycin A^,
Bleomycin A,.
Bleomycin B|<
Bleomycin B?
E eomycin 6j
Bleomycinoic Acid
1-Bleomycinoic acid sulfate
Bleomycin A!
A rl -[3-(Methylsulfinyl)propyl]bleomycinamide sulfate
Bleomycin Demethyl-A 2
A fl -[3-(Methylsulfanyl)propyl]bleomycinamide sulfate
Bleomycin A 2
A fl -[3-(Dimethylsulfonium)propyl]bleomycinamide
sulfate
Bleomycin A 2 ._ a
N 1 -(4-Aminobutyl)bleomycinamide sulfate
Bleomycin A 2 ._ b
./V'-^-Aminopropyrjbleomycinamide sulfate
Bleomycin A 5
A rl -{3-[(4-Aminobutyl)amino]propyl}bleomycinamide
sulfate
Bleomycin B { '
Bleomycinamide sulfate
Bleomycin B 2
N 1 -(4-Guanidinobutyl)bleomycinamide sulfate
Bleomycin B 4
A n -{4-[3-(4-Guanidinobutyl)guanidino]butyl}-
bleomycinamide sulfate
[9041-93-4, Bleomycin Sulfate]
Bleomycin Sulfate is the sulfate of a mixture of sub-
stances having antitumor activity produced by the
growth of Streptomyces verticillus.
JPXV
Official Monographs / Bleomycin Sulfate 373
It contains not less than 1400 ,ug (potency) and not
more than 2000 fig (potency) per mg, calculated on the
dried basis. The potency of Bleomycin Sulfate is ex-
pressed as mass (potency) of bleomycin A 2 (C 55 H 84 C1N
17 21 S 3 : 1451.00).
Description Bleomycin Sulfate occurs as a white to yellow-
ish white powder.
It is freely soluble in water, and slightly soluble in ethanol
(95).
It is hygroscopic.
Identification (1) To 4 mg of Bleomycin Sulfate add 5 fiL
of copper (II) sulfate TS, and dissolve in water to make
100 mL. Determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of
Bleomycin Sulfate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) A solution of Bleomycin Sulfate (1 in 200) responds to
the Qualitative Tests <1.09> (1) and (2) for sulfate.
pH <2.54> The pH of a solution obtained by dissolving 10
mg of Bleomycin Sulfate in 20 mL of water is between 4.5
and 6.0.
Content ratio of the active principle Dissolve 10 mg of
Bleomycin Sulfate in 20 mL of water, and use this solution as
the sample solution. Perform the test with 20 /iL of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the peak area
of bleomycin A 2 (the first principal peak) is between 55% and
70%, that of bleomycin B 2 (the second principal peak) is be-
tween 25 % and 32% , the total peak area of bleomycin A 2 and
bleomycin B 2 is not less than 85%, the peak area of demethyl-
bleomycin A 2 (a peak having the relative retention time of 1 .5
- 2.5 against bleomycin A 2 ) is not more than 5.5%, and the
total area of the rest peaks is not more than 9.5%.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /im in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase stock solution: Dissolve 0.96 g of sodium 1-
pentanesulfonate and 1.86 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in 1000 mL of water
and 5 mL of acetic acid (100), and adjust the pH to 4.3 with
ammonia TS.
Mobile phase A: A mixture of the mobile phase stock
solution and methanol (9:1).
Mobile phase B: A mixture of the mobile phase stock solu-
tion and methanol (3:2).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-60
60-75
100^0
0^ 100
100
Flow rate: About 1.2mL/min
Time span of measurement: Twenty minutes after elution
of the peak of demethylbleomycin A 2 beginning after the sol-
vent peak.
System suitability —
System performance: When the procedure is run with
20 fiL of the sample solution under the above operating con-
ditions, bleomycin A 2 and bleomycin B 2 are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
20 fiL of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of
bleomycin A 2 is not more than 2.0%.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 80 mg of Bleomycin Sulfate in 4 mL
of water is clear and colorless.
(2) Copper — Dissolve exactly 75 mg of Bleomycin Sul-
fate in 10 mL of diluted nitric acid (1 in 100), and use this
solution as the sample solution. Separately, to exactly 15 mL
of Standard Copper Solution add diluted nitric acid (1 in 100)
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with the sample solution and stan-
dard solution as directed under Atomic Absorption Spec-
trophotometry <2.23> according to the following conditions:
the absorbance of the sample solution is not more than that
of the standard solution (not more than 200 ppm).
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Copper hollow-cathode lamp
Wavelength: 324.8 nm
Loss on drying <2.41> Not more than 3.0% (60 mg, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours). Take the sample
to be tested while avoiding moisture absorption.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Mycrobacterium smegmatis ATCC
607
(ii) Agar medium for seed, base layer and transferring the
test organism
Glycerin 10.0 g
Peptone 10.0 g
Meat extract 10.0 g
Sodium chloride 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the components and sterilize. Adjust the pH after
sterilization to 6.9 - 7.1 with sodium hydroxide TS.
(iii) Liquid media for suspending the test organism
Glycerin 10.0 g
Peptone 10.0 g
Meat extract 10.0 g
Sodium chloride 3.0 g
374 Boric Acid / Official Monographs
JP XV
Water 1000 mL
Mix all the components and sterilize. Adjust the pH after
sterilization to 6.9-7.1 with sodium hydroxide TS.
(iv) Preparation of seeded agar layer - Cultivate the test
organism on the slant of the agar medium for transferring the
test organism at 27°C for 40 to 48 hours, then inoculate the
test organism thus obtained in 100 mL of the liquid media for
suspending the test organism, cultivate with shaking at be-
tween 25 °C and 27 °C for 5 days, and use this as the suspen-
sion of test organism. Store the suspension of test organism
at a temperature not exceeding 5°C, and use within 14 days.
Add 0.5 mL of the suspension of test organism in 100 mL of
the agar medium for seed previously kept at 48 °C, mix thor-
oughly, and use as the seeded agar layer.
(v) Preparation of cylinder-agar plate — Proceed as
directed in 7. Preparation of cylinder-agar plate under the
Microbial Assay for Antibiotics, dispensing 5.0 mL of agar
medium for base layer and 8.0 mL of the agar medium for
seed into the Petri dish.
(vi) Standard solutions — Weigh accurately an amount of
Bleomycin A 2 Hydrochloride Reference Standard, previously
dried under reduced pressure not exceeding 0.67 kPa at an or-
dinary temperature for 3 hours, equivalent to about 15 mg
(potency), dissolve in 0.1 mol/L phosphate buffer solution,
pH 6.8 to make exactly 100 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
5°C or below, and use within 30 days. Take exactly a suitable
amount of the standard stock solution before use, add 0.1
mol/L phosphate buffer solution, pH 6.8 to make solutions
so that each mL contains 30 /xg (potency) and 15 ^g (poten-
cy), and use these solutions as the high concentration stan-
dard solution and low concentration standard solution, re-
spectively.
(vii) Sample solutions — Weigh accurately an amount of
Bleomycin Sulfate, equivalent to about 15 mg (potency), dis-
solve in 0.1 mol/L phosphate buffer solution, pH 6.8 to make
exactly 100 mL. Take exactly a suitable amount of this solu-
tion, add 0.1 mol/L phosphate buffer solution, pH 6.8 to
make solutions so that each mL contains 30 /ug (potency) and
15 fig (potency), and use these solutions as the high concen-
tration sample solution and low concentration sample
solution, respectively.
Containers and storage Containers — Tight containers.
Boric Acid
**a
H 3 B0 3 : 61.83
Boric Acid, when dried,
99.5% of H3BO3.
contains not less than
Description Boric Acid occurs as colorless or white crystals
or crystalline powder. It is odorless, and has a slight, charac-
teristic taste.
It is freely soluble in warm water, in hot ethanol (95) and in
glycerin, soluble in water and in ethanol (95), and practically
insoluble in diethyl ether.
The pH of a solution of Boric Acid (1 in 20) is between 3.5
and 4.1.
Identification A solution of Boric Acid (1 in 20) responds to
the Qualitative Tests <1.09> for borate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Boric Acid in 25 mL of water or in 10 mL of hot ethanol (95):
the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Boric
Acid according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 0.40 g
of Boric Acid according to Method 1, and perform the test
(not more than 5 ppm).
Loss on drying <2.41> Not more than 0.5% (2 g, silica gel,
5 hours).
Assay Weigh accurately about 1.5 g of Boric Acid, previ-
ously dried, add 15 g of D-sorbitol and 50 mL of water, and
dissolve by warming. After cooling, titrate <2.50> with 1
mol/L sodium hydroxide VS (indicator: 2 drops of
phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 61.83 mg of H3BO3
Containers and storage Containers — Well-closed contain-
ers.
Freeze-dried Botulism Antitoxin,
Equine
Freeze-dried Botulism Antitoxin, Equine, is a prepa-
ration for injection which is dissolved before use.
It contains botulism antitoxin type A, botulism an-
titoxin type B, botulism antitoxin type E and botulism
antitoxin type F in immunoglobulin of horse origin. It
may contain one, two or three of these four antitoxins.
It conforms to the requirements of Freeze-dried
Botulism Antitoxin, Equine, in the Minimum Require-
ments for Biological Products.
Description Freeze-dried Botulism Antitoxin, Equine,
becomes a colorless or yellow-brown, clear liquid or a slightly
white-turbid liquid on the addition of solvent.
Bromazepam
~/n "7-tf/\°A
C 14 H 10 BrN3O: 316.15
7-Bromo-5-(pyridin-2-yl)-l,3-dihydro-2//-l,4-
benzodiazepin-2-one [1812-30-2]
JPXV
Official Monographs / Bromhexine Hydrochloride
375
Bromazepam, when dried, contains not less than
99.0% and not more than 101.0% of C 14 H 10 BrN 3 O.
Description Bromazepam occurs as white to light yellowish
white crystals or crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
methanol, in ethanol (99.5) and in acetone, and practically
insoluble in water.
Melting point: about 245°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Bromazepam in ethanol (99.5) (1 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Bromazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bromazepam in a platinum crucible according to Method 4,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 50 mg of Bromazepam
in 5 mL of a mixture of acetone and methanol (3:2), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, and add the mixture of acetone and methanol (3:2)
to make exactly 50 mL. Pipet 5 mL of this solution, add the
mixture of acetone and methanol (3:2) to make exactly 50
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 20 /uL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, ammonia solution (28)
and ethanol (99.5) (38:1:1) to a distance of about 12 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution and the spot of the starting point
are not more than 2, and not more intense than the spot from
the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, 105°C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Bromazepam, previ-
ously dried, dissolve in 80 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.62 mg of C 14 H 10 BrN 3 O
Containers and storage Containers — Well-closed contain-
ers.
Bromhexine Hydrochloride
7*nA^+->>±£lgi£
Br..
.-;'-"■
Xi
^
•HCI
C 14 H 20 Br 2 N 2 .HCl: 412.59
2-Amino-3,5-dibromo-/V-cyclohexyl-/V-
methylbenzylamine monohydrochloride [611-75-6]
Bromhexine Hydrochloride, when dried, contains
not less than 98.5% of C 14 H 20 Br 2 N 2 .HCl.
Description Bromhexine Hydrochloride occurs as white
crystals or crystalline powder.
It is freely soluble in formic acid, sparingly soluble in
methanol, and slightly soluble in water and in ethanol (95).
The pH of its saturated solution is between 3.0 and 5.0.
Melting point: about 239°C (with decomposition).
Identification (1) Dissolve 3 mg of Bromhexine Hydro-
chloride in 0.01 mol/L hydrochloric acid TS to make 100
mL. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Brom-
hexine Hydrochloride as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Infrared Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) Add 20 mL of water to 1 g of Bromhexine Hydro-
chloride. After thorough shaking, add 3 mL of sodium
hydroxide TS, and extract with four 20-mL portions of
diethyl ether. Neutralize the water layer with dilute nitric
acid: the solution responds to the Qualitative Tests <1.09> (2)
for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Bromhexine Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve 50
mg of Bromhexine Hydrochloride in 10 mL of methanol, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, and add the mobile phase to make exactly 20
mL. Pipet 1 mL of this solution, add the mobile phase to
make exactly 25 mL, and use this solution as the standard so-
lution. Perform the test with exactly 5 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: each peak area other than bromhexine is not
larger than the peak area of bromhexine of the standard
solution.
376 Bromocriptine Mesilate / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 245 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 Lira in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of potassium dihydrogen
phosphate in 900 mL of water, adjust the pH to 7.0 with 0.5
mol/L sodium hydroxide TS, and add water to make 1000
mL. To 200 mL of this solution add 800 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
bromhexine is about 6 minutes.
Selection of column: To 0.05 g of bamethane sulfate add
0.5 mL of the sample solution, and add the mobile phase to
make 10 mL. Proceed with 5 iiL of this solution under the
above operating conditions, and calculate the resolution. Use
a column giving elution of bamethane and bromhexine in this
order with the resolution between these peaks being not less
than 7.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of bromhexine from 5 iiL of the standard
solution is between 5 mm and 15 mm.
Time span of measurement: About 2 times as long as the
retention time of bromhexine beginning after the solvent
peak.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Bromhexine
Hydrochloride, previously dried, dissolve in 2 mL of formic
acid, add 60 mL of acetic anhydride, and warm in a water
bath at 50°C for 15 minutes. After cooling, titrate <2.50> with
0.1 mol/L perchloric acid VS until the color of the solution
changes from purple through blue-green to yellow-green (in-
dicator: 2 drops of crystal violet TS). Perform a blank deter-
mination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 41.26 mg of C 14 H 20 Br 2 N 2 .HCl
Containers and storage Containers — Well-closed containers.
Storage — Light-resistant.
Bromocriptine Mesilate
7n=E?U -/*> / -> ;ug±£
• H,,C-SO a H
C 32 H4 BrN 5 O5.CH 4 O3S: 750.70
(5 ' S)-2-Bromo- 1 2' -hydroxy-2' -(1 -methylethyl)-5 ' -
(2-methylpropyl)ergotaman-3 ' ,6' , 1 8-trione
monomethanesulfonate [22260-51-1 ]
Bromocriptine Mesilate contains not less than 98.0%
of C 3 2H4oBrN505.CH40 3 S, calculated on the dried
basis.
Description Bromocriptine Mesilate occurs as a white to
pale yellowish white or pale brownish white, crystalline pow-
der. It is odorless, or has a faint characteristic odor.
It is very soluble in acetic acid (100), freely soluble in
methanol, sparingly soluble in ethanol (95), very slightly
soluble in acetic anhydride, in dichloromethane and in chlo-
roform, and practically insoluble in water and in diethyl
ether.
It is gradually colored by light.
Identification (1) Dissolve 2 mg of Bromocriptine Mesi-
late in 1 mL of methanol, add 2 mL of 4-dimethylaminoben-
zaldehyde-ferric chloride TS, and shake: a purplish blue
color develops.
(2) Determine the absorption spectrum of a solution of
Bromocriptine Mesilate in methanol (3 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Bromocriptine Mesilate as directed in the paste method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(4) Perform the test with Bromocriptine Mesilate as
directed under Flame Coloration Test <1.04> (2): a green
color appears.
Optical rotation <2.49> [a]™: +95 - +105° [0.1 g, calculat-
ed on the dried basis, a mixture of methanol and
dichloromethane (1:1), 10 mL, 100 mm].
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Bromocriptine Mesilate in 10 mL of methanol: the
solution is clear, and has no more color than the following
control solution.
Control solution: To 2.5 mL of Cobalt (II) Chloride
Colorimetric Stock Solution, 6.0 mL of Iron (III) Chloride
Colorimetric Stock Solution and 1.0 mL of Copper (II) Sul-
fate Colorimetric Stock Solution add diluted hydrochloric
acid (1 in 40) to make exactly 100 mL.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Bromocriptine Mesilate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
(3) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Bromocriptine Mesilate in 10 mL of a mixture of
methanol and chloroform (1:1), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add a
mixture of methanol and chloroform (1:1) to make exactly
200 mL, and use this solution as the standard solution (1).
Pipet 10 mL of the standard solution (1), add a mixture of
methanol and chloroform (1:1) to make exactly 20 mL, and
use this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 iiL each of the sample solution
JPXV
Official Monographs / Bucillamine 377
and standard solutions (1) and (2), as a band with 1 cm in
width, on a plate of silica gel for thin-layer chromatography.
Develop the plate immediately with a mixture of
dichloromethane, 1,4-dioxane, ethanol (95) and ammonia
solution (28) (1800:150:50:1) to a distance of about 10 cm,
and dry the plate under reduced pressure for 30 minutes.
Spray evenly Dragendorff's TS for spraying on the plate, then
spray evenly hydrogen peroxide TS, cover the plate with a
glass plate, and examine: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution (1), and the spot other than
the principal spot, which is more intense than the spot from
the standard solution (2), is not more than one.
Loss on drying <2.41> Not more than 3.0% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 80°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Bromocriptine
Mesilate, dissolve in 80 mL of a mixture of acetic anhydride
and acetic acid (100) (7:1), and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 75.07 mg of C 32 H4 BrN5O5.CH 4 O3S
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding — 18°C.
Bromovalerylurea
-/□^huujkss
CH 3 O
H,C
and enantiomer
C 6 H„BrN 2 2 : 223.07
(2/?5')-(2-Bromo-3-methylbutanoyl)urea
[496-67-3]
Bromovalerylurea, when dried, contains not less
than 98.0% of C 6 H„BrN 2 2 .
Description Bromovalerylurea occurs as colorless or white
crystals or crystalline powder. It is odorless, and has a slight-
ly bitter taste.
It is soluble in ethanol (95), sparingly soluble in diethyl
ether, and very slightly soluble in water.
It dissolves in sulfuric acid, in nitric acid and in
hydrochloric acid, and precipitates are produced on the addi-
tion of water.
It dissolves in sodium hydroxide TS.
Identification (1) Boil 0.2 g of Bromovalerylurea with 5
mL of a solution of sodium hydroxide (1 in 10): the gas
evolved changes moistened red litmus paper to blue. Boil this
solution with an excess of dilute sulfuric acid: the odor of
valeric acid is perceptible.
(2) To 0. 1 g of Bromovalerylurea add 0.5 g of anhydrous
sodium carbonate, and decompose thoroughly by gentle
heating. Dissolve the residue in 5 mL of hot water, cool,
acidify with acetic acid (31), and filter: the filtrate responds to
the Qualitative Tests <1.09> (2) for bromide.
Melting point <2.60> 151 - 155°C
Purity (1) Acidity or alkalinity — To 1.5 g of
Bromovalerylurea add 30 mL of water, shake for 5 minutes,
and filter: the filtrate is neutral.
(2) Chloride <1.03>— Perform the test with a 10-mL por-
tion of the filtrate obtained in (1). Prepare the control solu-
tion with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not
more than 0.028%).
(3) Sulfate <1.14>— Perform the test with 10 mL of the
filtrate obtained in (1). Prepare the control solution with 0.40
mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of
Bromovalerylurea according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(5) Arsenic <1.U> — Dissolve 0.5 g of Bromovalerylurea
in 5 mL of sodium hydroxide TS, use this solution as the test
solution, and perform the test (not more than 4 ppm).
(6) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Bromovalerylurea: the solution is not more
colored than Matching Fluid A.
Loss on drying <2.41> Not more than 0.5% (1 g, 80°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Bromovalerylurea,
previously dried, in a 300-mL conical flask, add 40 mL of
sodium hydroxide TS, and boil gently for 20 minutes under a
reflux condenser. Cool, wash the lower part of the reflux con-
denser and the mouth of the flask with 30 mL of water, and
combine the washings with the solution in the conical flask.
Add 5 mL of nitric acid and exactly 30 mL of 0.1 mol/L sil-
ver nitrate VS, and titrate <2.50> the excess silver nitrate with
0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of
ammonium iron (III) sulfate TS). Perform a blank determi-
nation.
Each mL of 0.1 mol/L silver nitrate VS
= 22.31 mg of C 6 H„BrN 2 2
Containers and storage Containers — Well-closed contain-
ers.
Bucillamine
7->7; >
HS """X
H NH
C0 2 H
SH
H.,C CH 3
C 7 H 13 N0 3 S 2 : 223.31
(2i?)-2-(2-Methyl-2-sulfanylpropanoylamino)-3-
sulfanylpropanoic acid
[65002-17-7]
Bucillamine, when dried, contains not less than
98.5% and not more than 101.0% of C 7 H 13 N0 3 S 2 .
Description Bucillamine occurs as white, crystals or crystal-
378
Bucumolol Hydrochloride / Official Monographs
JP XV
line powder.
It is freely soluble in methanol and in ethanol (95), and
slightly soluble in water.
Identification (1) To 5 mL of a solution of Bucillamine (1
in 250) add 2 mL of sodium hydroxide TS and 2 drops of so-
dium pentacyanonitrosylferrate (III) TS: the solution reveals
a red-purple color.
(2) Determine the infrared absorption spectrum of Bucil-
lamine as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a£°: +33.0 - +36.5° (after
drying, 2 g, ethanol (95), 50 mL, 100 mm).
Melting point <2.60> 136 - 140°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bucillamine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bucillamine according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 60 mg of Bucillamine in
20 mL of a mixture of water and methanol (1:1), and use this
solution as the sample solution. Pipet 3 mL of the sample so-
lution, add the mixture of water and methanol (1:1) to make
exactly 200 mL, and use this solution as the standard solu-
tion. Immediately perform the test with exactly 20 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine each peak area by the automatic
integration method: the peak areas of related substances,
having the relative retention time of about 2.3 and 3.1 with
respect to the peak of bucillamine, obtained from the sample
solution are not larger than 8/15 times and 2/5 times the peak
area of bucillamine from the standard solution, respectively,
and the area of the peak other than the peaks of bucillamine
and two of the related substances mentioned above from the
sample solution is not larger than 1/5 times the peak area of
bucillamine from the standard solution. The total area of the
peaks other than bucillamine from the sample solution is not
larger than the peak area of bucillamine from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.01 mol/L citric acid TS and
methanol (1:1).
Flow rate: Adjust the flow rate so that the retention time of
bucillamine is about 5 minutes.
Time span of measurement: About 7 times as long as the
retention time of bucillamine beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mixture of water and methanol (1 : 1) to
make exactly 10 mL. Confirm that the peak area of bucilla-
mine obtained with 20 [iL of this solution is equivalent to 7 to
13% of that obtained with 20 /xL of the standard solution.
System performance: Dissolve 0.10 g of bucillamine and 10
mg of 4-fluorobenzoic acid in 100 mL of methanol. To 10 mL
of this solution add water to make exactly 50 mL. When the
procedure is run with 20 /xL of this solution under the above
operating conditions, bucillamine and 4-fluorobenzoic acid
are eluted in this order with the resolution between these
peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
bucillamine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 6 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Bucillamine, dis-
solve in 35 mL of methanol, add 15 mL of water, and titrate
<2.50> with 0.05 mol/L iodine VS (potentiometric titration).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.05 mol/L iodine VS
= 11.17 mg of C 7 H 13 N0 3 S 2
Containers and storage Containers — Tight containers.
Bucumolol Hydrochloride
-fi^u-ntsm^
H OH
•HCI
and enantiomer
C 17 H 23 N0 4 .HC1: 341.83
8-{(2/?5)-3-[(l,l-Dimethylethyl)amino]-2-
hydroxypropyloxy}-5-methylchromen-2-one
monohydrochloride [36556-75-9]
Bucumolol Hydrochloride, when dried, contains not
less than 99.0% of C 17 H 23 N0 4 .HC1.
Description Bucumolol Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in water, sparingly soluble in methanol
and in ethanol (95), slightly soluble in acetic acid (100), and
practically insoluble in diethyl ether.
Melting point: about 228°C (with decomposition).
Identification (1) Dissolve 0.01 g of Bucumolol
Hydrochloride in 10 mL of diluted ethanol (95) (1 in 2), and
observe under ultraviolet light (main wavelength: 365 nm):
the solution shows a yellow-green fluorescence. Render this
solution alkaline by adding sodium hydroxide TS: the
fluorescence disappears. Acidify the solution by adding dilute
hydrochloric acid: the fluorescence reappears.
(2) Dissolve 0.1 g of Bucumolol Hydrochloride in 5 mL
JP XV
Official Monographs / Bufetolol Hydrochloride
379
of water, and add 5 drops of Reinecke salt TS: a light red
precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Bucumolol Hydrochloride (1 in 60,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of
Bucumolol Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(5) A solution of Bucumolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Absorbance <2.24> E& (296 nm): 330 - 360 (after drying,
40 mg, water, 2500 mL).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Bucumolol Hydrochloride in 20 mL of water: the solution is
clear and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Bucumolol Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bucumolol Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Bucumolol
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, and
add methanol to make exactly 50 mL. Pipet 5 mL of this
solution, add methanol to make exactly 25 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of methanol and ammonia-ammonium chloride buffer solu-
tion, pH 10.7, (30: 1) to a distance of about 12 cm, and air-d-
ry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Bucumolol
Hydrochloride, previously dried, add 45 mL of acetic acid
(100), dissolve by warming at 60°C, and cool. Add 105 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.18 mg of C 17 H 23 N0 4 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Bufetolol Hydrochloride
^7i r-n-;U£lg±£
C 18 H 29 N0 4 .HC1: 359.89
l-(l,l-Dimethylethyl)amino-3-[2-(tetrahydrofuran-
2-ylmethoxy)phenoxy]propan-2-ol monohydrochloride
[35108-88-4]
Bufetolol Hydrochloride, when dried, contains not
less than 98.5% of C 18 H 29 N0 4 .HC1.
Description Bufetolol Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in water and in methanol, soluble in
ethanol (95) and in acetic acid (100), and practically insoluble
in diethyl ether.
A solution of Bufetolol Hydrochloride (1 in 10) is optically
inactive.
Identification (1) To 5 mL of a solution of Bufetolol
Hydrochloride (1 in 100) add 5 drops of Reinecke salt TS: a
light red precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Bufetolol Hydrochloride (1 in 20,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Bufetolol Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Bufetolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 153 - 157 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Bufetolol Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.5 g of
Bufetolol Hydrochloride. Prepare the control solution with
0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.038%).
(3) Heavy metals <1. 07>— Proceed with 2.0 g of Bufetolol
Hydrochloride according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Related substances — Dissolve 0.20 g of Bufetolol
Hydrochloride in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
380
Bufexamac / Official Monographs
JP XV
10 nL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, acetone, ethanol (95) and ammonia solution (28)
(40:20:5:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Bufetolol
Hydrochloride, previously dried, dissolve in 10 mL of acetic
acid (100), add 50 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 35.99 mg of C 18 H 29 N0 4 .HC1
Containers and storage Containers — Tight containers.
Bufexamac
■/7i*t7^
C 12 H 17 N0 3 : 223.27
2-(4-Butyloxyphenyl)-/V-hydroxy acetamide
[2438-72-4]
Bufexamac, when dried,
98.0% of C 12 H 17 N0 3 .
contains not less than
Description Bufexamac occurs as white to pale yellowish
white crystals or crystalline powder. It has a faint, charac-
teristic odor, and is tasteless.
It is freely soluble in 7V,./V-dimethylformamide, sparingly
soluble in methanol and in ethanol (95), and practically in-
soluble in water and in diethyl ether.
Melting point: about 162°C (with decomposition).
Identification (1) To 5 mL of a solution of Bufexamac in
methanol (1 in 5000) add 1 drop of iron (III) chloride-
methanol TS, and shake: a dark red color develops.
(2) Determine the absorption spectrum of a solution of
Bufexamac in ethanol (95) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Bufexamac as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Bufexamac in 20 mL of ethanol (95): the solution is clear
and colorless.
(2) Heavy metals </. 07>— Proceed with 2.0 g of Bufex-
amac according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bufexamac according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Bufexamac in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Use a plate of sili-
ca gel with fluorescent indicator for thin-layer chro-
matography, moisten the surface of the plate evenly by
spraying with 0.1 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate TS, and dry at 110°C for about 30 minutes.
Spot 15 /xL each of the sample solution and standard solution
on the plate. Develop the plate with a mixture of chloroform,
cyclohexane, methanol and acetic acid (100) (6:4:1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Bufexamac, previ-
ously dried, dissolve in 40 mL of iV,./V-dimethylformamide,
and titrate <2.50> with 0.1 mol/L tetramethylammonium
hydroxide-methanol VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide-methanol VS
= 22.33 mg of C 12 H 17 N0 3
Containers and storage Containers — Tight containers.
Bufexamac Cream
Bufexamac Cream contains not less than 90% and
not more than 1 10% of the labeled amount of bufex-
amac (C 12 H 17 N0 3 : 223.27).
Method of preparation
ments, with Bufexamac.
Prepared as directed under Oint-
Description Bufexamac Cream is white.
pH: 4.0-6.0
Identification To a quantity of Bufexamac Cream, equiva-
lent to 0.05 g of Bufexamac according to the labeled amount,
add 10 mL of tetrahydrofuran, shake well, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 0.05 g of bufexamac for assay in 10 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Use a plate of silica gel for thin-layer
JP XV
Official Monographs / Bufexamac Ointment
381
chromatography, moisten the surface of the plate evenly by
spraying with 0.1 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate TS, and dry the plate at 110°C for about 30
minutes. Spot 5 fiL each of the sample solution and standard
solution on the plate. Develop the plate with a mixture of
pentane, ethyl acetate and acetic acid (100) (7:4:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly iron
(III) chloride TS on the plate: the spot from the sample solu-
tion and that from the standard solution show a red-brown
color and the same Rf value.
Assay Weigh accurately a quantity of Bufexamac Cream,
equivalent to about 50 mg of bufexamac (Q2IL7NO3), dis-
solve in 40 mL of methanol, and add methanol to make ex-
actly 50 mL. Pipet 10 mL of this solution, add exactly 5 mL
of the internal standard solution and add the mobile phase to
make 100 mL, filter, and use the filtrate as the sample solu-
tion. Separately, weigh accurately about 50 mg of bufexamac
for assay, previously dried at 105°C for 4 hours, and dissolve
in methanol to make exactly 50 mL. Pipet 10 mL of this solu-
tion, add exactly 5 mL of the internal standard solution, add
the mobile phase to make 100 mL, and use this solution as the
standard solution. Perform the test with 20 /iL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of bufexamac to that of the internal standard, respectively.
Amount (mg) of bufexamac (C I2 H 17 N0 3 )
= W s x (Q T /Q S )
W s : Amount (mg) of bufexamac for assay
Internal standard solution — A solution of diphenylimidazole
in methanol (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.5 g of sodium 1 -octane sulfonate
and 0.6 g of disodium dihydrogen ethylenediamine tetraa-
cetate dihydrate in 850 mL of water, and add 400 mL of
methanol, 400 mL of acetonitrile and 8 mL of acetic acid
(100).
Flow rate: Adjust the flow rate so that the retention time of
bufexamac is about 6 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, bufexamac and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of bufexamac to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Bufexamac Ointment
Bufexamac Ointment contains not less than 90%
and not more than 110% of the labeled amount of
bufexamac (C 12 H 17 N0 3 : 223.27).
Method of preparation Prepare as directed under Oint-
ments, with Bufexamac.
Identification To a quantity of Bufexamac Ointment,
equivalent to 0.05 g of Bufexamac according to the labeled
amount, add 5 mL of tetrahydrofuran, shake well, add 5 mL
of ethanol (99.5), shake, centrifuge, and use the supernatant
liquid as the sample solution. Separately, dissolve 0.05 g of
bufexamac for assay in 10 mL of methanol, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Use a plate of silica gel for thin-layer chromatography,
moisten the surface of the plate evenly by spraying with 0.1
mol/L disodium dihydrogen ethylenediamine tetraacetate
TS, and dry the plate at 110°C for about 30 minutes. Spot 5
/xL each of the sample solution and standard solution on the
plate. Develop the plate with a mixture of pentane, ethyl
acetate and acetic acid (100) (7:4:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly iron (III) chloride TS
on the plate: the spot from the sample solution and that from
the standard solution show a red-brown color and the same
Rf value.
Assay Weigh accurately a quantity of Bufexamac Oint-
ment, equivalent to about 50 mg of bufexamac (Q2IL7NO3),
add 40 mL of tetrahydrofuran, warm to 40°C, dissolve by
shaking, cool, and add tetrahydrofuran to make exactly 50
mL. Pipet 10 mL of this solution, add exactly 5 mL of the in-
ternal standard solution and the mobile phase to make 100
mL, and filter, if necessary, through a membrane filter of
0.45-^m porosity. Discard the first 20 mL of the filtrate, and
use the subsequent filtrate as the sample solution. Separately,
weigh accurately about 50 mg of bufexamac for assay, previ-
ously dried at 105 °C for 4 hours, and dissolve in tetra-
hydrofuran to make exactly 50 mL. Pipet 10 mL of this solu-
tion, add exactly 5 mL of the internal standard solution and
the mobile phase to make 100 mL, and use this solution as the
standard solution. Perform the test with 20 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, g T and Q s , of the peak area
of bufexamac to that of the internal standard, respectively.
Amount (mg) of bufexamac (C 12 H 17 N0 3 )
= W s x (Q T /Q S )
W s : Amount (mg) of bufexamac for assay
Internal standard solution — A solution of diphenylimidazole
in methanol (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
382 Bumetanide / Official Monographs
JP XV
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.5 g of sodium 1 -octane sulfonate
and 0.6 g of disodium dihydrogen ethylenediamine tetraa-
cetate dihydrate in 850 mL of water, and add 400 mL of
methanol, 400 mL of acetonitrile and 8 mL of acetic acid
(100).
Flow rate: Adjust the flow rate so that the retention time of
bufexamac is about 6 minutes.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, bufexamac and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of bufexamac to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Bumetanide
-f**-Y
C 17 H 20 N 2 O 5 S: 364.42
3-Butylamino-4-phenoxy-5-sulfamoylbenzoic acid
[28395-03-1]
Bumetanide, when dried, contains not less than
98.5% of Ci 7 H 2 oN 2 5 S.
Description Bumetanide occurs as white crystals or crystal-
line powder.
It is freely soluble in pyridine, soluble in methanol and in
ethanol (95), slightly soluble in diethyl ether, and practically
insoluble in water.
It dissolves in potassium hydroxide TS.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Bumetanide in 1 mL
of pyridine, add 2 drops of copper (II) sulfate TS, shake, add
3 mL of water and 5 mL of chloroform, shake, and allow to
stand: a light blue color develops in the chloroform layer.
(2) Dissolve 0.04 g of Bumetanide in 100 mL of phos-
phate buffer solution, pH 7.0, and dilute 10 mL of the solu-
tion with water to make 100 mL. Determine the absorption
spectrum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Bumetanide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 232 - 237 °C
Purity (1) Clarity and color of solution — Dissolve 50 mg
of Bumetanide in 2 mL of a solution of potassium hydroxide
(1 in 30) and 8 mL of water: the solution is clear, and is not
more colored than the following control solution.
Control solution: Pipet 0.5 mL each of Cobalt (II) Chlo-
ride Colorimetric Stock Solution, Iron (III) Chloride Colori-
metric Stock Solution and Copper (II) Sulfate Colorimetric
Stock Solution, mix them, and add diluted hydrochloric acid
(1 in 40) to make exactly 100 mL.
(2) Chloride <1.03>— Mix well 0.5 g of Bumetanide with
0.7 g of potassium nitrate and 1.2 g of anhydrous sodium
carbonate, transfer, in small portions, to a red-hot platinum
crucible, and heat to red-hot until the reaction is complete.
After cooling, to the residue add 14 mL of dilute sulfuric acid
and 6 mL of water, boil for 5 minutes, filter, wash the residue
with 10 mL of water, combine the filtrate and the washing,
and add 6 mL of dilute nitric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.30 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.021%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of
Bumetanide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bumetanide according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Bumetanide in 10 mL of methanol, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, and add methanol to make exactly 100 mL. Pipet 2
mL of this solution, add methanol to make exactly 10 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, acetic acid (100), cyclohexane
and methanol (32:4:4:1) to a distance of about 12 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Bumetanide, previ-
ously dried, dissolve in 50 mL of ethanol (95), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 36.44 mg of C I7 H 20 N 2 O 5 S
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Bupranolol Hydrochloride
383
Storage — Light-resistant.
Bunazosin Hydrochloride
-?->->/ ~>>ikmik
o
CHj
H 3 C
H 3 C^
N Y N
N
NH ?
•HCI
C 19 H 27 N 5 3 .HC1: 409.91
4-Amino-2-(4-butanoyl-l,4-diazepan-l-yl)-6,7-
dimethoxyquinazoline monohydrochloride [72712-76-2]
Bunazosin Hydrochloride, when dried, contains not
less than 98.0% of C 19 H 27 N 5 3 .HC1.
Description Bunazosin Hydrochloride occurs as a white
crystalline powder.
It is very soluble in formic acid, slightly soluble in water
and in methanol, very slightly soluble in ethanol (99.5), and
practically insoluble in diethyl ether.
Melting point: about 273 °C (with decomposition).
Identification (1) Dissolve 0.1 g of Bunazosin Hydrochlo-
ride in 10 mL of 0.2mol/L hydrochloric acid TS, and boil
for 3 minutes over a flame: butylic acid like odor is percepti-
ble.
(2) Determine the infrared absorption spectrum of
Bunazosin Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Bunazosin Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Bunazosin Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.05 g of Bunazosin
Hydrochloride in 50 mL of the mobile phase, and use this so-
lution as the sample solution. To exactly 1 mL of the sample
solution add the mobile phase to make exactly 200 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of both solutions by the automatic integration method:
the total area of the peaks other than the peak of bunazosin
from the sample solution is not larger than the peak area of
bunazosin from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /xm in parti-
cle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 1.44 g of sodium lauryl sulfate in a
suitable amount of water, add 10 mL of acetic acid (100), 500
mL of acetonitrile and water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
bunazosin is about 5 minutes.
Selection of column: Proceed with 20 /uL of a mixture of
the standard solution and a solution of procaine hydrochlo-
ride in the mobile phase (1 in 20,000) (1:1) under the above
operating conditions, and calculate the resolution. Use a
column giving elution of procaine and bunazosin in this or-
der with the resolution between these peaks being not less
than 3.0.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of bunazosin obtained from 20 uL of the
standard solution is 20 to 60% of the full-scale.
Time span of measurement: About 6 times of the retention
time of bunazosin.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Bunazosin
Hydrochloride, previously dried, dissolve in 6 mL of formic
acid, add exactly 15 mL of 0.1 mol/L perchloric acid, and
heat for 20 minutes on a water bath. After cooling, add 20
mL of acetic acid (100), and titrate <2.50> the excess perchlor-
ic acid with 0.1 mol/L sodium acetate VS (potentiometric
titration). Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 40.99 mg of C 19 H 27 N 5 3 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Bupranolol Hydrochloride
V PH H
U JL HaC CH 3
and enantiomer
C 14 H 22 C1N0 2 .HC1: 308.24
(2i?5>3-(2-Chloro-5-methylphenoxy)-l -(1 , 1 -
dimethylethyl)aminopropan-2-ol monohydrochloride
[15148-80-8]
Bupranolol Hydrochloride, when dried, contains not
less than 98.0% of C 14 H 22 C1N0 2 .HC1.
Description Bupranolol Hydrochloride occurs as a white,
crystalline powder.
It is sparingly soluble in methanol, slightly soluble in
water, in ethanol (95) and in acetic acid (100), very slightly
soluble in acetic anhydride, and practically insoluble in
diethyl ether.
The pH of a solution of Bupranolol Hydrochloride (1 in
384
Busulfan / Official Monographs
JP XV
1000) is between 5.2 and 6.2.
Identification (1) Take 0.01 g of Bupranolol Hydrochlo-
ride in a test tube, mix with 25 mg of potassium iodide and 25
mg of oxalic acid dihydrate, cover the mouth of the test tube
with filter paper moistened with a solution of 2,6-dibromo-/V-
chloro-l,4-benzoquinone monoimine in ethanol (95) (1 in
100), and heat gently for several minutes. Expose the filter
paper to ammonia gas: the filter paper acquires a blue color.
(2) Determine the absorption spectrum of a solution of
Bupranolol Hydrochloride in 0.1 mol/L hydrochloric acid
TS (1 in 10,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Bupranolol Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Bupranolol Hydrochloride (1 in 200)
responds to the Qualitative Tests <1.09> for chloride.
Absorbance <2.24> £}* (275 nm): 57 - 60 (after drying, 50
mg, 0.1 mol/L hydrochloric acid TS, 500 mL).
Melting point <2.60> 223 - 226°C
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Bupranolol Hydrochloride in 15 mL of water: the solution
is clear and colorless.
(2) Acidity — Dissolve 0.10 g of Bupranolol Hydrochlo-
ride in 15 mL of freshly boiled and cooled water, and add 1
drop of methyl red TS: a light red color develops. To this so-
lution add 0.05 mL of 0.01 mol/L sodium hydroxide VS: the
color changes to yellow.
(3) Sulfate <1.14>— Perform the test with 0.10 g of
Bupranolol Hydrochloride. Prepare the control solution with
0.35 mL of 0.005 mol/L sulfuric acid VS (not more than
0.168%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Bupranolol Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Bupranolol Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(6) Related substances — Dissolve 0.30 g of Bupranolol
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of polyamide with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
methanol, ammonia solution (28) and water (16:4:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.18 g of Bupranolol
Hydrochloride, previously dried, dissolve in 60 mL of a mix-
ture of acetic anhydride and acetic acid (100) (2:1) by warm-
ing, cool, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.82 mg of C 14 H 22 C1N0 2 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Busulfan
~/X)1~7t>
C 6 H 14 6 S 2 : 246.30
Tetramethylenedimethanesulf onate [55-95-7 ]
Busulfan contains not less than
C 6 H 14 6 S 2 , calculated on the dried basis.
98.5'
of
Description Busulfan occurs as a white, crystalline powder.
It is slightly soluble in diethyl ether, very slightly soluble in
ethanol (95), and practically insoluble in water.
Identification (1) To 0.1 g of Busulfan add 10 mL of
water and 5 mL of sodium hydroxide TS, dissolve by heating,
and use this solution as the sample solution.
(i) To 7 mL of the sample solution add 1 drop of potassi-
um permanganate TS: the red-purple color of potassium per-
manganate TS changes from blue-purple through blue to
green.
(ii) Acidify 7 mL of the sample solution with dilute sul-
furic acid, and add 1 drop of potassium permanganate TS:
the color of potassium permanganate TS remains.
(2) Determine the infrared absorption spectrum of Busul-
fan, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Melting point <2.60> 115-118°C
Purity (1) Sulfate <1.14>— To 1.0 g of Busulfan add 40
mL of water, and dissolve by heating. Cool in ice for 15
minutes, and filter. Wash the residue with 5 mL of water,
combine the washings with the filtrate, and add 1 mL of
dilute hydrochloric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 0.40 mL of 0.005 mol/L sulfuric acid
VS (not more than 0.019%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Busulfan
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
JP XV
Official Monographs / Butropium Bromide
385
phosphorus (V) oxide, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Busulfan, add 40
mL of water, and boil gently under a reflux condenser for 30
minutes. Cool, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.32 mg of C 6 H 14 6 S 2
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Butropium Bromide
7 r- n tf^AJUk*
-.£■
C 28 H3 8 BrN0 4 : 532.51
(l/?,3r,5S)-8-(4-Butoxybenzyl)-3-[(2S)-hydroxy-2-
phenylpropanoyloxy]-8-methyl-8-azoniabicyclo[3.2.1]octane
bromide [29025-14-7]
Butropium Bromide, when dried, contains not less
than 98.0% of C 28 H 3 8BrN0 4 .
Description Butropium Bromide occurs as white crystals or
crystalline powder.
It is very soluble in formic acid, freely soluble in methanol,
soluble in ethanol (95), slightly soluble in water, and practi-
cally insoluble in diethyl ether and in acetic anhydride.
Identification (1) To 1 mg of Butropium Bromide add 3
drops of fuming nitric acid, and evaporate on a water bath to
dryness. Dissolve the residue in 1 mL of 7V,./V-dimethylfor-
mamide, and add 5 to 6 drops of tetraethylammonium
hydroxide TS: a red-purple color develops.
(2) Determine the absorption spectrum of a solution of
Butropium Bromide in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum 1: both
spectra exhibit similar intensities of absorption at the same
wavelengths. Separately, determine the absorption spectrum
of a solution of Butropium Bromide in methanol (1 in 5000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum 2: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(3) A solution of Butropium Bromide in methanol (1 in
20) responds to the Qualitative Tests <1.09> (1) for bromide.
Optical rotation <2.49> [a]p : - 14.0
drying, 0.5 g, methanol, 20 mL, 100 mm).
17.0° (after
Purity (1) Heavy metals <1.07> — Dissolve 1.0 g of Butro-
pium Bromide in 40 mL of ethanol (95), add 2 mL of dilute
acetic acid and water to make 50 mL. Perform the test, using
this solution as the test solution. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 20
ppm).
(2) Related substances — Dissolve 50 mg of Butropium
Bromide in 10 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 5 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine each peak area of
both solutions by the automatic integration method: the peak
area having a ratio of the retention time about 0.5 to butropi-
um from the sample solution is not larger than 1/4 of the
peak area from the standard solution, and the total area of all
peaks other than the peak eluted first, the peak having a ratio
of the retention time to butropium about 0.5 and butropium
peak from the sample solution is not larger than the peak
area from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.15 g of sodium lauryl sulfate in
1000 mL of a mixture of acetonitrile and 0.005 mol/L sulfur-
ic acid (3:2).
Flow rate: Adjust the flow rate so that the retention time of
butropium is about 5 minutes.
Selection of column: Dissolve 0.50 g of Butropium
Bromide in 9 mL of ethanol (99.5) and 1 mL of 0.1 mol/L
potassium hydroxide-ethanol TS, and heat at 70 °C for 15
minutes. After cooling, to 1 mL of this solution add the
mobile phase to make 100 mL. Proceed with 5 juL of this
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of the peak of
butropium and the peak having a ratio of the retention time
about 0.7 to butropium with the resolution between these
peaks being not less than 2.5.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of the butropium obtained from 5 /uL of
the standard solution is between 10 mm and 30 mm.
Time span of measurement: About twice as long as the
retention time of butropium.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.8 g of Butropium
Bromide, previously dried, dissolve in 5 mL of formic acid,
add 100 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid-dioxane VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid-dioxane VS
= 53.25 mg of C 28 H 38 BrN0 4
Containers and storage Containers — Well-closed contain-
ers.
386
Butyl Parahydroxybenzoate / Official Monographs
JP XV
Storage — Light-resistant.
Butyl Parahydroxybenzoate
CH 3
C u H 14 3 : 194.23
Butyl 4-hydroxybenzoate [94-26-8]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦)
Butyl Parahydroxybenzoate contains not less than
98.0% and not more than 102.0% of C„H 14 03.
♦Description Butyl Parahydroxybenzoate occurs as color-
less crystals or white, crystalline powder.
It is freely soluble in ethanol (95) and in acetone, and prac-
tically insoluble in water. »
Identification (1) The melting point <2.60> of Butyl Para-
hydroxybenzoate is between 68 °C and 71 C C.
*(2) Determine the infrared absorption spectrum of
Butyl Parahydroxybenzoate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.*
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Butyl Parahydroxybenzoate in 10 mL of ethanol (95): the so-
lution is clear and not more intensely colored than the follow-
ing control solution.
Control solution: To 5.0 mL of cobalt (II) chloride colori-
metric stock solution, 12.0 mL of iron (III) chloride colori-
metric stock solution and 2.0 mL of cupper (II) sulfate colori-
metric stock solution add water to make 1000 mL.
(2) Acidity — Dissolve 0.20 g of Butyl Parahydroxybenzo-
ate in 5 mL of ethanol (95), add 5 mL of freshly boiled and
cooled water and 0.1 mL of bromocresol green-sodium
hydroxide-ethanol TS, then add 0.1 mL of 0.1 mol/L sodium
hydroxide VS: the solution shows a blue color.
*(3) Heavy metals <1.07> — Dissolve 1.0 g of Butyl Para-
hydroxybenzoate in 25 mL of acetone, add 2 mL of dilute
acetic acid and water to make 50 mL, and perform the test
using this solution as the test solution. Prepare the control so-
lution as follows: to 2.0 mL of Standard Lead Solution add
25 mL of acetone, 2 mL of dilute acetic acid, and water to
make 50 mL (not more than 20 ppm).»
(4) Related substances — Dissolve 0.10 g of Butyl Para-
hydroxybenzoate in 10 mL of acetone, and use this solution
as the sample solution. Pipet 0.5 mL of the sample solution,
add acetone to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
[iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
methanol, water and acetic acid (100) (70:30:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot other than the
principal spot is not more intense than the spot obtained with
the standard solution.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Butyl Parahydrox-
ybenzoate, add exactly 20 mL of 1 mol/L sodium hydroxide
VS, heat at about 70°C for 1 hour, and immediately cool in
ice. Titrate <2.50> the excess sodium hydroxide with 0.5
mol/L sulfuric acid VS up to the second equivalent point
(potentiometric titration). Perform a blank determination.
Each mL of 1 mol/L sodium hydroxide VS
= 194.2 mg of C U H 14 03
♦Containers and storage Containers — Well-closed contain-
ers. »
Cacao Butter
Oleum Cacao
Cacao Butter is the fat obtained from the seed of
Theobroma cacao Linne (Sterculiaceae).
Description Cacao Butter occurs as a yellowish white, hard,
brittle mass. It has a slight, chocolate-like odor, and has no
odor of rancidity.
It is freely soluble in diethyl ether and in petroleum ether,
soluble in boiling ethanol (99.5), and very slightly soluble in
ethanol (95).
Congealing point of the fatty acids: 45 - 50°C
Melting point 31-35°C (Cram the sample into a capillary
tube without melting the sample).
Specific gravity <7.75> df Q : 0.895 - 0.904
Acid value <1.13> Not more than 3.0.
Saponification value <1.13> 188 - 195
Iodine value <7.75> 35 - 43
Containers and storage Containers — Well-closed contain-
ers.
Anhydrous Caffeine
l^?!^ >
CH 3
C 8 H 10 N 4 O 2 : 194.19
l,3,7-Trimethyl-l//-purine-2,6-(37 : /,77 I /)-dione
[58-08-2]
Anhydrous Caffeine, when dried, contains not less
JPXV
Official Monographs / Caffeine Hydrate
387
than 98.5% of CgH^NA,.
Description Anhydrous Caffeine occurs as white crystals or
powder. It is odorless, and has a bitter taste.
It is freely soluble in chloroform, sparingly soluble in
water, in acetic anhydride and in acetic acid (100), and slight-
ly soluble in ethanol (95) and in diethyl ether.
The pH of a solution of Anhydrous Caffeine (1 in 100) is
between 5.5 and 6.5
Identification (1) To 2 mL of a solution of Anhydrous
Caffeine (1 in 500) add tannic acid TS dropwise: a white
precipitate, which dissolves upon the dropwise addition of
tannic acid TS, is produced.
(2) To 0.01 g of Anhydrous Caffeine add 10 drops of
hydrogen peroxide TS and 1 drop of hydrochloric acid, and
evaporate on a water bath to dryness: the residue acquires a
yellow-red color. Invert the residue over a vessel containing 2
to 3 drops of ammonia TS: the color turns red-purple, and
disappears upon the addition of 2 to 3 drops of sodium
hydroxide TS.
(3) Dissolve 0.01 g of Anhydrous Caffeine in water to
make 50 mL. To 5 mL of this solution add 3 mL of diluted
acetic acid (31) (3 in 100) and 5 mL of pyridine (1 in 10), mix,
add 2 mL of diluted sodium hypochlorite TS (1 in 5), and
allow to stand for 1 minute. Add 2 mL of sodium thiosulfate
TS and 5 mL of sodium hydroxide TS to the solution: a yel-
low color develops.
Melting point <2.60> 235 - 238°C
Purity (1) Chloride <1.03> — Dissolve 2.0 g of Anhydrous
Caffeine in 80 mL of hot water, cool rapidly to 20°C, add
water to make 100 mL, and use this solution as the sample so-
lution. To 40 mL of the sample solution add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.011%).
(2) Sulfate <1.14>— To 40 mL of the sample solution ob-
tained in (1) add 1 mL of dilute hydrochloric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 0.40 mL of
0.005 mol/L sulfuric acid VS (not more than 0.024%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Anhy-
drous Caffeine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Related substances — Dissolve 0.10 g of Anhydrous
Caffeine in 10 mL of chloroform, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
chloroform to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add chloroform to make exactly 10 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform and ethanol (95) (9:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
(5) Readily carbonizable substances <1.15> — Perform the
test using 0.5 g of Anhydrous Caffeine: the solution is not
more colored than Matching Fluid D.
Loss on drying <2.4I> Not more than 0.5% (1 g, 80°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.4 g of Anhydrous
Caffeine, previously dried, dissolve in 70 mL of a mixture of
acetic anhydride and acetic acid (100) (6:1), and titrate <2.50>
with 0.1 mol/L perchloric acid VS until the solution changes
from purple through green to yellow (indicator: 3 drops of
crystal violet TS). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.42 mg of C 8 H 10 N 4 O 2
Containers and storage Containers — Tight containers.
Caffeine Hydrate
;b7i-i >7kW$i
•H 2
C 8 H 10 N 4 O 2 .H 2 O: 212.21
l,3,7-Trimethyl-l/f-purine-2,6-(3i7,7//)-dione
monohydrate [5743-12-4]
Caffeine Hydrate, when dried, contains not less than
98.5% of caffeine (C 8 H 10 N 4 O 2 : 194.19).
Description Caffeine Hydrate occurs as white, soft crystals
or powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in chloroform, sparingly soluble in
water, in acetic acid (100) and in acetic anhydride, slightly
soluble in ethanol (95), and very slightly soluble in diethyl
ether.
The pH of a solution of Caffeine Hydrate (1 in 100) is be-
tween 5.5 and 6.5.
It effloresces in dry air.
Identification (1) To 2 mL of a solution of Caffeine Hy-
drate (1 in 500) add tannic acid TS dropwise: a white
precipitate, which dissolves upon the dropwise addition of
tannic acid TS, is produced.
(2) To 0.01 g of Caffeine Hydrate add 10 drops of hydro-
gen peroxide TS and 1 drop of hydrochloric acid, and
evaporate to dryness on a water bath: the residue acquires a
yellow-red color. Invert the residue over a vessel containing 2
to 3 drops of ammonia TS: the color turns red-purple, and
disappears upon the addition of 2 to 3 drops of sodium
hydroxide TS.
(3) Dissolve 0.01 g of Caffeine Hydrate in water to make
50 mL. To 5 mL of this solution add 3 mL of diluted acetic
acid (31) (3 in 100) and 5 mL of a solution of pyridine (1 in
10), mix, add 2 mL of diluted sodium hypochlorite TS (1 in
5), and allow to stand for 1 minute. Add 2 mL of sodium
thiosulfate TS and 5 mL of sodium hydroxide TS to the solu-
tion: a yellow color develops.
Melting point <2.60> 235 - 238°C (after drying).
388
Caffeine and Sodium Benzoate / Official Monographs
JP XV
Purity (1) Chloride <1.03>— Dissolve 2.0 g of Caffeine
Hydrate in 80 mL of hot water, cool rapidly to 20°C, add
water to make 100 mL, and use this solution as the sample so-
lution. To 40 mL of the sample solution add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.011%).
(2) Sulfate <1.14>— To 40 mL of the sample solution ob-
tained in (1) add 1 mL of dilute hydrochloric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 0.40 mL of
0.005 mol/L sulfuric acid VS (not more than 0.024%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Caffeine
Hydrate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(4) Related substances — Dissolve 0.10 g of Caffeine Hy-
drate in 10 mL of chloroform, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
chloroform to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add chloroform to make exactly 10 mL, and use this so-
lution as the standard solution. Perform the test as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of chloroform
and ethanol (95) (9:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
(5) Readily carbonizable substances <1J5> — Perform the
test using 0.5 g of Caffeine Hydrate: the solution is not more
colored than Matching Fluid D.
Loss on drying <2.41> 0.5 - 8.5% (1 g, 80°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.4 g of Caffeine Hydrate,
previously dried, dissolve in 70 mL of a mixture of acetic an-
hydride and acetic acid (100) (6:1), and titrate <2.50> with 0.1
mol/L perchloric acid VS until the solution changes from
purple through green to yellow (indicator: 3 drops of crystal
violet TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.42 mg of C 8 H 10 N 4 O 2
Containers and storage Containers — Tight containers.
Caffeine and Sodium Benzoate
S*tith l J'>A*7i'f>
Caffeine and Sodium Benzoate, when dried, contains
not less than 48.0% and not more than 50.0% of
caffeine (C 8 H 10 N 4 O 2 : 194.19), and not less than 50.0%>
and not more than 52.0% of sodium benzoate
(C 7 H 5 Na0 2 : 144.10).
Description Caffeine and Sodium Benzoate occurs as a
white powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in water, soluble in acetic acid (100) and
in acetic anhydride, sparingly soluble in ethanol (95), and
practically insoluble in diethyl ether.
Identification (1) Dissolve 1 g of Caffeine and Sodium
Benzoate in 10 mL of water in a separator, add 1 drop of
phenolphthalein TS, and add carefully 0.01 mol/L sodium
hydroxide VS dropwise until a faint red color develops. Ex-
tract with three 20-mL portions of chloroform by thorough
shaking, and separate the chloroform layer from the water
layer. [Use the water layer for test (2).] Filter the combined
chloroform extracts, evaporate the filtrate to dryness on a
water bath, and proceed the following tests with the residue:
(i) To 2 mL of a solution of the residue (1 in 500) add tan-
nic acid TS dropwise: a white precipitate, which dissolves
upon the dropwise addition of tannic acid TS, is produced.
(ii) To 0.01 g of the residue add 10 drops of hydrogen
peroxide TS and 1 drop of hydrochloric acid, evaporate to
dryness on a water bath: the residue acquires a yellow-red
color. Invert the residue over a vessel containing 2 to 3 drops
of ammonia TS: the color turns red-purple, and disappears
upon the addition of 2 to 3 drops of sodium hydroxide TS.
(iii) Dissolve 0.01 g of the residue in water to make 50
mL. To 5 mL of this solution add 3 mL of diluted acetic acid
(31) (3 in 100) and 5 mL of a solution of pyridine (1 in 10),
mix, add 2 mL of diluted sodium hypochlorite TS (1 in 5),
and allow to stand for 1 minute. Add 2 mL of sodium
thiosulfate TS and 5 mL of sodium hydroxide TS to the
solution: a yellow color develops.
(2) To 5 mL of the water layer obtained in (1) add 5 mL
of water: the solution responds to the Qualitative Tests <1.09>
(2) for benzoate.
(3) Heat Caffeine and Sodium Benzoate: white fumes are
evolved. Ignite furthermore, and to the residue add
hydrochloric acid: bubbles are produced, and the solution
responds to the Qualitative Tests <1.09> (1) for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Caffeine and Sodium Benzoate in 5 mL of water: the solution
is clear and colorless.
(2) Alkalinity — Dissolve 1.0 g of Caffeine and Sodium
Benzoate in 20 mL of water, and add 1 or 2 drops of
phenolphthalein TS: no red color develops.
(3) Chloride <1.03>— Dissolve 0.5 g of Caffeine and Sodi-
um Benzoate in 10 mL of water, and add 30 mL of ethanol
(95), 6 mL of dilute nitric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.70 mL of 0.01 mol/L
hydrochloric acid VS, 30 mL of ethanol (95) and water to
make 50 mL (not more than 0.050%).
(4) Chlorinated compounds — Dissolve 1.0 g of Caffeine
and Sodium Benzoate in 40 mL of water, add 10 mL of dilute
sulfuric acid, and extract with two 20-mL portions of diethyl
ether. Allow the combined diethyl ether extracts to evaporate
at room temperature to dryness. Place this residue and 0.7 g
of calcium carbonate in a crucible, mix with a small amount
of water, and dry. Ignite at about 600°C, dissolve the residue
in 20 mL of dilute nitric acid, and filter. Wash the residue
with 15 mL of water, combine the filtrate and the washings,
and add water to make 50 mL. To this solution add 0.5 mL
of silver nitrate TS: the solution is not more turbid than the
following control solution to which 0.5 mL of silver nitrate
TS has been added.
JP XV
Official Monographs / Precipitated Calcium Carbonate
389
Control solution: Dissolve 0.7 g of calcium carbonate in 20
mL of dilute nitric acid, and filter. Wash the residue with 15
mL of water, combine the nitrate and the washings, and add
1.2 mL of 0.01 mol/L hydrochloric acid VS and water to
make 50 mL.
(5) Heavy metals <1.07> — Dissolve 2.0 g of Caffeine and
Sodium Benzoate in 47 mL of water, add slowly, with
vigorous stirring, 3 mL of dilute hydrochloric acid, and filter.
Discard the first 5 mL of the filtrate, neutralize the subse-
quent 25 mL of the filtrate with ammonia TS, and add 2 mL
of dilute acetic acid and water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the
control solution with 2.0 mL of Standard Lead Solution by
adding 2 mL of dilute acetic acid and water to make 50 mL
(not more than 20 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Caffeine and Sodium Benzoate according to Method 1,
and perform the test (not more than 2 ppm).
(7) Phthalic acid— To 0.10 g of Caffeine and Sodium
Benzoate add 1 mL of water and 1 mL of resorcinol-sulfuric
acid TS, and heat the mixture in an oil bath heated at a tem-
perature between 120°C and 125 °C to evaporate the water,
then heat the residue for further 90 minutes, cool, and
dissolve in 5 mL of water. To 1 mL of the solution add 10 mL
of a solution of sodium hydroxide (43 in 500), shake, then
examine under light at a wavelength between 470 nm and 490
nm: the green fluorescence of the solution is not more intense
than that of the following control solution.
Control solution: Dissolve 61 mg of potassium hydrogen
phthalate in water to make exactly 1000 mL. Pipet exactly 1
mL of the solution, add 1 mL of resorcinol-sulfuric acid TS,
and proceed as directed above.
(8) Readily carbonizable substances <1.15> — Proceed
with 0.5 g of Caffeine and Sodium Benzoate, and perform the
test: the solution is not more colored than Matching Fluid A.
Loss on drying <2.41> Not more than 3.0% (2 g, 80 C C,
4 hours).
Assay (1) Sodium benzoate — Weigh accurately about
0.2 g of Caffeine and Sodium Benzoate, previously dried, dis-
solve by warming in 50 mL of a mixture of acetic anhydride
and acetic acid for nonaqueous titration (6:1), cool, and
titrate <2.50> with 0.1 mol/L perchloric acid-dioxane VS to
the first equivalence point (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid-dioxane VS
= 14.41 mg of C 7 H 5 Na0 2
(2) Caffeine— Continue the titration <2.50> in (1) with 0. 1
mol/L perchloric acid-dioxane VS from the first equivalence
point to the second equivalence point (potentiometric titra-
tion).
Each mL of 0.1 mol/L perchloric acid-dioxane VS
= 19.42 mg of C 8 H 10 N 4 O 2
Containers and storage Containers — Well-closed contain-
ers.
Precipitated Calcium Carbonate
>*»J*i!*lJU>"}A
CaC0 3 : 100.09
Precipitated Calcium Carbonate, when dried, con-
tains not less than 98.5% of calcium carbonate
(CaC0 3 ).
Description Precipitated Calcium Carbonate occurs as a
white, fine crystalline powder. It is odorless and tasteless.
It is practically insoluble in water, but its solubility in water
is increased in the presence of carbon dioxide.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves with effervescence in dilute acetic acid, in dilute
hydrochloric acid and in dilute nitric acid.
Identification (1) Dissolve 0.5 g of Precipitated Calcium
Carbonate in 10 mL of dilute hydrochloric acid, boil, then
cool, and neutralize with ammonia TS: the solution responds
to the Qualitative Tests <1.09> for calcium salt.
(2) Precipitated Calcium Carbonate responds to the
Qualitative Tests <1.09> (1) for carbonate.
Purity (1) Acid-insoluble substances — To 5.0 g of
Precipitated calcium Carbonate add 50 mL of water, then
add 20 mL of hydrochloric acid dropwise with stirring, boil
for 5 minutes, cool, add water to make 200 mL, and filter
through filter paper for quantitative analysis. Wash the
residue until the last washing shows no turbidity with silver
nitrate TS, and ignite the residue together with the filter
paper: the mass of the residue is not more than 10.0 mg.
(2) Heavy metals <1.07>— Mix 2.0 g of Precipitated Calci-
um Carbonate with 5 mL of water, add slowly 6 mL of dilute
hydrochloric acid, and evaporate on a water bath to dryness.
Dissolve the residue in 50 mL of water, and filter. To 25 mL
of the filtrate add 2 mL of dilute acetic acid, 1 drop of ammo-
nia TS and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: evaporate 3 mL of hydrochloric acid on a water
bath to dryness, and add 2 mL of dilute acetic acid, 2.0 mL
of Standard Lead Solution and water to make 50 mL (not
more than 20 ppm).
(3) Barium — Mix 1.0 g of Precipitated Calcium Car-
bonate with 10 mL of water, add dropwise 4 mL of
hydrochloric acid with stirring, boil for 5 minutes, cool, add
water to make 40 mL, and filter. With the filtrate, perform
the test as directed under Flame Coloration Test <1.04> (1):
no green color appears.
(4) Magnesium and alkali metals — Dissolve 1.0 g of
Precipitated Calcium Carbonate in a mixture of 20 mL of
water and 10 mL of dilute hydrochloric acid, boil, neutralize
with ammonia TS, and add ammonium oxalate TS until
precipitation of calcium oxalate is completed. Heat the mix-
ture on a water bath for 1 hour, cool, dilute with water to 100
mL, shake well, and filter. To 50 mL of the filtrate add 0.5
mL of sulfuric acid, evaporate to dryness, and ignite at
600°C to constant mass: the mass of the residue is not more
than 5.0 mg.
390
Calcium Chloride Hydrate / Official Monographs
JP XV
(5) Arsenic <1.11>— Moisten 0.40 g of Precipitated Calci-
um Carbonate with 1 mL of water, then dissolve in 4 mL of
dilute hydrochloric acid, use this solution as the test solution,
and perform the test (not more than 5 ppm).
Loss on drying <2.41>
4 hours).
Not more than 1.0% (1 g, 180°C,
Assay Weigh accurately about 0.12 g of Precipitated Calci-
um Carbonate, previously dried, and dissolve in 20 mL of
water and 3 mL of dilute hydrochloric acid. Add 80 mL of
water, 15 mL of a solution of potassium hydroxide (1 in 10)
and 0.05 g of NN indicator, and titrate <2.50> immediately
with 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS until the color of the solution changes from
red-purple to blue.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 5.004 mg of CaCC-3
Containers and storage Containers — Tight containers.
bidity or precipitate is produced.
(6) Barium — Dissolve 0.5 g of Calcium Chloride Hydrate
in 5 mL of water, add 2 drops of dilute hydrochloric acid and
2 mL of potassium sulfate TS, and allow to stand for 10
minutes: no turbidity is produced.
(7) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Calcium Chloride Hydrate according to Method 1, and
perform the test (not more than 2 ppm).
Assay Weigh accurately about 0.4 g of Calcium Chloride
Hydrate, and dissolve in water to make exactly 200 mL.
Measure exactly 20 mL of this solution, add 40 mL of water,
2 mL of 8 mol/L potassium hydroxide TS and 0.1 g of NN
indicator, and titrate <2.50> immediately with 0.02 mol/L
disodium dihydrogen ethylenediamine tetraacetate VS until
the color of the solution changes from red-purple to blue.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.940 mg of CaCl 2 .2H 2
Containers and storage Containers — Tight containers.
Calcium Chloride Hydrate
Calcium Chloride Injection
i£{t*UUv^A>3E#+>ft
CaCl 2 .2H 2 0: 147.01
Calcium Chloride Hydrate contains not less than
96.7% and not more than 103.3% of CaCl 2 .2H 2 0.
Description Calcium Chloride Hydrate occurs as white
granules or masses. It is odorless.
It is very soluble in water, and soluble in ethanol (95), and
practically insoluble in diethyl ether.
It is deliquescent.
Identification A solution of Calcium Chloride Hydrate (1 in
10) responds to the Qualitative Tests <1.09> for calcium salt
and for chloride.
pH <2.54> The pH of a solution of 1.0 g of Calcium Chlo-
ride Hydrate in 20 mL of freshly boiled and cooled water is
between 4.5 and 9.2.
Purity (1) Clarity and color of solution — A solution of
1.0 g of Calcium Chloride Hydrate in 20 mL of water is clear
and colorless.
(2) Sulfate <1.14>— Take 1.0 g of Calcium Chloride Hy-
drate, and perform the test. Prepare the control solution with
0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.024%).
(3) Hypochlorite — Dissolve 0.5 g of Calcium Chloride
Hydrate in 5 mL of water, add 2 to 3 drops of dilute
hydrochloric acid and 2 to 3 drops of zinc iodide-starch TS:
no blue color develops immediately.
(4) Heavy metals <1.07> — Proceed with 2.0 g of Calcium
Chloride Hydrate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(5) Iron, aluminum or phosphate — Dissolve, in a Nessler
tube, 1.0 g of Calcium Chloride Hydrate in 20 mL of water
and 1 drop of dilute hydrochloric acid, boil, then cool, add 3
drops of ammonia TS, and heat the solution to boil: no tur-
Calcium Chloride Injection is an aqueous solution
for injection.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of calcium chloride
(CaCl 2 : 110.98).
The concentration of Calcium Chloride Injection is
expressed as the quantity of calcium chloride (CaCl 2 ).
Method of preparation Prepare as directed under Injection,
with Calcium Chloride Hydrate.
Description Calcium Chloride Injection is a clear, colorless
liquid.
Identification Calcium Chloride Injection responds to the
Qualitative Tests <1.09> for calcium salt and for chloride.
pH <2.54> 4.5-7.5
Bacterial endotoxins <4.01> Less than 0.30EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Calcium Chloride In-
jection, equivalent to about 0.4 g of calcium chloride
(CaCl 2 ), and proceed as directed in the Assay under Calcium
Chloride Hydrate.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.220 mg of CaCl 2
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
JPXV
Official Monographs / Calcium Gluconate Hydrate 391
Calcium Folinate
Calcium Leucovorin
H,N N H
H
CT H
u
SrW
H CO;
CO? ca ! '
C 20 H 2l CaN 7 O 7 : 511.50
Monocalcium A f -{4-[(2-amino-5-formyl-4-
oxo- 1,4, 5,6,7, 8-hexahydropteridin-6-
yl)methylamino]benzoyl } -L-glutamate [1492-18-8 ]
Calcium Folinate contains not less than 95.0% and
not more than 102.0% of C 2 oH2iCaN 7 07, calculated on
the anhydrous basis.
Description Calcium Folinate occurs as a light yellow to
yellow powder. It is odorless and tasteless.
It is very soluble in water, freely soluble in acetic acid
(100), and practically insoluble in ethanol (95) and in diethyl
ether.
It is gradually affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Calcium Folinate (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Calcium Folinate Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Calci-
um Folinate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Calcium Folinate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Calcium Folinate (1 in 100) responds to
the Qualitative Tests <1.09> (2), (3) and (4) for calcium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Calcium Folinate in 10 mL of water: the solution is clear and
yellow.
(2) Heavy metals <1.07> — Proceed with 0.40 g of Calcium
Folinate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 50 ppm).
Water <2.48> Weigh accurately about 0.2 g of Calcium
Folinate in a dried titration flask, and dissolve in 25 mL of
acetic acid (100). Add 10.0 mL of Standard Water-Methanol
Solution, titrate with Karl Fischer TS to the end point and
perform the test: it is not more than 17.0%. Perform a blank
determination, and make any necessary correction.
Assay Weigh accurately about 20 mg of Calcium Folinate,
dissolve in the mobile phase to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 17.5 mg of Calcium Folinate Reference
Standard, calculated on the anhydrous basis, dissolve in the
mobile phase to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 20 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of folinate in each solution.
Amount (mg) of C 2 oH 21 CaN 7 7
= W s x (A T /A S )
W s : Amount (mg) of Calcium Folinate Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside
diameter and about 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 jura in
particle diameter).
Column temperature: Room temperature.
Mobile phase: To 860 mL of water add 100 mL of acetoni-
trile and 15 mL of tetrabutylammonium hydroxide-methanol
TS, adjust the pH to 7.5 with 2mol/L sodium dihydrogen-
phosphate TS, and add water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
folinate is about 10 minutes.
Selection of column: Dissolve 17.5 mg of folic acid in 100
mL of the mobile phase, and to 5 mL of this solution add 20
mL of the standard solution. Proceed with 20 /xh of this solu-
tion under the above operating conditions, and calculate the
resolution. Use a column giving elution of folinate and folic
acid in this order with the resolution of these peaks being not
less than 3.6.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of each peak area
of folinate is not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Calcium Gluconate Hydrate
HO H H OH
«>^ J^ J< ^.COj
HO H H OH
Ca^ • HjO
C 12 H 22 Ca0 14 .H 2 0: 448.39
Monocalcium di-D-gluconate monohydrate [299-28-5]
Calcium Gluconate Hydrate, when dried, contains
not less than 99.0% and not more than 104.0% of
C 12 H 22 Ca0 14 .H 2 0.
Description Calcium Gluconate Hydrate occurs as a white,
crystalline powder or granules.
It is soluble in water, and practically insoluble in ethanol
(99.5).
392 Calcium Hydroxide / Official Monographs
JP XV
Identification (1) To separately 10 mg each of Calcium
Gluconate Hydrate and calcium gluconate for thin-layer
chromatography add 1 mL of water, dissolve by warming,
and use these solutions as the sample solution and standard
solution, respectively. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
[iL each of the sample solution and standard solution on a
plate of silica gel for thin -layer chromatography. Develop
the plate with a mixture of ethanol (95), water, ammonia so-
lution (28) and ethyl acetate (5:3:1:1) to a distance of about
10 cm, air-dry the plate, and heat the plate at 110°C for 20
minutes. After cooling, spray evenly hexaammonium hep-
tamolybdate-cerium (IV) sulfate TS on the plate, air-dry, and
heat at 110°C for 10 minutes: the spots with the sample solu-
tion and the standard solution are the same in the Rf value
and color tone.
(2) A solution of Calcium Gluconate Hydrate (1 in 40)
responds to the Qualitative Tests <1.09> (1), (2) and (3) for
calcium salt.
Optical rotation <2.49> [ a ]p°: +6 - +11° (after drying, 0.5
g, water, warming, after cooling, 25 mL, 100 mL).
pH <2.54> Dissolve 1.0 g of Calcium Gluconate Hydrate in
20 mL of water by warming: the pH of the solution is be-
tween 6.0 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Calcium Gluconate Hydrate in 50 mL of water by warming:
the solution is clear and colorless.
(2) Chloride <1.03>— Take 0.40 g of Calcium Gluconate
Hydrate, and perform the test. Prepare the control solution
with 0.80 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.071%).
(3) Sulfate <7. 14>— Take 1 .0 g of Calcium Gluconate Hy-
drate, and perform the test. Prepare the control solution with
1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048
%).
(4) Heavy metals <1.07> — Dissolve 1.0 g of Calcium
Gluconate Hydrate in 30 mL of water and 2 mL of dilute
acetic acid by warming, cool, and add water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion, 2 mL of dilute acetic acid, and water to make 50 mL
(not more than 20 ppm).
(5) Arsenic <1.11> — Dissolve 0.6 g of Calcium Gluconate
Hydrate in 5 mL of water by warming, add 5 mL of dilute
sulfuric acid and 1 mL of bromine TS, and concentrate on a
water bath to 5 mL. Perform the test with this solution as the
test solution (not more than 3.3 ppm).
(6) Sucrose and reducing sugars — To 0.5 g of Calcium
Gluconate Hydrate add 10 mL of water and 2 mL of dilute
hydrochloric acid, and boil the solution for 2 minutes. After
cooling, add 5 mL of sodium carbonate TS, allow to stand
for 5 minutes, add water to make 20 mL, and filter. To 5 mL
of the filtrate add 2 mL of Fehling's TS, and boil for 1
minute: no orange-yellow to red precipitate is formed imme-
diately.
Loss on drying <2.41> Not more than 1.0% (1 g, 80 C C,
2 hours).
Assay Weigh accurately about 0.4 g of Calcium Gluconate
Hydrate, previously dried, dissolve in 100 mL of water, add 2
mL of 8 mol/L potassium hydroxide TS and 0.1 g of NN in-
dicator, and titrate <2.50> immediately with 0.05 mol/L diso-
dium dihydrogen ethylenediamine tetraacetate VS until the
color of the solution changes from red-purple to blue.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 22.42 mg of C 12 H 22 CaOi 4 .H 2
Containers and storage Containers — Well-closed contain-
ers.
Calcium Hydroxide
Slaked Lime
Ca(OH) 2 : 74.09
Calcium Hydroxide contains not less than 90.0% of
Ca(OH) 2 .
Description Calcium Hydroxide occurs as a white powder.
It has a slightly bitter taste.
It is slightly soluble in water, very slightly soluble in boiling
water, and practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves in dilute acetic acid, in dilute hydrochloric acid
and in dilute nitric acid.
It absorbs carbon dioxide from air.
Identification (1) Mix Calcium Hydroxide with 3 to 4
times its mass of water: the mixture is slushy and is alkaline.
(2) Dissolve 1 g of Calcium Hydroxide in 30 mL of dilute
acetic acid, and boil. After cooling, neutralize with ammonia
TS: the solution responds to the Qualitative tests <1.09> (2)
and (3) for calcium salt.
Purity (1) Acid-insoluble substances — To 5 g of Calcium
Hydroxide add 100 mL of water, add hydrochloric acid drop-
wise with strring until the solution becomes acidic, and fur-
ther add 1 mL of hydrochloric acid. Boil this solution for 5
minutes, cool, and filter through a tared glass filter (G4).
Wash the residue with boiling water until the last washing ex-
hibits no turbidity upon addition of silver nitrate TS, and dry
at 105°C to constant mass: the mass is not more than 25 mg.
(2) Heavy metals <1.07> — Dissolve 1.0 g of Calcium
Hydroxide in 10 mL of dilute hydrochloric acid, evaporate
on a water bath to dryness, dissolve the residue in 40 mL of
water, and filter. To 20 mL of the filtrate add 2 mL of dilute
acetic acid and water to make 50 mL, and perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion as follows: evaporate 5 mL of dilute hydrochloric acid
on a water bath to dryness, and add 2 mL of dilute acetic
acid, 2.0 mL of Standard Lead Solution and water to make
50 mL (not more than 40 ppm).
(3) Magnesium and alkali metals — Dissolve 1.0 g of
Calcium Hydroxide in a mixture of 20 mL of water and 10
mL of dilute hydrochloric acid, boil, neutralize with ammo-
nia TS, and precipitate calcium oxalate completely by adding
dropwise ammonium oxalate TS. Heat the mixture on a
water bath for 1 hour, cool, dilute with water to 100 mL,
shake, and filter. To 50 mL of the filtrate add 0.5 mL of sul-
JP XV
Official Monographs / Calcium Oxide 393
furic acid, evaporate to dryness, and ignite at 600°C to con-
stant mass: the mass of the residue does not exceed 24 mg.
(4) Arsenic </.//> — Dissolve 0.5 g of Calcium Hydroxide
in 5 mL of dilute hydrochloric acid, and perform the test with
this solution as the test solution (not more than 4 ppm).
Assay Weigh accurately about 1 g of Calcium Hydroxide,
dissolve by adding 10 mL of dilute hydrochloric acid, and
add water to make 100 mL. Measure 10 mL of this solution,
add 90 mL of water and 1.5 mL of 8 mol/L potassium
hydroxide TS, shake, allow to stand for 3 to 5 minutes, and
then add 0.1 g of NN indicator. Titrate <2.50> immediately
with 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS, until the red-purple color of the solution
changes to blue.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 3.705 mg of Ca(OH) 2
Containers and storage Containers — Tight containers.
Calcium Lactate Hydrate
H OH
X
•SHjO
and enantiomer
C 6 H 10 CaO 6 .5H 2 O: 308.29
Monocalcium bis[(2/?5')-2-hydroxypropanonate]
pentahydrate [63690-56-2]
Calcium Lactate Hydrate, when dried, contains not
less than 97.0% of calcium lactate (C 6 H 10 CaO 6 :
218.22).
Description Calcium Lactate Hydrate occurs as white pow-
der or granules. It is odorless, and has a slightly acid taste.
A 1 g portion of it dissolves gradually in 20 mL of water,
and it is slightly soluble in ethanol (95), and practically in-
soluble in diethyl ether.
It is partly efflorescent at ordinary temperature, and yields
the anhydride at 120°C.
Identification A solution of Calcium Lactate Hydrate (1 in
20) responds to the Qualitative Tests <1.09> for calcium salt
and for lactate.
Purity (1) Clarity of solution — Dissolve 1.0 g of Calcium
Lactate Hydrate in 20 mL of water by warming: the solution
is clear.
(2) Acidity or alkalinity — To the solution obtained in (1)
add 2 drops of phenolphthalein TS: no red color is produced.
Then add 0.50 mL of 0.1 mol/L sodium hydroxide VS: a red
color develops.
(3) Heavy metals <1.07> — Dissolve 1.0 g of Calcium Lac-
tate Hydrate in 30 mL of water and 5 mL of dilute acetic acid
by warming, cool, add water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution from 2.0 mL of Standard Lead Solution and
2 mL of dilute acetic acid, and dilute with water to 50 mL
(not more than 20 ppm).
(4) Magnesium or alkali metals — Dissolve 1.0 g of Calci-
um Lactate Hydrate in 40 mL of water, add 0.5 g of ammoni-
um chloride, boil, then add 20 mL of ammonium oxalate TS.
Heat the mixture on a water bath for 1 hour, cool, dilute with
water to 100 mL, and filter. To 50 mL of the filtrate add 0.5
mL of sulfuric acid, evaporate to dryness, and ignite between
450°C and 550°C to constant mass: the mass of the residue is
not more than 5 mg.
(5) Arsenic </.//> — Dissolve 0.5 g of Calcium Lactate
Hydrate in 2 mL of water and 3 mL of hydrochloric acid, and
perform the test with this solution as the test solution (not
more than 4 ppm).
(6) Volatile fatty acid — Warm 1.0 g of Calcium Lactate
Hydrate with 2 mL of sulfuric acid: an odor of acetic acid or
butyric acid is not perceptible.
Loss on drying <2.41> 25.0 - 30.0% (1 g, 80°C, 1 hour at
first, then 120°C, 4 hours).
Assay Weigh accurately about 0.5 g of Calcium Lactate,
previously dried, add water, dissolve by heating on a water
bath, cool, and add water to make exactly 100 mL. Pipet 20
mL of this solution, then 80 mL of water and 1.5 mL of 8
mol/L potassium hydroxide TS, and allow to stand for 3 to 5
minutes. Add 0.1 g of NN indicator, and titrate <2.50> im-
mediately with 0.02 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS until the color of the solution changes
from red to blue.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.364 mg of C 6 H I0 CaO 6
Containers and storage Containers — Tight containers.
Calcium Oxide
Quick Lime
CaO: 56.08
Calcium Oxide, when incinerated, contains not less
than 98.0% of CaO.
Description Calcium Oxide occurs as hard, white masses,
containing a powder. It is odorless.
It is very slightly soluble in boiling water, and practically
insoluble in ethanol (95).
One gram of Calcium Oxide dissolves almost completely in
2500 mL of water.
It slowly absorbs moisture and carbon dioxide from air.
Identification (1) Moisten Calcium Oxide with water: heat
is generated and a white powder is obtained. Mix the powder
with about 5 times its mass of water: the mixture is alkaline.
(2) Dissolve 1 g of Calcium Oxide in 20 mL of water by
adding a few drops of acetic acid (31): the solution responds
to the Qualitative Tests <1.09> for calcium salt.
Purity (1) Acid-insoluble substances — Disintegrate 5.0 g
of Calcium Oxide with a small amount of water, add 100 mL
of water, add dropwise hydrochloric acid with stirring until
the solution becomes acidic, and further add 1 mL of
394 Calcium Pantothenate / Official Monographs
JP XV
hydrochloric acid. Boil the solution for 5 minutes, cool, filter
through a glass filter (G4), wash the residue with boiling
water until no turbidity is produced when silver nitrate TS is
added to the last washing, and dry at 105 °C to constant mass:
the mass of the residue is not more than 10.0 mg.
(2) Carbonate — Disintegrate 1 .0 g of Calcium Oxide with
a small amount of water, mix thoroughly with 50 mL of
water, allow to stand for a while, remove most of the super-
natant milky liquid by decantation, and add an excess of di-
lute hydrochloric acid to the residue: no vigorous efferves-
cence is produced.
(3) Magnesium and alkali metals — Dissolve 1.0 g of
Calcium Oxide in 75 mL of water by adding dropwise
hydrochloric acid, and further add 1 mL of hydrochloric
acid. Boil for 1 to 2 minutes, neutralize with ammonia TS,
add dropwise an excess of hot ammonium oxalate TS, heat
the mixture on a water bath for 2 hours, cool, add water to
make 200 mL, mix thoroughly, and filter. Evaporate 50 mL
of the filtrate with 0.5 mL of sulfuric acid to dryness, and
heat the residue strongly at 600°C to constant mass: the mass
of the residue is not more than 15 mg.
Loss on ignition <2.43> Not more than 10.0% (1 g, 900°C,
constant mass).
Assay Weigh accurately about 0.7 g of Calcium Oxide,
previously incinerated at 900°C to constant mass and cooled
in a desiccator (silica gel), and dissolve in 50 mL of water and
8 mL of diluted hydrochloric acid (1 in 3) by heating. Cool,
and add water to make exactly 250 mL. Pipet 10 mL of the
solution, add 50 mL of water, 2 mL of 8 mol/L potassium
hydroxide TS and 0.1 g of NN indicator, and titrate <2.50>
with 0.02 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS, until the red-purple color of the solution
changes to blue.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 1.122 mg of CaO
Containers and storage Containers — Tight containers.
Calcium Pantothenate
M°> r-x>gt*i;L-v^A
H 3 C CH 3
HO H
^\^,cor
C 18 H 32 CaN 2 O 10 : 476.53
Monocalcium bis{3-[(2/?)-2,4-dihydroxy-3,3-
dimethylbutanoylamino]propanoate} [13 7-08-6]
Calcium Pantothenate, when dried, contains not less
than 5.7% and not more than 6.0% of nitrogen (N:
14.01), and not less than 8.2% and not more than
8.6%, of calcium (Ca: 40.08).
Description Calcium Pantothenate occurs as a white
powder. It is odorless, and has a bitter taste.
It is freely soluble in water, very slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
The pH of a solution of Calcium Pantothenate (1 in 20) is
between 7.0 and 9.0.
It is hygroscopic.
Identification (1) Dissolve 0.05 g of Calcium Pan-
tothenate in 5 mL of sodium hydroxide TS, and filter. To the
filtrate add 1 drop of copper (II) sulfate TS: a deep blue color
develops.
(2) To 0.05 g of Calcium Pantothenate add 5 mL of sodi-
um hydroxide TS, and boil for 1 minute. After cooling, add
diluted hydrochloric acid (1 in 10) to adjust the solution to a
pH between 3 and 4, and add 2 drops of iron (III) chloride
TS: a yellow color is produced.
(3) A solution of Calcium Pantothenate (1 in 10)
responds to the Qualitative Tests <1.09> for calcium salt.
Optical rotation <2.49> [a] J,: +25.0
drying, 1 g, water, 20 mL, 100 mm).
+ 28.5° (after
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Calcium Pantothenate in 20 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Calcium
Pantothenate according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Alkaloids — Dissolve 0.05 g of Calcium Pantothenate
in 5 mL of water, add 0.5 mL of hexaammonium hep-
tamolybdate TS and 0.5 mL of a solution of phosphoric acid
(1 in 10): no white turbidity is produced.
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C,
4 hours).
Assay (1) Nitrogen — Proceed with about 50 mg of Calci-
um Pantothenate, previously dried and accurately weighed,
as directed under Nitrogen Determination <1.08>.
(2) Calcium — Weigh accurately about 0.4 g of Calcium
Pantothenate, previously dried, and dissolve in 30 mL of
water by warming. After cooling, add exactly 25 mL of 0.05
mol/L disodium dihydrogen ethylenediamine tetraacetate
VS, then 10 mL of ammonia-ammonium chloride buffer so-
lution, pH 10.7, and 0.04 g of eriochrome black T-sodium
chloride indicator, and titrate <2.50> the excess disodium di-
hydrogen ethylenediamine tetraacetate with 0.05 mol/L mag-
nesium chloride VS until the color of the solution changes
from blue-purple to red-purple. Perform a blank determina-
tion.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.004 mg of Ca
Containers and storage Containers — Tight containers.
Calcium Paraaminosalicylate
Granules
Pas-calcium Granules
Calcium Paraaminosalicylate Granules contain not
less than 95.0% and not more than 105.0% of the la-
beled amount of calcium paraaminosalicylate hydrate
JP XV
Official Monographs / Calcium Paraaminosalicylate Hydrate
395
(C 7 H 5 CaN0 3 .3 1/2H 2 0: 254.25).
Method of preparation Prepare as directed under Granules,
with Calcium Paraaminosalicylate Hydrate.
Identification Powder Calcium Paraaminosalicylate Gran-
ules, weigh a portion of the powder, equivalent to 50 mg of
Calcium Paraaminosalicylate Hydrate according to the la-
beled amount, add 100 mL of water, shake, and filter. To 10
mL of the filtrate add 1 mL of 1 mol/L hydrochloric acid TS,
shake, and add 1 drop of iron (III) chloride TS: a red-purple
color develops.
Particle size <6.03> It meets the requirement.
Assay Powder Calcium Paraaminosalicylate Granules,
weigh accurately a portion of the powder, equivalent to
about 0.2 g of calcium paraaminosalicylate hydrate (C 7 H 5
CaN0 3 .3 1/2H 2 0), add 60 mL of water and 0.75 mL of di-
lute hydrochloric acid, and dissolve by heating on a water
bath. After cooling, add water to make exactly 100 mL, and
filter. Pipet 30 mL of the filtrate, transfer to an iodine flask,
and proceed as directed in the Assay under Calcium Para-
aminosalicylate Hydrate.
Each mL of 0.05 mol/L bromine VS
= 4.238 mg of C 7 H 5 CaN0 3 .3 1/2H 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Calcium Paraaminosalicylate
Hydrate
Pas-calcium Hydrate
A77J /^U^JU^JUv^ATkMfcl
C02~
C 7 H 5 CaN0 3 -3 1/2 H 2 0: 254.25
Monocalcium 4-amino-2-oxidobenzoate hemiheptahydrate
[133-15-3, anhydride]
Calcium Paraaminosalicylate Hydrate contains not
less than 97.0% and not more than 103.0% of calcium
para-aminosalicylic acid (C 7 H 5 CaN0 3 : 191.20), calcu-
lated on the anhydrous basis.
Description Calcium Paraaminosalicylate Hydrate occurs
as a white to slightly colored powder. It has a slightly bitter
taste.
It is very slightly soluble in water, and practically insoluble
in methanol and in ethanol (99.5).
It is gradually colored to brown by light.
Identification (1) To 50 mg of Calcium Paraaminosalicy-
late Hydrate add 100 mL of water, shake well, and filter. To
10 mL of the filtrate add 1 mL of 1 mol/L hydrochloric acid
TS, shake, and add 1 drop of iron (III) chloride TS: a red-
purple color develops.
(2) Determine the infrared absorption spectrum of Calci-
um Paraaminosalicylate Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) To 3 g of Calcium Paraaminosalicylate Hydrate add
15 mL of ammonium chloride TS and 15 mL of water, heat
on a water bath until almost dissolved, and filter after cool-
ing: the filtrate responds to the Qualitative Tests <1.09> (1),
(2) and (3) for calcium salt.
Purity (1) Chloride <1.03>— Dissolve 1.0 g of Calcium
Paraaminosalicylate Hydrate in 15 mL of dilute nitric acid,
and add water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution with
0.70 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.025%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Calcium
Paraaminosalicylate Hydrate according to method 3, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Dissolve 0.40 g of Calcium Para-
aminosalicylate Hydrate in 20 mL of 0.1 mol/L hydrochloric
acid TS by warming on a water bath, use this solution as the
test solution, and perform the test (not more than 5 ppm).
(4) 3-Aminophenol — To 0.10 g of Calcium Para-
aminosalicylate Hydrate add 5 mL of 0.1 mol/L disodium di-
hydrogen ethylenediamine tetraacetate TS, previously cooled
in ice-water, and dissolve by shaking vigorously. Add imme-
diately 3 mL of ammonia-ammonium chloride buffer solu-
tion, pH 11.0, previously cooled in ice water, and shake. Add
2 mL of 4-amino-iV,A L diethylanilme sulfate TS, shake, add
10.0 mL of cyclohexane and 4 mL of diluted potassium hex-
acyanoferrate (III) TS (1 in 10), and shake immediately for 20
seconds. Centrifuge this solution, wash the separated cyclo-
hexane layer with two 5-mL portions of diluted ammonia TS
(1 in 14), add 1 g of anhydrous sodium sulfate, shake, and al-
low to stand for 5 minutes: the clear cyclohexane layer is not
more colored than the following control solution.
Control solution: Dissolve 50 mg of 3-aminophenol in
water, and dilute with water to exactly 500 mL. Measure ex-
actly 20 mL of this solution, and add water to make exactly
100 mL. Take 5.0 mL of this solution, add 3 mL of am-
monia-ammonium chloride buffer solution, pH 11.0, previ-
ously cooled in ice-water, and treat this solution in the same
manner as the sample.
Water <2.48> 23.3-26.3% (0.1 g, volumetric titration,
direct titration)
Assay Weigh accurately about 0.2 g of Calcium Para-
aminosalicylate Hydrate, dissolve in 60 mL of water and 0.75
mL of dilute hydrochloric acid by warming on a water bath.
After cooling, add water to make exactly 100 mL, and use
this solution as the sample solution. Measure exactly 30 mL
of the sample solution, transfer to an iodine flask, and add
exactly 25 mL of 0.05 mol/L bromine VS and 20 mL of a so-
lution of potassium bromide (1 in 4). Add immediately 14
mL of a mixture of acetic acid (100) and hydrochloric acid
(5:2), stopper the flask immediately, and allow to stand for 10
minutes with occasional shaking. Add cautiously 6 mL of
potassium iodide TS, and shake gently. After 5 minutes, ti-
trate <2.50> the produced iodine with 0.1 mol/L sodium
thiosulfate VS (indicator: 1 mL of starch TS). Perform a
396
Dibasic Calcium Phosphate Hydrate / Official Monographs
JP XV
blank determination.
Each mL of 0.05 mol/L bromine VS
= 3.187 mg of C 7 H 5 CaN0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dibasic Calcium Phosphate Hydrate
CaHP0 4 .2H 2 0: 172.09
Dibasic Calcium Phosphate Hydrate, when dried,
contains not less than 98.0% of dibasic calcium phos-
phate (CaHP0 4 : 136.06).
Description Dibasic Calcium Phosphate Hydrate occurs as
a white, crystalline powder. It is colorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute nitric
acid.
Identification (1) Dissolve 0.1 g of Dibasic Calcium Phos-
phate Hydrate in 10 mL of diluted hydrochloric acid (1 in 6)
by warming, add 2.5 mL of ammonia TS dropwise with shak-
ing, and add 5 mL of ammonium oxalate TS: a white
precipitate is produced.
(2) Dissolve 0.1 g of Dibasic Calcium Phosphate Hydrate
in 5 mL of dilute nitric acid, and add 2 mL of hexaammoni-
um heptamolybdate TS after warming for 1 to 2 minutes at
70°C: a yellow precipitate is produced.
Purity (1) Acid-insoluble substance — Dissolve 5.0 g of
Dibasic Calcium Phosphate Hydrate in 40 mL of water and
10 mL of hydrochloric acid, and boil for 5 minutes. After
cooling, collect the insoluble substance using filter paper for
assay. Wash with water until no more turbidity of the wash-
ing is produced when silver nitrate is added. Ignite to inciner-
ate the residue and filter paper: the mass is not more than 2.5
mg (not more than 0.05%).
(2) Chloride <1.03>— Dissolve 0.20 g of Dibasic Calcium
Phosphate Hydrate in 20 mL of water and 13 mL of dilute
nitric acid, add water to make 100 mL, and filter, if necessa-
ry. Perform the test using a 50-mL portion of this solution as
the test solution. Prepare the control solution with 0.70 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.248%).
(3) Sulfate <1.14> — Dissolve by warming 1.0 g of Dibasic
Calcium Phosphate Hydrate in 5 mL of water and 5 mL of
dilute hydrochloric acid, add water to make 100 mL, and
filter, if necessary. Take 30 mL of the filtrate, and add 1 mL
of dilute hydrochloric acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution with 1.0 mL of 0.005 mol/L sulfuric acid
VS (not more than 0.160%).
(4) Carbonate — Mix 1.0 g of Dibasic Calcium Phosphate
Hydrate with 5 mL of water, and add immediately 2 mL of
hydrochloric acid: no effervescence occurs.
(5) Heavy metals <1.07> — Dissolve 0.65 g of Dibasic Cal-
cium Phosphate Hydrate in a mixture of 5 mL of water and 5
mL of dilute hydrochloric acid by warming, cool, and add
ammonia TS until precipitates begin to form in the solution.
Dissolve the precipitates by adding a small amount of dilute
hydrochloric acid dropwise, add 10 mL of hydrochloric acid-
ammonium acetate buffer solution, pH3.5, and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution as follows: to 10
mL of hydrochloric acid-ammonium acetate buffer solution,
pH 3.5, add 2.0 mL of Standard Lead Solution and water to
make 50 mL (not more than 31 ppm).
(6) Barium — Heat 0.5 g of Dibasic Calcium Phosphate
Hydrate with 10 mL of water, add 1 mL of hydrochloric acid
dropwise with stirring, and filter, if necessary. Add 2 mL of
potassium sulfate TS to the filtrate, and allow to stand for 10
minutes: no turbidity forms.
(7) Arsenic <1.11> — Dissolve 1.0 g of Dibasic Calcium
Phosphate Hydrate in 5 mL of dilute hydrochloric acid, and
perform the test with this solution as the test solution (not
more than 2 ppm).
Loss on drying <2.4I> 19.5 - 22.0% (1 g, 200°C, 3 hours).
Assay Weigh accurately about 0.4 g of Dibasic Calcium
Phosphate Hydrate, previously dried, dissolve in 12 mL of
dilute hydrochloric acid, and add water to make exactly 200
mL. Pipet 20 mL of this solution, add exactly 25 mL of 0.02
mol/L disodium dihydrogen ethylenediamine tetraacetate
VS, 50 mL of water and 5 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.7, and titrate <2.50> the excess dis-
odium dihydrogen ethylenediamine tetraacetate with 0.02
mol/L zinc acetate VS (indicator: 25 mg of eriochrome black
T-sodium chloride indicator). Perform a blank determina-
tion.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.721 mg of CaHP0 4
Containers and storage Containers — Well-closed contain-
ers.
Anhydrous Dibasic Calcium
Phosphate
*7kU>^7K»7^UV^A
CaHP0 4 : 136.06
Anhydorous Dibasic Calcium Posphate, when dried,
contains not less than 98.0% of CaHP0 4 .
Description Anhydrous Dibasic Calcium Phosphate occurs
as white, crystalline powder or granules. It is odorless and
tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute nitric
acid.
Identification (1) Dissolve 0.1 g of Anhydrous Dibasic
Calcium Phosphate in 10 mL of diluted hydrochloric acid (1
in 6) by warming, add 2.5 mL of ammonia TS dropwise with
shaking, and add 5 mL of ammonium oxalate TS: a white
precipitate is produced.
JPXV
Official Monographs / Monobasic Calcium Phosphate Hydrate
397
(2) Dissolve 0.1 g of Anhydrous Dibasic Calcium Phos-
phate in 5 mL of dilute nitric acid, and add 2 mL of hexaam-
monium heptamolybdate TS after warming for 1 to 2 minutes
at 70°C: a yellow precipitate is produced.
Purity (1) Acid-insoluble substances — Dissolve 5.0 g of
Anhydrous Dibasic Calcium Phosphate in 40 mL of water
and 10 mL of hydrochloric acid, and boil for 5 minutes, Af-
ter cooling, collect the insoluble substance using filter paper
for assay. Wash with water until no more turbidity of the
washing is produced when silver nitrate is added. Ignite to in-
cinerate the residue with the filter paper: the mass is not more
than 2.5 mg (not more than 0.05%).
(2) Chloride <1.03>— Dissolve 0.20 g of Anhydrous Di-
basic Calcium Phosphate in 20 mL of water and 13 mL of di-
lute nitric acid, add water to make 100 mL, and filter, if
necessary. Perform the test using a 50-mL portion of this so-
lution as the test solution. Prepare the control solution with
0.70 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.248%).
(3) Sulfate <I.14> — Dissolve by warming 0.80 g of Anhy-
drous Dibasic Calcium Phosphate in 5 mL of water and 5 mL
of dilute hydrochloric acid, add water to make 100 mL, and
filter, if necessary. Take 30 mL of the filtrate, and add 1 mL
of dilute hydrochloric acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution with 1.0 mL of 0.005 mol/L sulfuric acid
VS (not more than 0.200%).
(4) Carbonate — Mix 1.0 g of Anhydrous Dibasic Calci-
um Phosphate with 5 mL of water, and add immediately 2
mL of hydrochloric acid: no effervescence occurs.
(5) Heavy metals <1.07> — Dissolve 0.65 g of Anhydrous
Dibasic Calcium Phosphate in a mixture of 5 mL of water
and 5 mL if dilute hydrochloric acid by warming, cool, and
add ammonia TS until precipitates begin to form in the solu-
tion. Dissolve the precipitates by adding a small amount of
dilute hydrochloric acid dropwise, add 10 mL of hydrochlor-
ic acid-ammonium acetate buffer solution, pH 3.5, and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution as follows: to
10 mL of hydrochloric acid-ammonium acetate buffer solu-
tion, pH 3.5, add 2.0 mL of Standard Lead Solution and
water to make 50 mL (not more than 31 ppm).
(6) Barium — Heat 0.5 g of Anhydrous Dibasic Calcium
Phosphate with 10 mL of water, add 1 mL of hydrochloric
acid dropwise with stirring, and filter, if necessary. Add 2 mL
of potassium sulfate TS to the filtrate, and allow to stand for
10 minutes: no turbidity forms.
(7) Arsenic <1.11> — Dissolve 0.5 g of Anhydrous Dibasic
Calcium Phosphate in 5 mL of dilute hydrochloric acid, and
perform the test with this solution as the test solution (not
more than 2 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, 200°C,
3 hours).
Assay Weigh accurately about 0.4 g of Anhydrous Dibasic
Calcium Phosphate, previously dried, dissolve in 12 mL of
dilute hydrochloric acid, and add water to make exactly 200
mL. Pipet 20 mL of this solution, add exactly 25 mL of 0.02
mol/L disodium dihydrogen ethylenediamine tetraacetate
VS, 50 mL of water and 5 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.7, and titrate <2.50> the excess dis-
odium dihydrogen ethylenediamine tetraacetate with 0.02
mol/L zinc acetate VS (indicator: 0.025 g of eriochrome
black T-sodium chloride indicator). Perform a blank deter-
mination.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.721 mg of CaHPC-4
Containers and storage Containers — Well-closed contain-
ers.
Monobasic Calcium Phosphate
Hydrate
Ca(H 2 P0 4 ) 2 -H 2 0: 252.07
Monobasic Calcium Phosphate Hydrate, when
dried, contains not less than 90.0% of Ca(H 2 P0 4 )2.H2
O.
Description Monobasic Calcium Phosphate Hydrate occurs
as white crystals or crystalline powder. It is odorless and has
an acid taste.
It is sparingly soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute nitric
acid.
It is slightly deliquescent.
Identification (1) Dissolve 0.1 g of Monobasic Calcium
Phosphate Hydrate in 10 mL of diluted hydrochloric acid (1
in 6) by warming, add 2.5 mL of ammonia TS dropwise with
shaking, and add 5 mL of ammonium oxalate TS: a white
precipitate is produced.
(2) Dissolve 0.1 g of Monobasic Calcium Phosphate Hy-
drate in 5 mL of dilute nitric acid, and add 2 mL of hexaam-
monium heptamolybdate TS after warming for 1 to 2 minutes
at 70°C: a yellow precipitate is produced.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Monobasic Calcium Phosphate Hydrate in 19 mL of water
and 2 mL of diluted hydrochloric acid (3 in 4), and heat on a
water bath for 5 minutes with occasional shaking: the solu-
tion is clear and colorless.
(2) Dibasic phosphate and acid — Triturate 1.0 g of
Monobasic Calcium Phosphate Hydrate with 3 mL of water,
and add 100 mL of water and 1 drop of methyl orange TS: a
red color develops. Then add 1.0 mL of 1 mol/L sodium
hydroxide VS: the color changes to yellow.
(3) Chloride <1.03>— Dissolve 1.0 g of Monobasic Calci-
um Phosphate Hydrate in 20 mL of water and 12 mL of di-
lute nitric acid, add water to make exactly 100 mL, and filter,
if necessary. Perform the test using 50 mL of this solution as
the test solution. Prepare the control solution with 0.25 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.018%).
(4) Sulfate <1.14> — Dissolve 1.0 g of Monobasic Calcium
Phosphate Hydrate in 20 mL of water and 1 mL of
hydrochloric acid, add water to make 100 mL, and filter, if
necessary. Perform the test using 50 mL of this solution as
the test solution. Prepare the control solution with 0.50 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.048%).
398
Calcium Polystyrene Sulfonate / Official Monographs
JP XV
(5) Heavy metals <1.07> — Dissolve 0.65 g of Monobasic
Calcium Phosphate Hydrate in a mixture of 5 mL of water
and 5 mL of dilute hydrochloric acid by warming, cool, and
add ammonia TS until precipitates begin to form in the solu-
tion. Dissolve the precipitates by adding a small amount of
dilute hydrochloric acid dropwise, add 10 mL of hydrochlor-
ic acid-ammonium acetate buffer solution, pH 3.5, and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution as follows: to
10 mL of hydrochloric acid-ammonium acetate buffer solu-
tion, pH 3.5, add 2.0 mL of Standard Lead Solution and
water to make 50 mL (not more than 31 ppm).
(6) Arsenic <1.11> — Dissolve 1 .0 g of Monobasic Calcium
Phosphate Hydrate in 5 mL of dilute hydrochloric acid, and
perform the test with this solution as the test solution (not
more than 2 ppm).
Loss on drying <2.41> Not more than 3.0% (1 g, silica gel,
24 hours).
Assay Weigh accurately about 0.4 g of Monobasic Calcium
Phosphate Hydrate, previously dried, dissolve in 3 mL of di-
lute hydrochloric acid, and add water to make exactly 100
mL. Pipet 20 mL of this solution, add exactly 25 mL of 0.02
mol/L disodium dihydrogen ethylenediamine tetraacetate
VS, 50 mL of water and 5 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.7, and titrate <2.50> the excess dis-
odium dihydrogen ethylenediamine tetraacetate with 0.02
mol/L zinc acetate VS (indicator: 25 mg of eriochrome black
T-sodium chloride indicator). Perform a blank determina-
tion.
Each mL of 0.02 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 5.041 mg of Ca(H 2 P0 4 ) 2 .H 2
Containers and storage Containers — Tight containers.
Calcium Polystyrene Sulfonate
7fCUX5 1 U>;UU*>gg*iJL.v^A
Calcium Polystyrene Sulfonate is a cation exchange
resin prepared as the calcium form of the sulfonated
styrene divinylbenzene copolymer.
When dried, it contains not less than 7.0% and not
more than 9.0% of calcium (Ca: 40.08).
Each g of Calcium Polystyrene Sulfonate, when
dried, exchanges with 0.053 to 0.071 g of potassium (K:
39.10).
Description Calcium Polystyrene Sulfonate occurs as a pale
yellowish white to light yellow powder. It is odorless and
tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Identification (1) Determine the infrared absorption spec-
trum of Calcium Polystyrene Sulfonate, previously dried, as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(2) Mix 0.5 g of Calcium Polystyrene Sulfonate with 10
mL of dilute hydrochloric acid, filter, and neutralize the
filtrate with ammonia TS: the solution responds to the
Qualitative Tests <1.09> for calcium salt.
Purity (1) Ammonium — Place 1.0 g of Calcium Polysty-
rene Sulfonate in a flask, add 5 mL of sodium hydroxide TS,
cover the flask with a watch glass having a moistened strip of
red litmus paper on the underside, and boil for 15 minutes:
the gas evolved does not change the red litmus paper to blue
(not less than 5 ppm).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Calcium
Polystyrene Sulfonate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Calcium Polystyrene Sulfonate according to Method 3,
and perform the test (not more than 2 ppm).
(4) Styrene — To 10.0 g of Calcium Polystyrene Sulfonate
add 10 mL of acetone, shake for 30 minutes, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 10 mg of styrene in acetone to make exactly 100 mL.
Pipet 1 mL of this solution, dilute with acetone to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 5 /xL each of the sample solu-
tion and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions.
Determine the peak heights, H T and H s , of styrene in each so-
lution: H-y is not larger than H s .
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A stainless steel column 3 mm in inside diameter
and 2 m in length, having polyethylene glycol 20 M coated at
the ratio of 15% on siliceous earth for gas chromatography
(150 to 180 /um in particle diameter).
Column temperature: A constant temperature of about
90°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
styrene is about 9 minutes.
System suitability —
System performance: Mix 10 mg of styrene with 1000 mL
of acetone. When the procedure is run with 5 fiL of this
solution under the above operating conditions, the number of
theoretical plates and the symmetry factor of the peak of sty-
rene are not less than 800 and 0.8 to 1.2, respectively.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak heights of
styrene is not more than 5%.
(5) Sodium — Pipet 2 mL of the 50-mL solution obtained
in the Assay (1), add 0.02 mol/L hydrochloric acid TS to
make exactly 500 mL, and use this solution as the sample so-
lution. Separately, weigh accurately 0.2542 g of sodium chlo-
ride, previously dried at 130°C for 2 hours, and dissolve in
0.02 mol/L hydrochloric acid TS to make exactly 1000 mL.
Pipet a suitable volume of this solution, and dilute with 0.02
mol/L hydrochloric acid TS to make a solution containing 1
to 3 n% of sodium (Na: 22.99) per mL, and use these solutions
as the standard solutions. Perform the test with the sample
solution and standard solutions according to the Atomic Ab-
sorption Spectrophotometry <2.23> under the following con-
ditions, and determine the amount of sodium in the sample
solution using the calibration curve obtained from the stan-
JPXV
Official Monographs / Calcium Polystyrene Sulfonate
399
i>0mm
Actual volume to the mark of 20 cm at which the sedimentation
tube is inserted: 550 ml.
Single suction volume: 10 ml.
A: Mark of pi pet bulb
B: Pi pet bulb for suction
C: Two-way stopcock
D: Vent-hole
K: Suction part of pi pot
F: Mark of 20 cm
G: Base line of cm
H: Outlet of pipct
1: Capillary tube of pipct
j: Sedimentation tube
Fig. Andreasen pipet
dard solutions: the amount of sodium is not more than 1%.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A sodium hollow-cathode lamp
Wavelength: 589.0 nm
Loss on drying <2.41> Not more than i0.0% (1 g, in vacu-
um, 80°C, 5 hours).
Microparticles (i) Apparatus: Use an apparatus as shown
in the illustration.
(ii) Procedure: Weigh accurately about 5.5 g of Calcium
Polystyrene Sulfonate, previously dried, add 300 mL of
water of 25 C C, and mix for 5 minutes. Transfer this turbid
solution to the sedimentation tube J, keeping a temperature
at 25°C, add water of 25°C to 2 mm below the mark F of 20
cm of the sedimentation tube J, and then insert the pipet.
Open the two-way stopcock C, exhaust air, add exactly water
from the vent-hole D to the mark F of 20 cm, and close the
two-way stopcock C. Shake the apparatus well vertically and
horizontally, disperse Calcium Polystyrene Sulfonate in
water, and then open the two-way stopcock, and allow to
stand at 25 ± i°C for 5 hours and 15 minutes.
Then, draw exactly the meniscus of the turbid solution in
sedimentation tube J up to the mark of pipet bulb A by suc-
tion, open the two-way stopcock C to the outlet of pipet H,
and transfer exactly measured 20 mL of the turbid solution to
a weighing bottle. Repeat the procedure, and combine exactly
measured 20 mL of the turbid solution. Evaporate 20 mL of
this turbid solution on a water bath to dryness, dry to con-
stant mass at 105°C, and weigh the residue as W s (g). Pipet
20 mL of used water, and weigh the residue in the same man-
ner as IV B (g). Calculate the difference mi (g) between W s and
W B , and calculate the amount of microparticles (S) by the
following equation: the amount of microparticles is not more
than 0.1%.
S (%) = {(mi x F)/(20 x W T )} x 100
W T : Amount (g) of Calcium Polystyrene Sulfonate
V: Actual volume (mL) to the mark of 20 cm at which the
suction part of pipet is inserted.
Assay (1) Calcium — Weigh accurately about 1 g of Calci-
um Polystyrene Sulfonate, previously dried, and disperse in 5
mL of 3 mol/L hydrochloric acid TS. Transfer this mixture,
and wash out completely with the aid of a small quantity of 3
mol/L hydrochloric acid TS to a column 12 mm in inside di-
ameter and 70 mm in length, packed with a pledged of fine g-
lass wool in the bottom of it, placing a 50-mL volumetric
flask as a receiver under the column. Then collect about 45
mL of eluate, adding 3 mol/L hydrochloric acid TS to the
column, and add water to make exactly 50 mL. Pipet 20 mL
of this solution, adjust with ammonia TS to a pH of exactly
10. Titrate <2.50> immediately with 0.05 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS until the red-pur-
ple color of the solution disappears, and a blue color de-
velops (indicator: 0.04 g eriochrome black T-sodium chloride
indicator). Perform a blank determination, and make any
necessary correction.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.004 mg of Ca
(2) Potassium exchange capacity — Pipet 50 mL of Stan-
dard Potassium Stock Solution into a glass-stoppered flask
containing about 1.0 g of dried Calcium Polystyrene Sul-
fonate, accurately weighed, stir for 120 minutes, filter, and
discard the first 20 mL of the filtrate. Pipet 5 mL of the subse-
quent filtrate, and add 0.02 mol/L hydrochloric acid TS to
make exactly 100 mL. Pipet 10 mL of this solution, add 0.02
mol/L hydrochloric acid TS to make exactly 1000 mL, and
use this solution as the sample solution. Separately, measure
exactly a suitable volume of Standard Potassium Stock Solu-
tion, dilute with 0.02 mol/L hydrochloric acid TS to make
solutions containing 0.5 to 2.5 /xg of potassium (K: 39.10) per
mL, and use these solutions as the standard solutions. Per-
form the test with the sample solution and standard solutions
as directed under Atomic Absorption Spectrophotometry
<2.23> according to the following conditions, and determine
the amount, Y (mg), of potassium in 1000 mL of the sample
solution, using the calibration curve obtained from the stan-
dard solutions. The exchange quantity for potassium per g of
dried Calcium Polystyrene Sulfonate is 53 to 71 mg, calculat-
ing by the following equation.
Exchange quantity (mg) for potassium (K) per g of
dried Calcium Polystyrene Sulfonate
= (X - 100 Y)/W
X: The amount (mg) of potassium in 50 mL of Standard
400 Calcium Stearate / Official Monographs
JP XV
Potassium Stock Solution before exchange.
W: The amount (g) of dried Calcium Polystyrene Sul-
fonate.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A potassium hollow-cathode lamp
Wavelength: 766.5 nm
Containers and storage Containers — Tight containers.
Calcium Stearate
*x7U>S*JjU$">A
Calcium Stearate mainly consists of calcium salts of
stearic acid (C 18 H 36 2 : 284.48) and palmitic acid
(C 16 H 32 2 : 256.42).
Calcium Stearate, when dried, contains not less than
6.4% and not more than 7.1% of calcium (Ca: 40.08).
Description Calcium Stearate occurs as a white, light, bulky
powder. It feels smooth when touched, and is adhesive to the
skin. It is odorless or has a faint, characteristic odor.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Identification (1) Shake vigorously 3 g of Calcium
Stearate with 20 mL of diluted hydrochloric acid (1 in 2) and
30 mL of diethyl ether for 3 minutes, and allow to stand: the
separated aqueous layer responds to the Qualitative Tests
<1.09> (1), (2) and (4) for calcium salt.
(2) Wash the diethyl ether layer obtained in (1) with 20
mL and 10 mL of dilute hydrochloric acid and 20 mL of
water successively, and evaporate the diethyl ether on a water
bath: the residue melts <1.13> at a temperature not below
54°C.
Purity (1) Heavy metals <1.07>— Heat gently 1.0 g of Cal-
cium Stearate with caution at the beginning, and heat fur-
ther, gradually raising the temperature, to incineration. After
cooling, add 2 mL of hydrochloric acid, evaporate on a water
bath to dryness, warm the residue with 20 mL of water and 2
mL of dilute acetic acid for 2 minutes, cool, filter, and wash
the residue with 15 mL of water. Combine the filtrate and the
washings, add water to make 50 mL, and perform the test
using this solution as the test solution. Prepare the control
solution by evaporating 2 mL of hydrochloric acid on a water
bath to dryness and by adding 2 mL of dilute acetic acid, 2.0
mL of Standard Lead Solution and water to make 50 mL (not
more than 20 ppm).
(2) Arsenic <1.11> — To 1.0 g of Calcium Stearate add 5
mL of diluted hydrochloric acid (1 in 2) and 20 mL of chlo-
roform, shake vigorously for 3 minutes, allow to stand, and
separate the water layer. Perform the test with the water layer
as the test solution (not more than 2 ppm).
Loss on drying <2.41> Not more than 4.0% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 0.5 g of Calcium Stearate,
previously dried, heat gently with caution at first, and then ig-
nite gradually to ash. Cool, add 10 mL of dilute hydrochloric
acid to the residue, warm for 10 minutes on a water bath, and
transfer the contents to a flask with the aid of 10-mL, 10-mL,
and 5-mL portions of hot water. Add sodium hydroxide TS
until the solution becomes slightly turbid, and then add 25
mL of 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS, 10 mL of ammonia-ammonium chloride
buffer solution, pH 10.7, 4 drops of eriochrome black T TS
and 5 drops of methyl yellow TS, and titrate <2.50> rapidly
the excess disodium dihydrogen ethylenediamine tetraacetate
with 0.05 mol/L magnesium chloride VS, until the green
color of the solution disappears and a red color develops.
Perform a blank determination.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.004 mg of Ca
Containers and storage Containers — Well-closed contain-
ers.
Camellia Oil
Oleum Camelliae
Camellia Oil is the fixed oil obtained from the peeled
seeds of Camellia japonica Linne (Theaceae).
Description Camellia Oil is a colorless or pale yellow, clear
oil. It is nearly odorless and tasteless.
It is miscible with diethyl ether and with petroleum ether.
It is slighthy soluble in ethanol (95).
It congeals partly at — 10°C, and completely at - 15 C C.
Specific gravity d\\: 0.910 - 0.914
Identification To 2 mL of Camellia Oil add dropwise 10 mL
of a mixture of fuming nitric acid, sulfuric acid, and water
(1:1:1), previously cooled to room temperature: a bluish
green color develops at the zone of contact.
Acid value <1.13> Not more than 2.8.
Saponification value <1.13> 188 - 194
Unsaponifiable matters <1.13> Not more than 1.0%.
Iodine value <1.13> 78 - 83
Containers and storage Containers — Tight containers.
Camostat Mesilate
NH
X.
H,N N
H
^K
CHj
CH-i
■ H 3 C— SO a H
C 20 H 22 N4O5.CH 4 O3S: 494.52
Dimethylcarbamoylmethyl
4-(4-guanidinobenzoyloxy)phenylacetate
monomethanesulfonate [59721-29-8]
Camostat Mesilate, when dried, contains not less
JPXV
Official Monographs / rf-Camphor
401
than 98.5% of QoH^Oj.CH^S.
Description Camostat Mesilate occurs as white crystals or
crystalline powder.
It is sparingly soluble in water, slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
Identification (1) To 4 mL of a solution of Camostat
Mesilate (1 in 2000) add 2 mL of 1-naphthol TS and 1 mL of
diacetyl TS, and allow to stand for 10 minutes: a red color
develops.
(2) Determine the absorption spectrum of a solution of
Camostat Mesilate (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Camostat Mesilate Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) To 0.1 g of Camostat Mesilate add 0.2 g of sodium
hydroxide, fuse by gentle heating, and continue to heat for 20
to 30 seconds. After cooling, add 0.5 mL of water and 3 mL
of dilute hydrochloric acid, and heat: the gas evolved changes
moistened potassium iodate-starch paper to blue.
Melting point <2.60> 194 - 198°C
Purity (1) Heavy metals <1.07> — Dissolve 1.0 g of
Camostat Mesilate in 40 mL of water by warming, and add 2
mL of dilute acetic acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 2.0 mL of Standard Lead Solution and
2 mL of dilute acetic acid (not more than 20 ppm).
(2) Arsenic <I.II> — Dissolve 2.0 g of Camostat Mesilate
in 20 mL of 2mol/L hydrochloric acid TS by heating in a
water bath, and continue to heat for 20 minutes. After cool-
ing, centrifuge, take 10 mL of the supernatant liquid, and use
this solution as the test solution. Perform the test (not more
than 2 ppm).
(3) Related substances — Dissolve 30 mg of Camostat
Mesilate in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
ethanol (95) to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, water and acetic acid
(100) (3:1:1) to a distance of about 10 cm, and air-dry the
plate. Allow the plate to stand overnight in iodine vapor: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 1.0% (1 g, silica gel,
105°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 50 mg each of Camostat
Mesilate and Camostat Mesilate Reference Standard, previ-
ously dried, and dissolve each in water to make exactly 50
mL. Pipet 5 mL each of these solutions, add exactly 5 mL of
the internal standard solution, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 2//L each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qt and Q s , of the peak area of camostat to that of the inter-
nal standard.
Amount (mg) of QoH^CvCH^S
= W s x (Q T /Q S )
W s : Amount (mg) of Camostat Mesilate Reference Stan-
dard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in ethanol (95) (1 in 1500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol, a solution of sodi-
um 1-heptane sulfonate (1 in 500), a solution of sodium
lauryl sulfate (1 in 1000) and acetic acid (100) (200:100:50:1).
Flow rate: Adjust the flow rate so that the retention time of
camostat is about 10 minutes.
System suitability —
System performance: When the procedure is run with 2 /iL
of the standard solution under the above operating condi-
tions, camostat and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of camostat to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
^/-Camphor
d-t)>7)l
C 10 H 16 O: 152.23
(li?,4i?)-l,7,7-Trimethylbicyclo[2.2.1]heptan-2-one
[464-49-3]
cf-Camphor contains not less than 96.0% of
CioH 16 0.
Description rf-Camphor occurs as colorless or white, trans-
lucent crystals, crystalline powder or masses. It has a charac-
teristic, agreeable odor, and a slightly bitter taste, followed
by a pleasant, cooling sensation.
It is freely soluble in ethanol (95), in diethyl ether and in
carbon disulfide, and slightly soluble in water.
It slowly volatilizes at room temperature.
402
(//-Camphor / Official Monographs
JP XV
Identification Dissolve 0.1 g of d-Camphor in 2 mL of
methanol, add 1 mL of 2,4-dinitrophenylhydradine TS, and
heat for 5 minutes on a water bath: an orange-red precipitate
is formed.
Optical rotation <2.49> [a]™: +41.0 - +43.0° (5 g, ethanol
(95), 50 mL, 100 mm).
Melting point <2.60> 111 - 182°C
Purity (1) Water— Shake 1 .0 g of d-Camphor with 10 mL
of carbon disulfide: the solution is clear.
(2) Chlorinated compounds — Mix 0.20 g of finely pow-
dered d-Camphor with 0.4 g of sodium peroxide in a dried
porcelain crucible. Heat the crucible gently by the open flame
until the incineration is complete. Dissolve the residue in 20
mL of warm water, acidify with 12 mL of dilute nitric acid,
and filter the solution into a Nessler tube. Wash the filter
paper with three 5-mL portions of hot water, adding the
washings to the filtrate. After cooling, add water to make 50
mL, then add 1 mL of silver nitrate TS, mix well, and allow
to stand for 5 minutes: the turbidity of the solution does not
exceed that of the following control solution.
Control solution: Prepare in the same manner as described
above, using 0.20 mL of 0.01 mol/L hydrochloric acid VS.
(3) Non-volatile residue — Heat 2.0 g of d-Camphor on a
water bath until sublimation is complete, then dry the residue
at 105°C for 3 hours: the mass of the residue does not exceed
1.0 mg.
Assay Weigh accurately about 0.1 g each of d-Camphor
and d-Camphor Reference Standard, add exactly 5 mL each
of the internal standard solution, dissolve in dehydrated
methanol to make 100 mL, and use these solutions as the
sample solution and the standard solution. Perform the test
with 2//L each of these solutions as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, Q T and Q s , of the peak area of
d-camphor to that of the internal standard.
Amount (mg) of C I0 H 16 O
= W s x (Q T /Q S )
W s : Amount (mg) of d-Camphor Reference Standard
Internal standard solution — A solution of methyl salicylate in
ethanol (99.5) (1 in 25).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 3 m
in length, which is packed with 10% of polyethylene glycol 20
M for gas chromatography supported on 180 to 250 /xm mesh
silanized siliceous earth for gas chromatography.
Column temperature: A constant temperature of about
160°C
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
d-camphor is about 6 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, d-camphor and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of d-camphor to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
^/-Camphor
Synthetic Camphor
d/-+J>7JU
H^C .CH 3
and enantiomer
C 10 H 16 O: 152.23
(li?S,4flS>l,7,7-Trimethylbicyclo[2.2.1]heptan-2-one
[76-22-2]
dV-Camphor contains not less than 96.0% of
CioH 16 0.
Description d/-Camphor occurs as colorless or white, trans-
lucent crystals, crystalline powder or masses. It has a charac-
teristic, agreeable odor, and has a slightly bitter taste fol-
lowed by a pleasant, cooling sensation.
It is freely soluble in ethanol (95), in diethyl ether and in
carbon disulfide, and slightly soluble in water.
It slowly volatilizes at room temperature.
Identification Dissolve 0.1 g of d/-Camphor in 2mL of
methanol, add 1 mL of 2,4-dinitrophenylhydradine TS, and
heat for 5 minutes on a water bath: an orange-red precipitate
is formed.
1.5- +1.5° (5g, ethanol
Optical rotation <2.49> [«]£>:
(95), 50 mL, 100 mm).
Melting point <2.60> 175 - 180°C
Purity (1) Water— Shake 1 .0 g of d/-Camphor with 10 mL
of carbon disulfide: the solution is clear.
(2) Chlorinated compounds — Mix 0.20 g of finely pow-
dered d/-Camphor with 0.4 g of sodium peroxide in a dried
porcelain crucible. Heat the crucible gently by the open flame
until the incineration is complete. Dissolve the residue in 20
mL of warm water, acidify with 12 mL of dilute nitric acid,
and filter the solution into a Nessler tube. Wash the filter
paper with three 5-mL portions of hot water, adding the
washings to the filtrate. After cooling, add water to make 50
mL, then add 1 mL of silver nitrate TS, mix well, and allow
to stand for 5 minutes: the turbidity of the solution does not
exceed that of the following control solution.
Control solution: Prepare in the same manner as described
above, using 0.20 mL of 0.01 mol/L hydrochloric acid VS.
(3) Non-volatile residue — Heat 2.0 g of d/-Camphor on a
water bath until sublimation is complete, then dry the residue
at 105°C for 3 hours: the mass of the residue does not exceed
l.Omg.
Assay Weigh accurately about 0.1 g each of d/-Camphor
and d/-Camphor Reference Standard, add exactly 5 mL each
of the internal standard solution, dissolve in dehydrated
methanol to make 100 mL, and use these solutions as the
JPXV
Official Monographs / Captopril
403
sample solution and standard solution, respectively. Perform
the test with 2 //L each of these solutions as directed under
Gas Chromatography <2.02> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of (//-camphor to that of the internal standard.
Amount (mg) of Ci H 16 O
= W s x (Qj/Q s )
W s : Amount (mg) of (//-Camphor Reference Standard
Internal standard solution — A solution of methyl salicylate in
ethanol (99.5) (1 in 25).
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A glass column 3 mm in inside diameter and 3
m in length, which is packed with 10% of polyethylene
glycol 20 M for gas chromatography supported on 180 to
250 [im mesh silanized siliceous earth for gas chro-
matography.
Column temperature: A constant temperature of about
160°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
(//-camphor is about 6 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, (//-camphor and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of (//-camphor to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Capsules
Capsules are made of gelatin or a suitable material,
and their shape is a pair of cylinders with one end
closed which can be overlapped on each other.
Method of preparation Dissolve Gelatin or the like in water
by warming, add Glycerin or D-Sorbitol, emulsifier, preser-
vatives, coloring substances and so forth, if necessary, to
make a thick gluey solution, and form into capsules while
warm.
Capsules may be coated with a lubricant, if necessary.
Description Capsules are odorless and elastic.
Purity Odor, solubility, and acidity or alkalinity — Place,
without overlapping of the parts, 1 piece (1 pair) of Capsules
in a 100-mL conical flask, add 50 mL of water, and shake
often, keeping the temperature at 37 ± 2°C. Perform this test
5 times: they all dissolve within 10 minutes. All these solu-
tions are odorless, and neutral or slightly acidic.
Containers and storage Containers — Well-closed contain-
ers.
Captopril
H CH 3
N ,.C0 2 rt
C 9 H 15 N0 3 S: 217.29
(2S)-l-[(25')-2-Methyl-3-sulfanylpropanonyl]pyrrolidine-2-
carboxylic acid [62571-86-2]
Captopril contains not less than 98.0% of
C 9 H 15 N0 3 S, calculated on the dried basis.
Description Captopril occurs as white crystals or crystalline
powder.
It is very soluble in methanol, freely soluble in ethanol
(99.5), and soluble in water.
Identification Determine the infrared absorption spectrum
of Captopril as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]^ 5 : -125- -134° (after drying,
0.1 g, ethanol (99.5), 10 mL, 100 mm).
Melting point <2.60> 105-110°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Captopril according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Captopril according to Method 1, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.20 g of Captopril in
methanol to make exactly 10 mL, and use this solution as the
sample solution. Separately, dissolve 15 mg of 1,1'-
[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-diproline in metha-
nol to make exactly 250 mL, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10,mL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop with a
mixture of toluene and acetic acid (100) (13:7) to a distance of
about 15 cm, and air-dry the plate. Place the plate in a cham-
ber filled with iodine vapor, and allow to stand for 30
minutes: the number of the spots other than the spot corre-
sponding to that from the standard solution and the principal
spot from the sample solution is not more than two, and they
are not more intense than the spot from the standard solu-
tion.
(4) 1 , 1 ' - [3 ,3 ' -Dithiobis(2-methyl- 1 -oxopropyl)] -L-
diproline — Dissolive 0. 10 g of Captopril in methanol to make
exactly 20 mL, and use this solution as the sample solution.
Separately, dissolve 25 mg of l,l'-[3,3'-dithiobis(2-methyl-l-
oxopropyl)]-L-diproline in methanol to make exactly 250 mL,
and use this solution as the standard solution. Perform the
404
Carbamazepine / Official Monographs
JP XV
test with exactly 20 iuL each of these solutions as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the peak area, A T and A s , of
l,r-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-diproline of
these solutions: A T is not larger than ^4 S .
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, methanol and phos-
phoric acid (1000:1000:1).
Flow rate: Adjust the flow rate so that the retention time of
l,l'-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-diproline is
about 10 minutes.
System suitability —
System performance: Dissolve 25 mg each of Captopril
and l,l'-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-diproline
in 200 mL of methanol. When the procedure is run with 20
/uL of this solution under the above operating conditions,
captopril and l,l'-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-
diproline are eluted in this order with the resolution between
these peaks being not less than 3.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of l,l'-[3,3'-dithiobis(2-methyl-l-oxopropyl)]-L-diproline is
not more than 2.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Captopril, dissolve
in 100 mL of water, add 20 mL of dilute sulfuric acid and 1 g
of potassium iodide, and shake. Titrate <2.50> with 1/60 mol
/L potassium iodate VS (indicator: 2 mL of starch TS). Per-
form a blank determination in the same manner, and make
any necessary correction.
Each mL of 1/60 mol/L potassium iodate VS
= 21.73 mg of C 9 H 15 N0 3 S
Containers and storage Containers — Tight containers.
Carbamazepine
C 15 H 12 N 2 0: 236.27
5//-Dibenz[£>,/]azepine-5-carboxamide [298-46-4]
Carbamazepine, when dried, contains not less than
97.0% and not more than 103.0%, of C 15 H 12 N 2 0.
Description Carbamazepine occurs as a white to slightly
yellowish white powder. It is odorless and tasteless at first,
and leaves a slightly bitter aftertaste.
It is freely soluble in chloroform, sparingly soluble in
ethanol (95) and in acetone, and very slightly soluble in water
and in diethyl ether.
Identification (1) To 0.1 g of Carbamazepine add 2 mL of
nitric acid, and heat on a water bath for 3 minutes: an oran-
ge-red color is produced.
(2) To 0.1 g of Carbamazepine add 2 mL of sulfuric acid,
and heat on a water bath for 3 minutes: a yellow color is
produced with a green fluorescence.
(3) Examine Carbamazepine under ultraviolet light: the
solution shows an intense blue fluorescence.
(4) Determine the absorption spectrum of the solution
obtained in the Assay as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
Melting point <2.60> 189 - 193 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Carbamazepine in 10 mL of chloroform: the solution is clear
and colorless to pale yellow.
(2) Acidity — To 2.0 g of Carbamazepine add exactly 40
mL of water, stir well for 15 minutes, and filter through a g-
lass filter (G3). To 10 mL of this filtrate add 1 drop of
phenolphthalein TS and 0.50 mL of 0.01 mol/L sodium
hydroxide VS: a red color is produced.
(3) Alkalinity — To 10 mL of the filtrate obtained in (2)
add 1 drop of methyl red TS and 0.50 mL of 0.01 mol/L
hydrochloric acid VS: a red color is produced.
(4) Chloride <1.03> — Dissolve 0.25 g of Carbamazepine
in 30 mL of acetone, add 6 mL of dilute nitric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.20 mL of 0.01 mol/L hydrochloric acid VS add 30 mL of
acetone, 6 mL of dilute nitric acid and water to make 50 mL
(not more than 0.028%).
(5) Heavy metals <1. 07>— Proceed with 2.0 g of Car-
bamazepine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(6) Related substances — Dissolve 0.25 g of Carbamaze-
pine in 10 mL of chloroform, and use this solution as the
sample solution. Separately, dissolve 5.0 mg of iminodiben-
zyl in chloroform to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of toluene and methanol (19:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly potassium dichromate-sulfuric acid TS on the plate:
the spots other than the principal spot obtained from the
sample solution is not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 50 mg of Carbamazepine, previously
JPXV
Official Monographs / Carbazochrome Sodium Sulfonate Hydrate
405
dried and accurately weighed, in ethanol (95) to make exactly
250 mL. Dilute 5 mL of this solution with ethanol (95) to ex-
actly 100 mL. Perform the test as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and determine the absor-
bance A of this solution at the wavelength of maximum
absorption at about 285 nm.
Amount (mg) of C 15 H 12 N 2 = (A/490) x 50,000
Containers and storage Containers — Tight containers.
Carbazochrome Sodium Sulfonate
Hydrate
CHs
Y^
o
and enantiomer
C 1 oH 11 N 4 Na0 5 S.3H 2 0: 376.32
Monosodium (2/?5)-l-methyl-6-oxo-5-semicarbazono-
2,3,5,6-tetrahydroindole-2-sulfonate trihydrate
[52422-26-5, anhydride]
Carbazochrome Sodium Sulfonate Hydrate contains
not less than 98.0% and not more than 102.0% of car-
bazochrome sodium sulfonate (C 10 H n N4NaO 5 S:
322.27), calculated on the anhydrous basis.
Description Carbazochrome Sodium Sulfonate Hydrate oc-
curs as orange-yellow, crystals or crystalline powder.
It is sparingly soluble in water, very slightly soluble in
methanol and in ethanol (95), and practically insoluble in
diethyl ether.
A solution of Carbazochrome Sodium Sulfonate (1 in 100)
shows no optical rotation.
Melting point: about 210°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Carbazochrome Sodium Sulfonate Hydrate (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Car-
bazochrome Sodium Sulfonate Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Carbazochrome Sodium Sulfonate Hy-
drate (1 in 100) responds to the Qualitative Tests <1.09> (1)
for sodium salt.
pH <2.54> Dissolve 0.8 g of Carbazochrome Sodium Sul-
fonate Hydrate in 50 mL of water by warming, and cool: the
pH of this solution is between 5.0 and 6.0.
Purity (1) Clarity of solution — Dissolve 1.0 g of Carbazo
chrome Sodium Sulfonate Hydrate in 50 mL of water by
warming, and allow to cool: the solution is clear. Perform the
test with this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: the absorbance at 590 nm is not
more than 0.070.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Car-
bazochrome Sodium Sulfonate Hydrate according to Method
2, and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 50 mg of Carbazo-
chrome Sodium Sulfonate Hydrate in 100 mL of water, and
use this solution as the sample solution. Pipet 2 mL of the
sample solution, add water to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of these solutions by the automatic integration method:
the total area of the peaks other than the peak of car-
bazochrome sulfonate from the sample solution is not larger
than the peak area of carbazochrome sulfonate from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 360 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.2 g of ammonium dihydrogen
phosphate in 1000 mL of water, and filter through a mem-
brane filter (0.4 /um in pore size) if necessary. To 925 mL of
this solution add 75 mL of ethanol (95), shake, and adjust the
pH to 3 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
carbazochrome sulfonate is about 7 minutes.
Time span of measurement: About 3 times as long as the
retention time of carbazochrome sulfonate beginning after
the solvent peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of carbazochrome sulfonate
obtained from 1 /xL of this solution is equivalent to 7 to 1 3 %
of that of carbazochrome sulfonate obtained from 10 /uL of
the standard solution.
System performance: Dissolve 10 mg each of Car-
bazochrome Sodium Sulfonate and carbazochrome in
100 mL of water by warming. When the procedure is run with
10 fiL of this solution under the above operating conditions,
carbazochrome sulfonate and carbazochrome are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
carbazochrome sulfonate is not more than 2.0%.
Water <2.48> 13.0 - 16.0% (0.3 g, direct titration).
Assay Weigh accurately about 0.25 g of Carbazochrome
Sodium Sulfonate Hydrate, dissolve in 50 mL of water, apply
to a chromatographic column, 10 mm in diameter, previously
prepared with 20 mL of strongly acidic ion exchange resin for
column chromatography (type H), and allow to flow at a rate
406
Carbidopa Hydrate / Official Monographs
JP XV
of 4 mL per minute. Wash the column with 150 mL of water,
combine the washing and the former effluent solution, and
titrate <2.50> with 0.05 mol/L sodium hydroxide VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.05 mol/L sodium hydroxide VS
= 16.11 mg of C 10 H 11 N 4 NaO 5 S
Containers and storage Containers — Well-closed contain-
ers.
Carbidopa Hydrate
ti ii tf k n°7kft ti
CO s H
*^ y HaC N-NH 2 * H 2°
C 10 H 14 N 2 O 4 .H 2 O: 244.24
(25')-2-(3,4-Dihydroxybenzyl)-2-hydrazinopropanoic
acid monohydrate [38821-49-7]
Carbidopa Hydrate contains not less than 98.0% of
C 10 H 14 N 2 O 4 .H 2 O.
Description Carbidopa Hydrate occurs as a white to yellow-
ish white powder.
It is sparingly soluble in methanol, slightly soluble in
water, very slightly soluble in ethanol (95), and practically in-
soluble in diethyl ether.
Melting point: about 197°C (with decomposition).
Identification (1) Dissolve 0.01 g of Carbidopa Hydrate in
250 mL of a solution of hydrochloric acid in methanol (9 in
1000). Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>
at the wavelengths between 240 nm and 300 nm, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Carbidopa Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Car-
bidopa Hydrate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Carbidopa Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a£°: -21.0 - -23.5° (1 g, alumi-
num (III) chloride TS, 100 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Carbidopa Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 50 mg of Carbidopa
Hydrate in 70 mL of the mobile phase, by warming and using
ultrasonication, if necessary. After cooling, add the mobile
phase to make 100 mL, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add the mobile
phase to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area from both solu-
tions by the automatic integration method: the total area of
all peaks other than the peak of carbidopa from the sample
solution is not larger than the peak area of carbidopa from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of carbidopa.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of carbidopa obtained from
20 [iL of this solution is equivalent to 7 to 13% of that of
carbidopa obtained from 20,mL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Loss on drying <2.41> 6.9 - 7.9% (1 g, in vacuum not ex-
ceeding 0.67 kPa, 100°C, 6 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Carbidopa
Hydrate and Carbidopa Reference Standard (determine
separately the loss on drying <2.41> in the same menner as
Carbidopa Hydrate), and dissolve each in 70 mL of the mo-
bile phase, by warming and using ultrasonication if necessa-
ry. After cooling, add the mobile phase to make exactly 100
mL, and use these solutions as the sample solution and stan-
dard solution, respectively. Perform the test with exactly 20
IxL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of carbidopa in each solution.
Amount (mg) of C 10 H 14 N 2 O 4 .H 2 O
= W s x (Aj/A s ) x 1.0796
W s : Amount (mg) of Carbidopa Reference Standard, cal-
culated on the dried basis
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (7//m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 950 mL of 0.05 mol/L sodium dihydro-
gen phosphate TS add 50 mL of ethanol (95), and adjust the
pH to 2.7 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
carbidopa is about 6 minutes.
System suitability —
System performance: Dissolve 50 mg each of Carbidopa
and methyldopa in 100 mL of the mobile phase. When the
procedure is run with 20 /xh of this solution under the above
operating conditions, methyldopa and carbidopa are eluted
in this order with the resolution between these peaks being
not less than 0.9.
JPXV
Official Monographs / Carbon Dioxide
407
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
carbidopa is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
L-Carbocisteine
l-^JUtK'vXx-I' >
HCfcC
h' nh 2
CO;H
C 5 H 9 N0 4 S: 179.19
(2/?)-2-Amino-3-carboxymethylsulfanylpropanoic acid
[638-23-3]
L-Carbocisteine, when dried, contains not less than
98.5% of C 5 H 9 N0 4 S.
Description L-Carbocisteine occurs as a white crystalline
powder. It is odorless, and has a slightly acid taste.
It is very slightly soluble in water, and practically insoluble
in ethanol (95).
It dissolves in dilute hydrochloric acid or in sodium
hydroxide TS.
Melting point: about 186°C (with decomposition).
Identification (1) To 0.2 g of L-Carbocisteine add 1 mL of
lead acetate TS and 3 mL of water, shake, add 0.2 g of sodi-
um hydroxide, and heat over a flame for 1 minute: a dark
brown to black precipitate is formed.
(2) Determine the infrared absorption spectrum of L-Car-
bocisteine as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [«]£?: -33.5- -36.5° Weigh ac-
curately about 5 g of L-Carbocisteine, previously dried, dis-
solve in 20 mL of water and a suitable amount of a solution
of sodium hydroxide (13 in 100), and adjust the pH with 1
mol/L hydrochloric acid TS or 0.1 mol/L hydrochloric acid
TS to 6.0, and add water to make exactly 50 mL. Determine
the optical rotation of this solution in a 100-mm cell.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
L-Carbocisteine in 10 mL of sodium hydroxide TS: the solu-
tion is clear and colorless.
(2) Chloride <1.03>— Dissolve 0.20 g of L-Carbocisteine
in 10 mL of water and 20 mL of nitric acid, and add water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution as follows. To 0.40 mL
of 0.01 mol/L hydrochloric acid VS add 20 mL of nitric acid
and water to make 50 mL (not more than 0.071%).
(3) Ammonium <1.02> — Perform the test with 0.25 g of
L-Carbocisteine using the distillation under reduced pressure.
Prepare the control solution with 5.0 mL of Standard Am-
monium Solution (not more than 0.02%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of L-Car-
bocisteine according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of L-Carbocisteine according to Method 3, and perform the
test (not more than 2 ppm).
(6) Related substances — Dissolve 0.30 g of L-Car-
bocisteine in 10 mL of 0.2 mol/L sodium hydroxide TS, and
use this solution as the sample solution. Pipet 2 mL of the
sample solution, and add 0.2 mol/L sodium hydroxide TS to
make exactly 100 mL. Pipet 1 mL of this solution, and add
0.2 mol/L sodium hydroxide TS to make exactly 10 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution, in 15 mm length along the starting line
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of 1-butanol, water and acetic
acid (100) (3:1:1) to a distance of about 10 cm, and dry the
plate at 80 °C for 30 minutes. Spray evenly a solution of nin-
hydrin in acetone (1 in 50) on the plate, and heat at 80°C for
5 minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of L-Carbocisteine,
previously dried, dissolve in exactly 20 mL of 0.1 mol/L per-
chloric acid VS and 50 mL of acetic acid (100), and titrate
<2.50> the excess perchloric acid with 0.1 mol/L sodium
acetate VS (potentiometric titration). Perform a blank deter-
mination.
Each mL of 0.1 mol/L perchloric acid VS
= 17.92 mg of C 5 H 9 N0 4 S
Containers and storage Containers — Tight containers.
Carbon Dioxide
-mitmm
C0 2 : 44.01
Carbon Dioxide contains not less than 99.5 vol% of
co 2 .
Description Carbon Dioxide is a colorless gas at room tem-
perature and under atmospheric pressure. It is odorless.
A 1 mL volume of Carbon Dioxide dissolves in 1 mL of
water, and the solution is slightly acid.
1000 mL of Carbon Dioxide at 0°C and under a pressure of
101.3 kPa weighs about 1.978 g.
Identification (1) Put a flaming wood splinter into Carbon
Dioxide: the flame is extinguished immediately.
(2) Pass Carbon Dioxide into calcium hydroxide TS: a
white precipitate is produced. Collect the precipitate, and add
acetic acid (31): it dissolves with effervescence.
Purity Maintain containers of Carbon Dioxide between
18 C C and 22°C for not less than 6 hours prior to the test, and
correct the volume of Carbon Dioxide to 20°C and under a
408
Carmellose / Official Monographs
JP XV
pressure of 101.3 kPa.
(1) Acidity — Place 50 mL of freshly boiled and cooled
water in a Nessler tube, and pass 1000 mL of Carbon Dioxide
into it for 15 minutes through an introducing tube about 1
mm in diameter extending to 2 mm from the bottom of the
Nessler tube, then add 0.10 mL of methyl orange TS: the so-
lution is not more colored than the following control solu-
tion.
Control solution: To 50 mL of freshly boiled and cooled
water in a Nessler tube add 0.10 mL of methyl orange TS and
1.0 mL of 0.01 mol/L hydrochloric acid VS.
(2) Hydrogen phosphide, hydrogen sulfide or reducing
organic substances — Place 25 mL of silver nitrate-ammonia
TS and 3 mL of ammonia TS in each of two Nessler tubes A
and B, and designate the solution in each tube as solution A
and solution B, respectively. Pass 1000 mL of Carbon Di-
oxide into solution A in the same manner as directed in (1):
the turbidity and color of this solution are the same as that of
solution B.
(3) Carbon monoxide — Introduce 5.0 mL of Carbon Di-
oxide into a gas-cylinder or a syringe for gas chromatography
from a metal cylinder holding gas under pressure and fitted
with a pressure-reducing valve, through a directly connected
polyvinyl tube. Perform the test with this according to the
Gas Chromatography <2.02> under the following conditions:
no peak is observed at the same retention time as that of car-
bon monoxide.
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with 300 to 500 fiva. zeolite for
gas chromatography (0.5 nm in porous size).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
carbon monoxide is about 20 minutes.
Selection of column: To 0.1 mL each of carbon monoxide
and air in a gas mixer add carrier gas to make 100 mL, and
mix well. Proceed with 5.0 mL of the mixed gas under the
above operating conditions. Use a column giving elution of
oxygen, nitrogen and carbon monoxide in this order with a
well-resolving of their peaks.
Detection sensitivity: Adjust the sensitivity so that the peak
height of carbon monoxide obtained from 5.0 mL of the mix-
ed gas used in the selection of column is about 10 cm.
(4) Oxygen and nitrogen — Introduce 1.0 mL of Carbon
Dioxide into a gas-measuring tube or syringe for gas chro-
matography from a metal cylinder under pressure with a
pressure-reducing valve through a directly connected poly-
vinyl chloride tube. Perform the test as directed under Gas
Chromatography <2.02> according to the following condi-
tions, and determine the peak area A T of air. Separately, in-
troduce 0.50 mL of nitrogen into the gas mixer, draw carrier
gas into the mixer to make exactly 100 mL, allow to mix
thoroughly, and use this mixture as the standard gas mixture.
Perform the test with 1.0 mL of this mixture in the same
manner as directed in the case of Carbon Dioxide, and deter-
mine the peak area A s of nitrogen: A T is smaller than A s , and
no other peak appears.
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with silica gel for gas chro-
matography (300 to 500 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
nitrogen is about 2 minutes.
Selection of column: Collect 0.5 mL of nitrogen in a gas
mixer, add Carbon Dioxide to make 100 mL, mix well, and
proceed with 1.0 mL of the mixture under the above operat-
ing conditions. Use a column giving well-resolved peaks of
nitrogen and Carbon Dioxide in this order.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of nitrogen obtained from 1 .0 mL of the
standard gas mixture composes about 50% of the full scale.
Assay For the withdrawing of Carbon Dioxide, proceed as
directed in the Purity. Place 125 mL of a solution of potassi-
um hydroxide (1 in 2) in a gas pipet of suitable capacity.
Measure exactly about 100 mL of Carbon Dioxide in a
100-mL gas buret filled with water. Force the entire volume
of gas into the gas pipet, and shake for 5 minutes. Draw some
of the unabsorbed gas into the gas buret, measure the
volume, force the residual back upon the surface of the liquid
in the gas pipet, and repeat this procedure until a constant
volume of the residual reading is obtained. Determine the
volume K(mL) of the residual gas, and correct its volume V
to 20°C and under a pressure of 101.3 kPa.
Volume (mL) of C0 2
= calculated volume (mL) of the sample
— calculated volume V (mL)
Containers and storage Containers — Pressure resistant
metal cylinders.
Storage— Not exceeding 40°C.
Carmellose
Carboxymethylcellulose
CMC
Carmellose is a polycarboxymethylether of cellulose.
Description Carmellose occurs as a white powder. It is
odorless and tasteless.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It swells with water to form suspension.
It becomes viscid in sodium hydroxide TS.
The pH of a suspension, obtained by shaking 1.0 g of Car-
mellose with 100 mL of water, is between 3.5 and 5.0.
It is hygroscopic.
Identification (1) Shake well 0.1 g of Carmellose with 10
mL of water, add 2 mL of sodium hydroxide TS, shake, and
allow to stand for 10 minutes. Use this solution as the sample
solution. To 1 mL of the sample solution add water to make 5
mL. To 1 drop of this solution add 0.5 mL of concentrated
disodium chlomotropate TS, and heat in a water bath for 10
minutes: a red-purple color develops.
(2) Shake 5 mL of the sample solution obtained in (1)
JPXV
Official Monographs / Carmellose Calcium
409
with 10 mL of acetone: a white, fiocculent precipitate is
produced.
(3) Shake 5 mL of the sample solution obtained in (1)
with 1 mL of iron (III) chloride TS: a brown, flocculent
precipitate is produced.
Purity (1) Chloride <1.03>— Shake well 0.8 g of Carmel-
lose with 50 mL of water, dissolve in 10 mL of sodium
hydroxide TS, and add water to make 100 mL. Heat 20 mL
of this solution with 10 mL of dilute nitric acid on a water
bath until a flocculent precipitate is produced, cool, cen-
trifuge, and take out the supernatant liquid. Wash the
precipitate with three 10-mL portions of water by centrifug-
ing each time, combine the supernatant liquid and the wash-
ings, and add water to make 100 mL. Take 25 mL of this so-
lution, and add 6 mL of dilute nitric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.40 mL of 0.01 mol/
L hydrochloric acid VS (not more than 0.360%).
(2) Sulfate <1.14>— Shake well 0.40 g of Carmellose with
25 mL of water, dissolve in 5 mL of sodium hydroxide TS,
and add 20 mL of water. Heat this solution with 2.5 mL of
hydrochloric acid in a water bath until a flocculent precipitate
is produced. Cool, centrifuge, and take out the supernatant
liquid. Wash the precipitate with three 10-mL portions of
water by centrifuging each time, combine the supernatant liq-
uid and the washings, and add water to make 100 mL. Filter
this solution, discard 5 mL of the first filtrate, take 25 mL of
the subsequent filtrate, and add 1 mL of dilute hydrochloric
acid and water to make 50 mL. Perform the test using this
solution as the test solution. Prepare the control solution
with 1.5 mL of 0.005 mol/L sulfuric acid VS (not more than
0.720%).
(3) Silicate — Weigh accurately about 1 g of Carmellose,
ignite in a platinum dish, add 20 mL of dilute hydrochloric
acid, cover with a watch glass, and boil gently for 30 minutes.
Remove the watch glass, and evaporate on a water bath to
dryness with the aid of a current of air. Continue heating fur-
ther for 1 hour, add 10 mL of hot water, stir well, and filter
through a filter paper for quantitative analysis. Wash the
residue with hot water, dry the residue together with the filter
paper when no turbidity is produced on the addition of silver
nitrate TS to the last washing, and ignite to constant mass:
the amount of residue is not more than 0.5%.
(4) Heavy metals <1.07> — Proceed with 1.0 g of Carmel-
lose according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(5) Arsenic <1.11> — Take 1.0 g of Carmellose, prepare
the test solution according to Method 3, and perform the test
(not more than 2 ppm).
Loss on drying <2.41> Not more than 8.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 1.5% (after
drying, 1 g).
Containers and storage Containers — Tight containers.
Carmellose Calcium
Carboxymethylcellulose Calcium
CMC Calcium
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Carmellose Calcium is the calcium salt of a polycar-
boxymethyl ether of cellulose.
♦Description Carmellose Calcium occurs as a white to
yellowish white powder.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It swells with water to form a suspension.
The pH of a suspension, obtained by shaking 1.0 g of
Carmellose Calcium with 100 mL of water, is between 4.5
and 6.0.
It is hygroscopic*
Identification (1) Shake thoroughly 0.1 g of Carmellose
Calcium with 10 mL of water, followed by 2 mL of sodium
hydroxide TS, allow to stand for 10 minutes, and use this
solution as the sample solution. To 1 mL of the sample
solution add water to make 5 mL. To 1 drop of this solution
add 0.5 mL of chromotropic acid TS, and heat in a water
bath for 10 minutes: a red-purple color develops.
(2) Shake 5 mL of the sample solution obtained in (1)
with 10 mL of acetone: a white, flocculent precipitate is
produced.
(3) Shake 5 mL of the sample solution obtained in (1)
with 1 mL of iron (III) chloride TS: a brown, flocculent
precipitate is produced.
(4) Ignite 1 g of Carmellose Calcium to ash, dissolve the
residue in 10 mL of water and 6 mL of acetic acid (31), and
filter, if necessary. Boil the filtrate, cool, and neutralize with
ammonia TS: the solution responds to the Qualitative Tests
<1.09> (1) and (3) for calcium salt.
Purity (1) Alkalinity — Shake thoroughly 1.0 g of Carmel-
lose Calcium with 50 mL of freshly boiled and cooled water,
and add 2 drops of phenolphthalein TS: no red color de-
velops.
(2) Chloride <1.03>— Shake thoroughly 0.80 g of Carmel-
lose Calcium with 50 mL of water, add 10 mL of sodium
hydroxide TS to dissolved, add water to make 100 mL, and
use this solution as the sample solution. Heat 20 mL of the
sample solution with 10 mL of 2 mol/L nitric acid TS on a
water bath until a flocculent precipitate is produced. After
cooling, centrifuge, and take out the supernatant liquid.
Wash the precipitate with three 10-mL portions of water by
centrifuging each time, combine the supernatant and the
washings, and add water to make 100 mL. Take 25 mL of
this solution, and add 1 mL of nitric acid and water to make
50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.40 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.36%).
(3) Sulfate <1.14> — Heat 10 mL of the sample solution
410
Carmellose Sodium / Official Monographs
JP XV
obtained in (2) with 1 mL of hydrochloric acid in a water bath
until a flocculent precipitate is produced. Cool, centrifuge,
and take out the supernatant liquid. Wash the precipitate
with three 10-mL portions of water by centrifuging each
time, combine the supernatant and the washings, and add
water to make 100 mL. Perform the test with 25 mL this
solution as the test solution. Prepare the control solution
with 0.42 mL of 0.005 mol/L sulfuric acid VS. To the test
solution and the control solution add 1 mL of 3 mol/L
hydrochloric acid and 3 mL of barium chloride TS, then add
water to make 50 mL, and mix. Allow to stand for 10
minutes, and compare the turbidity of these solutions: the
turbidity obtained with the test solution is not more than that
obtained with the control solution (not more than 1.0%).
*(4) Heavy metals <1.07> — Proceed with 1.0 g of Car-
mellose Calcium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).»
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> 10 - 20% (after drying 1 g).
♦Containers and storage Containers — Tight containers. ♦
Carmellose Sodium
Carboxymethylcellulose Sodium
CMC Sodium
till/ P-Xth U^7A
Carmellose Sodium is the sodium salt of a polycar-
boxymethylether of cellulose.
It, when dried, contains not less than 6.5% and not
more than 8.5% of sodium (Na: 22.99).
Description Carmellose Sodium occurs as a white to yellow-
ish white powder or granules. It has no taste.
It is practically insoluble in methanol, in ethanol (95), in
acetic acid (100) and in diethyl ether.
It forms a viscid solution in water and in warm water.
It is hygroscopic.
Identification (1) Dissolve 0.2 g of Carmellose Sodium in
20 mL of warm water with stirring, cool, and use this solu-
tion as the sample solution. To 1 mL of the sample solution
add water to make 5 mL. To 1 drop of this solution add 0.5
mL of concentrated disodium chlomotropate TS, and heat in
a water bath for 10 minutes: a red-purple color develops.
(2) To 10 mL of the sample solution obtained in test (1)
add 1 mL of copper (II) sulfate TS: a blue flocculent
precipitate is produced.
(3) To 3 g of Carmellose Sodium add 20 mL of methanol
and 2 mL of dilute hydrochloric acid, boil gently on a water
bath for 5 minutes, and filter. Evaporate the filtrate to dry-
ness, and add 20 mL of water to the residue: the solution
responds to the Qualitative Tests <1.09> for sodium salt.
pH <2.54> Add 1.0 g of Carmellose Sodium in small por-
tions to 100 mL of warm water with stirring, dissolve, and
cool: the pH of this solution is between 6.0 and 8.0.
Purity (1) Clarity and color of solution — Firmly attach a
glass plate of good quality 2 mm in thickness, to the bottom
of a glass column 250 mm in height, 25 mm in inner diameter
and 2 mm in thickness. This is used as an outer tube. Similar-
ly prepare an inner tube by attaching a glass plate of good
quality 2 mm in thickness to the bottom of a glass column 300
mm in height, 15 mm in inner diameter and 2 mm in thick-
ness. Dissolve 1.0 g of Carmellose Sodium in 100 mL of
water, pour this solution into the outer tube, and place on a
piece of white paper on which 15 parallel black lines 1 mm in
width and 1 mm in interval are drawn. Moving the inner tube
up and down and observing from the upper part, determine
the height of the solution up to the lower edge of the inner
tube when the distinction of the lines becomes impossible.
Repeat the operation 3 times, and calculate the mean value: it
is larger than that calculated from the similar operation, us-
ing the following control solution.
Control solution: To 5.50 mL of 0.005 mol/L sulfuric acid
VS add 1 mL of dilute hydrochloric acid, 5 mL of ethanol
(95) and water to make 50 mL. Add 2 mL of barium chloride
TS, mix well, and allow to stand for 10 minutes. Shake well
this solution before use.
(2) Chloride <1.03>— Dissolve 0.5 g of Carmellose Sodi-
um in 50 mL of water, and use this solution as the sample so-
lution. Shake 10 mL of the sample solution with 10 mL of di-
lute nitric acid, heat to produce a flocculent precipitate in a
water bath, cool, and centrifuge. Separate the supernatant
liquid, wash the precipitate with three 10-mL portions of
water, centrifuging each time, combine the supernatant liq-
uid with the washings, and dilute with water to 200 mL. Per-
form the test using 50 mL of this solution as the test solution.
Prepare the control solution with 0.45 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.640%).
(3) Sulfate <1.14>— Add 1 mL of hydrochloric acid to 10
mL of the sample solution obtained in (2), shake well, heat to
produce a flocculent precipitate in a water bath, cool, and
centrifuge. Separate the supernatant liquid, wash the
precipitate with three 10-mL portions of water, centrifuging
each time, combine the washings with the supernatant liquid
mentioned above, and dilute to 50 mL with water. Take 10
mL of this solution, dilute with water to 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution with 0.40 mL of 0.005 mol/L sulfuric acid
VS (not more than 0.960%).
(4) Silicate — Weigh accurately about 1 g of Carmellose
Sodium, ignite in a platinum dish, add 20 mL of dilute
hydrochloric acid, cover with a watch glass, and boil gently
for 30 minutes. Remove the watch glass, and evaporate on a
water bath to dryness with the aid of a current of air. Con-
tinue heating for further 1 hour, add 10 mL of hot water, stir
well, and filter through a filter paper for quantitative analy-
sis. Wash the residue with hot water, dry together with the
filter paper after no turbidity is produced on the addition of
silver nitrate TS to the last washing, and then ignite to con-
stant mass: the mass of the residue is not more than 0.5%.
(5) Heavy metals <1.07> — Proceed with 1.0 g of Carmel-
lose Sodium according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(6) Arsenic <1.11> — To 1.0 g of Carmellose Sodium add
20 mL of nitric acid, heat gently until it becomes fluid, cool,
add 5 mL of sulfuric acid, and heat until white fumes are
evolved. Add, if necessary, 5 mL of nitric acid after cooling,
and heat again. Repeat this operation until the solution
JPXV
Official Monographs / Carnauba Wax
411
becomes colorless or slightly yellow. After cooling, add 15
mL of a saturated solution of ammonium oxalate monohy-
drate, and heat until white fumes are evolved again, cool, and
dilute with water to 25 mL. Take 5 mL of this solution as the
test solution, and perform the test. The solution has no more
color than the following standard stain.
Standard stain: Without using Carmellose Sodium, pro-
ceed in the same manner, then transfer 5 mL of this solution
to a generator bottle, add exactly 2 mL of Standard Arsenic
Solution, and proceed as directed for the test with the test so-
lution (not more than lOppm).
(7) Starch — Add 2 drops of iodine TS to 10 mL of the
sample solution obtained in (2): no blue color develops.
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C,
4 hours).
Assay Weigh accurately about 0.5 g of Carmellose Sodium,
previously dried, add 80 mL of acetic acid (100), connect with
a reflux condenser, and heat in an oil bath maintained at
130°C for 2 hours. Cool, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 2.299 mg of Na
Containers and storage Containers — Tight containers.
Carmofur
t))l=E7-)l
C u H 16 FN 3 3 : 257.26
5-Fluoro-l-(hexylaminocarbonyl)uracil [61422-45-5]
Carmofur, when dried, contains not less than 98.0%
of C n H 16 FN 3 3 .
Description Carmofur occurs as a white crystalline powder.
It is very soluble in 7V,7V-dimethylformamide, freely solu-
ble in acetic acid (100), soluble in diethyl ether, sparingly
soluble in methanol and in ethanol (99.5), and practically in-
soluble in water.
Melting point: about 111°C (with decomposition).
Identification (1) Proceed with 5 mg of Carmofur as
directed under Oxygen Flask Combustion Method <1.06>, us-
ing a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS
and 20 mL of water as the absorbing liquid, and prepare the
test solution: the test solution responds to the Qualitative
Tests <1.09> (2) for fluoride.
(2) Determine the absorption spectrum of a solution of
Carmofur in a mixture of methanol and phosphoric acid-
acetic acid-boric acid buffer solution, pH 2.0, (9:1) (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Determine the infrared absorption spectrum of Car-
mofur, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Heavy metals <1.07>— Proceed with 2.0 g of
Carmofur according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve 0.20 g of Carmofur in
10 mL of a mixture of methanol and acetic acid (100) (99:1),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add a mixture of methanol and acetic
acid (100) (99:1) to make exactly 500 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 15 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of toluene
and acetone (5:3) to a distance of about 12 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution. After exposure of the plate to bromine
vapor for 30 second, spray evenly a solution of fluorescein in
ethanol (95) (1 in 2500): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
50°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Carmofur, previ-
ously dried, dissolve in 20 mL of A^Af-dimethylformamide,
and titrate <2.50> with 0.1 mol/L tetramethylammonium
hydroxide-methanol VS until the color of the solution
changes from yellow through blue-green to blue (indicator: 3
drops of thymol blue-dimethylformamide TS).
Each mL of 0.1 mol/L tetramethylammonium
hydroxide-methanol VS
= 25.73 mg of C n H 16 FN 3 3
Containers and storage Containers — Tight containers.
Carnauba Wax
Cera Carnauba
Carnauba Wax is the wax obtained from the leaves
of Copernicia cerifera Mart (Palmae).
Description Carnauba Wax occurs as light yellow to light
brown, hard and brittle masses or white to light yellow pow-
der. It has a slight, characteristic odor. It is tastelss.
It is practically insoluble in water, in ethanol (95), in
diethyl ether and in xylene.
Specific gravity df : 0.990 - 1.002
412 Carteolol Hydrochloride / Official Monographs
JP XV
Melting point: 80 - 86°C
Acid value <1.13> Not more than 10.0. Use a mixture of xy-
lene and ethanol (95) (2:1) as solvent.
Saponification value <1.13> 78-95 Weigh accurately
about 3 g of Carnauba Wax in a 300-mL flask, add 25 mL of
xylene, and dissolve by warming. To this solution add 50 mL
of ethanol (95) and exactly 25 mL of 0.5 mol/L potassium
hydroxide-ethanol VS, and proceed as directed in the
Saponification value. The time of heating should be 2 hours
and the titration should be done by warming.
Iodine value <1.13> 5-14 (Dissolve the sample by shaking a
glass-stoppered flask in warm water.)
Containers and storage Containers — Well-closed contain-
ers.
Carteolol Hydrochloride
,CH 3
^5S/
H3C CH 3
• HCI
and enantiomer
5- [(IRSyi -( 1 , 1 -Dimethylethyl)amino-
2-hydroxypropyloxy]-3,4-dihydroquinolin-2(l//)-one
monohydrochloride [51781-21-6]
Carteolol Hydrochloride, when dried, contains not
less than 99.0% of C 16 H 24 N 2 3 .HC1.
Description Carteolol Hydrochloride occurs as white crys
tals or crystalline powder.
It is soluble in water, sparingly soluble in methanol, very
slightly soluble in ethanol (95) and in acetic acid (100), and
practically insoluble in diethyl ether.
The pH of a solution of Carteolol Hydrochloride (1 in 100)
is between 5.0 and 6.0.
The solution of Carteolol Hydrochloride (1 in 20) shows no
optical rotation.
Melting point: about 277°C (with decomposition).
Identification (1) Dissolve 0.1 g of Carteolol Hydrochlo-
ride in 5 mL of water, and add 5 drops of Reinecke salt TS: a
light red precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Carteolol Hydrochloride (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of Car-
teolol Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(4) A solution of Carteolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Carteolol Hydrochloride in 30 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Carteolol
Hydrochloride according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Carteolol Hydrochloride according to Method 3, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Carteolol
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 2 mL of the sample solution, and
add methanol to make exactly 100 mL. Pipet 1 mL of this
solution, add methanol to make exactly 10 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 fiL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol and ammonia solution (28)
(50:20:1) to a distance of about 12 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Carteolol
Hydrochloride, previously dried, add 30 mL of acetic acid
(100), dissolve by heating on a water bath, and cool. After
adding 70 mL of acetic anhydride, titrate <2.50> with 0.1 mol
/L perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.88 mg of C 16 H 24 N 2 3 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Carumonam Sodium
j] )],=£-)- Ai-\> VO A
CO s Na
N ^>— N O
w-i J o h h
C 12 H 12 N 6 Na 2 O 10 S 2 : 510.37
Disodium (Z)-{(2-aminothiazol-4-yl)[(2S,3S)-2-
carbamoyloxymethyl-4-oxo-l-sulfonatoazetidin-3-
ylcarbamoyl]methyleneaminooxy}acetate [86832-68-0]
Carumonam Sodium contains not less than 850 fig
(potency) and not more than 920 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Carumonam Sodium is expressed as mass (potency) of
carumonam (C 12 H 14 N 6 O 10 S 2 : 466.40).
JPXV
Official Monographs / Carumonam Sodium 413
Description Carumonam Sodium occurs as a white to yel-
lowish white, crystals or crystalline powder.
It is freely soluble in water, soluble in formamide, very
slightly soluble in methanol, and practically insoluble in
acetic acid (100) and in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Carumonam Sodium (3 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Carumonam Sodium Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of
Carumonam Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Carumonam Sodium Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Carumonam
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropionate-d 4 for nuclear magnetic resonance
spectroscopy as an internal reference compound: it exhibits a
double signal A at around <55.5 ppm, and a single signal B at
around 61. ppm. The ratio of the integrated intensity of
these signals, A:B, is about 1:1.
(4) Carumonam Sodium responds to the Qualitative
Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [a]™: +18.5- +21.0° (0.1 g calculat-
ed on the anhydrous basis, water, 10 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Carumonam Sodium in 10 mL of water is between 5.0 and
6.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Carumonam Sodium in 5 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Carumonam Sodium according to Method 2, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 15 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Carumonam Sodium according to Method 4, and perform
the test (not more than 1 ppm).
(4) Related substance 1 — Weigh accurately about 0.1 g of
Carumonam Sodium, and dissolve in the mobile phase to
make exactly 50 mL. Pipet 5 mL of this solution, add the mo-
bile phase to make exactly 25 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.1 g of
Carumonam Sodium Reference Standard, and dissolve in the
mobile phase to make exactly 50 mL. Pipet 5 mL of this solu-
tion, and add the mobile phase to make exactly 25 mL. Pipet
1 mL of this solution, add the mobile phase to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 10 iiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method. Calculate the amount of the related substances by
the following equation: the amount of the related substance
having the relative retention time of 0.7 to the peak of
carumonam is not more than 4.0%, and each amount of the
related substances other than the related substance having the
relative retention time of 0.7 to the peak of carumonam is not
more than 1.0%.
Amount (%) of related substance
= (W s /Wj) x (At/ A s )
W s : Amount (g) of Carumonam Sodium Reference Stan-
dard
W T : Amount (g) of the sample
^4 S : Peak area of carumonam from the standard solution
A T : Each peak area other than carumonam from the sam-
ple solution
Operating conditions-
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of carumonam.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add the mobile phase to make
exactly 50 mL. Confirm that the peak area of carumonam ob-
tained from 10 iiL of this solution is equivalent to 7 to 13%
of that from 10 iiL of the standard solution.
System performance: Dissolve 40 mg of Carumonam Sodi-
um in 20 mL of the mobile phase. To 5 mL of this solution
add 5 mL of a solution of resorcinol in the mobile phase (9 in
1000) and the mobile phase to make 25 mL. When the proce-
dure is run with 10 /uL of this solution under the above oper-
ating conditions, resorcinol and carumonam are eluted in this
order with the resolution between these peaks being not less
than 2.5.
System repeatability: When the test is repeated 3 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
carumonam is not more than 2.0%.
(5) Related substance 2 — Weigh accurately about 0.1 g of
Carumonam Sodium, and dissolve in the mobile phase to
make exactly 50 mL. Pipet 5 mL of this solution, add the mo-
bile phase to make exactly 25 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.1 g of
Carumonam Sodium Reference Standard, and dissolve in the
mobile phase to make exactly 50 mL. Pipet 5 mL of this solu-
tion, and add the mobile phase to make exactly 25 mL. Pipet
1 mL of this solution, add the mobile phase to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 10 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method. Calculate the amount of the related substances by
the following equation: the amount of each related substance
is not more than 1.0%.
Amount (%) of related substance
= (W s /Wj) X (At/As)
W s : Amount (g) of Carumonam Sodium Reference Stan-
dard
414
Carumonam Sodium / Official Monographs
JP XV
Wj\ Amount (g) of the sample
^4 S : Peak area of carumonam from the standard solution
A T : Each area of the peaks appeared after the peak of
carumonam from the sample solution
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Assay.
Mobile phase: A mixture of a solution of ammonium sul-
fate (1 in 10,000), methanol and acetic acid (100) (74:25:1).
Flow rate: Dissolve 0.01 g of phthalic acid in the mobile
phase to make 100 mL. Adjust the flow rate so that the reten-
tion time of phthalic acid is about 6.5 minutes when the
procedure is run with 10 /xh of this solution.
Time span of measurement: About 10 times as long as the
retention time of carumonam.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add the mobile phase to make
exactly 50 mL. Confirm that the peak area of carumonam
obtained from 10,mL of this solutionis equivalent to 7 to 13%
of that from 10 juL of the standard solution.
System performance: Dissolve 40 mg of Carumonam Sodi-
um in 20 mL of the mobile phase. To 5 mL of this solution
add 5 mL of a solution of resorcinol in the mobile phase (9 in
1000) and the mobile phase to make 25 mL. When the proce-
dure is run with 10 iiL of this solution under the above oper-
ating conditions, resorcinol and carumonam are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 3 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
carumonam is not more than 2.0%.
(6) Total amount of related substances - The total of the
amounts of the related substances obtained in the Related
substance 1 and the Related substance 2 is not more than
6.0%.
Water <2.48> Not more than 2.0% (0.2 g, volumetric titra-
tion, direct titration; Use a mixture of formamide for water
determination and methanol for water determination (3:1) in-
stead of methanol for water determination).
Assay Weigh accurately an amount of Carumonam Sodium
and Carumonam Sodium Reference Standard, equivalent to
about 40 mg (potency), and dissolve each in the mobile phase
to make exactly 20 mL. Measure exactly 5 mL each of these
solutions, add exactly 5 mL of the internal standard solution
and the mobile phase to make 25 mL, and use these solutions
as the sample solution and standard solution. Perform the
test with 10 iiL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qj and Q s , of the peak area of carumonam to that of the
internal standard.
Amount [ug (potency)] of carumonam (C 12 H 14 N 6 OioS2)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Carumonam Sodium
Reference Standard
Internal standard solution — A solution of resorcinol in the
mobile phase (9 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 [im in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of ammonium sul-
fate (1 in 10,000), methanol and acetic acid (100) (97:2:1).
Flow rate: Adjust the flow rate so that the retention time of
carumonam is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, the internal standard and carumonam are eluted
in this order with the resolution between these peaks being
not less than 2.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of carumonam to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Castor Oil
Oleum Ricini
Castor Oil is the fixed oil obtained by compression
from the seeds of Ricinus communis Linne (Euphor-
biaceae).
Description Castor Oil is a colorless or pale yellow, clear,
viscous oil. It has a slight, characteristic odor, and has a
bland at first, and afterwards slightly acrid taste.
It is miscible with ethanol (99.5) and with diethyl ether.
It is freely soluble in ethanol (95), and practically insoluble
in water.
When cooled to 0°C, it becomes more viscous, and turbidi-
ty is gradually formed.
Identification To 3 g of Castor Oil add 1 g of potassium
hydroxide, and heat the mixture carefully to fuse: a charac-
teristic odor is perceptible. Dissolve the fused matter in 30
mL of water, add an excess of magnesium oxide, and filter.
Acidify the filtrate with hydrochloric acid: white crystals is
produced.
Specific gravity <7.75> df 5 : 0.953 - 0.965
Acid value <1.13> Not more than 1.5.
Saponification value <1.13> 176 - 187
Hydroxyl value <I.I3> 155 - 177
Iodine value <7.75> 80 - 90
Purity Adulteration — Shake to mix 1 .0 g of Castor Oil with
4.0 mL of ethanol (95): it dissolves clearly. Add 15 mL of
ethanol (95): no turbidity is produced.
Containers and storage Containers — Tight containers.
JP XV
Official Monographs / Cefaclor 415
Aromatic Castor Oil
Method of preparation
Castor Oil
Orange Oil
Mentha Oil
990 mL
5mL
5mL
To make 1000 mL
Mix the above ingredients.
Description Aromatic Castor Oil is a colorless or yellowish,
clear, viscous liquid. It has an aromatic odor.
Identification To 3 g of Aromatic Castor Oil add 1 g of
potassium hydroxide, and heat the mixture carefully to fuse:
a characteristic odor is perceptible. Dissolve the fused matter
in 30 mL of water, add an excess of magnesium oxide, and
filter. Acidify the filtrate with hydrochloric acid: white crys-
tals are produced.
Containers and storage Containers — Tight containers.
Cefaclor
t7?^nji
COjH
w
C 15 H 14 C1N304S: 367.81
(6/?,7i?)-7-[(2/?)-2-Amino-2-phenylacetylamino]-3-
chloro-8-oxo-5-thia- 1 -azabicyclo [4.2.0] oct-2-ene-2-
carboxylic acid [53994-73-3]
Cefaclor contains not less than 950 fig (potency) and
not more than 1020 fig (potency) per mg, calculated on
the anhydrous basis. The potency of Cefaclor is ex-
pressed as mass (potency) of cefaclor (C^rl^O^C^S).
Description Cefaclor occurs as a white to yellowish white
crystalline powder.
It is slightly soluble in water and in methanol, and practi-
cally insoluble in TV, TV-dim ethylformamide and in ethanol
(99.5).
Identification (1) Determine the absorption spectrum of a
solution of Cefaclor (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefaclor as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(3) Dissolve 40 mg of Cefaclor in 0.5 mL of heavy water
for nuclear magnetic resonance spectroscopy and 1 drop of
deuterated hydrochloric acid for nuclear magnetic resonance
spectroscopy, and determine the spectrum of this solution as
directed under Nuclear Magnetic Resonance Spectroscopy
<2.21> ('H), using sodium 3-trimethylsilylpropanesulfonate
for nuclear magnetic resonance spectroscopy as an internal
reference compound: it exhibits an AB type quartet signal A
at around <53.7 ppm, and a single signal or a sharp multiple
signal B at around <57.6 ppm. The ratio of the integrated in-
tensity of each signal, A:B, is about 2:5.
(4) Perform the test with Cefaclor as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Optical rotation <2.49> [a]™: + 105 - + 120° (0.1 g calculated
on the anhydrous basis, water, 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefaclor according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution by suspend-
ing 1.0 g of Cefaclor in 10 mL of /V,/V-dimethylformamide,
and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Cefaclor in 10
mL of sodium dihydrogen phosphate TS, pH 2.5, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add sodium dihydrogen phosphate TS, pH 2.5 to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine each peak area by the automatic
integration method: the peak areas other than cefaclor from
the sample solution are not more than 1/2 of the peak area of
cefaclor from the standard solution, and the total of the peak
areas other than cefaclor from the sample solution is not
more than 2 times of the peak area of cefaclor from the stan-
dard solution. If necessary, proceed with 20 fiL of sodium di-
hydrogen phosphate TS, pH 2.5 in the same manner as above
to compensate the base line.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 7.8 g of sodium dihydrogen
phosphate dihydrate in 1000 mL of water, and adjust the pH
to 4.0 with phosphoric acid.
Mobile phase B: To 550 mL of the mobile phase A add 450
mL of acetonitrile for liquid chromatography.
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-30
30-45
45-55
95^75
75^
5^ 25
25 -* 100
100
416
Cefaclor Capsules / Official Monographs
JP XV
Flow rate: 1.0 mL per minute.
Time span of measurement: About 2.5 times as long as the
retention time of cefaclor beginning after the solvent peak.
System suitability-
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add sodium dihydrogen phosphate
TS, pH 2.5 to make exactly 20 mL. Confirm that the peak
area of cefaclor obtained from 20 /xL of this solution is
equivalent to 4 to 6% of that from 20 /xL of the standard
solution.
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, the number of theoretical plates and the sym-
metry factor of the peak of cefaclor are not less than 40,000
steps and 0.8 to 1.3, respectively.
System repeatability: When the test is repeated 3 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviations of the peak areas
and the retention times of cefaclor are not more than 2.0%,
respectively.
Water <2.48> Not more than 6.5% (0.2 g, volumetric titra-
tion, back titration).
Assay Weigh accurately an amount of Cefaclor and
Cefaclor Reference Standard, equivalent to about 0.1 g
(potency), and dissolve each in 0.1 mol/L phosphate buffer
solution, pH 4.5 to make exactly 100 mL. Pipet 10 mL each
of these solutions, add exactly 10 mL of the internal standard
solution, add 0.1 mol/L phosphate buffer solution, pH 4.5 to
make 50 mL, and use these solutions as the sample solution
and the standard solution. Perform the test with 10 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the ratios, Q T and Q s , of the
peak area of cefaclor to that of the internal standard.
Amount [jug (potency)] of C^H^Cl^C^S
= Ws x (St/Ss) x 1000
W s : Amount [mg (potency)] of Cefaclor Reference
Standard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen
phosphate in 1000 mL of water, and adjust the pH to 3 .4 with
diluted phosphoric acid (3 in 500). To 940 mL of this solution
add 60 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefaclor is about 7 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, cefaclor and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefaclor to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefaclor Capsules
Cefaclor Capsules contain not less than 90.0% and
not more than 110.0% of the labeled amount of
cefaclor (QsHhCINjC^S: 367.81).
Method of preparation Prepare as directed under Capsules,
with Cefaclor.
Identification Shake vigorously a quantity of the contents
of one capsule of Cefaclor Capsules, equivalent to 20 mg
(potency) of Cefaclor according to the labeled amount, with
10 mL of water, centrifuge, and use the supernatant liquid as
the sample solution. Separately, dissolve 20 mg of Cefaclor
Reference Standard in 10 mL of water, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 2 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of acetoni-
trile, water, ethyl acetate and formic acid (30:10:10:1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): the principal
spot from the sample solution and the spot from the standard
solution show the same Rf value.
Purity Related substances — Weigh accurately not less than
5 Cefaclor Capsules, open the capsules and carefully take out
the contents, mix well, and powder, if necessary. Wash the
empty capsules with a little amount of diethyl ether, if neces-
sary, allow the capsules to stand at room temperature to
vaporize adhering diethyl ether, and weigh accurately the
capsules to calculate the mass of the contents. Weigh ac-
curately a quantity of the contents, equivalent to about 0.25 g
(potency) of Cefaclor, shake with 40 mL of 0.1 mol/L phos-
phate buffer solution, pH 4.5 for 10 minutes, add the same
buffer solution to make exactly 50 mL, and filter through a
0.45-,wm pore-size membrane filter. Discard the first 1 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. Separately, weigh accurately about 20 mg (potency) of
Cefaclor Reference Standard, and dissolve in 0.1 mol/L
phosphate buffer solution, pH 4.5 to make exactly 20 mL.
Pipet 2.5 mL of this solution, add the same buffer solution to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with exactly 20 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas by the automatic integra-
tion method. Calculate the amount of each related substance
by the following equation: the amount of each related sub-
JP XV
Official Monographs / Cefaclor Fine Granules 417
stance is not more than 0.5%, and the total amount of the
related substances is not more than 2.5%. If necessary, cor-
rect the fluctuation of the base line by performing the test in
the same way with 20 ,mL of 0. 1 mol/L phosphate buffer solu-
tion, pH 4.5.
Amount (%) of each related substance
= (W S /W T ) x (A Ti /A s ) x (W M /C) x (25/2)
Total amount (%) of the related substances
= (W s /Wj) x (2M Tn A4 s ) x (W w /C) x (25/2)
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
W T : Amount (mg) of the contents of Cefaclor Capsules
W M : Average mass (mg) of the contents in 1 capsule
A Ti : Area of each peak other than cefaclor and solvent
from the sample solution
^4 S : Peak area of cefaclor from the standard solution
C: Labeled potency [mg (potency)] of Cefaclor in 1 capsule
Operating conditions —
Proceed as directed in the operating conditions in the Puri-
ty (3) under Cefaclor.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add 0.1 mol/L phosphate buffer solution, pH
4.5 to make exactly 20 mL. Confirm that the peak area of
cefaclor obtained with 20 /xL of this solution is equivalent to
3.5 to 6.5% of that of cefaclor obtained with 20,mL of the
standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Purity (3) under
Cefaclor.
Water <2.48> Not more than 8.0% (0.2 g, volumetric titra-
tion, back titration).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with one Cefaclor Capsules at 50 revolu-
tions per minute according to the Paddle method using 900
mL of water as the dissolution medium. Withdraw not less
than 20 mL of the dissolution medium 15 minutes after start
of the test, and filter through a membrane filter with pore size
of not more than 0.5 //m. Discard the first 10 mL of the
filtrate, pipet KmL of the subsequent filtrate, add water to
make exactly V mL so that each mL contains about 20 n%
(potency) of Cefaclor according to the labeled amount, and
use this solution as the sample solution. Separately, weigh ac-
curately about 20 mg (potency) of Cefaclor Reference Stan-
dard, and dissolve in water to make exactly 20 mL. Pipet 1
mL of this solution, add water to make exactly 50 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A T and A s , at 265 nm of the sample solution and
standard solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>. The dissolution rate in 15 minutes is
not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
cefaclor (CuH^ClNjC^S)
= W s x (Aj/A s ) x (V'/V) x (l/Q x 90
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
C: Labeled amount [mg (potency)] of Cefaclor in 1 capsule
Assay Weigh accurately not less than 5 Cefaclor Capsules,
open the capsules and carefully take out the contents, mix
well, and powder, if necessary. Wash the empty capsules with
a little amount of diethyl ether, if necessary, allow the cap-
sules to stand at room temperature to vaporize adhering
diethyl ether, and weigh accurately the capsules to calculate
the mass of the contents. Weigh accurately a quantity of the
contents, equivalent to about 0.1 g (potency) of Cefaclor ac-
cording to the labeled amount, shake vigorously with 60 mL
of 0.1 mol/L phosphate buffer solution, pH 4.5 for 10
minutes, add the same buffer solution to make exactly 100
mL, and centrifuge. Pipet 10 mL of the supernatant liquid,
add exactly 10 mL of the internal standard solution and the
same buffer solution to make 50 mL, and use this solution as
the sample solution. Separately, weigh accurately about 50
mg (potency) of Cefaclor Reference Standard, and dissolve in
0.1 mol/L phosphate buffer solution, pH 4.5 to make exactly
50 mL. Pipet 10 mL of this solution, add exactly 10 mL of
the internal standard solution and the same buffer solution to
make 50 mL, and use this solution as the standard solution.
Proceed as directed in the Assay under Cefaclor.
Amount [mg (potency)] of cefaclor (Q5LL4CIN3O4S)
= W s x (Q T /Q S ) x 2
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefaclor Fine Granules
■\z~7 7 <7 a )\,mi&
Cefaclor Fine Granules contain not less than 90.0%
and not more than 110.0% of the labeled amount of
cefaclor (C15H14CIN3O4S: 367.81).
Method of preparation Prepare fine granules as directed
under Powders, with Cefaclor.
Identification Shake vigorously a quantity of Cefaclor Fine
Granules, equivalent to 20 mg (potency) of Cefaclor accord-
ing to the labeled amount, with 10 mL of water, centrifuge,
and use the supernatant liquid as the sample solution.
Separately, dissolve 20 mg (potency) of Cefaclor Reference
Standard in 10 mL of water, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 2//L each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography, develop the plate with a mixture of acetonitrile,
water, ethyl acetate and formic acid (30:10:10:1) to a distance
of about 10 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the principal spot from
the sample solution and the spot from the standard solution
show the same Rf value.
418
Cefaclor Fine Granules / Official Monographs
JP XV
Purity Related substances — Weigh accurately a quantity of
Cefaclor Fine Granules after powdered if necessary, equiva-
lent to about 0.1 g (potency) of Cefaclor according to the la-
beled amount, shake with 40 mL of 0.1 mol/L phosphate
buffer solution, pH 4.5 for 10 minutes, add the same buffer
solution to make exactly 50 mL, and filter through a 0.45-//m
pore-size membrane filter. Discard the first 1 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 20 mg (potency) of
Cefaclor Reference Standard, and dissolve in 0.1 mol/L
phosphate buffer solution, pH 4.5 to make exactly 20 mL.
Pipet 2 mL of this solution, add the same buffer solution to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 50 /xL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas by the automatic integra-
tion method. Calculate the amount of each related substance
by the following equation: the amount of each related sub-
stance is not more than 0.5%, and the total amount of the
related substances is not more than 3.0%. If necessary, cor-
rect the fluctuation of the base line by performing the test in
the same way with 50 fiL of 0. 1 mol/L phosphate buffer solu-
tion, pH 4.5.
Amount (%) of each related substance
= (W s /Wj) x (A T /A S ) x (l/Q x 5
Total amount (%) of the related substances
= (W S /W T ) x (ZAj/As) x (l/Q x 5
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
W T : Amount (g) of sample
A T : Area of the peak other than cefaclor and the solvent
from the sample solution
^4 S : Peak area of cefaclor from the standard solution
C: Labeled potency [mg (potency)] of cefaclor
(C 15 H 14 C1N 3 4 S) per g of the sample
Operating conditions —
Proceed as directed in the operating conditions in the Puri-
ty (3) under Cefaclor.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add 0.1 mol/L phosphate buffer solution, pH
4.5 to make exactly 20 mL. Confirm that the peak area of
cefaclor obtained with 50 /xL of this solution is equivalent to
3.5 to 6.5% of that of cefaclor obtained with 50 /uL of the
standard solution.
System performance: When the procedure is run with 50
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefaclor are not less than 40,000 and be-
tween 0.8 and 1.3, respectively.
System repeatability: When the test is repeated 3 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefaclor is not more than 2.0%.
Water <2.48> Not more than 1.5% (1 g, volumetric titra-
tion, back titration).
Uniformity of dosage units <6.02> The granules in single-
unit container meet the requirement of the Mass variation
test.
Dissolution <6J0> Perform the test according to the follow-
ing method: It meets requirement.
Perform the test with an accurately weighed quantity of
Cefaclor Fine Granules, equivalent to about 0.25 g (potency)
of Cefaclor according to the labeled amount, at 50 revolu-
tions per minute according to the Paddle method using 900
mL of water as the dissolution medium. Withdraw not less
than 20 mL of the dissolution medium 15 minutes after start
of the test, and filter through a membrane filter with a pore
size not exceeding 0.5 /xm. Discard the first 10 mL of the
filtrate, pipet KmL of the subsequent filtrate, add water to
make exactly V mL so that each mL contains about 20 /xg
(potency) of Cefaclor according to the labeled amount, and
use this solution as the sample solution. Separately, weigh ac-
curately about 20 mg (potency) of Cefaclor Reference Stan-
dard, and dissolve in water to make exactly 20 mL. Pipet 1
mL of this solution, add water to make exactly 50 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A T and A s , at 265 nm of the sample solution and
standard solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>. The dissolution rate in 15 minutes is
not less than 85%.
Dissolution rate (%) with respect to the labeled amount of
cefaclor (CijHmCINjC^S)
= (W S /W T ) x (A T /A S ) x (V'/V) x (l/Q x 90
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
W T : Amount [mg (potency)] of sample
C: Labeled amount [mg (potency)] of cefaclor
(Q5FL4CIN3O4S) per g of Cefaclor Fine Granules
Particle size <6.03> It meets the requirement of fine granules
of the Powders.
Assay Weigh accurately a quantity of Cefaclor Fine Gran-
ules after powdered if necessary, equivalent to about 0.1 g
(potency) of Cefaclor according to the labeled amount, shake
vigorously with 60 mL of 0.1 mol/L phosphate buffer solu-
tion, pH 4.5 for 10 minutes, add the same buffer solution to
make exactly 100 mL, and centrifuge. Pipet 10 mL of the su-
pernatant liquid, add exactly 10 mL of the internal standard
solution and the same buffer solution to make 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg (potency) of Cefaclor Reference Stan-
dard, and dissolve in 0.1 mol/L phosphate buffer solution,
pH 4.5 to make exactly 50 mL. Pipet 10 mL of this solution,
add exactly 10 mL of the internal standard solution and the
same buffer solution to make 50 mL, and use this solution as
standard solution. Proceed as directed in the Assay under
Cefaclor.
Amount [mg (potency)] of cefaclor (Q5FL4CIN3O4S)
= W s x (Q T /Q S ) x 2
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Cefaclor Compound Granules 419
Cefaclor Compound Granules
Cefaclor Compound Granules contain gastric gran-
ules and enteric granules in one pack.
It contains cefaclor (Q5H14CIN3O4S: 367.81) equiva-
lent to not less than 90.0% and not more than 110.0%
of the labeled total potency and the labeled potency of
gastric granule, respectively.
Method of preparation Prepare as directed under Granules,
with Cefaclor, and divide into packs.
Identification Shake vigorously a quantity of Cefaclor
Compound Granules, equivalent to 20 mg (potency) of
Cefaclor according to the labeled total potency, with 10 mL
of water, centrifuge, and use the supernatant liquid as the
sample solution. Separately, dissolve 20 mg of Cefaclor
Reference Standard in 10 mL of water, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 2//L each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of acetoni-
trile, water, ethyl acetate and formic acid (30:10:10:1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): the principal
spot from the sample solution and the spot from the standard
solution show the same Ri value.
Purity Related substances — Weigh accurately not less than
5 Cefaclor Compound Granules, transfer their total contents
to a mortar, add a little amount of 0.1 mol/L phosphate
buffer solution, pH 4.5, grind well, add the same buffer solu-
tion to make exactly KmL so that each mL contains about 2
mg (potency) of Cefaclor according to the labeled total
potency, and filter through a 0A5-/um pore-size membrane
filter. Discard the first 1 mL of the filtrate, and use the subse-
quent filtrate as the sample solution. Separately, weigh ac-
curately about 20 mg (potency) of Cefaclor Reference Stan-
dard, and dissolve in 0.1 mol/L phosphate buffer solution,
pH 4.5 to make exactly 20 mL. Pipet 2 mL of this solution,
add the same buffer solution to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 50 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method. Calculate the
amount of each related substance by the following equation:
the amount of each related substance is not more than 0.6%,
and the total amount of the related substances is not more
than 2.8%. If necessary, correct the fluctuation of the base
line by performing the test in the same way with 50 /uL of 0.1
mol/L phosphate buffer solution, pH 4.5.
Amount (%) of each related substance
= W s x (A Ti /A s ) x {V/(C x 7)} x 1/10
Total amount (%) of the related substances
= W s x (2M Tn A4 s ) x {V/(C x 7)} x 1/10
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
A Ti : Area of each peak other than cefaclor, solvent and ex-
cipient from the sample solution
^4 S : Peak area of cefaclor from the standard solution
C: Labeled total potency [mg (potency)] of cefaclor in 1
pack
T: Number (pack) of sample
Operating conditions —
Proceed as directed in the operating conditions in the Puri-
ty (3) under Cefaclor.
System suitability —
Test for required detectability: Pipet 1 mL of standard so-
lution, and add 0.1 mol/L phosphate buffer solution, pH 4.5
to make exactly 20 mL. Confirm that the peak area of
cefaclor obtained with 50 /uL of this solution is equivalent to
3.5 to 6.5% of that of cefaclor obtained with 50 /xL of the
standard solution.
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefaclor are not less than 40,000 and be-
tween 0.8 and 1.3, respectively.
System repeatability: When the test is repeated 3 times with
50 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefaclor is not more than 2.0%.
Water <2.48> Not more than 5.5% (0.3 g, volumetric titra-
tion, back titration).
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
(1) Total potency — Take out the total contents of 1
Cefaclor Compound Granules, add a little amount of 0.1
mol/L phosphate buffer solution, pH 4.5, grind well, add the
same buffer solutions to make exactly KmL so that each mL
contains about 3.8 mg (potency) of Cefaclor according to the
labeled total potency after shaking vigorously for 10 minutes,
and centrifuge. Pipet 3 mL of the supernatant liquid, add ex-
actly 10 mL of the internal standard solution and the same
buffer solution to make 50 mL, and use this solution as the
sample solution. Separately, weigh accurately an amount of
Cefaclor Reference Standard, equivalent to about 50 mg
(potency), and dissolve in 0.1 mol/L phosphate buffer solu-
tion, pH 4.5 to make exactly 50 mL. Pipet 10 mL of this solu-
tion, add exactly 10 mL of the internal standard solution and
the same buffer solution to make 50 mL, and use this solu-
tion as the standard solution. Then, proceed as directed in the
Assay under Cefaclor.
Amount [mg (potency)] of cefaclor (QsH^ClNjC^S)
= W s x (Q T /Q S ) x (V/15)
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
(2) Potency of gastric granule — Stir gentry the total con-
tents of 1 Cefaclor Compound Granules with 60 mL of 0.1
mol/L phosphate buffer solution, pH 4.5 for 5 minutes, add
the same buffer solution to make exactly V mL so that each
mL contains about 1.5 mg (potency) of Cefaclor according to
420
Cefaclor Compound Granules / Official Monographs
JP XV
the labeled potency of gastric granule, and centrifuge. Pipet 7
mL of the supernatant liquid, add exactly 10 mL of the inter-
nal standard solution and the same buffer solution to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately an amount of Cefaclor Reference Standard,
equivalent to about 50 mg (potency), and dissolve in 0.1
mol/L phosphate buffer solution, pH 4.5 to make exactly 50
mL. Pipet 10 mL of this solution, add exactly 10 mL of the
internal standard solution and the same buffer solution to
make 50 mL, and use this solution as the standard solution.
Then, proceed as directed in the Assay under Cefaclor.
Amount [mg (potency)] of cefaclor (Q5I-L4C1N3O4S)
= W s x (Or/Qg) x (K/35)
fV s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 Cefaclor Compound Granules at 50
revolutions per minute according to the Paddle method using
900 mL of the 1st fluid for dissolution test as the dissolution
medium. Withdraw not less than 20 mL of the dissolution
medium 60 minutes after start of the test, and filter through a
membrane filter with a pore size not exceeding 0.5 fim. Dis-
card the first 10 mL of the filtrate, pipet FmL of the subse-
quent filtrate, add the 1st fluid for dissolution test to make ex-
actly V mL so that each mL contains about 20 n% (potency)
of Cefaclor according to the labeled potency of gastric gran-
ule, and use this solution as the sample solution. Separately,
weigh accurately an amount of Cefaclor Reference Standard,
equivalent to about 20 mg (potency), and dissolve in the 1st
fluid for dissolution test to make exactly 20 mL. Pipet 2 mL
of this solution, add the 1st fluid for dissolution test to make
exactly 100 mL, and use this solution as the standard solu-
tion. Determine the absorbances, A T and A s , at 265 nm of
the sample solution and standard solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>. The dissolu-
tion rate in 60 minutes is between 35% and 45%.
Dissolution rate (%) of cefaclor (CisHmCINjCmS) with
respect to the labeled potency
= W s x (A T /A S ) x (V'/V) x (I/O x 90
fV s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
C: Labeled total potency [mg (potency)] of Cefaclor in 1
pack
Separately, perform the test with 1 Cefaclor Compound
Granules at 50 revolutions per minute according to the Pad-
dle method using 900 mL of 2nd fluid for dissolution method
as the dissolution medium. Withdraw not less than 20 mL of
the dissolution medium 60 minutes after start of the test, and
filter through a membrane filter with pore size of not more
than 0.5 /um. Discard the first 10 mL of the filtrate, pipet V
mL of the subsequent filtrate, add 0.01 mol/L hydrochloric
acid TS to make exactly V mL so that each mL contains
about 20 n% (potency) of Cefaclor according to the labeled
total potency, and use this solution as the sample solution.
Separately, weigh accurately an amount of Cefaclor Refer-
ence Standard, equivalent to about 20 mg (potency), dissolve
in 2nd fluid for dissolution test to make exactly 100 mL, and
warm at 37 °C for 60 minutes. Pipet 2 mL of this solution,
add 0.01 mol/L hydrochloric acid TS to make exactly 20 mL,
and use this solution as the standard solution. Determine the
absorbances, A T and A s , at 265 nm of the sample solution
and standard solution as directed under Ultraviolet-visible
Spectrophotometry <2.24> using 0.01 mol/L hydrochloric
acid TS as the blank. The dissolution rate in 60 minutes is not
less than 70%.
Dissolution rate (%) of cefaclor (C5IL4CIN3O4S) with
respect to the labeled potency
= W s x (A T /A S ) x (V'/V) x (1/Q x 90
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
C: Labeled total potency [mg (potency)] of Cefaclor in 1
pack
Particle size <6.03> It meets the requirement.
Assay (1) Total potency — Take out the total contents of
not less than 5 Cefaclor Compound Granules, add a small
amount of 0.1 mol/L phosphate buffer solution, pH 4.5,
grind well, add the same buffer solution to make a solution
containing about 5 mg (potency) of Cefaclor per mL accord-
ing to the labeled total potency after shaking vigorously for
10 minutes, and centrifuge. Pipet 2 mL of the supernatant
liquid, add exactly 10 mL of the internal standard solution
and the same buffer solution to make 50 mL, and use this so-
lution as the sample solution. Separately, weigh accurately an
amount of Cefaclor Reference Standard, equivalent to about
50 mg (potency), and dissolve in 0.1 mol/L phosphate buffer
solution, pH 4.5 to make exactly 50 mL. Pipet 10 mL of this
solution, add exactly 10 mL of the internal standard solution
and the same buffer solution to make 50 mL, and use this so-
lution as the standard solution. Then, proceed as directed in
the Assay under Cefaclor.
Amount [mg (potency)] of cefaclor (G5PL4CIN3O4S)
= W s x (Q T /Q S ) x 2
W s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
(2) Potency of gastric granule — Stir gentry the total con-
tents of not less than 5 Cefaclor Compound Granules with
about 100 mL of 0.1 mol/L phosphate buffer solution, pH
4.5 for 5 minutes, the same buffer solution so that each mL
containing about 2 mg (potency) of Cefaclor according to the
labeled potency of gastric granule, and centrifuge. Pipet 5
mL of the supernatant liquid, add exactly 10 mL of the inter-
nal standard solution and the same buffer solution to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately an amount of Cefaclor Reference Standard,
equivalent to about 50 mg (potency), and dissolve in 0.1
mol/L phosphate buffer solution, pH 4.5 to make exactly 50
mL. Pipet 10 mL of this solution, add exactly 10 mL of the
internal standard solution and the same buffer solution to
make 50 mL, and use this solution as the standard solution.
Then, proceed as directed in the Assay under Cefaclor.
Amount [mg (potency)] of cefaclor (C I5 H 14 C1N304S)
JPXV
Official Monographs / Cefadroxil 421
= W s x (Qj/Qs) x 2
fV s : Amount [mg (potency)] of Cefaclor Reference Stan-
dard
Internal standard solution — A solution of 4-aminoacetophe-
none in 0.1 mol/L phosphate buffer solution, pH 4.5 (1 in
700).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefadroxil
t7 7 HP+yJl
CO ? H
"CjTO"
C 16 H 17 N 3 5 S: 363.39
(6jR,7«)-7-[(2i?)-2-Amino-2-(4-
hydroxyphenyl)acetylamino]-3-methyl-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
[50370-12-2]
Cefadroxil contains not less than 950 fig (potency)
and not more than 1020 fig (potency) per mg, calculat-
ed on the anhydrous basis. The potency of Cefadroxil
is expressed as mass (potency) of cefadroxil
(C 16 H 17 N 3 O s S).
Description Cefadroxil occurs as a white to light yellow-
white powder.
It is sparingly soluble in water, slightly soluble in
methanol, and very slightly soluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cefadroxil (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Cefadroxil Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefadroxil as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Cefadroxil Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
(3) Determine the spectrum of a solution of Cefadroxil in
a mixture of heavy water for nuclear magnetic resonance
spectroscopy and deuterated hydrochloric acid (3:1) (1 in 10),
using sodium 3-(trimethylsilyl)propionate-d 4 for nuclear
magnetic resonance spectroscopy as an internal reference
compound, as directed under Nuclear Magnetic Resonance
Spectroscopy <2.21> ('H): it exhibits a single signal A at
around 5 2.1 ppm, a double signal B at around 8 7.0 ppm,
and a double signal C at around 3 7.5 ppm. The ratio of
integrated intensity of each signal, A:B:C, is about 3:2:2.
Optical rotation <2.49> [ a ]^ 5 : +164- +182° (0.6 g calcu-
lated on the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefadroxil in 200 mL of water:
pH of the solution is between 4.0 and 6.0.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefadroxil according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 0.1 g of Cefadroxil in 4
mL of a mixture of ethanol (99.5), water and diluted
hydrochloric acid (1 in 5) (75:22:3), and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add a
mixture of ethanol (99.5), water and diluted hydrochloric
acid (1 in 5) (75:22:3) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 2^L each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop with a mixture of ethyl acetate, water,
ethanol (99.5) and formic acid (14:5:5:1) to a distance of
about 12 cm, and air-dry the plate. Spray evenly ninhydrin-
citric acid-acetic acid TS on the plate, and heat at 100°C for
10 minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Water <2.48> Not less than 4.2% and not more than 6.0%
(0.5 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Cefadroxil and
Cefadroxil Reference Standard equivalent to about 50 mg
(potency), dissolve each in water to make exactly 500 mL,
and use these solutions as the sample solution and standard
solution, respectively. Perform the test with exactly 10 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the peak areas, A T and A s ,
of cefadroxil of the solutions.
Amount [fig (potency)] of C 16 H 17 N 3 5 S
= W s x {Aj/A s )x 1000
W s : amount [mg (potency)] of Cefadroxil Reference Stan-
dard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 262 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of a solution of potassium di-
hydrogenphosphate (17 in 12,500) and methanol (17:3).
Flow rate: Adjust the flow rate so that the retention time of
cefadroxil is about 5 minutes.
System suitability —
System performance: Dissolve about 5 mg (potency) of
cefadroxil and about 10 mg (potency) of propylene glycol
cefatrizine in 50 mL of water. When the procedure is run with
10 fiL of this solution under the above operating conditions,
cefadroxil and cefatrizine are eluted in this order with the
resolution between these peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
422
Cefalexin / Official Monographs
JP XV
H 2 N H
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefadroxil is not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefalexin
■\Ll 7 U + v>
CO,H
H H
C 16 H 17 N 3 4 S: 347.39
(6/?,7#)-7-[(2#)-2-Amino-2-phenylacetylamino]-3-
methyl-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid [15686-71-2]
Cefalexin contains not less than 950 fig (potency)
and not more than 1030 fig (potency) per mg, calculat-
ed on the anhydrous basis. The potency of Cefalexin is
expressed as mass (potency) of cefalexin
(C 16 H 17 N 3 4 S).
Description Cefalexin occurs as a white to light yellowish
white, crystals or crystalline powder.
It is sparingly soluble in water, slightly soluble in
methanol, and practically insoluble in ethanol (95) and in
A r .,A r -dimethylformamide.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Cefalexin (3 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefalexin as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(3) Determine the spectrum of a solution of Cefalexin in
heavy water for nuclear magnetic resonance spectroscopy (1
in 200) as directed under Nuclear Magnetic Resonance Spec-
troscopy <2.21> ('H), using sodium 3-trimethylsilyl-
propanesulfonate for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits a sin-
gle signal A at around <5 1.8 ppm, and a single or a sharp mul-
tiple signal B at around 3 7.5 ppm. The ratio of integrated in-
tensity of these signals, A:B, is about 3:5.
Optical rotation <2.49> [a] D °: + 144 - + 158° (0.125 g calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefalexin according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefalexin by suspending in 10 mL of TV^A^-dimethylfor-
mamide, and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve about 25 mg of
Cefalexin in a solution of potassium dihydrogenphosphate (9
in 500) to make 5 mL, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add a solution of
potassium dihydrogenphosphate (9 in 500) to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 20 fiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the areas of each peak by the automatic in-
tegration method. If necessary, correct the change of the
base-line due to the potassium dihydrogenphosphate solution
by proceeding in the same manner with 20 fiL of a solution of
potassium dihydrogenphosphate (9 in 500): each peak area
other than cefalexin from the sample solution is not more
than the peak area of cefalexin from the standard solution,
and the total of the peak areas which are bigger than 1/50 of
the peak area of cefalexin from the standard solution and
those other than cefalexin from the sample solution is not
more than 5 times of the peak area of cefalexin from the stan-
dard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 1.0 g of sodium 1-pentanesul-
fonate in 1000 mL of water, add 15 mL of triethylamine, and
adjust to pH 2.5 with phosphoric acid.
Mobile phase B: Dissolve 1.0 g of sodium 1-pentanesul-
fonate in 300 mL of water, add 15 mL of triethylamine, and
adjust to pH 2.5 with phosphoric acid. To this solution add
350 mL of acetonitrile and 350 mL of methanol.
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile A and B as directed in the following table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0- 1
100
1 -34.5
100^0
0^100
34.5-35.5
100
Flow rate: 1.0 mL per minute
Time span of measurement: About 2 times as long as the
retention time of cefalexin beginning after the solvent peak.
System suitability —
Test for required detection: Pipet 2 mL of the standard so-
lution, add a solution of potassium dihydrogenphosphate (9
in 500) to make exactly 100 mL. Confirm that the peak area
of cefalexin obtained from 20 fiL of this solution is equiva-
lent to 1.8 to 2.2% of that of cefalexin obtained from 20 fiL
of the standard solution.
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefalexin are not less than 150,000 steps
and between 0.8 and 1.3, respectively.
JPXV
Official Monographs / Cefalotin Sodium
423
System repeatability: When the test is repeated 3 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the retention
time and the peak areas of cefalexin are not more than 2.0%,
respectively.
Water <2.48> Not more than 8.0% (0.2 g, volumetric titra-
tion, back titration).
Assay Weigh accurately an amount of Cefalexin and
Cefalexin Reference Standard, equivalent to about 0.1 g
(potency), dissolve each in 0.1 mol/L phosphate buffer solu-
tion, pH 4.5 to make exactly 100 mL. Pipet 10 mL of these
solutions, add exactly 5 mL of the internal standard solution,
then add 0.1 mol/L phosphate buffer solution, pH 4.5 to
make 50 mL, and use these solutions as the sample solution
and standard solution. Perform the test with 10 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Q T and Q s , of the peak
area of cefalexin to that of the internal standard.
Amount [fig (potency)] of Ci 6 H 17 N 3 04S
= W s x (Q T /Q S ) x 1000
W s : amount [mg (potency)] of Cefalexin Reference Stan-
dard
Internal standard solution — A solution of m-hydroxy-
acetophenone in 0.1 mol/L phosphate buffer solution, pH
4.5 (1 in 1500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen-
phosphate in 1000 mL of water, adjust to pH 3.0 with diluted
phosphoric acid (3 in 500). To 800 mL of this solution add
200 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
cefalexin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, cefalexin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 5 times with
10 /iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefalexin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Cefalotin Sodium
CO ? Na O
»H
C 16 H 15 N 2 Na0 6 S 2 : 418.42
Monosodium (6i?,7i?)-3-acetoxymethyl-8-oxo-7-[2-
(thiophen-2-yl)acetylamino]-5-thia-l-azabicyclo[4.2.0]oct-
2-ene-2-carboxylate [58-71-9]
Cefalotin Sodium contains not less than 910 fig
(potency) and not more than 980 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Cefalotin Sodium is expressed as mass (potency) of
cefalotin (C 16 H 16 N 2 6 S 2 : 396.44).
Description Cefalotin Sodium occurs as white to light yel-
lowish white, crystals or crystalline powder.
It is freely soluble in water, slightly soluble in methanol,
very slightly soluble in ethanol (95), and practically insoluble
in acetonitrile.
Identification (1) Determine the absorption spectrum of a
solution of Cefalotin Sodium (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cefalotin Sodium Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same wave-
lengths.
(2) Determine the infrared absorption spectrum of
Cefalotin Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cefalotin Sodium Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefalotin
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a single signal A at around 32. 1 ppm, a single or
sharp multiple signal B at around <53.9 ppm, and a multiple
signal C at around <57.0 ppm. The ratio of the integrated in-
tensity of these signals, A:B:C, is about 3:2:2.
(4) Cefalotin Sodium responds to the Qualitative Tests
<].09> (1) for sodium salt.
Optical rotation <2.49> [a] 2 ^ ■ + 124 - + 134° (5 g, water, 100
mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Cefalotin Sodium in 10 mL of water is between 4.5 and
7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
424 Cefapirin Sodium / Official Monographs
JP XV
Cefalotin Sodium in 5 mL of water: the solution is clear and
light yellow.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Cefalotin
Sodium according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefalotin Sodium according to Method 3, and perform the
test (not more than 2 ppm).
(4) Related substances — Pipet 1 mL of the standard
solution obtained in the Assay, add the mobile phase to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with exactly 10,mL each of the sample
solution obtained in the Assay and the standard solution pre-
pared here as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the peak area
other than cefalotin from the sample solution is not more
than the peak area of cefalotin from the standard solution,
and the total area of the peaks other than cefalotin from the
sample solution is not more than 3 times the peak area of
cefalotin from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of cefalotin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of cefalotin
obtained from 10 [iL of this solutionis equivalent to 7 to 13%
of that from 10 juL of the standard solution.
System performance: Heat the standard solution in a water
bath of 90°C for 10 minutes, and cool. Measure exactly 2.5
mL of this solution, and add the mobile phase to make ex-
actly 100 mL. When the procedure is run with 10 liL of this
solution under the above operating conditions, the resolution
between the peak of cefalotin and the peak, having the rela-
tive retention time of about 0.5 with respect to cefalotin, is
not less than 9, and the symmetry factor of the peak of
cefalotin is not more than 1.8.
System repeatability: When the test is repeated 3 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefalotin is not more than 2.0%.
Water <2.48> Not more than 1.0% (0.5 g, volumetric titra-
tion, back titration).
Assay Weigh accurately an amount of Cefalotin Sodium
and Cefalotin Sodium Reference Standard, equivalent to
about 25 mg (potency), and dissolve each in the mobile phase
to make exactly 25 mL, and use these solutions as the sample
solution and standard solution. Perform the test with exactly
10 iiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of cefalotin of the solutions.
Amount [tig (potency)] of cefalotin (C 16 H 16 N 2 6 S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefalotin Sodium Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 17 g of sodium acetate trihydrate in
790 mL of water, and add 0.6 mL of acetic acid (100). If
necessary adjust the pH to 5.9 ± 0.1 with 0.1 mol/L sodium
hydrochloride TS or acetic acid (100). To this solution add
150 mL of acetonitrile and 70 mL of ethanol (95).
Flow rate: Adjust the flow rate so that the retention time of
cefalotin is about 12 minutes.
System suitability —
System performance: Heat the standard solution in a water
bath of 90°C for 10 minutes, and cool. Measure exactly 2.5
mL of this solution, and add the mobile phase to make
exactly 100 mL. When the procedure is run with 10 iiL of this
solution under the above operating conditions, the resolution
between the peak of cefalotin and the peak, having the rela-
tive retention time of about 0.5 with respect to cefalotin is not
less than 9, and the symmetry factor of the peak of cefalotin
is not more than 1.8.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefalotin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefapirin Sodium
-tz7 7ti°U >i~\- U^A
COjNa O
-A.
Ch,
H H
O
C 17 H 16 N 3 Na0 6 S 2 : 445.45
Monosodium (6i?,7i?)-3-acetoxymethyl-8-oxo-7-
[2-(pyridin-4-ylsulfanyl)acetylamino]-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[24356-60-3]
Cefapirin Sodium contains not less than 865 Lig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefapirin Sodium is expressed as mass
(potency) of cefapirin (C 17 H 17 N 3 6 S 2 : 423.46).
Description Cefapirin Sodium occurs as a white to yellow-
ish white powder.
It is freely soluble in water, sparingly soluble in methanol,
slightly soluble in ethanol (95), and practically insoluble in
acetone.
Identification (1) Determine the absorption spectrum of a
solution of Cefapirin Sodium (3 in 200,000) as directed under
JPXV
Official Monographs / Cefatrizine Propylene Glycolate
425
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cefapirin Sodium Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Cefapirin Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cefapirin Sodium Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefapirin
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10), using sodium 3-(trimethylsilyl)pro-
pionate-d 4 for nuclear magnetic resonance spectroscopy as an
internal reference compound, as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H): it exhibits a
single signal A at around d 2.2 ppm, and multiple signals, B
and C, at around <5 7.3 ppm and at around 5 8.3 ppm, respec-
tively. The ratio of integrated intensity of these signals,
A:B:C, is about 3:2:2.
(4) Cefapirin Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]g: + 157 - + 175° (2 g calculated
as the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefapirin Sodium in 10 mL of
water: pH of the solution is between 6.5 and 8.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefapirin Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefapirin Sodium according to Method 3, and perform the
test (not more than 2 ppm). Use a solution of magnesium ni-
trate hexahydrate in ethanol (95) (1 in 25).
(3) Related substances — Dissolve 0.1 g of Cefapirin Sodi-
um in 5 mL of a mixture of acetone and water (3:1), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add a mixture of acetone and water (3:1) to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop with a mixture of ethyl acetate, acetone, water and
acetic acid (100) (5:2:1:1) to a distance of about 10 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
and other than the spot at the original point from the sample
solution are not more intense than the spot from the standard
solution.
Water <2.48> Not more than 2.0% (0.7 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefapirin Sodium
and Cefapirin Sodium Reference Standard equivalent to
about 0.1 g (potency), dissolve each in phosphate buffer solu-
tion, pH 6.0 to make exactly 100 mL. Pipet 5 mL of each so-
lution, add exactly 5 mL of the internal standard solution
and phosphate buffer solution, pH 6.0 to make 100 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 20 liL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of cefapirin to that of the internal standard.
Amount [wg (potency)] of cefapirin (C 17 H 17 N 3 6 S2)
= Ws x (Qt/Qs) x 1000
W s : Amount [mg (potency)] of Cefapirin Sodium Refer-
ence Standard
Internal standard solution — A solution of vanillin (1 in
1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L sodium di-
hydrogenphosphate TS, pH 2.6 and acetonitrile (93:7).
Flow rate: Adjust the flow rate so that the retention time of
cefapirin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
liL of the standard solution under the above operating condi-
tions, cefapirin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefapirin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Cefatrizine Propylene Glycolate
-tz7 7 r- U-/>^ntiU>^U=l-JU
H;M H
H 2 C
x^
Or I
C 18 H 18 N 6 5 S 2 .C 3 H 8 2 : 538.60
(6i?,7fl)-7-[(2i?)-2-Amino-2-(4-
hydroxyphenyl)acetylamino]-8-oxo-3-[2-(l//-l,2,3-
triazol-4-yl)sulfanylmethyl]-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
monopropane-l,2-diolate (1/1)
[51627-14-6, Cefatrizine]
Cefatrizine Propylene Glycolate contains not less
than 785 tig (potency) and not more than 876 tig
426
Cefazolin Sodium / Official Monographs
JP XV
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefatrizine Propylene Glycolate is ex-
pressed as mass (potency) of cefatrizine (C lg H 18 N 6 O s S 2 :
462.50).
Description Cefatrizine Propylene Glycolate occurs as a
white to yellowish white powder.
It is sparingly soluble in water, and practically insoluble in
methanol and in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cefatrizine Propylene Glycolate (1 in 50,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Cefatrizine Propylene Glycolate
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefatrizine Propylene Glycolate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Cefatrizine Propylene Glycolate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Determine the spectrum of a solution of Cefatrizine
Propylene Glycolate in a mixture of heavy water for nuclear
magnetic resonance spectroscopy and deuterated hydrochlor-
ic acid for nuclear magnetic resonance spectroscopy (3:1) (1
in 10), using sodium 3-(trimethylsilyl)propionate-d 4 for
nuclear magnetic resonance spectroscopy as an internal
reference compound, as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> (>H): it exhibits a double sig-
nal A at around <5 1.2 ppm, a double signal B at around <5 7.0
ppm, a double signal C at around d 7.5 ppm and a single sig-
nal D at around 5 8.3 ppm. The ratio of integrated intensity
of these signals, A:B:C:D, is about 3:2:2:1.
Optical rotation <2.49> [a]g": + 52 - +58° (2.5 g calculated
on the anhydrous bases, 1 mol/L hydrochloric acid TS, 50
mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefatrizine Propylene Glycolate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefatrizine Propylene Glycolate according to Method 3,
and perform the test (not more than 2 ppm). Use a solution
of magnesium nitrate hexahydrate in ethanol (1 in 25).
(3) Related substances — Dissolve 25 mg of Cefatrizine
Propylene Glycolate in 5 mL of water, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add water to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop with a
mixture of 1-butanol, water and acetic acid (100) (3:1:1) to a
distance of about 12 cm, and air-dry the plate. Spray evenly
ninhydrin-citric acid-acetic acid TS on the plate, and heat at
100°C for 10 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Water <2.48> Not more than 2.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefatrizine Propy-
lene Glycolate and Cefatrizine Propylene Glycolate Refer-
ence Standard equivalent to about 0.1 g (potency), dissolve
each in water to make exactly 500 mL, and use these solutions
as the sample solution and standard solution, respectively.
Perform the test with exactly 10 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the peak areas, A T and A s , of cefatrizine of
these solutions
Amount L"g (potency)] of cefatrizine (C 18 H 18 N 6 5 S 2 )
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefatrizine Propylene
Glycolate Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 270 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of a solution of potassium di-
hydrogenphosphate (17 in 12,500) and methanol (17:3).
Flow rate: Adjust the flow rate so that the retention time of
cefatrizine is about 11 minutes.
System suitability —
System performance: Dissolve about 5 mg (potency) of
Cefadroxil and about 10 mg (potency) of Cefatrizine Propy-
lene Glycolate in 50 mL of water. When the procedure is run
with 10 /uL of this solution under the above operating condi-
tions, cefadroxil and cefatrizine are eluted in this order with
the resolution between these peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of peak areas of
cefatrizine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefazolin Sodium
-tz7r7U >i-\- U^A
C0 2 Na N
N I H H
C 14 H 13 N 8 Na04S 3 : 476.49
Monosodium (6i?,7i?)-3-(5-methyl-l,3,4-thiadiazol-2-
ylsulfanylmethyl)-8-oxo-7-[2-(l//-tetrazol-l-
yl)acetylamino]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate [27164-46-1]
Cefazolin Sodium contains not less than 900 fig
(potency) and not more than 975 Lig (potency) per mg,
JPXV
Official Monographs / Cefazolin Sodium
427
calculated on the anhydrous basis. The potency of
Cefazolin Sodium is expressed as mass (potency) of
cefazolin (CnHnNgC^: 454.51).
Description Cefazolin Sodium occurs as a white to light yel-
low-white, crystals or crystalline powder.
It is freely soluble in water and in formamide, slightly solu-
ble in methanol, and practically insoluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cefazolin Sodium (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Cefazolin Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefazolin
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10), using sodium 3-trimethylsilylpropionate-d 4
for nuclear magnetic resonance spectroscopy as an internal
reference compound, as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H): it exhibits single sig-
nals, A and B, at around d 2.7 ppm and at around d 9.3 ppm,
respectively. The ratio of integrated intensity of these signals,
A:B, is about 3:1.
(4) Cefazolin Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [a]™: - 19 - -23° (2.5 g calculated
as the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefazolin Sodium in 10 mL of
water: pH of the solution is between 4.8 and 6.3.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefazolin Sodium in 10 mL of water: the solution is clear and
colorless to pale yellow, and its absorbance at 400 nm deter-
mined as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24> is not more than 0.35. The test should be performed
within 10 minutes after preparing of the solution.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Cefazo-
lin Sodium according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Cefazolin Sodium according to Method 3, and perform
the test. When prepare the test solution, add 1.5 mL of
hydrogen peroxide (30) after addition of 10 mL of a solution
of magnesium nitrate hexahydrate in ethanol (95) (1 in 50),
and then ignite (not more than 1 ppm).
(4) Related substances — Dissolve 0.10 g of Cefazolin So-
dium in 20 mL of 0.1 mol/L phosphate buffer solution, pH
7.0 and use this solution as the sample solution. Prepare the
sample solution before use. Perform the test with 5 /uL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and measure
the areas of a peak appeared at the relative retention time of
about 0.2 to the retention time of cefazolin and peaks other
than cefazolin by the automatic integration method, and
calculate the amounts of these peak areas by the area percen-
tage method: the amount of each peak area is not more than
1.5%, and the total area of the peaks other than cefazolin is
not more than 2.5%. The area of the peak appeared at the
relative retention time of about 0.2 to the retention time of
cefazolin obtained here is used after multiplying by its sen-
sitivity coefficient, 1.43.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of cefazolin beginning after the solvent peak.
System suitability —
Test for required detection: Dissolve about 80 mg of
Cefazolin Reference Standard in 0.1 mol/L phosphate buffer
solution, pH 7.0 to make 100 mL, and use this solution as the
solution for system suitability test. Pipet 1 mL of the solu-
tion, and add 0.1 mol/L phosphate buffer solution, pH 7.0 to
make exactly 20 mL. Confirm that the peak area of cefazolin
obtained from 5 /uL of this solution is equivalent to 3 to 7%
of that of cefazolin obtained from 5 fiL of the solution for
system suitability test.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of cefazolin is not more than 1.0%.
Water <2.48> Not more than 2.5% (1.0 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1) in-
stead of methanol for water determination).
Assay Weigh accurately an amount of Cefazolin Sodium
and Cefazolin Reference Standard, equivalent to about 0.1 g
(potency), dissolve each in the internal standard solution to
make exactly 100 mL, and use these solutions as the sample
solution and standard solution, respectively. Perform the test
with 5 /uL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
cefazolin to that of the internal standard.
Amount \jug (potency)] of cefazolin (C 14 H I4 N 8 4 S3)
= Ws x (Qt/Qs) x 1000
W s : Amount [mg (potency)] of Cefazolin Reference Stan-
dard
Internal standard solution — A solution of j9-acetoanisidide in
0.1 mol/L phosphate buffer solution, pH 7.0 (11 in 20,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (lO^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.27 g of disodium hydrogen phos-
phate dodecahydrate and 0.47 g of citric acid monohydrate in
water to make 935 mL, and add 65 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefazolin is about 8 minutes.
428
Cefazolin Sodium Hydrate / Official Monographs
JP XV
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, cefazolin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 4.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of cefazolin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Cefazolin Sodium Hydrate
-fe777"U>1-r- U7A7Kftt)
C0 2 Na
>.-N
rN ^ N i-hs
N I H H
.A,
•SHjO
C l4 H l3 N 8 Na0 4 S 3 .5H 2 0: 566.57
Monosodium (6/?,7/?)-3-(5-methyl-l,3,4-thiadiazol-2-
ylsulfanylmethyl)-8-oxo-7-[2-(l//-tetrazol-
l-yl)acetylamino]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate pentahydrate [115850-11-8]
Cefazolin Sodium Hydrate contains not less than
920 fig (potency) and not more than 975 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Cefazolin Sodium Hydrate is expressed as mass
(potency) of cefazolin (C 14 H 14 N 8 4 S 3 : 454.51).
Description Cefazolin Sodium Hydrate occurs as white to
pale yellowish white crystals.
It is freely soluble in water, sparingly soluble in methanol,
slightly soluble in ethanol (95), and practically insoluble in
diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Cefazolin Sodium Hydrate (1 in 50,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 270 nm and 274 nm.
(2) Determine the infrared absorption spectrum of
Cefazolin Sodium Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
1761cm" 1 , 1667 cm" 1 , 1599 cm" 1 , 1540 cm" 1 and
1389 cm" 1 .
(3) Determine the spectrum of a solution of Cefazolin So-
dium Hydrate in heavy water for nuclear magnetic resonance
spectroscopy (1 in 10), using sodium 3-trimethylsilyl-
propionate-d 4 for nuclear magnetic resonance spectroscopy
as an internal reference compound, as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ( 1 H): it exhibits sin-
gle signals, A and B, at around 3 2.7 ppm and at around 8 9.3
ppm. The ratio of integrated intensity of each signal, A:B, is
about 3:1.
(4) Cefazolin Sodium Hydrate responds to the Qualita-
tive Tests <1.09> (1) for sodium salt.
Absorbance <2.24> E/° m (272 nm): 272 - 292 (80 mg calcu-
lated on the anhydrous basis, water, 5000 mL).
Optical rotation <2.49> [ a ]g>: -20 - -25° (2.5 g calculated
on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefazolin Sodium Hydrate in
10 mL of water: the pH of the solution is between 4.8 and
6.3.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Cefazo-
lin Sodium Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic — Being specified separately.
(4) Related substances — Being specified separately.
(5) Residual solvents — Being specified separately.
Water <2.48> Not less than 13.7% and not more than
16.0% (0.1 g, volumetric titration, direct titration. Use a
mixture of formamide for water determination and methanol
for water determination (2:1) instead of methanol for water
determination).
Bacterial endotoxins <4.01> Less than 0.10 EU/mg (poten-
cy).
Assay Weigh accurately an amount of Cefazolin Sodium
Hydrate and Cefazolin Reference Standard, equivalent to
about 0.1 g (potency), dissolve in the internal standard solu-
tion to make exactly 100 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
5 fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, g T and Q s ,
of the peak area of cefazolin to that of the internal standard.
Amount [fig (potency)] of cefazolin (C 14 H 14 N 8 04S3)
= Ws x (Gt/Gs) x 1000
W s : Amount [mg (potency)] of Cefazolin Reference Stan-
dard
Internal standard solution — A solution of j9-acetoanisidide in
0.1 mol/L phosphate buffer solution, pH 7.0 (11 in 20,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.27 g of disodium hydrogen phos-
phate dodecahydrate and 0.47 g of citric acid monohydrate in
water to make 935 mL. To this solution, add 65 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefazolin is about 8 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, cefazolin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 4.
JPXV
Official Monographs / Cefbuperazone Sodium 429
System repeatability: When the test is repeated 5 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
cefazolin is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Cefbuperazone Sodium
t77"^7'/>tr U^A
°Vy° «
HjC v _N
CO;Na ■ !
O HN H
H 3 C
xY t
H OH o
C 22 H 28 N 9 Na0 9 S 2 : 649.63
Monosodium (6RJS)-7-{(2R,3S)-2-[(4-ethyl-2,3-
dioxopiperazine-l-carbonyl)amino]-3-
hydroxybutanoylamino } -7-methoxy-3-(l -methyl- 1 H-
tetrazol-5-ylsulfanylmethyl)-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[76648-01-6]
Cefbuperazone Sodium contains not less than 870 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefbuperazone Sodium is expressed as
mass (potency) of cefbuperazone (C22H29N9O9S2:
627.65).
Description Cefbuperazone Sodium occurs as white to light
yellowish white, powder or masses.
It is very soluble in water, freely soluble in methanol and in
pyridine, sparingly soluble in ethanol (95), and very slightly
soluble in acetonitrile.
Identification (1) Determine the absorption spectrum of a
solution of Cefbuperazone Sodium (1 in 50,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Dissolve 0.1 g of Cefbuperazone Sodium in 0.5 mL of
deuterated pyridine for nuclear magnetic resonance spec-
troscopy and 1 drop of heavy water for nuclear magnetic
resonance spectroscopy, and determine the spectrum of this
solution as directed under Nuclear Magnetic Resonance Spec-
troscopy <2.21> ('H), using tetramethylsilane for nuclear
magnetic resonance spectroscopy as an internal reference
compound: it exhibits a triplet signal A at around 31. 1 ppm,
and two doublet signals, B and C, at around <51.6 ppm and at
around <55.1 ppm, respectively. The ratio of the integrated in-
tensity of each signal, A:B:C, is about 3:3:1.
(3) Cefbuperazone Sodium responds to the Qualitative
Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [a]™: +48- +56° (0.4 g calculated
on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 1 .0 g of Cefbuperazone Sodium in 4 mL
of water: the pH of the solution is between 4.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefbuperazone Sodium in 4 mL of water: the solution is clear
and light yellow.
(2) Heavy metals </. 07>— Proceed with 2.0 g of Cef-
buperazone Sodium according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefbuperazone Sodium according to Method 4, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Cefbupera-
zone Sodium in 100 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with exactly 25 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method. Calculate the
percentages of each peak area of related substances from the
sample solution against 50 times of the peak area of
cefbuperazone from the standard solution; the amount of
related substance I having the relative retention time of about
0.2 to cefbuperazone is not more than 2.0%, the amount of
related substance II having the relative retention time of
about 0.6 to cefbuperazone is not more than 4.5% and the
amount of related substance III having the relative retention
time of about 1.6 to cefbuperazone is not more than 1.0%,
and the total amount of these related substances is not more
than 6.0%. For these calculations, use the values of the peak
areas of the related substances I and III obtained by the
automatic integration method after multiplying by each sen-
sitivity coefficient, 0.72 and 0.69, respectively.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of cefbuperazone.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of cefbuperazone
obtained from 25 fiL of this solution is equivalent to 7 to 1 3 %
of that from 25 fiL of the standard solution.
System performance: When the procedure is run with
25 fiL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of cefbuperazone are not less than 5000
steps and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
25 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefbuperazone is not more than 2.0%.
Water <2.48> Not more than 1.0% (3 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefbuperazone
Sodium and Cefbuperazone Reference Standard, equivalent
to about 0.1 g (potency), and dissolve each in the mobile
phase to make exactly 100 mL. Measure exactly 10 mL each
of these solutions, add exactly 10 mL of the internal standard
430
Cefcapene Pivoxil Hydrochloride Hydrate / Official Monographs
JP XV
solution and the mobile phase to make 50 mL, and use these
solutions as the sample solution and standard solution. Per-
form the test with 10 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of cefbuperazone to
that of the internal standard.
Amount [/xg (potency)] of cefbuperazone (C 2 2H29N 9 9 S2)
= Ws x (Qj/Qs) x 1000
fV s : Amount [mg (potency)] of Cefbuperazone Reference
Standard
Internal standard solution — A solution of acetonitrile in the
mobile phase (1 in 4000).
Operating conditions—
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.0 g of tetra-M-propylammonium
bromide in 1000 mL of a mixture of water, acetonitrile and
acetic acid-sodium acetate buffer solution, pH 5.0 (83:13:4).
Flow rate: Adjust the flow rate so that the retention time of
cefbuperazone is about 16 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the internal standard and cefbuperazone are elut-
ed in this order with the resolution between these peaks being
not less than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefbuperazone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place.
Cefcapene Pivoxil Hydrochloride
Hydrate
H3C ,CHg
H H
O
NH 2
C 23 H 29 Nj0 8 S 2 .HCl.H 2 0: 622.11
2,2-Dimethylpropanoyloxymethyl (6i?,7jR)-7-[(2Z)-2-(2-
aminothiazol-4-yl)pent-2-enoylamino]-3-
carbamoyloxymethyl-8-oxo-5 -thia- 1 -azabicyclo [4.2.0] oct-2-
ene-2-caboxylate monohydrochloride monohydrate
[147816-24-8]
Cefcapene Pivoxil Hydrochloride Hydrate contains
not less than 722 [xg (potency) and not more than 764
[xg (potency) per mg, calculated on the anhydrous ba-
sis. The potency of Cefcapene Pivoxil Hydrochloride
Hydrate is expressed as mass (potency) of cefcapene (C
17 H 19 N 5 6 S 2 : 453.49).
Description Cefcapene Pivoxil Hydrochloride Hydrate oc-
curs as a white to pale yellowish white, crystalline powder or
mass. It has slightly a characteristic odor.
It is freely soluble in JV,7V-dimethylformamide and in
methanol, soluble in ethanol (99.5), slightly soluble in water,
and practically insoluble in diethyl ether.
Identification (1) Determine the infrared absorption spec-
tra of Cefcapene Pivoxil Hydrochloride Hydrate and Cefca-
pene Pivoxil Hydrochloride Reference Standard as directed
in the paste method under Infrared Spectrophotometry
<2.25>, and compare these spectra: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(2) Determine the spectrum of a solution of Cefcapene
Pivoxil Hydrochloride Hydrate in deuterated methanol for
nuclear magnetic resonance spectroscopy (1 in 50) as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
('H), using tetramethylsilane for nuclear magnetic resonance
spectroscopy as an internal reference compound: it exhibits a
triplet signal A at around 5 6.3 ppm, and a single signal B at
around 5 6.7 ppm, and the ratio of integrated intensity of
each signal, A:B, is about 1:1.
(3) Dissolve 10 mg of Cefcapene Pivoxil Hydrochloride
Hydrate in 2 mL of a mixture of water and methanol (1:1),
and add 1 drop of silver nitrate TS: a white precipitate is
formed.
Absorbance <2.24> E& (265 nm): 255 - 285 (30 mg calcu-
lated on the anhydrous basis, a mixture of acetate buffer solu-
tion, pH 5.5 and methanol (1:1), 2000 mL).
Optical rotation <2.49> [ a ]£°: + 51 - + 54° (0.1 g calculated
on the anhydrous basis, methanol, 10 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefcapene Pivoxil Hydrochloride Hydrate according to
Method 4, and perform the test. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 10
ppm).
(2) Related substance I — Dissolve an amount of Cefca-
pene Pivoxil Hydrochloride Hydrate, equivalent to about 10
mg (potency), in 2 mL of methanol, add a mixture of water
and methanol (1:1) to make 50 mL, and use this solution as
the sample solution. Perform the test with 30 /xL of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method. If necessary,
compensate the base-line by performing in the same manner
as the test with 30 /xL of a mixture of water and methanol
(1:1). Measure the amount of the peak other than cefcapene
pivoxil by the area percentage method: the amounts of the
peaks, having the relative retention times of about 1.5 and
about 1.7 with respect to cefcapene pivoxil, are not more
than 0.2%, respectively. The amount of the peak other than
the peaks mentioned above is not more than 0.1%, and the
total of them is not more than 1.5%.
Operating conditions —
JPXV
Official Monographs / Cefcapene Pivoxil Hydrochloride Hydrate
431
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase A: Dissolve 5.99 g of potassium dihydrogen
phosphate in water to make 1100 mL. To this solution add a
solution prepared by dissolving 1.89 g of tetra-w-pentylam-
monium bromide in methanol to make 1000 mL.
Mobile phase B: A mixture of methanol and water (22:3).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phase A and B as directed in the following ta-
ble.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-20
20-40
40-50
98 -► 50
50
2
2^ 50
50
Flow rate: 0.8 mL per minute.
Time span of measurement: About 2.5 times as long as the
retention time of cefcapene pivoxil.
System suitability —
Test for required detectability: To exactly 1 mL of the sam-
ple solution add a mixture of water and methanol (1:1) to
make exactly 100 mL, and use this solution as the solution for
system suitability test. Pipet 1 mL of the solution for system
suitability test, and add the mixture of water and methanol
(1:1) to make exactly 10 mL. Confirm that the peak area of
cefcapene pivoxil obtained from 30 /uL of this solution is e-
quivalent to 7 to 13% of that of cefcapene pivoxil obtained
from 30 /uL of the solution for system suitability test.
System performance: Dissolve 10 mg of Cefcapene Pivoxil
Hydrochloride Hydrate and 10 mg of propyl parahydrox-
ybenzoate in 25 mL of methanol, and add water to make 50
mL. To 5 mL of this solution add the mixture of water and
methanol (1:1) to make 50 mL. When the procedure is run
with 30 /uL of this solution under the above operating condi-
tions, cefcapene pivoxil and propyl parahydroxybenzoate are
eluted in this order with the resolution between these peaks
being not less than 7.
System repeatability: When the test is repeated 3 times with
30 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of cefcapene pivoxil is not more than 4.0%.
(3) Related substance II — Dissolve an amount of Cefca-
pene Pivoxil Hydrochloride Hydrate, equivalent to about 2
mg (potency), in A^A^dimethylformamide for liquid chro-
matography to make 20 mL, and use this solution as the sam-
ple solution. Perform the test with 20 /xL of the sample solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the total area of
the peaks which appear earlier than cefcapene pivoxil is not
more than 1.7% of the total area of the peaks other than the
solvent.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 7.8 mm in inside di-
ameter and 30 cm in length, packed with styrene-divinylben-
zene copolymer for liquid chromatography.
Column temperature: A constant temperature of about
25°C.
Mobile phase: A solution of lithium bromide in N,N-
dimethylformamide for liquid chromatography (13 in 5000).
Flow rate: Adjust the flow rate so that the retention time of
cefcapene pivoxil is about 22 minutes.
Time span of measurement: About 1.8 times as long as the
retention time of cefcapene pivoxil.
System suitability —
Test for required detection: To exactly 1 mL of the sample
solution add AfA^dimethylformamide for liquid chro-
matography to make exactly 100 mL, and use this solution as
the solution for system suitability test. Pipet 3 mL of the so-
lution for system suitability test, and add /V,/V-dimethylfor-
mamide for liquid chromatography to make exactly 10 mL.
Conform that the peak area of cefcapene pivoxil obtained
from 20 /xL of this solution is equivalent to 20 to 40% of that
of cefcapene pivoxil obtained from 20 /xL of the solution for
system suitability test.
System performance: When the procedure is run with 20
/xL of the sample solution under the above operating condi-
tions, the number of theoretical plates of the peak of cefca-
pene pivoxil is not less than 12,000 steps.
System repeatability: When the test is repeated 6 times with
20 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of cefcapene pivoxil is not more than 4.0% .
Water <2.48> Not less than 2.8% and not more than 3.7%
(0.5 g, volumetric titration, back titration).
Assay Weigh accurately an amount of Cefcapene Pivoxil
Hydrochloride Hydrate and Cefcapene Pivoxil Hydrochlo-
ride Reference Standard, equivalent to about 20 mg (poten-
cy), and dissolve each in a mixture of water and methanol
(1:1) to make exactly 50 mL. Pipet 10 mL each of these solu-
tions, add exactly 10 mL of the internal standard solution
and the mixture of water and methanol (1 : 1) to them to make
50 mL, and use these solutions as the sample solution and
standard solution, respectively. Perform the test with 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Qt and Q s , of
the peak area of cefcapene pivoxil to that of the internal stan-
dard of these solutions.
Amount \jxg (potency)] of cefcapene (Ci7H 19 N 5 6 S2)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Cefcapene Pivoxil
Hydrochloride Reference Standard
Internal standard solution — A solution of p-benzylphenol in
a mixture of water and methanol (1:1) (7 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 3.0 mm in inside di-
ameter and 7.5 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3 /xm in particle di-
ameter).
432
Cefcapene Pivoxil Hydrochloride Fine Granules / Official Monographs
JP XV
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.56 g of sodium dihydrogen-
phosphate dihydrate and 1.22 g of sodium 1-decanesulfonate
in water to make 1000 mL. To 700 mL of this solution add
300 mL of acetonitrile and 100 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
cefcapene pivoxil is about 5 minutes.
System suitability —
System performance: Dissolve 0.2 g of Cefcapene Pivoxil
Hydrochloride Hydrate in 10 mL of methanol, and warm in a
water bath at 60°C for 20 minutes. After cooling, pipet 1 mL
of this solution, and add exactly 10 mL of the internal stan-
dard solution and the mixture of water and methanol (1:1) to
make 50 mL. When the procedure is run with 10 jxL of this
solution under the above operating conditions, cefcapene
pivoxil, fra«s-cefcapene pivoxil and the internal standard are
eluted in this order, the ratios of the retention time of trans-
cefcapene pivoxil and the internal standard to that of cefca-
pene pivoxil are about 1.7 and 2.0, respectively, and the reso-
lution between the peaks of fraws-cefcapene pivoxil and the
internal standard is not less than 1.5.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of cefcapene pivoxil to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at a temperature not exceeding
5°C.
Cefcapene Pivoxil Hydrochloride
Fine Granules
^77]^:
t°7K'+->;u±£i^«5
Cefcapene Pivoxil Hydrochloride Fine Granules
contains not less than 90.0% and not more than
110.0% of cefcapene (C 17 H 19 N 5 6 S 2 : 453.49).
Method of preparation Prepare to finely granulated form as
directed under Powders, with Cefcapene Pivoxil Hydrochlo-
ride Hydrate.
Identification Powder Cefcapene Pivoxil Hydrochloride
Fine Granules. To a portion of the powder, equivalent to 10
mg (potency) of Cefcapene Pivoxil Hydrochloride Hydrate,
add 40 mL of methanol, shake vigorously, and add methanol
to make 50 mL. To 4 mL of this solution add methanol to
make 50 mL, and filter through a membrane filter with a pore
size of 0.45 fim. Determine the absorption spectrum of the
filtrate as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 264 nm and 268
nm.
Purity (1) Related substances I — Powder Cefcapene
Pivoxil Hydrochloride Fine Granules. To a portion of the
powder, equivalent to 5 mg (potency) of Cefcapene Pivoxil
Hydrochloride Hydrate, add 1 mL of methanol, and shake.
Add 25 mL of a mixture of water and methanol (1:1), shake
vigorously for 5 minutes, and filter through a membrane filter
with a pore size of 0.45 /xm. Discard the first 3 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Perform the test with 30 /xL of the sample solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine each peak area by the automat-
ic integration method. If necessary, compensate the base-line
by performing in the same manner as the test with 30 /xL of a
mixture of water and mothod (1:1). Calculate the amount of
the peaks other than the peak of cefcapene pivoxil by the area
percentage method: the amount of the substance, having the
relative retention time of about 1 .3 with respect to cefcapene
pivoxil, is not more than 0.4%, the amount of the trans-
isomer of cefcapene pivoxil, having the relative retention
time of about 1.5, is not more than 1.1%, the amount of the
substance other than that mentioned above is not more than
0.3%, and the total of these substances is not more than 2.8
%.
Operating conditions —
Proceed as directed in the Purity (2) under Cefcapene
Pivoxil Hydrochloride Hydrate.
System suitability —
Proceed as directed in the Purity (2) under Cefcapene
Pivoxil Hydrochloride Hydrate.
(2) Related substances II — Powder Cefcapene Pivoxil
Hydrochloride Fine Granules. To a portion of the powder,
equivalent to 2 mg (potency) of Cefcapene Pivoxil
Hydrochloride Hydrate, add 20 mL of A^TV-dimethylfor-
mamide for liquid chromatography, shake vigorously for 10
minutes, and filter through a membrane filter with a pore size
of 0.45 jxm. Discard the first 3 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Perform the test
with 20 /xL of the sample solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the total area of the peaks eluted before that of
cefcapene pivoxil is not more than 4.0% of the total area of
all peaks other than the solvent peak.
Operating conditions —
Proceed as directed in the Purity (3) under Cefcapene
Pivoxil Hydrochloride Hydrate.
System suitability —
Proceed as directed in the Purity (3) under Cefcapene
Pivoxil Hydrochloride Hydrate.
Water <2.48> Not more than 1.4% (0.5 g, volumetric titra-
tion, back titration). Perform the test without pulverizing the
sample, and handling the sample under a relative humidity of
less than 30%.
Uniformity of dosage units <6.02> The granules in single-u-
nit container meet the requirement of the Mass variation test.
Dissolution Being specified separately.
Particle size <6.03> It meets the requirement of the fine
granules of the Powders.
Assay Weigh accurately an amount of Cefcapene Pivoxil
Hydrochloride Fine Granules, equivalent to about 0.2 g
(potency) of and Cefcapene Pivoxil Hydrochloride Hydrate,
add 100 mL of the mixture of water and methanol (1:1),
shake vigorously for 10 minutes, add the mixture of water
and methanol (1:1) to make exactly 200 mL, and centrifuge at
3000 rpm for 5 minutes. Filter the supernatant liquid through
a membrane filter with a pore size of 0.45 /xm, discard the first
1 mL of the filtrate, pipet the subsequent 2 mL of the filtrate,
JPXV
Official Monographs / Cefcapene Pivoxil Hydrochloride Tablets
433
add exactly 5 mL of the internal standard solution and the
mixture of water and methanol (1:1) to make 25 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 20 mg (potency) of Cefcapene Pivoxil
Hydrochloride Reference Standard, and dissolve in the mix-
ture of water and methanol (1:1) to make exactly 50 mL.
Pipet 10 mL of this solution, add exactly 10 mL of the inter-
nal standard solution and the mixture of water and methanol
(1 : 1) to make 50 mL, and use this solution as the standard so-
lution. Proceed as directed in the Assay under Cefcapene
Pivoxil Hydrochloride Hydrate.
Amount [mg (potency)] of cefcapene (C 17 H 19 N50 6 S2)
= W s x (Qj/Qs) x 10
fV s : Amount [mg (potency)] of Cefcapene Pivoxil
Hydrochloride Reference Standard
Internal standard solution — A solution of /?-benzylphenol in
the mixture of water and methanol (1:1) (7 in 4000).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefcapene Pivoxil Hydrochloride
Tablets
Cefcapene Pivoxil Hydrochloride Tablets contain
not less than 90.0% and not more than 105.0% of the
labeled amount of cefcapene (C 17 H 19 N 5 6 S2: 453.49).
Method of preparation Prepare as directed under Tablets,
with Cefcapene Pivoxil Hydrochloride Hydrate.
Identification To an amount of powdered Cefcapene Pivox-
il Hydrochloride Tablets, equivalent to about 10 mg (poten-
cy) of Cefcapene Pivoxil Hydrochloride Hydrate according
to the labeled amount, add 40 mL of methanol, shake
vigorously, and add methanol to make 50 mL. To 4 mL of
this solution add methanol to make 50 mL, filter through a
membrane filter with pore size of 0.45 pm, and use the filtrate
as the sample solution. Determine the absorption spectrum of
the sample solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
263 nm and 267 nm.
Purity (1) Related substances I — To an amount of pow-
dered Cefcapene Pivoxil Hydrochloride Tables, equivalent to
about 5 mg (potency) of Cefcapene Pivoxil Hydrochloride
Hydrate according to the labeled amount, add 1 mL of
methanol, and shake. Add 25 mL of a mixture of water and
methanol (1:1), shake vigorously for 5 minutes, and filter
through a membrane filter with pore size of 0.45 fim. Discard
the first 3 mL of the filtrate, and use the subsequent filtrate as
the sample solution. Perform the test with 30 /xh of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method. If necessary,
proceed with 30 /xL of the mixture of water and methanol
(1:1) in the same manner as the sample solution to compen-
sate the base line. Calculate the amounts of the peaks other
than cefcapene pivoxil by the area percentage method: the
peak, having the relative retention time of about 1.3 with
respect to cefcapene pivoxil, is not more than 0.4%, the peak
of cefcapene pivoxil trans-isomer, having the relative reten-
tion time of about 1.5, is not more than 0.5%, any other
peaks are not more than 0.3%, respectively, and the total of
these peaks is not more than 2.0%.
Operating conditions —
Proceed as directed in the Purity (2) under Cefcapene
Pivoxil Hydrochloride Hydrate.
System suitability —
Proceed as directed in the Purity (2) under Cefcapene
Pivoxil Hydrochloride Hydrate.
(2) Related substances II — To an amount of powdered
Cefcapene Pivoxil Hydrochloride Tablets, equivalent to 2 mg
(potency) of Cefcapene Pivoxil Hydrochloride Hydrate ac-
cording to the labeled amount, add 20 mL of A r ,A^-dimethyl-
formamide for liquid chromatography, shake vigorously for
10 minutes, and filter through a membrane filter with pore
size of 0.45 ftm. Discard the first 3 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Perform the
test with 20 liL of the sample solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine each peak area by the automatic in-
tegration method: the total area of the peaks which are eluted
before cefcapene pivoxil is not more than 3.3% of the total
area of the peaks other than the solvent peak.
Operating conditions —
Proceed as directed in the Purity (3) under Cefcapene
Pivoxil Hydrochloride Hydrate.
System suitability —
Proceed as directed in the Purity (3) under Cefcapene
Pivoxil Hydrochloride Hydrate.
Water <2.48> Not more than 3.9% (0.5 g, volumetric titra-
tion, back titration). Powdering of the sample tablets and
handling of the powder are performed under the relative hu-
midity of not exceeding 30%.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Cefcapene Pivoxil Hydrochloride Tablets
add 5 mL of water, and shake vigorously for 5 minutes to dis-
integrate. Add 20 mL of methanol, shake vigorously for 5
minutes, add a mixture of methanol and water (4:1) to make
exactly 50 mL, and centrifuge at 3000 rpm for 5 minutes.
Filter the supernatant liquid through a membrane filter with
pore size of 0.45 fim, and discard the first 1 mL of the filtrate.
Pipet FmL of the subsequent filtrate, equivalent to about 6
mg (potency) of Cefcapene Pivoxil Hydrochloride Hydrate
according to the labeled amount, add exactly 15 mL of the in-
ternal standard solution, add a mixture of water and
methanol (1:1) to make 75 mL, and use this solution as the
sample solution. Hereinafter, proceed as directed in the As-
say.
Amount [mg (potency)] of cefcapene (CnHjgNsOA)
= W s x (Q T /Q S ) x (15/ F)
W s : Amount [mg (potency)] of Cefcapene Pivoxil
Hydrochloride Reference Standard
Internal standard solution — A solution of j9-benzylphenol in
a mixture of water and methanol (1:1) (7 in 4000).
Dissolution Being specified separately.
434
Cefdinir / Official Monographs
JP XV
Assay To an amount of Cefcapene Pivoxil Hydrochloride
Tablets, equivalent to about 0.6 g (potency) of Cefcapene
Pivoxil Hydrochloride Hydrate according to the labeled
amount, add 20 mL of water, and shake for 5 minutes to dis-
integrate. Add 80 mL of methanol, shake vigorously for 5
minutes, add a mixture of methanol and water (4:1) to make
exactly 200 mL, and centrifuge at 3000 rpm for 5 minutes.
Filter the supernatant liquid through a membrane filter with
pore size of 0.45 fim, and discard the first 1 mL of the filtrate.
Pipet 2 mL of the subsequent filtrate, add exactly 15 mL of
the internal standard solution, add the mixture of water and
methanol (1:1) to make 75 mL, and use this solution as the
sample solution. Separately, weigh accurately an amount of
Cefcapene Pivoxil Hydrochloride Reference Standard,
equivalent to about 20 mg (potency), and dissolve in the mix-
ture of water and methanol (1:1) to make exactly 50 mL.
Pipet 10 mL of this solution, add exactly 10 mL of the inter-
nal standard solution, add the mixture of water and methanol
(1:1) to make 50 mL, and use this solution as the standard so-
lution. Proceed as directed in the Assay under Cefcapene
Pivoxil Hydrochloride Hydrate.
Amount [mg (potency)] of cefcapene (C 17 H 19 N506S2)
= W s x (Q T /Q S ) x 30
W s : Amount [mg (potency)] of Cefcapene Pivoxil
Hydrochloride Reference Standard
Internal standard solution — A solution of />-benzylphenol in
the mixture of water and methanol (1:1) (7 in 4000).
Containers and storage Containers — Tight containers.
Cefdinir
-b7-x-JU
COjH
H 2 N^ J II H H
s
C 14 H 13 N505S 2 : 395.41
(6fl,7.R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-
2-(hydroxyimino)acetylamino]-8-oxo-3-vinyl-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
[91832-40-5]
Cefdinir contains not less than 930 fig (potency) and
not more than 1020 fig (potency) per mg. The potency
of Cefdinir is expressed as mass (potency) of cefdinir
(C 14 H 13 N 5 5 S 2 ).
Description Cefdinir occurs as a white to light yellow crys-
talline powder.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in 0.1 mol/L phosphate buffer solution, pH
7.0.
Identification (1) Determine the absorption spectra of so-
lutions of Cefdinir and Cefdinir Reference Standard in 0.1
mol/L phosphate buffer solution, pH 7.0 (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare these spectra: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectra of Cefdinir
and Cefdinir Reference Standard as directed in the paste
method under Infrared Spectrophotometry <2.25>, and com-
pare these spectra: both spectra exhibit similar intensities of
absorption at the same wave numbers.
(3) Determine the spectrum of a solution of Cefdinir in a
mixture of deuterated dimethyl sulfoxide and heavy water for
nuclear magnetic resonance spectroscopy (4:1) (1 in 10), us-
ing tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound, as directed un-
der Nuclear Magnetic Resonance Spectroscopy <2.21> ('H): it
exhibits multiple signals, A at around d 5.0 - 6.1 ppm and B
at around 5 6.4 - 7.5 ppm. The ratio of integrated intensity
of each signal, A:B is about 2:1.
Absorbance <2.24> £}*, (287 nm): 570-610 (50 mg, 0.1
mol/L phosphate buffer solution, pH 7.0, 5000 mL).
Optical rotation <2.49> [«]£": -58- -66° (0.25 g, 0.1
mol/L phosphate buffer solution, pH 7.0, 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefdinir according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve about 0.1 g of Cefdinir
in 10 mL of 0.1 mol/L phosphate buffer solution, pH 7.0.
Pipet 3 mL of this solution, add tetramethylammonium
hydroxide TS, pH 5.5 to make exactly 20 mL, and use this
solution as the sample solution. Perform the test with 10,mL
of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions, de-
termine the areas of each peak by the automatic integration
method, and calculate the amounts of their peaks by the area
percentage method: the amount of E-isomer having the rela-
tive retention time 1.5 to cefdinir is not more than 0.8%, and
the amount of total peak areas other than cefdinir is not more
than 3.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase A: To 1000 mL of tetramethylammonium
hydroxide TS, pH 5.5, add 0.4 mL of 0.1 mol/L disodium di-
hydrogen ethylenediamine tetraacetate TS.
Mobile phase B: To 500 mL of tetramethylammonium
hydroxide TS, pH 5.5 add 300 mL of acetonitrile for liquid
chromatography and 200 mL of methanol, and add 0.4 mL
of 0.1 mol/L disodium dihydrogen ethylenediamine tetraa-
cetate TS.
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile A and B as directed in the following table.
JPXV
Official Monographs / Cefdinir Capsules 435
Time after injection
Mobile phase
Mobile phase
of the sample (min)
A (vol%)
B (vol%)
0-2
95
5
2-22
95^75
5^25
22-32
75^50
25^50
32-37
50
50
37-38
50^95
50^5
38-58
95
5
Flow rate: 1.0 mL per minute. The retention time of cef-
dinir is about 22 minutes under this condition.
Time span of measurement: About 40 minutes after injec-
tion of the sample solution.
System suitability —
Test for required detection: Pipet 1 mL of the sample solu-
tion, add tetramethylammonium hydroxide TS, pH 5.5 to
make exactly 100 mL, and use this solution as the test solu-
tion for system suitability. Pipet 1 mL of the test solution for
system suitability, add tetramethylammonium hydroxide TS,
pH 5.5 to make exactly 10 mL. Confirm that the peak area of
cefdinir obtained from 10 /xL of this solution is equivalent to
7 to 13% of that obtained from 10 /xL of the test solution for
system suitability.
System performance: Dissolve 0.03 g of Cefdinir Reference
Standard and 2 mg of cefdinir lactam ring-cleavage lactones
in 3 mL of 0.1 mol/L phosphate buffer solution, pH 7.0, add
tetramethylammonium hydroxide TS, pH 5.5, to make 20
mL. When the procedure is run with 10 [iL of this solution
under the above operating conditions, peak 1 and peak 2 of
cefdinir lactam ring-cleavage lactones separated into 4 peaks,
cefdinir, peak 3 and peak 4 of remaining cefdinir lactam ring-
cleavage lactones are eluted in this order. Relative retention
time of peak 3 of cefdinir lactam ring-cleavage lactone to the
retention time of cefdinir is not less than 1 .09. The number of
theoretical plates and the symmetry factor of the peak of cef-
dinir are not less than 7000 steps and not more than 3.0,
respectively.
System repeatability: When the test is repeated 3 times with
10 /xL of the test solution for system suitability under the
above operating conditions, the relative standard deviation
of the peak areas of cefdinir is not more than 2.0%.
Water <2.48> Not more than 2.0% (1 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1) in-
stead of methanol for water determination).
Assay Weigh accurately an amount of Cefdinir and Cef-
dinir Reference Standard equivalent to about 20 mg (poten-
cy), dissolve each in 0.1 mol/L phosphate buffer solution, pH
7.0 to make exactly 100 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
exactly 5 fiL of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the peak areas, A T
and A s , of cefdinir of the solutions.
Amount [/ug (potency)] of Ci 4 H 13 N505S2
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefdinir Reference Stan-
dard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 1000 mL of tetramethylammonium
hydroxide TS, pH 5.5, add 0.4 mL of 0.1 mol/L disodium di-
hydrogen ethylenediamine tetraacetate TS. To 900 mL of this
solution add 60 mL of acetonitrile for liquid chromatography
and 40 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
cefdinir is about 8 minutes.
System suitability —
System performance: Dissolve 2 mg of Cefdinir Reference
Standard and 5 mg of cefdinir lactam ring-cleavage lactones
in 10 mL of 0.1 mol/L phosphate buffer solution, pH 7.0.
When the procedure is run with 5 /xL of this solution under
the above operating conditions, peak 1 and peak 2 of cefdinir
lactam ring-cleavage lactones separated into 4 peaks, cef-
dinir, peak 3 and peak 4 of remaining cefdinir lactam ring-
cleavage lactones are eluted in this order. The resolution be-
tween the peak 2 of cefdinir lactam ring-cleavage lactone and
that of cefdinir is not less than 1 .2. The number of theoretical
plates and the symmetry factor of the peak of cefdinir are not
less than 2000 steps and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
5 ixL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
cefdinir is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefdinir Capsules
Cefdinir Capsules contain not less than 90.0% and
not more than 110.0% of the labeled amount of cef-
dinir (C 14 H 13 N505S 2 : 395.41).
Method of preparation
with Cefdinir.
Prepare as directed under Capsules,
Identification To an amount of the contents of Cefdinir
Capsules, equivalent to 10 mg (potency) of Cefdinir accord-
ing to the labeled amount, add 100 mL of 0.1 mol/L phos-
phate buffer solution, pH 7.0, exposure to ultrasonic waves
for 1 minute, and filter. To 2 mL of the filtrate add 0.1 mol/L
phosphate buffer solution, pH 7.0 to make 20 mL, and deter-
mine the absorption spectrum of this solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>; it exhibits
maxima between 221 nm and 225 nm and between 285 nm
and 289 nm.
Uniformity of dosage unit <6.02>
of the Mass variation test.
It meets the requirement
Dissolution <6.10> Perform the test according to the follow-
436
Cefdinir Fine Granules / Official Monographs
JP XV
ing method: it meets the requirement.
Perform the test with 1 capsule of Cefdinir Capsules at 50
revolutions per minute according to the Paddle method using
a sinker, using 900 mL of 2nd fluid for dissolution test as the
dissolution medium. Withdraw 20 mL or more of the dissolu-
tion medium 30 minutes after starting the test for a 50-mg
capsule, or 45 minutes after for a 100-mg capsule, and filter
through a membrane filter with pore size of not more than 0.5
/urn. Discard the first 10 mL of the filtrate, pipet FmL of the
subsequent filtrate, add 2nd fluid for dissolution test to make
exactly V mL so that each mL contains about 56 [ig (poten-
cy) of Cefdinir according to the labeled amount, and use this
solution as the sample solution. Separately, weigh accurately
about 28 mg (potency) of Cefdinir Reference Standard, and
dissolve in 2nd fluid for dissolution test to make exactly 100
mL. Pipet 4 mL of this solution, add 2nd fluid for dissolution
test to make exactly 20 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 20 /xh each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01>, and determine the peak areas, A
T and A s , of cefdinir. The dissolution rate of a 50-mg capsule
in 30 minutes is not less than 80% and that of a 100-mg cap-
sule in 45 minutes is not less than 75%.
Dissolution rate (%) with respect to the labeled amount of
cefdinir (C 14 H 13 N50 5 S 2 )
= W s x (Aj/As) x (V'/V) x (l/Q x 180
W s : Amount [mg (potency)] of Cefdinir Reference Stan-
dard
C: Labeled amount [mg (potency)] of cefdinir
(C 14 H 13 N505S2) in 1 capsule
Operating conditions-
Proceed as directed in the Assay under Cefdinir.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefdinir are not less than 2000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 liL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefdinir is not more than 1.0%.
Assay Weigh accurately not less than 5 Cefdinir Capsules,
take out the contents, and powder. Wash the empty capsules
with a little amount of diethyl ether, if necessary, allow to
stand at a room temperature to vaporize the adhering diethyl
ether, and weigh accurately the mass of the capsules to calcu-
late the mass of the contents. Weigh accurately an amount of
the contents, equivalent to about 0.1 g (potency) of Cefdinir
according to the labeled amount, add 70 mL of 0.1 mol/L
phosphate buffer solution, pH 7.0, shake for 30 minutes, and
add 0.1 mol/L phosphate buffer solution, pH 7.0 to make ex-
actly 100 mL. Centrifuge this solution at 3000 revolutions per
minute for 10 minutes, pipet 4 mL of the supernatant liquid,
add 0.1 mol/L phosphate buffer solution, pH 7.0 to make ex-
actly 20 mL, and use this solution as the sample solution.
Separately, weigh accurately an amount of Cefdinir Refer-
ence Standard, equivalent to about 20 mg (potency), dissolve
in 0.1 mol/L phosphate buffer solution, pH 7.0 to make ex-
actly 100 mL, and use this solution as the standard solution.
Proceed as directed in the Assay under Cefdinir.
Amount [mg (potency)] of cefdinir (Q4FL3N5O5S2)
= W s x (A T /A S ) x 5
W s : Amount [mg (potency)] of Cefdinir Reference Stan-
dard
Containers and storage Containers — Tight containers.
Cefdinir Fine Granules
Cefdinir Fine Granules contain not less than 93.0%
and not more than 107.0% of the labeled amount of
cefdinir (CuHnNsOsS;,: 395.41).
Method of preparation Prepare to finely granulated form as
directed under Powders, with Cefdinir.
Identification To an amount of Cefdinir Fine Granules,
equivalent to 10 mg (potency) of Cefdinir according to the la-
beled amount, add 100 mL of 0.1 mol/L phosphate buffer so-
lution, pH 7.0, exposure to ultrasonic waves for 1 minute,
and filter. To 2 mL of the filtrate add 0.1 mol/L phosphate
buffer solution, pH 7.0 to make 20 mL, and determine the ab-
sorption spectrum of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 221 nm and 225 nm and between 285 nm and 289
nm.
Uniformity of dosage units <6.02> The granules in single-
unit container meet the requirement of the Mass variation
test.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with an accurate amount of Cefdinir Fine
Granules, equivalent to about 0.1 g (potency) of Cefdinir ac-
cording to the labeled amount, at 50 revolutions per minute
according to the Paddle method using 900 mL of 2nd fluid
for dissolution test as the dissolution medium. Withdraw 20
mL or more of the dissolution medium 30 minutes after start-
ing the test, and filter through a membrane filter with a pore
size not exceeding 0.5 Lira. Discard the first 10 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 28 mg (potency) of Cef-
dinir Reference Standard, and 2nd fluid for dissolution test to
make exactly 50 mL. Pipet 4 mL of this solution, add 2nd
fluid for dissolution test to make exactly 20 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 iiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01>, and deter-
mine the peak areas, A T and A s , of cefdinir. The dissolution
rate in 30 minutes is not less than 75%.
Dissolution rate (%) with respect to the labeled amount of
cefdinir (C 14 H 13 N50 5 S 2 )
= (W S /W T ) x (A T /A S ) x (l/Q x 360
W s : Amount [mg (potency)] of Cefdinir Reference Stan-
dard
W T : Amount (g) of sample
C: Labeled amount [mg
(C 14 H 13 N 5 5 S 2 ) in 1 g
(potency)] of cefdinir
JPXV
Official Monographs / Cefditoren Pivoxil 437
Operating conditions —
Proceed as directed in the Assay under Cefdinir.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefdinir are not less than 2000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefdinir is not more than 1.0%.
Particle size <6.03> It meets the requirement of fine granules
of the Powders.
Assay Powder, if necessary, and weigh accurately an
amount of Cefdinir Fine Granules, equivalent to about 0.1 g
(potency) of Cefdinir according to the labeled amount, add
70 mL of 0.1 mol/L phosphate buffer solution, pH 7.0, shake
for 30 minutes, and add 0.1 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 100 mL. Centrifuge at 3000
revolutions per minute for 10 minutes, pipet 4 mL of the su-
pernatant liquid, add 0.1 mol/L phosphate buffer solution,
pH 7.0 to make 20 mL, and use this solution as the sample so-
lution. Separately, weigh accurately an amount of Cefdinir
Reference Standard, equivalent to about 20 mg (potency),
dissolve in 0.1 mol/L phosphate buffer solution, pH 7.0 to
make exactly 100 mL, and use this solution as the standard
solution. Proceed as directed in the Assay under Cefdinir.
Amount [mg (potency)] of cefdinir (Q4FL3N5O5S2)
= W s x (A T /A S ) x 5
W s : Amount [mg (potency)] of Cefdinir Reference Stan-
dard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefditoren Pivoxil
-tz7vr-U> tf** + ->JU
C 25 H 28 N 6 7 S 3 : 620.72
2,2-Dimethylpropanoyloxymethyl (6R,lR)-l-[{Z)-2-{2-
aminothiazol-4-yl)-2-(methoxyimino)acetylamino]-3-
[(lZ)-2-(4-methylthiazol-5-yl)ethenyl]-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate [117467-28-4]
Cefditoren Pivoxil contains not less than 11 '0 fig
(potency) and not more than 820 fig (potency) per mg,
calculated on the anhydrous basis. The potency of Cef-
ditoren Pivoxil is expressed as mass (potency) of cef-
ditoren (C 19 H 18 N 6 5 S 3 : 506.58).
Description Cefditoren Pivoxil occurs as a light yellowish
white to light yellow crystalline powder.
It is sparingly soluble in methanol, slightly soluble in
acetonitrile and in ethanol (95), very slightly sobuble in
diethyleter and practically insoluble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Dissolve 5 mg of Cefditoren Pivoxil in 3
mL of hydroxylammonium chloride-ethanol TS, allow to
stand for 5 minutes, add 1 mL of acidic ammonium iron (III)
sulfate TS and shake: a red-brown color develops.
(2) Dissolve 1 mg of Cefditoren Pivoxil in 1 mL of dilute
hydrochloric acid and 4 mL of water, add 3 drops of sodium
nitrite TS under ice-cooling, shake, and allow to stand for 2
minutes. Then add 1 mL of ammonium amidosulfate TS,
shake well, and allow to stand for 1 minute, and add 1 mL of
7V,A r -diethyl-/V'-l-naphthylethylenediamine oxalate TS: a
purple color develops.
(3) Determine the absorption spectrum of a solution of
Cefditoren Pivoxil in methanol (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of a solution of Cefditoren Pivoxil Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(4) Determine the spectrum of a solution of Cefditoren
Pivoxil in deuterated chloroform for nuclear magnetic
resonance spectroscopy (1 in 50), using tetramethylsilane for
nuclear magnetic resonance spectroscopy as an internal refer-
ence compound, as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H): it exhibits single signals
A, B and C, at around J 1.1 ppm, at around <5 2.4 ppm and at
around 5 4.0 ppm, double signals D and E, at around 5 6.4
ppm and at around 8 6.7 ppm, and a single signal F at around
5 8.6 ppm. The ratio of integrated intensity of each signal
A:B:C:D:E:F, is about 9:3:3:1:1:1.
Absorbance <2.24> E\°j m (231 nm): 340-360 (50 mg,
methanol, 2500 mL).
Optical rotation <2.49> [ a ] 2 ^: -45- -52° (50 mg,
methanol, 10 mL, 100 mm).
Purity (1) Heavy metals <l.07> — Proceed with 2.0 g of
Cefditoren Pivoxil according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Being specified separately.
(3) Residual solvents — Being specified separately.
Water <2.48> Not more than 1.5% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition Being specified separately.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately an
amount of Cefditoren Pivoxil and Cefditoren Pivoxil Refer-
ence Standard, equivalent to about 40 mg (potency), dissolve
in 40 mL of acetonitrile, add exactly 10 mL each of the inter-
nal standard solution, and add acetonitrile to make 100 mL,
and use these solutions as the sample solution and standard
solution. Perform the test with 1 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
438
Cefditoren Pivoxil Fine Granules / Official Monographs
JP XV
and calculate the ratios, Q T and Q s , of the peak area of cef-
ditoren pivoxil to that of the internal standard.
Amount [/xg (potency)] of cefditoren (C I9 H 18 N 6 5 S3)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Cefditoren Pivoxil Refer-
ence Standard
Internal standard solution — A solution of propyl p-hydrox-
ybenzoate in acetonitrile (1 in 200).
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.58 g of ammonium formate in
900 mL of water, adjust to pH 6.0 with diluted formic acid (1
in 250), and add water to make 1000 mL. To 450 mL of this
solution add 275 mL of acetonitrile and 275 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
cefditoren pivoxil is about 15 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the internal standard and cefditoren pivoxil are eluted
in this order with the resolution between these peaks being
not less than 5.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefditoren pivoxil to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Uniformity of dosage units <6.02> The granules in single-
unit container meet the requirement of the Mass variation
test.
Dissolution Being specified separately.
Particle size <6.03> It meets the requirement of fine granules
of the Powders.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately an
amount of powdered Cefditoren Pivoxil Fine Granules,
equivalent to about 40 mg (potency) of Cefditoren Pivoxil ac-
cording to the labeled amount, add 70 mL of diluted acetoni-
trile (3 in 4), and shake vigorously. To this solution add ex-
actly 10 mL of the internal standard solution, then add
acetonitrile to make 100 mL, filter, and use the filtrate as the
sample solution. Separately, weigh accurately about 20 mg
(potency) of Cefditoren Pivoxil Reference Standard, dissolve
in 20 mL of acetonitrile, add exactly 5 mL of the internal
standard solution, then add acetonitrile to make 50 mL, and
use this solution as the standard solution. Proceed as directed
in the Assay under Cefditoren Pivoxil.
Amount [mg (potency)] of cefditoren (C 19 H 18 N 6 5 S3)
= W s x (Q T /Q S ) x 2
W s : Amount [mg (potency)] of Cefditoren Pivoxil Refer-
ence Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in acetonitrile (1 in 200)
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefditoren Pivoxil Tablets
Cefditoren Pivoxil Fine Granules
t7vH/> t°7K' + ->;i4H6
Cefditoren Pivoxil Fine Granules contain not less
than 90.0% and not more than 1 10.0% of the labeled
amount of cefditoren (Ci 9 H 18 N 6 5 S 3 : 506.58).
Method of preparation Prepare in the form of very fine
granules as directed under Powders, with Cefditoren Pivoxil.
Identification To an amount of powdered Cefditoren
Pivoxil Fine Granules, equivalent to 0.1 g (potency) of Cef-
ditoren Pivoxil according to the labeled amount, add 10 mL
of acetonitrile, shake vigorously, and filter. To 1 mL of the
filtrate add acetonitrile to make 50 mL. To 1 mL of this solu-
tion add acetonitrile to make 20 mL, and determine the ab-
sorption spectrum of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 230 nm and 234 nm.
Purity Related substances — Being specified separately.
Loss on drying <2.41> Not more than 4.5% (0.5 g, reduced
Cefditoren Pivoxil Tablets contain not less than
90.0% and not more than 110.0%, of the labeled
amount of cefditoren (C 19 H 18 N 6 5 S 3 : 506.58),
Method of preparation Prepare as directed under Tablets,
with Cefditoren Pivoxil.
Identification To an amount of powdered Cefditoren
Pivoxil Tablets, equivalent to 35 mg (potency) of Cefditoren
Pivoxil according to the labeled amount, add 100 mL of
methanol, shake, and filter. To 5 mL of the filtrate add
methanol to make 100 mL, and determine the absorption
spectrum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
229 nm and 233 nm.
Purity Related substances — Being specified separately.
Loss on drying <2.41> Not more than 4.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Cefditoren Pivoxil Tablets add 12.5 mL of
the 1st fluid for disintegration test, shake vigorously, add
JPXV
Official Monographs / Cefepime Dihydrochloride Hydrate
439
about 25 mL of acetonitrile, shake again, and add acetoni-
trile to make exactly 50 mL. Pipet KmL of this solution,
equivalent to about 20 mg (potency) of Cefditoren Pivoxil ac-
cording to the labeled amount, add exactly 5 mL of the inter-
nal standard solution, then add diluted acetonitrile (3 in 4) to
make 50 mL, filter, and use the filtrate as the sample solution.
Separately, weigh accurately about 20 mg (potency) of Cef-
ditoren Pivoxil Reference Standard, dissolve in 20 mL of
acetonitrile, add exactly 5 mL of the internal standard solu-
tion, then add acetonitrile to make 50 mL, and use this solu-
tion as the standard solution. Proceed as directed in the
Assay under Cefditoren Pivoxil.
Amount [mg (potency)] of cefditoren (C 19 H 18 N 6 5 S3)
= Ws x (Qj/Qs) x (50/ V)
W s : Amount [mg (potency)] of Cefditoren Pivoxil Refer-
ence Standard
Internal standard solution — A solution of propyl para-
hydroxybezoate in acetonitrile (1 in 200)
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Cefditoren Pivoxil Tablets
at 50 revolutions per minute according to the Paddle method
using 900 mL of the 1st fluid for dissolution test as the disso-
lution medium. Withdraw 20 mL or more of the dissolution
medium 20 minutes after starting the test, and filter through a
membrane filter with pore size of not more than 0.45 ^m.
Discard the first 10 mL of the filtrate, pipet the subsequent V
mL, add water to make exactly V mL so that each mL con-
tains about 11 fi% (potency) of Cefditoren Pivoxil according
to the labeled amount, and use this solution as the sample so-
lution. Separately, weigh accurately about 22 mg (potency)
of Cefditoren Pivoxil Reference Standard, dissolve in 20 mL
of diluted acetonitrile (3 in 4), then add the 1st fluid for disso-
lution test to make exactly 200 mL. Pipet 2 mL of this solu-
tion, add water to make exactly 20 mL, and use this solution
as the standard solution. Determine the absorbances, A T and
A s , at 272 nm of the sample solution and standard solution as
directed under Ultraviolelt-visible Spectrophotometry <2.24>
using water as the control. The dissolution rate in 20 minutes
is not less than 85%.
Dissolution rate (%) with respect to the labeled amount
= W s x (Aj/As) x (V'/V) x (1/C) x 45
W s : Amount [mg (potency)] of Cefditoren Pivoxil Refer-
ence Standard
C: Labeled amount [mg (potency)] of cefditoren pivoxil
(C 25 H 28 N 6 7 S 3 ) in 1 tablet
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. To an amount of Cefdito-
ren Pivoxil Tablets, equivalent to 0.5 g (potency) of Cefdito-
ren Pivoxil according to the labeled amount, add 63 mL of
the 1st fluid for disintegration test, shake vigorously, add
about 125 mL of acetonitrile, shake again, and add acetoni-
trile to make exactly 250 mL. Pipet 10 mL of this solution,
add exactly 5 mL of the internal standard solution, then add
diluted acetonitrile (3 in 4) to make 50 mL, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 20 mg (potency) of Cefditoren Pivoxil Reference Stan-
dard, dissolve in 20 mL of acetonitrile, add exactly 5 mL of
the internal standard solution, then add acetonitrile to make
50 mL, and use this solution as the standard solution. Pro-
ceed as directed in the Assay under Cefditoren Pivoxil.
Amount [mg (potency)] of cefditoren (C 19 H 18 N 6 5 S 3 )
= W s x (Q T /Q S ) x 25
W s : Amount [mg (potency)] of Cefditoren Pivoxil Refer-
ence Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in acetonitrile (1 in 200)
Containers and storage Containers — Tight containers.
Cefepime Dihydrochloride Hydrate
-tz-7itfAJgg^7kMfci
CH,,
CO;.
N
" I
'3
■ 2HCI ■ HjO
C 19 H 2 4N 6 5 S 2 .2HC1.H 2 0: 571.50
(6i?,7i?)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-
(methoxyimino)acetylamino]-3-(l-methylpyrrolidinium-l-
ylmethyl)-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate dihydrochloride monohydrate
[123171-59-5]
Cefepime Dihydrochloride Hydrate contains not less
than 835 fig (potency) and not more than 886 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefepime Dihydrochloride Hydrate is
expressed as mass (potency) of cefepime (C 19 H 2 4N 6 5
S 2 : 480.56).
Description Cefepime Dihydrochloride Hydrate occurs as a
white to yellowish white, crystals or crystalline powder.
It is freely soluble in water and in methanol, and slightly
soluble in ethanol (95), and practically in soluble in
diethylether.
Identification (1) Dissolve 0.02 g of Cefepime Di-
hydrochloride Hydrate in 2 mL of water, add 1 mL of a solu-
tion of hydroxylammonium chloride (1 in 10) and 2 mL of
sodium hydroxide TS, allow to stand for 5 minutes, then add
3 mL of 1 mol/L hydrochloric acid TS and 3 drops of iron
(III) chloride TS: a red-brown color develops.
(2) Determine the absorption spectra of solutions (1 in
20,000) of Cefepime Dihydrochloride Hydrate and Cefepime
Dihydrochloride Reference Standard as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
these spectra: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(3) Determine the infrared absorption spectra of
Cefepime Dihydrochloride Hydrate and Cefepime Di-
hydrochloride Reference Standard as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare these spectra: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(4) Determine the spectrum of a solution of Cefepime Di-
hydrochloride Hydrate in heavy water for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
440
Cefepime Dihydrochloride Hydrate / Official Monographs
JP XV
Magnetic Resonance Spectroscopy <2.21> ('H), using sodium
3-trimethylsilylpropionate-d 4 for nuclear magnetic resonance
spectroscopy as an internal reference compound: it exhibits
single signals, A and B, at around d 3.1 ppm and at around 5
7.2 ppm, respectively, and the ratio of integrated intensity of
each signal, A:B, is about 3:1.
(5) Dissolve 15 mg of Cefepime Dihydrochloride Hydrate
in 5 mL of water, and add 2 drops of silver nitrate TS: a white
turbidity is produced.
Absorbance <2.24> E\ v ° m (259 nm): 310 - 340 (50 mg calcu-
lated on the anhydrous basis, water, 1000 mL).
Optical rotation <2.49> [a]™: +39 - +47° (60 mg calculat-
ed on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 0.1 g of Cefepime Dihydrochloride Hy-
drate in 10 mL of water: the pH of this solution is between
1.6 and 2.1.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Cefepime
Dihydrochloride Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) A r -Methylpyrrolidine — Weigh accurately an amount
of Cefepime Dihydrochloride Hydrate equivalent to about 80
mg (potency), dissolve in diluted nitric acid (2 in 3125) to
make exactly 10 mL, and use this solution as the sample solu-
tion. Separately, put 30 mL of water in a 100-mL volumetric
flask, weigh accurately the mass of flask, then add about
0.125 g of A r -methylpyrrolidine, weigh accurately the mass of
the flask again, and add water to make exactly 100 mL. Pipet
4 mL of this solution, add diluted nitric acid (2 in 3125) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 100 iiL each of the
sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the peak areas, A T and A s , of N-
methylpyrrolidine by the automatic integration method. Cal-
culate the amount of 7V-methylpyrrolidine per 1 mg (potency)
of Cefepime Dihydrochloride Hydrate by the following equa-
tion: not more than 0.5%. The sample solution must be test-
ed within 20 minutes after preparation.
Amount (%) of /V-methylpyrrolidine
= i(W s x f)/W T ) x (A T /A S ) x (1/250)
W s : Amount (mg) of /V-methylpyrrolidine
W T : Amount [mg (potency)] of sample
/: Purity (%) of /V-methylpyrrolidine
Operating conditions-
Detector: An electric conductivity detector
Column: A plastic tube 4.6 mm in inside diameter and 5
cm in length, packed with hydrophilic silica gel for liquid
chromatography carrying sulfonic acid groups having the
exchange capacity of about 0.3 meq per g (5/«n in particle
diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 990 mL of diluted nitric acid (2 in 3125)
add 10 mL of acetonitrile.
Flow rate: 1.0 mL per minute.
System suitability —
System performance: To 20 mL of a solution of sodium
chloride (3 in 1000) add 0.125 g of N-methylpyrrolidine, and
add water to make 100 mL. To 4 mL of this solution add
diluted nitric acid (2 in 3125) to make 100 mL. When the
procedure is run with 100 fiL of this solution under the above
operating conditions, sodium and /V-methylpyrrolidine are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 5 times with
100 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of 7V-methylpyrrolidine is not more than 4.0%.
(4) Related substances — Dissolve about 0.1 g of
Cefepime Dihydrochloride Hydrate in the mobile phase A to
make 50 mL, and use this solution as the sample solution.
Perform the test with 5 liL of the sample solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the area of each peak by
the automatic integration method. Calculate the total of the
peak areas other than cefepime: not more than 0.5%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 0.57 g of ammonium di-
hydrogenphosphate in 1000 mL of water.
Mobile phase B: Acetonitrile
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phase A and B as directed in the following
table.
Time after injection
of the sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-25
100^75
0^25
Flow rate: Adjust the flow rate so that the retention time of
cefepime is about 9.5 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of cefepime.
System suitability —
Test for required detection: To 1 mL of the sample solu-
tion add the mobile phase A to make 10 mL, and use this
solution as the solution for system suitability test. To 1 mL of
the solution for system suitability test add the mobile phase A
to make 10 mL, and use this solution as the solution for test
for required detection. Pipet 1 mL of the solution for test for
required detection, add the mobile phase A to make exactly
10 mL. Conform that the peak area of cefepime obtained
from 5 [iL of this solution is equivalent to 7 to 1 3% of that of
cefepime obtained from 5 [iL of the solution for test for
required detection.
System performance: When the procedure is run with 5 fiL
of the solution for system suitability test under the above
operating conditions, the number of theoretical plates of the
peak of cefepime is not less than 6000 steps.
System repeatability: When the test is repeated 3 times with
JPXV
Official Monographs / Cefepime Dihydrochloride for Injection
441
5 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of cefepime is not more than 2.0%.
Water <2.48> Not less than 3.0% and not more than 4.5%
(Weigh accurately about 50 mg, and add exactly 2 mL of
methanol for water determination to dissolve. Use exactly 0.5
mL of this solution as the test solution; coulometric titra-
tion).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Bacterial endotoxins <4.01> Less than 0.04 EU/mg (poten-
cy).
Assay Weigh accurately an amount of Cefepime Di-
hydrochloride Hydrate and Cefepime Dihydrochloride
Reference Standard, equivalent to about 60 mg (potency),
dissolve in the mobile phase to make exactly 50 mL, and use
these solutions as the sample solution and standard solution,
respectively. Perform the test with exactly 10,mL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas, A T and A s , of
cefepime of each solution.
Amount [/ug (potency)] of cefepime (Ci9H 2 4N 6 5 S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefepime Dihydrochloride
Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust a solution of sodium 1-pentanesul-
fonate (261 in 100,000) to pH 3.4 with acetic acid (100), then
adjust this solution to pH 4.0 with a solution of potassium
hydroxide (13 in 20). To 950 mL of this solution add 50 mL
of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefepime is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates of the peak of
cefepime is not less than 1500 steps.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefepime is not more than 2.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Cefepime Dihydrochloride for
Injection
Cefepime Dihydrochloride for Injection is a prepa-
ration for injection, which is dissolved before use.
It contains not less than 95.0% and not more
than 110.0% of the labeled amount of cefepime
(C 19 H 24 N 6 5 S 2 : 480.56).
Method of preparation Prepare as directed under Injec-
tions, with Cefepime Dihydrochloride Hydrate.
Description Cefepime Dihydrochloride for Injection occurs
as a white to pale yellow powder.
Identification (1) Dissolve 40 mg of Cefepime Di-
hydrochloride in 2 mL of water, add 1 mL of a solution of
hydroxylammonium chloride (1 in 10) and 2 mL of sodium
hydroxide TS, allow to stand for 5 minutes, then add 3 mL of
1 mol/L hydrochloric acid TS and 3 drops of iron (III) chlo-
ride TS: a red-brown color develops.
(2) Determine the absorption spectrum of a solution of
Cefepime Dihydrochloride for Injection (1 in 12,500) as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 233 nm and 237 nm and between
255 nm and 259 nm.
pH <2.54> The pH of a solution obtained by dissolving an
amount of Cefepime Dihydrochloride for Injection, equiva-
lent to 0.5 g (potency) of Cefepime Dihydrochloride Hydrate
according to the labeled amount, in 5 mL of water is between
4.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve an
amount of Cefepime Dihydrochloride for Injection, equiva-
lent to 0.5 g (potency) of Cefepime Dihydrochloride Hydrate
according to the labeled amount, in 5 mL of water: the solu-
tion is clear and colorless or light yellow. The color is not dar-
ker than Matching Fluid I.
(2) 7V-Methylpyrrolidine — Weigh accurately an amount
of Cefepime Dihydrochloride for Injection, equivalent to
about 0.2 g (potency) of Cefepime Dihydrochloride Hydrate
according to the labeled amount, dissolve in diluted nitric
acid (2 in 625) to make exactly 20 mL, and use this solution as
the sample solution. Separately, transfer 30 mL of water into
a 100-mL volumetric flask, weigh accurately the mass of the
flask, add about 0.125 g of 7V-methylpyrrolidine, then weigh
accurately the mass, and add water to make exactly 100 mL.
Pipet 4 mL of this solution, add diluted nitric acid (2 in 3 125)
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 100 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following
conditions, and determine the peak areas of 7V-methylpyrroli-
dine, A T and A s , by the automatic integration method within
20 minutes after the sample solution is prepared. Calculate
the amount of 7V-methylpyrrolidine per mg (potency) of
Cefepime Dihydrochloride for Injection by the following for-
mula: not more than 1.0%.
Amount (%) of Af-methylpyrrolidine
442
Cefixime / Official Monographs
JP XV
= {{W s xf)/W T } x (A T /A S ) x (1/125)
W s : Amount (mg) of Af-methylpyrrolidine
W T ; Amount [mg (potency)] of cefepime in the sample
/: Purity (%) of Af-methylpyrrolidine
Operating conditions —
Proceed as directed in the operating conditions in the Puri-
ty (3) under Cefepime Dihydrochloride Hydrate.
System suitability —
Proceed as directed in the system suitability in the Purity
(3) under Cefepime Dihydrochloride Hydrate.
Water <2.48> Not more than 4.0% (Weigh accurately about
50 mg of Cefepime Dihydrochloride for Injection, dissolve in
exactly 2 mL of methanol for Karl Fischer method, and
perform the test with exactly 0.5 mL of this solution. Coulo-
metric titration).
Bacterial endotoxins <4.01> Less than 0.06 EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Cefepime Dihydrochloride for Injection. Weigh ac-
curately an amount of the content, equivalent to about 60 mg
(potency) of Cefepime Dihydrochloride Hydrate according to
the labeled amount, dissolve in the mobile phase to make
exactly 50 mL, and use this solution as the sample solution.
Separately, weigh accurately an amount of Cefepime
Dihydrochloride Reference Standard, equivalent to about
60 mg (potency), dissolve in the mobile phase to make exactly
50 mL, and use this solution as the standard solution. Pro-
ceed as directed in the Assay under Cefepime Dihydrochlo-
ride Hydrate.
Amount L"g (potency)] of cefepime (C 19 H24N 6 5 S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefepime Dihydrochloride
Reference Standard
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Cefixime
■b7^->A
XOjH
f COjH
-Hi
HsN_ i J o H H
C 16 H 15 N 5 7 S 2 : 453.45
(6R , 1R )-7- [(Z )-2-(2- Aminothiazol-4-yl)-2-
(carboxymethoxyimino)acetylamino]-8-oxo-3-vinyl-5-thia-
1 -azabicyclo [4 .2 .0] oct-2-ene-2-carboxylic acid
[79350-37-1]
Cefixime contains not less than 930 fig (potency) and
not more than 1020 ^g (potency) per mg, calculated on
the anhydrous basis. The potency of Cefixime is ex-
pressed as mass (potency) of cefixime (C 16 Hi5N50 7 S 2 ).
Description Cefixime occurs as a white to light yellow crys-
talline powder.
It is freely soluble in methanol and in dimethylsulfoxide,
sparingly soluble in ethanol (99.5), and practically insoluble
in water.
Identification (1) Determine the absorption spectrum of a
solution of Cefixime in 0.1 mol/L phosphate buffer solution,
pH 7.0 (1 in 62,500) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of
Cefixime Reference Standard prepared in the same manner as
the sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefixime as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Cefixime Reference Standard: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
(3) Dissolve 0.05 g of Cefixime in 0.5 mL of a mixture of
deuterated dimethylsulfoxide for nuclear magnetic resonance
spectroscopy and heavy water for nuclear magnetic
resonance spectroscopy (4:1). Determine the spectrum of this
solution, using tetramethylsilane for nuclear magnetic
resonance spectroscopy as an internal reference compound,
as directed under Nuclear Magnetic Resonance Spectroscopy
<2.21> ('H): it exhibits a single signal A at around 5 A.l ppm,
and a multiple signal B between d 6.5 ppm and S 7.4 ppm.
The ratio of integrated intensity of these signals, A:B, is
about 1:1.
Optical rotation <2.49> [a]™: -75 - -88° (0.45 g calculat-
ed on the anhydrous bases, a solution of sodium hydrogen
carbonate (1 in 50), 50 mL, 100 mm).
Purity Dissolve 0.1 g of Cefixime in 100 mL of 0.1 mol/L
phosphate buffer solution, pH 7.0, and use this solution as
the sample solution. Perform the test with 10 juL of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, measure the areas of
the peaks by the automatic integration method, and calculate
the amounts of these peak areas by the area percentage
method: the amount of each peak area other than cefixime is
not more than 1.0%, and the total area of the peaks other
than cefixime is not more than 2.5%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of cefixime beginning after the solvent peak.
System suitability —
Test for required detection: Pipet 1 mL of the sample solu-
tion, and add 0.1 mol/L phosphate buffer solution, pH 7.0 to
JPXV
Official Monographs / Cefmenoxime Hydrochloride 443
make exactly 100 mL. Confirm that the peak height of
cefixime obtained from 10 liL of this solution is equivalent to
20 to 60 mm.
System performance: Dissolve about 2 mg of Cefixime
Reference Standard in 200 mL of 0.1 mol/L phosphate buffer
solution, pH 7.0, and use this solution as the solution for sys-
tem suitability test. When the procedure is run with 10 tiL of
the solution according to the above operating conditions, the
number of theoretical plates and the symmetry factor of the
peak of cefixime are not less than 4000 and not more than
2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 iiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of cefixime is not more than 2.0%.
Water <2.48> Not less than 9.0 and not more than 12.0%
(0.1 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Cefixime and
Cefixime Reference Standard, equivalent to about 0.1 g
(potency), and dissolve in 0.1 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 100 mL each. Pipet 10 mL each
of these solutions, add 0.1 mol/L phosphate buffer solution,
pH 7.0 to make exactly 50 mL each, and use these solutions
as the sample solution and standard solution, respectively.
Perform the test with exactly 10 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the peak areas, A T and A s , of cefixime of
these solutions.
Amount \p.g (potency)] of C 16 H 15 N 5 07S2
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefixime Reference Stan-
dard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 125 mm in length, packed with octadecylsilanized silica
gel for liquid chromatography (4^m in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 25 mL of a solution of tetrabutylammo-
nium hydroxide TS (10 in 13) add water to make 1000 mL,
adjust to pH 6.5 with diluted phosphoric acid (1 in 10). To
300 mL of this solution add 100 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefixime is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefixime are not less than 4000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of peak areas of
cefixime is not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefmenoxime Hydrochloride
ir7// + ->Ai£i^
,0
C0 2 H
M'° v«'
H=N^ J II H H
N
J*
,-N
rHCI
C 16 H 17 N 9 5 S 3 .|hC1: 529.79
(6/?,7/?)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-
(methoxyimino)acetylamino]-3-(l -methyl- l//-tetrazol-5-
ylsulfanylmethyl)-8-oxo-5-thia-l-azabicyclo [4.2.0] oct-2-
ene-2-carboxylic acid hemihydrochloride [75738-58-8]
Cefmenoxime Hydrochloride contains not less than
890 fig (potency) and not more than 975 Lig (potency)
per mg, calculated on the dehydrated basis. The poten-
cy of Cefmenoxime Hydrochloride is expressed as mass
(potency) of cefmenoxime (C 16 H 17 N 9 5 S3: 511.56).
Description Cefmenoxime Hydrochloride occurs as white
to light orange-yellow crystals or crystalline powder.
It is freely soluble in formamide and in dimethylsulfoxide,
slightly soluble in methanol, very slightly soluble in water,
and practically insoluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cefmenoxime Hydrochloride in 0.1 mol/L phos-
phate buffer solution, pH 6.8 (3 in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cefmenoxime Hydrochloride Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefmenoxime Hydrochloride as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Cefmenoxime Hydrochloride Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
(3) Determine the spectrum of a solution of Cef-
menoxime Hydrochloride in deuterated dimethylsulfoxide
for nuclear magnetic resonance spectroscopy (1 in 10) as
directed under Nuclear Magnetic Resonance Spectroscopy
<2.21> ('H), using tetramethylsilane for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits two single signals, A and B, at around <53.9ppm,
and a single signal C at around S6.S ppm. The ratio of the
integrated intensity of each signal, A:B:C, is about 3:3:1.
(4) Dissolve 10 mg of Cefmenoxime Hydrochloride in
1 mL of diluted sodium carbonate TS (1 in 20), add 5 mL of
acetic acid (100) and 2 drops of silver nitrate TS: a white
precipitate is formed.
Optical rotation <2.49> [«]™: -27- -35° (1 g, 0.1 mol/L
phosphate buffer solution, pH 6.8, 100 mL, 100 mm).
444
Cefmenoxime Hydrochloride / Official Monographs
JP XV
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Cefmenoxime Hydrochloride in 150 mL of water is be-
tween 2.8 and 3.3.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 1.0 g of Cefmenoxime Hydrochloride in
10 mL of diluted sodium carbonate TS (1 in 4) is clear and
colorless to light yellow.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Cef-
menoxime Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefmenoxime Hydrochloride according to Method 4 and
adding 10 mL of dilute hydrochloric acid to the residue after
cooling, and perform the test (not more than 2 ppm).
(4) Related substances — Weigh accurately about 0.1 g
of Cefmenoxime Hydrochloride, dissolve in 20 mL of
0.1 mol/L phosphate buffer solution, pH 6.8, and add the
mobile phase to make exactly 100 mL. Pipet 4 mL of this
solution, add the mobile phase to make exactly 50 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of l-methyl-l//-tetrazol-5-thiol, and
dissolve in the mobile phase to make exactly 100 mL. Pipet
4 mL of this solution, add the mobile phase to make exactly
250 mL, and use this solution as the standard solution (1).
Weigh accurately about 0.1 g of Cefmenoxime Hydrochlo-
ride Reference Standard, dissolve in 20 mL of 0.1 mol/L
phosphate buffer solution, pH 6.8, and add the mobile phase
to make exactly 100 mL. Pipet 1 mL of this solution, add the
mobile phase to make exactly 250 mL, and use this solution
as the standard solution (2). Perform the test immediately af-
ter preparation of these solutions with exactly 10 /uL each of
the sample solution and standard solutions (1) and (2) as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area obtained
from the chromatograms of these solutions by the automatic
integration method, and calculate the amounts of 1-methyl-
l//-tetrazol-5-thiol and the total related substance by the fol-
lowing formula: the amount of l-methyl-l//-tetrazol-5-thiol
is not more than 1.0%, and the total related substance is not
more than 3.0%.
Amount (%) of 1 -methyl- l//-tetrazol-5-thiol
= (W S!i /W T ) x G4 Ta A4 Sa ) x 20
Amount (%) of total related substances
= UWsz/Wj) x 04 Ta A4 Sa ) x 20}
+ {(^sb/^i) + (S T A4 Sb ) x 5}
W Sr : Amount (g) of l-methyl-l//-tetrazol-5-thiol
W sb : Amount (g) of Cefmenoxime Hydrochloride Refer-
ence Standard
W T : Amount (g) of the sample
A Sa : Peak area of l-methyl-l//-tetrazol-5-thiol from the
standard solution (1)
A sb : Peak area of cefmenoxime from the standard solution
(2)
A Ja : Peak area of l-methyl-l//-tetrazol-5-thiol from the
sample solution
S T : Total area of the peaks other than 1-methyl-l//-
tetrazol-5-thiol and other than cefmenoxime from the
sample solution
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2.5 times as long as the
retention time of cefmenoxime.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution (1), add the mobile phase to make
exactly 100 mL. Confirm that the peak area of 1-methyl-l//-
tetrazol-5-thiol obtained from 10 fiL of this solution is
equivalent to 4.5 to 5.5% of that from the standard solution
(1). Then, measure exactly 2 mL of the standard solution (2),
add the mobile phase to make exactly 100 mL. Confirm that
the peak area of cefmenoxime obtained from 10 fiL of this
solution is equivalent to 1.5 to 2.5% of that from the stan-
dard solution (2).
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution (1) under the above operating
conditions, the relative standard deviation of the peak area of
l-methyl-l//-tetrazol-5-thiol is not more than 1.0%.
Water <2.48> Not more than 1.5% (1 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for Karl
Fisher method and methanol for Karl Fisher method (2:1)).
Assay Weigh accurately an amount of Cefmenoxime
Hydrochloride and Cefmenoxime Hydrochloride Reference
Standard, equivalent to about 50 mg (potency), dissolve each
in 10 mL of 0.1 mol/L phosphate buffer solution, pH 6.8,
and add the mobile phase to make exactly 50 mL. Pipet 4 mL
each of these solutions, add exactly 20 mL of the internal
standard solution and the mobile phase to make 50 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 10 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak area
of cefmenoxime to that of the internal standard.
Amount L«g (potency)] of cefmenoxime (C I6 H 17 N 9 5 S3)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Cefmenoxime Hydrochlo-
ride Reference Standard
Internal standard solution — A solution of phthalimide in
methanol (3 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ftm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (50:10:1).
Flow rate: Adjust the flow rate so that the retention time of
cefmenoxime is about 8 minutes.
System suitability —
System performance: When the procedure is run with
10,mL of the standard solution under the above operating
conditions, cefmenoxime and the internal standard are eluted
JPXV
Official Monographs / Cefmetazole Sodium 445
in this order with the resolution between these peaks being
not less than 2.3.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak areas of cefmenoxime to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Cefmetazole Sodium
3 "Y
o ft-
C 15 H 16 N 7 Na05S3: 493.52
Monosodium (6R,1R)-1-
{[(cyanomethylsulfanyl)acetyl]amino}-7-methoxy-3-
(1 -methyl- l//-tetrazol-5-ylsulfanylmethyl)-8-oxo-5-thia-
l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[56796-20-4]
Cefmetazole Sodium contains not less than 860 fig
(potency) and not more than 965 fig (potency) per mg,
calculated on the anhydrous basis. The potency of Cef-
metazole Sodium is expressed as mass (potency) of cef-
metazole (Ci 5 H 17 N 7 5 S3: 471.53).
Description Cefmetazole Sodium occurs as a white to light
yellowish white, powder or mass.
It is very soluble in water, freely soluble in methanol,
slightly soluble in ethanol (95), and very slightly soluble in
tetrahydrofuran.
Identification (1) Determine the absorption spectrum of a
solution of Cefmetazole Sodium (1 in 40,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Cef-
metazole Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefmetazole
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals, A, B and C, at around 5 3.6 ppm, at
around 5 4. 1 ppm and at around 5 5.2 ppm, respectively. The
ratio of integrated intensity of each signal, A:B:C, is about
3:3:1.
(4) Cefmetazole Sodium responds to the Qualitative
Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [«]£: +73- +85° (0.25 g, water,
25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefmetazole Sodium in 10 mL
of water: the pH of the solution is between 4.2 and 6.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefmetazole Sodium in 10 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Cefmeta-
zole Sodium according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefmetazole Sodium according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Cefmetazole
Sodium in 2 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 25 mL, and use this solution as the standard so-
lution (1). Separately, dissolve 0.10 g of 1-methyl-l//-
tetrazole-5-thiol in water to make exactly 100 mL, and use
this solution as the standard solution (2). Immediately per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 1 fiL each of the sample so-
lution and standard solutions (1) and (2) on a plate of silica
gel for thin-layer chromatography. Develop with a mixture of
1-buthanol, water and acetic acid (100) (4:1:1) to a distance
of about 12 cm, and air-dry the plate. Allow the plate to
stand in iodine vapor: the spot obtained from the sample so-
lution corresponding to the spot from the standard solution
(2) is not more intense than the spot from the standard solu-
tion (2), and the spots other than this spot and other than the
principal spot are not more intense than the spot from the
standard solution (1).
Water <2.48> Not more than 1.0% (1 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefmetazole Sodium
and Cefmetazole Reference Standard, equivalent to about 50
mg (potency), and dissolve each in the mobile phase to make
exactly 25 mL. Pipet 1 mL each of these solutions, add exact-
ly 10 mL of the internal standard solution, and use these solu-
tions as the sample solution and standard solution, respec-
tively. Perform the test with 10 fiL each of these solutions as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Q T and Q s ,
of the peak area of cefmetazole to that of the internal stan-
dard of each solution.
Amount [fig (potency)] of cefmetazole (C^HnNyOsSs)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Cefmetazole Reference
Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in the mobile phase (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 214 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
446
Cefminox Sodium Hydrate / Official Monographs
JP XV
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.75 g of ammonium dihydrogen-
phosphate in 700 mL of water, add 280 mL of methanol, 20
mL of tetrahydrofuran and 3.2 mL of 40% tetrabutylammo-
nium hydroxide TS, and adjust to pH 4.5 with phosphoric
acid.
Flow rate: Adjust the flow rate so that the retention time of
cefmetazole is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, cefmetazole and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 5 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefmetazole to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Hermetic containers.
Cefminox Sodium Hydrate
H0 2 C
H NH; o
HaC v N
CO.Na N * N
O H
C l6 H 20 N 7 NaO 7 S 3 .7H 2 O: 667.66
Monosodium (6i?,7/S)-7-{2-[(2/S)-2-amino-2-
carboxyethylsulfanyl]acetylamino}-7-methoxy-3-(l-methyl-
l//-tetrazol-5-ylsulfanylmethyl)-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate heptahydrate
[75498-96-3]
Cefminox Sodium Hydrate contains not less than
900 fig (potency) and not more than 970 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Cefminox Sodium Hydrate is expressed as mass
(potency) of cefminox (C l6 H 2l N 7 7 S 3 : 519.58).
Description Cefminox Sodium Hydrate occurs as a white to
light yellow crystalline powder.
It is freely soluble in water, sparingly soluble in methanol,
and practically insoluble in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Cefminox Sodium Hydrate (1 in 50,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Cefminox Sodium Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Cef-
minox Sodium Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Cefminox Sodium Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Cefminox
Sodium Hydrate in heavy water for nuclear magnetic
resonance spectroscopy (1 in 30) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using sodium
3-trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a multiple signal, A, at around 5 3.2 ppm, a single
signal, B, at around 5 3.5 ppm, a single signal, C, at around 8
4.0 ppm, and a single signal, D, at around 5 5.1 ppm. The
ratio of integrated intensity of each signal, A:B:C:D, is about
2:3:3:1.
(4) Cefminox Sodium Hydrate responds to the Qualita-
tive Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [a]™: +62- +72° (50 mg, water, 10
mL, 100 mm).
pH <2.54> Dissolve 0.70 g of Cefminox Sodium Hydrate in
10 mL of water: the pH of the solution is between 4.5 and
6.0.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefminox Sodium Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Cefminox Sodium Hydrate according to Method 3, and
perform the test (not more than 1 ppm).
Water <2.48> Not less than 18.0% and not more than
20.0% (0.1 g, volumetric titration, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Escherichia coli NIHJ
(ii) Culture medium — Use the medium iii in 3) under (1)
Agar media for seed and base layer. Adjust the pH of the
medium so that it will be 6.5 to 6.6 after sterilization.
(iii) Standard solution — Weigh accurately an amount of
Cefminox Sodium Reference Standard, equivalent to about
40 mg (potency), dissolve in 0.05 mol/L phosphate buffer so-
lution, pH 7.0 to make exactly 50 mL, and use this solution
as the standard stock solution. Keep the standard stock solu-
tion at 5°C or below and use within 7 days. Take exactly a
suitable amount of the standard stock solution before use,
add 0.05 mol/L phosphate buffer solution, pH 7.0 to make
solutions so that each mL contains 40 fig (potency) and 20 fig
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solution — Weigh accurately an amount of
Cefminox Sodium Hydrate equivalent to about 40 mg
(potency), dissolve in 0.05 mol/L phosphate buffer solution,
pH 7.0 to make exactly 50 mL. Take exactly a suitable
amount of this solution, add 0.05 mol/L phosphate buffer
solution, pH 7.0 to make solutions so that each mL contains
40 fig (potency) and 20 fig (potency), and use these solutions
as the high concentration sample solution and low concentra-
tion sample solution, respectively.
(v) Procedure — Incubate between 32°C and 35°C.
Containers and storage Containers — Hermetic containers.
JPXV
Official Monographs / Cefodizime Sodium
447
Cefodizime Sodium
■te^Tt^-JAi-Y- U^A
CH3
.-
C0 2 Na
?"\
Vn"
HjN-
trW
-COjNa
-CH 3
C 20 H 18 N 6 Na 2 O 7 S 4 : 628.63
Disodium (6i?,7/?)-7-[(Z)-2-(2-aminothiazol-4-yl)-
2-(methoxyimino)acetylamino]-3-[(5-carboxylatomethyl-
4-methylthiazol-2-yl)sulfanylmethyl]-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate [86329-79-5]
Cefodizime Sodium contains not less than 890 fig
(potency) per mg, calculated on the anhydrous basis
and corrected by the ethanol amount. The potency of
Cefodizime Sodium is expressed as mass (potency) of
cefodizime (C 2 oH2oN 6 7 S4: 584.67).
Description Cefodizime Sodium occurs as a white to light
yellowish white crystalline powder.
It is very soluble in water, and practically insoluble in
acetonitrile and in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Cefodizime Sodium (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of a solution of Cefodizime Sodium Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefodizime Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Cefodizime Sodium Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Cefodizime
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals, A, B and C, at around 32. 3 ppm, at
around <54.0 ppm, and at around <57.0 ppm. The ratio of the
integrated intensity of these signals, A:B:C, is about 3:3:1.
(4) Cefodizime Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: -56- -62° (0.2 g calculated
on the anhydrous basis and corrected by the ethanol amount,
water, 20 mL, 100 mm).
pH <2.54> Dissolve 1 .0 g of Cefodizime Sodium in 10 mL of
water: the pH of the solution is between 5.5 and 7.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefodizime Sodium in 10 mL of water: the solution is clear
and pale yellow to light yellow.
(2) Heavy metals <1.07>— Weigh 1.0 g of Cefodizime So-
dium in a crucible, cover loosely, and carbonize by gentle
heating. After cooling, add 2 mL of sulfuric acid, heat grad-
ually until the white fumes are no longer evolved, and ignite
between 500°C and 600°C. Proceed according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefodizime Sodium according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 30 mg of Cefodizime
Sodium in 10 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 5 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area by
the automatic integration method: the peak area other than
cefodizime from the sample solution is not more than the
peak area of cefodizime from the standard solution, and the
total area of the peaks other than cefodizime from the sample
solution is not more than 3 times the peak area of cefodizime
from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of cefodizime beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mobile phase to make
exactly 20 mL. Confirm that the peak area of cefodizime
obtained from 5 /uL of this solution is equivalent to 7 to 13%
of that from 5 /uL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
(5) Ethanol — Weigh accurately about 1 g of Cefodizime
Sodium, and dissolve in water to make exactly 10 mL. Pipet 2
mL of this solution, add exactly 2 mL of the internal stan-
dard solution, and use this solution as the sample solution.
Separately, weigh accurately about 2 g of ethanol for gas
chromatography, and add water to make exactly 1000 mL.
Pipet 2 mL of this solution, add exactly 2 mL of the internal
standard solution, and use this solution as the standard solu-
tion. Perform the test with 10 /iL each of the sample solution
and the standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, Q T and Q s , of the peak area of
ethanol to that of the internal standard: the amount of
ethanol is not more than 2.0%.
Amount (%) of ethanol = (W S /W T ) x (Q T /Q S )
W s : Amount (g) of ethanol for gas chromatography
W T : Amount (g) of the sample
Internal standard solution — A solution of 1-propanol (1 in
400).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
448
Cefoperazone Sodium / Official Monographs
JP XV
Column: A glass column 3.2 mm in inside diameter and
3 m in length, packed with tetrafluoroethylene polymer for
gas chromatography (180 - 250 /xm in particle diameter) coat-
ed in 15% with polyethylene glycol 20 M.
Column temperature: A constant temperature of about
100°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethanol is about 3 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, ethanol and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 2.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ethanol to that of the internal standard is not
more than 2.0%.
Water <2.48> Not more than 4.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefodizime Sodium
and Cefodizime Sodium Reference Standard, equivalent to
about 50 mg (potency), add exactly 10 mL of the internal
standard solution to dissolve, add water to make 100 mL,
and use these solutions as the sample solution and standard
solution. Perform the test with 10 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of
cefodizime to that of the internal standard.
Amount [Mg (potency)] of cefodizime (C 2 oH 2 oN 6 07S 4 )
= Ws x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Cefodizime Sodium Ref-
erence Standard
Internal standard solution — A solution of anhydrous caffeine
(3 in 400).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.80 g of potassium dihydrogen
phosphate and 0.20 g of anhydrous disodium hydrogen phos-
phate in a suitable amount of water, and add 80 mL of
acetonitrile and water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
cefodizime is about 5 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, cefodizime and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 6.
System repeatability: When the test is repeated 6 times with
10 /uh of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefodizime to that of the internal standard is not
more than 2.0%.
Containers and storage Containers — Tight containers.
Cefoperazone Sodium
■b7*^7'/>th U^A
CO?Na N sV
%_ n -4^ s ^n
C 25 H 26 N 9 Na0 8 S 2 : 667.65
Monosodium (6i?,7i?)-7-{(2.R)-2-[(4-ethyl-2,3-
dioxopiperazine-l-carbonyl)amino]-2-(4-
hydroxyphenyl)acetylamino } -3-(l -methyl- 1 //-tetrazol-
5-ylsulf anylmethyl)-8-oxo-5-thia- 1 -azabicyclo [4.2.0] oct-
2-ene-2-carboxylate [62893-20-3]
Cefoperazone Sodium contains not less than 871 Lig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefoperazone Sodium is expressed as
mass (potency) of cefoperazone ^st^NgO^:
645.67).
Description Cefoperazone Sodium occurs as a white to yel-
lowish white crystalline powder.
It is very soluble in water, soluble in methanol, and slightly
soluble in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Cefoperazone Sodium (1 in 50,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the spectrum of a solution of Cefopera-
zone Sodium in heavy water for nuclear magnetic resonance
spectroscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('Ft), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a triplet signal A at around 5 1.2 ppm, and double
signals, B and C, at around 5 6.8 ppm and at around 5 7.3
ppm. The ratio of integrated intensity of these signals,
A:B:C, is about 3:2:2.
(3) Cefoperazone Sodium responds to the Qualitative
Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [a] D °: - 15 - -25° (1 g, water, 100
mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefoperazone Sodium in 4 mL
of water: the pH of the solution is between 4.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefoperazone Sodium in 10 mL of water: the solution is clear
and pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cefoper-
JPXV
Official Monographs / Cefotaxime Sodium
449
azone Sodium according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefoperazone Sodium according to Method 4, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.1 g of Cefoperazone
Sodium in 100 mL of water, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add water to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with exactly 25 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the areas of each peak by the automatic
integration method. Calculate the percentages of each peak
area from the sample solution to 50 times of the peak area of
cefoperazone from the standard solution: the related sub-
stance I with the retention time of about 8 minutes is not
more than 5.0%, the related substance II with that of about
17 minutes is not more than 1.5%, and the total of all related
substances is not more than 7.0%. Use the peak areas of the
related substances I and II after multiplying by their relative
response factor, 0.90 and 0.75, respectively.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of cefoperazone beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 1 mL of the standard so-
lution, and add the mobile phase to make exactly 20 mL.
Confirm that the peak area of cefoperazone obtained from 25
/uL of this solution is equivalent to 3.5 to 6.5% of that of
cefoperazone obtained from 25 /xL of the standard solution.
System performance: When the procedure is run with 25
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of cefoperazone are not less than 5000 steps
and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
25 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefoperazone is not more than 2.0%.
Water <2.48> Not more than 1.0% (3 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefoperazone Sodi-
um equivalent to about 0.1 g (potency), and dissolve in water
to make exactly 100 mL. Pipet 5 mL of this solution, add ex-
actly 5 mL of the internal standard solution, and use this so-
lution as the sample solution. Separately, weigh accurately an
amount of Cefoperazone Reference Standard equivalent to
about 0.1 g (potency), dissolve in 5 mL of 0.1 mol/L phos-
phate buffer solution, pH 7.0, and add water to make exactly
100 mL. Pipet 5 mL of this solution, add exactly 5 mL of the
internal standard solution, and use this solution as the stan-
dard solution. Perform the test with 10 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
cefoperazone to that of the internal standard.
Amount \jxg (potency)] of cefoperazone ^sL^NgOA)
= W s x (Qj/Q s ) x 1000
W s : Amount [mg (potency)] of Cefoperazone Reference
Standard
Internal standard solution — A solution of acetanilide in a
mixture of water and acetonitrile (43:7) (3 in 8000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 57 mL of acetic acid (100) add 139 mL of
triethylamine and water to make 1000 mL. To 20 mL of this
solution add 835 mL of water, 140 mL of acetonitrile and 5
mL of dilute acetic acid.
Flow rate: Adjust the flow rate so that the retention time of
cefoperazone is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the internal standard and cefoperazone are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefoperazone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place.
Cefotaxime Sodium
CO;.Na
H;N"
(jV'ii
«
o
CH-,
C 16 H 16 N 5 Na0 7 S 2 : 477.45
Monosodium (6i?,7i?)-3-acetoxymethyl-7-[(Z)-2-(2-
aminothiazol-4-yl)-2-(methoxyimino)acetylamino]-8-oxo-5-
thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[64485-93-4]
Cefotaxime Sodium contains not less than 916 [xg
(potency) per mg, calculated on the dried basis. The
potency of Cefotaxime Sodium is expressed as mass
(potency) of cefotaxime (C 16 H 17 N 5 7 S 2 : 455.47).
Description Cefotaxime Sodium occurs as white to light
yellowish white crystalline powder.
It is freely soluble in water, sparingly soluble in methanol,
and very slightly soluble in ethanol (95).
450
Cefotaxime Sodium / Official Monographs
JP XV
Identification (1) Dissolve 2 mg of Cefotaxime Sodium in
0.01 mol/L hydrochloric acid TS to make 100 mL. Deter-
mine the absorption spectrum of this solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave-
lengths.
(2) Determine the infrared absorption spectrum of
Cefotaxime Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Cefotaxime
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 125) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits three single signals, A, B and C, at around 32. 1
ppm, at around <54.0 ppm and at around 51 . ppm. The ratio
of the integrated intensity of each signal, A:B:C, is about
3:3:1.
(4) Cefotaxime Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: +58 - +64° (0.25 g calculated
on the dried basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Cefotaxime Sodium in 10 mL of water is between 4.5 and
6.5.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 1.0 g of Cefotaxime Sodium in 10 mL
of water is clear and light yellow.
(2) Sulfate <1.14>— Dissolve 2.0 g of Cefotaxime Sodium
in 40 mL of water, add 2 mL of dilute hydrochloric acid and
water to make 50 mL, shake well, and filter. Discard first
10 mL of the filtrate, and to the subsequent 25 mL of the
filtrate add water to make 50 mL. Perform the test with this
solution as the test solution. Prepare the control solution as
follows: To 1.0 mL of 0.005 mol/L sulfuric acid VS add
1 mL of dilute hydrochloric acid and water to make 50 mL
(not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Cefotaxime Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefotaxime Sodium according to Method 3, and perform
the test (not more than 2 ppm).
(5) Related substances — Perform the test with 10 fiL of
the sample solution obtained in the Assay as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine each peak area obtained from the
chromatogram by the automatic integration method: the
each peak area other than cefotaxime is not more than 1.0%
and the total of these peak areas is not more than 3.0%.
Operating conditions —
Detector, column, column temperature, mobile phase A,
mobile phase B, flowing of the mobile phase, and flow rate:
Proceed as directed in the operating conditions in the Assay.
Time span of measurement: About 3.5 times as long as the
retention time of cefotaxime beginning after the solvent
peak.
System suitability —
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mobile phase A to make
exactly 100 mL. Pipet 2 mL of this solution, and add the
mobile phase A to make exactly 20 mL. Confirm that the
peak area of cefotaxime obtained from 10 /uL of this solution
is equivalent to 0.15 to 0.25% of that obtained from 10 /uL of
the standard solution.
Loss on drying <2.41> Not more than 3.0% (1 g, 105°C, 3
hours).
Assay Weigh accurately an amount of Cefotaxime Sodium
and Cefotaxime Reference Standard, equivalent to about
40 mg (potency), dissolve each in the mobile phase A to make
exactly 50 mL, and use these solutions as the sample solution
and standard solution. Perform the test with exactly 10 \xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the
following conditions, and determine the peak areas, A T and
A s , of cefotaxime of these solutions.
Amount [ug (potency)] of cefotaxime (C 16 H 17 N 5 7 S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefotaxime Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 235 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase A: To 0.05 mol/L disodium hydrogen
phosphate TS add phosphoric acid to adjust the pH to 6.25.
To 860 mL of this solution add 140 mL of methanol.
Mobile phase B: To 0.05 mol/L disodium hydrogen
phosphate TS add phosphoric acid to adjust the pH to 6.25.
To 600 mL of this solution add 400 mL of methanol.
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0- 7
100
7- 9
100^80
0^ 20
9-16
80
20
16-45
80^
20^100
45-50
100
Flow rate: Adjust the flow rate so that the retention time of
cefotaxime is about 14 minutes (about 1.3 mL/min).
System suitability —
System performance: To 1 mL of the standard solution
add 7.0 mL of water and 2.0 mL of methanol, mix, then add
JPXV
Official Monographs / Cefotetan 451
25 mg of sodium carbonate decahydrate, and shake. After
allowing to stand for 10 minutes, add 3 drops of acetic acid
(100) and 1 mL of the standard solution, and mix. When the
procedure is run with 10 ,mL of this solution under the above
operating conditions, desacetyl cefotaxime with the relative
retention time being about 0.3 to cefotaxime and cefotaxime
are eluted in this order with the resolution between these
peaks being not less than 20, and the symmetry factor of the
peak of cefotaxime is not more than 2.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefotaxime is not more than 2.0%.
Containers and storage Containers — Tight containers.
Cefotetan
t7*x5'>
COeH
° s Y it-
C 17 H 17 N 7 8 S 4 : 575.62
(6/?,7/?)-7-{[4-(Carbamoylcarboxymethylidene)-l,3-
dithietane-2-carbonyl] amino } -7-methoxy-3-(l -methyl- 1 H-
tetrazol-5-ylsulfanylmethyi)-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
[69712-56-7]
Cefotetan contains not less than 960 fig (potency)
and not more than 1010 fig (potency) per mg, calculat-
ed on the anhydrous basis. The potency of Cefotetan
is expressed as mass (potency) of cefotetan
(C 17 H 17 N 7 8 S 4 ).
Description Cefotetan occurs as white to light yellowish
white powder.
It is sparingly soluble in methanol, and slightly soluble in
water and in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Cefotetan in phosphate buffer solution for
antibiotics, pH 6.5 (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Cefotetan Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefotetan as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Cefotetan Reference Standard: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(3) Dissolve 50 mg of Cefotetan in 0.5 mL of a solution
of sodium hydrogen carbonate in heavy water for nuclear
magnetic resonance spectroscopy (1 in 25). Determine the
spectrum of this solution as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium
3-trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals, A, B, C and D, at around (53.6 ppm,
at around SA. ppm, at around 35. 1 ppm and at around 65.2
ppm, respectively. The ratio of the integrated intensity of
each signal, A:B:C:D, is about 3:3:1:1.
Optical rotation <2.49> [a]™: + 112 - + 124° (0.5 g calculated
on the anhydrous basis, a solution of sodium hydrogen
carbonate (1 in 200), 50 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefotetan in 10 mL of a solution of sodium hydrogen
carbonate (1 in 30): the solution is clear, and colorless or light
yellow.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Cefote-
tan according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Related substances — Weigh accurately about 0.1 g of
Cefotetan, dissolve in a suitable amount of methanol, add ex-
actly 2 mL of the internal standard solution and methanol to
make 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 3 mg of 1-methyl-l//-
tetrazole-5-thiol for liquid chromatography, previously dried
in a desiccator (in vacuum, silica gel) for 2 hours, and about 2
mg of Cefotetan Reference Standard, calculated on the anhy-
drous basis, dissolve in methanol to make exactly 20 mL.
Pipet 2 mL of this solution, add exactly 2 mL of the internal
standard solution and methanol to make 20 mL, and use this
solution as the standard solution. Perform the test with 5 fiL
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the ratios, Q Ta , Qib, Gtc, Qta,
<2 Te and Q Tt , of the peak areas of l-methyl-l//-tetrazole-5-
thiol, cefotetan lactone having the relative retention time of
about 0.5 with respect to cefotetan, Zl 2 -cefotetan having the
relative retention time of about 1.2 with respect to cefotetan,
isothiazole substance having the relative retention time of
about 1 .3 with respect to cefotetan, each of other related sub-
stances and the total of other related substances, to the peak
area of the internal standard, respectively, obtained from the
sample solution, and the ratios, Qsa and Q sb , of the peak
areas of 1 -methyl- l//-tetrazole-5-thiol and cefotetan, to the
peak area of the internal standard, respectively, obtained
from the standard solution. Calculate the amount of 1-
methyl-l//-tetrazole-5-thiol, cefotetan lactone, zl 2 -cefotetan,
isothiazole substance, each of other related substances and
the total of other related substances from the following equa-
tions: the amount of l-methyl-l//-tetrazole-5-thiol is not
more than 0.3%, cefotetan lactone is not more than 0.3%,
Zl 2 -cefotetan is not more than 0.5%, isothiazole substance is
not more than 0.5%, each of other related substances is not
more than 0.2% and the total of other related substances is
not more than 0.4%.
l-Methyl-l//-tetrazole-5-thiol(%)
= (WsJWj) x (e Ta /gsa) x (1/100)
Cefotetan lactone (%)
= (W sb /W T ) x (Q Tb /g sb ) x (1/100)
Zl 2 -Cefotetan (%)
= (W sb /W T ) x (Q Tc /e sb ) x (1/100)
Isothiazole substance (%)
452
Cefotetan / Official Monographs
JP XV
= (W sb /W T ) x (Q Td /Q sb ) x (1/100)
Each of other related substances (%)
= (W sb /Wj) x (e Te /e sb ) x (1/100)
Total of other related substances (%)
= (W sb /Wj) x (g Tf /Q sb ) x (1/100)
W Sa : Amount (mg) of l-methyl-l//-tetrazole-5-thiol
W sb : Amount (mg) of Cefotetan Reference Standard, cal-
culated on the anhydrous basis
W T : Amount (g) of the sample
Internal standard solution — A solution of anhydrous caffeine
in methanol (3 in 10,000).
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3.5 times as long as the
retention time of cefotetan.
System suitability —
Test for required detectability: Measure exactly 15 mL of
the standard solution, and add methanol to make exactly 100
mL. Confirm that the peak area of cefotetan obtained from
5 fxL of this solution is equivalent to 12 to 18% of that from
5 fiL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of cefotetan to that of the internal standard is not
more than 2.0%.
Water <2.48> Not more than 2.5% (1 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Isomer ratio Dissolve 10 mg of Cefotetan in 20 mL of
methanol, and use this solution as the sample solution. Per-
form the test with 5 /iL of the sample solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method. Calculate the amount of the adjacent
two peaks appeared at around the retention time of 40
minutes, one having shorter retention time is /-substance and
another having longer retention time is rf-substance, by the
area percentage method: the amount of /-substance is not less
than 35% and not more than 45%.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /um in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.1 mol/L phosphate buffer
solution, pH 7.0, water and a solution of tetrabutylammoni-
um hydrogensulfate in acetonitrile (1 in 150) (9:9:2).
Flow rate: Adjust the flow rate so that the retention time of
/-substance is about 40 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the sample solution under the above operating conditions,
/-substance and (/-substance are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: To exactly 1 mL of the sample solu-
tion add methanol to make exactly 10 mL. When the test is
repeated 6 times with 5 fiL of this solution under the above
operating conditions, the relative standard deviation of the
peak area of /-substance is not more than 5.0%.
Assay Weigh accurately an amount of Cefotetan and
Cefotetan Reference Standard, equivalent to about 50 mg
(potency), and dissolve each in phosphate buffer solution for
antibiotics, pH 6.5 to make exactly 50 mL. Pipet 15 mL each
of these solutions, add exactly 10 mL of the internal standard
solution and phosphate buffer solution for antibiotics, pH
6.5 to make 50 mL, and use these solutions as the sample so-
lution and standard solution. Perform the test with 5 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the ratios, Q T and Q s , of the
peak area of cefotetan to that of the internal standard.
Amount [fig (potency)] of C] 7 H 17 N 7 8 S 4
= Ws x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Cefotetan Reference Stan-
dard
Internal standard solution — A solution of anhydrous caffeine
(1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 11.53 g of phosphoric acid in
1000 mL of water. To 850 mL of this solution add 50 mL of
acetonitrile, 50 mL of acetic acid (100) and 50 mL of
methanol.
Flow rate: Adjust the flow rate so that the retention time of
cefotetan is about 17 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, the internal standard and cefotetan are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 5 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of cefotetan to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and at a temperature not exceed-
ing 5°C.
JPXV
Official Monographs / Cefotiam Hexetil Hydrochloride
453
Cefotiam Hexetil Hydrochloride
CH 3
rrYf
■2-1 CI
C 27 H 37 N 9 7 S 3 .2HC1 : 768.76
(li?S)-l-CycIohexyloxycarbonyloxyethyl (6R,1R)-1-
[2-(2-aminothiazol-4-yl)acetylamino]-3-[l-(2-
dimethylaminoethyl)-l//-tetrazol-5-ylsulfanylm ethyl] -
8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
dihydrochloride [95789-30-3]
Cefotiam Hexetil Hydrochloride contains not less
than 615 fig (potency) and not more than 690 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefotiam Hexetil Hydrochloride is ex-
pressed as mass (potency) of cefotiam (C18H23N9O4S3:
525.63).
Description Cefotiam Hexetil Hydrochloride occurs as a
white to light yellow powder.
It is very soluble in water, in methanol and in ethanol (95),
freely soluble in dimethylsulfoxide, and slightly soluble in
acetonitrile.
It dissolves in 0.1 mol/L hydrochloric acid TS.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Cefotiam Hexetil Hydrochloride in 0.1 mol/L
hydrochloric acid TS (3 in 125,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Cefotiam Hexetil Hydrochloride Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the spectrum of a solution of Cefotiam
Hexetil Hydrochloride in deuterated dimethylsulfoxide for
nuclear magnetic resonance spectroscopy (1 in 20) as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
('H), using tetramethylsilane for nuclear magnetic resonance
spectroscopy as an internal reference compound: it exhibits
two single signals, A and B, at around <52.8 ppm and at
around 36. 6 ppm, and a multiple signal, C, at around 36.9
ppm. The ratio of the integrated intensity of each signal,
A:B:C, is about 6:1:1.
(3) To a solution of Cefotiam Hexetil Hydrochloride
(1 in 200) add 2 mL of dilute nitric acid and 1 mL of silver ni-
trate TS, and mix: a white precipitate is formed.
Optical rotation <2.49> [a]™: +52- +60° (0.1 g calculated
on the anhydrous basis, 0.1 mol/L hydrochloric acid TS, 10
mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefotiam Hexetil Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Cefotiam Hexetil Hydrochloride according to Method 3,
and perform the test, using a solution of magnesium nitrate
hexahydrate in ethanol (95) (1 in 5) (not more than 1 ppm).
(3) Related substance 1 — Weigh accurately about 50 mg
of Cefotiam Hexetil Hydrochloride, and dissolve in a mixture
of diluted phosphoric acid (1 in 100) and acetonitrile (4:1) to
make exactly 50 mL. Pipet 10 mL of this solution, add a mix-
ture of diluted phosphoric acid (1 in 100) and acetonitrile
(4:1) to make exactly 25 mL, and use this solution as the sam-
ple solution. Separately, weigh accurately about 50 mg of
Cefotiam Hexetil Hydrochloride Reference Standard, and
dissolve in a mixture of diluted phosphoric acid (1 in 100) and
acetonitrile (4:1) to make exactly 50 mL. Pipet 1 mL of this
solution, add a mixture of diluted phosphoric acid (1 in 100)
and acetonitrile (4:1) to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with exactly
10 fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method. Calculate the amount of
the related substances by the following equation: the amount
of the related substance having the relative retention time of
about 1.2 to one of the peaks of cefotiam hexetil, which has
the larger retention time, is not more than 2.0%, and each
amount of the other related substances is not more than
0.5%. For this calculation, use the value of the peak area ob-
tained by the automatic integration method of the related
substance having the relative retention time of about 1 .2 to
one of the peaks of cefotiam hexetil, which has the larger
retention time, after multiplying by its relative response fac-
tor, 0.78.
Amount (%) of each related substance
= (W S /W T ) x (A T /A S ) x 5
W s : Amount (g) of Cefotiam Hexetil Hydrochloride
Reference Standard
W T : Amount (g) of sample
A s : Total of two peak areas of cefotiam hexetil from the
standard solution
A T : Each peak area of related substance from the sample
solution
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of diluted 0.2 mol/L potassi-
um dihydrogen phosphate TS (1 in 2), acetonitrile and acetic
acid (100) (72:28:1).
Mobile phase B: A mixture of acetonitrile, diluted
0.2 mol/L potassium dihydrogen phosphate TS (1 in 2) and
acetic acid (100) (60:40:1).
Flowing of the mobile phase: Adjust so that the mixing
rate of the mobile phase A and the mobile phase B is changed
lineally from 1:0 to 0:1 for 30 minutes.
454
Cefotiam Hexetil Hydrochloride / Official Monographs
JP XV
Flow rate: 0.7 mL per minute.
Time span of measurement: As long as about 3 times of the
retention time of one of the cefotiam hexetil peaks, which
appears first, beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add a mixture of diluted phos-
phoric acid (1 in 100) and acetonitrile (4:1) to make exactly 50
mL. Confirm that each area of the two peaks of cefotiam
hexetil obtained from 10 liL of this solution is equivalent to
1.6 to 2.4% of that from 10,mL of the standard solution.
System performance: When the procedure is run with
10 iiL of the standard solution under the above operating
conditions, the resolution between the two peaks of cefotiam
hexetil is not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the total of the
two peak areas of cefotiam hexetil is not more than 2.0%.
(4) Related substance 2 — Weigh accurately about 20 mg
of Cefotiam Hexetil Hydrochloride, dissolve in exactly 2 mL
of methanol, add a mixture of a solution of diammonium
hydrogen phosphate (79 in 20,000) and acetic acid (100)
(200:3) to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 25 mg of
Cefotiam Hydrochloride Reference Standard, and dissolve in
the mobile phase to make exactly 50 mL. Pipet 2 mL of this
solution, add the mobile phase to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with exactly 10,mL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method. Calculate the
amount of the related substances by the following equation:
the amounts of the related substances having the relative
retention time of about 0.1 and 0.9 to cefotiam are not more
than 1.0%, respectively, and each amount of the related sub-
stances other than the related substances having the relative
retention time of about 0.1 and 0.9 to cefotiam is not more
than 0.5%. For this calculation, use the value of the peak
area of the related substance having the relative retention
time of about 0.9 to cefotiam after multiplying by its sensitiv-
ity coefficient, 0.76.
Amount (%) of each related substance
= (W S /W T ) x (A T /A S ) x 4
W s : Amount (g) of Cefotiam Hydrochloride Reference
Standard
W T : Amount (g) of the sample
^4 S : Peak area of cefotiam from the standard solution
A T : Each peak area from the sample solution
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of diammonium
hydrogen phosphate (79 in 20,000), methanol and acetic acid
(100) (200:10:3).
Flow rate: Adjust the flow rate so that the retention time of
cefotiam is about 15 minutes.
Time span of measurement: As long as about 2 times of the
retention time of cefotiam beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 50 mL. Confirm that the peak area of cefotiam
obtained from 10 /uL of this solution is equivalent to 1.6 to
2.4% of that from 10 /uL of the standard solution.
System performance: To 1 mL of a solution of
acetaminophen in the mobile phase (1 in 50,000) add 3 mL of
the standard solution, and mix well. When the procedure is
run with 10 iiL of this solution under the above operating
conditions, acetaminophen and cefotiam are eluted in this
order with the resolution between these peaks being not less
than 4.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefotiam is not more than 2.0%.
(5) Total amount of related substances — The total of the
amount of related substances obtained in the Related sub-
stance 1 and the Related substance 2 is not more than 6.5%.
Water <2.48> Not more than 3.5% (0.1 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Isomer ratio Proceed the test with 20 juL of the sample
solution obtained in the Assay as directed under Liquid
Chromatography <2.01> according to the conditions directed
in the Assay, and determine the areas of the two peaks, A a
for the faster peak and A b for the later peak, closely appeared
each other at the retention time of around 10 minutes:
AJ(A^ + A b ) is not less than 0.45 and not more than 0.55.
Assay Weigh accurately an amount of Cefotiam Hexetil
Hydrochloride and Cefotiam Hexetil Hydrochloride Refer-
ence Standard, equivalent to about 30 mg (potency), and dis-
solve each in a mixture of diluted phosphoric acid (1 in 100)
and acetonitrile (4:1) to make exactly 50 mL. Measure exactly
5 mL each of these solutions, add exactly 5 mL of the internal
standard solution and a mixture of diluted phosphoric acid (1
in 100) and acetonitrile (4:1) to make exactly 50 mL, and use
these solutions as the sample solution and standard solution.
Perform the test with 20 liL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of cefotiam hexetil to
that of the internal standard. For this calculation, the total of
the areas of the two peaks appeared closely each other at the
retention time of around 10 minutes is used as the peak area
of cefotiam hexetil.
Amount [Mg (potency)] of cefotiam (Ci 8 H 2 3N 9 4 S3)
= Ws x (Qt/Qs) x 1000
W s : Amount [mg (potency)] of Cefotiam Hexetil Hydro-
chloride Reference Standard
Internal standard solution — A solution of benzoic acid in a
mixture of diluted phosphoric acid (1 in 100) and acetonitrile
(4:1) (7 in 10,000).
Operating conditions —
JPXV
Official Monographs / Cefotiam Hydrochloride
455
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted 0.2 mol/L potassium
dihydrogen phosphate TS (1 in 2), acetonitrile and acetic acid
(100) (72:28:1).
Flow rate: Adjust the flow rate so that the retention time of
the faster peak of cefotiam hexetil is about 9 minutes.
System suitability —
System performance: When the procedure is run with
20 fiL of the standard solution under the above operating
conditions, the internal standard and cefotiam hexetil are
eluted in this order with the resolution between the two peaks
of cefotiam hexetil being not less than 2.0.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefotiam hexetil to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefotiam Hydrochloride
t7 * ^7 A£££
CH 3
CO;H N-" N
H S N-
N
<* I
S
H H
N
• 2HCI
C 18 H 23 N 9 4 S3.2HC1: 598.55
(6/?,7i?)-7-[2-(2-Aminothiazol-4-yl)acetylamino]-3-
[ 1 -(2-dimethylaminoethyl)- 1 //-tetrazol-5-ylsulf anylmethyl] -
8-oxo-5-thia- 1 -azabicy clo [4 .2 .0] oct-2-ene-2-carboxylic acid
dihydrochloride [66309-69-1]
Cefotiam Hydrochloride contains not less than 810
fig (potency) and not more than 890 fig (potency) per
mg, calculated on the anhydrous basis. The potency of
Cefotiam Hydrochloride is expressed as mass (potency)
of cefotiam (C 18 H 23 N 9 4 S 3 : 525.63).
Description Cefotiam Hydrochloride occurs as white to
light yellow, crystals or crystalline powder.
It is freely soluble in water, in methanol and in formamide,
slightly soluble in ethanol (95), and practically insoluble in
acetonitrite.
Identification (1) Determine the absorption spectrum of a
solution of Cefotiam Hydrochloride (1 in 50,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Cefotiam Hydrochloride Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefotiam Hydrochloride as directed in the potassium chlo-
ride disk method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum or
the spectrum of Cefotiam Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Determine the spectrum of a solution of Cefotiam
Hydrochloride in heavy water for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using sodium
3-trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals at around <5 3.1 ppm and at around 3
6.7 ppm, respectively. The ratio of integrated intensity of
each signal is about 6:1.
(4) Dissolve 0.1 g of Cefotiam Hydrochloride in 5 mL of
dilute nitric acid, and immediately add 1 mL of silver nitrate
TS: a white precipitate is formed.
Optical rotation <2.49> [a]™: +60- +72° (1 g calculated
on the anhydrous bases, water, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefotiam Hydrochloride in 10
mL of water: the pH of the solution is between 1.2 and 1.7.
Purity (1) Clarity of solution — Dissolve 1.0 g of Cefotiam
Hydrochloride in 10 mL of water: the solution is clear, and
colorless to yellow.
(2) Heavy metals <1.07>— To 1.0 g of Cefotiam
Hydrochloride add 1 mL of sulfuric acid, and heat gently to
carbonize. After cooling, add 10 mL of a solution of mag-
nesium nitrate hexahydrate in ethanol (95) (1 in 10), fire the
ethanol to burn, then heat gradually to incinerate. If a car-
bonized residue still retains, moisten the residue with a little
amount of sulfuric acid, and ignite again to incinerate. After
cooling, add 2 mL of hydrochloric acid to the residue, heat
on a water bath to dissolve, then heat to dryness. Add 10 mL
of water, and heat to dissolve. After cooling, add ammonia
TS dropwise to adjust to pH 3 - 4, if necessary, filter, wash
the residue on the filter with 10 mL of water, transfer the
filtrate and washings into a Nessler tube, add water to make
50 mL, and use this solution as the test solution. Prepare the
control solution with 2.0 mL of Standard Lead Solution in
the same manner as for preparation of the test solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Incinerate 1.0 g of Cefotiam
Hydrochloride according to Method 4. After cooling, add 10
mL of dilute hydrochloric acid to the residue, heat to dissolve
on the water bath, and use this solution as the test solution.
Perform the test (not more than 2 ppm).
Water <2.48> Not more than 7.0% (0.25 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1) in-
stead of methanol for water determination).
Assay Weigh accurately an amount of Cefotiam
Hydrochloride and Cefotiam Hydrochloride Reference Stan-
dard, equivalent to about 0.1 g (potency), and dissolve each
in the mobile phase to make exactly 100 mL, and use these so-
lutions as the sample solution and standard solution, respec-
tively. Perform the test with exactly 10 fiL each of the sample
456
Cefotiam Hydrochloride for Injection / Official Monographs
JP XV
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the peak areas, A T and A s , of cefotiam
of these solutions.
Amount [fig (potency)] of cefotiam (C 18 H 2 3N 9 4 S3)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefotiam Hydrochloride
Reference Standard
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 125 mm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 800 mL of 0.05 mol/L disodium
hydrogenphosphate TS add 0.05 mol/L potassium di-
hydrogenphosphate TS to adjust the pH to 7.7. To 440 mL of
this solution add 60 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefotiam is about 14 minutes.
System suitability —
System performance: Dissolve 0.04 g of orcine in 10 mL of
the standard solution. When the procedure is run with 10,mL
of the standard solution under the above operating condi-
tions, orcine and cefotiam are eluted in this order with the
resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefotiam is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Cefotiam Hydrochloride for
Injection
Cefotiam Hydrochloride for Injection is a prepara-
tion for injection which is dissolved before use.
It contains not less than 90.0% and not more
than 110.0% of the labeled amount of cefotiam
(C 18 H 23 N 9 04S 3 : 525.63).
Method of Preparation Prepare as directed under Injec-
tion, with Cefotiam Hydrochloride.
Description Cefotiam Hydrochloride for Injection occurs
as a white to light yellow powder.
Identification (1) Determine the absorption spectrum of a
solution of Cefotiam Hydrochloride for Injection (1 in
50,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 257 nm and 261
nm.
(2) Dissolve 50 mg of Cefotiam Hydrochloride for Injec-
tion in 0.5 mL of heavy water for nuclear magnetic resonance
spectroscopy, and determine the spectrum of this solution as
directed under Nuclear Magnetic Resonance Spectroscopy
<2.21> ('H): it exhibits a single signal A between 6 2.7 ppm
and d 3.0 ppm, and a single signal B at around S 6.5 ppm.
The ratio of the integrated intensity of each signal, A:B, is
about 6:1.
pH <2.54> The pH of a solution prepared by dissolving an
amount of Cefotiam Hydrochloride for Injection, equivalent
to 0.5 g (potency) according to the labeled amount, in 5 mL
of water is between 5.7 and 7.2.
Purity Clarity and color of solution — Dissolve an amount
of Cefotiam Hydrochloride for Injection, equivalent to 1.0 g
(potency) of Cefotiam Hydrochloride according to the la-
beled potency, in 10 mL of water: the solution is clear, and
the absorbance of this solution, determined at 450 nm 10
minutes after dissolving as directed under Ultraviolet-visible
Spectrophotometry <2.24>, is not more than 0.20.
Loss on drying <2.41> Not more than 6.0% (0.5 g, in vacu-
um, 60°C, 3 hours).
Bacterial endotoxins <4.01> Less than 0.125 EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the contents of not less than 10
Cefotiam Hydrochloride for Injection. Weigh accurately an
amount of the content, equivalent to about 50 mg (potency)
of Cefotiam Hydrochloride according to the labeled amount,
dissolve in the mobile phase to make exactly 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg (potency) of Cefotiam Hydrochloride
Reference Standard, dissolve in the mobile phase to make ex-
actly 50 mL, and use this solution as the standard solution.
Proceed as directed in the Assay under Cefotiam Hydrochlo-
ride.
Amount [wg (potency)] of cefotiam (C 18 H 2 3N 9 4 S3)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefotiam Hydrochloride
Reference Standard
Operating conditions-
Proceed as directed in the Assay under Cefotiam
Hydrochloride.
System Suitability —
Proceed as directed in the Assay under Cefotiam
Hydrochloride.
Containers and storage Containers — Hermetic containers.
Polyethylene or polypropylene containers for aqueous injec-
tions may be used.
JPXV
Official Monographs / Cefozopran Hydrochloride
457
Cefozopran Hydrochloride
iz7*7''/ : 7>^^±g
CM,
co 2
S H H
'HCI
H ? N
C 19 H 17 N 9 5 S 2 .HC1: 551.99
(6jR,7jR)-7-[(Z)-2-(5-Amino-l,2,4-thiadiazol-3-yl)-2-
(methoxyimino)acetylamino]-3-(l//-
imidazo [1,2-6] pyridazin-4-ium- 1 -ylmethyl)-8-oxo-
5 -thia- 1 -azabicyclo [4 . 2 . 0] oct-2-ene-2-carb oxylate
monohydrochloride [113359-04-9, Cefozopran]
Cefozopran Hydrochloride contains not less than
860 fig (potency) and not more than 960 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Cefozopran Hydrochloride is expressed as mass
(potency) of cefozopran (C 19 H 17 N905S2: 515.53).
Description Cefozopran Hydrochloride occurs as a white to
pale yellow, crystals or crystalline powder.
It is freely soluble in dim ethylsulf oxide and in formamide,
slightly soluble in water, in methanol and in ethanol (95), and
practically insoluble in acetonitrile and diethyleter.
Identification (1) Dissolve 0.02 g of Cefozopran
Hydrochloride in 10 mL of water, add 1 mL of a solution of
hydroxylammonium chloride (1 in 10) and 2 mL of sodium
hydroxide TS, allow to stand for 5 minutes, then add 3 mL of
1 mol/L hydrochloric acid TS and 3 drops of iron (III) chlo-
ride TS, and mix: a red-purple color develops.
(2) Determine the absorption spectra of solutions of
Cefozopran Hydrochloride and Cefozopran Hydrochloride
Reference Standard in a mixture of sodium chloride TS and
methanol (3:2) (1 in 100,000) as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare these spectra:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the spectrum of a solution of Cefozopran
Hydrochloride in deuterated dimethylsulfoxide for nuclear
magnetic resonance spectroscopy (1 in 20) as directed under
Nuclear Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits a sin-
gle signal A at around 5 3.9 ppm, a double signal B at around
8 5.2 ppm, and a quartet signal C at around 5 8.0 ppm, and
the ratio of integrated intensity of each signal, A:B:C, is
about 3:1:1.
(4) Dissolve 0.01 g of Cefozopran Hydrochloride in 1 mL
of water and 2 mL of acetic acid (100), add 2 drops of silver
nitrate TS, and mix: a white turbidity is formed.
Absorbance <2.24> E\ v ° m (238 nm): 455 - 485 (50 mg calcu-
lated on the anhydrous basis, a mixture of sodium chloride
TS and methanol (3:2), 5000 mL).
Optical rotation <2.49> [a]™: -73 - -78° (0.1 g calculated
on the anhydrous basis, a mixture of sodium chloride TS and
methanol (3:2), 10 mL, 100 mm).
Purity (1) Clarity and color of solution - Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Cefozopran Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Arsenic — Being specified separately.
(4) Related substances - Being specified separately.
Water <2.48> Not more than 2.5% (0.5 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1) in-
stead of methanol for water determination).
Residue on ignition Being specified separately.
Bacterial endotoxins <4.01> Less than 0.05 EU/mg (poten-
cy).
Assay Weigh accurately an amount of Cefozopran
Hydrochloride and Cefozopran Hydrochloride Reference
Standard, equivalent to about 50 mg (potency), and dissolve
each in the mobile phase to make exactly 50 mL. Pipet 10 mL
each of these solutions, add exactly 10 mL of the internal
standard solution and the mobile phase to make 25 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 10 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Qt and Q s , of the peak area
of cefozopran to that of the internal standard of these solu-
tions.
Amount [fig (potency)] of cefozopran (C I9 H 17 N 9 5 S 2 )
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Cefozopran Hydrochloride
Reference Standard
Internal standard solution — A solution of 2,4-dihydroxyben-
zoic acid in the mobile phase (1 in 1250).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Mix 0.366 g of diethylamine with water to
make 1000 mL, and add 60 mL of acetonitrile and 5 mL of
acetic acid (100).
Flow rate: Adjust the flow rate so that the retention time of
cefozopran is about 9 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, cefozopran and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefozopran to that of the internal standard is not
458
Cefozopran Hydrochloride for Injection / Official Monographs
JP XV
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Cefozopran Hydrochloride for
Injection
Cefozopran Hydrochloride for Injection is a prepa-
ration for injection which is dissolved before use.
It contains not less than 90.0% and not more than
115.0% of the labeled potency of cefozopran
(Ci 9 H 17 N 9 5 S 2 : 515.53).
Method of Preparation Prepare as directed under the Injec-
tions, with Cefozopran Hydrochloride.
Description Cefozopran Hydrochloride for Injection oc-
curs as a white to light yellow, powder or masses.
Identification (1) Determine the absorption spectrum of a
solution of Cefozopran Hydrochloride for Injection (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 236 nm and 241
nm.
(2) To 50 mg of Cefozopran Hydrochloride for Injection
add 0.8 mL of deuterated dimethylsulfoxide for nuclear mag-
netic resonance spectroscopy, and filter after shaking, and
determine the spectrum of the filtrate as directed under
Nuclear Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits a sin-
gle signal A at around 5 3.9 ppm, a double signal B at around
8 5.0 ppm, and a quartet signal C at around 5 8.0 ppm. The
ratio of the integrated intensity of each signal, A:B:C, is
about 3:1:1.
pH <2.54> Dissolve an amount of Cefozopran Hydrochlo-
ride for Injection, equivalent to 0.5 g (potency) of
Cefozopran Hydrochloride according to the labeled amount,
in 5 mL of water: the pH of this solution is between 7.5 and
9.0.
Purity (1) Clarity and color of solution — Dissolve an
amount of Cefozopran Hydrochloride for Injection, equiva-
lent to 1 g (potency) of Cefozopran Hydrochloride according
to the labeled amount, in 10 mL of water: the solution is clear
and has no more color than Matching Fluid N.
(2) Related substances — Being specified separately.
Water <2.48> Not more than 2.5% (0.5 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for Karl
Fischer method and methanol for Karl Fischer method (2:1)
instead of methanol for Karl Fischer method).
Bacterial endotoxins <4.01> Less than 0.05 EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Cefozopran Hydrochloride for Injection. Weigh ac-
curately an amount of the contents, equivalent to about 0.5 g
(potency) of Cefozopran Hydrochloride according to the la-
beled amount, and add water to make exactly 100 mL. Pipet
2 mL of this solution, add exactly 10 mL of the internal stan-
dard solution, add the mobile phase to make 25 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately an amount of Cefozopran Hydrochloride Reference
Standard, equivalent to about 50 mg (potency), and dissolve
in the mobile phase to make exactly 50 mL. Pipet 10 mL of
this solution, add exactly 10 mL of the internal standard so-
lution, add the mobile phase to make 25 mL, and use this so-
lution as the standard solution. Proceed as directed in the
Assay under Cefozopran Hydrochloride.
Amount [mg (potency)] of cefozopran (Ci9H 17 N 9 5 S2)
= W s x (Qj/Qs) x 10
W s : Amount [mg (potency)] of Cefozopran Hydrochloride
Reference Standard
Internal standard solution — A solution 2,4-dihydroxybenzoic
acid in the mobile phase (1 in 1250).
Containers and storage Containers — Hermetic containers.
Polyethylene or polypropylene containers for aqueous injec-
tions may be used.
Storage — Light-resistant.
Cefpiramide Sodium
C 25 H 23 N 8 Na0 7 S 2 : 634.62
Monosodium (6RJR )-!-{{2R )-2-[(4-hydroxy-6-
methylpyridine-3-carbonyl)amino]-2-(4-
hydroxyphenyl)acetylamino } -3-(l -methyl- 1 //-tetrazol-
5-ylsulf anylmethyl)-8-oxo-5-thia- 1 -azabicyclo [4.2.0] oct-
2-ene-2-carboxylate [74849-93-7]
Cefpiramide Sodium contains not less than 900 fig
(potency) and not more than 990 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Cefpiramide Sodium is expressed as mass (potency) of
cefpiramide (C 25 H 24 N 8 7 S 2 : 612.64).
Description Cefpiramide Sodium occurs as white to yellow-
ish white powder.
It is very soluble in dimethylsulfoxide, freely soluble in
water, sparingly soluble in methanol, and slightly soluble in
ethanol (95).
Identification (1) Determine the absorption spectrum of a
JPXV
Official Monographs / Cefpiramide Sodium 459
solution of Cefpiramide Sodium in 0.05 mol/L phosphate
buffer solution, pH 7.0 (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the spectrum of a solution of Cefpiramide
Sodium in deuterated dimethylsulfoxide for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using tetra-
methylsilane for nuclear magnetic resonance spectroscopy as
an internal reference compound: it exhibits single signals, A,
B and C, at around <52.3 ppm, at around <53.9 ppm and at
around <58.2 ppm, respectively. The ratio of the integrated in-
tensity of each signal, A:B:C, is about 3:3:1.
(3) Cefpiramide Sodium responds to the Qualitative
Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: -33 - -40° (0.2 g calculated
on the anhydrous basis, 0.05 mol/L phosphate buffer solu-
tion, pH 7.0, 10 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 2.0 g
of Cefpiramide Sodium in 20 mL of water is between 5.5 and
8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefpiramide Sodium in 10 mL of 0.05 mol/L phosphate
buffer solution, pH 7.0: the solution is clear, and colorless or
light yellow.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Cef-
piramide Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Weigh accurately about 25 mg
of Cefpiramide Sodium, dissolve in 0.03 mol/L phosphate
buffer solution, pH 7.5 to make exactly 50 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 25 mg of l-methyl-l//-tetrazole-5-thiol for liquid chro-
matography, previously dried in a desiccator (in vacuum, sili-
ca gel) for 2 hours, and an amount of Cefpiramide Reference
Standard, equivalent to about 75 mg (potency), dissolve them
in 0.03 mol/L phosphate buffer solution, pH 7.5 to make ex-
actly 100 mL. Pipet 2 mL of this solution, add 0.03 mol/L
phosphate buffer solution, pH 7.5 to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 5 /xL of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method. Calculate the
amount of l-methyl-l//-tetrazole-5-thiol, each of the other
related substances and the total of the other related sub-
stances by the following equations: the amount of 1-methyl-
l//-tetrazole-5-thiol, each of the other related substances and
the total of the other related substances are not more than 1 .0
%, not more than 1.5% and not more than 4.0%, respec-
tively.
Amount (%) of l-methyl-l.ff-tetrazole-5-thiol (C 2 H 4 N 4 S)
= (W S JW T ) x fc4 T .A4sJ
Amount (%) of each of other related substances
= (W sb /W T ) x (A T JA sb )
W Sa : Amount (mg) of l-methyl-l//-tetrazole-5-thiol
W sb : Amount [mg (potency)] of Cefpiramide Reference
Standard
W T : Amount (mg) of the sample
^4 Sa : Peak area of 1 -methyl- l//-tetrazole-5-thiol from the
standard solution
A sh : Peak area of cefpiramide from the standard solution
;4 Ta : Peak area of 1 -methyl- l//-tetrazole-5-thiol from the
sample solution
A Tc : Area of each peak other than l-methyl-l//-tetrazole-
5-thiol and cefpiramide from the sample solution
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octylsilanized silica gel for
liquid chromatography (lO^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.03 mol/L phosphate buffer
solution, pH 7.5 and methanol (3:1).
Flow rate: Adjust the flow rate so that the retention time of
cefpiramide is about 11 minutes.
Time span of measurement: About 2 times as long as the
retention time of cefpiramide.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add 0.03 mol/L phosphate buffer
solution, pH 7.5 to make exactly 50 mL. Confirm that the
peak area of l-methyl-l//-tetrazole-5-thiol obtained from
5 /xL of this solution is equivalent to 8 to 12% of that from
5 fXL of the standard solution.
System performance: Dissolve 25 mg of Cefpiramide
Reference Standard and 7 mg of cinnamic acid in the mobile
phase to make 50 mL. When the procedure is run with 5 ftL
of this solution under the above operating conditions, cin-
namic acid and cefpiramide are eluted in this order with the
resolution between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
5 ,uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of 1-
methyl-l//-tetrazole-5-thiol is not more than 2.0%.
Water <2.48> Not more than 7.0% (0.35 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefpiramide Sodium
and Cefpiramide Reference Standard, equivalent to about 50
mg (potency), add exactly 5 mL of the internal standard solu-
tion to dissolve, then add the mobile phase to make 100 mL,
and use these solutions as the sample solution and standard
solution. Perform the test with 5 [iL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of cef-
piramide to that of the internal standard.
Amount [Mg (potency)] of cefpiramide (C 2 5H24N 8 7 S2)
= W s x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Cefpiramide Reference
Standard
Internal standard solution — A solution of 4-dimethylamino-
antipyrine (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
460
Cefpirome Sulfate / Official Monographs
JP XV
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octylsilanized silica gel for
liquid chromatography (10 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.01 mol/L phosphate
buffer solution, pH 6.8, acetonitrile, methanol and tetra-
hydrofuran (22:1:1:1).
Flow rate: Adjust the flow rate so that the retention time of
cefpiramide is about 7 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, cefpiramide and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of cefpiramide to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and at a temperature not exceed-
ing 5°C.
Cefpirome Sulfate
-tz7fc?nAfilEK£
H ? N
•H a SOi
C 22 H 22 N 6 5 S 2 .H 2 S0 4 : 612.66
(6i?,7i?)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-
(methoxyimino)acetylamino]-3-(6,7-dihydro-5//-
cyclopenta[d]pyridinium-l-ylmethyl)-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate monosulfate
[98753-19-6]
Cefpirome Sulfate contains not less than 760 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefpirome Sulfate is expressed as mass
(potency) of cefpirome (C 2 2H22N 6 5 S2: 514.58).
Description Cefpirome Sulfate occurs as a white to pale yel-
lowish white crystalline powder, and has a slight, characteris-
tic ordor.
It is soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
It is hygroscopic.
Identification (1) Dissolve 10 mg of Cefpirome Sulfate in
2 mL of water, add 3 mL of hydroxylammonium
hydrochloride-ethanol TS, allow to stand for 5 minutes, add
1 mL of acidic ammonium iron (III) sulfate TS, and shake: a
red-brown color develops.
(2) Dissolve 1 mg of Cefpirome Sulfate in 4 mL of water,
add 1 mL of dilute hydrochloric acid while cooling in ice, add
1 mL of a freshly prepared solution of sodium nitrite (1 in
100), and allow to stand for 2 minutes. Add 1 mL of ammo-
nium amidosulfuric acid TS while cooling in ice bath, allow
to stand for 1 minute, and add 1 mL of a solution of N-\-
naphthylethylene dihydrochloride (1 in 1000): a purple color
develops.
(3) Take 5 mg of Cefpirome Sulfate, dissolve in 1 mL of
ethanol (95) and 1 mL of water, add 100 mg of l-chloro-2,4-
dinitrobenzene, and heat on a water bath for 5 minutes. After
cooling, add 2 or 3 drops of a solution of sodium hydroxide
(1 in 10) and 3 mL of ethanol (95): a red-brown color de-
velops.
(4) Determine the absorption spectra of solutions of Cef-
pirome Sulfate and Cefpirome Sulfate Reference Standard in
0.01 mol/L hydrochloric acid TS (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectra: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(5) Determine the spectrum of a solution of Cefpirome
Sulfate in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 25) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a single signal A at around <5 4.1 ppm, a double sig-
nal B at around d 5.9 ppm, a single signal C at around 5 7.1
ppm, and a multiple signal D at around <5 7.8 ppm. The ratio
of integrated intensity of each signal, A:B:C:D, is about
3:1:1:1.
(6) A solution of Cefpirome Sulfate (1 in 250) responds
to the Qualitative Tests <1.09> (1) for sulfate salt.
Absorbance <2.24> E\ 0/ ° m (270 nm): 405 - 435 (50 mg calcu-
lated on the anhydrous basis, 0.01 mol/L hydrochloric acid
TS, 2500 mL).
Optical rotation <2.49> [ a ]g>: -27 - -33° (50 mg calculat-
ed on the anhydrous basis, a solution prepared by addition of
water to 25 mL of actonitrile to make 50 mL, 20 mL, 100
mm).
pH <2.54> Dissolve 0.1 g of Cefpirome Sulfate in 10 mL of
water: the pH of the solution is between 1.6 and 2.6.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cefpi-
rome Sulfate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic — Being specified separately.
(4) Related substances — Being specified separately.
(5) Residual solvents — Being specified separately.
Water <2.48> Not more than 2.5% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition Being specified separately.
Bacterial endotoxins <4.01> Less than 0.10 EU/mg (poten-
cy).
Assay Weigh accurately an amount of Cefpirome Sulfate
and Cefpirome Sulfate Reference Standard, equivalent to
about 50 mg (potency), dissolve each in water to make exactly
100 mL. Pipet 5 mL of these solutions, add each in water to
make exactly 20 mL, and use these solutions as the sample so-
lution and standard solution. Perform the test with exactly 20
JPXV
Official Monographs / Cefpodoxime Proxetil
461
fiL of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the peak areas, A T and A s ,
of cefpirome of each solution.
Amount [fig (potency)] of cefpirome (C22H 2 2N 6 5 S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefpirome Sulfate Refer-
ence Standard
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 270 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 3.45 g of ammonium dihydrogen-
phosphatein 1000 mL of water, and adjust the pH to 3.3 with
phosphoric acid. To 800 mL of this solution add 100 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefpirome is about 7.5 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates of the peak of cefpi-
rome is not less than 3600.
System repeatability: When the test is repeated 5 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefpirome is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — At a temperature between 2 and 8°C.
Cefpodoxime Proxetil
*VYY
Chj
CH S
CK^.0
CHj
H H and epimer al C*
C2iH 27 N 5 9 S2: 557.60
(lflSH-Kl-MethylethyfJcarbonyloxylethyl
(6/?,7/?)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetylamino]-3-methoxymethyl-8-oxo-5-
thia- 1 -azabicyclo [4 . 2 . 0] oct-2-ene-2-carboxy late
[87239-81-4]
Cefpodoxime Proxetil contains not less than 706 fig
(potency) and not more than 774 fig (potency) per mg,
calculated on the anhydrous basis. The potency of Cef-
podoxime Proxetil is expressed as mass (potency) of
cefpodoxime (C 15 H 17 N 5 6 S 2 : 427.46).
Description Cefpodoxime Proxetil occurs as a white to light
brownish white powder.
It is very soluble in acetonitrile, in methanol and in chlo-
roform, freely soluble in ethanol (99.5), and very slightly
soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Cefpodoxime Proxetil in acetonitrile (3 in
200,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Cefpodoxime
Proxetil Reference Standard prepared in the same manner as
the sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Cef-
podoxime Proxetil as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cefpodoxime Proxetil Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Cefpodoxime
Proxetil in deuterochloroform for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits dou-
ble signals, A and B, at around <51.3 ppm and at around <5 1 .6
ppm, and single signals, C and D, at around <53.3 ppm and at
around <54.0ppm. The ratio of the integrated intensity of
these signals, A:B:C:D, is about 2:1:1:1.
Optical rotation <2.49> [a]^: +24.0 - +31.4° (0.1 g calculat-
ed on the anhydrous basis, acetonitrile, 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefpodoxime Proxetil according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 50 mg of Cefpodoxime
Proxetil in 50 mL of a mixture of water, acetonitrile and
acetic acid (100) (99:99:2), and use this solution as the sample
solution. Perform the test with 20 fiL of the sample solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. If necessary, perform the test in
the same manner with 20 fiL of the mixture of water, acetoni-
trile and acetic acid (100) (99:99:2) to compensate for the
base line. Determine each peak area by the automatic
integration method, and calculate the amounts of them by
the area percentage method: the peak, having the relative
retention time of about 0.8 with respect to the isomer B of
cefpodoxime proxetil, is not more than 2.0%, the peak other
than cefpodoxime proxetil is not more than 1.0%, and the
sum of the peaks other than cefpodoxime proxetil is not more
than 6.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
22°C.
Mobile phase A: A mixture of water, methanol and a solu-
462
Cefroxadine Hydrate / Official Monographs
JP XV
tion of formic acid (1 in 50) (11:8:1).
Mobile phase B: A mixture of methanol and a solution of
formic acid (1 in 50) (19:1).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-65
95
5
65 - 145
95^ 15
5^85
145- 155
15
85
Flow rate: Adjust the flow rate so that the retention time of
the isomer B of cefpodoxime proxetil is about 60 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of the isomer B of cefpodoxime proxetil begin-
ning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the sample solution, add the mixture of water, acetonitrile
and acetic acid (100) (99:99:2) to make exactly 200 mL, and
use this solution as the solution for required detectability test.
Pipet 2 mL of the solution for required detectability test, and
add the mixture of water, acetonitrile and acetic acid (100)
(99:99:2) to make exactly 100 mL. Confirm that the peak
areas of the isomer A and the isomer B of cefpodoxime prox-
etil obtained from 20 fiL of this solution are equivalent to 1 .4
to 2.6% of them from 20 fiL of the solution for required
detectability test, respectively.
System performance: Dissolve 1 mg of Cefpodoxime Prox-
etil in 100 mL of the mixture of water, acetonitrile and acetic
acid (100) (99:99:2). When the procedure is run with 20 fiL of
this solution under the above operating conditions, the
isomer A and the isomer B of cefpodoxime proxetil are eluted
in this order with the resolution between these peaks being
not less than 6.
System repeatability: Dissolve 1 mg of Cefpodoxime Prox-
etil in 100 mL of the mixture of water, acetonitrile and acetic
acid (100) (99:99:2). When the test is repeated 5 times with 20
fiL of this solution under the above operating conditions, the
relative standard deviations of the peak areas of the isomer A
and the isomer B are not more than 2.0%, respectively.
Water <2.48> Not more than 2.5% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Isomer ratio Perform the test with 5 fiL of the sample solu-
tion obtained in the Assay as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A^ and A b , of the two isomers
of cefpodoxime proxetil, having the smaller and larger reten-
tion times, respectively, by the automatic integration
method: A b /(A„ + A b ) is between 0.50 and 0.60.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
System suitability —
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Assay Weigh accurately an amount of Cefpodoxime Prox-
etil and Cefpodoxime Proxetil Reference Standard, equiva-
lent to about 60 mg (potency), dissolve in 80 mL of acetoni-
trile, add exactly 4 mL of the internal standard solution, add
acetonitrile to make 100 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
5 fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Q T1 , Q sl ,
Qt2 and Q S 2, of the areas of the two peaks of the isomers of
cefpodoxime proxetil to the peak area of the internal stan-
dard.
Amount [fig (potency)] of cefpodoxime (C 15 H 17 N50 6 S2)
= W s x {(Q T1 + Q T2 )/(Q sl + Q S2 )} x 1000
W s : Amount [mg (potency)] of Cefpodoxime Proxetil
Reference Standard
Internal standard solution — Dissolve 0.3 g of ethyl para-
hydroxybenzoate in a solution of citric acid in acetonitrile (1
in 2000) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and methanol (11:9),
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 11 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, the internal standard, the isomer A and the isomer B
are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 5 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of the isomer B of cefpodoxime proxetil to that of
the internal standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefroxadine Hydrate
o
H L .N H
C 16 H 19 N 3 5 S.2H 2 0: 401.43
(6RJR )-l-[(2R )-2-Amino-2-cyclohexa-l ,4-
dienylacetylamino]-3-methoxy-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid dihydrate
[51762-05-1, anhydride]
Cefroxadine Hydrate contains not less than 930 fig
JPXV
Official Monographs / Cefroxadine Hydrate
463
(potency) and not more than 1020 ,ug (potency) per mg,
calculated on the dehydrated basis. The potency of
Cefroxadine Hydrate is expressed as mass (potency) of
cefroxadine (C 16 H 19 N 3 5 S: 365.40).
Description Cefroxadine Hydrate occurs as pale yellowish
white to light yellow, crystalline particles or powder.
It is very soluble in formic acid, slightly soluble in water
and in methanol, and very slightly soluble in acetonitrile and
in ethanol (95).
It dissolves in 0.001 mol/L hydrochloric acid TS and in
dilute acetic acid.
Identification (1) Determine the absorption spectrum of a
solution of Cefroxadine Hydrate in 0.001 mol/L hydrochlor-
ic acid TS (1 in 50,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of
Cefroxadine Reference Standard prepared in the same man-
ner as the sample solution: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(2) Determine the spectrum of a solution of Cefroxadine
Hydrate in deuterated formic acid for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits three
sharp single signals, A, B and C, at around 32. 8 ppm, at
around <54.1 ppm and at around <56.3 ppm. The ratio of the
integrated intensity of each signal, A:B:C, is about 4:3:1.
Optical rotation <2.49> [ a ]™: +95 - + 108° (0.1 g calculated
on the dehydrated basis, diluted acetic acid (100) (3 in 25),
100 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Weigh 1.0 g of Cefroxa-
dine Hydrate in a porcelain crucible, add 10 mL of a solution
of magnesium nitrate hexahydrate in ethanol (95) (1 in 10),
mix, burn the ethanol, and carbonize by gently heating. After
cooling, add 2 mL of nitric acid, heat carefully, and inciner-
ate by ignition at 500 - 600°C. If a carbonized substance is
still remained, moisten it with a small amount of nitric acid,
and incinerate again by ignition. After cooling, add 6 mL of
hydrochloric acid, and evaporate on a water bath to dryness.
Moisten the residue with 3 drops of hydrochloric acid, and
add 10 mL of hot water to dissolve the residue by heating on
a water bath. After cooling, adjust the pH between 3 and 4
with ammonia TS, add 2 mL of dilute acetic acid, filter if
necessary, transfer to a Nessler tube, wash the crucible with
10 mL of water, and add the washing and water to the tube to
make 50 mL. Perform the test with this solution. Prepare the
control solution as follows: Put 2.0 mL of Standard Lead So-
lution and 10 mL of a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 10) in a porcelain crucible, and pro-
ceed as directed for the preparation of the test solution (not
more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefroxadine Hydrate according to Method 4, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 10 mg of Cefroxadine
Hydrate in 100 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 40 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area
obtained from the chromatograms of these solutions by the
automatic integration method: the areas of the peaks ap-
peared at the relative retention times of 0.07, 0.6 and 0.8
against the peak of cefroxadine from the sample solution are
not more than 2 times, 4 times and 1 time of the peak area of
cefroxadine from the standard solution, respectively, and any
peak area other than cefroxadine and other than the peaks
mentioned above from the sample solution is not more than
1/2 of the peak area of cefroxadine from the standard solu-
tion, and the total area of the peaks other than cefroxadine
from the sample solution is not more than 6 times of the peak
area of cefroxadine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.4 g of sodium perchlorate in 1000
mL of a mixture of water and acetonitrile (489:11).
Flow rate: Adjust the flow rate so that the retention time of
cefroxadine is about 20 minutes.
Time span of measurement: About 2 times as long as the
retention time of cefroxadine.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mobile phase to make
exactly 20 mL. Confirm that the peak area of cefroxadine ob-
tained from 40 /xh of this solution is equivalent to 7 to 13%
of that obtained from 40 jXL of the standard solution.
System performance: Dissolve 3 mg of Cefroxadine Hy-
drate and 15 mg of orcin in 100 mL of the mobile phase.
When the procedure is run with 40 /iL of this solution under
the above operating conditions, orcin and cefroxadine are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
40 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefroxadine is not more than 2.0%.
Water <2.48> Not less than 8.5% and not more than 12.0%
(0.1 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Cefroxadine Hy-
drate and Cefroxadine Reference Standard, equivalent to
about 50 mg (potency), dissolve each in a suitable amount of
a mixture of dilute acetic acid and phosphoric acid (500:1),
add exactly 5 mL of the internal standard solution and a mix-
ture of dilute acetic acid and phosphoric acid (500:1) to make
200 mL, and use these solutions as the sample solution and
standard solution. Perform the test with 10 /xh each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak area
of cefroxadine to that of the internal standard.
Amount [ug (potency)] of cefroxadine (C 16 H 19 N 3 05S)
= W s x (Q T /Q S ) x 1000
464
Cefsulodin Sodium / Official Monographs
JP XV
W s : Amount [mg (potency)] of Cefroxadine Reference
Standard
Internal standard solution— Dissolve 1.6 g of vanillin in 5 mL
of methanol, and add a mixture of dilute acetic acid and
phosphoric acid (500:1) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of ammonium sul-
fate (1 in 50) and acetonitrile (97:3).
Flow rate: Adjust the flow rate so that the retention time of
cefroxadine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, cefroxadine and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak areas of cefroxadine to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Cefsulodin Sodium
t7XPy>th U^A
co,-
H H
C 22 H 19 N 4 Na0 8 S 2 : 554.53
Monosodium (6/?,7/?)-3-(4-carbamoylpyridinium-l-
ylmethyl)-8-oxo-7-[(2/?)-2-phenyl-2-sulfonatoacetylamino]-
5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[52152-93-9]
Cefsulodin Sodium contains not less than 900 tig
(potency) and not more than 970 Lig (potency) per mg,
calculated on the anhydrous basis. The potency of Cef-
sulodin Sodium is expressed as mass (potency) of cef-
sulodin (C 22 H 20 N 4 O 8 S 2 : 532.55).
Description Cefsulodin Sodium occurs as white to light yel-
low, crystals or crystalline powder.
It is freely soluble in water and in formamide, slightly solu-
ble in methanol, and very slightly soluble in ethanol (95).
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Cefsulodin Sodium (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cefsulodin Sodium Reference Standard pre-
pared in the same manner as sample solution: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Cef-
sulodin Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cefsulodin Sodium Reference Standard: both spec-
tra exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefsulodin
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a multiple signal A between 5 7.3 ppm and 6 1.1
ppm, and double signals, B and C, at around 5 8.4 ppm and
at around 5 9.1 ppm, respectively. The ratio of integrated in-
tensity of these signals, A:B:C, is about 5:2:2.
(4) Cefsulodin Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [«]£>: + 16.5 - +20.0° (0.1 g calcu-
lated on the anhydrous basis, water, 10 mL, 100 mm).
pH <2.54> Dissolve 1 .0 g of Cefsulodin Sodium in 10 mL of
water: the pH of the solution is not less than 3.3 and not
more than 4.8.
Purity (1) Clarity of solution — Dissolve 1.0 g of Cef-
sulodin Sodium in 10 mL of water: the solution is clear.
(2) Heavy metals <1. 07>— To 1.0 g of Cefsulodin Sodium
add 10 mL of a solution of magnesium nitrate hexahydrate in
ethanol (95) (1 in 5), mix, fire the ethanol to burn, then heat
gradually to carbonize. After cooling, add 2 mL of nitric
acid, heat carefully, then heat at 500 - 600°C to incinerate. If
a carbonized residue still retains, add a little amount of nitric
acid, and heat again to incinerate. After cooling, add 6 mL of
hydrochloric acid to the residue, heat to dryness on a water
bath, then moisten the residue with 3 drops of hydrochloric
acid, add 10 mL of hot water, and heat on a water bath to
dissolve. Add ammonia TS dropwise to adjust to pH 3-4,
and add 2 mL of dilute acetic acid. If necessary, filter, wash
the crucible and residue on the filter with 10 mL of water,
transfer the filtrate and washings into a Nessler tube, add
water to make 50 mL, and use this solution as the test solu-
tion. Prepare the control solution as follows: To 2.0 mL of
Standard Lead Solution add 10 mL of a solution of magnesi-
um nitrate hexahydrate in ethanol (95) (1 in 5), fire the
ethanol to burn. After cooling, add 2 mL of nitric acid, heat
carefully, then heat at 500 - 600 C C. After cooling, add 6 mL
of hydrochloric acid, then proceed in the same manner as for
the preparation of the test solution (not more than 20 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Cefsulodin Sodium according to Method 3, using a solu-
tion of magnesium nitrate hexahydrate in ethanol (95) (1 in 5)
and 15 mL of dilute hydrochloric acid instead of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 50) and 3
mL of hydrochloric acid, and perform the test (not more than
2 ppm).
JPXV
Official Monographs / Cefsulodin Sodium
465
(4) Related substances — Weigh accurately about 0.10 g
of Cefsulodin Sodium, dissolve in water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 20 mg of isonicotinic acid amide and
about 20 mg of Cefsulodin Sodium Reference Standard
(separately determine the water <2.48> in the same manner as
Cefsulodin Sodium), and dissolve in water to make exactly
100 mL. Pipet 10 mL of this solution, add water to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the areas of each peak by the automatic in-
tegration method. Calculate the amount of the related sub-
stances by the following formula: the amount of isonicotinic
acid amide is not more than 1.0%, and the total of other
related substances is not more than 1.2%.
Amount (%) of isonicotinic acid amide
= (A/Bj) x (W x /Wi) x 5
Total amount (%) of the other related substances
= (B/B s ) x (W S /W T ) x 5
A: Peak area of isonicotinic acid amide from the sample
solution
B: Total peak area other than cefsulodin and other than
isonicotinic acid amide from the sample solution
B\. Peak area of isonicotinic acid amide from the standard
solution
B s : Peak area of cefsulodin from the standard solution
W T : Amount (g) of the sample
W s : Amount (g) of Cefsulodin Sodium Reference Stan-
dard
W t : Amount (g) of isonicotinic acid amide
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of a solution of ammonium
sulfate (1 in 100) and acetonitrile (97:3).
Mobile phase B: A mixture of a solution of ammonium sul-
fate (1 in 100) and acetonitrile (92:8).
Flowing of the mobile phase: Change the mobile phase A
to B at 14 minutes after the injection of sample.
Flow rate: Adjust the flow rate so that the retention time of
cefsulodin is about 9 minutes.
Time span of measurement: About 4 times as long as the
retention time of cefsulodin.
System suitability —
Test for required detection: Pipet 1 mL of the standard so-
lution, add water to make exactly 10 mL. Confirm that the
peak areas of isonicotinic acid amide and cefsulodin obtained
from 10 /uL of this solution are equivalent to 7 to 13% of
those of isonicotinic acid amide and cefsulodin obtained
from 10 /xL of the standard solution.
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, isonicotinic acid amide and cefsulodin are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 5 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefsulodin is not more than 1.0%.
Water <2.48> Not more than 5.0% (1 g, volumetric titra-
tion, direct titration, avoiding moisture absorption of the
sample, using a mixture of formamide for water determina-
tion and methanol for water determination (2:1) instead of
methanol for water determination).
Assay Weigh accurately an amount of Cefsulodin Sodium
and Cefsulodin Sodium Reference Standard, equivalent to
about 0.1 g (potency), dissolve each in water to make exactly
50 mL, and use these solutions as the sample solution and
standard solution. Perform the test with exactly 10 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the peak areas, A T and ^4 S , of
cefsulodin of each solution.
Amount L"g (potency)] of cefsulodin (C22H20N4O8S2)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Cefsulodin Sodium Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of ammonium sul-
fate (1 in 100) and acetonitrile (97:3).
Flow rate: Adjust the flow rate so that the retention time of
cefsulodin is about 9 minutes.
System suitability —
System performance: Dissolve 40 mg of isonicotinic acid
amide in 25 mL of the standard solution. When the proce-
dure is run with 10 /xL of this solution under the above oper-
ating conditions, isonicotinic acid amide and cefsulodin are
eluted in this order with the resolution between these peaks
being not less than 5.
System repeatability: When the test is repeated 5 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefsulodin is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
466
Ceftazidime Hydrate / Official Monographs
JP XV
Ceftazidime Hydrate
h 3 c-V
H2N-
• SH z O
C 22 H 22 N 6 7 S 2 .5H 2 0: 636.65
(6/?,7/?)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(l-carboxy-
l-methylethoxyimino)acetylamino]-3-(pyridinium-l-
ylmethyl)-8-oxo-5-thia-l-azabicyclo [4.2.0] oct-2-ene-2-
carboxylate pentahydrate [78439-06-2]
Ceftazidime Hydrate contains not less than 950 fig
(potency) and not more than 1020 fig (potency) per mg,
calculated on the dried basis. The potency of Ceftazi-
dime Hydrate is expressed as mass (potency) of ceftazi-
dime (C 22 H 22 N 6 7 S 2 : 546.58).
Description Ceftazidime Hydrate occurs as a white to light
yellowish white crystalline powder.
It is slightly soluble in water, and very slightly soluble in
acetonitrile and in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Ceftazidime Hydrate in phosphate buffer solu-
tion, pH 6.0 (1 in 100,000) as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum or the spectrum of a solution of
Ceftazidime Reference Standard prepared in the same man-
ner as the sample solution: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Ceftazidime Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Ceftazidime Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) To 0.05 g of Ceftazidime Hydrate add 5 mg of dried
sodium carbonate, and add 0.5 mL of heavy water for
nuclear magnetic resonance spectroscopy to dissolve. Deter-
mine the spectrum of this solution as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using sodium
3-trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals, A and B, at around S 1.5 ppm and at
around 5 6.9 ppm, and a multiple signal C between S 7.9 ppm
and d 9.2 ppm. The ratio of integrated intensity of each sig-
nal, A:B:C, is about 6:1:5.
Optical rotation <2.49> [a]™: -28 - -34° (0.5 g calculated
on the dried bases, phosphate buffer solution, pH 6.0, 100
mL, 100 mm).
pH <2.54> Dissolve 0.5 g of Ceftazidime Hydrate in 100 mL
of water: the pH of the solution is between 3.0 and 4.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ceftazidime Hydrate in 10 mL of a solution obtained by dis-
solving 5 g of anhydrous disodium hydrogenphosphate and 1
g of potassium dihydrogenphosphate in water to make 100
mL: the solution is clear, and its absorbance <2.24> at 420 nm
is not more than 0.20.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Ceftazi-
dime Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ceftazidime Hydrate according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances (i) Trityl-f-butyl substance and t-
butyl substance — Dissolve 0.10 g of Ceftazidime Hydrate in 2
mL of diluted disodium hydrogenphosphate TS (1 in 3), and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add diluted disodium hydrogenphosphate
TS (1 in 3) to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop with a mixture of w-butyl acetate,
acetic acid (100), acetate buffer solution, pH 4.5 and 1-
butanol (16:16:13:3) to a distance of about 12 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots which appear upper in position than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
(ii) Other related substances — Dissolve 20 mg of Ceftazi-
dime Hydrate in 10 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
exactly 5 fiL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the areas
of each peak by the automatic integration method: each peak
area other than ceftazidime from the sample solution is not
more than that of ceftazidime from the standard solution,
and the total of peak areas other than ceftazidime from the
sample solution is not more than 5 times of the peak area of
ceftazidime from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.0 g of ammonium dihydrogen-
phosphate in 750 mL of water, adjust to pH 3.5 with phos-
phoric acid, and add water to make 870 mL. To this solution
add 130 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ceftazidime is about 4 minutes.
Time span of measurement: About 3 times as long as the
retention time of ceftazidime beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 1 mL of the standard so-
JP XV
Official Monographs / Ceftazidime Hydrate
467
lution, add the mobile phase to make exactly 5 mL, and con-
firm that the peak area of ceftazidime obtained from 5 /uL of
this solution is equivalent to 15 to 25% of that of ceftazidime
obtained from 5 /iL of the standard solution.
System performance: Dissolve about 0.01 g each of
Ceftazidime Hydrate and acetanilide in 20 mL of the mobile
phase. When the procedure is run with 5 fiL of this solution
under the above operating conditions, ceftazidime and
acetanilide are eluted in this order with the resolution be-
tween these peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
ceftazidime is not more than 2.0%.
(5) Free pyridine — Weigh accurately about 50 mg of
Ceftazidime Hydrate, dissolve in the mobile phase to make
exactly 10 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.1 g of pyridine, and add
the mobile phase to make exactly 100 mL. Pipet 1 mL of this
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the peak
height, H T and H s , of pyridine of each solution: the amount
of free pyridine is not more than 0.3%.
Amount (mg) of free pyridine
= W s x (H T /H S ) x (1/1000)
W s : Amount (mg) of pyridine
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.88 g of ammonium dihydrogen-
phosphate in 500 mL of water, add 300 mL of acetonitrile
and water to make 1000 mL, and adjust to pH 7.0 with am-
monia solution (28).
Flow rate: Adjust the flow rate so that the retention time of
pyridine is about 4 minutes.
System suitability —
Test for required detection: Confirm that the peak height
of pyridine obtained from 10 /uL of the standard solution is
equivalent to 50% of the full scale.
System performance: Dissolve 5 mg of Ceftazidime Hy-
drate in 100 mL of a solution of pyridine in the mobile phase
(1 in 20,000). When the procedure is run with 10 iiL of this
solution under the above operating conditions, ceftazidime
and pyridine are eluted in this order with the resolution be-
tween these peaks being not less than 9.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the heights of
pyridine is not more than 5.0%.
Loss on dryness <2.41> Not less than 13.0% and not more
than 15.0% (0.1 g, in vacuum not exceeding 0.67 kPa, 60°C,
3 hours).
Assay Weigh accurately an amount of Ceftazidime Hydrate
and Ceftazidime Reference Standard, equivalent to about 0.1
g (potency), and dissolve each in 0.05 mol/L phosphate
buffer solution, pH 7.0 to make exactly 100 mL. Pipet 10 mL
each of these solutions, add exactly 5 mL of the internal stan-
dard solution, then add 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make 50 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 5 /uL each of these solutions as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Qt and Q s , of the peak
area of ceftazidime to that of the internal standard of each
solution.
Amount [fig (potency)] of ceftazidime (C22H22N 6 7 S 2 )
= W s x (Qj/Qs) x 1000
W s : Aamount [mg (potency)] of Ceftazidime Reference
Standard
Internal standard solution — A solution of dimedon in 0.05
mol/L phosphate buffer solution, pH 7.0 (11 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with hexasilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 4.26 g of anhydrous disodium
hydrogenphosphate and 2.72 g of potassium dihydrogen-
phosphate in 980 mL of water, and add 20 mL of acetoni-
trile.
Flow rate: Adjust the flow rate so that the retention time of
ceftazidime is about 4 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, the internal standard and ceftazidime are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of ceftazidime to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
468
Cefteram Pivoxil / Official Monographs
JP XV
Cefteram Pivoxil
H 3 C CH 3
CH 3
r
■y^
Hrfi-<T
H H
CM,,
C 22 H 27 N 9 7 S 2 : 593.64
2,2-Dimethylpropanoyloxymethyl (6R,lR)-l-[(Z)-2-
(2-aminothiazol-4-yl)-2-(methoxyimino)acetylamino]-3-
(5-methyl-2//-tetrazol-2-ylmethyl)-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[82547-58-8, Cefteram]
Cefteram Pivoxil contains not less than 743 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefteram Pivoxil is expressed as mass
(potency) of cefteram (C 16 H 17 N 9 5 S 2 : 479.49).
Description Cefteram Pivoxil occurs as a white to pale
yellowish white powder.
It is very soluble in acetonitrile, freely soluble in methanol,
in ethanol (95) and in chloroform, and practically insoluble
in water.
Identification (1) Determine the absorption spectrum of a
solution of Cefteram Pivoxil in 0.05 mol/L hydrochloric
acid-methanol TS (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the spectrum of a solution of Cefteram
Pivoxil in deuterated chloroform for nuclear magnetic
resonance spectroscopy (1 in 10) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits single
signals A, B and C, at around <51.2 ppm, at around <52.5 ppm
and at around 34. ppm, respectively. The ratio of the in-
tegrated intensity of these signals, A:B:C, is about 3:1:1.
Optical rotation <2.49> [ a ]™: +35- +43° (0.4 g calculated
on the anhydrous basis, methanol, 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cefteram Pivoxil according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefteram Pivoxil according to Method 4, and perform the
test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Cefteram
Pivoxilin in 50 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 50 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area by
the automatic integration method: the area of the peak, hav-
ing the relative retention time of about 0.9 with respect to
cefteram pivoxil from the sample solution is not more than
1 .25 times the peak area of cefteram pivoxil from the stan-
dard solution, the area of the peak, having the relative reten-
tion time of about 0.1 with respect to cefteram pivoxil from
the sample solution is not more than 0.25 times the peak area
of cefteram pivoxil from the standard solution, and the total
area of the peaks other than cefteram pivoxil from the sample
solution is not more than 2.75 times the peak area of cefter-
am pivoxil from the standard solution. For the above calcula-
tion, use the area of the peak, having the relative retention
time of about 0.1, after multiplying by its relative response
factor, 0.74.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of cefteram pivoxil.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of cefteram pivox-
il obtained from 10 /uL of this solution is equivalent to 7 to
13% of that from 10 fiL of the standard solution.
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of cefteram pivoxil are not less than
5000 and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cefteram pivoxil is not more than 3.0%.
Water <2.48> Not more than 3.0% (0.3 g, coulometric titra-
tion).
Assay Weigh accurately an amount of Cefteram Pivoxil
and Cefteram Pivoxil Mesitylenesulfonate Reference Stan-
dard, equivalent to about 40 mg (potency), dissolve each in
20 mL of diluted acetonitrile (1 in 2), add exactly 5 mL of the
internal standard solution and diluted acetonitrile (1 in 2) to
make 50 mL, and use these solutions as the sample solution
and standard solution. Perform the test with 10 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the ratios, g T and Q s , of the peak
area of cefteram pivoxil to that of the internal standard.
Amount \jug (potency)] of cefteram (C 16 H I7 N 9 5 S 2 )
= W s x (<2 T /<2s) x 1000
W s : Amount [mg (potency)] of Cefteram Pivoxil Mesity-
lenesulfonate Reference Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in diluted acetonitrile (1 in 2) (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
JPXV
Official Monographs / Cefteram Pivoxil Fine Granules
469
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 100 mL of acetic acid-sodium acetate
buffer solution, pH 5.0 add 375 mL of acetonitrile and water
to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
cefteram pivoxil is about 14 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the internal standard and cefteram pivoxil are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cefteram pivoxil to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Cefteram Pivoxil Fine Granules
Cefteram Pivoxil Fine Granules contain not less than
90.0% and not more than 110.0% of the labeled
amount of cefteram (C 16 H 17 N 9 5 S 2 : 479.49).
Method of preparation Prepare to finely granulated form as
directed under Powders, with Cefteram Pivoxil.
Identification Powder Cefteram Pivoxil Fine Granules. To
a portion of the powder, equivalent to 0.1 g (potency) of
Cefteram Pivoxil according to the labeled amount, add 20
mL of methanol, shake well, and filter. To 1 mL of the
filtrate add 0.05 mol/L hydrochloric acid-methanol TS to
make 500 mL, and determine the absorption spectrum as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 262 nm and 266 nm.
Purity Related substances — Powder Cefteram Pivoxil Fine
Granules, if necessary. To a portion, equivalent to 0.1 g
(potency) of Cefteram Pivoxil according to the labled
amount, add diluted acetonitrile (1 in 2) to make 100 mL, dis-
perse the particle with the aid of ultrasonic waves, then filter,
and use the filtrate as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 50
mL, and use this solution as the standard solution. Perform
the test with exactly 10 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
each peak area by the automatic integration method: the area
of the peak, having the relative retention time of about 0.9
with respect to cefteram pivoxil, is not larger than 1.75 times
the peak area of cefteram pivoxil from the standard solution,
the area of the peak, having the relative retention time of
about 0.1, is not larger than 0.68 times the peak area of
cefteram pivoxil from the standard solution, and the total
area of the peaks other than cefteram pivoxil is not larger
than 3.7 times the peak area of cefteram pivoxil from the
standard solution. For this calculation, use the peak area for
the peak having the relative retention time of about 0.1 after
multiplying by its relative response factor, 0.74.
Operating conditions —
Proceed as directed in tne Purity (3) under Cefteram
Pivoxil.
System suitability—
Proceed as directed in the Purity (3) under Cefteram
Pivoxil.
Water <2.48> Not more than 0.3% (0.1 g (potency), coulo-
metric titration).
Uniformity of dosage units <6.02> The Granules in single-u-
nit container meet the requirement of the Mass variation test.
Dissolution Being specified separately.
Particle size <6.03> It meets the requirement of fine granules
of the Powders.
Assay Powder Cefteram Pivoxil Fine Granules, if necessa-
ry, and use as the sample. Weigh accurately an amount of the
sample, equivalent to about 0.3 g (potency) of Ceteram
Pivoxil according to the labled amount, add exactly 30 mL of
the internal standard solution and diluted acetonitrile (1 in 2)
to make 300 mL. Disperse the particle with the aid of ultra-
sonic waves, then filter, and use the filtrate as the sample so-
lution. Separately, weigh accurately about 50 mg (potency) of
Cefteram Pivoxil Mesitylenesulfonate Reference Standard,
dissolve in 20 mL of diluted acetonitrile (1 in 2), add exactly 5
mL of the internal standard solution and diluted acetonitrile
(1 in 2) to make 50 mL, and use this solution as the standard
solution. Perform the test with 1 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of
cefteram pivoxil to that of the internal standard.
Amount [mg (potency)] of cefteram (C I6 H 17 N 9 05S2)
= W s x (Q T /Q S ) x 6
W s : Amount [mg (potency)] of Cefteram Pivoxil
Mesitylenesulfonate Reference Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in diluted acetonitrile (1:2) (1 in 1000).
Operating conditions —
Proceed as directed in the Assay under Cefteram Pivoxil.
System suitability —
Proceed as directed in the Assay under Cefteram Pivoxil.
Containers and storage Containers — Tight containers.
470
Ceftibuten Hydrate / Official Monographs
JP XV
Ceftibuten Hydrate
CO;H C0 2 H
"4-0 * 2H2 °
H H
O
C 15 H 14 N 4 6 S 2 .2H 2 0: 446.46
(6fl,7fl)-7-[(2Z)-2-(2-Aminothiazol-4-yl)-4-carboxybut-2-
enoylamino]-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid dihydrate [118081-34-8]
Ceftibuten Hydrate contains not less than 900 fig
(potency) and not more than 1020 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Ceftibuten Hydrate is expressed as mass (potency) of
ceftibuten (C 15 H 14 N40 6 S 2 : 410.42).
Description Ceftibuten Hydrate occurs as a white to pale
yellowish white crystalline powder and has a slight, charac-
teristic odor.
It is freely soluble in 7V,/V-dimethylformamide and in
dimethyl sulfoxide, and practically insoluble in water, in
ethanol (95) and in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Ceftibuten Hydrate in 0.1 mol/L phosphate
buffer solution for antibiotics, pH 8.0 (1 in 50,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 261 nm and 265 nm.
(2) Determine the infrared absorption spectrum of
Ceftibuten Hydrate as directed in the paste method under the
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3249 cm -1 , 1772 cm -1 , 1700 cm
-', 1651 cm" 1 and 1544 cm" 1 .
(3) Determine the spectrum of a solution of Ceftibuten
Hydrate in deuterated dimethyl sulfoxide for nuclear magnet-
ic resonance spectroscopy (1 in 30), using tetramethylsilane
for nuclear magnetic resonance spectroscopy as an internal
reference compound, as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H): it exhibits double sig-
nals A and B, at around 5 3.2 ppm and at around 5 5.1 ppm,
a quartet signal C, at around 5 5.8 ppm, and a single signal
D, at around 5 6.3 ppm. The ratio of integrated intensity of
each signal except the signal at around 8 3.2 ppm, B:C:D is
about 1:1:1.
Absorbance <2.24>
(263 nm): 320 - 345 (20 mg calcu-
lated on the anhydrous basis, 0.1 mol/L phosphate buffer so-
lution for antibiotics, pH 8.0, 1000 mL).
Optical rotation <2.49> [a]™: + 135 - + 155° (0.3 g calculated
on the anhydrous basis, 0.1 mol/L phosphate buffer solution
for antibiotics, pH 8.0, 50 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Ceftibuten Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Being specified separately.
Water <2.48> Not less than 8.0% and not more than 13.0%
(0.2 g, volumetric titration, direct titration. Use a mixture of
pyridine for water determination and ethylene glycol for
water determination (5:1) instead of methanol for water de-
termination).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Ceftibuten Hydrate
and Ceftibuten Hydrochloride Reference Standard, equiva-
lent to about 10 mg (potency), dissolve each in 36 mL of
0.1 mol/L phosphate buffer solution for antibiotics, pH 8.0,
add exactly 4 mL each of the internal standard solution,
shake, and use these solutions as the sample solution and
standard solution. Perform the test with 5 fiL of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
ceftibuten to that of the internal standard. Keep the sample
solution and the standard solution at 5°C or below and use
within 2 hours.
Amount [fig (potency)] of ceftibuten (C^H^^O^)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Ceftibuten Hydrochloride
Reference Standard
Internal standard solution — A solution of methyl /i-hydrox-
ybenzoate in acetonitrile (3 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 263 nm).
Column: A stainless steel column 4 mm in inside diameter
and 20 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (7 ftm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.005 mol/L M-decyl
trimethylammonium bromide TS and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
ceftibuten is about 10 minutes.
System suitability —
System performance: Dissolve 5 mg of Ceftibuten Hydrate
in 1 mol/L Hydrochloric acid TS to make 50 mL, and allow
to stand for 4 hours at room temperature. To 10 mL of this
solution add 0.1 mol/L phosphate buffer solution for
antibiotics, pH 8.0 to make 25 mL. When the procedure is
run with 5 fiL of this solution under the above operating con-
ditions, trans-isomer and ceftibuten are eluted in this order
with the resolution between these peaks being not less than
1.5.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of ceftibuten to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 5°C.
JPXV
Official Monographs / Ceftizoxime Sodium 471
Ceftizoxime Sodium
t7f'/+vAth U^A
CH,
CO z Na
H,N-
C 13 H 12 N 5 Na05S 2 : 405.38
Monosodium (6i?,7i?)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetylamino] -8-oxo-5-thia- 1 -
azabicyclo[4.2.0]oct-2-ene-2-carboxylate [68401-82-1]
Ceftizoxime Sodium contains not less than 925 fig
(potency) and not more than 965 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Ceftizoxime Sodium is expressed as mass (potency) of
ceftizoxime (C 13 H 13 N505S 2 : 383.40).
Description Ceftizoxime Sodium occurs as a white to light
yellow, crystals or crystalline powder.
It is very soluble in water, sparingly soluble in methanol,
and practically insoluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Ceftizoxime Sodium (1 in 63,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Ceftizoxime Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Ceftizoxime
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropionate-d 4 for nuclear magnetic resonance
spectroscopy as an internal reference compound: it exhibits a
single signal A at around d 4.0 ppm, a multiple signal B
around d 6.3 ppm, and a single signal C at around S 7.0 ppm.
The ratio of integrated intensity of each signal, A:B:C, is
about 3:1:1.
(4) Ceftizoxime Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [«]*>: + 125 - + 145° (0.25 g calcu-
lated on the anhydrous bases, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Ceftizoxime Sodium in 10 mL
of water: the pH of the solution is between 6.0 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ceftizoxime Sodium in 10 mL of water: the solution is clear,
and colorless to light yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Ceftizoxime Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Ceftizoxime Sodium according to Method 3, and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve 0.11 g of Ceftizoxime
Sodium in 100 mL of 0.1 mol/L phosphate buffer solution,
pH 7.0, and use this solution as the sample solution. Perform
the test with 5 fiL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the areas of each peak by the auto-
matic integration method: each peak area other than
ceftizoxime is not more than 0.5% of the peak area of
ceftizoxime, and the total of peak areas other than
ceftizoxime is not more than 1.0% of that of ceftizoxime.
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Assay.
Mobile phase: Dissolve 2.31 g of disodium hydrogen-
phosphate dodecahydrate and 1.42 g of citric acid monohy-
drate in 1000 mL of water, adjust to pH 3.6 with diluted
phosphoric acid (1 in 10) or dilute sodium hydroxide TS. To
200 mL of this solution add 10 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ceftizoxime is about 12 minutes.
Time span of measurement: About 5 times as long as the
retention time of ceftizoxime beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 1 mL of the sample solu-
tion, add 0.1 mol/L phosphate buffer solution, pH 7.0 to
make exactly 100 mL, and use this solution as the solution for
test for required detection. Pipet 1 mL of the solution, add
0.1 mol/L phosphate buffer solution, pH 7.0 to make exactly
10 mL, and confirm that the peak area of ceftizoxime ob-
tained from 5 fiL of this solution is equivalent to 7 to 13% of
that of ceftizoxime obtained from 5 fiL of the solution for
test for required detection.
System performance: Dissolve about 10 mg of Ceftizoxime
Reference Standard in 100 mL of 0.1 mol/L phosphate buffer
solution, pH 7.0, and use this solution as the solution for sys-
tem suitability test. When the procedure is run with 5 fiL of
this solution under the above operating conditions, the num-
ber of theoretical plates and the symmetry factor of the peak
of ceftizoxime are not less than 4000 steps and not more than
2.0, respectively.
System repeatability: When the test is repeated 6 times with
5 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of ceftizoxime is not more than 2.0%.
Water <2.48> Not more than 8.5% (0.4 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Ceftizoxime Sodium
and Ceftizoxime Reference Standard, equivalent to about 0.1
g (potency), and dissolve each in 0.1 mol/L phosphate buffer
solution, pH 7.0 to make exactly 20 mL. Pipet 2 mL each of
these solutions, add exactly 10 mL of the internal standard
solution, then add 0.1 mol/L phosphate buffer solution, pH
7.0 to make 20 mL, and use these solutions as the sample
solution and standard solution, respectively. Perform the test
with 5 ftL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
472 Ceftriaxone Sodium Hydrate / Official Monographs
JP XV
ceftizoxime to that of the internal standard of each solution.
Amount [wg (potency)] of ceftizoxime (C 13 H 13 N505S2)
= Ws x (Qt/Qs) x 1000
W s : Amount [mg (potency)] of Ceftizoxime Reference
Standard
Internal standard solution — A solution of 3-hydroxybenzoic
acid in 0.1 mol/L phosphate buffer solution, pH 7.0 (3 in
500).
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 2.31 g of disodium hydrogen-
phosphate dodecahydrate and 1.42 g of citric acid monohy-
drate in 1000 mL of water, and adjust to pH 3.6 with diluted
phosphoric acid (1 in 10) or dilute sodium hydroxide TS. To
450 mL of this solution add 50 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ceftizoxime is about 4 minutes.
System suitability —
System performance: When the procedure is run with 5 /uL
of the standard solution under the above operating condi-
tions, ceftizoxime and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.0 and the symmetry factor of each peak is not more
than 2.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of ceftizoxime to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ceftriaxone Sodium Hydrate
t7 r- U 7+7 >i- h U ^A7Kfttl
CHa
C0 2 Na
N \— N
II H H
J
N
I
CH
ONa
C 18 H 16 N 8 Na 2 7 S 3 .3|H 2 0: 661.60
Disodium (6R ,1R )-7- [(Z )-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetylamino]-3-(6-hydroxy-2-methyl-5-oxo-
2,5-dihydro-l,2,4-triazin-3-ylsulfanylmethyl)-8-oxo-5-
thia- 1 -azabicyclo [4 . 2 . 0] oct-2-ene-2-carboxy late
hemiheptahydrate [104376-79-6]
Ceftriaxone Sodium Hydrate contains not less than
905 /ug (potency) and not more than 935 /ug (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Ceftriaxone Sodium Hydrate is expressed as mass
(potency) of ceftriaxone (C 18 H 18 N 8 7 S 3 : 554.58).
Description Ceftriaxone Sodium Hydrate occurs as a white
to yellowish white crystalline powder.
It is freely soluble in water and in dimethylsulfoxide, spar-
ingly soluble in methanol, very slightly soluble in ethanol
(99.5), and practically insoluble in acetonitrile.
Identification (1) Determine the absorption spectrum of a
solution of Ceftriaxone Sodium Hydrate (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Ceftriaxone Sodium Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the spectrum of a solution of Ceftriaxone
Sodium Hydrate in deuterated dimethylsulfoxide for nuclear
magnetic resonance spectroscopy (1 in 10), using tetramethyl-
silane for nuclear magnetic resonance spectroscopy as an in-
ternal reference compound, as directed under Nuclear Mag-
netic Resonance Spectroscopy <2.21> (*H): it exhibits single
signals, A, B, C and D, at around 5 3.5 ppm, at around 5 3.8
ppm, at around 5 6.7 ppm and at around 5 7.2 ppm, respec-
tively. The ratio of integrated intensity of each signal, A: B:
C: D, is about 3:3:1:2. When the signal at around 5 3.5 ppm
overlaps with the signal of water, perform the measurement
in the probe kept at about 50°C.
(3) Ceftriaxone Sodium Hydrate responds to the Qualita-
tive Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: - 153 - - 170° (50 mg calculat-
ed on the anhydrous basis, water, 2.5 mL, 20 mm).
pH <2.54> Dissolve 0.6 g of Ceftriaxone Sodium Hydrate in
5 mL of water: the pH of the solution is between 6.0 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 0.6 g of
Ceftriaxone Sodium Hydrate in 5 mL of water: the solution is
clear and light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Ceftriaxone Sodium Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ceftriaxone Sodium Hydrate according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances 1 — Dissolve 20 mg of Ceftriaxone
Sodium Hydrate in 10 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add a mixture of water and acetonitrile for liquid
chromatography (11:9) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine each peak area by
the automatic integration method: the peak areas of the
impurity 1 having the relative retention time of about 0.5 and
the impurity 2 having the relative retention time of about 1.3
to ceftriaxone from the sample solution are not more than the
peak area of ceftriaxone from the standard solution. In this
case, these peak areas for the impurity 1 and the impurity 2
are used after multiplying by 0.9 and 1.2, respectively.
JPXV
Official Monographs / Ceftriaxone Sodium Hydrate
473
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.796 g of anhydrous disodium
hydrogen phosphate and 3.522 g of potassium dihydrogen
phosphate in water to make exactly 1000 mL, and use this so-
lution as the solution A. Separately, dissolve 20.256 g of
citric acid monohydrate and 7.840 g of sodium hydroxide in
water to make exactly 1000 mL, and use this solution as the
solution B. Dissolve 4.00 g of tetra-w-heptylammonium
bromide in 450 mL of acetonitrile for liquid chromato-
graphy, add 55 mL of the solution A, 5 mL of the solution B
and 490 mL of water.
Flow rate: Adjust the flow rate so that the retention time of
ceftriaxone is about 7 minutes.
Time span of measurement: About 2 times as long as the
retention time of ceftriaxone.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the sample solution, add a mixture of water and acetonitrile
for liquid chromatography (11:9) to make exactly 200 mL,
and use this solution as the solution for system suitability
test. Pipet 1 mL of the solution for system suitability test,
and add a mixture of water and acetonitrile for liquid chro-
matography (11:9) to make exactly 100 mL. Confirm that the
peak area of ceftriaxone obtained from 10 /uL of this solution
is equivalent to 0.9 to 1.1% of that from 10//L of the solu-
tion for system suitability test.
System performance: Dissolve 10 mg of Ceftriaxone
Sodium Hydrate in a mixture of water and acetonitrile for
liquid chromatography (11:9) to make 5 mL, add 5 mL of a
solution of diethyl terephthalate in a mixture of water and
acetonitrile for liquid chromatography (11:9) (9 in 5000), and
add a mixture of water and acetonitrile for liquid chro-
matography (11:9) to make 200 mL. When the procedure is
run with 10 [iL of this solution under the above operating
conditions, ceftriaxone and diethyl terephthalate are eluted in
this order, with the resolution between these peaks being not
less than 6.
System repeatability: When the test is repeated 6 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of ceftriaxone is not more than 1.0%.
(5) Related substances 2 — Dissolve 10 mg of Ceftriaxone
Sodium Hydrate in 10 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add a mixture of acetonitrile for liquid chro-
matography and water (23:11) to make exactly 100 mL, and
use this solution as standard solution. Perform the test with
exactly 10 [iL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the each
peak area of the impurities which appear after the peak of
ceftriaxone from the sample solution is not more than the
peak area of ceftriaxone from the standard solution, and the
total peak area of these impurities is not more than 2.5 times
of the peak area from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.796 g of anhydrous disodium
hydrogen phosphate and 3.522 g of potassium dihydrogen
phosphate in water to make exactly 1000 mL, and use this so-
lution as the solution A. Separately, dissolve 20.256 g of
citric acid monohydrate and 7.840 g of sodium hydroxide in
water to make exactly 1000 mL, and use this solution as the
solution B. Dissolve 4.00 g of tetra-M-heptylammonium
bromide in 450 mL of acetonitrile for liquid chro-
matography, and add 55 mL of the solution A, 5 mL of the
solution B, 490 mL of water and 700 mL of acetonitrile for
liquid chromatography.
Flow rate: Adjust the flow rate so that the retention time of
ceftriaxone is about 3 minutes.
Time span of measurement: About 10 times as long as the
retention time of ceftriaxone.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the sample solution, add a mixture of acetonitrile for liquid
chromatography and water (23:11) to make exactly 100 mL,
and use this solution as the solution for system suitability
test. Measure exactly 1 mL of the solution for system suitabil-
ity test, and add a mixture of acetonitrile for liquid chro-
matography and water (23:11) to make exactly 100 mL. Con-
firm that the peak area of ceftriaxone obtained from 10 /uL of
this solution is equivalent to 0.9 to 1.1% of that from 10 /xL
of the solution for system suitability test.
System performance: Dissolve 10 mg of Ceftriaxone
Sodium Hydrate in a mixture of acetonitrile for liquid chro-
matography and water (23:11) to make 5 mL, add 5 mL of a
solution of diethyl terephthalate in a mixture of water and
acetonitrile for liquid chromatography (11:9) (9 in 5000), and
add a mixture of acetonitrile for liquid chromatography and
water (23:11) to make 200 mL. When the procedure is run
with 10 /xh of this solution under the above operating condi-
tions, ceftriaxone and diethyl terephthalate are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of ceftriaxone is not more than 1.0%.
Water <2.48> Not less than 8.0% and not more than 1 1 .0%
(0.15 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Ceftriaxone Sodium
Hydrate and Ceftriaxone Sodium Reference Standard, e-
quivalent to about 0.1 g (potency), dissolve each in a mixture
of water and acetonitrile for liquid chromatography (1 1 :9) to
make exactly 50 mL. Pipet 5 mL of each solution, add ex-
actly 5 mL of the internal standard solution and a mixture of
water and acetonitrile for liquid chromatography (11:9) to
make 200 mL, and use these solutions as the sample solution
and standard solution, respectively. Perform the test with
474
Cefuroxime Axetil / Official Monographs
JP XV
10 /uh each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
ceftriaxone to that of the internal standard.
Amount [fig (potency)] of ceftriaxone (C 18 H 18 N 8 7 S3)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Ceftriaxone Sodium Ref-
erence Standard
Internal standard solution — A solution of diethyl terephtha-
late in a mixture of water and acetonitrile for liquid chro-
matography (11:9) (9 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 fim in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 5.796 g of anhydrous disodium
hydrogen phosphate and 3.522 g of potassium dihydrogen
phosphate in water to make exactly 1000 mL, and use this so-
lution as solution A. Dissolve 20.256 g of citric acid monohy-
drate and 7.840 g of sodium hydroxide in water to make ex-
actly 1000 mL, and use this solution as solution B. Dissolve
4.00 g of tetra-w-heptylammonium bromide in 450 mL of
acetonitrile for liquid chromatography, and add 490 mL of
water, 55 mL of solution A, and 5 mL of solution B.
Flow rate: Adjust the flow rate so that the retention time of
ceftriaxone is about 7 minutes.
System suitability —
System performance: When the procedure is run with
10 fiL of the standard solution under the above operating
conditions, ceftriaxone and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 6.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ceftriaxone to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefuroxime Axetil
t7n+->A 7+-k^;u
H 3 C O CH 3
ch, ^ o
NK
■^
C 20 H 22 N 4 O 10 S: 510.47
( IRS)- 1 -Acetoxy ethyl (6R ,7i?)-3-carbamoyloxymethyl-7-
[(Z)-2-furan-2-yl-2-(methoxyimino)acetylamino]-8-oxo-5-
thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[64544-07-6]
Cefuroxime Axetil contains not less than 800 fig
(potency) and not more than 850 fig (potency) per mg,
calculated on the anhydrous basis and corrected by
the amount of acetone. The potency of Cefuroxime
Axetil is expressed as mass (potency) of cefuroxime
(C 16 H 16 N 4 8 S: 424.39).
Description Cefuroxime Axetil occurs as white to yellowish
white, non-crystalline powder.
It is freely soluble in dimethylsulfoxide, soluble in
methanol, sparingly soluble in ethanol (95), and very slightly
soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Cefuroxime Axetil in methanol (3 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Cefuroxime Axetil Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cefuroxime Axetil as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cefuroxime Axetil Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Cefuroxime
Axetil in deuterated dimethylsulfoxide for nuclear magnetic
resonance spectroscopy (1 in 20) as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> ('H), using
tetramethylsilane for nuclear magnetic resonance spec-
troscopy as an internal reference compound: it exhibits a
double signal or a pair of double signals A at around
(51.5 ppm, a pair of single signals B at around <52.1 ppm, and
a single signal C at around <53.9 ppm. The ratio of the in-
tegrated intensity of each signal, A:B:C, is about 1:1:1.
Optical rotation <2.49> [a]™: +41 - +47° (0.5 g, methanol,
50 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cefuroxime Axetil according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Put 1.0 g of Cefuroxime Axetil in a
crucible, add 10 mL of a solution of magnesium nitrate hexa-
hydrate in ethanol (95) (1 in 10), burn the ethanol, then heat
gradually to incinerate. If a carbonized substance remains,
moisten with a small amount of nitric acid, and ignite to in-
cinerate. Cool, add 10 mL of dilute hydrochloric acid to the
residue, dissolve by warming on a water bath, and perform
the test using this solution as the test solution (not more than
2 ppm).
(3) Related substance — Dissolve 25 mg of Cefuroxime
Axetil in 4 mL of methanol, add a solution of ammonium di-
hydrogen phosphate (23 in 1000) to make 10 mL, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add 40 mL of methanol and a solution of ammonium
dihydrogen phosphate (23 in 1000) to make exactly 100 mL,
and use this solution as the standard solution. Perform the
JPXV
Official Monographs / Cefuroxime Axetil 475
test with exactly 2 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
each peak area by the automatic integration method: the area
of the peak other than cefuroxime axetil obtained from the
sample solution is not more than 1.5 times the sum area of
two peaks of cefuroxime axetil obtained from the standard
solution, and the sum area of the peaks other than
cefuroxime axetil from the sample solution is not more than 4
times the sum area of two peaks of cefuroxime axetil from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of the peak having the larger retention time of
the two peaks of cefuroxime axetil beginning after the solvent
peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add 4 mL of methanol and a solu-
tion of ammonium dihydrogen phosphate (23 in 1000) to
make exactly 10 mL. Confirm that the sum area of the two
peaks of cefuroxime axetil obtained from 2//L of this solu-
tion is equivalent to 7 to 13% of that obtained from 2^L of
the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
2 jXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the sum area of the
two peaks of cefuroxime axetil is not more than 2.0%.
(4) Acetone — Weigh accurately about 1 g of Cefuroxime
Axetil, add exactly 0.2 mL of the internal standard solution
and dimethylsulfoxide to make exactly 10 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 0.5 g of acetone, and add dimethylsulfoxide to make
exactly 100 mL. Pipet 0.2 mL of this solution, add exactly 0.2
mL of the internal standard solution and dimethylsulfoxide
to make exactly 10 mL, and use this solution as the standard
solution. Perform the test with 1 /xL each of the sample solu-
tion and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions, de-
termine each peak area by the automatic integration method,
and calculate the ratios, Q T and Q s , of the peak area of ace-
tone to that of the internal standard: the amount of acetone is
not more than 1.3%.
Amount (%) of acetone = (W S /W T ) x (Q T /g s ) X 0.2
W s : Amount (g) of acetone
Wj\ Amount (g) of the sample
Internal standard solution — A solution of 1-propanol in
dimethylsulfoxide (1 in 200).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with siliceous earth for gas chromato-
graphy coated with a mixture of polyethylene glycol 600 for
gas chromatography and polyethylene glycol 1500 for gas
chromatography (1:1) in the ratio of 20% (125 - 150 /um in
particle diameter).
Column temperature: A constant temperature of about
90°C.
Temperature of injection port: A constant temperature of
about 115°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 4 minutes.
System suitability —
System performance: When the procedure is run with 1 /uL
of the standard solution under the above operating condi-
tions, acetone and the internal standard are eluted in this ord-
er with the resolution between these peaks being not less than
5.
System repeatability: When the test is repeated 6 times with
1 iiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of acetone to that of the internal standard is not
more than 5.0%.
Water <2.48> Not more than 2.0% (0.4 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Isomer ratio Perform the test with 10 iiL of the sample so-
lution obtained in the Assay as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the area, A E , of the peak having the smaller
retention time and the area, A b , of the peak having the bigger
retention time of the two peaks of cefuroxime axetil:
A b /(A a + A b ) is between 0.48 and 0.55.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
System suitability —
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Assay Weigh accurately an amount of Cefuroxime Axetil
and Cefuroxime Axetil Reference Standard, equivalent to
about 50 mg (potency), and dissolve each in methanol to
make exactly 50 mL. Pipet 10 mL each of these solutions,
add exactly 5 mL of the internal standard solution, 5 mL of
methanol and a solution of ammonium dihydrogen phos-
phate (23 in 1000) to make 50 mL, and use these solutions as
the sample solution and the standard solution. Perform the
test with 10 iiL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine the
ratios, Qt and Q s , of the sum area of the two peaks of
cefuroxime axetil to the peak area of the internal standard.
Amount [fig (potency)] of cefuroxime (C 16 H 16 N 4 8 S)
= W s x (g T /g s ) x 1000
W s : Amount [mg (potency)] of Cefuroxime Axetil Refer-
ence Standard
Internal standard solution — A solution of acetanilide in
methanol (27 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 278 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with trimethylsilanized
476
Cefuroxime Sodium / Official Monographs
JP XV
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of ammonium
dihydrogen phosphate (23 in 1000) and methanol (5:3).
Flow rate: Adjust the flow rate so that the retention time of
the peak having the smaller retention time of the two peaks of
cefuroxime axetil is about 8 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the internal standard and cefuroxime axetil are
eluted in this order with the resolution between the two peaks
of cefuroxime axetil being not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
sum area of the two peaks of cefuroxime axetil to the peak
area of the internal standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cefuroxime Sodium
-fe7P*vA1-r- U^7A
CH 3
Vn''
:°rY"tt
jl
CT^NHs.
C l6 H l5 N 4 Na0 8 S: 446.37
Monosodium (6/?,7/?)-3-carbamoyloxymethyl-7-[(Z)-2-
furan-2-yl-2-(methoxyimino)acetylamino]-8-oxo-5-thia-l-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate [56238-63-2]
Cefuroxime Sodium contains not less than 875 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Cefuroxime Sodium is expressed as
mass (potency) of cefuroxime (C l6 H l6 N 4 8 S: 424.39).
Description Cefuroxime Sodium occurs as a white to light
yellowish white, crystals or crystalline powder.
It is freely soluble in water, soluble in methanol, and very
slightly soluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cefuroxime Sodium (1 in 100,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of a solution of Cefuroxime Sodium Reference Stan-
dard prepared in the same manner as sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Cefuroxime Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Cefuroxime Sodium Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Determine the spectrum of a solution of Cefuroxime
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a single signal A at around 5 4.0 ppm, a quartet sig-
nal B at around 5 6.6 ppm, and double signals, C and D, at
around 5 6.9 ppm and around 5 1.1 ppm, respectively. The
ratio of integrated intensity of each signal, A:B:C:D, is about
3:1:1:1.
(4) Cefuroxime Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [«]£>: + 59 - + 66° (0.5 g calculated
on the anhydrous bases, water, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cefuroxime Sodium in 10 mL
of water: the pH of the solution is between 6.0 and 8.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cefuroxime Sodium in 10 mL of water: the solution is clear,
and its absorbance <2.24> at 450 nm is not more than 0.25.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Cefuroxime Sodium according to Method 2, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 30 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cefuroxime Sodium according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 25 mg of Cefuroxime
Sodium in 25 mL of water, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the areas of each peak by the automatic
integration method: each peak area other than cefuroxime
from the sample solution is not more than the peak area of
cefuroxime from the standard solution, and the total of the
peak areas other than cefuroxime from the sample solution is
not more than 3 times of the peak area of cefuroxime from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of cefuroxime beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 1 mL of the standard so-
lution, add water to make exactly 10 mL, and confirm that
the peak area of cefuroxime obtained from 20 iuL of this solu-
tion is equivalent to 7 to 13% of that of cefuroxime obtained
from 20 fiL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefuroxime is not more than 2.0%.
JPXV
Official Monographs / Macrocrystalline Cellulose 477
Water <2.48> Not more than 4.0% (0.4 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Cefuroxime Sodium
and Cefuroxime Sodium Reference Standard, equivalent to
about 25 mg (potency), and dissolve each in water to make
exactly 25 mL, and use these solutions as the sample solution
and standard solution, respectively. Perform the test with ex-
actly 20 /uL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the peak area, A T and A s , of cefuroxime
of each solution.
Amount [Mg (potency)] of cefuroxime (C 16 H 16 N 4 8 S)
= W s x (Aj/As) x 1000
W s : Amount [mg (potency)] of Cefuroxime Sodium Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 273 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 125 mm in length, packed with hexasilanized sili-
ca gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.68 g of sodium acetate trihydrate
in 900 mL of water, adjust to pH 3.4 with acetic acid (100),
and add water to make 1000 mL. To 990 mL of this solution
add 10 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
cefuroxime is about 8 minutes.
System suitability —
System performance: Allow the sample solution to stand at
60°C for 10 minutes. When the procedure is run with 20 /xL
of this solution soon after cooling under the above operating
conditions, the resolution between the peak of cefuroxime
and the peak corresponding to the retention time of about 0.7
to the peak of cefuroxime is being not less than 2.0.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of cefuroxime is not more than 1.0%.
Containers and storage Containers — Tight containers.
Microcrystalline Cellulose
$SbHz;up-x
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Microcrystalline Cellulose is purified, partially
depolymerized a-cellulose, obtained as a pulp from fi-
brous plant material, with mineral acids.
♦The label indicates the degree of polymerization,
loss on drying, and bulk density values with the
range. ♦
♦Description Microcrystalline Cellulose occurs as a white
crystalline powder having fluidity.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It swells with sodium hydroxide TS on heating. »
Identification (1) Dissolve 20 g of zinc chloride and 6.5 g
of potassium iodide in 10.5 mL of water, add 0.5 g of iodine,
and shake for 15 minutes. Place about 10 mg of
Microcrystalline Cellulose on a watch glass, and disperse in 2
mL of this solution: the substance develops a blue-violet
color.
♦(2) Sieve 20 g of Microcrystalline Cellulose for 5
minutes on an air-jet sieve equipped with a screen (No. 391,
200 mm in inside diameter) having 38-^m openings. If more
than 5% is retained on the screen, mix 30 g of Microcrystal-
line Cellulose with 270 mL of water; otherwise, mix 45 g with
255 mL of water. Perform the mixing for 5 minutes in a high-
speed (18,000 revolutions per minute or more) power blen-
der. Transfer 100 mL of the dispersion to a 100-mL graduat-
ed cylinder, and allow to stand for 3 hours: a white, opaque,
bubble-free dispersion, which does not form a supernatant
liquid at the surface, is obtained.*
(3) Transfer 1.3 g of Microcrystalline Cellulose, ac-
curately weighed, to a 125-mL conical flask, and add exactly
25 mL each of water and 1 mol/L cupriethylenediamine TS.
Immediately purge the solution with nitrogen, insert the stop-
per, and shake on a suitable mechanical shaker to dissolve.
Perform the test with a suitable amount of this solution,
taken exactly, according to Method 1 under Viscosity Deter-
mination <2.53> using a capillary viscometer having the vis-
cosity constant (K) of approximately 0.03, at 25 ±0.1°C, and
determine the kinematic viscosity, v. Separately, perform the
test with a mixture of exactly 25 mL each of water and 1
mol/L cupriethylenediamine TS in the same manner as
above, using a capillary viscometer having K of approximate-
ly 0.01, and determine the kinematic viscosity, v .
Calculate the relative viscosity, n rei , of Microcrystalline
Cellulose by the formula:
>7rel=V/V
Obtain the product, [n]C, of intrinsic viscosity [>7](mL/g)
and concentration C (g/100 mL) from the value n If:1 of the Ta-
ble. When calculate the degree of polymerization, P, by the
following formula, P is not more than 350 ♦and within the
labeled range. ♦
P = (95)[n]C/W T
W T : Amount (g) of the sample, calculated on the dried
basis
pH <2.54> Shake 5.0 g of Microcrystalline Cellulose with 40
mL of water for 20 minutes, and centrifuge: the pH of the su-
pernatant liquid is between 5.0 and 7.5.
Purity ^(1) Heavy metals <1.07> — Proceed with 2.0 g of
Microcrystalline Cellulose according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).»
(2) Water-soluble substances — Shake 5.0 g of
Microcrystalline Cellulose with 80 mL of water for 10
minutes, filter with the aid of vacuum through a filter paper
for quantitative analysis (5C) into a vacuum flask. Evaporate
the clear filtrate in a tared evaporating dish to dryness
without charring, dry at 105 °C for 1 hour, cool in a desicca-
tor, and weigh: the difference between the mass of the residue
478
Microcrystalline Cellulose / Official Monographs
JP XV
and the mass obtained from a blank determination does not
exceed 12.5 mg.
(3) Diethyl ether-soluble substances — Place 10.0 g of
Microcrystalline Cellulose in a column having an internal di-
ameter of about 20 mm, and pass 50 mL of peroxide-free
diethyl ether through the column. Evaporate the eluate to
dryness in a previously dried and tared evaporation dish. Dry
the residue at 105 °C for 30 minutes, allow to cool in a desic-
cator, and weigh: the difference between the mass of the
residue and the mass obtained from a blank determination
does not exceed 5.0 mg.
Conductivity <2.51> Perform the test as directed in the Con-
ductivity Measurement with the supernatant liquid obtained
in the pH as the sample solution, and determine the conduc-
tivity *at 25±0.1°C.» Determine in the same way the con-
Table for Conversion of Relative Viscosity (ri rel ) into the Product of Limiting Viscosity and Concentration ([rj]C)
n,n
0.00
0.01
0.02
0.03
MC
0.04
0.05
0.06
0.07
0.08
0.09
1.1
0.098
0.106
0.115
0.125
0.134
0.143
0.152
0.161
0.170
0.180
1.2
0.189
0.198
0.207
0.216
0.225
0.233
0.242
0.250
0.259
0.268
1.3
0.276
0.285
0.293
0.302
0.310
0.318
0.326
0.334
0.342
0.350
1.4
0.358
0.367
0.375
0.383
0.391
0.399
0.407
0.414
0.422
0.430
1.5
0.437
0.445
0.453
0.460
0.468
0.476
0.484
0.491
0.499
0.507
1.6
0.515
0.522
0.529
0.536
0.544
0.551
0.558
0.566
0.573
0.580
1.7
0.587
0.595
0.602
0.608
0.615
0.622
0.629
0.636
0.642
0.649
1.8
0.656
0.663
0.670
0.677
0.683
0.690
0.697
0.704
0.710
0.717
1.9
0.723
0.730
0.736
0.743
0.749
0.756
0.762
0.769
0.775
0.782
2.0
0.788
0.795
0.802
0.809
0.815
0.821
0.827
0.833
0.840
0.846
2.1
0.852
0.858
0.864
0.870
0.876
0.882
0.888
0.894
0.900
0.906
2.2
0.912
0.918
0.924
0.929
0.935
0.941
0.948
0.953
0.959
0.965
2.3
0.971
0.976
0.983
0.988
0.994
1.000
1.006
1.011
1.017
1.022
2.4
1.028
1.033
1.039
1.044
1.050
1.056
1.061
1.067
1.072
1.078
2.5
1.083
1.089
1.094
1.100
1.105
1.111
1.116
1.121
1.126
1.131
2.6
1.137
1.142
1.147
1.153
1.158
1.163
1.169
1.174
1.179
1.184
2.7
1.190
1.195
1.200
1.205
1.210
1.215
1.220
1.225
1.230
1.235
2.8
1.240
1.245
1.250
1.255
1.260
1.265
1.270
1.275
1.280
1.285
2.9
1.290
1.295
1.300
1.305
1.310
1.314
1.319
1.324
1.329
1.333
3.0
1.338
1.343
1.348
1.352
1.357
1.362
1.367
1.371
1.376
1.381
3.1
1.386
1.390
1.395
1.400
1.405
1.409
1.414
1.418
1.423
1.427
3.2
1.432
1.436
1.441
1.446
1.450
1.455
1.459
1.464
1.468
1.473
3.3
1.477
1.482
1.486
1.491
1.496
1.500
1.504
1.508
1.513
1.517
3.4
1.521
1.525
1.529
1.533
1.537
1.542
1.546
1.550
1.554
1.558
3.5
1.562
1.566
1.570
1.575
1.579
1.583
1.587
1.591
1.595
1.600
3.6
1.604
1.608
1.612
1.617
1.621
1.625
1.629
1.633
1.637
1.642
3.7
1.646
1.650
1.654
1.658
1.662
1.666
1.671
1.675
1.679
1.683
3.8
1.687
1.691
1.695
1.700
1.704
1.708
1.712
1.715
1.719
1.723
3.9
1.727
1.731
1.735
1.739
1.742
1.746
1.750
1.754
1.758
1.762
4.0
1.765
1.769
1.773
1.777
1.781
1.785
1.789
1.792
1.796
1.800
4.1
1.804
1.808
1.811
1.815
1.819
1.822
1.826
1.830
1.833
1.837
4.2
1.841
1.845
1.848
1.852
1.856
1.859
1.863
1.867
1.870
1.874
4.3
1.878
1.882
1.885
1.889
1.893
1.896
1.900
1.904
1.907
1.911
4.4
1.914
1.918
1.921
1.925
1.929
1.932
1.936
1.939
1.943
1.946
4.5
1.950
1.954
1.957
1.961
1.964
1.968
1.971
1.975
1.979
1.982
4.6
1.986
1.989
1.993
1.996
2.000
2.003
2.007
2.010
2.013
2.017
4.7
2.020
2.023
2.027
2.030
2.033
2.037
2.040
2.043
2.047
2.050
4.8
2.053
2.057
2.060
2.063
2.067
2.070
2.073
2.077
2.080
2.083
4.9
2.087
2.090
2.093
2.097
2.100
2.103
2.107
2.110
2.113
2.116
5.0
2.119
2.122
2.125
2.129
2.132
2.135
2.139
2.142
2.145
2.148
5.1
2.151
2.154
2.158
2.160
2.164
2.167
2.170
2.173
2.176
2.180
5.2
2.183
2.186
2.190
2.192
2.195
2.197
2.200
2.203
2.206
2.209
5.3
2.212
2.215
2.218
2.221
2.224
2.227
2.230
2.233
2.236
2.240
5.4
2.243
2.246
2.249
2.252
2.255
2.258
2.261
2.264
2.267
2.270
5.5
2.273
2.276
2.279
2.282
2.285
2.288
2.291
2.294
2.297
2.300
5.6
2.303
2.306
2.309
2.312
2.315
2.318
2.320
2.324
2.326
2.329
5.7
2.332
2.335
2.338
2.341
2.344
2.347
2.350
2.353
2.355
2.358
5.8
2.361
2.364
2.367
2.370
2.373
2.376
2.379
2.382
2.384
2.387
5.9
2.390
2.393
2.396
2.400
2.403
2.405
2.408
2.411
2.414
2.417
6.0
2.419
2.422
2.425
2.428
2.431
2.433
2.436
2.439
2.442
2.444
JPXV
Official Monographs / Macrocrystalline Cellulose 479
ductivity of water used for the preparation of the sample so-
lution: the deference between these conductivities is not more
than 75 //S-cm -1
Loss on drying <2.41> Not more than 7.0% *and within a
range as specified on the label* (1 g, 105°C. 3 hours).
Residue on ignition <2.44> Not more than 0.1% *(2 g).»
Bulk density (i) Apparatus — Use a volumeter shown in
the figure. Put a No. 8. 6 sieve (2000 /urn) on the top of the
volumeter. A funnel is mounted over a baffle box, having
four glass baffle plates inside which the sample powder slides
as it passes. At the bottom of the baffle box is a funnel that
collect the powder, and allows it to pour into a sample receiv-
ing cup mounted directly below it.
(ii) Procedure — Weigh accurately the mass of a brass or
stainless steel cup, which has a capacity of 25.0 ±0.05 mL
MC
n,n
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.09
6.1
2.447
2.450
2.453
2.456
2.458
2.461
2.464
2.467
2.470
2.472
6.2
2.475
2.478
2.481
2.483
2.486
2.489
2.492
2.494
2.497
2.500
6.3
2.503
2.505
2.508
2.511
2.513
2.516
2.518
2.521
2.524
2.526
6.4
2.529
2.532
2.534
2.537
2.540
2.542
2.545
2.547
2.550
2.553
6.5
2.555
2.558
2.561
2.563
2.566
2.568
2.571
2.574
2.576
2.579
6.6
2.581
2.584
2.587
2.590
2.592
2.595
2.597
2.600
2.603
2.605
6.7
2.608
2.610
2.613
2.615
2.618
2.620
2.623
2.625
2.627
2.630
6.8
2.633
2.635
2.637
2.640
2.643
2.645
2.648
2.650
2.653
2.655
6.9
2.658
2.660
2.663
2.665
2.668
2.670
2.673
2.675
2.678
2.680
7.0
2.683
2.685
2.687
2.690
2.693
2.695
2.698
2.700
2.702
2.705
7.1
2.707
2.710
2.712
2.714
2.717
2.719
2.721
2.724
2.726
2.729
7.2
2.731
2.733
2.736
2.738
2.740
2.743
2.745
2.748
2.750
2.752
7.3
2.755
2.757
2.760
2.762
2.764
2.767
2.769
2.771
2.774
2.776
7.4
2.779
2.781
2.783
2.786
2.788
2.790
2.793
2.795
2.798
2.800
7.5
2.802
2.805
2.807
2.809
2.812
2.814
2.816
2.819
2.821
2.823
7.6
2.826
2.828
2.830
2.833
2.835
2.837
2.840
2.842
2.844
2.847
7.7
2.849
2.851
2.854
2.856
2.858
2.860
2.863
2.865
2.868
2.870
7.8
2.873
2.875
2.877
2.879
2.881
2.884
2.887
2.889
2.891
2.893
7.9
2.895
2.898
2.900
2.902
2.905
2.907
2.909
2.911
2.913
2.915
8.0
2.918
2.920
2.922
2.924
2.926
2.928
2.931
2.933
2.935
2.937
8.1
2.939
2.942
2.944
2.946
2.948
2.950
2.952
2.955
2.957
2.959
8.2
2.961
2.963
2.966
2.968
2.970
2.972
2.974
2.976
2.979
2.981
8.3
2.983
2.985
2.987
2.990
2.992
2.994
2.996
2.998
3.000
3.002
8.4
3.004
3.006
3.008
3.010
3.012
3.015
3.017
3.019
3.021
3.023
8.5
3.025
3.027
3.029
3.031
3.033
3.035
3.037
3.040
3.042
3.044
8.6
3.046
3.048
3.050
3.052
3.054
3.056
3.058
3.060
3.062
3.064
8.7
3.067
3.069
3.071
3.073
3.075
3.077
3.079
3.081
3.083
3.085
8.8
3.087
3.089
3.092
3.094
3.096
3.098
3.100
3.102
3.104
3.106
8.9
3.108
3.110
3.112
3.114
3.116
3.118
3.120
3.122
3.124
3.126
9.0
3.128
3.130
3.132
3.134
3.136
3.138
3.140
3.142
3.144
3.146
9.1
3.148
3.150
3.152
3.154
3.156
3.158
3.160
3.162
3.164
3.166
9.2
3.168
3.170
3.172
3.174
3.176
3.178
3.180
3.182
3.184
3.186
9.3
3.188
3.190
3.192
3.194
3.196
3.198
3.200
3.202
3.204
3.206
9.4
3.208
3.210
3.212
3.214
3.215
3.217
3.219
3.221
3.223
3.225
9.5
3.227
3.229
3.231
3.233
3.235
3.237
3.239
3.241
3.242
3.244
9.6
3.246
3.248
3.250
3.252
3.254
3.256
3.258
3.260
3.262
3.264
9.7
3.266
3.268
3.269
3.271
3.273
3.275
3.277
3.279
3.281
3.283
9.8
3.285
3.287
3.289
3.291
3.293
3.295
3.297
3.298
3.300
3.302
9.9
3.304
3.305
3.307
3.309
3.311
3.313
3.316
3.318
3.320
3.321
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
10
3.32
3.34
3.36
3.37
3.39
3.41
3.43
3.45
3.46
3.48
11
3.50
3.52
3.53
3.55
3.56
3.58
3.60
3.61
3.63
3.64
12
3.66
3.68
3.69
3.71
3.72
3.74
3.76
3.77
3.79
3.80
13
3.80
3.83
3.85
3.86
3.88
3.89
3.90
3.92
3.93
3.95
14
3.96
3.97
3.99
4.00
4.02
4.03
4.04
4.06
4.07
4.09
15
4.10
4.11
4.13
4.14
4.15
4.17
4.18
4.19
4.20
4.22
16
4.23
4.24
4.25
4.27
4.28
4.29
4.30
4.31
4.33
4.34
17
4.35
4.36
4.37
4.38
4.39
4.41
4.42
4.43
4.44
4.45
18
4.46
4.47
4.48
4.49
4.50
4.52
4.53
4.54
4.55
4.56
19
4.57
4.58
4.59
4.60
4.61
4.62
4.63
4.64
4.65
4.66
480
Powdered Cellulose / Official Monographs
JP XV
^11. H.fi *'n:vu
Powder junnel
IjjadiT]^ funnel
Kaffle assembly
Class baffles
Sample
ratcivi]!^ cu|j — * | j
SlillK!
and an inside diameter of 30.0 ±2.0 mm, and put the cup di-
rectly below the funnel of the volumeter. Slowly pour
Microcrystalline Cellulose 5.1 cm height from the upper part
of the powder funnel through the sieve, at a rate suitable to
prevent clogging, until the cup overflows. If the clogging oc-
curs, take out the sieve. Level the excess powder with the aid
of a slide glass, weigh the filled cup, and weigh accurately the
content of the cup, and then calculate the bulk density by the
following expression: the bulk density is within the labeled
specification.
Bulk density (g/cm 3 )=,4/25
A: Measured mass (g) of the content of the cup
♦Microbial limit <4.05> The total aerobic microbial count is
not more than 1000 per g, the total count of fungi and yeast is
not more than 100 per g, and yeast is not more than 100 per
g, and Escherichia coli, Salmonella species, Pseudomonas
aeruginosa and Staphylococcus aureus are not observed. »
♦Containers and storage Containers — Tight containers. ♦
Powdered Cellulose
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Powdered Cellulose is a purified, mechanically disin-
tegrated alpha cellulose * obtained as a pulp, after par-
tial hydrolysis as occasion demands ♦, from fibrous
plant materials.
The label indicates the mean degree of polymeriza-
tion value with a range.
♦Description Powdered Cellulose occurs as a white pow-
der.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether. ♦
Identification (1) Dissolve 20 g of zinc chloride and 6.5 g
of potassium iodide in 10.5 mL of water, add 0.5 g of iodine,
and shake for 15 minutes. Place about 10 mg of Powdered
Cellulose on a watch glass, and disperse in 2 mL of this solu-
tion: the substance develops a blue-violet color.
♦(2) Mix 30 g of Powdered Cellulose with 270 mL of
water in a high-speed (18,000 revolutions per minute or more)
blender for 5 minutes, transfer 100 mL of the dispersion to a
100-mL graduated cylinder, and allow to stand for 1 hour: a
supernatant liquid appears above the layer of the cellulose. »
(3) Transfer 0.25 g of Powdered Cellulose, accurately
weighed, to a 125-mL conical flask, add exactly 25 mL each
of water and 1 mol/L cupriethylenediamine TS, and proceed
as directed in the Identification (3) under Microcrystalline
Cellulose. The mean degree of polymerization, P, is not less
than 440 and is within the labeled specification.
pH <2.54> Mix 10 g of Powdered Cellulose with 90 mL of
water, and allow to stand for 1 hour with occasional stirring:
the pH of the supernatant liquid is between 5.0 and 7.5.
Purity *(1) Heavy metals <1.07> — Proceed with 2.0 g of
Powdered Cellulose according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).»
(2) Water-soluble substances — Shake 6.0 g of Powdered
Cellulose with 90 mL of recently boiled and cooled water,
and allow to stand for 10 minutes with occasional shaking.
Filter, with the aid of vacuum through a filter paper, discard
the first 10 mL of the filtrate, and pass the subsequent filtrate
through the same filter, if necessary, to obtain a clear filtrate.
Evaporate a 15.0-mL portion of the filtrate in a tared
evaporating dish to dryness without charring, dry at 105°C
for 1 hour, and weigh after allowing to cool in a desiccator:
the difference between the mass of the residue and the mass
obtained from a blank determination does not exceed 15.0
mg.
(3) Diethyl ether-soluble substances — Place 10.0 g of
Powdered Cellulose in a column having an internal diameter
of about 20 mm, and pass 50 mL of peroxide-free diethyl
ether through the column. Evaporate the eluate to dryness in
a previously dried and tared evaporation dish. Dry the
residue at 105°C for 30 minutes, and weigh after allowing to
cool in a desiccator: the difference between the mass of the
residue and the mass obtained from a blank determination
does not exceed 15.0 mg.
Loss on drying <2.41> Not more than 6.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.3% (1 g calculat-
ed on the dried basis).
♦Microbial limit <4.05> The total aerobic microbial count
does not exceed 1000 per g, the total combined fungus and
yeast count does not exceed 100 per g, and Escherichia coli,
Salmonella species, Pseudomonas aeruginosa and
Staphylococcus aureus are not observed. »
♦Containers and storage Containers — Tight containers. ♦
Cellacefate
Cellulose Acetate Phthalate
Cellulose acetate benzene-l,2-dicarboxylate
[9004-38-0]
This monograph is harmonized with the European
JPXV
Official Monographs / Celmoleukin (Genetical Recombination)
481
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Cellacefate is a reaction product of phthalic anhy-
dride and partially acetylated cellulose.
Cellacefate, calculated on the anhydrous and free
acid-free basis, contains not less than 21.5% and not
more than 26.0% of acetyl group (-COCH3: 43.04),
and not less than 30.0% and not more than 36.0% of
carboxybenzoyl group (-COC 6 H 4 COOH: 149.12).
♦Description Cellacefate occurs as a white powder or grain.
It is freely soluble in acetone, and practically insoluble in
water, in methanol and in ethanol (99.5). ♦
Identification Determine the infrared absorption spectrum
of Cellacefate directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with *the Reference Spectrum or* spectrum of Cel-
lacefate Reference Standard: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Viscosity <2.53> Weigh accurately a quantity of Cellacefate,
equivalent to 15 g calculated on the anhydrous basis, dissolve
in 85 g of a mixture of acetone and water (249: 1 in mass),
and perform the test with this solution at 25 ± 0.2°C as direct-
ed in Method 1 to obtain the kinematic viscosity v. Separate-
ly, determine the density, p, of Cellacefate as directed under
Determination of Specific Gravity and Density <2.56>, and
calculate the viscosiy, r\, as r\ = pv : not less than 45 mPa-s
and not more than 90mPa-s.
Purity (1) *Heavy metals <1.07> — Proceed with 2.0 g of
Cellacefate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).»
(2) Free acids — Weigh accurately about 3 g of Cellace-
fate, put in a glass-stoppered conical flask, add 100 mL of
diluted methanol (1 in 2), stopper tightly, and filter after
shaking for 2 hours. Wash both the flask and residue with
two 10-mL portions each of diluted methanol (1 in 2), com-
bine the washes to the filtrate, and titrate <2.50> with 0.1 mol
/L sodium hydroxide VS (indicator: 3 drops of
phenolphthalein TS). Perform the blank determination with
120 mL of diluted methanol (1 in 2), and make any necessary
correction.
Amount (%) of free acids = (0.8306 xA)/W
A: amount (mL) of 0.1 mol/L sodium hydroxide con-
sumed
W: amount (g) of the test sample, calculated on the anhy-
drous basis
The amount of free acids is not more than 3.0%, calculat-
ed as phthalic acid (C 8 H 6 4 : 166.13).
Water <2.48> Not more than 5.0% (1 g, volumetric titra-
tion, direct titration, using a mixture of ethanol (99.5) and
dichloromethane (3:2) instead of methanol for Karl Fischer
method).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Carboxybenzoyl group — Weigh accurately
about 1 g of Cellacefate, dissolve in 50 mL of a mixture of
ethanol (95) and acetone (3: 2), and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (indicator: 2 drops of
phenolphthalein TS). Perform a blank determination, and
make any necessary correction.
Content (%) of carboxybenzoyl group (C 8 H 5 3 )
1.491 x.4
W
- 1.795X5
X100
100-B
A: Amount (mL) of 0.1 mol/L sodium hydroxide con-
sumed
B: Amount (%) of free acids obtained in the Purity (2) Free
acids
W: Amount (g) of the test sample, calculated on the anhy-
drous basis
Assay (2) Acetyl group — Weigh accurately about 0.1 g of
Cellacefate, put in a glass-stoppered conical flask, add ex-
actly 25 mL of 0. 1 mol/L sodium hydroxide VS, and boil for
30 minutes under a reflux condenser. After cooling, add 5
drops of phenolphthalein TS, and titrate <2.50> with 0.1
mol/L hydrochloric acid VS. Perform a blank determina-
tion.
Content (%) of free acids and bound acetyl group (C 2 H 3 0)
= 0.4305^4/1^
A: Amount (mL) of 0.1 mol/L sodium hydroxide con-
sumed, corrected by the blank determination
W: Amount (g) of the test sample, calculated on the
anhydrous basis
Content (%) of acetyl group (C 2 H 3 0)
= [ {100 x (P - 0.51825)} /(100 - B)] - 0.5772C
B: Amount (%) of free acids obtained in the Purity (2) Free
acids
C: Content (%) of carboxybenzoyl group
P: Content (%) of free acids and bound acetyl group
(C 2 H 3 0)
Containers and storage Containers — Tight containers.
Celmoleukin (Genetical
Recombination)
-tzJl^E p -f * > Glfc^ffiJ&x.)
Ala-Pro -Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-GEn-LeiJ-Glu-His-LeiJ-Leu- Leu- Asp>-
Leu-Gln-MeHle-Leu-A&n-Gly-lie-Asn Asrt Tyr Lys Asri Pro Lys Leu Thr Arg Mat Lou
Thr-Prie-Lys-Phe-Tyr Mai Pra-Lys Lys AlaThr-Glu Leu Lys His Leu -Gin -Cys- Leu -Glu -
Glu - GFu - Leu - Lys - Pro - Leu - G lu - Glu- Val -Leu- Asn ■ Leu - Ala-Gin - Ser - Lys - Asrt ■ Phe -His- Leu -
Arg - P ro- Arg - Asp- Leu-He - Ser - Asn- 1 le- Asn - Val - 1 le -Val- Leu- Glu- Leu- Lys - Qly - Ser - Glu -
Thr -Thr - Phe - Mel - Cys - Glu -Tyr -Ala -Asp - G lu -Thr - Ala -Thr-lle - Val - Gl u - Phe - Leu- Asn - Arg -
Trp-llaThr Phe Cys Gin -Ser -lie -lie Ser Tnr-Leu-Thr
C693HnigN 178 O203S7: 15415.82
[94218-72-1]
The desired product of Celmoleukin (Genetical
Recombination) is a protein consisting of 133 amino
482
Celmoleukin (Genetical Recombination) / Official Monographs
JP XV
acid residues manufactured by E. coli through expres-
sion of human interleukin-2 cDNA.
It is a solution having a T-lymphocyte activating
effect.
It contains not less than 0.5 and not more than 1.5
mg of protein per mL, and 1 mg of this protein con-
tains potency not less than 8.0 x 10 6 units.
Description Celmoleukin (Genetical Recombination) oc-
curs as a colorless, clear liquid.
Identification (1) To 1 mL of Celmoleukin (Genetical
Recombination) add 0.05 mL of diluted copper (II) sulfate
TS (1 in 10), shake, add 0.9 g of potassium hydroxide, and
shake. When 0.3 mL of ethanol (99.5) is added to this solu-
tion and shaken, the ethanol layer exhibits a violet color.
(2) Based on the results of the Assay (1), place an amount
of Celmoleukin (Genetical Recombination) equivalent to
about 50 /xg of protein in two hydrolysis tubes, and evaporate
to dryness under vacuum. To one of the tubes add 100 fiL of
a mixture of diluted hydrochloric acid (59— > 125), mercapto a-
cetic acid and phenol (100:10:1), and shake. Place this
hydrolysis tube in a vial and humidify the inside of the vial
with 200 /xL of the mixture of diluted hydrochloric acid (59— ►
125), mercapto acetic acid and phenol (100:10:1). Replace the
vial interior with inert gas or reduce the pressure, and heat
for 24 hours at about 115°C. After drying under vacuum,
dissolve in 0.5 mL of 0.02 mol/L hydrochloric acid TS, and
use this solution as the sample solution (1). To the other
hydrolysis tube, add 100 fiL of ice cold performic acid, oxi-
dize for 1.5 hours on ice, add 50 jxL of hydrobromic acid, and
dry under vacuum. Add 200 /xL of water, repeat the dry un-
der vacuum procedure two more times, place the hydrolysis
tube in a vial, and humidify the inside of the vial with 200 fiL
of diluted hydrochloric acid (59— >125). Replace the vial in-
terior with inert gas or reduce the pressure (vacuum), and
heat for 24 hours at about 115°C. After drying under vacu-
um, dissolve in 0.5 mL of 0.02 mol/L hydrochloric acid TS,
and use this solution as the sample solution (2). Separately,
accurately weigh 60 mg of L-aspartic acid, 100 mg of L-glu-
tamic acid, 17 mg of L-alanine, 23 mg of L-methionine, 21 mg
of L-tyrosine, 24 mg of L-histidine hydrochloride monohy-
drate, 58 mg of L-threonine, 22 mg of L-proline, 14 mg of L-
cystine, 45 mg of L-isoleucine, 37 mg of L-phenylalanine, 32
mg of L-arginine hydrochloride, 32 mg of L-serine, 6 mg of
glycine, 18 mg of L-valine, 109 mg of L-leucine, 76 mg of L-
lysine hydrochloride, and 8 mg of L-tryptophan, add 0.1
mol/L hydrochloric acid TS to dissolve to make 500 mL, add
40 /xL of this solution to two hydrolysis tubes, and evaporate
to dryness under vacuum to make the standard solutions (1)
and (2) processed in the same way for each respective sample
solution. Perform the test with 250 jxL each of the sample so-
lution and standard solution as directed under Liquid Chro-
matography <2.01> and from the peak areas for each amino
acid obtained from the sample solutions and standard solu-
tions determine the molar number of the amino acids con-
tained in 1 mL of the sample solutions. Furthermore, when
calculating the number of amino acids assuming there are 22
leucine residues in one mole of Celmoleukin (Genetical
Recombination), there are 17 or 18 glutamic acid (or gluta-
mine), 11 to 13 threonine, 11 or 12 aspartic acid (or
asparagine), 11 lysine, 7 or 8 isoleucine, 6 to 9 serine, 6
phenylalanine, 5 alanine, 5 or 6 proline, 4 arginine and 4
methionine, 3 or 4 cysteine, 3 or 4 valine, 3 tyrosine, 3 histi-
dine, 2 glycine, and 1 tryptophan.
(3) Molecular mass Based on the results of the Assay (1),
add buffer for celmoleukin and dilute to prepare a sample so-
lution so that there is about 0.5 mg of protein per mL. To
vertical uncontinuous buffer SDS-polyacrylamide gel pre-
pared from resolving gel for celmoleukin and stacking gel for
celmoleukin add 20 /xL of the sample solution or 20 /xh of
molecular weight marker for celmoleukin to each stacking gel
well, and perform the electrophoresis. The molecular weight
of the main electrophoretic band is between the range of
12500 and 13800 when the band is stained by immersion in
Coomassie staining TS.
(4) Add 100 /xL of protein digestive enzyme TS to 100 /xL
of Celmoleukin (Genetical Recombination), shake, leave
standing for 18 to 24 hours at 37°C, and then add 2//L of 2-
mercaptoethanol. Leave for a further 30 minutes at 37 °C,
and add 5 /xL of trifluoroacetic acid solution (1 in 10). This is
the sample solution. Separately, process celmoleukin for liq-
uid chromatography using the same method. This is the stan-
dard solution. Perform the test using 50 /xL of each both the
sample and standard solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. When comparing the chromatograms obtained from
the sample and standard solutions, the retention times for the
sample and standard solutions are identical, and the peak
heights are similar.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm)
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (particle size: 5 /xm).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A solution of trifluoroacetic acid (1 in
1000).
Mobile phase B: A solution of trifluoroacetic acid in a mix-
ture of acetonitrile and water (17:3) (1 in 1000).
Mobile phase flow: The concentration gradient is con-
trolled by changing the ratio of mobile phases A and B as
shown in the table below.
Time after injection
Mobile phase
Mobile phase
of sample (min)
A (vol%)
B (vol%)
0-5
100
5-45
100^60
0^40
45-75
60^0
40^100
75-85
100
Flow: Adjust so that the retention time of celmoleukin is
about 70 minutes.
System suitability —
System performance: Add 2//L of 2-mercaptoethanol to
100 //L of celmoleukin for liquid chromatography, leave for
2 hours at 37 °C, and then run this solution under the above
conditions. Under these conditions, celmoleukin and its
reduced form are eluted in this order with the resolution be-
tween these peaks being not less than 1.5.
(5) Accurately measure an appropriate amount of Cel-
moleukin (Genetical Recombination), dilute by adding cul-
ture medium for celmoleukin, and prepare a sample solution
JPXV
Official Monographs / Celmoleukin (Genetical Recombination)
483
containing 800 units per mL. Add 25 /uh of this sample solu-
tion to 2 holes (A and B) of a flat-bottomed microtest plate
for tissue culture, and then add 25 /uL of reference anti-inter-
leukin-2 antiserum solution diluted with culture medium for
celmoleukin to hole A and 25 /uL of culture medium for cel-
moleukin to hole B. Add 50 iiL of culture medium for cel-
moleukin to another hole (hole C). After shaking the
microtest plate, warm for 30 minutes to 2 hours at 37 °C in air
containing 5% carbon dioxide. Next, add to each hole 50 /uL
of culture medium for celmoleukin containing the interleu-
kin-2 dependent mouse natural killer cells NKC3 and culture
for 16 to 24 hours at 37°C. Add 3-(4,5-dimethylthiazole-2-
yl)-2,5-diphenyl-2//-tetrazolium bromide TS, culture for 4 to
6 hours at 37°C, and add sodium lauryl sulfate TS and leave
for 24 to 48 hours. When the absorbance at 590 nm of the so-
lution in each hole is measured as directed under Ultraviolet-
visible Spectrophotometry <2.24>, the difference in absor-
bance between the solutions from holes A and C is not more
than 3% of the difference in absorbance between the solu-
tions from holes B and C.
pH <2.54> 4.5-5.5
Purity (1) Host cell-derived protein — Prepare a sample
solution by the accurate two times stepwise dilution of Cel-
moleukin (Genetical Recombination) with phosphate buffer-
sodium chloride TS (hereinafter referred to as PBS) contain-
ing bovine serum. Prepare a series of 5 standard solutions by
accurately diluting E. coli protein (hereinafter referred to as
ECP) over a range of 0.25 to 6 ng per mL with PBS contain-
ing bovine serum. Pipet 100 iiL of goat anti-ECP antibody
TS into each hole of a flat-bottomed microtest plate, leave for
16 to 24 hours at 4°C, and then remove the liquid. Wash
three times with PBS, add 200 fiL of PBS containing bovine
serum albumin, leave for at least 3 hours at room tempera-
ture, and then wash three more times with PBS. Pipet 100 //L
of the sample solution and each standard solution into each
hole, leave for 16 to 24 hours at 4°C, and then wash 5 times
with PBS. Add 100 juL of peroxidase-labeled rabbit anti-ECP
antibody Fab' TS, leave for at least 4 hours at room tempera-
ture, and wash 5 times with PBS. Next, add 100 //L of sub-
strate buffer for celmoleukin, allow to react for 5 to 25
minutes at room temperature in a dark place, and then add
100 /uL of diluted sulfuric acid (3 in 25). Measure the absor-
bances of these flat-bottomed microtest plates by Ultraviolet-
visible Spectrophotometry <2.24> at a wavelength of 492 nm.
Separately, using 100 /uL of PBS containing bovine serum,
perform a blank test using the same method and correct. De-
termine the absorbance of each standard solution, prepare a
calibration curve, determine the amount of ECP per mL of
the sample solution, and multiply by the sample solution di-
lution factor. When determining the concentration of ECP
per unit of protein in the sample solution, there is not more
than 0.02% (0.2//g/mg of protein).
(2) Polymers — Dilute (at least 4 steps) the sample solu-
tion prepared in the Identification (3) with buffer solution for
celmoleukin so that the protein content is within the range of
about 2 to 32 iig per mL to prepare a series of standard solu-
tions. Pipet 20 /uL of the sample solution or each of the stan-
dard solutions into the stacking gel well, and perform vertical
uncoupled buffer SDS-polyacrylamide gel electrophoresis fol-
lowed by immersion in Coomassie staining TS. Each elec-
trophoretic band is stained blue. Next, determine the peak
area of the electrophoretic bands obtained from each stan-
dard solution using a densitometer and calculate the protein
content using the calibration curve mentioned above. When
determining the polymer proteins derived from celmoleukin,
other than celmoleukin monomer, the amount is not more
than 2% in relation to the total protein.
(3) Related substances — Perform the test with 10 iiL each
of Celmoleukin (Genetical Recombination) and 0.01 mol/L
acetic acid buffer solution, pH 5.0, as directed under Liquid
Chromatography <2.01> under the following conditions, and
measure the area of each peak by an automatic integration
method. When the amount of related substances other than
celmoleukin is determined by the area percent method, the
total amount is not more than 5%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm)
Column: Stainless steel tube with an inside diameter of 4
mm and a length of 30 cm packed with octadecylsilanized sili-
ca gel for liquid chromatography (particle size: 5 iim).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A solution of trifluoroacetic acid in a mix-
ture of acetic acid and water (3:2) (1 in 1000)
Mobile phase B: A solution of trifluoroacetic acid in a mix-
ture of acetic acid and water (13:7) (1 in 1000)
Mobile phase flow: The concentration gradient is con-
trolled by changing the ratio of mobile phases A and B as
shown in the table below.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-60
70^10
30^90
Flow: Adjust so that the retention time of celmoleukin is
about 50 minutes.
Time span of measurement: About 1.3 times as long as the
retention time of celmoleukin beginning after the solvent
peak.
System suitability —
Test for required detectability: Measure exactly 0.5 mL of
Celmoleukin (Genetical Recombination), and add 0.01
mol/L acetic acid buffer solution, pH 5.0, to make exactly 50
mL. Confirm that the celmoleukin peak area obtained from
10^<L of this solution is 0.9 to 1.1% of the peak area ob-
tained from 10 iiL of Celmoleukin (Genetical Recombina-
tion).
System performance: Add 2 iiL of 2-mercaptoethanol to
100 iiL of Celmoleukin (Genetical Recombination), leave for
2 hours at 37 °C, and then run this solution under the above
conditions. Under these conditions, celmoleukin and its
reduced form are eluted in this order with the resolution be-
tween these peaks being not less than 3.0.
Ammonium acetate Measure exactly 0.1 mL of Celmoleu-
kin (Genetical Recombination), and add water to make ex-
actly 10 mL. This is the sample solution. Separately, ac-
curately weigh about 0.1 g of ammonium chloride, and add
water to make exactly 100 mL. Measure exactly 5 mL of this
solution, and add water to make exactly 100 mL. This is the
standard stock solution. Measure exactly 3 mL of the stan-
dard stock solution, and add water to make exactly 50 mL.
484
Cetanol / Official Monographs
JP XV
This is the standard solution. Perform the test with exactly 25
liL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. When determining the area of the
ammonium ion peak A T and A s , Celmoleukin (Genetical
Recombination) contains from 0.28 to 0.49 mg of ammoni-
um acetate per mL.
Amount (mg) of ammonium acetate (CH 3 COONH 4 ) per mL
= A-j/As x W s x 0.003 x 1.4410
W s : Amount (mg) of ammonium chloride
0.003: Dilution correction coefficient
1.4410: Molecular weight conversion coefficient for con-
verting ammonium chloride to ammonium acetate
Operating conditions —
Detector: Electronic conductivity detector
Column: Resin column with an inside diameter of 5 mm
and a length of 25 cm packed with weakly acidic ion exchange
resin for liquid chromatography (particle size: 5.5 /xm). As a
guard column, connect to a column with an inside diameter
of 5 mm and a length of 5 cm packed with weakly acidic ion
exchange resin for liquid chromatography.
Column temperature: A constant temperature of about
40°C.
Mobile phase: Diluted 0.1 mol/L methanesulfonic acid TS
(3 in 10).
Flow: Adjust the flow so that the retention time of ammo-
nium is about 8 minutes.
System suitability —
System performance: Measure exactly 1 mL of Standard
Sodium Stock Solution and 0.2 mL of Standard Potassium
Stock Solution, and then add water to make exactly 100 mL.
Measure exactly 5 mL of this solution and 3 mL of the stan-
dard stock solution, and then add water to make exactly 5
mL. When 25 fiL of this solution is run under the above con-
ditions, sodium, ammonium and potassium are eluted in this
order with the resolution between the peaks of sodium and
ammonium being not less than 3.0.
System repeatability: When the test is repeated 5 times with
25 /xL of the standard solution under the above conditions,
the relative standard deviation of the ammonium peak area is
not more than 10%.
Bacterial endotoxins <4.01> Less than lOOEU/mL
Sterility <4.06> Perform the test according to the Direct
method: it meets the requirement. In the test, add 0.5 mL of
Celmoleukin (Genetical Recombination) to 8 test tubes and
1.0 mL of Celmoleukin (Genetical Recombination) to 8 test
tubes containing 15 mL of thioglycol acid I for sterility test,
as well as 1.0 mL of Celmoleukin (Genetical Recombination)
to 8 test tubes containing 15 mL of soybean-casein digest
medium.
Assay (1) Total protein content — Measure accurately 1
mL of Celmoleukin (Genetical Recombination) and add
water to make exactly 10 mL. This is the sample solution.
Separately, weigh accurately about 50 mg of bovine serum al-
bumin for assay in water to prepare standard dilution solu-
tions of 50, 100, and 150,Mg/mL. Measure exactly 1 mL of
the sample solution and each standard dilution solution, add
exactly 2.5 mL of alkaline copper TS for protein content de-
termination, shake, and leave for 15 minutes. Next, add ex-
actly 2.5 mL of water and 0.5 mL of dilute Folin's TS, and
leave for 30 minutes at 37°C. Measure the absorbances of
these solutions at 750 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using 1 mL of water processed in
the same way as control. Using the calibration curve prepared
from the absorbance of the standard dilution solution, deter-
mine the protein content of Celmoleukin (Genetical Recom-
bination).
(2) Specific activity — Measure exactly 0.1 mL of Cel-
moleukin (Genetical Recombination) and add exactly 0.9 mL
of culture medium for celmoleukin to make the sample solu-
tion. Separately, take one Interleukin-2 Reference Standard
and add exactly 1 mL of water to dissolve. This is the stan-
dard solution. Accurately serially dilute the sample and stan-
dard solutions in two-fold steps with culture medium for cel-
moleukin, and add equal volumes of interleukin-2 dependent
mouse natural killer NKC3 cells to the serially diluted solu-
tions. The control solution is a mixture of equal volumes of
NKC3 and culture medium for celmoleukin. Incubate these
solutions for 16 to 24 hours at 37 °C. Following this, add a
volume of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2//-
tetrazolium bromide TS that is 1/5 that of the volume of cul-
ture medium for celmoleukin, incubate for 4 to 6 hours at 37°
C, add a volume of sodium lauryl sulfate TS equivalent to the
volume of the culture medium for celmoleukin, and leave for
24 to 48 hours. After eluting the blue-colored pigment gener-
ated, perform the test on these solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and measure
the absorbance at 590 nm. Taking the absorbance obtained
when 1000 to 2000 units of celmoleukin per mL are added as
100% and the absorbance of the control solution as 0%, de-
termine the dilution factor (A) of the Interleukin-2 Reference
Standard that shows an absorbance of 50% and dilution fac-
tor of Celmoleukin (Genetical Recombination) (B). Multiply
the B/A value by the unit number of the Interleukin-2 Refer-
ence Standard to determine the biological activity of 1 mL of
Celmoleukin (Genetical Recombination). Calculate the ratio
of biological activity in relation to protein content deter-
mined in the total protein content test.
Containers and storage Containers — Sterilized, tight con-
tainers.
Storage — Store at -20°C or lower.
Cetanol
Cetanol is a mixture of solid alcohols, and consists
chiefly of C 16 H 34 0: 242.44.
Description Cetanol occurs as unctuous, white flakes, gran-
ules, or masses. It has a faint, characteristic odor. It is taste-
less.
It is very soluble in pyridine, freely soluble in ethanol (95),
in ethanol (99.5) and in diethyl ether, very slightly soluble in
acetic anhydride, and practically insoluble in water.
Melting point <1.13> 47-53°C Prepare the sample ac-
cording to Method 2, then attach tightly a capillary tube to
the bottom of the thermometer by means of a rubber band or
by any suitable means, and make the bottom of the capillary
tube equal in position to the lower end of the thermometer.
JPXV
Official Monographs / Cetraxate Hydrochloride
485
Insert this thermometer into a test tube 17 mm in inside di-
ameter and about 170 mm in height, fasten the thermometer
with cork stopper so that the lower end of the thermometer is
about 25 mm distant from the bottom of the test tube. Sus-
pend the test tube in a beaker containing water, and heat the
beaker with constant stirring until the temperature rises to
5°C below the expected melting point. Then regulate the rate
of increase to 1°C per minute. The temperature at which the
sample is transparent and no turbidity is produced is taken as
the melting point.
Acid value <1.13> Not more than 1.0.
Ester value <1.13> Not more than 2.0.
Hydroxyl value <7.73> 210 - 232
Iodine value <1.13> Not more than 2.0.
Purity (1) Clarity of solution — Dissolve 3.0 g of Cetanol
in 25 mL of ethanol (99.5) by warming: the solution is clear.
(2) Alkalinity — To the solution obtained in (1) add 2
drops of phenolphthalein TS: no red color develops.
Residue on ignition <2.44> Not more than 0.05% (2 g).
Containers and storage Containers — Well-closed contain-
ers.
Cetraxate Hydrochloride
■feh : 7*-y— h£K£
H
CDjH
■HCI
C 17 H 23 N0 4 .HC1: 341.83
3-{4-[?ra«6 - -4-(Aminomethyl)cyclohexylcarbonyloxy]-
phenyl} propanoic acid monohydrochloride [27724-96-5]
Cetraxate Hydrochloride, when dried, contains not
less than 98.5% of C 17 H 23 N0 4 .HC1.
Description Cetraxate Hydrochloride occurs as white crys-
tals or crystalline powder.
It is soluble in methanol, sparingly soluble in water and in
ethanol (95), and practically insoluble in diethyl ether.
Melting point: about 236°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Cetraxate Hydrochloride in methanol (1 in 2500)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Dissolve 0.5 g of Cetraxate Hydrochloride in 5 mL of
a mixture of water and 2-propanol (1:1) by warming, cool to
below 25 C C. Filter, dry the formed crystals in vacuum for 4
hours, and further dry at 105°C for 1 hour. Determine the in-
frared absorption spectrum of the dried matter as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Cetraxate Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> (2) for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cetraxate Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cetraxate Hydrochloride according to Method 3, and per-
form the test with a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 5) (not more than 2 ppm).
(3) cis Isomer — Dissolve 0.10 g of Cetraxate Hydrochlo-
ride in 10 mL of water, and use this solution as the sample so-
lution. To exactly 5 mL of the sample solution add water to
make exactly 100 mL. To exactly 2 mL of this solution add
water to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /xL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine each peak area of both solu-
tions by the automatic integration method: the area of the
peak which has a retention time 1.3 to 1.6 times that of
cetraxate from the sample solution is not larger than the peak
area of cetraxate from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 220 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Adjust the pH of a mixture of water,
methanol and 0.5 mol/L ammonium acetate TS (15:10:4) to
6.0 with acetic acid (31).
Flow rate: Adjust the flow rate so that the retention time of
cetraxate is about 10 minutes.
System suitability —
System performance: Dissolve 0.02 g of Cetraxate
Hydrochloride and 0.01 g of phenol in 100 mL of water. To 2
mL of this solution add water to make 20 mL. When the
procedure is run with 10 fiL of this solution under the above
operating conditions, cetraxate and phenol are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cetraxate is not more than 2.0%.
(4) 3-(/>Hydroxyphenyl)propionic acid — To 0.10 g of
Cetraxate Hydrochloride add exactly 2 mL of the internal
standard solution and methanol to make 10 mL, and use this
solution as the sample solution. Separately, dissolve 25 mg of
3-(/>hydroxyphenyl)propionic acid in methanol to make ex-
actly 100 mL. To exactly 2 mL of this solution add exactly 2
mL of the internal standard solution and methanol to make
10 mL, and use this solution as the standard solution. Per-
form the test with 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Qs, of the peak area of 3-(/>hydrox-
yphenyl)propionic acid to that of the internal standard: Q T is
not larger than Q s .
486
Chenodeoxycholic Acid / Official Monographs
JP XV
Internal standard solution — A solution of caffeine in
methanol (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of a mixture of water,
methanol and 0.5 mol/L ammonium acetate TS (15:5:2) to
5.5 with acetic acid (31).
Flow rate: Adjust the flow rate so that the retention time of
3-(/?-hydroxyphenyl)propionic acid is about 7 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, 3-(j9-hydroxyphenyl)propionic acid and the inter-
nal standard are eluted in this order with the resolution be-
tween these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of 3-(/?-hydroxyphenyl)propionic acid to that of
the internal standard is not more than 1.0%.
(5) Related substances — Dissolve 0.10 g of Cetraxate
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol and acetic acid
(100) (20:4:3) to a distance of about 10 cm, and air-dry the
plate. Spray evenly ninhydrin TS on the plate, and heat at
90°C for 10 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Chenodeoxycholic Acid
Loss on drying <2.41>
hours).
Not more than 0.5% (0.5 g, 105°C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Cetraxate
Hydrochloride, previously dried, dissolve in 100 mL of
water, and adjust the pH of this solution to between 7.0 and
7.5 with dilute sodium hydroxide TS. To this solution add 10
mL of formaldehyde solution, stir for about 5 minutes, and
titrate <2.50> with 0.1 mol/L sodium hydroxide VS by taking
over about 20 minutes (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 34.18 mg of C 17 H 23 N0 4 .HC1
Containers and storage Containers — Tight containers.
$-/-r**->=i-;uK
H 3 C J
H 3 C„ 1
HjC 1
H [
■~H
„C0 2 H
! H
I "
HO'
H H
H
C 2 4H4o0 4 : 392.57
3a,7a-Dihydroxy-5/?-cholan
[474-25-9]
-24-oic
acid
Chenodeoxycholic Acid, when dried, contains not
less than 98.0% and not more than 101.0% of
C24H40O4.
Description Chenodeoxycholic Acid occurs as white, crys-
tals, crystalline powder or powder.
It is freely soluble in methanol and in ethanol (99.5), solu-
ble in acetone, and practically insoluble in water.
Identification Determine the infrared absorption spectrum
of Chenodeoxycholic Acid, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: + 11.0 -+ 13.0° (after
drying, 0.4 g, ethanol (99.5), 20 mL, 100 mm).
Melting point <2.60> 164 - 169°C
Purity (1) Chloride < 1.03 > —Dissolve 0.36 g of
Chenodeoxycholic Acid in 30 mL of methanol, add 10 mL of
dilute nitric acid and water to make 50 mL, and perform the
test with this solution. Prepare the control solution as fol-
lows: To 1.0 mL of 0.01 mol/L hydrochloric acid VS add 30
mL of methanol, 10 mL of dilute nitric acid and water to
make 50 mL (not more than 0.1%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Chenodeoxycholic Acid according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
(3) Barium — To 2.0 g of Chenodeoxycholic Acid add 100
mL of water, and boil for 2 minutes. To this solution add 2
mL of hydrochloric acid, boil for 2 minutes, filter after cool-
ing, and wash the filter with water until to get 100 mL of the
filtrate. To 10 mL of the filtrate add 1 mL of dilute sulfuric
acid: no turbidity is appeared.
(4) Related substances — Dissolve 0.20 g of Chenodeoxy-
cholic Acid in a mixture of acetone and water (9:1) to make
exactly 10 mL, and use this solution as the sample solution.
Separately, dissolve 10 mg of lithocholic acid for thin-layer
chromatography in the mixture of acetone and water (9:1) to
make exactly 10 mL. Pipet 2 mL of this solution, add the
mixture of acetone and water (9:1) to make exactly 100 mL,
and use this solution as the standard solution (1). Separately,
dissolve 10 mg of ursodeoxycholic acid in the mixture of ace-
tone and water (9:1) to make exactly 100 mL, and use this so-
JPXV
Official Monographs / Chloramphenicol
487
lution as the standard solution (2). Separately, dissolve 10 mg
of cholic acid for thin-layer chromatography in the mixture
of acetone and water (9:1) to make exactly 100 mL, and use
this solution as the standard solution (3). Pipet 1 mL of the
sample solution, and add the mixture of acetone and water
(9:1) to make exactly 20 mL. Pipet 0.5 mL, 1 mL, 2mL, 3
mL and 5 mL of this solution, add the mixture of acetone and
water (9:1) to each of them to make exactly 50 mL, and desig-
nate these solutions as standard solution A, standard solution
B, standard solution C, standard solution D and standard so-
lution E, respectively. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution, standard solutions (1), (2), (3)
and standard solutions A, B, C, D and E on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 4-methyl-2-pentanone, toluene and formic acid
(16:6:1) to a distance of about 15 cm, air-dry the plate, and
further dry at 120°C for 30 minutes. Immediately, spray
evenly a solution of phosphomolybdic acid «-hydrate in
ethanol (95) (1 in 5) on the plate, and heat at 120°C for 2 to 3
minutes: the spot corresponding to the spot with the standard
solution (1) is not more intense than the spot with the stan-
dard solution (1), the spot corresponding to the spot with the
standard solution (2) is not more intense than the spot with
the standard solution (2), and the spot corresponding to the
spot with the standard solution (3) is not more intense than
the spot with the standard solution (3).
As compared to the spots with the standard solutions A, B,
C, D and E, the spots other than the principal spot and other
than the spots mentioned above are not more intense than the
spot with the standard solution E, and the total amount of
them is not more than 1.5%.
Loss on drying <2.4I> Not more than 1.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Chenodeoxycholic
Acid, previously dried, dissolve in 40 mL of ethanol (95) and
20 mL of water, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (potentiometric titration). Perform a blank de-
termination in the same manner, and make any necessary
correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 39.26 mg of C 24 H 40 O4
Containers and storage Containers — Tight containers.
Chloral Hydrate
HO OH
CI
CI CI
C 2 H 3 C1 3 2 : 165.40
2,2,2-Trichloroethane-l , 1 -diol [302-1 7-0]
Chloral Hydrate contains not less than 99.5% of
C 2 H 3 C1 3 2 .
Description Chloral Hydrate occurs as colorless crystals. It
has a pungent odor and an acrid, slightly bitter taste.
It is very soluble in water, and freely soluble in ethanol (95)
and in diethyl ether.
It slowly volatilizes in air.
Identification (1) Dissolve 0.2 g of Chloral Hydrate in 2
mL of water, and add 2 mL of sodium hydroxide TS: the tur-
bidity is produced, and it separates into two clear layers by
warming.
(2) Heat 0.2 g of Chloral Hydrate with 3 drops of aniline
and 3 drops of sodium hydroxide TS: the disagreeable odor
of phenylisocyanide (poisonous) is perceptible.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Chloral Hydrate in 2 mL of water: the solution is clear and
colorless.
(2) Acidity— Dissolve 0.20 g of Chloral Hydrate in 2 mL
of water, and add 1 drop of methyl orange TS: a yellow color
develops.
(3) Chloride <1.03>— Perform the test with 1 .0 g of Chlo-
ral Hydrate. Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.011%).
(4) Chloral alcoholate— Warm 1.0 g of Chloral Hydrate
with 10 mL of sodium hydroxide TS, filter the upper layer,
add iodine TS to the filtrate until a yellow color develops, and
allow the solution to stand for 1 hour: no yellow precipitate is
produced.
(5) Benzene — Warm the solution obtained in (1) with 3
mL of water: no odor of benzene is perceptible.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 4 g of Chloral Hydrate in a
glass-stoppered flask, add 10 mL of water and exactly 40 mL
of 1 mol/L sodium hydroxide VS, and allow the mixture to
stand for exactly 2 minutes. Titrate <2.50> the excess sodium
hydroxide immediately with 0.5 mol/L sulfuric acid VS (indi-
cator: 2 drops of phenolphthalein TS). Perform a blank de-
termination, and make any necessary correction.
Each mL of 1 mol/L sodium hydroxide VS
= 165.4 mg of C 2 H 3 C1 3 2
Containers and storage Containers — Tight containers.
Chloramphenicol
^n^A7x-=i-ji.
C U H, 2 C1 2 N 2 5 : 323.13
2,2-Dichloro-7V-[(l.R,2.R)-l,3-dihydroxy-l-
(4-nitrophenyl)propan-2-yl] acetamide [56- 75-7]
Chloramphenicol contains not less than 980 fig
(potency) and not more than 1020 fig (potency) per mg,
calculated on the dried basis. The potency of Chloram-
phenicol is expressed as mass (potency) of chloram-
phenicol (C„H 12 C1 2 N 2 5 ).
Description Chloramphenicol occurs as white to yellowish
488
Chloramphenicol Palmitate / Official Monographs
JP XV
white, crystals or crystalline powder.
It is freely soluble in methanol and in ethanol (99.5), and
slightly soluble in water.
Identification (1) Determine the absorption spectrum of
the sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Chloramphenicol Reference Standard prepared
in the same manner as the sample solution: both spectra
exhibit similar intensities of absorption at the same wave-
lengths.
(2) Determine the infrared absorption spectrum of
Chloramphenicol as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Chloramphenicol Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
+ 18.5- +21.5° (1.25 g,
Optical rotation <2.49> [a]™'-
ethanol (99.5), 25 mL, 100 mm).
Melting point <2.60> 150 - 155°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Chloramphenicol according to Method 2, and perform the
test. Prepare the control solution with 2.5 mL of Standard
Lead Solution (not more than 25 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Chloramphenicol according to Method 4, and perform the
test (not more than 1 ppm).
(3) Related substances — Dissolve 0.10 g of Chloram-
phenicol in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution (1). Pipet 10 mL of the standard solu-
tion (1), add methanol to make exactly 20 mL, and use this
solution as the standard solution (2). Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 20 [iL each of the sample solution and standard
solutions (1) and (2) on a plate of silica gel with fluorescent
indicator for thin-layer chromatography, develop the plate
with a mixture of chloroform, methanol and acetic acid (100)
(79:14:7) to a distance of about 15 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot and the spot on the
original obtained from the sample solution are not more in-
tense than the spot from the standard solution (1), and the
total amount of these spots is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Chloramphenicol
and Chloramphenicol Reference Standard, equivalent to
about 0.1 g (potency), dissolve each in 20 mL of methanol,
and add water to make exactly 100 mL. Pipet 20 mL each of
these solutions, and add water to make exactly 100 mL. Pipet
10 mL each of these solutions, add water to make exactly 100
mL, and use these solutions as the sample solution and stan-
dard solution. Determine the absorbances, A T and A s , at 278
nm of the sample solution and standard solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>.
Amount [Mg (potency)] of CnH 12 Cl 2 N 2 5
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Chloramphenicol Refer-
ence Standard
Containers and storage Containers — Tight containers.
Chloramphenicol Palmitate
CV,N
CH 3
C 27 H 42 C1 2 N 2 6 : 561.54
(2i?,3i?)-2-(Dichloroacetyl)amino-3-hydroxy-3-
(4-nitrophenyl)propan-l-yl palmitate [530-43-8]
Chloramphenicol Palmitate contains not less than
558 fig (potency) and not more than 587 fig (potency)
per mg, calculated on the dried basis. The potency of
Chloramphenicol Palmitate is expressed as mass
(potency) of chloramphenicol (CnH^C^^CV
323.13).
Description Chloramphenicol Palmitate occurs as a white
to grayish white, crystalline powder.
It is freely soluble in acetone, sparingly soluble in methanol
and in ethanol (99.5), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Chloramphenicol Palmitate in ethanol (99.5) (1 in
33,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Chloramphenicol
Palmitate Reference Standard prepared in the same manner
as the sample solution: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Dissolve 5 mg each of Chloramphenicol Palmitate
and Chloramphenicol Palmitate Reference Standard in 1 mL
of acetone, and use these solutions as the sample solution and
the standard soution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of acetone and
cyclohexane (1:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the principal spot obtained from the sample solu-
tion has the same Rf value as the spot from the standard solu-
tion.
Optical rotation <2.49> [ a ] 2 D 5 : +21 - +25° (1 g calculated on
the dried basis, ethanol (99.5), 20 mL, 100 mm).
Melting point <2.60> 91 - 96°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Chloramphenicol Palmitate according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
JPXV
Official Monographs / Chloramphenicol Sodium Succinate
489
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Chloramphenicol Palmitate according to Method 3, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Chloram-
phenicol Palmitate in 50 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 [iL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. The test should be performed
within 30 minutes after the sample solution and standard so-
lution are prepared. Determine each peak area by the
automatic integration method: the total area of the peaks
other than the peak of chloramphenicol palmitate from the
sample solution is not more than 3.5 times the peak area of
chloramphenicol palmitate from the standard solution. For
this calculation, use the peak areas for chloramphenicol, hav-
ing the relative retention time of about 0.5 with respect to
chloramphenicol palmitate, and for chloramphenicol
dipalmitate, having the relative retention time of about 5.0
with respect to chloramphenicol palmitate, after multiplying
by their relative response factors, 0.5 and 1.4, respectively.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 270 nm).
Column: A stainless steel column 6.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fxva in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: Methanol
Flow rate: Adjust the flow rate so that the retention time of
chloramphenicol palmitate is about 5 minutes.
Time span of measurement: About 6 times as long as the
retention time of chloramphenicol palmitate.
System suitability —
Test for required detectability: Dissolve 50 mg of Chlo-
ramphenicol Palmitate in 50 mL of methanol. Pipet 1 mL of
this solution, add methanol to make exactly 100 mL, and use
this solution as the solution for system suitability test. Pipet 5
mL of the solution for system suitabillity test, and add
methanol to make exactly 50 mL. Confirm that the peak area
of chloramphenicol palmitate obtained from 20 /uL of this so-
lution is equivalent to 7 to 13% of that obtained from 20 /uL
of the solution for system suitability test.
System performance: When the procedure is run with
20 /xL of the solution for system suitability test under the
above operating conditions, the number of theoretical plates
of the peak of chloramphenicol palmitate is not less than
5000.
System repeatability: When the test is repeated 6 times with
20 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of chloramphenicol palmitate is not more
than 1.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Weigh accurately an amount of Chloramphenicol
Palmitate and Chloramphenicol Palmitate Reference Stan-
dard, equivalent to about 37 mg (potency), dissolve each in
40 mL of methanol and exactly 1 mL of acetic acid (100), and
add methanol to make exactly 50 mL. Pipet 10 mL each of
these solutions, add the mobile phase to make exactly 25 mL,
and use these solutions as the sample solution and standard
solution. Perform the test with exactly 10 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s .
Amount [/ug (potency)] of chloramphenicol (CnH^Cys^Os)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Chloramphenicol Palmi-
tate Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /on in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of methanol, water and acetic
acid (100) (172:27:1).
Flow rate: Adjust the flow rate so that the retention time of
chloramphenicol palmitate is about 7 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, the number of theoretical plates of the peak of
chloramphenicol palmitate is not less than 2400.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
chloramphenicol palmitate is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chloramphenicol Sodium Succinate
-?PvA7x-=l-Jl.=l/\^|gi7,x;U1- r- U^A
co 2 Na
C l5 H l5 Cl 2 N 2 Na0 8 : 445.18
Monosodium (2R,3R )-2-(dichloroacetyl)amino-3-hydroxy-
3-(4-nitrophenyl)propan-l-yl succinate [982-57-0]
Chloramphenicol Sodium Succinate contains not
less than 71 1 Lig (potency) per mg, calculated on the an-
hydrous basis. The potency of Chloramphenicol
Sodium Succinate is expressed as mass (potency) of
chloramphenicol (C n H 12 Cl 2 N 2 5 : 323.13).
Description Chloramphenicol Sodium Succinate occurs as
white to yellowish white, crystals or crystalline powder.
490
Chlordiazepoxide / Official Monographs
JP XV
It is very soluble in water, and freely soluble in methanol
and in ethanol (99.5).
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Chloramphenicol Sodium Succinate (1 in 50,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Chlo-
ramphenicol Sodium Succinate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Chloramphenicol Sodium Succinate responds to the
Qualitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ] 2 D 5 : +5 - +8° (1.25 g calculated on
the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .4 g
of Chloramphenicol Sodium Succinate in 5 mL of water is
between 6.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Chloramphenicol Sodium Succinate in 10 mL of water: the
solution is clear and colorless to yellowish.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Chlo-
ramphenicol Sodium Succinate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Chloramphenicol Sodium Succinate according to Method
1, and perform the test (not more than 2 ppm).
Water <2.48> Not more than 2.0% (1.0 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Chloramphenicol
Sodium Succinate, equivalent to about 20 mg (potency), dis-
solve in water to make exactly 1000 mL, and use this solution
as the sample solution. Separately, weigh accurately an
amount of Chloramphenicol Succinate Reference Standard,
equivalent to about 20 mg (potency), add about 50 mL of
water to make a suspension, and add gradually about 7 mL
of 0.01 mol/L sodium hydroxide TS while stirring to adjust
the pH to 7.0. To this solution add water to make exactly
1000 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , at 276 nm of the
sample solution and standard solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>.
Amount L"g (potency)] of chloramphenicol (CnH^Cy^Os)
= W s x (Aj/A s ) x 1000
W s : Amount [mg (potency)] of Chloramphenicol Suc-
cinate Reference Standard
Containers and storage Containers — Hermetic containers.
Chlordiazepoxide
Ci 6 H 14 ClN 3 0: 299.75
7-Chloro-2-methylamino-5-phenyl-3//-l,4-benzodiazepin-
4-oxide [58-25-3]
Chlordiazepoxide, when dried,
than 98.5% of C 16 H 14 C1N 3 0.
contains not less
Description Chlordiazepoxide occurs as white to light yel-
low crystals or crystalline powder.
It is freely soluble in acetic acid (100), sparingly soluble in
ethanol (95), very slightly soluble in diethyl ether, and practi-
cally insoluble in water.
It dissolves in dilute hydrochloric acid.
It is gradually affected by light.
Melting point: about 240°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Chlordiazepoxide in 0.1 mol/L hydrochloric acid
TS (1 in 200,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum or the spectrum of a solution of Chlor-
diazepoxide Reference Standard prepared in the same man-
ner as the sample solution: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectra of Chlor-
diazepoxide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of dried Chlordiazepoxide Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) Proceed with Chlordiazepoxide as directed under
Flame Coloration Test <1.04> (2), and perform the test: a
green color develops.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Chlordiazepoxide according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.20 g of Chlordiazepoxide in exactly 10 mL of a mixture of
methanol and ammonia TS (97:3), and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add a
mixture of methanol and ammonia TS (97:3) to make exactly
200 mL, and use this solution as the standard solution (1).
Separately, dissolve 10 mg of 2-amino-5-chlorobenzophe-
none for thin-layer chromatography in methanol to make ex-
actly 200 mL, and use this solution as the standard solution
(2). Perform the test with the these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 25 /uL of the
JPXV
Official Monographs / Chlordiazepoxide Powder
491
sample solution and 5 fiL each of the standard solutions (1)
and (2) on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate and ethanol (99.5) (19:1) to a distance of
about 12 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution (1). Spray evenly a
solution of sodium nitrite in 1 mol/L hydrochloric acid TS (1
in 100) on the plate, allow to stand for 1 minute, and spray
evenly Af-(l-naphthyl)-/V'-diethylethylenediamine oxalate-
acetone TS on the plate: the spots from the sample solution
are not more intense than the spots from the standard solu-
tion (2).
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Chlordiazepoxide,
previously dried, and dissolve in 50 mL of acetic acid (100).
Titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the supernatant liquid changes from purple through
blue-purple to blue (indicator: 3 drops of crystal violet TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 29.98 mg of C 16 H 14 C1N 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlordiazepoxide Powder
?njUv7-tf7K*v- Hi
Chlordiazepoxide Powder contains not less than 93
% and not more than 107% of the labeled amount of
chlordiazepoxide (C 16 H 14 C1N 3 0: 299.75).
Method of preparation Prepare as directed under Powder,
with Chlordiazepoxide.
Identification (1) Weigh a portion of Chlordiazepoxide
Powder, equivalent to 0.01 g of Chlordiazepoxide according
to the labeled amount, add 100 mL of 0.1 mol/L hydrochlor-
ic acid TS, shake, and filter. To 5 mL of the filtrate add 0.1
mol/L hydrochloric acid TS to make 100 mL, and determine
the absorption spectrum as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 244
nm and 248 nm and between 306 nm and 310 nm, and a mini-
mum between 288 nm and 292 nm.
(2) Weigh a portion of Chlordiazepoxide Powder,
equivalent to 0.02 g of Chlordiazepoxide according to the
labeled amount, add 10 mL of methanol, shake for 5
minutes, then filter by suction through a glass filter (G4),
evaporate the filtrate with the aid of a current of air to dry-
ness, and dry the residue in vacuum at 60°C for 1 hour. De-
termine the infrared absorption spectrum of the residue as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 1625 cm -1 , 1465 cm -1 , 1265
cm 1 , 850 cm ' and 765 cm '.
Purity Conduct this procedure without exposure to day-
light, using light-resistant vessels. To a portion of Chlordia-
zepoxide Powder, equivalent to 50 mg of Chlordiazepoxide
according to the labeled amount, add exactly 5 mL of a mix-
ture of methanol and ammonia TS (97:3), shake, centrifuge,
and use the supernatant liquid as the sample solution.
Separately, dissolve 50 mg of Chlordiazepoxide Reference
Standard in a mixture of methanol and ammonia TS (97:3) to
make exactly 50 mL, and use this solution as the standard so-
lution (1). Dissolve 5.0 mg of 2-amino-5-chlorobenzophe-
none for thin-layer chromatography in methanol to make ex-
actly 200 mL, and use this solution as the standard solution
(2). Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 25 /uL of the sample
solution and 10 /uL each of the standard solutions (1) and (2)
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Proceed as directed in the Purity (2) under
Chlordiazepoxide .
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately a quanti-
ty of Chlordiazepoxide Powder, equivalent to about 0.1 g of
Chlordiazepoxide (C 16 H 14 C1N 3 0), transfer to a glass-stop-
pered flask, wet with exactly 10 mL of water, add exactly 90
mL of methanol, stopper, shake vigorously for 15 minutes,
and centrifuge. Pipet 10 mL of the supernatant liquid, add
exactly 5 mL of the internal standard solution, add methanol
to make exactly 100 mL, and use this solution as the sample
solution. Separately, weigh accurately about 0.1 g of Chlor-
diazepoxide Reference Standard, previously dried in a desic-
cator (in vacuum, phosphorus (V) oxide, 60°C) for 4 hours,
and dissolve in exactly 10 mL of water and 90 mL of
methanol. Pipet 10 mL of this solution, add exactly 5 mL of
the internal standard solution, add methanol to make 100
mL, and use this solution as the standard solution. Perform
the test with 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and calculate the ra-
tios, Qt and Q s , of the peak area of chlordiazepoxide to that
of the internal standard.
Amount (mg) of chlordiazepoxide (C 16 H 14 C1N 3 0)
= W s x (Qj/Q s )
W s : Amount (mg) of Chlordiazepoxide Reference
Standard
Internal standard solution — A solution of isobutyl salicylate
in methanol (1 in 20).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (lO^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and 0.02 mol/L am-
monium dihydrogen phosphate TS (7:3).
Flow rate: Adjust the flow rate so that the retention time of
chlordiazepoxide is about 5 minutes.
System suitability —
System performance: When the procedure is run with
492 Chlordiazepoxide Tablets / Official Monographs
JP XV
10 /xL of the standard solution under the above operating
conditions, chlordiazepoxide and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of chlordiazepoxide to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage-Light-resistant.
Chlordiazepoxide Tablets
^QJU^T-feftf+v Kffi
Chlordiazepoxide Tablets contain not less than 93%
and not more than 107% of the labeled amount of
chlordiazepoxide (C 16 H 14 C1N 3 0: 299.75).
Method of preparation Prepare as directed under Tablets,
with Chlordiazepoxide.
Identification (1) Weigh a portion of powdered Chlordia-
zepoxide Tablets, equivalent to 0.01 g of Chlordiazepoxide
according to the labeled amount, add 100 mL of 0.1 mol/L
hydrochloric acid TS, shake, and filter. To 5 mL of the
filtrate add 0.1 mol/L hydrochloric acid TS to make 100 mL,
and determine the absorption spectrum as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 244 nm and 248 nm and between 306 nm and
310 nm, and a minimum between 288 nm and 292 nm.
(2) Weigh a portion of powdered Chlordiazepoxide
Tablets, equivalent to 0.01 g of Chlordiazepoxide according
to the labeled amount, add 10 mL of diethyl ether, shake
vigorously, and centrifuge. Evaporate 5 mL of the super-
natant liquid by warming on a water bath to dryness. Deter-
mine the infrared absorption spectrum of the residue as
directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 1625 cm -1 , 1465 cm -1 , 1265 cm
-', 850 cm" 1 and 765 cm" 1 .
Purity Related substances — Conduct this procedure
without exposure to daylight, using light-resistant vessels. To
a portion of powdered Chlordiazepoxide Tablets, equivalent
to 50 mg of Chlordiazepoxide according to the labeled
amount, add exactly 5 mL of a mixture of methanol and am-
monia TS (97:3), shake, centrifuge, and use the supernatant
liquid as the sample solution. Separately, dissolve 50 mg of
Chlordiazepoxide Reference Standard in a mixture of
methanol and ammonia TS (97:3) to make exactly 50 mL,
and use this solution as the standard solution (1). Dissolve 5.0
mg of 2-amino-5-chlorobenzophenone for thin-layer chro-
matography in methanol to make exactly 200 mL, and use
this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 25 /uL of the sample solution and 10
[iL each of the standard solutions (1) and (2) on a plate of sili-
ca gel with fluorescent indicator for thin-layer chro-
matography. Proceed as directed in the Purity (2) under
Chlordiazepoxide.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Chlordiazepoxide Tablets
at 100 revolutions per minute according to the Paddle
method, using 900 mL of 2nd fluid for dissolution test as the
test solution. Take 30 mL or more of the dissolved solution
60 minutes after start of the test, and filter through a mem-
brane filter with a pore size not exceeding 0.8 nm. Discard the
first 10 mL of the filtrate, pipet the subsequent FmL, add
2nd fluid for dissolution test to make exactly V mL so that
each mL contains about 3.7 /ug of chlordiazepoxide (C 16 H I4
C1N 3 0) according to the labeled amount, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 12 mg of chlordiazepoxide for assay, previously dried
in a desiccator for 4 hours (in vacuum, phosphorus (V) oxide,
60°C), and dissolve in 2nd fluid for dissolution test to make
exactly 200 mL. Pipet 3 mL of this solution, add 2nd fluid for
dissolution test to make exactly 50 mL, and use this solution
as the standard solution. Determine the absorbances, A T and
^4 S , of the sample solution and standard solution at 260 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Chlordiazepoxide Tablets in 60
minutes is not less than 70%.
Dissolution rate (%) with respect to the labeled
amount of chlordiazepoxide (Ci 6 H 14 ON 3 0)
= W s x (Aj/A s ) x (V'/V) x (1/C) x 27
W s : Amount (mg) of chlordiazepoxide for assay.
C: Labeled amount (mg) of chlordiazepoxide
(C 16 H 14 C1N 3 0) in 1 tablet.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately a quanti-
ty of Chlordiazepoxide Tablets, equivalent to about 0.1 g of
Chlordiazepoxide (C 16 H 14 C1N 3 0), transfer to a glass-stop-
pered flask, add 10 mL of water, and shake well to disinte-
grate. Add 60 mL of methanol, shake well, add methanol to
make exactly 100 mL, and centrifuge. Pipet 10 mL of the su-
pernatant liquid, add exactly 5 mL of the internal standard
solution, add methanol to make exactly 100 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 10 mg of Chlordiazepoxide Reference Standard, previ-
ously dried in a desiccator (in vacuum, phosphorus (V) oxide,
60°C) for 4 hours, dissolve in 1 mL of water and a suitable
amount of methanol, add exactly 5 mL of the internal stan-
dard solution, add methanol to make 100 mL, and use this
solution as the standard solution. Perform the test with 10 /iL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of chlordiazepoxide to that of the internal stan-
dard.
Amount (mg) of chlordiazepoxide (C 16 H 14 C1N 3 0)
= W s x (g T /Q s ) x 10
W s : Amount (mg) of Chlordiazepoxide Reference
Standard
Internal standard solution — A solution of isobutyl salicylate
in methanol (1 in 20).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
JPXV
Official Monographs / Chlorhexidine Hydrochloride
493
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and 0.02 mol/L am-
monium dihydrogen phosphate TS (7:3).
Flow rate: Adjust the flow rate so that the retention time of
chlordiazepoxide is about 5 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, chlordiazepoxide and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of chlordiazepoxide to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Chlorhexidine Gluconate Solution
O n )l^5->i>>?)lzi >|g±£;«
Chlorhexidine Gluconate Solution is a solution of
digluconate of chlorhexidine.
It contains not less than 19.0 w/v% and not more
than 21.0w/v% of chlorhexidine gluconate
(C 22 H 30 Cl 2 N 10 .2C 6 H 12 O 7 : 897.76).
Description Chlorhexidine Gluconate Solution is a clear,
colorless or pale yellow liquid. It is odorless, and has a bitter
taste.
It is miscible with water and with acetic acid (100). 1 mL of
Chlorhexidine Gluconate Solution is miscible with not more
than 5 mL of ethanol (99.5) and with not more than 3 mL of
acetone. By further addition of each of these solvents, a
white turbidity is formed.
It is gradually colored by light.
Specific gravity df : 1.06 - 1.07
Identification (1) To 0.05 mL of Chlorhexidine Gluconate
Solution add 5 mL of methanol, 1 mL of bromine TS and 1
mL of 8 mol/L sodium hydroxide TS: a deep red color is
produced.
(2) To 0.5 mL of Chlorhexidine Gluconate Solution add
10 mL of water and 0.5 mL of copper (II) sulfate TS: a white
precipitate is formed. Heat to boiling: the precipitate changes
to light purple.
(3) To 10 mL of Chlorhexidine Gluconate Solution add 5
mL of water, cool on ice, and add 5 mL of sodium hydroxide
TS dropwise with stirring: a white precipitate is formed. Col-
lect the precipitate by filtration, wash with water, recrystallize
from diluted ethanol (95) (7 in 10), and dry at 105 °C for 30
minutes: the crystals thus obtained melt <2.60> between 130°
C and 134°C.
(4) Neutralize the filtrate obtained in (3) with 5 mol/L
hydrochloric acid TS. To 5 mL of this solution add 0.65 mL
of acetic acid (100) and 1 mL of freshly distilled phenylhydra-
zine, and heat on a water bath for 30 minutes. After cooling,
scratch the inner wall of the vessel with a glass rod to induce
crystallization. Collect the crystals, dissolve in 10 mL of hot
water, add a small amount of activated charcoal, and filter.
Cool the filtrate, scratch the inner side of the vessel, collect
the formed crystals, and dry: the crystals thus obtained melt
<2.60> at about 195°C (with decomposition).
pH <2.54> To 5.0 mL of Chlorhexidine Gluconate Solution
add water to make 100 mL: the pH of the solution is between
5.5 and 7.0.
Purity p-Chloroaniline — To 2.0 mL of Chlorhexidine
Gluconate Solution add water to make exactly 100 mL. Pipet
5 mL of the solution, and add 20 mL of water and 5 mL of 1
mol/L hydrochloric acid TS. Add 0.3 mL of sodium nitrite
TS, shake, and allow to stand for 2 minutes. Add 4 mL of
ammonium amidosulfate TS, and then allow to stand for 1
minute. Add 5 mL of /V-(l-naphthyl)-/V'-diethylethylenedia-
mine oxalate-acetone TS, allow to stand for 10 minutes, add
1 mL of ethanol (95), and then add water to make 50 mL: the
color of the solution is not more intense than the following
control solution.
Control solution: Dissolve 20 mg of 4-chloroaniline in 10
mL of 1 mol/L hydrochloric acid TS, and add water to make
exactly 100 mL. Pipet 5 mL of the solution, and add water to
make exactly 100 mL. Pipet 5 mL of the solution, add 20 mL
of water and 5 mL of 1 mol/L hydrochloric acid TS, and pro-
ceed as directed for the preparation of the sample solution.
Residue on ignition <2.44> Not more than 0.1% (2 g, after
evaporation).
Assay Pipet 2 mL of Chlorhexidine Gluconate Solution,
evaporate to dryness on a water bath, dissolve the residue in
60 mL of acetic acid for nonaqueous titration, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 22.44 mg of C 22 H 30 Cl 2 N 10 .2C 6 H 12 O 7
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorhexidine Hydrochloride
<7Q)l'\lr'>it>mMik
N N N
Y Y
NH NH
NH NH
H H H
CI
•2HCI
C 22 H 30 C1 2 N I0 .2HC1: 578.37
1,1' -Hexamethylenebis [5-(4-chlorophenyl)biguanide]
dihydrochloride [3697-42-5]
Chlorhexidine Hydrochloride, when dried, contains
not less than 98.0% of C 22 H 30 C1 2 N 10 .2HC1.
Description Chlorhexidine Hydrochloride occurs as a
white, crystalline powder. It is odorless, and has a bitter
taste.
494
Chlorinated Lime / Official Monographs
JP XV
It is soluble in formic acid, slightly soluble in methanol and
in warm methanol, and practically insoluble in water, in
ethanol (95) and in diethyl ether.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Chlorhexidine
Hydrochloride in 5 mL of methanol by warming, and add 1
mL of bromine TS and 1 mL of 8 mol/L sodium hydroxide
TS: a deep red color is produced.
(2) Dissolve 0.3 g of Chlorhexidine Hydrochloride in 10
mL of 6 mol/L hydrochloric acid TS, cool in ice, and add 10
mL of 8 mol/L sodium hydroxide TS dropwise with stirring:
a white precipitate is produced. Collect the precipitate, wash
with water, recrystallize from diluted ethanol (95) (7 in 10),
and dry at 105°C for 30 minutes: the crystals so obtained
melt <2.60> between 130°C and 134°C.
(3) Dissolve 0.1 g of Chlorhexidine Hydrochloride in 50
mL of dilute nitric acid: the solution responds to the Qualita-
tive Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Chlorhexidine Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic < 1.1] > —To 1.0 g of Chlorhexidine
Hydrochloride in a crucible add 10 mL of a solution of mag-
nesium nitrate hexahydrate in ethanol (95) (1 in 10), fire the
ethanol (95) to burn, and heat gradually to incinerate. If a
carbonized substance remains, moisten with a small amount
of nitric acid, and ignite to incinerate. Cool, add 10 mL of di-
lute hydrochloric acid to the residue, dissolve by warming on
a water bath, use this solution as the test solution, and per-
form the test (not more than 2 ppm).
(3) /?-Chloroaniline — Dissolve 0.10 g of Chlorhexidine
Hydrochloride in 2 mL of formic acid, and add 15 mL of 1
mol/L hydrochloric acid TS and 20 mL of water immediate-
ly. Add 0.3 mL of sodium nitrite TS, shake, and allow to
stand for 2 minutes. Add 4 mL of ammonium amidosulfate
TS, and then allow to stand for 1 minute. Add 5 mL of 7V-(1-
naphthyl)-7V'-diethylethylenediamine oxalate-acetone TS,
allow to stand for 10 minutes, and add 1 mL of ethanol (95)
and water to make 50 mL: the solution has no more color
than the following control solution.
Control solution: Dissolve 20 mg of 4-chloroaniline in 10
mL of 1 mol/L hydrochloric acid TS, and add water to make
exactly 100 mL. Pipet 5 mL of the solution, and add water to
make exactly 100 mL. To 2.0 mL of the solution add 2 mL of
formic acid, 15 mL of 1 mol/L hydrochloric acid TS and 20
mL of water, and proceed in the same manner.
Loss on drying <2.41> Not more than 2.0% (1 g, 130°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Chlorhexidine
Hydrochloride, previously dried, dissolve in 2.0 mL of for-
mic acid, add 60 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 14.46 mg of C 22 H 30 C1 2 N 10 .2HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorinated Lime
Chlorinated Lime contains not less than 30.0% of
available chlorine (CI: 35.45).
Description Chlorinated Lime occurs as a white powder. It
has a chlorine-like odor.
It dissolves partially in water. The solution changes red lit-
mus paper to blue, then gradually decolorizes.
Identification (1) To Chlorinated Lime add dilute
hydrochloric acid: a gas, which has the odor of chlorine,
evolves, and the gas changes moistened starch-potassium
iodide paper to blue.
(2) Shake 1 g of Chlorinated Lime with 10 mL of water,
and filter: the filtrate responds to the Qualitative Tests <1.09>
(2) and (3) for calcium salt.
Assay Weigh accurately about 5 g of Chlorinated Lime,
transfer to a mortar, and triturate thoroughly with 50 mL of
water. Transfer to a 500-mL volumetric flask with the aid of
water, and add water to make 500 mL. Mix well, immediately
take exactly 50 mL of the mixture in an iodine flask, add 10
mL of potassium iodide TS and 10 mL of dilute hydrochloric
acid, and titrate <2.50> the liberated iodine with 0.1 mol/L
sodium thiosulfate VS (indicator: 3 mL of starch TS). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 3.545 mg of CI
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Chlormadinone Acetate
O CHj
C 23 H 29 C10 4 : 404.93
6-Chloro-3,20-dioxopregna-4,6-dien-17-yl acetate
[302-22-7]
Chlormadinone Acetate, when dried, contains not
less than 98.0% of C 23 H 29 C10 4 .
Description Chlormadinone Acetate occurs as white to light
yellow crystals or crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in acetonitrile,
slightly soluble in ethanol (95) and in diethyl ether, and prac-
tically insoluble in water.
JPXV
Official Monographs / Chlorobutanol 495
Identification (1) Dissolve 2 mg of Chlormadinone
Acetate in 1 mL of ethanol (95), and add 1 mL of 1,3-
dinitrobenzene TS and 1 mL of a solution of potassium
hydroxide (1 in 5): a red-purple color develops.
(2) To 0.05 g of Chlormadinone Acetate add 2 mL of
potassium hydroxide-ethanol TS, and boil on a water bath
for 5 minutes. After cooling, add 2 mL of diluted sulfuric
acid (2 in 7), and boil gently for 1 minute: the odor of ethyl
acetate is perceptible.
(3) Determine the infrared absorption spectrum of Chlor-
madinone Acetate, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum or the spectrum of previously dried Chlormadi-
none Acetate Reference Standard: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(4) Perform the test with Chlormadinone Acetate as
directed under Flame Coloration Test <1.04> (2): a green
color appears.
Optical rotation <2.49> [ a ]^:
10.0- -14.0° (after
drying, 0.2 g, acetonitrile, 10 mL, 100 mm).
Melting point <2. 60> 2 1 1 - 2 1 5 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Chlormadinone Acetate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Chlormadinone Acetate according to Method 3, and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 20 mg of Chlormadi-
none Acetate in 10 mL of acetonitrile, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
acetonitrile to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the total area of peaks other than
the peak of chlormadinone acetate from the sample solution
is not larger than the peak area of chlormadinone acetate
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 236 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of acetonitrile and water (13:7).
Flow rate: Adjust the flow rate so that the retention time of
chlormadinone acetate is about 10 minutes.
Time span of measurement: About 1.5 times as long as the
retention time of chlormadinone acetate beginning after the
solvent peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add acetonitorile to make exactly 50 mL. Con-
firm that the peak area of chlormadinone acetate obtained
from 10 /xL of this solution is equivalent to 7 to 13% of that
of chlormadinone acetate obtained from 10 juL of the stan-
dard solution.
System performance: Dissolve 8 mg of Chlormadinone
Acetate and 2 mg of butyl parahydroxybenzoate in 100 mL
of acetonitrile. When the procedure is run with 10 juL of this
solution under the above operating conditions, butyl para-
hydroxybenzoate and chlormadinone acetate are eluted in
this order with the resolution between these peaks being not
less than 8.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
chlormadinone acetate is not more than 1.0%.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 20 mg each of Chlormadi-
none Acetate and Chlormadinone Acetate Reference Stan-
dard, previously dried, and dissolve in ethanol (95) to make
exactly 100 mL. Pipet 5 mL each of these solutions, to each
add ethanol (95) to make exactly 100 mL, and use these solu-
tions as the sample solution and standard solution, respec-
tively. Perform the test with these solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and determine
the absorbances, A T and A s , at 285 nm.
Amount (mg) of C23H29CIO4
= W s x (A T /A S )
W s : Amount (mg) of Chlormadinone Acetate Reference
Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorobutanol
?n n~/?/— JU
H 3 C Ch\ 3
CI CI
C 4 H 7 C1 3 0: 177.46
1 , 1 , l-Trichloro-2-methylpropan-2-ol
[57-75-5]
Chlorobutanol contains not less than 98.0% of
C4H7CI3O, calculated on the anhydrous basis.
Description Chlorobutanol occurs as colorless or white
crystals. It has a camphoraceous odor.
It is very soluble in methanol, in ethanol (95) and in diethyl
ether, and slightly soluble in water.
It slowly volatilizes in air.
Melting point: not lower than about 76°C.
Identification (1) To 5 mL of a solution of Chlorobutanol
(1 in 200) add 1 mL of sodium hydroxide TS, then slowly add
3 mL of iodine TS: a yellow precipitate is produced and the
odor of iodoform is perceptible.
(2) To 0.1 g of Chlorobutanol add 5 mL of sodium
hydroxide TS, shake well the mixture, add 3 to 4 drops of
aniline, and warm gently: the disagreeable odor of phenyl
isocyanide (poisonous) is perceptible.
496
Chlorphenesin Carbamate / Official Monographs
JP XV
Purity (1) Acidity — Shake thoroughly 0.10 g of the pow-
der of Chlorobutanol with 5 mL of water: the solution is neu-
tral.
(2) Chloride <1.03>— Dissolve 0.5 g of Chlorobutanol in
25 mL of dilute ethanol, and add 6 mL of dilute nitric acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with 1 .0
mL of 0.01 mol/L hydrochloric acid VS by adding 25 mL of
dilute ethanol, 6 mL of dilute nitric acid and water to make
50 mL (not more than 0.071%).
Water <2.4S> Not more than 6.0% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Transfer about 0.1 g of Chlorobutanol, accurately
weighed, to a 200-mL conical flask, and dissolve in 10 mL of
ethanol (95). Add 10 mL of sodium hydroxide TS, boil under
a reflux condenser for 10 minutes, cool, add 40 mL of dilute
nitric acid and exactly 25 mL of 0.1 mol/L silver nitrate VS,
and shake well. Add 3 mL of nitrobenzene, and shake
vigorously until the precipitate is coagulated. Titrate <2.50>
the excess silver nitrate with 0.1 mol/L ammonium thioc-
yanate VS (indicator: 2 mL of ammonium iron (III) sulfate
TS). Perform a blank determination.
Each mL of 0.1 mol/L silver nitrate VS
= 5.915 mg of C 4 H 7 C1 3
Containers and storage Containers — Tight containers.
Chlorphenesin Carbamate
?njU7i*->>*),/U/U >IiXxJl
and enanttomer
C 10 H 12 ClNO 4 : 245.66
(2/?5)-3-(4-Chlorophenoxy)-2-hydroxypropyl carbamate
[886-74-8]
Chlorphenesin Carbamate, when dried, contains not
less than 98.0% and not more than 102.0% of
C 10 H 12 ClNO 4 .
Description Chlorphenesin Carbamate occurs as white crys-
tals or crystalline powder. It is odorless, and has a slightly
bitter taste.
It is freely soluble in methanol, in ethanol (95) and in pyri-
dine, soluble in 2-propanol, sparingly soluble in diethyl ether,
slightly soluble in water, and practically insoluble in hexane.
A solution of Chlorphenesin Carbamate in ethanol (95) (1
in 20) shows no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Chlorphenesin Carbamate in ethanol (95) (3 in
200,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Chlor-
phenesin Carbamate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Perform the test with Chlorphenesin Carbamate as
directed under Flame Coloration Test <1.04> (2): a green
color appears.
Melting point <2.60>
;-91°C
Purity (1) Heavy metals <1.07> — Dissolve 2.0 g of Chlor-
phenesin Carbamate in 20 mL of ethanol (95), and add 2 mL
of dilute acetic acid and water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 20 mL of ethanol (95), 2 mL of dilute acetic acid and
water to make 50 mL (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1 .0 g of
Chlorphenesin Carbamate according to Method 3, and per-
form the test (not more than 2 ppm).
(3) Related substances — (i) Chlorphenesin-2-carba-
mate: Dissolve 0.10 g of Chlorphenesin Carbamate in 20 mL
of a mixture of hexane for liquid chromatography and 2-
propanol (7:3), and use this solution as the sample solution.
Perform the test with 10 fiL of the sample solution as directed
under Liquid Chromatography <2.01> according to the
following conditions. Determine the peak area, A R , of chlor-
phenesin carbamate and the peak area, A b , of chlorphenesin-
2-carbamate by the automatic integration method: the ratio,
A h /(Az + A b ), is not larger than 0.007.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with silica gel for liquid chro-
matography (5 /um in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of hexane for liquid chro-
matography, 2-propanol and acetic acid (100) (700:300:1).
Flow rate: Adjust the flow rate so that the retention time of
chlorphenesin carbamate is about 9 minutes.
System suitability —
Test for required detection: Pipet 1 mL of the sample
solution, add a mixture of hexane for liquid chromatography
and 2-propanol (7:3) to make exactly 100 mL, and use this so-
lution as the solution for system suitability test. To exactly 5
mL of the solution for system suitability test add a mixture of
hexane for liquid chromatography and 2-propanol (7:3) to
make exactly 10 mL. Confirm that the peak area of chlor-
phenesin carbamate obtained from 10 /uL of this solution is
equivalent to 40 to 60% of that of chlorphenesin carbamate
obtained from 10,mL of the solution for system suitability
test.
System performance: Dissolve 0.1 g of Chlorphenesin Car-
bamate in 50 mL of methanol. To 25 mL of this solution add
25 mL of dilute sodium hydroxide TS, and warm at 60°C for
20 minutes. To 20 mL of this solution add 5 mL of 1 mol/L
hydrochloric acid TS, shake well with 20 mL of ethyl acetate,
and allow to stand to separate the ethyl acetate layer. When
the procedure is run with 10 /xL of this layer under the above
operating conditions, chlorphenesin, chlorphenesin carba-
mate and chlorphenesin-2-carbamate are eluted in this order,
with the ratios of the retention time of chlorphenesin and
JP XV
Official Monographs / Chlorpheniramine and Calcium Powder
497
chlorphenesin-2-carbamate with respect to chlorphenesin car-
bamate are about 0.7 and about 1.2, respectively, and with
the resolution between the peaks of chlorphenesin and chlor-
phenesin carbamate being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of chlorphenesin carbamate is not more than
2.0%.
(ii) Other related substances: Dissolve 0.10 g of Chlor-
phenesin Carbamate in 10 mL of ethanol (95), and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add ethanol (95) to make exactly 20 mL. Pipet 2 mL
of this solution, add ethanol (95) to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 50,aL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, methanol and ammonia solution (28) (17:2:1) to a
distance of about 10 cm, and air-dry the plate. Allow the
plate to stand in iodine vapor for 20 minutes: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, in vacu-
um, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Chlorphenesin Car-
bamate, previously dried, dissolve in 20 mL of pyridine, add
exactly 50 mL of 0.1 mol/L potassium hydroxide-ethanol
TS, and warm at 70°C for 40 minutes. After cooling, add 100
mL of ethanol (95), and titrate <2.50> the excess potassium
hydroxide with 0.1 mol/L hydrochloric acid VS until the
color of the solution changes from blue through blue-green to
yellow (indicator: 1 mL of thymol blue TS). Perform a blank
determination.
Each mL of 0.1 mol/L potassium hydroxide-ethanol TS
= 24.57 mg of C 10 H 12 ClNO 4
Containers and storage Containers — Tight containers.
Chlorpheniramine and Calcium
Powder
^n;u7.
■t>)l->OAWi
Chlorpheniramine and Calcium Powder contains
not less than 0.27% and not more than 0.33% of chlor-
pheniramine maleate (C^H^ClNj.QH^: 390.86).
Method of preparation
3g
800 g
Chlorpheniramine Maleate
Dibasic Calcium Phosphate Hydrate
Starch, Lactose Hydrate, or
their mixture a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Chlorpheniramine and Calcium Powder occurs
as a white powder.
Identification (1) Determine the absorption spectrum of
the sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 263 nm and 267 nm (chlorpheniramine
maleate).
(2) To 0.5 g of Chlorpheniramine and Calcium Powder
add 10 mL of dilute hydrochloric acid, shake well, and filter:
the filtrate responds to the Qualitative Tests <1.09> (3) for cal-
cium salt.
(3) To 0.5 g of Chlorpheniramine and Calcium Powder
add 10 mL of dilute nitric acid, shake well, and filter: the
filtrate responds to the Qualitative tests <1.09> (2) for phos-
phate.
(4) Shake 1 g of Chlorpheniramine and Calcium Powder
with 5 mL of methanol, filter, and use the filtrate as the sam-
ple solution. Separately, dissolve 0.01 g of chlorpheniramine
maleate in 17 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL each of the sample solution and standard solution on the
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, methanol, acetone and ammonia solution (28)
(73:15:10:2) to a distance of about 10 cm. Air-dry the plate,
and examine under ultraviolet light (main wavelength: 254
nm): the spots from the sample solution and from the stan-
dard solution show the same Rf value. Spray evenly Dragen-
dorff's TS for spraying upon the plate: the spot from the
standard solution and the corresponding spot from the sam-
ple solution reveal an orange color.
Assay Weigh accurately about 0.5 g of Chlorpheniramine
and Calcium Powder, transfer to a 30-mL glass-stoppered
centrifuge tube, add 20 mL of 0.05 mol/L sulfuric acid VS,
shake for 5 minutes, centrifuge, and collect the supernatant
liquid. Add 20 mL of 0.05 mol/L sulfuric acid VS to the
residue, and proceed twice in the same manner mentioned
above. Transfer all the supernatant liquid to a 200-mL sepa-
rator, add 30 mL of diethyl ether, shake, and allow to stand
for 5 minutes. Filter the water layer through dry filter paper
into another separator. Extract the diethyl ether layer with
two 10-mL portions of 0.05 mol/L sulfuric acid VS, filter the
extracts into the preceding separator containing the water
layer. Wash the filter paper with 5 mL of 0.05 mol/L sulfuric
acid VS, combine the washings with the water layer in the
preceding separator, and add 10 mL of ammonia TS. Extract
with two 50-mL portions of diethyl ether, combine the
diethyl ether layer, wash with 20 mL of water, and extract the
diethyl ether layer with two 20-mL and 5-mL portions of 0.25
mol/L sulfuric acid VS. Combine all the extracts, add 0.25
mol/L sulfuric acid VS to make exactly 50 mL, and use this
solution as the sample solution. Separately, dissolve about 75
mg of chlorpheniramine maleate for assay, previously dried
at 105°C for 3 hours and accurately weighed, in 10 mL of
0.05 mol/L sulfuric acid VS, and add 0.05 mol/L sulfuric
acid VS to make exactly 100 mL. Pipet 2 mL of the solution
into a 200-mL separator, add 58 mL of 0.05 mol/L sulfuric
acid VS and 30 mL of diethyl ether, and shake. Proceed in
the same manner as the sample solution, and use this solution
as the standard solution. Determine the absorbances, A T and
498
Chlorpheniramine Maleate / Official Monographs
JP XV
A s , of the sample solution and the standard solution at 265
nm as directed under Ultraviolet-visible Spectrophotometry <
2.24>, using 0.25 mol/L sulfuric acid VS as the blank.
Amount (mg) of chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 )
= ^s x G4tA4s)x(1/50)
W s : Amount (mg) of chlorpheniramine maleate for assay
Containers and storage Containers — Well-closed contain-
ers.
Chlorpheniramine Maleate
I
(
CO^H
C0 2 H
and enantiomer
C 16 H 19 C1N 2 .C4H 4 4 : 390.86
(3/?5')-3-(4-Chlorophenyl)-Af,iV-dimethyl-3-pyridin-2-
ylpropylamine monomaleate [113-92-8]
Chlorpheniramine Maleate, when dried, contains
not less than 98.0% and not more than 101.0% of dl-
chlorpheniramine maleate (C 16 H 19 C1N2.C4H 4 04).
Description Chlorpheniramine Maleate occurs as white,
fine crystals.
It is very soluble in acetic acid (100), freely soluble in water
and in methanol, and soluble in ethanol (99.5).
It dissolves in dilute hydrochloric acid.
A solution of Chlorpheniramine Maleate (1 in 20) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Chlorpheniramine Maleate in 0.1 mol/L
hydrochloric acid TS (3 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Chlorpheniramine Maleate Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Chlor-
pheniramine Maleate, previously dried, as directed in the
paste method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of previously dried Chlorpheniramine Maleate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Dissolve 0.10 g of Chlorpheniramine Maleate in 5 mL
of methanol, and use this solution as the sample solution.
Separately, dissolve 56 mg of maleic acid in 10 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 fiL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
diethyl ether, methanol, acetic acid (100) and water
(70:20:7:3) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): a spot among two of the spots obtained with the sample
solution shows the same intense and Rf value with the spot
with the standard solution.
pH <2.54> Dissolve 1.0 g of Chlorpheniramine Maleate in
100 mL of freshly boiled and cooled water: the pH of this so-
lution is between 4.0 and 5.5.
Melting point <2.60> 130 - 135°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Chlorpheniramine Maleate in 50 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Chlor-
pheniramine Maleate according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Chlorphenira-
mine Maleate in 100 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 3 mL of the sample solu-
tion, and add the mobile phase to make exactly 100 mL.
Pipet 2 mL of this solution, add the mobile phase to make ex-
actly 20 mL, and use this solution as the standard solution.
Perform the test with exactly 20 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the peak area other than maleic acid and chlor-
pheniramine is not larger than 2/3 times the peak area of
chlorpheniramine obtained with the standard solution, and
the total area of the peaks other than maleic acid and chlor-
pheniramine is not larger than the peak area of chlorphenira-
mine with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 8.57 g of ammonium dihydrogen
phosphate and 1 mL of phosphoric acid in water to make
1000 mL. To 800 mL of this solution add 200 mL of acetoni-
trile.
Flow rate: Adjust the flow rate so that the retention time of
chlorpheniramine is about 11 minutes.
Time span of measurement: About 4 times as long as the
retention time of chlorpheniramine after the solvent peak.
System suitability —
Test for required detectability: To exactly 2.5 mL of the
standard solution add the mobile phase to make exactly 25
mL. Confirm that the peak area of chlorpheniramine ob-
tained with 20 [iL of this solution is equivalent to 7 to 13% of
that with 20 iiL of the standard solution.
System performance: When the procedure is run with 20
iiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of chlorpheniramine are not less than 4000
and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
JP XV
Official Monographs / Chlorpheniramine Maleate Powder
499
20 nL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
chlorpheniramine is not more than 4.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 0.4 g of Chlorpheniramine Maleate,
previously dried and accurately weighed, in 20 mL of acetic
acid (100). Titrate <2.50> with 0.1 mol/L perchloric acid VS
until the color of the solution changes from purple through
blue-green to green (indicator: 2 drops of crystal violet TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 19.54 mg of C 16 H 19 C1N 2 .C 4 H 4 04
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorpheniramine Maleate
Injection
Chlorpheniramine Maleate Injection is an aqueous
solution for injection.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of cW-chlorpheniramine
maleate (C 16 H 19 C1N 2 .C4H 4 4 : 390.86).
Method of preparation Prepare as directed under Injec-
tions, with Chlorpheniramine Maleate.
Description Chlorpheniramine Maleate Injection is a clear,
colorless liquid.
pH: 4.5-7.0
Identification Take a volume of Chlorpheniramine Maleate
Injection, equivalent to 25 mg of Chlorpheniramine Maleate
according to the labeled amount, add 5 mL of dilute sodium
hydroxide TS, and extract with 20 mL of hexane. Wash the
hexane layer with 10 mL of water, shake with 0.5 g of anhy-
drous sodium sulfate for several minutes, and filter.
Evaporate the filtrate in a water bath at 50°C under a reduced
pressure, and determine the infrared absorption spectrum of
the residue as directed in the liquid film method under In-
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 2940 cm -1 , 2810 cm -1 , 2770
cm -1 , 1589 cm -1 , 1491cm -1 , 1470 cm -1 , 1434 cm -1 , 1091
cm -1 and 1015 cm -1 .
Bacterial endotoxins <4.01> Less than 8.8 EU/mg.
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Transfer an exactly measured volume of Chlor-
pheniramine Maleate Injection, equivalent to about 3 mg of
chlorpheniramine maleate (Ci 6 H 19 ClN 2 .C 4 H 4 4 ), to a
100-mL separator, add 20 mL of water and 2 mL of sodium
hydroxide TS, and extract with two 50-mL portions of
diethyl ether. Combine the diethyl ether extracts, wash with
20 mL of water, and then extract with 20-mL, 20-mL and
5-mL portions of 0.25 mol/L sulfuric acid TS successively.
Combine all acid extracts, and add 0.25 mol/L sulfuric acid
TS to make exactly 50 mL. Pipet 10 mL of this solution, add
0.25 mol/L sulfuric acid TS to make exactly 25 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 30 mg of Chlorpheniramine Maleate Refer-
ence Standard, previously dried at 105°C for 3 hours, and
dissolve in water to make exactly 200 mL. Pipet 20 mL of this
solution, transfer to a 100-mL separator, add 2 mL of sodi-
um hydroxide TS, and extract with two 50-mL portions of
diethyl ether. Proceed in the same manner as for the prepara-
tion of the sample solution, and use the solution so obtained
as the standard solution. Determine the absorbances A T and
A s of the sample solution and standard solution at a
wavelength of the maximum absorbance at about 265 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 )
= W s x (A T /A S ) x (1/10)
W s : Amount (mg) of Chlorpheniramine Maleate Refer-
ence Standard
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Chlorpheniramine Maleate Powder
? p )vi x - =7 5 > v u << >m^m
Chlorpheniramine Maleate Powder contains not less
than 93.0% and not more than 107.0% of the labeled
amount of ^-chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 : 390.86).
Method of preparation Prepare as directed under Powders,
with Chlorpheniramine Maleate.
Identification Weigh a portion of Chlorpheniramine Male-
ate Powder, equivalent to 50 mg of Chlorpheniramine Male-
ate according to the labeled amount, shake with 40 mL of 0.1
mol/L hydrochloric acid TS, and filter. Transfer the filtrate
to a separator, and wash with 40 mL of hexane. Add 10 mL
of sodium hydroxide TS, and extract with 20 mL of hexane.
Wash the hexane layer with 5 mL of water. Centrifuge, if
necessary, shake the hexane extract with 0.5 g of anhydrous
sodium sulfate for several minutes, and filter. Evaporate the
filtrate in a water bath under reduced pressure, and determine
the spectrum of the residue as directed in the liquid film
method under Infrared Spectrophotometry <2.25>: it exhibits
absorption at the wave number of about 2940 cm -1 , 2810
cm -1 , 2770 cm -1 , 1589 cm -1 , 1491cm -1 , 1470 cm -1 , 1434
cm -1 , 1091 cm -1 and 1015 cm -1 .
Particle size <6.03> It meets the requirement.
Assay Weigh accurately an amount of Chlorpheniramine
500
Chlorpheniramine Maleate Tablets / Official Monographs
JP XV
Maleate Powder, equivalent to about 4 mg of chlorphenira-
mine maleate (C 16 H 19 C1N2.C4H 4 04), add 70 mL of the inter-
nal standard solution, shake for 15 minutes, then add the in-
ternal standard solution to make exactly 100 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 20 mg of Chlorpheniramine Maleate Reference Stan-
dard, previously dried at 105 °C for 3 hours, and add the in-
ternal standard to make exactly 100 mL. Pipet 20 mL of this
solution, add the internal standard solution to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with 30 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of chlorpheniramine
to that of the internal standard.
Amount (mg) of chlorpheniramine maleate
(C I6 H 19 C1N 2 .C 4 H 4 4 )
= W s x (Qj/Qs) x ( 1 / 5 )
W s : Amount (mg) of Chlorpheniramine Maleate Refer-
ence Standard
Internal standard solution — To 7 mL of a solultion of methyl
parahydroxybenzoate in methanol (1 in 1000) add water to
make 1000 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 //m in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1 g of sodium 1-heptane sulfonate
in 900 mL of water, add 10 mL of acetic acid (100) and water
to make 1000 mL. To 650 mL of this solution add 350 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
chlorpheniramine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 30
/uL of the standard solution under the above operating condi-
tions, chlorpheniramine and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 2.0.
System repeatability: When the test is repeated 6 times with
30 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of chlorpheniramine to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Chlorpheniramine Maleate Tablets
Chlorpheniramine Maleate Tablets contain not less
than 93.0% and not more than 107. 0%> of the labeled
amount of ^/-chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 : 390.86).
Method of preparation Prepare as directed under Tablets,
with Chlorpheniramine Maleate.
Identification Weigh a portion of powdered Chlorphenira-
mine Maleate Tablets, equivalent to 50 mg of Chlorphenira-
mine Maleate according to the labeled amount, shake with 40
mL of 0.1 mol/L hydrochloric acid TS, and filter. Transfer
the filtrate to a separator, and wash with 40 mL of hexane.
Add 10 mL of sodium hydroxide TS, and extract with 20 mL
of hexane. Wash the hexane layer with 5 mL of water. Cen-
trifuge, if necessary, shake the hexane extract with 0.5 g of
anhydrous sodium sulfate for several minutes, and filter.
Evaporate the filtrate in a water bath at about 50°C under a
reduced pressure, and determine the infrared absorption
spectrum of the residue as directed on the film method under
Infrared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 2940 cm" 1 , 2810 cm -1 , 2770
cm" 1 , 1589 cm" 1 , 1491cm" 1 , 1470 cm" 1 , 1434 cm" 1 , 1091
cm" 1 and 1015 cm -1 .
Uniformity of dosage unit <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Chlorpheniramine Maleate Tablets add 10
mL of water, shake to disintegrate the tablet, then add water
to make exactly KmL of a solution containing about 80 //g of
chlorpheniramine maleate (C 16 Hi 9 ClN 2 .C 4 H 4 4 ) per mL, and
filter through a membrane filter with pore size of not more
than 0.5 fim. Pipet 5 mL of the filtrate, add exactly 2.5 mL of
the internal standard solution, add water to make 10 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 20 mg of Chlorpheniramine Maleate Refer-
ence Standard, previously dried at 105°C for 3 hours, and
add water to make exactly 100 mL. Pipet 20 mL of this solu-
tion, add exactly 25 mL of the internal standard solution,
add water to make 100 mL, and use this solution as the stan-
dard solution. Perform the test with 30 iuL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the conditions de-
scribed in the Assay, and determine the ratios, Q T and Q s , of
the peak area of chlorpheniramine to that of the internal
standard.
Amount (mg) of chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 )
= W s x (Q T /Q S ) x (K/250)
W s : Amount (mg) of Chlorpheniramine Maleate Refer-
ence Standard
Internal standard solution — To 7 mL of a solution of methyl
parahydroxybenzoate (1 in 250) add water to make 1000 mL.
Assay Weigh accurately the mass of not less than 20 Chlor-
pheniramine Maleate Tablets, and powder. Weigh accurately
a portion of the powder, equivalent to about 4 mg of chlor-
pheniramine maleate (C 16 H 19 C1N 2 .C 4 H 4 4 ), add 70 mL of
the internal standard solution, shake for 15 minutes, then
add the internal standard solution to make exactly 100 mL,
filter through a membrane filter with pore size of not more
than 0.5 /um, and use this solution as the sample solution.
Separately, weigh accurately about 20 mg of Chlorphenira-
mine Maleate Reference Standard, previously dried at 105°C
for 3 hours, and add the internal standard to make exactly
100 mL. Pipet 20 mL of this solution, add the internal stan-
JPXV
Official Monographs / (/-Chlorpheniramine Maleate
501
dard solution to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with 30 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the ratios, Q T and Q s , of the peak
area of chlorpheniramine to that of the internal standard.
Amount (mg) of chlorpheniramine maleate
(C I6 H 19 C1N 2 .C 4 H 4 4 )
= W s x (A T /A S ) x (1/5)
W s : Amount (mg) of Chlorpheniramine Maleate Refer-
ence Standard
Internal standard solution — To 7 mL of a solution of methyl
parahydroxybenzoate in methanol (1 in 1000) add water to
make 1000 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium 1-heptane sul-
fonate in 900 mL of water, add 10 mL of acetic acid (100)
and water to make 1000 mL. To 650 mL of this solution add
350 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
chlorpheniramine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 30
/uL of the standard solution under the above operating condi-
tions, the internal standard and chlorpheniramine are eluted
in this order with the resolution between these peaks being
not less than 2.0.
System repeatability: When the test is repeated 6 times with
30 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of chlorpheniramine to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
^/-Chlorpheniramine Maleate
rf-^nji,7i=7
--7H>K£
(
COjH
C 16 H 19 C1N 2 .C 4 H 4 04: 390.86
(3S')-3-(4-Chlorophenyl)-A^,A r -dimethyl-3-pyridin-
2-ylpropylamine monomaleate [2438-32-6]
(/-Chlorpheniramine Maleate, when dried, contains
not less than 99.0% and not more than 101.0% of
C 16 H 19 C1N 2 .C 4 H 4 4 .
Description (/-Chlorpheniramine Maleate occurs as a white,
crystalline powder.
It is very soluble in water, in methanol and in acetic acid
(100), and freely soluble in /V.A^dimethylformamide and in
ethanol (99.5).
It dissolves in dilute hydrochloric acid.
Identification (1) Determine the absorption spectrum of a
solution of (/-Chlorpheniramine Maleate in 0.1 mol/L
hydrochloric acid TS (3 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of (/-
Chlorpheniramine Maleate, previously dried, as directed in
the paste method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) Dissolve 0.10 g of (/-Chlorpheniramine Maleate in 5
mL of methanol, and use this solution as the sample solution.
Separately, dissolve 56 mg of maleic acid in 10 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
diethyl ether, methanol, acetic acid (100) and water
(70:20:7:3) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): a spot among two of the spots obtained with the sample
solution shows the same intense to the spot with the standard
solution, and its Rf value is about 0.4.
Optical rotation <2.49> [«]„: +39.5 - +43.0° (after drying,
0.5 g, A r ,/Y-dimethylformamide, 10 mL, 100 mm).
pH <2.54> Dissolve 1 .0 g of (/-Chlorpheniramine Maleate in
100 mL of freshly boiled and cooled water: the pH of this so-
lution is between 4.0 and 5.0.
Melting point <2.60> 111 - 115°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
(/-Chlorpheniramine Maleate in 50 mL of water: the solution
is clear and colorless.
(2) Heavy metals </. 07>— Proceed with 1.0 g of (/-Chlor-
pheniramine Maleate according to Method 4 and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of (/-Chlor-
pheniramine Maleate in 100 mL of the mobile phase, and use
this solution as the sample solution. Pipet 3 mL of the sample
solution, add the mobile phase to make exactly 100 mL. Pipet
2 mL of this solution, add the mobile phase to make exactly
20 mL, and use this solution as the standard solution. Per-
form the test with exactly 20 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the peak area other than maleic acid and (/-chlor-
pheniramine with the sample solution is not larger than 2/3
times the peak area of (/-chlorpheniramine with the standard
solution, and the total area of these peaks is not larger than
the peak area of (/-chlorpheniramine with the standard solu-
502
Chlorpromazine Hydrochloride / Official Monographs
JP XV
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 8.57 g of ammonium dihydrogen
phosphate and 1 mL of phosphoric acid in water to make
1000 mL. To 800 mL of this solution add 200 mL of acetic
acid.
Flow rate: Adjust the flow rate so that the retention time of
(/-chlorpheniramine is about 11 minutes.
Time span of measurement: About 4 times as long as the
retention time of (/-chlorpheniramine beginning after the sol-
vent peak.
System suitability —
Test for required detectability: To exactly 2.5 mL of the
standard solution add the mobile phase to make exactly 25
mL. Confirm that the peak area of (/-chlorpheniramine ob-
tained with 20 /xL of this solution is equivalent to 7 to 13% of
that with 20 /uL of the standard solution.
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of (/-chlorpheniramine are not less than 4000
and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
(/-chlorpheniramine is not more than 4.0%.
Loss on drying <2.41>
hours).
Not more than 0.5% (1 g, 65 °C, 4
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of (/-Chlorpheniramine
Maleate, previously dried, and dissolve in 20 mL of acetic
acid (100). Titrate <2.50> with 0.1 mol/L perchloric acid VS
until the color of the solution changes from purple through
blue-green to green (indicator: 2 drops of crystal violet TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 19.54 mg of C 16 H 19 C1N 2 .C 4 H 4 04
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorpromazine Hydrochloride
3-(2-Chloro- 1 0//-phenothiazin- 1 0-yl)-7V, N-
dimethylpropylamine monohydrochloride [69-09-0]
Chlorpromazine Hydrochloride, when dried,
tains not less than 99.0% of C 17 H 19 C1N 2 S.HC1.
con-
Description Chlorpromazine Hydrochloride occurs as a
white to pale yellow, crystalline powder. It is odorless, or has
a faint, characteristic odor.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), sparingly soluble in acetic anhydride, and
practically insoluble in diethyl ether.
It is gradually colored by light.
Identification (1) To 5 mL of a solution of Chlorproma-
zine Hydrochloride (1 in 1000) add 1 drop of iron (III) chlo-
ride TS: a red color develops.
(2) Dissolve 0.1 g of Chlorpromazine Hydrochloride in
20 mL of water and 3 drops of dilute hydrochloric acid, add
10 mL of 2,4,6-trinitrophenol TS, and allow to stand for 5
hours. Collect the resulting precipitate, wash with water,
recrystallize from a small portion of acetone, and dry at
105 °C for 1 hour: the crystals so obtained melt <2.60> be-
tween 175°C and 179°C.
(3) Dissolve 0.5 g of Chlorpromazine Hydrochloride in 5
mL of water, add 2 mL of ammonia TS, and heat on a water
bath for 5 minutes. Cool, filter, and render the filtrate acidic
with dilute nitric acid: the solution responds to the Qualita-
tive Tests <1.09> (2) for chloride.
Melting point <2.60> 194 - 198°C
pH <2.54> Dissolve 1 .0 g of Chlorpromazine Hydrochloride
in 20 mL of freshly boiled and cooled water, and measure
within 10 minutes: the pH of this solution is between 4.0 and
5.0.
Purity (1) Clarity and color of solution — A solution of
1.0 g of Chlorpromazine Hydrochloride in 20 mL of water,
when observed within 10 minutes, is clear and colorless to
pale yellow.
(2) Heavy metals <1. 07>— Proceed with 1.0 g of Chlor-
promazine Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
Loss on drying <2.41>
2 hours).
Not more than 0.5% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Chlorpromazine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 35.53 mg of Ci 7 H 19 ClN 2 S.HCl
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
•HC 1
C, 7 H„C1N 2 S.HC1: 355.33
JPXV
Official Monographs / Chlorpromazine Hydrochloride Tablets
503
Chlorpromazine Hydrochloride
Injection
Chlorpromazine Hydrochloride
Tablets
^Pi^P7v>M^t
Chlorpromazine Hydrochloride Injection is an aque-
ous solution for injection.
It contains not less than 95% and not more than 105
% of the labeled amount of chlorpromazine
hydrochloride (C 17 H 19 C1N 2 S.HC1: 355.33).
Method of preparation Prepare as directed under Injec-
tions, with Chlorpromazine Hydrochloride.
Description Chlorpromazine Hydrochloride Injection is a
clear, colorless or pale yellow liquid.
pH: 4.0-6.5
Identification (1) Proceed with a volume of Chlorproma-
zine Hydrochloride Injection, equivalent to 5 mg of Chlor-
promazine Hydrochloride according to the labeled amount,
as directed in the Identification (1) under Chlorpromazine
Hydrochloride.
(2) Proceed with a volume of Chlorpromazine
Hydrochloride Injection, equivalent to 0.1 g of Chlorproma-
zine Hydrochloride according to the labeled amount, as
directed in the Identification (2) under Chlorpromazine
Hydrochloride.
Extractable volume <6.05> It meets the requirement.
Assay Transfer an exactly measured volume of Chlor-
promazine Hydrochloride Injection, equivalent to about 0.15
g of chlorpromazine hydrochloride (C I7 H 19 C1N 2 S.HC1) to a
separator, add 30 mL of water and 10 mL of a solution of so-
dium hydroxide (1 in 5), and extract with two 30-mL portions
and three 20-mL portions of diethyl ether. Wash the com-
bined diethyl ether extracts with successive 10-mL portions of
water until the last washing shows no red color upon the ad-
dition of phenolphthalein TS. Concentrate the diethyl ether
extracts on a water bath to 20 mL, add 5 g of anhydrous sodi-
um sulfate, allow to stand for 20 minutes, and filter through a
pledget of absorbent cotton. Wash with diethyl ether, com-
bine the washings with the filtrate, and evaporate the diethyl
ether on a water bath. Dissolve the residue in 50 mL of ace-
tone and 5 mL of acetic acid (100), and titrate <2.50> with
0.05 mol/L perchloric acid VS until the color of the solution
changes from red-purple to blue-purple (indicator: 3 drops of
bromocresol green-methylrosaniline chloride TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 17.77 mg of C 17 H 19 C1N 2 S.HC1
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
-Hermetic containers,
Chlorpromazine Hydrochloride Tablets contain not
less than 93.0% and not more than 107.0% of the
labeled amount of chlorpromazine hydrochloride
(C 17 H 19 C1N 2 S.HC1: 355.33).
Method of preparation Prepare as directed under Tablets,
with Chlorpromazine Hydrochloride.
Identification (1) Shake a quantity of powdered Chlor-
promazine Hydrochloride Tablets, equivalent to 0.2 g of
Chlorpromazine Hydrochloride according to the labeled
amount, with 40 mL of 0.1 mol/L hydrochloric acid TS, and
filter. To 1 mL of the filtrate add 4 mL of water and 1 drop of
iron (III) chloride TS: a red color develops.
(2) To 20 mL of the filtrate obtained in (1) add 10 mL of
2,4,6-trinitrophenol TS dropwise, and proceed as directed in
the Identification (2) under Chlorpromazine Hydrochloride.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Chlorpromazine
Hydrochloride Tablet at 75 revolutions per minute according
to the Paddle method, using 900 mL of 2nd fluid for dissolu-
tion test as the test solution. Take 20 mL or more of the dis-
solved solution after 60 minutes from the start of the dissolu-
tion test, and filter through a membrane filter withn a pore
size not exceeding 0.8 /xm. Discard the first 10 mL of the
filtrate, pipet the subsequent FmL, add 2nd fluid for dissolu-
tion test to make exactly V mL so that each mL of the filtrate
contains about 5.6 fig of chlorpromazine hydrochloride (C 17
H I9 C1N 2 S.HC1) according to the labeled amount, and use this
solution as the sample solution. Separately, weigh accurately
about 0.09 g of chlorpromazine hydrochloride for assay,
previously dried at 105°C for 2 hours, dissolve in 2nd fluid
for dissolution test to make exactly 200 mL. Pipet 5 mL of
this solution, add 2nd fluid for dissolution test to make exact-
ly 100 mL, further pipet 5 mL of this solution, add 2nd fluid
for dissolution test to make exactly 20 mL, and use this solu-
tion as the standard solution. Determine the absorbances, A T
and A s , of the sample solution and standard solution at 254
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>.
The dissolution rate of Chlorpromazine Hydrochloride
Tablets in 60 minutes should be not less than 75%.
Dissolution rate (%) with respect to labeled amount
of chlorpromazine hydrochloride (Ci 7 H 19 ClN 2 S.HCl)
= W s x (A T /A S ) x (V'/V) x (1/C) x (45/8)
W s : Amount (mg) of chlorpromazine hydrochloride for
assay.
C: Labeled amount (mg) of chlorpromazine hydrochloride
(C 17 H 19 C1N 2 S.HC1) in 1 tablet.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately, and
powder not less than 20 Chlorpromazine Hydrochloride
Tablets. Weigh accurately a portion of the powder, equiva-
504
Chlorpropamide / Official Monographs
JP XV
lent to about 50 mg of chlorpromazine hydrochloride
(C 17 H 19 C1N 2 S.HC1), add 60 mL of a mixture of diluted phos-
phoric acid (1 in 500) and ethanol (99.5) (1:1), exposure to
ultrasonic waves for 5 minutes, then shake vigorously for 20
minutes, and add the mixture of diluted phosphoric acid (1 in
500) and ethanol (99.5) (1:1) to make exactly 100 mL. Filter
the solution through a membrane filter with a pore size not
exceeding 0.45 /um, and discard the first 3 mL of the filtrate.
To exactly 2.5 mL of the subsequent filtrate add exactly 5 mL
of the internal standard solution, then add the mixture of
diluted phosphoric acid (1 in 500) and ethanol (99.5) (1:1) to
make 25 mL, and use this solution as the sample solution.
Separately, weigh accurately about 25 mg of chlorpromazine
hydrochloride for assay, previously dried at 105°C for 2
hours, and dissolve in the mixture of diluted phosphoric acid
(1 in 500) and ethanol (99.5) (1:1) to make exactly 100 mL.
Pipet 5 mL of this solution, add exactly 5 mL of the internal
standard solution and the mixture of diluted phosphoric acid
(1 in 500) and ethanol (99.5) (1:1) to make 25 mL, and use
this solution as the standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Qj and Q
s, of the peak area of chlorpromazine to that of the internal
standard.
Amount (mg) of chlorpromazine hydrochloride
(C 17 H 19 C1N 2 S.HC1) = W s x (Q T /Q S ) x 2
W s : Amount (mg) of chlorpromazine hydrochloride for
assay
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in a mixture of diluted phosphoric acid (1 in 500)
and ethanol (99.5) (1:1) (1 in 4500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 256 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted 0.05 mol/L sodium di-
hydrogen phosphate TS (1 in 2) and acetonitrile (27:13).
Flow rate: Adjust the flow rate so that the retention time of
chlorpromazine is about 15 minutes.
System suitability-
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, the internal standard and chlorpromazine are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of chlorpromazine to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Chlorpropamide
o o
A.
^\^.CH!
C 10 H 13 ClN 2 O 3 S: 276.74
4-Chloro--/V-(propylcarbamoyi)benzenesulfonamide
[94-20-2]
Chlorpropamide, when dried, contains not less than
98.0% of C 10 H 13 ClN 2 O 3 S.
Description Chlorpropamide occurs as white, crystals or
crystalline powder.
It is freely soluble in methanol and in acetone, soluble in
ethanol (95), and slightly soluble in diethyl ether, and practi-
cally insoluble in water.
Identification (1) Dissolve 0.08 g of Chlorpropamide in 50
mL of methanol. To 1 mL of the solution add 0.01 mol/L
hydrochloric acid TS to make 200 mL. Determine the absorp-
tion spectrum of the solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Chlor-
propamide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Perform the test with Chlorpropamide as directed un-
der Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 127 - 131°C
Purity (1) Acidity — To 3.0 g Chlorpropamide add 150
mL of water, and warm at 70°C for 5 minutes. Allow to
stand in ice water for 1 hour, and filter. To 25 mL of the
filtrate add 2 drops of methyl red TS and 0.30 mL of 0.1 mol/
L sodium hydroxide VS: a yellow color develops.
(2) Chloride <1.03>— To 40 mL of the filtrate obtained in
(1) add 6 mL of dilute nitric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.25 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.011%).
(3) Sulfate <1.14>— To 40 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.35 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.021%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Chlor-
propamide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(5) Related substances — Dissolve 0.6 g of Chlor-
propamide in acetone to make exactly 10 mL, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add acetone to make exactly 300 mL, and use this solu-
JPXV
Official Monographs / Chlorpropamide Tablets
505
tion as the standard solution (1). Separately, dissolve 60 mg
of 4-chlorobenzene sulfonamide in acetone to make exactly
300 mL, and use this solution as the standard solution (2).
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 [iL each of the sample
solution and standard solutions (1) and (2) on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of cyclohexane, 3-methyl-l-butanol, methanol and
ammonia solution (28) (15:10:5:1) to a distance of about 10
cm, and air-dry the plate. After drying the plate at 100°C for
1 hour, spray evenly sodium hypochlorite TS on the plate,
and air-dry for 15 minutes. Then spray evenly potassium io-
dide-starch TS on the plate: the spot from the sample solu-
tion equivalent to the spot from the standard solution (2) is
not more intense than the spot from the standard solution
(2), and the spots other than the spot mentioned above and
other than the principal spot from the sample solution is not
more intense than the spot from the standard solution (1).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Chlorpropamide,
previously dried, dissolve in 30 mL of neutralized ethanol,
and add 20 mL of water. Titrate <2.50> with 0.1 mol/L sodi-
um hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 27.67 mg of C 10 H 13 ClN 2 O 3 S
Containers and storage Containers — Well-closed contain-
ers.
Chlorpropamide Tablets
^njl^nA°$ Kffi
Chlorpropamide Tablets contain not less than 95%
and not more than 105% of the labeled amount of
chlorpropamide (C 10 H 13 ClN 2 O3S: 276.74).
Method of preparation Prepare as directed under Tablets,
with Chlorpropamide.
Identification Take a quantity of powdered Chlor-
propamide Tablets, equivalent to 0.08 g of Chlorpropamide
according to the labeled amount, add 50 mL of methanol,
shake, and filter. To 1 mL of the filtrate add 0.01 mol/L
hydrochloric acid TS to make 200 mL, and determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 231 nm and 235 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Chlorpropamide Tablets
at 50 revolutions per minute according to the Paddle method,
using 900 mL of 2nd fluid for dissolution test as the test solu-
tion. Take 20 mL or more of the dissolved solution 45
minutes after starting the test, and filter through a membrane
filter with a pore size not exceeding 0.8 fim. Discard the first
10 mL of the filtrate, pipet the subsequent FmL of the
filtrate, add 2nd fluid for dissolution test to make exactly V
mL so that each mL contains about 10 tig of chlorpropamide
(C 10 H 13 ClN 2 O 3 S) according to the labeled amount, and use
this solution as the sample solution. Separately, weigh ac-
curately about 0.05 g of chlorpropamide for assay, previous-
ly dried at 105 °C for 3 hours, dissolve in 10 mL of methanol,
and add water to make exactly 50 mL. Pipet 1 mL of this so-
lution, add 2nd fluid for dissolution test to make exactly 100
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution at 232 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate of Chlor-
propamide Tablets in 45 minutes should be not less than 70
%.
Dissolution rate (%) with respect to the
labeled amount of chlorpropamide (C 10 H 13 ClN 2 O 3 S)
= W s x (A T /A S ) x (V'/V) x (1/C) x 18
W s : Amount (mg) of chlorpropamide for assay.
C: Labeled amount (mg) of chlorpropamide
(C 10 H 13 ClN 2 O 3 S) in 1 tablet.
Assay Weigh accurately and powder not less than 20 Chlor-
propamide Tablets. Weigh accurately a quantity of the pow-
der, equivalent to about 50 mg of chlorpropamide
(C I0 H 13 ClN 2 O 3 S), add 75 mL of the mobile phase, shake for
10 minutes, and add the mobile phase to make exactly 100
mL. Centrifuge this solution, pipet 10 mL of the supernatant
liquid, add the mobile phase to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of chlorpropamide for assay, previous-
ly dried at 105°C for 3 hours, dissolve in the mobile phase to
make exactly 100 mL. Pipet 10 mL of this solution, add the
mobile phase to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 20 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing operating conditions. Determine the peak areas, A T
and A s , of chlorpropamide of the sample solution and stan-
dard solution.
Amount (mg) of chlorpropamide (C 10 H, 3 ClN 2 O 3 S)
= W s x (A T /A S )
W s : Amount (mg) of chlorpropamide for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted acetic acid (100) (1 in
100) and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
chlorpropamide is about 5 minutes.
System suitability —
System performance: When the procedure is run with 20
iiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of chlorpropamide are not less than 1500 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
506
Cholera Vaccine / Official Monographs
JP XV
20 nL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
chlorpropamide is not more than 1.5%.
Containers and storage Containers — Well-closed contain-
ers.
Cholera Vaccine
Cholera Vaccine is a liquid for injection containing
inactivated Vibrio cholerae of the Ogawa and Inaba
strains.
Monotypic products may be manufactured, if neces-
sary.
It conforms to the requirements of Cholera Vaccine
in the Minimum Requirements for Biological
Products.
Description Cholera Vaccine is a white-turbid liquid.
Cholecalciferol
Vitamin D 3
ziU^JL-WiP-JU
C 27 H 4 40: 384.64
(3S,5Z,7£')-9,10-Secocholesta-5,7,10(19)-trien-3-ol
[67-97-0]
Cholecalciferol contains not less than 97.0% and not
more than 103.0% of C 27 H 44 0.
Description Cholecalciferol occurs as white crystals. It is
odorless.
It is freely soluble in ethanol (95), in chloroform, in diethyl
ether and in isooctane, and practically insoluble in water.
It is affected by air and by light.
Melting point: 84-88°C Transfer Cholecalciferol to a
capillary tube, and dry for 3 hours in a desiccator (in vacuum
at a pressure not exceeding 2.67 kPa). Immediately fireseal
the capillary tube, put it in a bath fluid, previously heated to a
temperature about 10°C below the expected melting point,
and heat at a rate of rise of about 3°C per minute, and read
the melting point.
Identification (1) Dissolve 0.5 mg of Cholecalciferol in 5
mL of chloroform, add 0.3 mL of acetic anhydride and 0.1
mL of sulfuric acid, and shake: a red color is produced, and
rapidly changes through purple and blue to green.
(2) Determine the infrared absorption spectrum of
Cholecalciferol as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Cholecalciferol Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
E i%
c lcm
Absorbance <2.24>
ethanol (95), 1000 mL).
Optical rotation <2.49>
(265 nm): 450-490 (10 mg,
r i20
+ 103- +112° (50 mg,
ethanol (95), 10 mL, 100 mm). Prepare the solution without
delay, using Cholecalciferol from a container opened not lon-
ger than 30 minutes, previously, and determine the rotation
within 30 minutes after the solution has been prepared.
Purity 7-Dehydrocholesterol — Dissolve 10 mg of Cholecal-
ciferol in 2.0 mL of diluted ethanol (95) (9 in 10), add a solu-
tion prepared by dissolving 20 mg of digitonin in 2.0 mL of
diluted ethanol (95) (9 in 10), and allow the mixture to stand
for 18 hours: no precipitate is formed.
Assay Proceed with the operation avoiding contact with air
or other oxidizing agents and using light-resistant containers.
Dissolve separately about 30 mg each of Cholecalciferol and
Cholecalciferol Reference Standard, accurately weighed, in
isooctane to make exactly 50 mL. Pipet 10 mL each of these
solutions, add 3 mL each of the internal standard solution,
then add the mobile phase to make 50 mL, and use these solu-
tions as the sample solution and standard solution. Perform
the test with 10 /xL each of the sample solution and the stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Qt and Q s , of the peak area of cholecalciferol to
that of the internal standard.
Amount (mg) of C27H44O
= W s x (Q T /Q S )
W s : Amount (mg) of Cholecalciferol Reference Standard
Internal standard solution — A solution of dimethyl phthalate
in isooctane (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 10 to 30 cm in length, packed with silica gel for
liquid chromatography (5 to 10 /xm in particle diameter).
Column temperature: Ordinary temperature.
Mobile phase: A mixture of hexane and w-amylalcohol
(997:3).
Flow rate: Adjust the flow rate so that the retention time of
cholecalciferol is about 25 minutes.
Selection of column: Dissolve 15 mg of Cholecalciferol
Reference Standard in 25 mL of isooctane. Transfer this so-
lution to a flask, heat under a reflux condenser in an oil bath
for 2 hours, and cool to room temperature rapidly. Transfer
this solution to a quartz test tube, and irradiate under a short-
wave lamp (main wavelength: 254 nm) and a long-wave lamp
(main wavelength: 365 nm) for 3 hours. To this solution add
the mobile phase to make 50 mL. Proceed with 10 /uL of this
solution under the above operating conditions. Use a column
with the ratios of the retention time of previtamin D 3 , trans-
vitamin D 3 and tachysterol 3 to that of cholecalciferol being
JPXV
about 0.5, about 0.6 and about 1.1, respectively, and with
resolution between previtamin D 3 and trans-vitamin D 3 , and
that between cholecalciferol and tachysterol 3 being not less
than 1.0.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, under nitrogen atmosphere, and
in a cold place.
Cholesterol
ziUXxn-jL-
C 27 H 46 0: 386.65
Cholest-5-en-3/?-ol
[57-88-5]
Description Cholesterol occurs as white to pale yellow crys-
tals or granules. It is odorless, or has a slight odor. It is taste-
less.
It is freely soluble in chloroform and in diethyl ether, solu-
ble in 1,4-dioxane, sparingly soluble in ethanol (99.5), and
practically insoluble in water.
It gradually changes to a yellow to light yellow-brown
color by light.
Identification (1) Dissolve 0.01 g of Cholesterol in 1 mL
of chloroform, add 1 mL of sulfuric acid, and shake: a red
color develops in the chloroform layer, and the sulfuric acid
layer shows a green fluorescence.
(2) Dissolve 5 mg of Cholesterol in 2 mL of chloroform,
add 1 mL of acetic anhydride and 1 drop of sulfuric acid, and
shake: a red color is produced, and it changes to green
through blue.
Optical rotation <2.49> [a]^: - 34
g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 147 - 150°C
■38° (after drying, 0.2
Purity (1) Clarity of solution — Place 0.5 g of Cholesterol
in a glass-stoppered flask, dissolve in 50 mL of warm ethanol
(95), and allow to stand at room temperature for 2 hours: no
turbidity or deposit is produced.
(2) Acidity — Place 1.0 g of Cholesterol in a flask, dis-
solve in 10 mL of diethyl ether, add 10.0 mL of 0.1 mol/L so-
dium hydroxide VS, and shake for 1 minute. Expel the
diethyl ether, and boil for 5 minutes. Cool, add 10 mL of
water, and titrate <2.50> with 0.05 mol/L sulfuric acid VS (in-
dicator: 2 drops of phenolphthalein TS). Perform a blank de-
termination.
The volume of 0.1 mol/L sodium hydroxide VS consumed
is not more than 0.30 mL.
Loss on drying <2.41> Not more than 0.30% (1 g, in vacu-
um, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Containers and storage Containers — Tight containers.
Official Monographs / Ciclacillin
Storage — Light-resistant.
Ciclacillin
507
•>^7-> l J>
H 2 N II H H
CH 3
CH 3
341.43
(2S,5i?,6i?)-6-[(l-Aminocyclohexanecarbonyl)amino]-
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-
carboxylic acid [3485-14-1]
Ciclacillin contains not less than 920 fig (potency)
and not more than 1010 fig (potency) per mg, calculat-
ed on the dehydrated basis. The potency of Ciclacillin
is expressed as mass (potency) of ciclacillin
(C 15 H 23 N 3 4 S).
Description Ciclacillin occurs as white to light yellowish
white crystalline powder.
It is sparingly soluble in water, slightly soluble in
methanol, and practically insoluble in acetonitrile and in
ethanol (99.5).
Identification Determine the infrared absorption spectrum
of Ciclacillin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Ciclacillin Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]™:
mL, 100 mm).
+ 300- +315° (2 g, water, 100
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Ciclacillin according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ciclacillin according to Method 3, and perform the test
(not more than 2 ppm).
Water <2.48> Not more than 2.0% (1 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Ciclacillin and
Ciclacillin Reference Standard, equivalent to about 50 mg
(potency), dissolve each in a suitable amount of the mobile
phase, add exactly 5 mL of the internal standard solution and
the mobile phase to make 50 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with 10 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Q T and
Qs, of the peak area of ciclacillin to that of the internal stan-
dard.
Amount [jug (potency)] of Q5H23N3O4S
= W s x (Qj/Q s ) x 1000
W s : Amount [mg (potency)] of Ciclacillin Reference
508
Ciclosporin / Official Monographs
JP XV
Standard
Internal standard solution — A solution of orcin in the mobile
phase (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.771 g of ammonium acetate in
about 900 mL of water, adjust the pH to 4.0 with acetic acid
(100), and add water to make 1000 mL. To 850 mL of this so-
lution add 150 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ciclacillin is about 4 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, ciclacillin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 8.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak areas of ciclacillin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Ciclosporin
Ciclosporin A
H ,CH 3
H 3 C
I — Ala-o-Ala-MeLeu'-MeLeu-MeVal — N
Abu -MeGly-MeLeu-Val-MeLeLj
CHs O
/ai-MeLeu — |
Abu = {2S )-2- Aminobutyric acid
MeGty = W-Methylglycine
MeLeu = W-Methylleucine
MeVgl = A/-Methylvaline
C 62 H 1U N U 12 : 1202.61
cyc/o{-[(2S,3i?,4i?,6£')-3-Hydroxy-4-methyl-2-
methylaminooct-6-enoyl]-L-2-aminobutanoyl-
A f -methylglycyl-A f -methyl-L-leucyl-L-valyl-/V-methyl-
L-leucyl-L-alanyl-D-alanyl-AT-methyl-L-leucyl-/V-methyl-
L-leucyWV-methyl-L-valyl-} [59865-13-3]
Ciclosporin contains not less than 98.5% and not
more than 101.5% of C 6 2H 111 N 11 12 , calculated on the
dried basis.
Description Ciclosporin occurs as a white powder.
It is very soluble in acetonitrile, in methanol and in ethanol
(95), freely soluble in diethyl ether, and practically insoluble
in water.
Identification Determine the infrared absorption spectrum
of Ciclosporin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™: - 185 - - 193° (0.1 g calcu-
lated on the dried basis, methanol, 20 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ciclosporin in 10 mL of ethanol (95): the solution is clear,
and has no more color than the following control solutions
(1), (2) or (3).
Control solution (1): To exactly 3.0 mL of Ferric Chloride
Stock CS and exactly 0.8 mL of Cobaltous Chloride Stock
CS add diluted hydrochloric acid (1 in 40) to make exactly
100 mL.
Control solution (2): To exactly 3.0 mL of Ferric Chloride
Stock CS, exactly 1.3 mL of Cobaltous Chloride Stock CS
and exactly 0.5 mL of Cupric Sulfate Stock CS add diluted
hydrochloric acid (1 in 40) to make exactly 100 mL.
Control solution (3): To exactly 0.5 mL of Iron (III) chlo-
ride Stock CS and exactly 1.0 mL of Cobaltous Chloride
Stock CS add diluted hydrochloric acid (1 in 40) to make ex-
actly 100 mL.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Ciclospo-
rin according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Related substances — Use the sample solution ob-
tained in the Assay as the sample solution. Pipet 2 mL of the
sample solution, add a mixture of water and acetonitrile (1:1)
to make exactly 200 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /uL each of the sam-
ple solution and standard soluton as directed under Liquid
Chromatography <2.01> according to the following
conditions. Determine each peak area of both solutions by
the automatic integration method: the area of the peak other
than the ciclosporin is not more than 0.7 times of the peak
area of ciclosporin from the standard solution, and the total
area of all peaks other than the ciclosporin is not more than
1.5 times of the peak area of ciclosporin from the standard
solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of ciclosporin beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add a mixture of water and acetonitrile (1:1) to
make exactly 20 mL. Confirm that the peak area of ciclospo-
rin obtained from 20 /xh of this solution is equivalent to 7 to
13% of that of ciclosporin obtained from 20 /uL of the
standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
JP XV
Official Monographs / Cilastatin Sodium
509
ciclosporin is not more than 3.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 30 mg each of Ciclosporin
and Ciclosporin Reference Standard (previously determine
the loss on drying <2.41> in the same manner as Ciclosporin),
and dissolve each in a mixture of water and acetonitrile (1:1)
to make exactly 25 mL, and use these solutions as the sample
solution and standard solution. Perform the test with exactly
20 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of ciclosporin.
Amount (mg) of C 62 H lu N n 12
= W s x (A T /A S )
W s : Amount (mg) of Ciclosporin Reference Standard,
calculated on the dried basis
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Connect the sample injection port and the column with a
stainless steel tube 0.3 mm in inside diameter and 1 m in
length.
Column temperature: A constant temperature of about
80°C (including the sample injection port and the connecting
tube).
Mobile phase: A mixture of water, acetonitrile, tert-butyl
methyl ether and phosphoric acid (520:430:50:1).
Flow rate: Adjust the flow rate so that the retention time of
ciclosporin is about 27 minutes.
System suitability —
System performance: Dissolve 3 mg of Ciclosporin U in
2.5 mL of a mixture of water and acetonitrile (1:1), and add
2.5 mL of the standard solution. When the procedure is run
with 20 /xL of this solution under the above operating
conditions, ciclosporin U and ciclosporin are eluted in this
order with the resolution between these peaks being not less
than 1.2.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ciclosporin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cilastatin Sodium
•>"7X93->1-h U^A
HOjC,
„CO;Na
Htx:
CH : ,
CH :i
C 16 H 25 N 2 Na0 5 S: 380.43
Monosodium (2Z)-7-{[(2i?)-2-amino-
2-carboxy ethyl] sulfanyl} -2-( {[(lS)-2,2-
dimethylcyclopropyl]carbonyl}amino)hept-2-enoate
[81129-83-1]
Cilastatin Sodium contains not less than 98.0% and
not more than 101.0% of C 16 H 2 5N2Na0 5 S, calculated
on the anhydrous basis and corrected on the amount of
the residual solvent.
Description Cilastatin Sodium occurs as a white to pale yel-
lowish white powder.
It is very soluble in water, freely soluble in methanol, and
slightly soluble in ethanol (99.5).
It is hygroscopic.
Identification (1) Determine the infrared absorption spec-
trum of Cilastatin Sodium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) A solution of Cilastatin Sodium (1 in 10) responds to
the Qualitative Tests <1.09> for sodium salt.
Optical rotation <2.49> [a]™: +41.5- +44.5° (0.1 g, cal-
culated on the anhydrous basis and corrected on the amount
of the residual solvent, a solution of hydrochloric acid in
methanol (9 in 1000), 10 mL, 100 mL).
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of Cilastatin Sodium in 100 mL of water is between 6.5 and
7.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cilastatin Sodium in 100 mL of water: the solution is clear
and the solution has no more color than the following control
solution.
Control solution: To a mixture of 2.4 mL of Iron (III)
Chloride Colorimetric Stock Solution and 0.6 mL of Cobalt
(II) Chloride Colorimetric Stock Solution add water to make
10 mL, pipet 5 mL of this solution, and add water to make
exactly 100 mL.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cilasta-
tin Sodium according to Method 2, and perform the test. Af-
ter carbonization, add 0.5 mL of sulfuric acid instead of
nitric acid. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
(3) Arsenic <1.11>— To 2.0 g of Cilastatin Sodium add 5
mL of nitric acid and 1 mL of sulfuric acid, and heat careful-
ly until white fumes are evolved. After cooling, heat with two
2-mL portions of nitric acid, then heat with several 2-mL por-
tions of hydrogen peroxide (30) until a colorless or pale yel-
510
Cilastatin Sodium / Official Monographs
JP XV
low solution is obtained. After cooling, heat again until white
fumes are evolved. After cooling, add water to make 5 mL,
and perform the test with this solution as the test solution: it
shows no more color than the following color standard.
Color standard: Prepare a solution according to the above
procedure without using Cilastatin Sodium, add exactly 2 mL
of Standard Arsenic Solution, and perform the test in the
same manner as the test solution (not more than 1 ppm).
(4) Related substances — Dissolve about 40 mg of Cilasta-
tin Sodium in 25 mL of water, and use this solution as the
sample solution. Pipet 3 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 /uL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the area of the peak other than cilasta-
tin is not larger than 1/6 times the peak area of cilastatin
from the standard solution, and the total area of the peaks
other than the peak of cilastatin is not larger than the peak
area of cilastatin from the standard solution.
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.5 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase A: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (7:3).
Mobile phase B: Diluted phosphoric acid (1 in 1000).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phase A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-30
30-40
15^100
100
85^0
Flow rate: 2.0 mL per minute.
Time span of measurement: 40 minutes.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add water to make exactly 30 mL. Confirm that
the peak area of cilastatin obtained with 20 /uL of this solu-
tion is equivalent to 2.3 to 4.5% of that obtained with 20 /xL
of the standard solution.
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the retention time of cilastatin is about 20 minutes, and
the number of theoretical plates and the symmetry factor of
the peak of cilastatin are not less than 10,000 and not more
than 2.5, respectively.
System repeatability: When the test is repeated 3 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cilastatin is not more than 2.0%.
(5) Residual solvents <2.46> — Weigh accurately about 0.2
g of Cilastatin Sodium, add exactly 2 mL of the internal stan-
dard solution, dissolve in water to make 10 mL, and use this
solution as the sample solution. Separately, measure exactly 2
mL of acetone, 0.5 mL of methanol and 0.5 mL of mesityl
oxide, and add water to make exactly 1000 mL. Pipet 2 mL
of this solution, add exactly 2 mL of the internal standard so-
lution, add water to make 10 mL, and use this solution as the
standard solution. Perform the test with 2 fXL each of the
sample solution and standard solution as directed under Gas
Chromatography <2.02> according to the following condi-
tions. Determine the ratios of the peak areas of acetone,
methanol and mesityl oxide and to the peak area of the inter-
nal standard, g Ta and Q Sa , Q Jh and Q sb , Gtc and Q Sc , and cal-
culate the amounts of acetone, methanol and mesityl oxide
by the following equation: they are not more than 1.0%, not
more 0.5% and not more than 0.4%, respectively.
Amount (%) of acetone (CH 3 COCH 3 )
= (1/^t) x (GTa/Gsa) x 400 x 0.79
Amount (%) of methanol (CH 3 OH)
= (1/Wt) x (STb/Gsb) x 100 x 0.79
Amount (%) of mesityl oxide (CH 3 COCH = C(CH 3 ) 2 )
= (1/Wt) x (Qtc/Qsc) x 100 x 0.86
W T : Amount (mg) of sample
0.79: Density (g/mL) of acetone and methanol
0.86: Density (g/mL) of mesityl oxide
Internal standard solution — To 0.5 mL of 1-propanol add
water to make 1000 mL.
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3.2 mm in inside diameter and 2.1
m in length, packed with teflon for gas chromatography (250
- 420 //m) coated with polyethylene glycol 20 M for gas chro-
matography at the ratio of 10%.
Column temperature: A constant temperature of about
70°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 5 minutes.
Time span of measurement: About 3 times as long as the
retention time of the internal standard.
System suitability —
System performance: When the procedure is run with the
standard solution under the above operating conditions, ace-
tone, methanol, 1-propanol and mesityl oxide are eluted in
this order, and these peaks completely separate each other.
System repeatability: When the test is repeated 6 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviations of the ratio of the
peak area of acetone, methanol and mesityl oxide to that of
the internal standard are not more than 4.0%, respectively.
Water <2.48> Not more than 2.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately about 0.3 g of Cilastatin Sodium,
dissolve in 30 mL of methanol, add 5 mL of water, and ad-
just to pH 3.0 with 0.1 mol/L hydrochloric acid TS. Titrate
<2.50> with 0.1 mol/L sodium hydroxide VS from the first
equivalence point to the third equivalence point (potentio-
metric titration).
Each mL of 0.1 mol/L sodium hydroxide VS
JPXV
Official Monographs / Cilostazol 511
= 19.02 mg of C 16 H 25 N 2 Na0 5 S
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Cilostazol
■nx9'/-)l
C 20 H 27 N 5 O 2 : 369.46
6-[4-(l-Cyclohexyl-l//-tetrazol-5-yl)butyloxy]-
3 ,4-dihydroquinolin-2(l//)-one
[73963-72-1]
Cilostazol, when dried, contains not less than
98.5% and not more than 101.5% of C20H27N5O;,.
Description Cilostazol occurs as white to pale yellowish
white, crystals or crystalline powder.
It is slightly soluble in methanol, in ethanol (99.5) and in
acetonitrile, and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Cilostazol in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Cilostazol Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Cilostazol as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Cilostazol Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Melting point <2.60> 158 - 162°C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Cilostazol according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve 25 mg of Cilostazol in
25 mL of acetonitrile, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add acetoni-
trile to make exactly 100 mL. Pipet 10 mL of this solution,
add acetonitrile to make exactly 50 mL, and use this solution
as the standard solution. Perform the test with exactly 10,mL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
cilostazol obtained with the sample solution is not larger than
0.7 times the peak area of cilostazol with the standard solu-
tion, and the total area of the peaks other than the peak of
cilostazol with the sample solution is not larger than 1 .2 times
the peak area of cilostazol with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with silica gel for liquid
chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of hexane, ethyl acetate and
methanol (10:9:1).
Flow rate: Adjust the flow rate so that the retention time of
cilostazol is about 7 minutes.
Time span of measurement: About 3 times as long as the
retention time of cilostazol beginning after the solvent peak.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add acetonitrile to make exactly 10 mL. Con-
firm that the peak area of cilostazol obtained with 10 /uL of
this solution is equivalent to 7 to 13% of that with 10 jiL of
the standard solution.
System performance: Pipet 1 mL of the sample solution,
add 1 mL of a solution prepared by dissolving 5 mg of 3,4-di-
hydro-6-hydroxy-2(l//)-quinolinone in 10 mL of acetonitrile
and acetonitrile to make exactly 100 mL. When the procedure
is run with 10 fXL of this solution under the above operating
conditions, 3,4-dihydro-6-hydroxy-2(l_fT)-quinolinone and
cilostazol are eluted in this order with the resolution between
these peaks being not less than 9.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cilostazol is not more than 2.0%.
Loss on drying <2.41> Not more than 0.1% (1 g, 105°C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Cilostazol and
Cilostazol Reference Standard, previously dried, dissolve
each in a suitable amount of methanol, add exactly 5 mL of
the internal standard solution and methanol to make 50 mL.
To 1 mL each of these solutions add methanol to make 10
mL, and use these solutions as the sample solution and the
standard solution, respectively. Perform the test with 10 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Qt and Q s , of
the peak area of cilostazol to that of the internal standard.
Amount (mg) of C 20 H 27 N 5 O 2 = W s x (g T /g s )
W s : Amount (mg) of Cilostazol Reference Standard
Internal standard solution — A solution of benzophenone in
methanol (1 in 250).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
512 Cilostazol Tablets / Official Monographs
JP XV
Mobile phase: A mixture of water, acetonitrile and
methanol (10:7:3).
Flow rate: Adjust the flow rate so that the retention time of
cilostazol is about 9 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, cilostazol and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 9.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of cilostazol to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Cilostazol Tablets
Cilostazol Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of
cilostazol (C 20 H 27 N 5 O 2 : 369.46).
Method of preparation Prepare as directed under Tablets,
with Cilostazol.
Identification Mix well an amount of powdered Cilostazol
Tablets, equivalent to 50 mg of Cilostazol according to the la-
beled amount, with 10 mL of acetone, centrifuge, and use the
supernatant liquid as the sample solution. Separately, dis-
solve 25 mg of Cilostazol Reference Standard in 5 mL of ace-
tone, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 6//L each of the sample solu-
tion and standard solution on a plate of silica gel for thin-lay-
er chromatography, develop the plate with a mixture of ethyl
acetate, acetonitrile, methanol and formic acid (75:25:5:1) to
a distance of about 12 cm, and air-dry the plate. Spray evenly
Dragendorff's TS for spraying on the plate: the principal spot
with the sample solution and the spot with the standard solu-
tion are orange in color and have the same Rf value.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Cilostazol Tablets add 2 mL of water to dis-
integrate the tablet, add the internal standard solution exactly
5 mL for a 50-mg tablet and exactly 10 mL for a 100-mg
tablet, and add methanol to make 50 mL. Shake for 10
minutes for the 50-mg tablet and for 20 minutes for the
100-mg tablet. To 1 mL of the solution add methanol to
make 10 mL for the 50-mg tablet and 20 mL for the 100-mg
tablet, filter through a membrane filter with pore size of not
more than 0.5 /urn, and use the filtrate as the sample solution.
Proceed as directed in the Assay.
Amount (mg) of cilostazol (C20H27N5O2)
= W s x «2 T /<2s) x (C/50)
W s : Amount (mg) of Cilostazol Reference Standard
C: Labeled amount (mg) of cilostazol (C20H27N5O2) in 1
tablet
Internal standard solution — A solution of benzophenone in
methanol (1 in 250).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Cilostazol Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of a solution of sodium lauryl sulfate (3 in 1000)
as the dissolution medium. Withdraw 20 mL or more of the
dissolution medium 45 minutes after starting the test for a
50-mg tablet and 60 minutes after starting the test for a
100-mg tablet, and filter through a membrane filter with a
pore size not exceeding 0.45 fim. Discard the first 10 mL of
the filtrate, pipet the subsequent KmL, add the solution of
sodium lauryl sulfate (3 in 1000) to make exactly V mL so
that each mL contains about 5.6 /ug of cilostazol (C20H27N5
2 ) according to the labeled amount, and use this solution as
the sample solution. Separately, weigh accurately about 28
mg of Cilostazol Reference Standard, previously dried at
105 °C for 2 hours, and dissolve in methanol to make exactly
100 mL. Pipet 4 mL of this solution, add the solution of sodi-
um lauryl sulfate (3 in 1000) to make exactly 200 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 257 nm as directed under Ultraviolet-visible Spec-
trophotometry using the solution of sodium lauryl sulfate (3
in 1000) as the control: the dissolution rates of a 50-mg tablet
in 45 minutes and a 100-mg tablet in 60 minutes are not less
than 75% and not less than 70%, respectively.
Dissolution rate (%) with respect to the labeled amount of
cilostazol (C20H27N5O2)
= W s x (A T /A S ) x (V'/V) x (l/Q x 18
W s : Amount (mg) of Cilostazol Reference Standard
C: Labeled amount (mg) of cilostazol (C20H27N5O2) in 1
tablet
Assay Weigh accurately, and powder not less than 20
Cilostazol Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 50 mg of cilostazol (C20H27N5O2),
add exactly 5 mL of the internal standard solution and
methanol to make 50 mL, and shake well for 10 minutes. To
1 mL of this solution add methanol to make 10 mL, filter
through a membrane filter with a pore size of not more than
0.5 /xm, and use the filtrate as the sample solution. Separate-
ly, weigh accurately about 50 mg of Cilostazol Reference
Standard, dissolve in a suitable amount of methanol, and add
exactly 5 mL of the internal standard solution and methanol
to make 50 mL. To 1 mL of this solution add methanol to
make 10 mL, and use this solution as the standard solution.
Perform the test with 10 /xh each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of cilostazol to that of
the internal standard.
Amount (mg) of cilostazol (C20H27N5O2)
= W s x (Q T /Q S )
W s : Amount (mg) of Cilostazol Reference Standard
Internal standard solution — A solution of benzophenone in
methanol (1 in 250).
JPXV
Official Monographs / Cisplatin 513
Operating conditions —
Proceed as directed in the operating conditions in the As-
say under Cilostazol.
System suitability —
System performance: Proceed as directed in the system
suitability in the Assay under Cilostazol.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of cilostazol to that of the internal standard is not
more than 1.5%.
Containers and storage Containers — Well-closed contain-
ers.
Cimetidine
->/^-^>
C 10 H 16 N 6 S: 252.34
2-Cyano-l-methyl-3-{2-[(5-methyl-l//-imidazol-4-
yl)methylsulfanyl]ethyl}guanidine [51481-61-9]
Cimetidine, when dried,
?.0% of C 10 H 16 N 6 S.
contains not less than
Description Cimetidine occurs as a white crystalline pow-
der. It is odorless, and has a bitter taste.
It is freely soluble in methanol and in acetic acid (100),
sparingly soluble in ethanol (95), slightly soluble in water,
and practically insoluble in diethyl ether.
It dissolves in dilute hydrochloric acid.
It is gradually colored by light.
Identification (1) To 0.1 mL of a solution of Cimetidine
in ethanol (95) (1 in 100) add 5 mL of citric acid-acetic anhy-
dride TS, and heat in a water bath for 15 minutes: a red-pur-
ple color develops.
(2) Determine the infrared absorption spectrum of
Cimetidine, previously dried, as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> Dissolve 0.5 g of Cimetidine in 50 mL of freshly
boiled and cooled water, shake for 5 minutes and filter: the
pH of the filtrate is between 9.0 and 10.5.
Melting point <2.60> 140 - 144°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cimetidine in 10 mL of methanol: the solution is clear and
colorless to pale yellow in color.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Cimeti-
dine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Arsenic <1.11> — Dissolve 1.0 g of Cimetidine in 5 mL
of dilute hydrochloric acid, and perform the test with this so-
lution (not more than 2 ppm).
(4) Related substances — Dissolve 0.5 g of Cimetidine in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL. Pipet 1 mL of this solution, add
methanol to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 4 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, methanol and ammonia solu-
tion (28) (21:2:2) to a distance of about 15 cm, air-dry the
plate, and then dry at 80°C for 30 minutes. Allow the plate to
stand in iodine vapor for 45 minutes: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.24 g of Cimetidine, previ-
ously dried, dissolve in 75 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 25.23 mg of C 10 H 16 N 6 S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Cisplatin
CI ^NHs
Pt
/ \
CI NH 3
Cl 2 H 6 N 2 Pt: 300.05
(SP-4-2)-Diamminedichloroplatinum
[15663-27-1]
Cisplatin, when dried, contains not less than 98.0%
and not more than 102.0%, of Cl 2 H 6 N 2 Pt.
Description Cisplatin occurs as a yellow crystalline powder.
It is sparingly soluble in iV,./V-dimethylformamide, slightly
soluble in water, and practically insoluble in ethanol (99.5).
Identification (1) To 5 mL of a solution of Cisplatin (1 in
2000) add 2 to 3 drops of a solution of tin (II) chloride
dihydrate (1 in 100): a brown precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Cisplatin in a solution of sodium chloride in 0.01 mol/L
hydrochloric acid TS (9 in 1000) (1 in 2000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cisplatin Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Cisplatin as directed in the potassium bromide disk method
514 Anhydrous Citric Acid / Official Monographs
JP XV
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Cisplatin Reference Standard: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
(4) A solution of Cisplatin (1 in 2000) responds to the
Qualitative Test <1.09> (1) for chloride.
Purity Ammonium amminetrichloroplatinate — Conduct
this procedure using light-resistant vessels. Dissolve 50 mg of
Cisplatin in a solution of sodium chloride (9 in 1000) to make
exactly 100 mL, and use this solution as the sample solution.
Separately, dissolve 10 mg of ammonium am-
minetrichloroplatinate for liquid chromatography, previous-
ly dried at 80°C for 3 hours, in the solution of sodium
chloride (9 in 1000) to make exactly 200 mL. Pipet 2 mL of
this solution, add the solution of sodium chloride (9 in 1000)
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with exactly 40 liL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following
conditions, and determine the peak area of ammonium
amminetrichloroplatinate by the automatic integration
method: the peak area from the sample solution is not more
than that from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 209 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with silica gel for liquid
chromatography having quaternary ammonium groups (10
/um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A solution of ammonium sulfate (1 in 800).
Flow rate: Adjust the flow rate so that the retention time of
ammonium amminetrichloroplatinate is about 8 minutes.
System suitability —
System performance: When the procedure is run with
40 iiL of the standard solution under the above operating
conditions, the number of theoretical plates and the
symmetry factor of the peak of ammonium am-
minetrichloroplatinate are not less than 1500 and not more
than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
40 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ammonium amminetrichloroplatinate is not more than 3.0%.
Loss on drying <2.41> Not more than 0.1% (1 g, 105°C, 4
hours).
Assay Conduct this procedure using light-resistant vessels.
Weigh accurately about 25 mg each of Cisplatin and
Cisplatin Reference Standard, previously dried, dissolve in
7V,./V-dimethylformamide to make exactly 25 mL, and use
these solutions as the sample solution and the standard
solution. Perform the test with exactly 40 iiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following
conditions, and determine the peak areas, A T and A s , of
cisplatin by the automatic integration method.
Amount (mg) of Cl 2 H 6 N 2 Pt = W s x (A T /A S )
W s : Amount (mg) of Cisplatin Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 310 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with aminopropyl-
silanized silica gel for liquid chromatography (5 /nm in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of ethyl acetate, methanol, water
and 7V,7V-dimethylformamide (25:16:5:5).
Flow rate: Adjust the flow rate so that the retention time of
cisplatin is about 4 minutes.
System suitability —
System performance: When the procedure is run with
40 [iL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of cisplatin are not less than 3000 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
40 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cisplatin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Anhydrous Citric Acid
HO CQ 2 H
C 6 H 8 7 : 192.12
2-Hydroxypropane-l,2,3-tricarboxylic acid
[77-92-9]
This monograph is harmonized with the European Phar-
macopoeia and the U.S. Pharmacopeia. The parts of the text
that are not harmonized are marked with symbol (* ♦).
Anhydrous Citric Acid contains not less than 99.5%
and not more than 100.5% of C 6 H 8 7 , calculated on
the anhydrous basis.
♦Description Anhydrous Citric Acid occurs as colorless
crystals, white granules or crystalline powder.
It is very soluble in water, and freely soluble in ethanol
(95)-
♦identification Determine the infrared absorption spec-
trum of Anhydrous Citric Acid, previously dried at 105 °C
for 2 hours, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.*
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
Anhydrous Citric Acid in water to make 10 mL: the solution
is clear and has no more color than the following control
solutions (1), (2) or (3).
Control solution (1): To 1.5 mL of Cobalt (II) Chloride
Colorimetric Stock Solution and 6.0 mL of Iron (III)
Chloride Colorimetric Stock Solution add water to make
JPXV
Official Monographs / Citric Acid Hydrate 515
1000 mL.
Control solution (2): To 0.15 mL of Cobalt (II) Chloride
Colorimetric Stock Solution, 7.2 mL of Iron (III) Chloride
Colorimetric Stock Solution and 0.15 mL of Copper (II) Sul-
fate Colorimetric Stock Solution add water to make
1000 mL.
Control solution (3): To 2.5 mL of Cobalt (II) Chloride
Colorimetric Stock Solution, 6.0 mL of Iron (III) Chloride
Colorimetric Stock Solution and 1 .0 mL of Copper (II)
Sulfate Colorimetric Stock Solution add water to make 1000
mL.
(2) Sulfates <1.14>— Dissolve 2.0 g of Anhydrous Citric
Acid in water to make 30 mL, and use this solution as the
sample solution. Separately, dissolve 0.181 g of potassium
sulfate in diluted ethanol (99.5) (3 in 10) to make exactly 500
mL. Pipet 5 mL of this solution, and add diluted ethanol
(99.5) (3 in 10) to make exactly 100 mL. To 4.5 mL of this so-
lution add 3 mL of a solution of barium chloride dihydrate (1
in 4), shake, and allow to stand for 1 minute. To 2.5 mL of
this solution add 15 mL of the sample solution and 0.5 mL of
acetic acid (31), and allow to stand for 5 minutes: the solution
has no more turbidity than the following control solution.
Control solution: Dissolve 0.181 g of potassium sulfate in
water to make exactly 500 mL. Pipet 5 mL of this solution,
add water to make exactly 100 mL, and proceed in the same
manner as above using this solution instead of the sample
solution.
(3) Oxalic acid — Dissolve 0.80 g of Anhydrous Citric
Acid in 4 mL of water, add 3 mL of hydrochloric acid and 1 g
of zinc, and boil for 1 minute. After allowing to stand for 2
minutes, take the supernatant liquid, add 0.25 mL of a solu-
tion of phenylhydrazinium hydrochloride (1 in 100), heat to
boil, and then cool quickly. To this solution add the equal
volume of hydrochloric acid and 0.25 mL of a solution of
potassium hexacyanoferrate (III) (1 in 20), mix, and allow to
stand for 30 minutes: the solution has no more color than the
following control solution prepared at the same time.
Control solution: To 4 mL of a solution of oxalic acid di-
hydrate (1 in 10,000) add 3 mL of hydrochloric acid and 1 g
of zinc, and proceed in the same manner as the test solution.
*(4) Heavy metals <1.07> — Proceed with 2.0 g of Anhy-
drous Citric Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).»
(5) Readily carbonizable substances <1J5> — Perform the
test with 0.5 g of Anhydrous Citric Acid, provided that the
solution is heated at 90°C for 1 hour and then cool quickly:
the solution has no more color than Matching Fluid K.
Water <2.48> Not more than 1.0% (2 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.55 g of Anhydrous Citric
Acid, dissolve in 50 mL of water, and titrate with 1 mol/L so-
dium hydroxide VS (indicator: 2 drops of phenotphthalein
TS).
Each mL of 1 mol/L sodium hydroxide VS
= 64.04 mg of C 6 H 8 7
♦Containers and storage Containers — Tight containers. ♦
Citric Acid Hydrate
HO COjH
C 6 H 8 7 .H 2 0: 210.14
2-Hydroxypropane-l,2,3-tricarboxylic acid monohydrate
[5949-29-1]
This monograph is harmonized with the European Phar-
macopoeia and the U.S. Pharmacopeia. The parts of the text
that are not harmonized are marked with symbol (* ♦).
Citric Acid Hydrate contains not less than 99.5%
and not more than 100.5% of anhydrous citric acid (C 6
H 8 7 : 192.12), calculated on the anhydrous basis.
♦Description Citric Acid Hydrate occurs as colorless crys-
tals, white granules or crystalline powder.
It is very soluble in water, and freely soluble in ethanol
(95).
It is efflorescent in dry air.»
♦identification Determine the infrared absorption spec-
trum of Citric Acid Hydrate, previously dried at 105 °C for 2
hours, as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers. ♦
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
Citric Acid Hydrate in water to make 10 mL: the solution is
clear and has no more color than the following control solu-
tions (1), (2) or (3).
Control solution (1): To 1.5 mL of Cobalt (II) Chloride
Colorimetric Stock Solution and 6.0 mL of Iron (III)
Chloride Colorimetric Stock Solution add water to make
1000 mL.
Control solution (2): To 0.15 mL of Cobalt (II) Chloride
Colorimetric Stock Solution, 7.2 mL of Iron (III) Chloride
Colorimetric Stock Solution and 0.15 mL of Copper (II) Sul-
fate Colorimetric Stock Solution add water to make
1000 mL.
Control solution (3): To 2.5 mL of Cobalt (II) Chloride
Colorimetric Stock Solution, 6.0 mL of Iron (III) Chloride
Colorimetric Stock Solution and 1.0 mL of Copper (II)
Sulfate Colorimetric Stock Solution add water to make
1000 mL.
(2) Sulfates <1.14>— Dissolve 2.0 g of Citric Acid Hy-
drate in water to make 30 mL, and use this solution as the
sample solution. Separately, dissolve 0.181 g of potassium
sulfate in diluted ethanol (99.5) (3 in 10) to make exactly 500
mL. Pipet 5 mL of this solution, and add diluted ethanol
(99.5) (3 in 10) to make exactly 100 mL. To 4.5 mL of this so-
lution add 3 mL of a solution of barium chloride dihydrate (1
in 4), shake, and allow to stand for 1 minute. To 2.5 mL of
this solution add 15 mL of the sample solution and 0.5 mL of
acetic acid (31), and allow to stand for 5 minutes: the solution
has no more turbidity than the following control solution.
Control solution: Dissolve 0.181 g of potassium sulfate in
water to make exactly 500 mL. Pipet 5 mL of this solution,
516 Clarithromycin / Official Monographs
JP XV
add water to make exactly 100 mL, and proceed in the same
manner as above using this solution instead of the sample
solution.
(3) Oxalic acid — Dissolve 0.80 g of Citric Acid Hydrate
in 4 mL of water, add 3 mL of hydrochloric acid and 1 g of
zinc, and boil for 1 minute. After allowing to stand for 2
minutes, take the supernatant liquid, add 0.25 mL of a solu-
tion of phenylhydrazinium hydrochloride (1 in 100), heat to
boil, and then cool quickly. To this solution add the equal
volume of hydrochloric acid and 0.25 mL of a solution of
potassium hexacyanoferrate (III) (1 in 20), mix, and allow to
stand for 30 minutes: the solution has no more color than the
following control solution prepared at the same time.
Control solution: To 4 mL of a solution of oxalic acid di-
hydrate (1 in 10,000) add 3 mL of hydrochloric acid and 1 g
of zinc, and proceed in the same manner as the test solution.
*(4) Heavy metals <1.07> — Proceed with 2.0 g of Citric
Acid Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).»
(5) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Citric Acid Hydrate, provided that the solu-
tion is heated at 90°C for 1 hour and then cool quickly: the
solution has no more color than Matching Fluid K.
Water <2.48> Not less than 7.5% and not more than 9.0%
(0.5 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.55 g of Citric Acid Hy-
drate, dissolve in 50 mL of water, and titrate <2.50> with 1
mol/L sodium hydroxide VS (indicator: 2 drops of
phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 64.04 mg of C 6 H 8 7
♦Containers and storage Containers — Tight containers. ♦
Clarithromycin
C 38 H 69 N0 13 : 747.95
(2R,3SAS,5R,6R,8R,l0R,llR,l2S,13R)-5-(3A,6-
Trideoxy-3-dimethylamino-y6-D-xy/o-hexopyranosyloxy)-3-
(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-n&o-
hexopyranosyloxy)- 1 1 , 1 2-dihydroxy-6-methoxy-
2,4,6,8,10,12-hexamethyl-9-oxopentadecan-13-olide
[81103-11-9]
Clarithromycin is a derivative of erythromycin.
It contains not less than 950 ,ug (potency) and not
more than 1050 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Clarithromycin is
expressed as mass (potency) of clarithromycin
(C 38 H 69 N0 13 ).
Description Clarithromycin occurs as a white crystalline
powder and has a bitter taste.
It is soluble in acetone and in chloroform, slightly soluble
in methanol and in ethanol (95), and practically insoluble in
water.
Identification (1) To 5 mg of Clarithromycin add 2 mL of
sulfuric acid, and shake gently: a red-brown color develops.
(2) Dissolve 3 mg of Clarithromycin in 2 mL of acetone,
and add 2 mL of hydrochloric acid: an orange color develops
and changes immediately to red to deep purple.
(3) Determine the infrared absorption spectra of
Clarithromycin and Clarithromycin Reference Standard as
directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>, and compare these spectra:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(4) Dissolve 10 mg each of Clarithromycin and
Clarithromycin Reference Standard in 4 mL of chloroform,
and use these solutions as the sample solution and standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop with a mixture of
chloroform, methanol and ammonia water (28) (100:5:1) to a
distance of about 15 cm, and air-dry the plate. Spray evenly
sulfuric acid on the plate, and heat at 105°C for 10 minutes:
the principal spot from the sample solution and the spot from
the standard solution show a dark purple color and have the
same Rf value.
Optical rotation <2.49> [a]™: - 87 - -97° (0.25 g calculat-
ed on the anhydrous basis, chloroform, 25 mL, 100 mm).
Melting point <2.60> 220 - 227 °C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Clarithromycin according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Arsenic — Prepare the test solution with 1.0 g of
Clarithromycin according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Weigh accurately about 0.1 g of
Clarithromycin, dissolve in the mobile phase to make exactly
20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of Clarithromycin
Reference Standard, dissolve in the mobile phase to make ex-
actly 20 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /uL each of the sample solu-
tion and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the each peak area by the automatic in-
tegration method: the amount of each related substance cal-
culated on the anhydrous basis is not more than 2.0%, and
the total of them is not more than 5.0%. Exclude any peak
with an area of less than 0.05%.
Amount (%) of each related substance calculated on the
anhydrous basis
JPXV
Official Monographs / Clarithromycin Tablets 517
= (W S /W T ) x (At/As) x 100
Total amount (%) of the related substances calculated on the
anhydrous basis
= (Ws/Wj) x (ZA T /A S ) x 100
W s : Amount (mg) of Clarithromycin Reference Standard
W T : Amount (mg) of the sample, calculated on the anhy-
drous basis
^4 S : Peak area of clarithromycin obtained with the stan-
dard solution
A T : Peak area of each related substance obtained with the
sample solution
2M T : Total area of the peaks other than clarithromycin ob-
tained with the sample solution
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 5 times as long as the
retention time of the main peak after 2 minutes of sample
injection.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 10 mL,
and use this solution as the solution for system suitability
test. Confirm that when the procedure is run with 10 fiL of
the solution for system suitability test, the peak area of
clarithromycin is equivalent to 14 - 26% of that obtained
from the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of clarithromycin is not more than 3.0%.
Water <2.48> Not more than 2.0% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately an amount of Clarithromycin and
Clarithromycin Reference Standard, equivalent to about 0.1
g (potency), and dissolve in the mobile phase to make exactly
20 mL. Pipet 2 mL each of these solutions, add exactly 2 mL
of the internal standard solution, add the mobile phase to
make 20 mL, and use these solutions as the sample solution
and the standard solution. Perform the test with 10,wL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the ratios, Qt and Q s , of the
peak area of clarithromycin to that of the internal standard.
Amount [//g (potency)] of C 38 H 69 N0 13
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Clarithromycin Reference
Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in the mobile phase (1 in 20,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of diluted 0.2 mol/L potassium
dihydrogenphosphate TS (1 in 3) and acetonitrile (13:7).
Flow rate: Adjust the flow rate so that the retention time of
clarithromycin is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, clarithromycin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of clarithromycin to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Well-closed contain-
ers.
Clarithromycin Tablets
Clarithromycin Tablets contain not less than 93.0%
and not more than 107.0% of the labeled amount of
clarithromycin (C 38 H 6 9N0 13 : 747.95).
Method of preparation Prepare as directed under Tablets,
with Clarithromycin.
Identification Shake a quantity of pulverized Clarithromy-
cin Tablets, equivalent to 60 mg (potency) of Clarithromycin
according to the labeled amount, with 40 mL of acetone for
10 minutes, and centrifuge at 4000 rpm for 5 minutes.
Evaporate 30 mL of the supernatant liquid, and determine
the infrared absorption spectrum of the residue so obtained
as directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 2980 cm - ', 2940 cm -1 , 1734
cm" 1 , 1693 cm" 1 , 1459 cm" 1 , 1379 cm" 1 and 1171 cm" 1 .
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Clarithromycin Tablets add exactly F/20
mL of the internal standard solution (1), then add the mobile
phase so that each mL contains about 5 mg (potency) of
clarithromycin (C 38 H 69 N0 13 ) to make KmL, and disperse to
fine particles with the aid of ultrasonic waves for 20 minutes
while occasional vigorous shaking. Centrifuge this solution at
4000 rpm for 15 minutes, and filter the supernatant liquid
through a membrane filter with a pore size of not more than
0.45 fim. Then, proceed as directed in the Assay.
Amount [mg (potency)] of clarithromycin (C 38 H 69 N0 13 )
= W s x (Q T /Q S ) x (K/10)
W s : Amount [mg (potency)] of Clarithromycin Reference
Standard
Internal standard solution (1) — A solution of butyl para-
518
Clarithromycin Tablets / Official Monographs
JP XV
hydroxybenzoate in the mobile phase (1 in 1000).
Internal standard solution (2) — To exactly 1 mL of the inter-
nal standard solution (1) add the mobile phase to make ex-
actly 20 mL.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Clarithromycin Tablets at
50 revolutions per minute according to the Paddle method,
using 900 mL of 0.05 mol/L disodium hydrogen phosphate-
citric acid buffer solution, pH 6.0 as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 30
minutes after starting the test, and filter through a membrane
filter with a pore size not exceeding 0.45 Lira. Discard the first
10 mL of the filtrate, pipet the subsequent FmL, add the mo-
bile phase to make exactly V mL so that each mL contains
about 28 fig (potency) of clarithromycin (C 38 H 69 N0 13 ) ac-
cording to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 28 mg
(potency) of Clarithromycin Reference Standard, and dis-
solve in acetonitrile for liquid chromatography to make ex-
actly 100 mL. Pipet 5 mL of this solution, add the mobile
phase to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with exactly 100 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and A s , of
clarithromycin. The dissolution rates in 30 minutes of a
50-mg tablet and a 200-mg tablet are not less than 80% and
not less than 75%, respectively.
Dissolution rate (%) with respect to the labeled amount of
clarithromycin (C 38 H 69 N0 13 )
= W s x (Aj/As) x {V'/V) x (1/Q x 90
W s : Amount [mg (potency)] of Clarithromycin Reference
Standard
C: Labeled amount [mg (potency)] of clarithromycin
(C 38 H 69 N0 13 ) in 1 tablet
Operating conditions-
Proceed as directed in the operating conditions in the As-
say.
System suitability —
System performance: When the procedure is run with 100
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of clarithromycin are not less than 3000 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
100 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
clarithromycin is not more than 2.0%.
Assay To not less than 5 Clarithromycin Tablets add dilut-
ed 0.2 mol/L potassium dihydrogen phosphate TS (1 in 3) so
that each mL contains about 8 mg (potency) of clarithromy-
cin (C 38 H 69 N0 13 ), disperse to fine particles with the aid of
ultrasonic waves, add exactly 1 mL of the internal standard
solution (1) per 100 mg (potency) of clarithromycin accord-
ing to the labeled amount, then add acetonitrile for liquid
chromatography so that each mL contains about 5 mg
(potency) of clarithromycin (C 38 H 69 N0 13 ), and disperse to
fine particles with the aid of ultrasonic waves for 10 minutes
while occasional vigorous shaking. Centrifuge of this solu-
tion at 4000 rpm for 15 minutes, and filter the supernatant
liquid through a membrane filter with a pore size of not more
than 0.45 /um. Discard the first 3 mL of the filtrate, to 2 mL
of the subsequent filtrate add the mobile phase to make 20
mL, and use this solution as the sample solution. Separately,
weigh accurately about 50 mg (potency) of Clarithromycin
Reference Standard, and dissolve in the mobile phase to
make exactly 10 mL. Pipet 2 mL of this solution, add exactly
2 mL of the internal standard solution (2) and the mobile
phase to make 20 mL, and use this solution as the standard
solution. Perform the test with 1 /xh each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of
clarithromycin to that of the internal standard.
Amount [mg (potency)] of clarithromycin (C 38 H 69 N0 13 )
= W s x (Q T /Q S ) x (1/5)
W s : Amount [mg (potency)] of Clarithromycin Reference
Standard
Internal standard solution (1) — A solution of butyl para-
hydroxybenzoate in the mobile phase (1 in 1000).
Internal standard solution (2) — To exactly 1 mL of the inter-
nal standard solution (1) add the mobile phase to make ex-
actly 20 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of diluted 0.2 mol/L potassium
dihydrogen phosphate TS (1 in 3) and acetonitrile for liquid
chromatography (13:7).
Flow rate: Adjust the flow rate so that the retention time of
clarithromycin is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
iiL of the standard solution under the above operating condi-
tions, clarithromycin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of clarithromycin to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Clindamycin Hydrochloride 519
Clemastine Fumarate
HsC , H H3C I
.^^.COjH
HC^C
C 21 H 26 C1N0.C 4 H 4 4 : 459.96
(2R )-2- {2-[( li? )- 1 -(4-Chlorophenyl)- 1 -
phenylethoxy]ethyl}-l-methylpyrrolidine monofumarate
[14976-57-9]
Clemastine Fumarate, when dried, contains not less
than 98.5% of C 21 H 26 C1N0.C 4 H 4 04.
Description Clemastine Fumarate occurs as a white, crys-
talline powder. It is odorless.
It is sparingly soluble in methanol and in acetic acid (100),
slightly soluble in ethanol (95), very slightly soluble in diethyl
ether, and practically insoluble in water.
Identification (1) To 5 mg of Clemastine Fumarate add 5
mL of sulfuric acid, and shake to dissolve: a yellow color de-
velops. Slowly drop this solution into 10 mL of water: the
yellow color immediately disappears.
(2) To 0.01 g of Clemastine Fumarate add 1 mL of fum-
ing nitric acid, and evaporate on a water bath to dryness.
Then add 2 mL of diluted hydrochloric acid (1 in 2) and 0.2 g
of zinc powder, heat for 10 minutes on a water bath, cool,
and filter. Add 20 mL of water to the filtrate. The solution
responds to the Qualitative Tests <1.09> for primary aromatic
amines.
(3) To 5 mL of a solution of Clemastine Fumarate (1 in
50,000), add 5 mL of 4-dimethylaminobenzaldehyde TS, and
warm for 10 minutes: a red-purple color develops.
(4) Perform the test with Clemastine Fumarate as direct-
ed under Flame Coloration Test <1.04> (2): a green color ap-
pears.
(5) Dissolve 0.04 g of Clemastine Fumarate and 0.01 g of
fumaric acid for thin-layer chromatography in 2 mL each of
a mixture of ethanol (95) and water (4:1) by gentle warming,
and use these solutions as the sample solution and the stan-
dard solution, respectively. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 fiL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
isopropyl ether, formic acid and water (90:7:3) to a distance
of about 10 cm, and air-dry the plate. Examine the plate un-
der ultraviolet light (main wavelength: 254 nm): the spot with
larger Rf value from the sample solution has the same Ri
value as the spot from the standard solution.
Optical rotation <2.49> [a]™: +16 -+18° (after drying,
0.1 g, methanol, 10 mL, 100 mm).
Melting point <2.60> 176 - 180°C (with decomposition).
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Clemastine Fumarate in 10 mL of methanol by warming: the
solution is clear and colorless.
(2) Heavy metals <1.07> — Perform the test with 1.0 g of
Clemastine Fumarate according to Method 2. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Take 1.0 g of Clemastine Fumarate,
prepare the test solution according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related Substances — Dissolve 0.10 g of Clemastine
Fumarate in 5 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 250 mL, and use this solution as
the standard solution (1). Pipet 5 mL of this solution, add
methanol to make exactly 10 mL, and use this solution as the
standard solution (2). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solutions (1) and (2)
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of chloroform, methanol and
ammonia solution (28) (90: 10: 1) to a distance of about 10 cm,
and air-dry the plate. After spraying evenly Dragendorff 's TS
on the plate, immediately spray evenly hydrogen peroxide
TS: the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution (1), and not more than 2 spots from the sample solu-
tion are more intense than the spot from the standard solu-
tion (2).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.4 g of Clemastine
Fumarate, previously dried, dissolved in 50 mL of acetic acid
(100), and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 46.00 mg of C 21 H 26 C1N0.C 4 H 4 4
Containers and storage Containers — Tight containers.
Clindamycin Hydrochloride
?y>*v 4 •»£!»£
HjC
,CH 3
N
h
A
CI
1
h
r
CHa
O HO
H
-OH
X>H
H 2
h
— r s.
■KCI
CHa
C 18 H 33 C1N 2 5 S.HC1: 461.44
Methyl 7-chloro-6,7 , 8-trideoxy-6- [(2S,4R )- 1 -methyl-4-
propylpyrrolidine-2-carboxamido] - 1 -thio-L-threo-a-O-
ga/acfo-octopyranoside monohydrochloride [21462-39-5]
Clindamycin Hydrochloride is the hydrochloride of
a derivative of lincomycin.
It contains not less than 759 fig (potency) and not
more than 902 fig (potency) per mg. The potency of
520
Clindamycin Hydrochloride Capsules / Official Monographs
JP XV
Clindamycin Hydrochloride is expressed as mass
(potency) of clindamycin (C 18 H3 3 C1N 2 5 S: 424.98).
Description Clindamycin Hydrochloride occurs as white to
grayish white, crystals or crystalline powder.
It is freely soluble in water and in methanol, and slightly
soluble in ethanol (95).
Identification Dissolve 0.1 g of Clindamycin Hydrochloride
in 5 mL of water, add 2 mL of sodium hydroxide TS, and
mix: a white turbidity is produced. To this solution add 0.3
mL of sodium pentacyanonitrosylferrate (III) TS, mix, allow
to stand at 60 to 65 °C for 10 minutes, and add 2 mL of dilute
hydrochloric acid: a blue-green color develops.
Optical rotation <2.49> [a]^ 5 : + 135 - + 150° (0.5 g calculated
on the anhydrous basis, water, 25 mL, 100 mm).
Water <2.48> Not more than 6.0% (0.3 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Micrococcus luteus ATCC 9341
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Weigh accurately an amount of
Clindamycin Hydrochloride Reference Standard, equivalent
to about 25 mg (potency), dissolve in 0.1 mol/L phosphate
buffer solution, pH 7.0 to make exactly 250 mL, and use this
solution as the standard stock solution. Keep the standard
stock solution at a temperature not exceeding 15 °C and use
within 14 days. Take exactly a suitable amount of the stan-
dard stock solution before use, add 0.1 mol/L phosphate
buffer solution, pH 7.0 to make solutions so that each mL
contains 2 fxg (potency) and 1 /xg (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Clindamycin Hydrochloride, equivalent to about 25 mg
(potency), and dissolve in 0.1 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 250 mL. Take exactly a suitable
amount of this solution, add 0.1 mol/L phosphate buffer
solution, pH 7.0 to make solutions so that each mL contains
2,«g (potency) and 1 /xg (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Clindamycin Hydrochloride
Capsules
/7lj>y T ,f
t^tl^iDl
Clindamycin Hydrochloride Capsules contain not
less than 90.0% and not more than 110.0% of the la-
beled amount of clindamycin (C lg H33ClN20 5 S:
424.98).
Method of preparation Prepare as directed under Capsules,
with Clindamycin Hydrochloride.
Identification To an amount of the contents of Clindamy-
cin Hydrochloride Capsules, equivalent to 10 mg (potency)
of Clindamycin Hydrochloride, add 2 mL of methanol,
shake well, centrifuge, and use the supernatant liquid as the
sample solution. Separately, dissolve 10 mg of Clindamycin
Hydrochloride Reference Standard in 2 mL of methanol, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of
methanol, toluene and ammonia solution (28) (140:60:3) to a
distance of about 12 cm, and air-dry the plate. Spray evenly a
mixture of 500 mL of a solution of L-tartaric acid (1 in 5) and
50 mL of bismuth subnitrate TS on the plate: the Rf values of
the principal spot with the sample solution and the spot with
the standard solution are not different each other.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 capsule of Clindamycin Hydrochloride Capsules add
a suitable amount of the mobile phase, shake for 30 minutes,
and add the mobile phase to make exactly KmL so that each
mL contains 0.75 mg (potency) of Clindamycin Hydrochlo-
ride according to the labeled amount. Centrifuge for 10
minutes, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 75 mg (potency) of
Clindamycin Hydrochloride Reference Standard, add the
mobile phase to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 20 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of clindamycin.
Amount [mg (potency)] of clindamycin (Qg^jClNjOsS)
= W s x (Aj/A s ) x (K/100)
W s : Amount [mg (potency)] of Clindamycin Hydrochlo-
ride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 7.5 with 8 mol/L potassium
hydroxide TS. To 550 mL of this solution add 450 mL of
acetonitrile for liquid chromatography.
Flow rate: Adjust the flow rate so that the retention time of
clindamycin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of clindamycin are not less than 3000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
JP XV
Official Monographs / Clindamycin Phosphate 521
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
clindamycin is not more than 1.0%.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Clindamycin Hydrochlo-
ride Capsules at 50 revolutions per minute according to the
Paddle method, using the sinker, using 900 mL of water as
the dissolution medium. Withdraw not less than 20 mL of the
dissolution medium 15 minutes after starting the test for a
75-mg capsule or 30 minutes after starting the test for a
150-mg capsule, and filter through a membrane filter with a
pore size not exceeding 0.45 //m. Discard the first 10 mL of
the filtrate, pipet KmL of the subsequent filtrate, add water
to make exactly V so that each mL contains about 83 /xm
(potency) of clindamycin hydrochloride, and use this solution
as the sample solution. Separately, weigh accurately about 17
mg (potency) of Clindamycin Hydrochloride Reference Stan-
dard, dissolve in water to make exactly 200 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01>, and deter-
mine the peak areas, A T and A s , of clindamycin. The dissolu-
tion rate of a 75-mg capsule in 15 minutes and that of a
150-mg capsule in 30 minutes are not less than 80%, respec-
tively.
Dissolution rate (%) with respect to the labeled amount of
clindamycin hydrochloride
= W s x (Aj/A s ) x (V'/V) x (I/O x 450
W s : Amount [mg (potency)] of Clindamycin Hydrochlo-
ride Reference Standard
C: Labeled amount [mg (potency)] of clindamycin
(C I8 H 33 C1N 2 5 S) in 1 tablet
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 7.5 with 8 mol/L potassium
hydroxide TS. To 550 mL of this solution add 450 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
clindamycin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of clindamycin are not less than 3000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
clindamycin is not more than 2.0%.
Assay Perform the test according to the Cylinder - plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, and standard
solutions — Proceed as directed in the Assay under Clindamy-
cin Hydrochloride.
(ii) Sample solutions — Weigh accurately the mass of not
less than 20 Clindamycin Hydrochloride Capsules, cut the
capsules and take out the contents, mix well, and powder, if
necessary. If necessary, wash the empty capsules with a small
amount of diethyl ether, allow to stand at room temperature
to dry the capsules, weigh their mass accurately, and calcu-
late the mass of the contents. Weigh accurately an amount of
the contents, equivalent to about 25 mg (potency) according
to the labeled amount, add a suitable amount of 0.1 mol/L
phosphate buffer solution, pH 7.0, shake vigorously, then
add 0.1 mol/L phosphate buffer solution to make a solution
so that each mL contains about 100 fig (potency), and filter,
if necessary. To exactly an amount of this solution add 0.1
mol/L phosphate buffer solution, pH 7.0 to make solutions
so that each mL contains 2,Mg (potency) and 1 /xg (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Clindamycin Phosphate
/7 Ij >y y"f ->> u >m^xT)i
C 18 H 34 C1N 2 8 PS: 504.96
Methyl 7-chloro-6,7,8-trideoxy-6-[(25',4/?)-l-methyl-4-
propylpyrrolidine-2-carboxamido]-l-thio-L-?/jreo-a-D-
ga/acfo-octopyranoside 2-dihydrogenphosphate
[24729-96-2]
Clindamycin Phosphate is a derivative of clindamy-
cin.
It contains not less than 800 /ug (potency) and not
more than 846 [xg (potency) per mg, calculated on the
anhydrous basis. The potency of Clindamycin Phos-
phate is expressed as mass (potency) of clindamycin
(Ci 8 H 33 ClN 2 5 S: 424.98).
Description Clindamycin Phosphate occurs as a white to
pale yellowish white crystalline powder.
It is freely soluble in water, sparingly soluble in methanol,
and practically insoluble in ethanol (95).
Identification Determine the infrared absorption spectrum
of Clindamycin Phosphate, previously dried at 100°C for 2
hours, as directed in the paste method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Clindamycin Phos-
phate Reference Standard previously dried at 100°C for 2
hours: both spectra exhibit similar intensities of absorption at
the same wave numbers.
522 Clinofibrate / Official Monographs
JP XV
Optical rotation <2.49> [a]™: + 115 - + 130° (0.25 g calculat-
ed on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> Dissolve 0.10 g of Clindamycin Phosphate in 10
mL of water. The pH of the solution is between 3.5 and 4.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Clindamycin Phosphate in 10 mL of freshly boiled and
cooled water: the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Clin-
damycin Phosphate according to Method 4, and perform the
test. Prepare the control solution with 1.0 mL of Standard
Lead Solution (not more than 5 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Clindamycin Phosphate according to Method 4, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.1 g of Clindamycin
Phosphate in 100 mL of the mobile phase, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 ,«L each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the peak area of
clindamycin, having the relative retention time of about 1.8
with respect to clindamycin phosphate, obtained from the
sample solution is not more than 1/2 of the peak area of clin-
damycin phosphate from the standard solution, and the total
area of the peaks other than clindamycin phosphate from the
sample solution is not more than 4 times the peak area of
clindamycin phosphate from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of clindamycin phosphate beginning after the
solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of clindamycin
phosphate obtained from 20 /uL of this solution is equivalent
to 7 to 13% of that from 20 [iL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Water <2.48> Not more than 6.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Clindamycin Phos-
phate and Clindamycin Phosphate Reference Standard,
equivalent to about 20 mg (potency), add exactly 25 mL of
the internal standard solution and the mobile phase to make
100 mL, and use these solutions as the sample solution and
standard solution. Perform the test with 20 /iL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, g T and Q s , of the peak area
of clindamycin phosphate to that of the internal standard.
Amount [/ug (potency)] of clindamycin (Q8H33CIN2O5S)
= Ws x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Clindamycin Phosphate
Reference Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in the mobile phase (3 in 50,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 210 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octylsilanized silica gel for
liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 10.54 g of potassium dihydrogen
phosphate in 775 mL of water, adjust the pH to 2.5 with
phosphoric acid, and add 225 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
clindamycin phosphate is about 8 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the standard solution under the above operating
conditions, clindamycin phosphate and the internal standard
are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of clindamycin phosphate to that of the internal
standard is not more than 2.5%.
Containers and storage Containers — Tight containers.
Clinofibrate
^'J/7^7- h
HO;C
CC^H
C 28 H 36 6 : 468.58
2,2'-(4,4'-Cyclohexylidenediphenoxy)-2,2'-
dimethyldibutanoic acid [30299-08-2]
Clinofibrate, when dried, contains not less than
98.5% of C 28 H 36 6 .
Description Clinofibrate occurs as a white to yellowish
white powder.
It is odorless and has no taste.
It is freely soluble in methanol, in ethanol (99.5), in ace-
tone and in diethyl ether, and practically insoluble in water.
A solution of Clinofibrate in methanol (1 in 20) shows no
optical rotation.
Melting point: about 146°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Clinofibrate in ethanol (99.5) (1 in 50,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
JPXV
Official Monographs / Clocapramine Hydrochloride Hydrate
523
(2) Determine the infrared absorption spectrum of
Clinofibrate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Clinofibrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Clinofibrate according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Clinofibrate
in 10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add acetone to
make exactly 50 mL. Pipet 5 mL of this solution, add acetone
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 50 //L each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform, cyclohexane
and acetic acid (100) (12:5:3) to a distance of about 12 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Isomer ratio To 50 mg of Clinofibrate add 0.4 mL of
thionyl chloride, stopper tightly, heat on a water bath of
60°C for 5 minutes with occasional shaking, and evaporate
the excess thionyl chloride at a temperature not exceeding
60°C under reduced pressure. Dissolve the residue in 2 mL of
toluene previously dried with synthetic zeolite for drying, add
2 mL of a solution of D-( + )-a-methylbenzylamine in toluene
previously dried with synthetic zeolite for drying (3 in 100),
mix gently, allow to stand for 10 minutes, and evaporate the
toluene at a temperature not exceeding 60 °C under reduced
pressure. Dissolve the residue in 5 mL of chloroform, and use
this solution as the sample solution. Perform the test with 5
/nL of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Determine each peak area, A 3 , A b and A c , of three peaks ap-
pear in order near the retention time of 40 minutes: a value,
U b /(A + A b + A c )} x 100, is between 40 and 70.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 30 cm in length, packed with silica gel for
liquid chromatography (5 /um in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of hexane and 2-propanol
(500:3).
Flow rate: Adjust the flow rate so that the retention time of
the peak appearing first is about 35 minutes.
Selection of column: Proceed with 5 ,aL of the sample solu-
tion under the above operating conditions. Use a column giv-
ing a complete separation of the three peaks.
Assay Weigh accurately about 0.45 g of Clinofibrate, previ-
ously dried, dissolve in 40 mL of ethanol (99.5), add 30 mL
of water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (indicator: 3 drops of phenolphthalein TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 23.43 mg of C 28 H 36 6
Containers and storage Containers — Tight containers.
Clocapramine Hydrochloride
Hydrate
• 8HCI ■ HjO
C 28 H 37 C1N 4 0.2HC1.H 2 0: 572.01
l'-[3-(3-Chloro-10,ll-dihydro-5//-dibenz[d,/]azepin-
5-yl)propyl]-l,4'-bipiperidine-4'-carboxamide
dihydrochloride monohydrate [60789-62-0]
Clocapramine Hydrochloride Hydrate, when dried,
contains not less than 98.0% of clocapramine
hydrochloride (C 28 H3 7 C1N 4 0.2HC1: 553.99).
Description Clocapramine Hydrochloride Hydrate occurs
as white crystals or crystalline powder. It is odorless, and has
a bitter taste.
It is freely soluble in acetic acid (100), sparingly soluble in
water and in methanol, slightly soluble in ethanol (95), in
chloroform and in isopropylamine, and practically insoluble
in acetic anhydride and in diethyl ether.
It is gradually colored by light.
Melting point: about 260°C (with decomposition, after
drying).
Identification (1) To 5 mL of a solution of Clocapramine
Hydrochloride Hydrate (1 in 2500) add 1 mL of nitric acid: a
blue color develops at first, and rapidly changes to deep blue,
and then changes to green to yellow-green.
(2) Determine the absorption spectrum of a solution of
Clocapramine Hydrochloride Hydrate in methanol (1 in
40,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>,, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Clocapramine Hydrochloride Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
524 Clofedanol Hydrochloride / Official Monographs
JP XV
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) Dissolve 0.1 g of Clocapramine Hydrochloride Hy-
drate in 10 mL of water by warming, and after cooling, add 2
mL of ammonia TS, and filter. Acidify the filtrate with dilute
nitric acid: the solution responds to the Qualitative Tests
<l.09> (2) for chloride.
Purity (1) Sulfate <1.14> — Dissolve 0.5 g of Clocapramine
Hydrochloride Hydrate in 40 mL of water by warming, after
cooling, and add 1 mL of dilute hydrochloric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 0.50 mL of
0.005 mol/L sulfuric acid VS (not more than 0.048%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Clocapramine Hydrochloride Hydrate according to Method
2, and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Clocapramine Hydrochloride Hydrate in 10 mL of
a mixture of chloroform and isopropylamine (99:1), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add a mixture of chloroform and isopropylamine
(99:1) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of diethyl
ether, ethyl acetate, methanol and ammonia solution (28)
(100:70:40:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> 2.0 - 3.5% (0.5 g, in vacuum at a
pressure not exceeding 0.67 kPa, phosphorus (V) oxide,
105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clocapramine
Hydrochloride Hydrate, previously dried, dissolve in 70 mL
of a mixture of acetic anhydride and acetic acid (100) (6:1),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 27.70 mg of C 28 H3 7 C1N 4 0.2HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Clofedanol Hydrochloride
^n7i^;-^lS
arid enantiomer
C 17 H 20 C1NO.HC1: 326.26
(li?5')-l-(2-Chlorophenyl)-3-dimethylamino-l-
phenylpropan-1-ol monohydrochloride [511-13-7]
Clofedanol Hydrochloride, when dried, contains not
less than 98.5% of C 17 H 20 C1NO.HC1.
Description Clofedanol Hydrochloride occurs as white
crystals or crystalline powder.
It is freely soluble in methanol, in ethanol (95) and in acetic
acid (100), sparingly soluble in water, and practically insolu-
ble in diethyl ether.
A solution of Clofedanol Hydrochloride in methanol
(1 in 20) does not show optical rotation.
Melting point: about 190°C (after drying, with decomposi-
tion).
Identification (1) Determine the absorption spectrum of a
solution of Clofedanol Hydrochloride in 0.01 mol/L
hydrochloric acid TS (1 in 2500) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Clofedanol Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Clofedanol Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Clofedanol Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.05 g of Clofedanol
Hydrochloride in 25 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 3 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area of both solutions
by the automatic integration method: the total area of all
peaks other than clofedanol from the sample solution is not
larger than the peak area of clofedanol from the standard so-
lution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column about 4 mm in inside di-
JPXV
Official Monographs / Clofibrate 525
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 jum in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.34 g of potassium methanesul-
fonate in diluted phosphoric acid (1 in 1000) to make 1000
mL, and to 650 mL of this solution add 350 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
clofedanol is about 9 minutes.
Selection of column: Dissolve 0.01 g each of Clofedanol
Hydrochloride and ethyl parahydroxybenzoate in methanol
to make 100 mL. Proceed with 3 ,«L of this solution under the
above operating conditions, and calculate the resolution. Use
a column giving elution of clofedanol and ethyl parahydrox-
ybenzoate in this order with the resolution of these peaks
being not less than 4.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of clofedanol obtained from 3 /uL of the
standard solution composes between 20% and 50% of the
full scale.
Time span of measurement: About three times as long as
the retention time of clofedanol beginning after the solvent
peak.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
silica gel, 80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clofedanol
Hydrochloride, previously dried, dissolve in 15 mL of acetic
acid (100), add 35 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 32.63 mg of C 17 H 20 C1NO.HC1
Containers and storage Containers — Tight containers.
Clofibrate
^□7^7-r
ci
xx:
■j
C CHs
C 12 H 15 C10 3 : 242.70
Ethyl 2-(4-chlorophenoxy)-2-methylpropanoate
[637-07-0]
Clofibrate, calculated on the unhydrous basis, con-
tains not less than 98.0% of C 12 H 15 C10 3 .
Description Clofibrate occurs as a colorless or light yellow,
clear, oily liquid. It has a characteristic odor and taste, which
is bitter at first, and subsequently sweet.
It is miscible with methanol, with ethanol (95), with
ethanol (99.5), with diethyl ether and with hexane, and prac-
tically insoluble in water.
It is gradually decomposed by light.
Identification (1) Determine the absorption spectrum of a
solution of Clofibrate in ethanol (99.5) (1 in 10,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum 1 or the
spectrum of a solution of Clofibrate Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths. Separately, determine the absorption spectrum
of a solution of Clofibrate in ethanol (99.5) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 2 or
the spectrum of a solution of Clofibrate Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Clofibrate as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum or the spectrum of Clofibrate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Refractive index <2.45> n™: 1.500 - 1.505
Specific gravity <2.56> df : 1.137-1.144
Purity (1) Acidity— Dissolve 2.0 g of Clofibrate in 100 mL
of neutralized ethanol, and add 1 drop of phenolphthalein TS
and 0.20 mL of 0.1 mol/L sodium hydroxide VS: the solu-
tion is red in color.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Clofibrate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11>— To 5.0 g of Clofibrate add 20 mL of
nitric acid and 5 mL of sulfuric acid, and heat until white
fumes are evolved. After cooling, if necessary, add further 5
mL of nitric acid, heat until white fumes are evolved, and
repeat this procedure until the solution is colorless to light
yellow. After cooling, add 15 mL of saturated ammonium
oxalate solution, and heat again until white fumes are
evolved. Cool, add water to make 25 mL, use 5 mL of this so-
lution as the test solution, and perform the test.
Color standard: Prepare a solution according to the above
procedure without using Clofibrate as the blank. Transfer 5
mL of the solution to a generator bottle, add 2.0 mL of Stan-
dard Arsenic Solution, and then proceed as directed in the
test solution (not more than 20 ppm).
(4) p-Chlorophenol — To 1 .0 g of Clofibrate add exactly 1
mL of the internal standard solution, then add the mobile
phase to make 5 mL, and use this solution as the sample solu-
tion. Separately, dissolve 0.010 g of 4-chlorophenol in a mix-
ture of hexane and 2-propanol (9:1) to make exactly 100 mL.
Pipet 10 mL of this solution, and add a mixture of hexane
and 2-propanol (9:1) to make exactly 50 mL. Pipet 6 mL of
this solution, add exactly 4 mL of the internal standard solu-
tion, then add the mobile phase to make 20 mL, and use this
solution as the standard solution. Perform the test with 20 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of 4-chlorophenol to that of the internal standard:
Q T is not greater than Qs-
Internal standard solution — A solution of 4-ethoxyphenol in
526 Clofibrate Capsules / Official Monographs
JP XV
the mobile phase (1 in 30,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 30 cm in length, packed with cyanopropyl-
silanized silica gel for liquid chromatography (5 to 10 fim in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of hexane, 2-propanol and acetic
acid (100) (1970:30:1).
Flow rate: Adjust the flow rate so that the retention time of
clofibrate is about 2 minutes.
Selection of column: Dissolve 10.0 g of clofibrate, 6 mg of
4-chlorophenol and 6 mg of 4-ethoxyphenol in 1000 mL of
hexane. Proceed with 20 iuL of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of clofibrate, 4-chlorophenol and 4-
ethoxyphenol in this order, with the resolution between the
peaks of clofibrate and 4-chlorophenol is not less than 5, and
with the resolution between the peaks of 4-chlorophenol and
4-ethoxyphenol is not less than 2.0.
Water <2.48> Not more than 0.2% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clofibrate, add ex-
actly 50 mL of 0.1 mol/L potassium hydroxide-ethanol VS,
and heat in a water bath under a reflux condenser with a car-
bon dioxide absorbing tube (soda-lime) for 2 hours with fre-
quent shaking. Cool, and titrate <2.50> immediately the ex-
cess potassium hydroxide with 0.1 mol/L hydrochloric acid
VS (indicator: 3 drops of phenolphthalein TS). Perform a
blank determination.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 24.27 mg of C 12 H 15 C10 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Clofibrate Capsules
^P7< 7*7- h *)7°-tz JU
Clofibrate Capsules contain not less than 93% and
not more than 107% of the labeled amount of
clofibrate (C 12 H 15 C10 3 : 242.70).
Method of preparation
with Clofibrate.
Prepare as directed under Capsules,
Identification Cut and open Clofibrate Capsules, and use
the contents as the sample. Determine the absorption spec-
trum of a solution of the sample in ethanol (99.5) (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 278 nm and 282
nm, and it exhibits a maximum between 224 nm and 228 nm
after diluting this solution 10 times with ethanol (99.5)
Purity /?-Chlorophenol — Cut and open not less than 20
Clofibrate Capsules, and proceed with 1.0 g of the well-mixed
contents as directed in the Purity (4) under Clofibrate.
Assay Weigh accurately not less than 20 Clofibrate Cap-
sules, cut and open the capsules, rinse the inside of the cap-
sules with a small amount of diethyl ether after taking out the
contents, evaporate the diethyl ether by allowing the capsules
to stand at room temperature, and weigh the capsules ac-
curately. Weigh accurately an amount of the contents,
equivalent to about 0.1 g of clofibrate (Q2FL5CIO3), dissolve
in acetonitrile to make exactly 100 mL. Pipet 5 mL of this so-
lution, add exactly 5 mL of the internal standard solution,
and use this solution as the sample solution. Separately,
weigh accurately about 0.1 g of Clofibrate Reference Stan-
dard, proceed in the same manner as directed for the sample
solution, and use the solution so obtained as the standard so-
lution. Perform the test with 10,mL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
clofibrate to that of the internal standard.
Amount (mg) of clofibrate (C^H^CIC^)
= W s x (Q T /Q S )
W s : Amount (mg) of Clofibrate Reference Standard, c alcu-
lated on the anhydrous basis
Internal standard solution — A solution of ibuprofen in the
mobile phase (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 /xm in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetonitrile and diluted phos-
phoric acid (1 in 1000) (3:2).
Flow rate: Adjust the flow rate so that the retention time of
clofibrate is about 10 minutes.
Selection of column: Dissolve 0.05 g of clofibrate and 0.3 g
of ibuprofen in 50 mL of acetonitrile. Proceed with 10 /xh of
this solution under the above operating conditions, and cal-
culate the resolution. Use a column giving elution of
ibuprofen and clofibrate in this order with the resolution be-
tween these peaks being not less than 6.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Clomifene Citrate
7PJ7i>7i>ffi
HO CO £ H
HOjfX )>C ^.COfH
C 26 H 28 C1N0.C 6 H 8 7 : 598.08
2-[4-(2-Chloro-l,2-diphenylvinyl)phenoxy]-JV,./V-
JP XV
Official Monographs / Clomifene Citrate Tablets 527
diethylethylamine monocitrate [50-41-9]
Clomifene Citrate, when dried, contains not less
than 98.0% of C 26 H 28 C1N0.C 6 H 8 7 .
Description Clomifene Citrate occurs as a white to pale yel-
lowish white powder. It is odorless.
It is freely soluble in methanol and in acetic acid (100),
sparingly soluble in ethanol (95), and practically insoluble in
diethyl ether.
It gradually changes in color by light.
Melting point: about 115°C
Identification (1) To 2 mL of a solution of Clomifene Ci-
trate in methanol (1 in 200) add 2 mL of Reinecke salt TS: a
light red precipitate is produced.
(2) Determine the absorption spectrum of a solution of
Clomifene Citrate in 0.1 mol/L hydrochloric acid TS (1 in
50,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Clomifene Citrate
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(3) A solution of Clomifene Citrate in methanol (1 in
200) responds to the Qualitative Tests <1.09> (1) and (2) for
citrate salt.
Purity (1) Clarity and color of solution — A solution of
1.0 g of Clomifene Citrate in 30 mL of methanol is clear and
colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Clomi-
fene Citrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
phosphorus (V) oxide, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Isomer ratio To 0.10 g of Clomifene Citrate add 10 mL of
water and 1 mL of sodium hydroxide TS, and extract with
three 15-mL portions of diethyl ether. Wash the combined
diethyl ether extracts with 20 mL of water, add 10 g of anhy-
drous sodium sulfate to the combined diethyl ether extracts,
shake for 1 minute, filter, and evaporate the diethyl ether of
the filtrate. Dissolve the residue in 10 mL of chloroform, and
use this solution as the sample solution. Perform the test with
2 /uL of the sample solution as directed under Gas Chro-
matography <2.02> according to the following conditions.
Determine the areas of two adjacent peaks, A a and A b , hav-
ing retention times of about 20 minutes, where A a is the peak
area of shorter retention time and A b is the peak area of
longer retention time: A b /(A !l + A b ) is between 0.3 and 0.5.
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A column 3 mm in inside diameter and 1 m in
length, having methylsilicone polymer coated at the ratio
of 1% on siliceous earth for gas chromatography (125 to
150 /um in particle diameter).
Column temperature: A constant temperature of about
195°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
the first peak of clomifene citrate is about 20 minutes.
System suitability —
System performance: When the procedure is run with 2 /iL
of the sample solution under the above operating conditions,
the resolution between the two peaks is not less than 1.3.
System repeatability: When the test is repeated 5 times with
2 /uL of the sample solution under the above operating condi-
tions, the relative standard deviation of A b /(A !l + A b ) is not
more than 5.0%.
Assay Weigh accurately about 1 g of Clomifene Citrate,
previously dried, dissolve in 50 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (indicator: 2
drops of crystal violet TS). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 59.81 mg of C 26 H 28 C1N0.C 6 H 8 7
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Clomifene Citrate Tablets
?n$7x>?i>|gt£ig
Clomifene Citrate Tablets contain not less than 93%
and not more than 107% of the labeled amount of the
clomifene citrate (C 26 H 28 C1N0.C 6 H 8 7 : 598.08).
Method of preparation Prepare as directed under Tablets,
with Clomifene Citrate.
Identification (1) Weigh a portion of powdered Clomifene
Citrate Tablets, equivalent to 1 g of Clomifene Citrate ac-
cording to the labeled amount, shake vigorously with 100 mL
of chloroform, and filter. Concentrate the filtrate on a water
bath, allow to stand at room temperature, collect the crystals
formed by filtration, and wash with a small quantity of chlo-
roform. Proceed with the crystals as directed in the Identifi-
cation (1) and (3) under Clomifene Citrate.
(2) Determine the absorption spectrum of a solution of
the crystals obtained in (1) in 0.1 mol/L hydrochloric acid TS
(1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 233 nm
and 237 nm, and between 290 nm and 294 nm.
Assay Weigh accurately, and powder not less than 20
Clomifene Citrate Tablets. Weigh accurately a portion of
the powder, equivalent to about 50 mg of clomifene citrate
(C 26 H 28 C1N0.C 6 H 8 7 ), add 50 mL of methanol, shake for 10
minutes, and add methanol to make exactly 100 mL. Cen-
trifuge a portion of this solution, pipet 4 mL of the super-
natant liquid, add methanol to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of Clomifene Citrate Reference Stan-
dard, previously dried in a desiccator (in vacuum, phospho-
rus (V) oxide) for 3 hours, and dissolve in methanol to make
exactly 100 mL. Pipet 4 mL of this solution, and dilute with
methanol to make exactly 100 mL, and use this solution as
the standard solution. Determine the absorbances, A T and
A s , of the sample solution and the standard solution, respec-
tively, at 295 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of clomifene citrate (C 26 H 28 C1N0.C 6 H 8 7 )
528
Clomipramine Hydrochloride / Official Monographs
JP XV
= W s x (Aj/A s )
W s : Amount (mg) of Clomifene Citrate Reference Stan-
dard
Containers and storage Containers — Tight containers.
Clomipramine Hydrochloride
<7u=.-f=7$.>imm
C 19 H 23 C1N 2 .HC1: 351.31
3-(3-Chloro-10,ll-dihydro-5i7-dibenz[&,/]azepin-
5-yl)-A r ,./V-dimethylpropylamine monohydrochloride
[17321-77-6]
Clomipramine Hydrochloride, when dried, contains
not less than 98.5% of C 19 H 23 C1N 2 .HC1.
Description Clomipramine Hydrochloride occurs as a white
to pale yellow, crystalline powder. It is odorless.
It is very soluble in acetic acid (100), freely soluble in
water, in methanol and in chloroform, soluble in ethanol
(95), sparingly soluble in acetic anhydride, slightly soluble in
acetone, and practically insoluble in ethyl acetate and in
diethyl ether.
Identification (1) Dissolve 3 mg of Clomipramine
Hydrochloride in 1 mL of nitric acid: a deep blue color de-
velops.
(2) Determine the absorption spectrum of a solution of
Clomipramine Hydrochloride in 0.1 mol/L hydrochloric acid
TS (3 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Take 1 g of Clomipramine Hydrochloride in a separa-
tor, dissolve in 10 mL of water, add 5 mL of sodium
hydroxide TS, and extract with two 30-mL portions of
diethyl ether [the water layer is used for Identification (4)].
Combine the diethyl ether extracts, add 20 mL of water, and
shake. Take diethyl ether layer, dry with a small portion of
anhydrous sodium sulfate, and filter. Evaporate the com-
bined extracts by warming on a water bath, and proceed the
test with the residue as directed under Flame Coloration Test
<1.04> (2): a green color appears.
(4) The solution neutralized by adding dilute nitric acid
to the water layer obtained in (3) responds to the Qualitative
Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Clomipramine Hydrochloride
in 10 mL of water: the pH of this solution is between 3.5 and
5.0.
Melting point <2.60> 192 - 196 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Clomipramine Hydrochloride in 10 mL of water: the solution
is clear and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Clomipramine Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Clomipramine Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Clomipra-
mine Hydrochloride in 10 mL of methanol, and use this solu-
tion as the sample solution. Separately, weigh 20 mg of Im-
ipramine Hydrochloride, dissolve in methanol to make exact-
ly 100 mL, and use this solution as the standard solution (1).
Then pipet 1 mL of the sample solution, and add methanol to
make exactly 50 mL. Pipet 5 mL of the solution, add
methanol to make exactly 50 mL, and use this solution as the
standard solution (2). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solutions (1) and (2)
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, acetone and
ammonia solution (28) (15:5:1) to a distance of about 10 cm,
and air-dry the plate. Spray evenly potassium dichromate-
sulfuric acid TS on the plate: the spot from the sample solu-
tion, corresponding to that from the standard solution (1), is
not more intense than the spot from the standard solution
(1). Each of the spots other than the principal spot and the
above spot from the sample solution is not more intense than
the spot from the standard solution (2).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clomipramine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 35.13 mg of C 19 H 23 C1N 2 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Clonazepam
<J,N
C 15 H 10 ClN 3 O 3 : 315.71
5-(2-Chlorophenyl)-7-nitro-l ,3-dihydro-2//-l ,4-
benzodiazepin-2-one [1622-61-3]
JPXV
Official Monographs / Clonidine Hydrochloride
529
Clonazepam, when dried, contains not less than
99.0% of C 15 H 10 ClN3O3.
Description Clonazepam occurs as white to light yellow,
crystals or crystalline powder.
It is sparingly soluble in acetic anhydride and in acetone,
slightly soluble in methanol and in ethanol (95), very slightly
soluble in diethyl ether, and practically insoluble in water.
It is gradually colored by light.
Melting point: about 240°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Clonazepam in methanol (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Clonazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Perform the test with Clonazepam as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Purity (1) Chloride <1.03>— To 1.0 g of Clonazepam add
50 mL of water, allow to stand for 1 hour with occasional
shaking, and filter. Discard the first 20 mL portion of the
filtrate, take the subsequent 20 mL portion of the filtrate, and
add 6 mL of dilute nitric acid and water to make 50 mL. Use
this solution as the test solution, and perform the test. Pre-
pare the control solution as follows: to 0.25 mL of 0.01
mol/L hydrochloric acid VS add 6 mL of dilute nitric acid
and water to make 50 mL (not more than 0.022%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Clonazepam according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.25 g of Clonazepam
in 10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add acetone to make
exactly 100 mL, then pipet 1 mL of this solution, add acetone
to make exactly 10 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 fXL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of nitromethane and ace-
tone (10:1) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clonazepam, previ-
ously dried, dissolve in 70 mL of acetic anhydride, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.57 mg of C 15 H 10 ClN 3 O3
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Clonidine Hydrochloride
tu-v>i&m.
H
ODD
*HCI
C 9 H 9 C1 2 N 3 .HC1: 266.55
2-(2,6-Dichlorophenylimino)imidazolidine
monohydrochloride [4205-91-8]
Clonidine Hydrochloride, when dried, contains not
less than 99.0% of C 9 H 9 C1 2 N 3 .HC1.
Description Clonidine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in methanol, soluble in water and in
ethanol (95), slightly soluble in acetic acid (100), and practi-
cally insoluble in acetic anhydride and in diethyl ether.
Identification (1) To 5 mL of a solution of Clonidine
Hydrochloride (1 in 1000) add 6 drops of Dragendorff's TS:
an orange precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Clonidine Hydrochloride in 0.01 mol/L hydrochloric acid TS
(3 in 10,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Cloni-
dine Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Clonidine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Clonidine Hydrochloride in 20
mL of water: the pH of this solution is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Clonidine Hydrochloride in 20 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Cloni-
dine Hydrochloride according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.5 g
of Clonidine Hydrochloride according to Method 3, and per-
form the test (not more than 4 ppm).
(4) Related substances — Dissolve 0.20 g of Clonidine
Hydrochloride in 2 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, and
add methanol to make exactly 100 mL. Pipet 1 mL and 2 mL
530
Cloperastine Hydrochloride / Official Monographs
JP XV
of this solution, to each add methanol to make exactly 20
mL, and use these solutions as the standard solution (1) and
the standard solution (2), respectively. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 2 /uL each of the sample solution and standard
solutions (1) and (2) on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of tol-
uene, 1,4-dioxane, ethanol (99.5) and ammonia solution (28)
(10:8:2:1) to a distance of about 12 cm, air-dry the plate, and
then dry at 100°C for 1 hour. Spray evenly sodium
hypochlorite TS on the plate, air-dry the plate for 15 minutes,
and then spray evenly potassium iodide starch TS on the
plate: the spots other than the principal spot and the spot of
the starting point from the sample solution are not more in-
tense than the spot from the standard solution (2), and the
numbers of spots other than the principal spot and the spot
of the starting point, which are more intense than the spot
from the standard solution (1), are not more than 3.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Clonidine
Hydrochloride, previously dried, and dissolve in 30 mL of
acetic acid (100) by warming. After cooling, add 70 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 26.66 mg of C 9 H 9 C1 2 N 3 .HC1
Containers and storage Containers — Tight containers.
Cloperastine Hydrochloride
^n<7Xf>MJS
>HCI
and anantiomer
C 20 H 24 C1NO.HC1: 366.32
l-{2-[(RS)-(4-
Chlorophenyl)phenylmethoxy] ethyl} piperidine
monohydrochloride [14984-68-0]
Cloperastine Hydrochloride, when dried, contains
not less than 98.5% of C 20 H 24 C1NO.HC1.
Description Cloperastine Hydrochloride occurs as white,
crystals or crystalline powder.
It is very soluble in water, in methanol, in ethanol (95) and
in acetic acid (100), and soluble in acetic anhydride.
A solution of Cloperastine Hydrochloride (1 in 10) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Cloperastine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 2500) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum 1: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Separately, determine the absorption spectrum of a solution
of Cloperastine Hydrochloride in 0.1 mol/L hydrochloric
acid TS (1 in 62,500) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum 2: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cloperastine Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Shake 10 mL of a solution of Cloperastine
Hydrochloride (1 in 100) with 2 mL of ammonia TS and 20
mL of diethyl ether, separate the water layer, wash the water
layer with 20 mL of diethyl ether, and filter. Acidify the
filtrate with dilute nitric acid: the solution responds to the
Qualitative Tests <1.09> for chloride.
Melting point <2.60> 148 - 152 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cloperastine Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 40 mg of Cloperastine
Hydrochloride in 50 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area of both solutions by the automatic integration method:
The areas of two peaks corresponding to the relative reten-
tion times about 0.8 and 3.0 to the retention time of cloper-
astine obtained from the sample solution are not larger than
the peak area from the standard solution, respectively, and
the area of the peak corresponding to the relative retention
time about 2.0 to cloperastine is not larger than 5/3 of the
peak area from the standard solution, and the areas of the
peaks other than cloperastine and other than the peaks men-
tioned above are all not larger than 3/5 of the peak area from
the standard solution. The total area of these peaks is not
larger than 2 times of the peak area from the standard solu-
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 222 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol, 0.1 mol/L mono-
basic potassium phosphate TS and perchloric acid
(500:250:1).
Flow rate: Adjust the flow rate so that the retention time of
cloperastine is about 7 minutes.
Selection of column: Dissolve 0.03 g of Cloperastine
JPXV
Official Monographs / Clotiazepam 531
Hydrochloride and 0.04 g of benzophenone in 100 mL of the
mobile phase. To 2.0 mL of this solution add the mobile
phase to make 50 mL. Perform the test with 20 iiL of this so-
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of cloperastine
and benzophenone in this order with the resolution between
these peaks being not less than 6.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of cloperastine obtained from 20 iiL of
the standard solution is about 30% of the full scale.
Time span of measurement: About 4 times as long as the
retention time of cloperastine, beginning after the solvent
peak.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Cloperastine
Hydrochloride, previously dried, dissolve in 70 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 36.63 mg of C 20 H 24 C1NO.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Clotiazepam
H,C
C 16 H 15 C1N 2 0S: 318.82
5-(2-Chlorophenyl)-7-ethyl-l-methyl-l,3-dihydro-2i/-
thieno[2,3-e][l,4]-diazepin-2-one [33671-46-4]
Clotiazepam, when dried, contains not less than
98.5% of C 16 H 15 ClN 2 OS.
Description Clotiazepam occurs as white to light yellowish
white crystals or crystalline powder. It is odorless, and has a
slightly bitter taste.
It is very soluble in chloroform, freely soluble in methanol,
in ethanol (95), in acetone, in acetic acid (100) and in ethyl
acetate, soluble in diethyl ether, and practically insoluble in
water.
It dissolves in 0.1 mol/L hydrochloric acid TS.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Clotiazepam in 3 mL
of sulfuric acid: the solution shows a light yellow fluorescence
under ultraviolet light (main wavelength: 365 nm).
(2) Determine the absorption spectrum of a solution of
Clotiazepam in 0.1 mol/L hydrochrolic acid TS (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Prepare the test solution with 0.01 g of Clotiazepam
as directed under Oxygen Flask Combustion Method <1.06>,
using 10 mL of diluted hydrogen peroxide (30) (1 in 5) as the
absorbing liquid. Apply a small amount of water to the upper
part of the Apparatus A, pull out C carefully, wash C, B and
the inner side of A with 15 mL of methanol, and use the ob-
tained solution as the test solution. Add 0.5 mL of dilute
nitric acid to 15 mL of the test solution: this solution
responds to the Qualitative Tests <1.09> (2) for chloride. The
remaining test solution responds to the Qualitative Tests
<1.09> (1) for sulfate.
Melting point <2.60> 106 - 109°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Clotiazepam in 10 mL of ethanol (95): the solution is clear
and is not more colored than the following control solution.
Control solution: To 5 mL of Matching Fluid C add 0.01
mol/L hydrochloric acid TS to make 10 mL.
(2) Chloride <1.03>— To 1.0 g of Clotiazepam add 50 mL
of water, shake for 30 minutes, and filter. To 30 mL of the
filtrate add 6 mL of dilute nitric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 0.25 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.015%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Clotia-
zepam according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Clotiazepam, according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.25 g of Clotiazepam
in 10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add acetone to make
exactly 20 mL, pipet 2 mL of this solution, add acetone to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 iiL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and acetone
(5:1) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.5% (1 g, 80°C,
3 hours).
Residue on ignition <2.44> not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Clotiazepam, previ-
ously dried, dissolve in 80 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid (potentiometric titra-
tion). Perform a blank determination in, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.88 mg of C 16 H 15 ClN 2 OS
Containers and storage Containers — Tight containers.
532 Clotrimazole / Official Monographs
Storage — Light-resistant.
Clotrimazole
?n r- IJ7'/-JI
C 22 H 17 C1N 2 : 344.84
1 - [(2-Chlorophenyl)(diphenyl)methyl] - l//-imidazole
[23593-75-1]
Clotrimazole, when dried, contains not less than
98.0% of C 2 2H 17 C1N 2 .
Description Clotrimazole occurs as a white, crystalline
powder. It is odorless and tasteless.
It is freely soluble in dichloromethane and in acetic acid
(100), soluble in 7V,./V-dimethylformamide, in methanol and
in ethanol (95), slightly soluble in diethyl ether, and practical-
ly insoluble in water.
Identification (1) To 0.1 g of Clotrimazole add 10 mL of 5
mol/L hydrochloric acid TS, dissolve by heating, and cool.
To this solution add 3 drops of Reinecke salt TS: a light red
precipitate is produced.
(2) Determine the absorption spectrum of a solution of
Clotrimazole in methanol (1 in 5000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Clotrimazole, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) Perform the test with Clotrimazole as directed under
Flame Coloration Test (2) <1.04>: a green color appears.
Melting point <2.60> 142 - 145°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Clotrimazole in 10 mL of dichloromethane: the solution is
clear and colorless.
(2) Chloride <1. 03>— Dissolve 1.0 g of Clotrimazole in 40
mL of A^TV-dimethylformamide, add 6 mL of dilute nitric
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution with
0.60 mL of 0.01 mol/L hydrochloric acid VS, 40 mL of N,N-
dimethylformamide, 6 mL of dilute nitric acid and water to
make 50 mL (not more than 0.021%).
(3) Sulfate <1.14>— Dissolve 0.5 g of Clotrimazole in 10
mL of methanol, and add 1 mL of dilute hydrochloric acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with
0.05 mL of 0.005 mol/L sulfuric acid VS, 10 mL of
JP XV
methanol, 1 mL of dilute hydrochloric acid and water to
make 50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of
Clotrimazole according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Clotrimazole according to Method 3, and perform the test
(not more than 2 ppm).
(6) Imidazole — Dissolve 0.10 g of Clotrimazole in exactly
10 mL of dichloromethane, and use this solution as the sam-
ple solution. Separately, dissolve 25 mg of imidazole for thin-
layer chromatography in dichloromethane to make exactly 50
mL. Pipet 5 mL of this solution, add dichloromethane to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of methanol and chloroform (3:2) to a distance of about
10 cm, and air-dry the plate. Spray evenly sodium hydrochlo-
ride TS on the plate, and air-dry the plate for 15 minutes,
then spray evenly potassium iodide-starch TS on the plate:
the spot from the sample solution, corresponding to that
from the standard solution, is not more intense than that
from the standard solution.
(7) (2-Chlorophenyl)-diphenylmethanol — Dissolve 0.20 g
of Clotrimazole in exactly 10 mL of dichloromethane, and
use this solution as the sample solution. Separately, dissolve
0.010 g of (2-chlorophenyl)-diphenylmethanol for thin-layer
chromatography in dichloromethane to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 10 /xh each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate and ammonia solution
(28) (50: 1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spot from the sample solution, corresponding to that
from the standard solution, is not more intense than that
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Clotrimazole,
previously dried, and dissolve in 80 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.48 mg of C 22 H 17 C1N 2
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
JPXV
Official Monographs / Cloxacillin Sodium Hydrate
533
Cloxacillin Sodium Hydrate
H H
,CO;,Na
CH 3
■HjO
C 19 H 17 ClN 3 Na0 5 S.H 2 0: 475.88
Monosodium (2S,5i?,6i?)-6-{[3-(2-chlorophenyl)-5-
methylisoxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-
thia-l-azabicyclo[3.2.0]heptane-2-carboxylate
monohydrate [7081-44-9]
Cloxacillin Sodium Hydrate contains not less than
900 fig (potency) and not more than 960 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Cloxacillin Sodium Hydrate is expressed as mass
(potency) of cloxacillin (C 19 H 18 C1N 3 5 S: 435.88).
Description Cloxacillin Sodium Hydrate occurs as white to
light yellowish white, crystals or crystalline powder.
It is freely soluble in water, in ./V,./V-dimethylformamide
and in methanol, and sparingly soluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Cloxacillin Sodium Hydrate in methanol (1 in
2500) as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Cloxacillin Sodium
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelength.
(2) Determine the infrared absorption spectrum of Clox-
acillin Sodium Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Cloxacillin Sodium Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Cloxacillin Sodium Hydrate responds to the Qualita-
tive Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: +163 - +171° (1 g calculated
on the anhydrous basis, water, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cloxacillin Sodium Hydrate in
10 mL of water: the pH of the solution is between 5.5 and
7.5.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 1 .0 g of Cloxacillin Sodium Hydrate
in 10 mL of water is clear and colorless to light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cloxacil-
lin Sodium Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Cloxacillin Sodium Hydrate according to Method 5, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Cloxacillin
Sodium Hydrate in 50 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 fiL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak other than cloxacillin obtained from the sample solution
is not more than the peak area of cloxacillin obtained from
the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 4.953 g of diammonium hydrogen
phosphate in 700 mL of water, and add 250 mL of acetoni-
trile. Adjust the pH to 4.0 with phosphoric acid, and add
water to make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
cloxacillin is about 24 minutes.
Time span of measurement: About 3 times as long as the
retention time of cloxacillin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of cloxacillin
obtained from 1 fiL of this solution is equivalent to 7 to 1 3 %
of that obtained from the standard solution.
System performance: Weigh accurately about 50 mg of
Cloxacillin Sodium Reference Standard, dissolve in a suitable
amount of the mobile phase, add 5 mL of a solution of
guaifenesin in the mobile phase (1 in 200), then add the
mobile phase to make exactly 50 mL, and use this solution as
the solution for system suitability test. When the procedure is
run with 10,mL of the solution for system suitability test un-
der the above operating conditions, guaifenesin and cloxacil-
lin are eluted in this order with the resolution between these
peaks being not less than 25.
System repeatability: When the test is repeated 6 times with
10 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the ratios of the peak area of cloxacillin to that of
guaifenesin is not more than 1.0%.
Water <2.48> 3.0 - 4.5% (0.2 g, volumetric titration, direct
titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer.
(iii) Standard solutions - Weigh accurately an amount of
Cloxacillin Sodium Reference Standard equivalent to about
20 mg (potency), and dissolve in 0.05 mol/L phosphate
buffer solution, pH 7.0 to make exactly 100 mL. Take exactly
a suitable amount of this solution, add 0.05 mol/L phos-
534 Cloxazolam / Official Monographs
JP XV
phate buffer solution, pH 7.0 to make solutions so that each
mL contains 20 fig (potency) and 5 fig (potency), and use
these solutions as the high concentration standard solution
and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Cloxacillin Sodium Hydrate equivalent to about 20 mg
(potency), and dissolve in 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 100 mL. Take exactly a suitable
amount of the solution, add 0.05 mol/L phosphate buffer so-
lution, pH 7.0 to make solutions so that each mL contains 20
fig (potency) and 5 fig (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Cloxazolam
and enanliomer
C 17 H 14 C1 2 N 2 2 : 349.21
(1 lbi?5)-10-Chloro-llb-(2-chlorophenyl)-2, 3,7,1 lb-
tetrahydro[l,3]oxazolo[3,2-rf][l,4]benzodiazepin-
6(5i/)-one [24166-13-0]
Cloxazolam, when dried, contains not less than
99.0% of C 17 H 14 C1 2 N 2 2 .
Description Cloxazolam occurs as white crystals or crystal-
line powder.
It is odorless and tasteless.
It is freely soluble in acetic acid (100), sparingly soluble in
dichloromethane, slightly soluble in ethanol (99.5) and in
diethyl ether, very slightly soluble in ethanol (95), and practi-
cally insoluble in water.
It dissolves in dilute hydrochloric acid.
It is gradually colored by light.
Melting point: about 200°C (with decomposition).
Identification (1) Dissolve 0.01 g of Cloxazolam in 10 mL
of ethanol (99.5) by heating, and add 1 drop of hydrochloric
acid: the solution shows a light yellow color and a yellow-
green fluorescence under ultraviolet light (main wavelength:
365 nm). Add 1 mL of sodium hydroxide TS to this solution:
the color and fluorescence of this solution disappear immedi-
ately.
(2) Dissolve 0.01 g of Cloxazolam in 5 mL of dilute
hydrochloric acid by heating in a water bath for 10 minutes.
After cooling, 1 mL of this solution responds to the Qualita-
tive Tests <1.09> for primary aromatic amines.
(3) Place 2 g of Cloxazolam in a 200-mL flask, add 50 mL
of ethanol (95) and 25 mL of sodium hydroxide TS, and boil
under a reflux condenser for 4 hours. After cooling, neutral-
ize with dilute hydrochloric acid, and extract with 30 mL of
dichloromethane. Dehydrate with 3 g of anhydrous sodium
sulfate, filter, and evaporate the dichloromethane of the
filtrate. Dissolve the residue in 5 mL of methanol by heating
on a water bath, and cool immediately in an ice bath. Collect
the crystals, and dry the crystals is vacuum at 60°C for 1
hour: it melts <2.60> between 87°C and 91 °C.
(4) Determine the absorption spectrum of a solution of
Cloxazolam in ethanol (99.5) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(5) Proceed with Cloxazolam as directed under Flame
Coloration Test <1.04> (2), and perform the test: a green
color appears.
Absorbance <2.24> E[ 0/ ° m (244 nm): 390 - 410 (after drying,
1 mg, ethanol (99.5), 100 mL).
Purity (1) Chloride <1.03>— To 1.0 g of Cloxazolam add
50 mL of water, allow to stand for 1 hour with occasional
shaking, and filter. To 25 mL of this filtrate add 6 mL of di-
lute nitric acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.20 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.014%).
(2) Heavy metals <1.07>— Proceed with 1.0 g of Clox-
azolam according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Place 1.0 g of Cloxazolam in a Kjel-
dahl flask, add 5 mL of sulfuric acid and 5 mL of nitric acid,
and heat gently. Repeat the addition of 2 to 3 mL of nitric
acid at times, and continue heating until a colorless to light
yellow solution is obtained. After cooling, add 15 mL of
saturated ammonium oxalate solution, and heat the solution
until dense white fumes are evolved, and evaporate to a
volume of 2 to 3 mL. After cooling, dilute with water to 10
mL, and perform the test with this solution as the test solu-
tion (not more than 2 ppm).
(4) Related substances — Dissolve 0.05 g of Cloxazolam
in 10 mL of dichloromethane, and use this solution as the
sample solution. Pipet 1 mL of this solution, add
dichloromethane to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 fiL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Immediately after air-drying, develop the
plate with a mixture of toluene and acetone (5: 1) to a distance
of about 10 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spots other than
the principal spot from the sample solution are not more in-
tense than that from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Cloxazolam, previ-
ously dried, and dissolve in 50 mL of acetic acid (100). Titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to blue-green
(indicator: 2 drops of crystal violet TS). Perform a blank de-
termination.
Each mL of 0.1 mol/L perchloric acid VS
= 34.92 mg of C 17 H 14 C1 2 N 2 2
JPXV
Official Monographs / Cocaine Hydrochloride
535
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cocaine Hydrochloride
C 17 H 21 N0 4 .HC1: 339.81
(l^,2/?,35,55)-2-Methoxycarbonyl-8-methyl-8-
azabicyclo[3.2.1]oct-3-yl benzoate monohydrochloride
[53-21-4]
Cocaine Hydrochloride, when dried, contains not
less than 98.0% of C 17 H 21 N0 4 .HC1.
Description Cocaine Hydrochloride occurs as colorless
crystals or a white crystalline powder.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), slightly soluble in acetic anhydride, and
practically insoluble in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Cocaine Hydrochloride in 0.01 mol/L
hydrochloric acid TS (1 in 10,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum 1 : both spectra exhibit
similar intensities of absorption at the same wavelengths.
Separately, determine the absorption spectrum of a solution
of Cocaine Hydrochloride in 0.01 mol/L hydrochloric acid
TS (1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum 2: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cocaine Hydrochloride, previously dried, as directed in the
potassium bromide disk method under the Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Cocaine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]™: - 70 - - 73° (after drying, 0.5
g, water, 20 mL, 100 mm).
Purity (1) Acidity — Dissolve 0.5 g of Cocaine Hydrochlo-
ride in 10 mL of water, add 1 drop of methyl red TS, and
neutralize with 0.01 mol/L sodium hydroxide VS: the con-
sumed volume is not more than 1.0 mL.
(2) Cinnamyl cocaine — Dissolve 0.10 g of Cocaine
Hydrochloride in 5 mL of water, and add 0.3 mL of diluted
sulfuric acid (1 in 20) and 0.10 mL of 0.02 mol/L potassium
permanganate VS: the red color does not disappear within 30
minutes.
(3) Isoatropyl cocaine — Dissolve 0.10 g of Cocaine
Hydrochloride in 30 mL of water in a beaker. Transfer 5 mL
of this solution to a test tube, add 1 drop of ammonia TS,
and mix. After the precipitate is coagulated, add 10 mL of
water, and transfer the mixture to the former beaker, to
which 30 mL of water has been added previously. Wash the
test tube with 10 mL of water, combine the washings with the
mixture in the beaker, add 3 drops of ammonia TS to the
combined mixture, and mix gently: a crystalline precipitate is
produced. Allow to stand for 1 hour: the supernatant liquid
is clear.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.5 g of Cocaine Hydrochlo-
ride, previously dried, dissolve in 50 mL of a mixture of acet-
ic anhydride and acetic acid (100) (7:3), and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 33.98 mg of C 17 H 21 N0 4 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Coconut Oil
Oleum Cocois
■Vv>S
Coconut oil is the fixed oil obtained from the seeds
of Cocos nucifera Linne (Palmae).
Description Coconut Oil is a white to light yellow mass or a
colorless or light yellow, clear oil. It has a slight, characteris-
tic odor and a mild taste.
It is freely soluble in diethyl ether and in petroleum ether.
It is practically insoluble in water.
At a temperature below 15 °C, it congeals to a hard and
brittle solid.
Melting point: 20-28 c C
Acid value <1.13> Not more than 0.2.
Saponification value <1.13> 246 - 264
Unsaponifiable matter <1.13> Not more than 1.0%.
Iodine value <1.I3> 7-11
Containers and storage Containers — Tight containers.
536
Codeine Phosphate Hydrate / Official Monographs
JP XV
Codeine Phosphate Hydrate
nfM >U>K£*tt*
• H a PO,
-HeO
HsC-
C 18 H 21 N0 3 .H 3 P0 4 .1/2H 2 0: 406.37
(5i?,6S)-4,5-Epoxy-3-methoxy-17-methyI-7,8-
didehydromorphinan-6-ol monophosphate hemihydrate
[41444-62-6]
Codeine Phosphate Hydrate contains not less than
98.0% of codeine phosphate (C 18 H 21 N03.H 3 P0 4 :
397.36), calculated on the anhydrous basis.
Description Codeine Phosphate Hydrate occurs as white to
yellowish white crystals or crystalline powder.
It is freely soluble in water and in acetic acid (100), slightly
soluble in methanol and in ethanol (95), and practically in-
soluble in diethyl ether.
The pH of a solution of Codeine Phosphate Hydrate (1 in
10) is between 3.0 and 5.0.
It is affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Codeine Phosphate Hydrate (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Codeine Phosphate Hydrate, previously dried at 105°C for 4
hours, as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(3) A solution of Codeine Phosphate Hydrate (1 in 20)
responds to the Qualitative Tests <1.09> (1) for phosphate.
Optical rotation <2.49> [a]™: -98 - - 102° (0.4 g calculat-
ed on the anhydrous basis, water, 20 mL, 100 mm).
Purity (1) Chloride <1.03>— Perform the test with 0.5 g of
Codeine Phosphate Hydrate. Prepare the control solution
with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.021%).
(2) Sulfate <1.14>— Perform the test with 0.20 g of
Codeine Phosphate Hydrate. Prepare the control solution
with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than
0.240%).
(3) Related substances — Dissolve 0.20 g of Codeine
Phosphate Hydrate in 10 mL of a mixture of 0.01 mol/L
hydrochloric acid TS and ethanol (99.5) (4:1), and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add a mixture of 0.01 mol/L hydrochloric acid TS and
ethanol (99.5) (4:1) to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethanol (99.5), toluene, acetone and ammonia solution
(28) (14:14:7:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Water <2.4S> 1.5 - 3.0% (0.5 g, direct titration).
Assay Dissolve about 0.5 g of Codeine Phosphate Hydrate,
accurately weighed, in 70 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to greenish
blue (indicator: 3 drops of crystal violet TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.74 mg of C I8 H 21 N0 3 .H 3 P0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
1% Codeine Phosphate Powder
3fM>U>K£»i%
10% Codeine Phosphate Powder contains not less
than 0.90% and not more than 1 .10% of codeine phos-
phate hydrate (Qgl^NOj.HjPCV/^O: 406.37).
Method of preparation
Codeine Phosphate Hydrate
Lactose Hydrate
10 g
a sufficient quantity
To make
1000 g
Prepare as directed under Powders,
gredients.
with the above in-
Identification Determine the absorption spectrum of a solu-
tion of 1% Codeine Phosphate Powder (1 in 100) as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 283 nm and 287 nm.
Assay Weigh accurately about 5g of 1% Codeine Phos-
phate Powder, dissolve in water to make exactly 100 mL,
then pipet 10 mL of this solution, add exactly 10 mL of the
internal standard solution, and use this solution as the sample
solution. Separately, weigh accurately about 50 mg of
codeine phosphate for assay (previously determine the water
<2.48> in the same manner as Codeine Phosphate Hydrate),
dissolve in water to make exactly 100 mL, then pipet 10 mL
of this solution, add exactly 10 mL of the internal standard
solution, and use this solution as the standard solution. Per-
form the test with 20 fiL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of codeine to that of
the internal standard.
Amount (mg) of codeine phosphate Hydrate
(C 18 H 21 N0 3 .H 3 P0 4 .i/ 2 H 2 0)
JPXV
Official Monographs / Codeine Phosphate Tablets 537
= W s x (Q T /Q S ) x 1.0227
fV s : Amount (mg) of codeine phosphate for assay, calcu-
lated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
codeine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, codeine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of codeine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
10% Codeine Phosphate Powder
10% Codeine Phosphate Powder contains not less
than 9.3% and not more than 10.7% of codeine phos-
phate Hydrate (QgH^NOj.HjPCvV^O: 406.37).
Method of preparation
Codeine Phosphate Hydrate
Lactose Hydrate
100 g
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above
ingredients.
Identification Determine the absorption spectrum of a solu-
tion of 10% Codeine Phosphate Powder (1 in 1000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 283 nm and 287 nm.
Assay Weigh accurately about 2.5 g of 10% Codeine Phos-
phate Powder, dissolve in water to make exactly 100 mL,
then pipet 2 mL of this solution, add exactly 10 mL of the
internal standard solution and water to make 20 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of codeine phosphate for assay (previ-
ously determine the water <2.48> in the same manner as
Codeine Phosphate Hydrate), dissolve in water to make ex-
actly 100 mL, then pipet 10 mL of this solution, add exactly
10 mL of the internal standard solution, and use this solution
as the standard solution. Perform the test with 20 /xL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Q T and Q s , of the peak
area of codeine to that of the internal standard:
Amount (mg) of codeine phosphate Hydrate
(C 18 H 21 N03.H 3 P0 4 .i/2H 2 0)
= W s x (g T /Qs) x 5 x 1.0227
W s : Amount (mg) of codeine phosphate for assay, calcu-
lated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
codeine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /uL of the standard solution under the above operating
conditions, codeine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 5 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of codeine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Codeine Phosphate Tablets
nfM >U>K££
Codeine Phosphate Tablets contain not less than
93% and not more than 107% of the labeled amount
of codeine phosphate hydrate (Q8H21NO3.H3PO4.V2H2
O: 406.37)
Method of preparation Prepare as directed under Tablets,
with Codeine Phosphate Hydrate.
Identification To a quantity of powdered Codeine Phos-
phate Tablets, equivalent to 0.1 g of Codeine Phosphate Hy-
drate according to the labeled amount, add 20 mL of water,
shake, and filter. To 2 mL of the filtrate add water to make
538
Cod Liver Oil / Official Monographs
JP XV
100 mL, and determine the absorption spectrum as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 283 nm and 287 nm.
Assay Weigh accurately and powder not less than 20
Codeine Phosphate Tablets. Weigh accurately a portion of
the powder, equivalent to about 0.1 g of codeine phosphate
hydrate (C 18 H 21 N03.H 3 P04. 1 /2H 2 0), add 30 mL of water,
shake, add 20 mL of diluted dilute sulfuric acid (1 in 20),
treat the mixture with ultrasonic waves for 10 minutes, and
add water to make exactly 100 mL. Filter the solution, then
pipet 5 mL of the filtrate, add exactly 10 mL of the internal
standard solution and water to make 20 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 50 mg of codeine phosphate for assay (previously de-
termine the water <2.48> in the same manner as Codeine
Phosphate Hydrate), dissolve in water to make exactly 100
mL, then pipet 10 mL of this solution, add exactly 10 mL of
the internal standard solution, and use this solution as the
standard solution. Perform the test with 20 \xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of codeine to that of the internal standard.
Amount (mg) of codeine phosphate hydrate
(C 18 H 21 N0 3 .H 3 P0 4 . 1 /2H 2 0)
= W s x (Q T /Q S ) x 2 x 1.0227
W s : Amount (mg) of codeine phosphate for assay, calcu-
lated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
codeine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, codeine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of codeine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Cod Liver Oil
Cod Liver Oil is the fatty oils obtained from fresh
livers and pyloric appendages of Gadus macrocephalus
Tilesius or Theragra chalco gramma Pallas (Gadidae).
Cod Liver Oil contains not less than 2000 Vitamin A
Units and not more than 5000 Vitamin A Units per g.
Description Cod Liver Oil is a yellow to orange oily liquid.
It has a characteristic, slightly fishy odor and a mild taste.
It is miscible with chloroform.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water.
It is decomposed by air or by light.
Identification Dissolve 0.1 g of Cod Liver Oil in 10 mL of
chloroform, and to 1 mL of this solution add 3 mL of an-
timony (III) chloride TS: a blue color develops immediately,
but the color fades rapidly.
Specific gravity <7.75> df : 0.918 - 0.928
Acid value <1.13> Not more than 1.7.
Saponification value <7.75> 180 - 192
Unsaponifiable matter <1.13> Not more than 3.0%.
Iodine value <7.75> 130-170
Purity Rancidity — No unpleasant odor of rancid oil is per-
ceptible on warming Cod Liver Oil.
Assay Proceed with about 0.5 g of Cod Liver Oil, accurate-
ly weighed, as directed in Method 2 under the Vitamin A De-
termination <2.5J>, and perform the test.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled, or under
nitrogen atmosphere.
Colchicine
zui£3->
CH 3
C 22 H 25 N0 6 : 399.44
A r -[(75)-(l,2,3,10-Tetramethoxy-9-oxo-
5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)]acetamide
[64-86-8]
Colchicine contains not less than 97.0% and not
more than 102.0% of C 2 2H 25 N0 6 , calculated on the an-
hydrous basis and corrected by the amount of ethyl
acetate.
Description Colchicine occurs as a yellowish white powder.
JPXV
Official Monographs / Colchicine 539
It is very soluble in methanol, freely soluble in N,N-
dimethylformamide, in ethanol (95) and in acetic anhydride,
and sparingly soluble in water.
It is colored by light.
Identification (1) Determine the absorption spectrum of a
solution of Colchicine in ethanol (95) (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) To 1 g of potassium bromide for infrared absorption
spectrum add 0.5 mL of a solution of Colchicine in methanol
(1 in 50), grind thoroughly, and dry in vacuum at 80°C for 1
hour. Determine the infrared absorption spectrum of this
powder as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]g>: -235 - -250° (0.1 g calculated
on the anhydrous basis and corrected by the amount of ethyl
acetate, ethanol (95), 10 mL, 100 mm).
Purity (1) Colchiceine — Dissolve 0.10 g of Colchicine in
10 mL of water, and to 5 mL of this solution add 2 drops of
iron (III) chloride TS: no definite green color develops.
(2) Chloroform and ethyl acetate — Weigh accurately
about 0.6 g of Colchicine, dissolve in exactly 2 mL of the in-
ternal standard solution, add ./V,7V-dimethylformamide to
make 10 mL, and use this solution as the sample solution.
Separately, weigh 0.30 g of chloroform using a 100-mL volu-
metric flask containing about 20 mL of A^Af-dimethylfor-
mamide, and add 7V,7V-dimethylformamide to make exactly
100 mL. Pipet 2 mL of this solution, add 7V,./V-dimethylfor-
mamide to make exactly 200 mL, and use this solution as the
standard solution (1). Separately, weigh accurately about 1.8
g of ethyl acetate using a 100-mL volumetric flask containing
about 20 mL of 7V,./V-dimethylformamide, and add N,N-
dimethylformamide to make exactly 100 mL. Pipet 2 mL of
this solution, add exactly 2 mL of the internal standard solu-
tion and A^TV-dimethylformamide to make 10 mL, and use
this solution as the standard solution (2). Perform the test
with 2 /uL each of the sample solution and standard solutions
(1) and (2) as directed under Gas Chromatography <2.02>
according to the following conditions: the peak area of
chloroform is not more than that from the standard solution
(1). Determine the ratios of the peak area of ethyl acetate to
that of the internal standard, Q T and Q s , of the sample solu-
tion and standard solution (2), and calculate the amount of
ethyl acetate by the following formula: the amount of ethyl
acetate is not more than 6.0%.
Amount (%) of ethyl acetate (C 4 H 8 2 )
= (W S /W T ) x (Q T /Q S ) x 2
W s : Amount (g) of ethyl acetate
W T : Amount (g) of the sample
Internal standard solution — A solution of 1-propanol in
Af,Af-dimethylformamide (3 in 200)
Operating conditions —
Detector: A hydrogen flame-ionization detector
Column: A fused silica column 0.53 mm in inside diameter
and 30 m in length, coated inside surface with polyethylene
glycol 20 M for gas chromatography 1.0 //m in thickness.
Column temperature: 60°C for 7 minutes, then up to
100°C at a rate of 40°C per minute if necessary, and hold at
100°C for 10 minutes.
Injection port temperature: A constant temperature of
about 130°C
Detector temperature: A constant temperature of about
200°C
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
ethyl acetate is about 3 minutes.
Split ratio: 1:20
System suitability —
Test for required detectability: Pipet 2 mL of the standard
solution (2), and add A^Af-dimethylformamide to make
exactly 25 mL. Pipet 1 mL of this solution, and add N,N-
dimethylformamide to make exactly 50 mL. Confirm that the
peak area of ethyl acetate obtained from 2 iiL of this solution
is equivalent to 0.11 to 0.21% of that obtained from 2^L of
the standard solution (2).
System performance: To 1 mL of chloroform add N,N-
dimethylformamide to make 10 mL. To 1 mL of this solution
add 2 mL of ethyl acetate and A^Af-dimethylformamide to
make 100 mL. To 2 mL of this solution add 2 mL of the in-
ternal standard solution and A^Af-dimethylformamide to
make 10 mL. When the procedure is run with 2 iiL of this
solution under the above operating conditions, ethyl acetate,
chloroform and the internal standard are eluted in this order
with the resolution between the peaks of chloroform and the
internal standard being not less than 2.0.
System repeatability: When the test is repeated 3 times with
2 jxL of the standard solution (2) under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of ethyl acetate to that of the internal standard is
not more than 3.0%.
(3) Related substances Dissolve 60 mg of Colchicine in
100 mL of diluted methanol (1 in 2). Pipet 1 mL of this
solution, add diluted methanol (1 in 2) to make exactly 100
mL, and use this solution as the sample solution. Perform the
test with 20 iiL of the sample solution as directed under Liq-
uid Chromatography <2.01> according to the following
conditions, and determine each peak area by the automatic
integration method. Calculate the total amount of the peaks
other than colchicine by the area percentage method: not
more than 5.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 450 mL of 0.05 mol/L potassium di-
hydrogen phosphate TS add methanol to make 1000 mL. Ad-
just the pH to 5.5 with diluted phosphoric acid (7 in 200).
Flow rate: Adjust the flow rate so that the retention time of
colchicine is about 7 minutes.
Time span of measurement: About 2 times as long as the
retention time of colchicine beginning after the solvent peak.
System suitability —
Test for required detectability: Pipet 1 mL of the sample
solution, and add diluted methanol (1 in 2) to make exactly
50 mL. Confirm that the peak area of colchicine obtained
540
Colistin Sodium Methanesulfonate / Official Monographs
JP XV
from 20 /xh of this solution is equivalent to 1.4 to 2.6% of
that obtained from 20 fiL of the sample solution.
System performance: When the procedure is run with
20 /xL of the sample solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of colchicine are not less than 6000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of
colchicine is not more than 2.0%.
Water <2.48> Not more than 2.0% (0.5 g, volumetric titra-
tion, back titration).
Assay Weigh accurately about 0.4 g of Colchicine, dissolve
in 25 mL of acetic anhydride, and titrate <2.50> with 0.05
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination in the same manner, and make any
necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 19.97 mg of C 22 H 25 N0 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Colistin Sodium Methanesulfonate
=i U X^>/9>X)l*>Mi- h U OA
I 1
H -Dbu Ttir-Dbu-Dbu Dbu D-Leu-LeuDbu^Dbu-Thr — '
111 II
N7.R Air-Ft AP-R APR N"-R
Colislin A Sodium Melhanesulfonale
Colislin B Sodium Melhanesulfonale
R = 6- Methyloctanic acid
Dbu = L- a , y - Diaminobutyric acid
ff.
"SO a Na
R = 6-Methylheptanaicacid
Dbu = L- a ,y- Diaminobutyric acid
[8068-28-8, Colistin Sodium Methanesulfonate]
Colistin Sodium Methanesulfonate is the sodium salt
of colistin derivatives, and is a mixture of colistin A
sodium methanesulfonate and colistin B sodium
methanesulfonate. It, when dried, contains not less
than 11,500 Units per mg. The unit of Colistin Sodium
Methanesulfonate is expressed as mass of colistin A
(R = 6-methyloctanic acid, R'=H; C53H 10 oN 16 13 :
1169.46).
Description Colistin Sodium Methanesulfonate occurs as a
white to light yellowish white powder.
It is freely soluble in water, and practically insoluble in
ethanol (95).
Identification (1) Dissolve 20 mg of Colistin Sodium
Methanesulfonate in 2 mL of water, add 0.5 mL of sodium
hydroxide TS, and add 5 drops of copper (II) sulfate TS while
shaking: a blue-purple color develops.
(2) Dissolve 40 mg of Colistin Sodium Methanesulfonate
in 1 mL of 1 mol/L hydrochloric acid TS, and add 0.5 mL of
dilute iodine TS: the color of iodine disappears.
(3) Determine the infrared absorption spectrum of
Colistin Sodium Methanesulfonate, previously dried, as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum or the spectrum of dried
Colistin Sodium Methanesulfonate Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(4) Colistin Sodium Methanesulfonate responds to the
Qualitative Tests <1.09> (1) for sodium salt.
pH <2.54> Dissolve 0.1 g of Colistin Sodium Methanesul-
fonate in 10 mL of water, and allow to stand for 30 minutes:
the pH of the solution is between 6.5 and 8.5.
Purity (1) Clarity and color of solution — Dissolve 0.16 g
of Colistin Sodium Methanesulfonate in 10 mL of water: the
solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Colistin
Sodium Methanesulfonate according to Method 4, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 30 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Colistin Sodium Methanesulfonate according to Method
4, and perform the test (not more than 2 ppm).
(4) Free colistin — Dissolve 80 mg of Colistin Sodium
Methanesulfonate in 3 mL of water, add 0.05 mL of a solu-
tion of silicotungstic acid 26-water (1 in 10), and immediately
compare the solution with the reference suspension described
under Test Methods for Plastic Containers <7.02>: the turbid-
ity is not greater than that of the reference suspension (not
more than 0.25%).
Loss on drying <2.41> Not more than 3.0% (0.1 g, reduced
pressure, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Escherichia coli NIHJ
(ii) Culture medium — To 10.0 g of peptone, 30.0 g of so-
dium chloride, 3.0 g of meat extract and 20.0 g of agar add
1000 mL of water, then add a suitable amount of sodium
hydroxide TS so that the pH of the medium is being 6.5 to 6.6
after sterilization, sterile, and use this as the seeded agar
medium and the agar medium for base layer.
(iii) Standard solutions — Weigh accurately an amount of
Colistin Sodium Methanesulfonate Reference Standard,
previously dried, dissolve in phosphate buffer solution, pH
6.0 to make a solution containing 100,000 Units per mL, and
use this solution as the standard stock solution. Keep the
standard stock solution at 10°C or below and use within 7
days. Take exactly a suitable amount of the standard stock
solution before use, and add phosphate buffer solution, pH
6.0 to make solutions so that each mL contains 10,000 Units
and 2500 Units, and use these solutions as the high concentra-
tion standard solution and low concentration standard solu-
tion, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Colistin Sodium Methanesulfonate, previously dried, dis-
solve in phosphate buffer solution, pH 6.0 to make a solution
containing about 100,000 Units per mL, and use this solution
as the sample stock solution. Take exactly a suitable amount
of the sample stock solution, add phosphate buffer solution,
pH 6.0 to make solutions so that each mL contains 10,000
Units and 2500 Units, and use these solutions as the high con-
JPXV
Official Monographs / Colistin Sulfate 541
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Tight containers.
Colistin Sulfate
=lUX5 1 >SSg^
1 n- ,
J — Dbu -Tnr. DbLj-Dbu-Dbu-D-Leu-Leu-Dbu-Dbu-Thr - 1 * 2r£ H^SO*
C0 2 H
Colistin ASulfate : R=CH, Dt, u =
Colistin BSulfale : R = H Dbu= .\
H NH 2
Colistin A Sulfate C 5 3H 1 ooN 16 1 3.2yH 2 S0 4 : 1414.66
Colistin B Sulfate C 52 H 98 N 16 Oi3.2yH 2 S0 4 : 1400.63
[1264-72-8]
Colistin Sulfate is the sulfate of a mixture of peptide
substances having antibacterial activity produced by
the growth of Bacillus polymyxa var. colistinus.
It, when dried, contains not less than 16,000 units
per mg. The potency of Colistin Sulfate is expressed
as unit calculated from the amount of colistin A
(C 5 3H 1 ooN 16 13 : 1169.46). One unit of Colistin Sulfate
is equivalent to 0.04 fig of colistin A (C 5 3H 100 N 16 O 13 ).
Description Colistin Sulfate occurs as a white to light yel-
lowish white powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
It is hygroscopic.
Identification (1) Dissolve 20 mg of Colistin Sulfate in
2 mL of water, add 0.5 mL of sodium hydroxide TS, then
add 5 drops of copper (II) sulfate TS while shaking: a purple
color develops.
(2) Dissolve 50 mg of Colistin Sulfate in 10 mL of diluted
hydrochloric acid (1 in 2). Transfer 1 mL of this solution in a
tube for hydrolysis, seal, and heat at 135 C C for 5 hours.
After cooling, open the tube, and evaporate the content to
dryness until the odor of hydrochloric acid is no more
perceptible. Dissolve the residue in 0.5 mL of water, and use
this solution as the sample solution. Separately, dissolve
20 mg each of L-leucine, L -threonine, phenylalanine and L-
serine in 10 mL of water, and use these solutions as the stan-
dard solution (1), the standard solution (2), the standard
solution (3) and the standard solution (4). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 1 /uL each of the sample solution
and standard solutions on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, acetic acid (100), water, pyridine and ethanol (99.5)
(60:15:10:6:5) to a distance of about 10 cm, and dry the plate
at 105°C for 10 minutes. Spray evenly ninhydrin TS on the
plate, and heat at 110°C for 5 minutes: three principal spots
are obtained from the sample solution, the Rf values of two
spots of them are the same with those of the corresponding
spots obtained from the standard solution (1) and the stan-
dard solution (2), and the Rf value of the rest principal spot is
about 0.1. No spot is observed at the position corresponding
to the spots obtained from the standard solution (3) and the
standard solution (4).
(3) A solution of Colistin Sulfate (1 in 20) responds to the
Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [a]™: -63 --73° (1.25 g, after
drying, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Colistin Sulfate in 10 mL of water is between 4.0 and 6.0.
Purity (1) Sulfuric acid — Weigh accurately about 0.25 g
of previously dried Colistin Sulfate, dissolve in a suitable
amount of water, adjust the pH to 1 1 with ammonia solution
(28), and add water to make 100 mL. To this solution add ex-
actly 10 mL of 0.1 mol/L barium chloride VS and 50 mL of
ethanol (99.5), and titrate with <2.50> 0.1 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS until the blue-pur-
ple color of the solution disappears (indicator: 0.5 mg of
phthalein purple): the amount of sulfuric acid (S0 4 ) is 16.0 to
18.0%.
Each mL of 0.1 mol/L barium chloride VS
= 9.606 mg of S0 4
(2) Related substances — Dissolve 50 mg of Colistin Sul-
fate in 10 mL of water, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add water to make
exactly 50 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 1 fiL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of pyridine, 1-butanol, water and acetic acid (100) (6:5:4:1)
to a distance of about 10 cm, and dry the plate at 100°C for
30 minutes. Spray evenly ninhydrin-butanol TS on the plate,
and heat at 100°C for about 20 minutes: the spot other than
the principal spot from the sample solution is not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 6.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Escherichia coli NIHJ
(ii) Culture medium — Dissolve 10.0 g of peptone, 30.0 g
of sodium chloride, 3.0 g of meat extract and 15.0 g of agar
in 1000 mL of water, adjust the pH with sodium hydroxide
TS so that the solution will be 6.5 to 6.6 after sterilization,
and use as the agar media for seed layer and for base layer,
(iii) Standard solutions — Weigh accurately an amount
of Colistin Sulfate Reference Standard, previously dried,
equivalent to about 1,000,000 units, dissolve in phosphate
buffer solution, pH 6.0 to make exactly 10 mL, and use this
solution as the standard stock solution. Keep the standard
stock solution at not exceeding 10°C, and use within 7 days.
Take exactly a suitable amount of the standard stock solution
before use, add phosphate buffer solution, pH 6.0 to make
solutions so that each mL contains 10,000 units and 2500
units, and use these solutions as the high concentration stan-
542 Corn Oil / Official Monographs
JP XV
dard solution and low concentration standard solution, re-
spectively.
(iv) Sample solutions — Weigh accurately an amount of
Colistin Sulfate, previously dried, equivalent to about
1,000,000 units, and dissolve in phosphate buffer solution,
pH 6.0 to make exactly 10 mL. Take exactly a suitable
amount of this solution, add phosphate buffer solution, pH
6.0 to make solutions so that each mL contains 10,000 units
and 2500 units, and use these solutions as the high concentra-
tion sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Corn Oil
Oleum Maydis
Corn Oil is the fixed oil obtained from the embryo of
Zea mays Linne (Gramineae).
Description Corn Oil is a clear, light yellow oil. It is odor-
less or has a slight odor, and a mild taste.
It is miscible with diethyl ether and with petroleum ether.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water.
At — 7°C, it congeals to an unguentary mass.
Specific gravity dg: 0.915 - 0.921
Acid value <1.13> Not more than 0.2.
Saponification value <7.73> 187-195
Unsaponiflable matter <1.13> Not more than 1.5%.
Iodine value <I.I3> 103 - 130
Containers and storage Containers — Tight containers.
Corn Starch
Amylum Maydis
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Corn Starch consists of starch granules derived from
the ripen seeds of Zea mays Linne (Gramineae).
♦Description Corn Starch occurs as white to pale yellowish
white masses or powder.
It is practically insoluble in water and in ethanol (99.5). ♦
Identification (1) Under a microscope, Corn Starch,
preserved in a mixture of water and glycerin (1:1), appears as
irregularly polygonal simple grains about 2-23 /xm in di-
ameter, or irregularly orbicular or spherical simple grains
about 25 - 35 /xm in diameter; hilum appears as distinct cave
or 2 - 5 radial clefts; concentric striation absent; a black
cross, its intersection point on hilum, is observed when grains
A Boiling flask (500 mL)
B: Funnel (100 mL)
C: Condenser
D: Test-lube
E:Tap
The figures are in mm.
are put between two nicol prisms fixed at right angle to each
other.
(2) To 1 g of Corn Starch add 50 mL of water, boil for 1
minute, and allow to cool: a subtle white-turbid, pasty liquid
is formed.
(3) To 1 mL of the pasty liquid obtained in (2) add 0.05
mL of diluted iodine TS (1 in 10): an orange-red to deep blue
color is formed and the color disappears by heating.
pH <2.54> Put 5.0 g of Corn Starch in a non-metal vessel,
add 25.0 mL of freshly boiled and cooled water, mix gently
for 1 minute, and allow to stand for 15 minutes: the pH of
the solution is between 4.0 and 7.0.
Purity (1) Iron — To 1.5 g of Corn Starch add 15 mL of 2
mol/L hydrochloric acid TS, mix, filter, and use the filtrate as
the test solution. To 2.0 mL of Standard Iron Solution add
water to make 20 mL, and use as the control solution. Put
10 mL each of the test solution and the control solution in
test tubes, add 2 mL of a solution of citric acid (1 in 5) and
0.1 mL of mercapto acetic acid, and mix. Alkalize with
ammonia solution (28) to litmus paper, add water to make 20
mL, and mix. Transfer 10 mL each of these solutions into test
tubes, allow to stand for 5 minutes, and compare the color of
these solutions against a white background: the color of the
test solution is not more intense than that of the control solu-
tion (not more than 10 ppm).
(2) Oxidizing substances — To 4.0 g of Corn Starch add
50.0 mL of water, shake for 5 minutes, and centrifuge. To
30.0 mL of the supernatant liquid add 1 mL of acetic acid
(100) and 0.5 to 1.0 g of potassium iodide, shake, and allow
to stand for 25 to 30 minutes at a dark place. Add 1 mL of
starch TS, and titrate <2.50> with 0.002 mol/L sodium
thiosulfate VS until the color of the solution disappears. Per-
form a blank determination and make any necessary correc-
tion: the volume of 0.002 mol/L sodium thiosulfate VS con-
sumed is not more than 1.4 mL (not more than 20 ppm, cal-
culated as hydrogen peroxide).
(3) Sulfur dioxide —
(i) Apparatus Use as shown in the following figure.
(ii) Procedure Introduce 150 mL of water into the
boiling flask, close the tap of the funnel, and pass carbon
dioxide through the whole system at a rate of 100 ± 5 mL per
minute. Pass cooling water through the condenser, and place
JPXV
Official Monographs / Cortisone Acetate
543
10 mL of hydrogen peroxide-sodium hydroxide TS in the
test-tube. After 15 minutes, remove the funnel without
interrupting the stream of carbon dioxide, and introduce
through the opening into the flask about 25 g of Corn Starch,
accurately weighed, with the aid of 100 mL of water. Apply
tap grease to the outside of the connection part of the funnel,
and load the funnel. Close the tap of the funnel, pour 80 mL
of 2 mol/L hydrochloric acid TS into the funnel, open the tap
to introduce the hydrochloric acid into the flask, and close
the tap while several mL of the hydrochloric acid remains, in
order to avoid losing sulfur dioxide. Place the flask in a water
bath, and heat the mixture for 1 hour. Transfer the contents
of the test-tube with the aid of a little water to a wide-necked
conical flask. Heat in a water bath for 15 minutes, and cool.
Add 0.1 mL of bromophenol blue TS, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS until the color changes from
yellow to violet-blue lasting for at least 20 seconds. Perform a
blank determination and make any necessary correction. Cal-
culate the amount of sulfur dioxide by applying the following
formula: it is not more than 50 ppm.
Amount (ppm) of sulfur dioxide = ( VI W) x 1000x3.203
W: Amount (g) of the sample
V: Amount (mL) of 0.1 mol/L sodium hydroxide VS
consumed
Loss on drying <2.41> Not more than 15.0% (1 g, 130°C, 90
minutes).
Reisdue on ignition <2.44> Not more than 0.6% (1 g).
*Containers and storage Containers — Well-closed contain-
ers. ♦
Cortisone Acetate
C 23 H 30 O 6 : 402.48
17,21 -Dihydroxypregn-4-ene-3 , 1 1 ,20-trione 21 -acetate
[50-04-4]
Cortisone Acetate, when dried, contains not less
than 97.0% and not more than 102.0%, of C 23 H 30 O 6 .
Description Cortisone Acetate occurs as white crystals or
crystalline powder.
It is sparingly soluble in methanol, slightly soluble in
ethanol (99.5), and practically insoluble in water.
Melting point: about 240°C (with decomposition).
Identification (1) To 2 mg of Cortisone Acetate add 2 mL
of sulfuric acid, and allow to stand for a while: a yellowish
green color is produced, and it gradually changes to yellow-
orange. Examine the solution under ultraviolet light: the so-
lution shows a light green fluorescence. Add carefully 10 mL
of water to this solution: the color of the solution is dis-
charged, and the solution remains clear.
(2) Determine the absorption spectrum of a solution of
Cortisone Acetate in methanol (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cortisone Acetate Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of Corti-
sone Acetate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Cortisone Acetate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve Cortisone Acetate and
Cortisone Acetate Reference Standard in acetone, respec-
tively, then evaporate the acetone to dryness, and repeat the
test on the residues.
Optical rotation <2.49> [a]™: +207 - +216° (after drying,
0.1 g, methanol, 10 mL, 100 mm).
Purity Related substances — Dissolve 25 mg of Cortisone
Acetate in 10 mL of a mixture of acetonitrile, water and acet-
ic acid (100) (70:30:1), and use this solution as the sample so-
lution. Pipet 1 mL of this solution add the mixture of
acetonitrile, water and acetic acid (100) (70:30:1) to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10,mL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the peak area other than cortisone acetate is not
larger than 1/2 times the peak area of cortisone acetate ob-
tained with the standard solution, and the total area of the
peak other than cortisone acetate is not larger than 1.5 times
the peak area of cortisone acetate with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of water and acetonitrile (7:3).
Mobile phase B: A mixture of acetonitrile and water (7:3).
Flowing of the mobile phase: Control the gradient by mixing
the mobile phases A and B as directed in the following table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-5
90
10
5-25
90^10
10^90
25-30
10
90
Flow rate: About 1 mL per minute.
Time span of measurement: About 3 times as long as the
retention time of cortisone acetate beginning after the solvent
peak.
544
Creosote / Official Monographs
JP XV
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add a mixture of acetonitrile, water and acetic
acid (100) (70:30:1) to make exactly 10 mL. Confirm that the
peak area of cortisone acetate obtained with 15 /uL of this so-
lution is equivalent to 8 to 12% of that with 15 ftL of the stan-
dard solution.
System performance: When the procedure is run with 15 /xL
of the sample solution under the above operating conditions,
the number of theoretical plates and the symmetry factor of
the peak of cortisone acetate are not less than 10,000 and not
more than 1.3, respectively.
System repeatability: When the test is repeated 3 times with
15 /iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
cortisone acetate is not more than 5.0%.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105°C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 10 mg each of Cortisone Acetate and
Cortisone Acetate Reference Standard, previously dried and
accurately weighed, in 50 mL of methanol, add exactly 5 mL
each of the internal standard solution, then add methanol to
make 100 mL, and use these solutions as the sample solution
and the standard solution. Perform the test with lO^L each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the ratios, Q T and Q s , of the
peak area of cortisone acetate to that of the internal stan-
dard.
Amount (mg) of C 2 3H 30 O 6
= W s x (Qj/Qs)
W s : Amount (mg) of Cortisone Acetate Reference Stan-
dard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (3 in 5000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (13:7).
Flow rate: Adjust the flow rate so that the retention time of
cortisone acetate is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, cortisone acetate and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of cortisone acetate to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Creosote
Creosote is a mixture of phenols obtained from
wood tar.
Description Creosote is a colorless or pale yellow, clear liq-
uid. It has a characteristic odor, and a burning taste.
It is miscible with ethanol (95) and with diethyl ether.
It is slightly soluble in water.
Its saturated solution is neutral.
It is highly refractive.
It gradually changes in color by light or by air.
Identification To 10 mL of a saturated solution of Creosote
add 1 drop of iron (III) chloride TS: a purple color develops,
and the solution becomes rapidly turbid, and changes
through blue and muddy green to brown.
Specific gravity <2.56> (ff a : not less than 1.076.
Purity (1) Bases and hydrocarbons — Shake 1.0 mL of
Creosote with 9 mL of sodium hydroxide TS: the solution is
clear and does not darken. On further addition of 50 mL of
water, the solution is practically clear.
(2) Phenol or coal-tar creosote — Shake Creosote with an
equal volume of collodion: no coagulum is produced.
(3) Other impurities — To 1.0 mL of Creosote add 2 mL
of petroleum benzin and 2 mL of barium hydroxide TS,
shake, and allow to stand: no blue or muddy brown color de-
velops in the upper layer of the mixture, and no red color de-
velops in the lower layer.
Distilling range <2.57> 200 - 220°C, not less than 85 vol%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cresol
C 7 H s O: 108.14
Cresol is a mixture of isomeric cresols.
Description Cresol is a clear, colorless or yellow to yellow-
brown liquid. It has a phenol-like odor.
It is miscible with ethanol (95) and with diethyl ether.
It is sparingly soluble in water.
It dissolves in sodium hydroxide TS.
A saturated solution of Cresol is neutral to bromocresol
purple TS.
It is a highly refractive liquid.
It becomes dark brown by light or on aging.
Identification To 5 mL of a saturated solution of Cresol
add 1 to 2 drops of dilute iron (III) chloride TS: a blue-purple
color develops.
Specific gravity <2.56> df : 1.032 - 1.041
JPXV
Official Monographs / Saponated Cresol Solution
545
Purity (1) Hydrocarbons — Dissolve 1.0 mL of Cresol in
60 mL of water: the solution shows no more turbidity than
that produced in the following control solution.
Control solution: To 54 mL of water add 6.0 mL of 0.005
mol/L sulfuric acid and 1.0 mL of barium chloride TS, and
after thorough shaking, allow to stand for 5 minutes.
(2) Sulfur compounds — Transfer 20 mL of Cresol in a
100-mL conical flask, place a piece of moistened lead (II)
acetate paper on the mouth of the flask, and warm for 5
minutes on a water bath: the lead (II) acetate paper may de-
velop a yellow color, but neither a brown nor a dark tint.
Distilling range <2.57> 196 - 206°C, not less than 90 vol%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cresol Solution
Cresol Solution contains not less than 1.25 vol% and
not more than 1.60 vol% of cresol.
Saponated Cresol Solution
Method of preparation
Saponated Cresol Solution
Water or Purified Water
30 mL
a sufficient quantity
To make 1000 mL
Prepare by mixing the above ingredients.
Description Cresol Solution is a clear or slightly turbid, yel-
low solution. It has the odor of cresol.
Identification Shake 0.5 mL of the oily layer obtained in the
Assay with 30 mL of water, filter, and perform the following
tests using this filtrate as the sample solution:
(1) To 5 mL of the sample solution add 1 to 2 drops of
iron (III) chloride TS: a blue-purple color develops.
(2) To 5 mL of the sample solution add 1 to 2 drops of
bromine TS: a light yellow, flocculent precipitate is
produced.
Assay Transfer 200 mL of Cresol Solution, exactly meas-
ured, to a 500-mL distilling flask. Add 40 g of sodium chlo-
ride and 3 mL of dilute sulfuric acid, and connect the distil-
ling apparatus with the distilling flask, and distil into a cassia
flask which contains 30 g of powdered sodium chloride and 3
mL of kerosene, exactly measured, until the distillate meas-
ures 90 mL. Draw off the water from the condenser, and con-
tinue the distillation until water vapor begins to come out of
the tip of the condenser. Shake often the cassia flask in warm
water to dissolve the sodium chloride, and allow to stand for
15 minutes. After cooling to 15°C, add a saturated solution
of sodium chloride, and allow to stand for more than 3 hours
with occasional shaking. Allow to stand for 1 to 2 minutes
with gentle shaking to combine the separated oil drops with
the oil layer. The difference between the number of mL of the
oil layer measured and 3 mL represents the amount (mL) of
cresol.
Containers and storage Containers — Tight containers.
?u7-;l-5^>;«
Saponated Cresol Solution contains not less than 42
vol% and not more than 52 vol% of cresol.
Method of preparation
Cresol
Fixed Oil
Potassium Hydroxide
Water or Purified Water
500 mL
300 mL
a suitable quantity
a sufficient quantity
To make 1000 mL
Dissolve Potassium Hydroxide, in required quantity for
saponification, in a sufficient quantity of Water or Purified
Water, add this solution to fixed oil, previously warmed, add
a sufficient quantity of Ethanol, if necessary, heat in a water
bath by thorough stirring, and continue the saponification.
After complete saponification, add Cresol, stir thoroughly
until the mixture becomes clear, and add sufficient Water or
Purified Water to make 1000 mL. A corresponding amount
of Sodium Hydroxide may be used in place of Potassium
Hydroxide.
Description Saponated Cresol Solution is a yellow-brown
to red-brown, viscous liquid. It has the odor of cresol.
It is miscible with water, with ethanol (95) and with glyce-
rin.
It is alkaline.
Identification Proceed as directed in the Identification un-
der Cresol, using the distillate in the Purity (3).
Purity (1) Alkalinity — Mix well 0.50 mL of Saponated
Cresol Solution with 10 mL of neutralized ethanol, add 2 to 3
drops of phenolphthalein TS and 0.10 mL of 1 mol/L
hydrochloric acid VS: no red color develops.
(2) Unsaponified matter — To 1.0 mL of Saponated
Cresol Solution add 5 mL of water, and shake: the solution is
clear.
(3) Cresol fraction — Transfer 180 mL of Saponated
Cresol Solution to a 2000-mL distilling flask, add 300 mL of
water and 100 mL of dilute sulfuric acid, and distil with
steam until the distillate becomes clear. Draw off the water
from the condenser, and continue the distillation until water
vapor begins to come out of the tip of the condenser. Cool
the condenser again, and continue distillation for 5 minutes.
Dissolve 20 g of sodium chloride per 100 mL of the distillate,
allow to stand, and collect the separated clear oil layer. After
adding about 15 g of powdered calcium chloride for drying in
small portions with frequent shaking, allow to stand for 4
hours. Filter, and distil exactly 50 mL of the filtrate: the dis-
tillate is not less than 43 mL between 196°C and 206°C.
Assay Transfer 5 mL of Saponated Cresol Solution, exactly
measured, to a 500-mL distilling flask, holding the pipet ver-
tically for 15 minutes to draw off the solution into the flask.
Add 200 mL of water, 40 g of sodium chloride and 3 mL of
dilute sulfuric acid, connect the distilling apparatus with the
distilling flask, and distil into a cassia flask which contains 30
g of powdered sodium chloride and exactly 3 mL of kerosene,
until the distillate reaches 90 mL. Draw off the water from the
546
Croconazole Hydrochloride / Official Monographs
JP XV
condenser, and continue the distillation until water vapor be-
gins to come out of the tip of the condenser. Allow the cassia
flask to stand in warm water for 15 minutes to dissolve the
sodium chloride with frequent shaking. Cool to 15°C, add a
saturated solution of sodium chloride, and allow to stand for
more than 3 hours with occasional shaking. Allow to stand
for 1 to 2 minutes with gentle shaking, and combine the sepa-
rated oil drops with the oil layer. The volume (mL) subtract-
ed 3 (mL) from the oil layer measured represents the amount
(mL) of cresol.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Croconazole Hydrochloride
Vn=\TV-)li£&i&
'<■>,
C!
N
• HCI
C 18 H 15 C1N 2 0.HC1: 347.24
1 - { 1 - [2-(3-Chlorobenzyloxy)phenyl] vinyl} - l//-imidazole
monohydrochloride [77174-66-4]
Croconazole Hydrochloride, when dried,
not less than 98.5% of C 18 H 15 ClN 2 O.HCl.
contains
Description Croconazole Hydrochloride occurs as white to
pale yellowish white crystals or crystalline powder.
It is very soluble in water, freely soluble in methanol, in
ethanol (95) and in acetic acid (100), and practically insoluble
in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Croconazole Hydrochloride in methanol (1 in
20,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Croconazole Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Dissolve 0.05 g of Croconazole Hydrochloride in 10
mL of water, add 2 mL of sodium hydroxide TS and 20 mL
of diethyl ether, and shake. Wash the separated aqueous lay-
er with two 10-mL portions of diethyl ether, and acidify the
solution with 2 mL of dilute nitric acid: the solution responds
to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 148 - 153°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Croconazole Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 50 mg of Croconazole
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 iuL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, hexane, methanol and ammonia solution (28)
(30:15:5:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot and other than
the spot of the starting point from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 60°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Croconazole
Hydrochloride, previously dried, dissolve in 10 mL of acetic
acid (100), add 40 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS [indicator: 1 to 2 drops of
a solution of malachite green oxalate in acetic acid (100) (1 in
100)] until the color of the solution changes from blue-green
through green to yellow-green. Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.72 mg of C, 8 H 15 ClN 2 O.HCl
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Croscarmellose Sodium
{7nx7]J^n-xth U^A
This monograph is harmonized with the European Phar-
macopoeia and the U.S. Pharmacopeia. The parts of the text
that are not harmonized are marked with symbol (* ♦).
Croscarmellose Sodium is the sodium salt of a cross-
linked poly carboxymethylether of cellulose.
♦Description Croscarmellose Sodium occurs as a white to
yellowish white powder.
It is practically insoluble in ethanol (99.5) and in diethyl
ether.
It swells with water and becomes a suspension.
It is hygroscopic. »
Identification (1) To 1 g of Croscarmellose Sodium add
100 mL of a solution of methylene blue (1 in 250,000), stir
well, and allow to stand: blue cotton-like precipitates appear.
(2) To 1 g of Croscarmellose Sodium add 50 mL of
water, and stir well to make a suspension. To 1 mL of this
suspension add 1 mL of water and 5 drops of fleshly prepared
solution of 1-naphtol in methanol (1 in 25), and gently add 2
mL of sulfuric acid along a wall of the vessel: a red-purple
color appears at the zone of contact.
(3) The suspension obtained in (2) responds to the
JPXV
Official Monographs / Croscarmellose Sodium
547
Qualitative Tests <1.09> (1) for sodium salt.
pH <2.54> To 1.0 g of Croscarmellose Sodium add 100 mL
of water, and stir for 5 minutes: the pH of the supernatant
liquid is between 5.0 and 7.0.
Purity *(1) Heavy metals <1.07> — Proceed with 2.0 g of
Croscarmellose Sodium according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).»
*(2) Sodium chloride and sodium glycolate — The total
amount of sodium chloride and sodium glycolate is not more
than 0.5%.
(i) Sodium chloride: Weigh accurately about 5 g of
Croscarmellose Sodium, add 50 mL of water and 5 mL of
hydrogen peroxide (30), and heat on a water bath for 20
minutes with occasional stirring. After cooling, add 100 mL
of water and 10 mL of nitric acid, and titrate <2.50> with 0.1
mol/L silver nitrate VS (potentiometric titration). Perform a
blank determination in the same manner and make any neces-
sary correction.
Each mL of 0.1 mol/L silver nitrate VS = 5.844 mg of NaCl
(ii) Sodium glycolate: Weigh accurately about 0.5 g of
Croscarmellose Sodium, add 2 mL of acetic acid (100) and 5
mL of water, and stir for 15 minutes. Add gradually 50 mL
of acetone with stirring, then add 1 g of sodium chloride, stir
for 3 minutes, and filter through a filter paper moistened with
acetone. Wash the residue thoroughly with 30 mL of acetone,
combine the filtrate and washings, add acetone to make ex-
actly 100 mL, and use this solution as the sample stock solu-
tion. Separately, dissolve 0.100 g of glycolic acid in water to
make 200 mL. Pipet 0.5 mL, 1 mL, 2 mL, 3 mL and 4 mL of
this solution, add water to make them exactly 5 mL, then add
5 mL of acetic acid (100) and acetone to make exactly 100
mL, and designate them standard stock solution (1), standard
stock solution (2), standard stock solution (3), standard stock
solution (4) and standard stock solution (5), respectively.
Pipet 2 mL each of the sample stock solution and the stan-
dard stock solutions (1), (2), (3), (4) and (5), and heat them in
a water bath for 20 minutes to evaporate acetone. After cool-
ing, add exactly 5 mL of 2,7-dihydroxynaphthalene TS, mix,
then add 15 mL of 2,7-dihydroxynaphthalene TS, mix, cover
the mouth of the vessels with aluminum foil, and heat in a
water bath for 20 minutes. After cooling, add sulfuric acid to
make exactly 25 mL, mix, and designate them sample solu-
tion, standard solution (1), standard solution (2), standard
solution (3), standard solution (4) and standard solution (5),
respectively. Separately, to 10 mL of a mixture of water and
acetic acid (100) (1:1) add acetone to make exactly 100 mL,
and proceed with exactly 2 mL of this solution in the same
manner for preparation of the sample solution, and use the
solution so obtained as the blank solution. Determine the ab-
sorbances, A T , A sl , A S2 , A si , A S4 and ^4 S5 , of the sample solu-
tion and the standard solutions (1), (2), (3), (4) and (5), re-
spectively, at 540 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using the blank solution as the
control. Determine the amount (g) of glycolic acid, X, in 100
ml of the sample solution from the calibration curve obtained
with the standard solutions, and calculate the amount of so-
dium glycolate by the following formula.
Amount (%) of sodium glycolate
= (X/W) x 100 x 1.2890
W: Amount (g) of sample*
*(3) Water-soluble substance — Weigh accurately about 10
g of Croscarmellose Sodium, disperse in 800 mL of water by
stirring for 1 minute every 10 minutes during 30 minutes, and
allow to stand for at most 1 hour to precipitate. Filter by suc-
tion or centrifuge the clear upper portion, and weigh ac-
curately the mass of about 150 mL of the filtrate or super-
natant liquid. Heat to concentrate this liquid avoiding to dry-
ness, then dry at 105°C for 4 hours, and weigh the mass of
the residue accurately. Calculate the amount of the water-
soluble substance by the following formula: not less than 1.0
% and not more than 10.0%.
Amount (%) of water-soluble substance
= 100 Wi (800 + W l )/W l W 1
Wi. Amount (g) of sample
W-i. Amount (g) of the filtrate or supernatant liquid of
about 150 mL
Wj,: Amount (g) of the residue*
Precipitation test Put 75 mL of water in a 100-mL glass-
stoppered graduated cylinder, and add portion by portion
with 1.5 g of Croscarmellose Sodium divided into three por-
tions while shaking vigorously at each time. Then, add water
to make 100 mL, shake until to get a homogenous dispersion,
and allow to stand for 4 hours: the volume of the settled layer
is not less than 10.0 mL and not more than 30.0 mL.
Degree of substitution Weigh accurately about 1 g of
Croscarmellose Sodium, put in a 500-mL glass-stoppered
conical flask, add 300 mL of sodium chloride TS, then add
25.0 mL of 0.1 mol/L sodium hydroxide VS, stopper, and al-
low to stand for 5 minutes with occasional shaking. Add 5
drops of w-cresol purple TS, then add exactly 15 mL of 0.1
mol/L hydrochloric acid VS using a buret, stopper the flask,
and shake. If the color of the solution is purple, add exactly
1-mL portions of 0.1 mol/L hydrochloric acid VS using the
buret, with shaking each time, until the color of the solution
changes to yellow, then titrate <2.50> with 0.1 mol/L sodium
hydroxide VS until the color changes from yellow to purple.
Perform a blank determination in the same manner. Calcu-
late the degrees of substitution of acid-carboxymethyl group
and sodium-carboxym ethyl group, A and S: A + S is not less
than 0.60 and not more than 0.85.
,4 = 1150M/(7102-412M-80C)
S = (162 + 58,4)C/(7102— 80C)
M: Amount (mmol) of sodium hydroxide needed to neu-
tralize 1 g of sample, calculated on the dried basis
C: The value (%) obtained in Residue on ignition
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C, 6
hours).
Residue on ignition <2.44> 14.0 - 28.0% (after drying, 1 g).
Containers and storage Containers — Tight containers.
548
Cyanamide / Official Monographs
JP XV
H 2 N-CN
Cyanamide
CH 2 N 2 : 42.04
Aminonitrile [420-04-2]
Cyanamide contains not less than 97.0% and not
more than 101.0% of CH 2 N 2 , calculated on the anhy-
drous basis.
Description Cyanamide occurs as white crystals or crystal-
line powder.
It is very soluble in water, in methanol, in ethanol (99.5)
and in acetone.
The pH of a solution of Cyanamide (1 in 100) is between
5.0 and 6.5.
It is hygroscopic.
Melting point: about 46°C
Identification (1) To 1 mL of a solution of Cyanamide (1
in 100) add 1 mL of potassium l,2-naphthoquinone-4-sul-
fonate TS and 0.2 mL of sodium hydroxide TS: a deep red
color develops.
(2) Drop one or two drops of a solution of Cyanamide in
acetone (1 in 100) onto a potassium bromide disk prepared as
directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>, and air-dry the disk. Deter-
mine the infrared absorption spectrum of the disk as directed
in the film method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cyanamide in 10 mL of water: the solution is clear and color-
less.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Cyanamide according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
Water <2.48> Not more than 1.0% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Cyanamide, and dis-
solve in water to make exactly 250 mL. Pipet 15 mL of this
solution, add 2 to 3 drops of dilute nitric acid, 10 mL of am-
monia TS and exactly 50 mL of 0.1 mol/L silver nitrate VS,
and allow to stand for 15 minutes with occasional shaking.
Add water to make exactly 100 mL, filter, discard the first 20
mL of the filtrate, and pipet the subsequent 50 mL. After
neutralizing this solution with dilute nitric acid, add 3 mL of
dilute nitric acid, and titrate <2.50> the excess silver nitrate
with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL
of ammonium iron (III) sulfate TS). Perform a blank deter-
mination.
Each mL of 0.1 mol/L silver nitrate VS
= 2.102 mg of CH 2 N 2
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Cyanocobalamin
Vitamin B 12
H;N
o
J-CH 3 </ }T
C 6 3H 88 CoN 14 14 P: 1355.37
Coa-[a-(5,6-Dimethyl-l//-benzoimidazol-l-yl)]-Co/?-
cyanocobamide [68-19-9]
Cyanocobalamin contains not less than 96.0% and
not more than 102.0% of C 63 H 8g CoN 14 1 4P, calculated
on the dried basis.
Description Cyanocobalamin occurs as dark red crystals or
powder.
It is sparingly soluble in water, and slightly soluble in
ethanol (99.5).
It is hygroscopic.
Identification (1) Determine the absorption spectrum of
the sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Cyanocobalamin Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Mix 1 mg of Cyanocobalamin with 0.05 g of potassi-
um hydrogen sulfate, and fuse by igniting. Cool, break up
the mass with a glass rod, add 3 mL of water, and dissolve by
boiling. Add 1 drop of phenolphthalein TS, then add drop-
wise sodium hydroxide TS until a light red color just
develops. Add 0.5 g of sodium acetate trihydrate, 0.5 mL of
dilute acetic acid and 0.5 mL of a solution of disodium 1-
nitroso-2-naphthol-3,6-disulfonate (1 in 500): a red to oran-
ge-red color is immediately produced. Then add 0.5 mL of
hydrochloric acid, and boil for 1 minute: the red color does
not disappear.
(3) Transfer 5 mg of Cyanocobalamin to a 50-mL distil-
ling flask, dissolve in 5 mL of water, and add 2.5 mL of
hypophosphorous acid. Connect the flask with a short con-
JPXV
Official Monographs / Cyclopentolate Hydrochloride
549
denser, and dips its tip into a test tube containing 1 mL of a
solution of sodium hydroxide (1 in 50). Heat gently for 10
minutes, then distil 1 mL into a test tube. To the test tube add
4 drops of a saturated solution of ammonium iron (II) sulfate
hexahydrate, shake gently, then add about 0.03 g of sodium
fluoride, and heat the contents to boil. Immediately add
dropwise diluted sulfuric acid (1 in 7) until a clear solution
results, then add 3 to 5 drops more of diluted sulfuric acid (1
in 7): a blue to blue-green color develops.
pH <2.54> Dissolve 0.10 g of Cyanocobalamin in 20 mL of
water: the pH of this solution is between 4.2 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 20 mg
of Cyanocobalamin in 10 mL of water: the solution is clear
and red in color.
(2) Pseudocyanocobalamin — Dissolve 1.0 mg of
Cyanocobalamin in 20 mL of water, transfer the solution to a
separator, add 5 mL of a mixture of m-cresol and carbon
tetrachloride (1:1), and shake vigorously for 1 minute. Allow
to separate, draw off the lower layer into another separator,
add 5 mL of diluted sulfuric acid (1 in 7), shake vigorously,
and allow to separate completely. If necessary, centrifuge the
mixture: the supernatant liquid is colorless or not more
colored than the following control solution.
Control solution: Dilute 0.6 mL of 0.02 mol/L potassium
permanganate VS with water to make 1000 mL.
Loss on drying <2.41> Not more than 12% (50 mg, in vacu-
um at a pressure not exceeding 0.67 kPa, phosphorus (V)
oxide, 100°C, 4 hours).
Assay Weigh accurately about 20 mg each of Cyanocobala-
min and Cyanocobalamin Reference Standard (previously
determine the loss on drying <2.41> in the same manner as
Cyanocobalamin), dissolve in water to make exactly 1000
mL, respectively, and use these solutions as the sample solu-
tion and the standard solution. Determine the absorbances,
Aj and A s , of the sample solution and the standard solution,
respectively, at 361 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>.
Amount (mg) of C 63 H 88 CoN 14 14 P
= W s X (At/As)
W s ; Amount (mg) of Cyanocobalamin Reference Stan-
dard, calculated on the dried basis
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cyanocobalamin Injection
Vitamin B 12 Injection
Cyanocobalamin Injection is an aqueous solution
for injection.
It contains not less than 95.0% and not more than
115.0% of the labeled amount of cyanocobalamin (C 63
H 88 CoN 14 14 P: 1355.37)
Method of preparation Prepare as directed under Injec-
tions, with Cyanocobalamin.
Description Cyanocobalamin Injection is a clear, light red
to red liquid
It is gradually affected by light.
pH: 4.0-5.5
Identification Determine the absorption spectrum of the
sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 277 nm and 279 nm, between 360 nm, and 362
nm and between 548 nm and 552 nm. Determine the absor-
bances, A x and A 2 , of this solution at the wavelengths of
maximum absorption between 360 nm and 362 nm, and be-
tween 548 nm and 552 nm, respectively: the ratio A 2 /Ai is
not less than 0.29 and not more than 0.32.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Cyanocobalamin Injec-
tion, equivalent to about 2 mg of cyanocobalamin
(C 63 H 88 CoN 14 14 P), add water to make exactly 100 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 20 mg of Cyanocobalamin Reference Stan-
dard (previously determine the loss on drying <2.41> in the
same manner as Cyanocobalamin), add water to make exact-
ly 1000 mL, and use this solution as the standard solution.
With these solutions, proceed as directed in the Assay under
Cyanocobalamin.
Amount (mg) of cyanocobalamin (C 63 H 88 CoN 14 14 P)
= W s x (A T /A S ) x (1/10)
W s : Amount (mg) of Cyanocobalamin Reference Stan-
dard, calculated on the dried basis
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Cyclopentolate Hydrochloride
•HCI
a.-c 9nantisner
C 17 H 25 N0 3 .HC1: 327.85
2-(Dimethylamino)ethyl (2RS)-2-(\ -
hydroxycyclopentyljphenylacetate monohydrochloride
[5870-29-1]
Cyclopentolate Hydrochloride, when dried, contains
not less than 98.5% of C 17 H 25 N0 3 .HC1.
Description Cyclopentolate Hydrochloride occurs as a
white, crystalline powder. It is odorless, or has a characteris-
tic odor.
It is very soluble in water, freely soluble in ethanol (95), in
acetic acid (100) and in chloroform, sparingly soluble in acet-
ic anhydride, and practically insoluble in diethyl ether.
Identification (1) To 1 mL of a solution of Cyclopentolate
Hydrochloride (1 in 100) add 1 mL of Reinecke salt TS: a
550
Cyclophosphamide Hydrate / Official Monographs
JP XV
light red precipitate is formed.
(2) Dissolve 0.2 g of Cyclopentolate Hydrochloride in 2
mL of water, add 2 mL of sodium hydroxide TS, and boil for
1 minute. After cooling, add 2 drops of nitric acid: a phenyla-
cetic acid-like odor is perceptible.
(3) Determine the infrared absorption spectrum of Cy-
clopentolate Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Cyclopentolate Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 0.20 g of Cyclopentolate Hydrochloride
in 20 mL of water: the pH of this solution is between 4.5 and
5.5.
Melting point <2.60> 135 - 138°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cyclopentolate Hydrochloride in 10 mL of water: the solu-
tion is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cy-
clopentolate Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Cyclopento-
late Hydrochloride in 10 mL of chloroform, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, and add chloroform to make exactly 20 mL. Pipet 1
mL of this solution, add chloroform to make exactly 10 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 2-
propanol, w-butyl acetate, water and ammonia solution (28)
(100:60:23:17) to a distance of about 10 cm, and air-dry the
plate. Spray evenly a solution of sulfuric acid in ethanol
(99.5) (1 in 10) on the plate, and heat at 120°C for 30
minutes. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.05% (1 g).
Assay Weigh accurately about 0.5 g of Cyclopentolate
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (4:1), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue-green to yel-
low-green (indicator: 2 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.79 mg of C 17 H 25 N0 3 .HC1
Containers and storage Containers — Tight containers.
Cyclophosphamide Hydrate
■<7 n *t.7t 5 h^kM
■HjO
C 7 H I5 C1 2 N 2 2 P.H 2 0: 279.10
iV,JV-Bis(2-chloroethyl)-3,4,5,6-tetrahydro-2//-l,3,2-
oxazaphosphorin-2-amine 2-oxide monohydrate
[6055-19-2]
Cyclophosphamide Hydrate contains not less than
97.0% of C 7 H 15 C1 2 N 2 2 P.H 2 0.
Description Cyclophosphamide Hydrate occurs as white
crystals or crystalline powder. It is odorless.
It is very soluble in acetic acid (100), freely soluble in
ethanol (95), in acetic anhydride and in chloroform, and
soluble in water and in diethyl ether.
Melting point: 45-53°C
Identification (1) Dissolve 0.1 g of Cyclophosphamide
Hydrate in 10 mL of water, and add 5 mL of silver nitrate
TS: no precipitate is produced. Then boil this solution: a
white precipitate is produced. Collect the precipitate, and add
dilute nitric acid to a portion of this precipitate: it does not
dissolve. Add excess ammonia TS to another portion of the
precipitate: it dissolves.
(2) Add 1 mL of diluted sulfuric acid (1 in 25) to 0.02 g of
Cyclophosphamide Hydrate, and heat until white fumes are
evolved. After cooling, add 5 mL of water, and shake. Neu-
tralize with ammonia TS, then acidify with dilute nitric acid:
this solution responds to the Qualitative Tests <1.09> (2) for
phosphate.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Cyclophosphamide Hydrate in 10 mL of water: the solu-
tion is clear and colorless.
(2) Chloride <1.03>— Perform the test with 0.40 g of Cyclo-
phosphamide Hydrate at a temperature not exceeding 20°C.
Prepare the control solution with 0.40 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(3) Heavy metals <1.07> — Proceed with 1.0 gof Cyclophos-
phamide Hydrate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Water <2.48> 5.5 - 7.0% (0.5 g, direct titration).
Assay Weigh accurately about 0.3 g of Cyclophosph-
amide Hydrate, add 15 mL of hydrogen chloride-ethanol TS,
and heat in a water bath under a reflux condenser for 3.5
hours while protecting from moisture. Distil the ethanol un-
der reduced pressure. Dissolve the residue in 40 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid-dioxane VS (indicator:
2 drops of crystal violet TS) until the color of the solution
changes from blue through green to yellow. Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid-dioxane VS
JPXV
Official Monographs / Cyproheptadine Hydrochloride Hydrate 551
= 13.96 mg of C 7 H 15 C1 2 N 2 2 P.H 2
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
Cycloserine
■+M ?Q-tzU >
r nh
h 2 n-t\
H
C 3 H 6 N 2 2 : 102.09
(AR )-4-Aminoisoxazolidin-3-one
[68-41-7]
Cycloserine contains not less than 950 /ig (potency)
and not more than 1020 /ug (potency) per mg, calculat-
ed on the dried basis. The potency of Cycloserine is ex-
pressed as mass (potency) of cycloserine (C 3 H 6 N 2 2 ).
Description Cycloserine occurs as white to light yellowish
white, crystals or crystalline powder.
It is soluble in water, and sparingly soluble in ethanol (95).
Identification Determine the infrared absorption spectrum
of Cycloserine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Cycloserine Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Optical rotation <2.49> [a]™: +108- +114° (2.5 g calcu-
lated on the dried basis, 2 mol/L sodium hydroxide TS, 50
mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Cycloserine in 20 mL of water:
the pH of the solution is between 5.0 and 7.4.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Cycloserine according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Condensation products — Dissolve 20 mg of Cycloser-
ine in sodium hydroxide TS to make exactly 50 mL, and de-
termine the absorbance of this solution at 285 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>: not
more than 0.8.
Loss on drying <2.41> Not more than 1.5% (0.5 g, reduced
pressure, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er. Adjust the pH of the medium so that it will be 6.0 to 6.1
after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Cycloserine Reference Standard, previously dried at 60°C for
3 hours under reduced pressure of not exceeding 0.67 kPa,
equivalent to about 40 mg (potency), dissolve in water to
make exactly 100 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at 5 C C or be-
low and use within 24 hours. Take exactly a suitable amount
of the standard stock solution before use, and add phosphate
buffer solution, pH 6.0 to make solutions so that each mL
contains 100 /ug (potency) and 50,Mg (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(4) Sample solutions — Weigh accurately an amount of
Cycloserine equivalent to about 40 mg (potency), dissolve in
water to make exactly 100 mL. Take exactly a suitable
amount of this solution, add phosphate buffer solution, pH
6.0 to make solutions so that each mL contains 100 /ug
(potency) and 50 /ug (potency), and use these solutions as the
high concentration sample solution and low concentration
sample solution, respectively.
Containers and storage Containers — Well-closed contain-
ers.
Cyproheptadine Hydrochloride
Hydrate
V
-f n s\79 v >£K£*Jfaft
•HCI
Hp
C 21 H 21 N.HCl.li/ 2 H 2 0: 350.88
4-(5//-Dibenzo[a,rf]cyclohepten-5-ylidene)-l-
methylpiperidine monohydrochloride sesquihydrate
[41354-29-4]
Cyproheptadine Hydrochloride Hydrate, when d-
ried, contains not less than 98.5% of cyproheptadine
hydrochloride (C 21 H 21 N.HC1: 323.86).
Description Cyproheptadine Hydrochloride Hydrate oc-
curs as a white to pale yellow, crystalline powder. It is odor-
less, and has a slightly bitter taste.
It is freely soluble in methanol and in acetic acid (100),
soluble in chloroform, sparingly soluble in ethanol (95),
slightly soluble in water, and practically insoluble in diethyl
ether.
Identification (1) Dissolve 0.1 g of Cyproheptadine
Hydrochloride Hydrate in 10 mL of methanol, apply 1 drop
of this solution on filter paper, air-dry, and examine under
ultraviolet light (main wavelength: 254 nm): the solution
shows a pale blue fluorescence.
(2) Weigh 0.1 g of Cyproheptadine Hydrochloride Hy-
drate, transfer to a separator, dissolve in 5 mL of chlo-
roform, add 4 mL of water and 1 mL of sodium carbonate
TS, and shake. Transfer the chloroform layer to another
separator, and wash with 4 mL of water by shaking well.
Filter the chloroform layer through absorbent cotton
moistened previously with chloroform, and evaporate the
552 Cytarabine / Official Monographs
JP XV
filtrate to dryness. Dissolve the residue in 8 mL of dilute
ethanol by warming at 65 °C. Rub the inner wall of the con-
tainer with a glass rod while cooling until crystallization be-
gins, and allow to stand for 30 minutes. Collect the crystals,
and dry at 80°C for 2 hours: the crystals melt <2.60> between
111°C and 115°C.
(3) Determine the absorption spectrum of a solution of
Cyproheptadine Hydrochloride Hydrate in ethanol (95) (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(4) A saturated solution of Cyproheptadine Hydrochlo-
ride Hydrate responds to the Qualitative Tests <1.09> (2) for
chloride.
Purity (1) Acidity — Dissolve 2.0 g of Cyproheptadine
Hydrochloride Hydrate in 25 mL of methanol, and add 1
drop of methyl red TS and 0.30 mL of 0.1 mol/L sodium
hydroxide VS: a yellow color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Cypro-
heptadine Hydrochloride Hydrate according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
Loss on drying <2.41> 7.0 - 9.0% (1 g, in vacuum at a pres-
sure not exceeding 0.67 kPa, 100°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Cyproheptadine
Hydrochloride Hydrate, previously dried, and dissolve in 20
mL of acetic acid (100) by warming at 50°C. After cooling,
add 40 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.39 mg of C 21 H 21 N.HC1
Containers and storage Containers — Well-closed contain-
ers.
Cytarabine
-&-?£>
OH H
C 9 H 13 N 3 5 : 243.22
1 -/?-D-Arabinof uranosylcytosine
[I47.94-4]
Cytarabine, when dried, contains not less than
98.5% and not more than 101.0% of CsH^NjCV
Description Cytarabine occurs as white crystals or crystal-
line powder.
It is freely soluble in water, soluble in acetic acid (100), and
very slightly soluble in ethanol (99.5).
It dissolves in 0.1 mol/L hydrochloric acid TS.
Melting point: about 214°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Cytarabine in 0.1 mol/L hydrochloric acid TS
(1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Cytarabine as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: + 154 - + 160° (after drying,
0.1 g, water, 10 mL, 100 mm).
pH <2.54> Dissolve 0.20 g of Cytarabine in 20 mL of water:
the pH of this solution is between 6.5 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Cytarabine in 10 mL of water: the solution is clear and color-
less.
(2) Chloride <1.03>— Perform the test with 1.0 g of
Cytarabine. Prepare the control solution with 0.25 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.009%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Cytara-
bine according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(4) Related substances — Dissolve 0.10 g of Cytarabine in
10 mL of water, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add water to make exactly
200 mL, and use this solution as the standard solution (1).
Pipet 10 mL of the standard solution (1), add water to make
exactly 25 mL and use this solution as the standard solution
(2). Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xh each of the
sample solution and standard solutions (1) and (2) on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with 1-butanol saturated with
water to a distance of about 12 cm, and air-dry the plate. Ex-
amine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solution
(1), and the number of them which are more intense than the
spot from the standard solution (2) is not more than two.
Spray evenly acidic potassium permanganate TS on the plate:
any spot other than the principal spot does not appear.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately about 0.2 g of Cytarabine, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.05 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 12.16 mg of C 9 H I3 N 3 5
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Daunorubicin Hydrochloride
553
Dantrolene Sodium Hydrate
C 14 H 9 N 4 Na0 5 .3i/2H 2 0: 399.29
Monosodium 3-[5-(4-nitrophenyl)furan-2-
ylmethylene]amino-2,5-dioxo-l,3-imidazolidinate
hemiheptahydrate [14663-23-1, anhydride]
Dantrolene Sodium Hydrate contains not less than
98.0% of dantrolene sodium (CuH^NaO;: 336.23),
calculated on the anhydrous basis.
Description Dantrolene Sodium Hydrate occurs as a yel-
lowish orange to deep orange, crystalline powder.
It is soluble in propylene glycol, sparingly soluble in
methanol, slightly soluble in ethanol (95), very slightly solu-
ble in water and in acetic acid (100), and practically insoluble
in acetone, in tetrahydrofuran and in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Dantrolene Sodium Hydrate in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Dan-
trolene Sodium Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) To 0. 1 g of Dantrolene Sodium Hydrate add 20 mL of
water and 2 drops of acetic acid (100), shake well, and filter:
the filtrate responds to the Qualitative Tests <1.09> (1) for so-
dium salt.
Purity (1) Alkalinity — To 0.7 g of Dantrolene Sodium
Hydrate add 10 mL of water, shake well, and centrifuge or
filter through a membrane filter. To 5 mL of the supernatant
or the filtrate add 45 mL of water, 3 drops of phenolphthalein
TS and 0.10 mL of 0.1 mol/L hydrochloric acid VS: a red
color is not produced.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Dantro-
lene Sodium Hydrate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Related Substances — Dissolve 50 mg of Dantrolene
Sodium Hydrate in 20 mL of tetrahydrofuran and 2 mL of a-
cetic acid (100), add ethanol (99.5) to make 100 mL, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add ethanol (99.5) to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 iiL of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area from
these solutions by the automatic integration method: the total
area of all peaks other than the peak of dantrolene from the
sample solution is not larger than the peak area of dantrolene
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 300 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with silica gel for
liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of hexane, acetic acid (100) and
ethanol (99.5) (90:10:9).
Flow rate: Adjust the flow rate so that the retention time of
dantrolene is about 8 minutes.
Selection of column: Dissolve 5 mg of Dantrolene Sodium
Hydrate and 0.1 g of theophylline in 20 mL of tetrahydrofu-
ran and 2 mL of acetic acid (100), and add ethanol (99.5) to
make 100 mL. To 10 mL of this solution add ethanol (99.5)
to make 100 mL. Proceed with 10 /uL of this solution under
the above operating conditions, and calculate the resolution.
Use a column giving elution of theophylline and dantrolene
in this order with the resolution between these peaks being
not less than 6.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of dantrolene from 10 iiL of the standard
solution is 10% to 40% of the full scale.
Time span of measurement: About twice as long as the
retention time of dantrolene beginning after the solvent peak.
Water <2.48> 14.5 - 17.0% (0.2 g, direct titration).
Assay Weigh accurately about 0.7 g of Dantrolene Sodium
Hydrate, dissolve in 180 mL of a mixture of propylene glycol
and acetone (1:1), and titrate <2.50> with 0.1 mol/L perchlor-
ic acid VS (potentiometric titration). Perform a blank deter-
mination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 33.624 mg of Ci 4 H 9 N 4 Na05
Containers and storage Containers — Tight containers.
Daunorubicin Hydrochloride
y^/JHfv>^^
CH 3
OH . HCI
NH ;
C 27 H 29 NOi .HCl: 563.98
(2S,4S)-2-Acetyl-4-(3-amino-2,3,6-trideoxy-a-L-/>'xo-
hexopyranosyloxy)-2,5,12-trihydroxy
-7-methoxy-l,2,3,4-tetrahydrotetracene-6,ll-dione
monohydrochloride [23541-50-6]
Daunorubicin Hydrochloride is the hydrochloride of
554 Deferoxamine Mesilate / Official Monographs
JP XV
an anthracycline substance having antitumor activity
produced by the growth of Streptomyces peucetius.
It contains not less than 940 fig (potency) and not
more than 1050 fig (potency) per mg, calculated on the
dried basis. The potency of Daunorubicin Hydrochlo-
ride is expressed as mass (potency) of daunorubicin
hydrochloride (C 27 H 29 NO 10 .HCl).
Description Daunorubicin Hydrochloride occurs as a red
powder.
It is soluble in water and in methanol, and slightly soluble
in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Daunorubicin Hydrochloride in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Daunorubicin
Hydrochloride Reference Standard prepared in the same
manner as the sample solution: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(2) A solution of Daunorubicin Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]™: +250 - +275° (15 mg calculat-
ed on the dried basis, methanol, 10 mL, 100 mm).
pH <2.54> Dissolve 0.15 g of Daunorubicin Hydrochloride
in 30 mL of water: the pH of the solution is between 4.5 and
6.0.
Purity (1) Clarity and color of solution — Dissolve 20 mg
of Daunorubicin Hydrochloride in 10 mL of water: the
solution is clear and red.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Daunorubicin Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 10 mg of Daunorubicin
Hydrochloride in 5 mL of methanol, and use this solution as
the sample solution. Pipet 3 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol, water and a-
cetic acid (100) (15:5:1:1) to a distance of about 10 cm, and
air-dry the plate. Examine the spots with the naked eye: the
spot other than the principal spot obtained from the sample
solution is not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 7.5% (0.1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Weigh accurately an amount of Daunorubicin
Hydrochloride and Daunorubicin Hydrochloride Reference
Standard, equivalent to about 20 mg (potency), dissolve each
in a suitable amount of the mobile phase, add exactly 4 mL of
the internal standard solution and the mobile phase to make
20 mL, and use these solutions as the sample solution and the
standard solution, respectively. Perform the test with 5 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Q T and Q s , of
the peak area of daunorubicin to that of the internal stan-
dard.
Amount [ag (potency)] of C27H29NO10.HCI
= Ws x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Daunorubicin Hydrochlo-
ride Reference Standard
Internal standard solution — A solution of 2-naphthalenesul-
fonic acid in the mobile phase (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /urn in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Adjust the pH of a mixture of water and
acetonitrile (31:19) to 2.2 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
daunorubicin is about 9 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, the internal standard and daunorubicin are eluted in
this order with the resolution between these peaks being not
less than 2.0.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of daunorubicin to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Tight containers.
Deferoxamine Mesilate
f7iPW
• H 3 C-SOsH
C 2 5H 48 N 6 8 .CH 4 03S: 656.79
N- [5-(Acetylhydroxyamino)pentyl] -N' -(5- { 3- [(5-
aminopentyl)hydroxycarbamoyl]propanoylamino}pentyl)-
N' -hydroxysuccinamide monomethanesulf onate
[138-14-7]
Deferoxamine Mesilate contains not less than 98.0%
and not more than 102.0% of Q^H^IWCH^S,
calculated on the anhydrous basis.
Description Deferoxamine Mesilate occurs as a white to
pale yellowish white, crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5), in 2-propanol and in diethyl ether.
Melting point: about 147°C (with decomposition).
JPXV
Official Monographs / Deferoxamine Mesilate
555
Identification (1) To 5 mL of a solution of Deferoxamine
Mesilate (1 in 500) add 1 drop of iron (III) chloride TS: a
deep red color develops.
(2) To 0.05 g of Deferoxamine Mesilate add 0.2 g of sodi-
um hydroxide, melt by heating over a small flame, and heat
further for 2 to 3 seconds. To the residue add 0.5 mL of
water, acidify with dilute hydrochloric acid, and warm: the
gas evolved changes moistened potassium iodate-starch paper
to blue.
(3) Determine the infrared absorption spectrum of
Deferoxamine Mesilate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Deferoxamine Mesilate Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
pH <2.54> Dissolve 1.0 g of Deferoxamine Mesilate in 10
mL of water: the pH of this solution is between 3.5 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Deferoxamine Mesilate in 10 mL of water: the solution is
clear and colorless to pale yellow.
(2) Chloride <1.03>— Perform the test with 1.0 g of
Deferoxamine Mesilate. Prepare the control solution with
0.90 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.032%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of
Deferoxamine Mesilate. Prepare the control solution with
0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.040%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Deferox-
amine Mesilate according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Deferoxamine Mesilate according to Method 3, and per-
form the test. Use a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 10) (not more than 2 ppm).
(6) Related substances — Dissolve 50 mg of Deferoxamine
Mesilata in 50 mL of the mobile phase, and use this solution
as the sample solution. Pipet 3 mL of the sample solution,
add the mobile phase to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with exactly
20 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area of both
solutions by the automatic integration method: the total area
of all peaks other than the peak of deferoxamine is not larger
than the peak area of deferoxamine from the standard solu-
tion.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4 mm in inside diameter
and 20 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /um in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.32 g of diammonium hydrogen
phosphate, 0.37 g of disodium dihydrogen ethylenediamine
tetraacetate dihydrate and 1.08 g of sodium 1-heptanesul-
fonate in 950 mL of water, and adjust the pH of this solution
to 2.8 with phosphoric acid. To 800 mL of this solution add
100 mL of 2-propanol.
Flow rate: Adjust the flow rate so that the retention time of
deferoxamine is about 15 minutes.
Time span of measurement: About two times as long as the
retention time of deferoxamine beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 100 mL.
Confirm that the peak area of deferoxamine obtained from
20 [iL of this solution is equivalent to 1.5 to 2.5% of that of
deferoxamine obtained from 20 /xL of the standard solution.
System performance: Dissolve 16 mg of Deferoxamine
Mesilate and 4 mg of methyl parahydroxybenzoate in 50 mL
of the mobile phase. When the procedure is run with 20 /xh of
this solution under the above operating conditions, deferoxa-
mine and methyl parahydroxybenzoate are eluted in this ord-
er with the resolution between these peaks being not less than
4.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of deferoxamine is not more than 3.0%.
Water <2.48> Not more than 2.0% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 60 mg of Deferoxamine
Mesilate and Deferoxamine Mesilate Reference Standard
(previously determine the water <2.4S> in the same manner as
Deferoxamine Mesilate), dissolve each in 20 mL of water,
add 10 mL of 0.05 mol/L sulfuric acid TS, and add water to
make exactly 50 mL. Pipet 5 mL each of these solutions, add
exactly 5 mL of 0.05 mol/L sulfuric acid TS and exactly 0.2
mL of iron (III) chloride TS, then add water to make exactly
50 mL, and use these solutions as the sample solution and the
standard solution, respectively. Perform the test with these
solutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using a solution prepared by adding 0.05 mol/
L sulfuric acid TS to 0.2 mL of iron (III) chloride TS to make
exactly 50 mL as the blank, and determine the absorbances,
A T and A s , of each solution from the sample solution and the
standard solution at 430 nm.
Amount (mg) of C^H^NgCvCH^S
= W s x (A T /A S )
W s : Amount (mg) of Deferoxamine Mesilate Reference
Standard, calculated on the anhydrous basis
Containers and storage Containers — Tight containers.
556 Dehydrocholic Acid / Official Monographs
JP XV
Dehydrocholic Acid
ft: Knzi-jL-gg
C0 2 H
C 24 H340 5 : 402.52
3,7,12-Trioxo-5/?-cholan-24-oic acid [81-23-2]
Dehydrocholic Acid, when dried, contains not less
than 98.5% of C 24 H 3 40 5 .
Description Dehydrocholic Acid occurs as a white, crystal-
line powder. It is odorless, and has a bitter taste.
It is sparingly soluble in 1,4-dioxane, slightly soluble in
ethanol (95), and practically insoluble in water and in diethyl
ether.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 5 mg of Dehydrocholic Acid in
1 mL of sulfuric acid and 1 drop of formaldehyde solution,
and allow to stand for 5 minutes. Add 5 mL of water to the
solution: the solution shows a yellow color and a blue-green
fluorescence.
(2) To 0.02 g of Dehydrocholic Acid add 1 mL of ethanol
(95), shake, add 5 drops of 1,3-dinitrobenzene TS and 0.5
mL of a solution of sodium hydroxide (1 in 8), and allow to
stand: a purple to red-purple color develops, and gradually
changes to brown.
Optical rotation <2.49> [a]™'- +29
g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 233 - 242°C
+ 32° (after drying, 0.2
Purity (1) Odor — To 2.0 g of Dehydrocholic Acid add
100 mL of water, and boil for 2 minutes: the solution is odor-
less.
(2) Clarity and color of solution — To 0.10 g of De-
hydrocholic Acid, previously powdered in a mortar, add 30
mL of ethanol (95), and dissolve by shaking for 10 minutes:
the solution is clear and colorless.
(3) Chloride <1.03>— To 2.0 g of Dehydrocholic Acid add
100 mL of water, shake for 5 minutes and filter, and use this
filtrate as the sample solution. To 25 mL of the sample solu-
tion add 6 mL of dilute nitric acid, heat in a water bath for 6
minutes, filter after cooling, and collect the clear filtrate.
Wash the residue with 10 mL of water, combine the washings
and the filtrate, dilute with water to 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.30 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.021%).
(4) Sulfate <1.14> — Add 1 mL of dilute hydrochloric acid
to 25 mL of the sample solution obtained in (3), heat in a
water bath for 6 minutes, filter after cooling, and collect the
clear filtrate. Wash the residue with 10 mL of water, combine
the washings and the filtrate, dilute with water to 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 0.50 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.048%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of De-
hydrocholic Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(6) Barium — To the solution obtained in (1) add 2 mL of
hydrochloric acid, and boil for 2 minutes. Cool, filter, and
wash with water until 100 mL of the filtrate is obtained. To 10
mL of the filtrate add 1 mL of dilute sulfuric acid: no turbidi-
ty is produced.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Dehydrocholic
Acid, previously dried, add 40 mL of neutralized ethanol and
20 mL of water, and dissolve by warming. Add 2 drops of
phenolphthalein TS, titrate <2.50> with 0.1 mol/L sodium
hydroxide VS, adding 100 mL of freshly boiled and cooled
water as the end point is approached, and continue the titra-
tion.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.25 mg of C 24 H 34 05
Containers and storage Containers — Well-closed contain-
ers.
Purified Dehydrocholic Acid
HUB* fc: Kna-;n
C0 2 H
C 24 H 34 5 : 402.52
3,7,12-Trioxo-5/?-cholan-24-oic acid [81-23-2]
Purified Dehydrocholic Acid, when dried, contains
not less than 99.0% of C 24 H 34 O s .
Description Purified Dehydrocholic Acid occurs as a white,
crystalline powder. It is odorless, and has a bitter taste.
It is sparingly soluble in 1,4-dioxane, slightly soluble in
ethanol (95), and practically insoluble in water and in diethyl
ether.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 5 mg of Purified Dehydrochol-
ic Acid in 1 mL of sulfuric acid and 1 drop of formaldehyde
solution, and allow to stand for 5 minutes. Add 5 mL of
water to the solution: the solution shows a yellow color and
blue-green fluorescence.
(2) To 0.02 g of Purified Dehydrocholic Acid add 1 mL
of ethanol (95), shake, add 5 drops of 1,3-dinitrobenzene TS
and 0.5 mL of a solution of sodium hydroxide (1 in 8), and
allow to stand: a purple to red-purple color develops, and
gradually changes to brown.
JPXV
Official Monographs / Dehydrocholic Acid Injection
557
Optical rotation <2.49> [a]™: + 29 - +32° (after drying, 0.2
g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 237 - 242°C
Purity (1) Odor — To 2.0 g of Purified Dehydrocholic
Acid add 100 mL of water, and boil for 2 minutes: the solu-
tion is odorless.
(2) Clarity and color of solution — Dissolve 0.10 g of
Purified Dehydrocholic Acid, previously powdered in a mor-
tar, in 30 mL of ethanol (95) by shaking for 10 minutes: the
solution is clear and colorless.
(3) Chloride <1.03>— To 2.0 g of Purified Dehydrocholic
Acid add 100 mL of water, shake for 5 minutes and filter, and
use this filtrate as the sample solution. To 25 mL of the sam-
ple solution add 6 mL of dilute nitric acid, heat in a water
bath for 6 minutes, filter after cooling, and collect the clear
filtrate. Wash the residue with 10 mL of water, combine the
washings and the filtrate, dilute with water to 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution with 0.30 mL of 0.01 mol/L hydrochlor-
ic acid VS (not more than 0.021%).
(4) Sulfate <1.14> — Add 1 mL of dilute hydrochloric acid
to 25 mL of the sample solution obtained in (3), heat in a
water bath for 6 minutes, filter after cooling, and collect the
clear filtrate. Wash the residue with 10 mL of water, combine
the washings and the filtrate, dilute with water to 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 0.50 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.048%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of Purified
Dehydrocholic Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(6) Barium — To the solution obtained in (1) add 2 mL of
hydrochloric acid, and boil for 2 minutes, cool, filter, and
wash the filter with water until 100 mL of the filtrate is ob-
tained. To 10 mL of the filtrate add 1 mL of dilute sulfuric
acid: no turbidity is produced.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Purified De-
hydrocholic Acid , previously dried, add 40 mL of neutral-
ized ethanol and 20 mL of water, and dissolve by warming.
Add 2 drops of phenolphthalein TS, then titrate <2.50> with
0.1 mol/L sodium hydroxide VS, adding 100 mL of freshly
boiled and cooled water as the end point is approached, and
continue the titration.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.25 mg of C 24 H3405
Containers and storage Containers — Well-closed contain-
ers.
Dehydrocholic Acid Injection
Dehydrocholate Sodium Injection
Dehydrocholic Acid Injection is an aqueous solution
for injection.
It contains not less than 95% and not more than 105
% of the labeled amount of dehydrocholic acid (C24H34
O s : 402.52).
Method of preparation Dissolve Purified Dehydrocholic
Acid in a solution of Sodium Hydroxide, and prepare as
directed under Injections.
Description Dehydrocholic Acid Injection is a clear, color-
less to light yellow liquid, and has a bitter taste.
pH: 9-11
Identification Transfer a volume of Dehydrocholic Acid In-
jection, equivalent to 0.1 g of Purified Dehydrocholic Acid
according to the labeled amount, to a separator, and add 10
mL of water and 1 mL of dilute hydrochloric acid: a white
precipitate is produced. Extract the mixture with three 15-mL
portions of chloroform, combine all the chloroform extracts,
evaporate the chloroform on a water bath, and dry the
residue at 105 °C for 1 hour: the residue so obtained melts
<2.60> between 235°C and 242°C.
Purity Heavy metals <1.07> — Evaporate a volume of De-
hydrocholic Acid Injection, equivalent to 1.0 g of Purified
Dehydrocholic Acid according to the labeled amount, on a
water bath to dryness. Proceed with the residue according to
Method 2, and perform the test. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 20
ppm).
Bacterial endotoxins <4.01> Less than 0.30EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay Transfer an exactly measured volume of De-
hydrocholic Acid Injection, equivalent to about 0.5 g of de-
hydrocholic acid (C24H34O5), to a 100-mL separator, and
add, if necessary, water to make 25 mL. Add 2 mL of
hydrochloric acid, and extract with 25-mL, 20-mL and
15-mL portions of chloroform successively. Combine the
chloroform extracts, wash with cold water until the washings
become negative to acid, and evaporate the chloroform on a
water bath. Dissolve the residue in 40 mL of neutralized
ethanol and 20 mL of water by warming. Add 2 drops of
phenolphthalein TS to this solution, titrate <2.50> with 0.1
mol/L sodium hydroxide VS, adding 100 mL of freshly
boiled and cooled water as the end point is approached, and
continue the titration.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.25 mg of Q4H34O5
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
558
Demethylchlortetracycline Hydrochloride / Official Monographs
JP XV
Demethylchlortetracycline
Hydrochloride
T"V T-)V7 a )IT r- v+M >7 U >M&k
NH,
■ HCI
C 21 H 21 C1N 2 8 .HC1: 501.31
(45 , ,4aS,5a5',6S,12a5')-7-Chloro-4-dimethylamino-
3,6,10,12,12a-pentahydroxy-l,ll-dioxo-
l,4,4a,5,5a,6,ll,12a-octahydrotetracene-2-carboxamide
monohydrochloride [64-73-3]
Demethylchlortetracycline Hydrochloride is the
hydrochloride of a tetracycline substance having an-
tibacterial activity produced by the growth of the
mutant of Streptomyces aureofaciens.
It contains not less than 900 fig (potency) and not
more than 1010 fig (potency) per mg, calculated on
the dried basis. The potency of Demethylchlortetracy-
cline Hydrochloride is expressed as mass (potency)
of demethylchlortetracycline hydrochloride
(C 21 H 21 C1N 2 8 .HC1).
Description Demethylchlortetracycline Hydrochloride oc-
curs as a yellow crystalline powder.
It is soluble in water, and slightly soluble in ethanol (99.5).
Identification (1) Dissolve 40 mg of Demethylchlor-
tetracycline Hydrochloride in 250 mL of water. To 10 mL of
this solution add 85 mL of water and 5 mL of a solution of
sodium hydroxide (1 in 5). Determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of De-
methylchlortetracycline Hydrochloride Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of De-
methylchlortetracycline Hydrochloride as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Demethylchlor-
tetracycline Hydrochloride Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) A solution of Demethylchlortetracycline Hydrochlo-
ride (1 in 100) responds to the Qualitative Tests <1.09> (2) for
chloride.
Optical rotation <2.49> [ a £°: -248 - -263° (0.25 g calculat-
ed on the dried basis, 0.1 mol/L hydrochloric acid TS, 25
mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Demethylchlortetracycline
Hydrochloride in 100 mL of water: the pH of the solution is
between 2.0 and 3.0.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Demethylchlortetracycline Hydrochloride according to
Method 2, and perform the test. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 20
ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Demethylchlortetracycline Hydrochloride according to
Method 4, and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 25 mg of Demethyl-
chlortetracycline Hydrochloride in 50 mL of 0.01 mol/L
hydrochloric acid TS, and use this solution as the sample so-
lution. Pipet 5 mL of the sample solution, add 0.01 mol/L
hydrochloric acid TS to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine each peak area
obtained from the chromatograms of these solutions by the
automatic integration method: the peak area other than
demethylchlortetracycline obtained from the sample solution
is not more than 6/5 times that of demethylchlortetracycline
from the standard solution, and the sum of the areas of the
peaks other than demethylchlortetracycline from the sample
solution is not more than 2 times the peak area of
demethylchlortetracycline from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of demethylchlortetracycline beginning after
the solvent peak.
System suitability —
Test for required detectability: Measure exactly 10 mL of
the standard solution, add 0.01 mol/L hydrochloric acid TS
to make exactly 50 mL, and use this solution as the solution
for system suitability test. Pipet 5 mL of the solution for sys-
tem suitability test, and add 0.01 mol/L hydrochloric acid TS
to make exactly 50 mL. Confirm that the peak area of de-
methylchlortetracycline obtained from 20 fiL of this solution
is equivalent to 7 to 13% of that from 20 fiL of the solution
for system suitability test.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
demethylchlortetracycline is not more than 1.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately an amount of Demethylchlor-
tetracycline Hydrochloride and Demethylchlortetracycline
Hydrochloride Reference Standard, equivalent to about
25 mg (potency), dissolve each in 0.01 mol/L hydrochloric
acid TS to make exactly 50 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with exactly 20 fiL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of demethylchlortetracycline.
Amount ["g (potency)] of C 21 H 2I C1N 2 8 .HC1
JPXV
Official Monographs / Deslanoside 559
= W s x (A T /A S ) x 1000
fV s : Amount [mg (potency)] of Demethylchlortetracycline
Hydrochloride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.1 mm in inside di-
ameter and 25 cm in length, packed with styrene-divinylben-
zene copolymer for liquid chromatography (10 [im in particle
diameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase: Dissolve 3.5 g of dipotassium hydrogen
phosphate, 1.5 g of tetrabutylammonium hydrogensulfate
and 0.4 g of disodium dihydrogen ethylenediamine tetraa-
cetate dihydrate in 300 mL of water, and adjust the pH to 8.5
with sodium hydroxide TS. To this solution add 75.0 g of t-
butanol and water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
demethylchlortetracycline is about 8 minutes.
System suitability —
System performance: Heat 10 mL of the standard solution
on a water bath for 60 minutes. When the procedure is run
with 20 /xL of this solution so obtained under the above oper-
ating conditions, 4-epidemethylchlortetracycline and de-
methylchlortetracycline are eluted in this order with the reso-
lution between these peaks being not less than 3. The relative
retention time of 4-epidemethylchlortetracycline with respect
to demethylchlortetracycline is about 0.7.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
demethylchlortetracycline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Deslanoside
?X7 J V K
HO ^
OH
C 4 7H 74 0, 9 : 943.08
3/?-[/?-D-Glucopyranosyl-(l->4)-2,6-dideoxy-
y§-D-n'do-hexopyranosyl-(l- > 4)-2,6-dideoxy-
/?-D-n&o-hexopyranosyl-(l->4)-2,6-dideoxy-/?-D-n'£>0-
hexopyranosyloxy]-12/?,14-dihydroxy-5/?,14/?-card-
20(22)-enolide [17598-65-1]
Deslanoside, when dried, contains not less than
90.0% and not more than 102.0% of C 47 H 74 19 .
Description Deslanoside occurs as colorless or white crys-
tals or a white, crystalline powder. It is odorless.
It is freely soluble in anhydrous pyridine, sparingly soluble
in methanol, slightly soluble in ethanol (95), and practically
insoluble in water and in diethyl ether.
It is hygroscopic.
Identification Transfer 1 mg of Deslanoside to a small test
tube about 10 mm in inside diameter, dissolve in 1 mL of a
solution of iron (III) chloride hexahydrate in acetic acid (100)
(1 in 1000), and underlay gently with 1 mL of sulfuric acid: at
the zone of contact of two liquids a brown ring is produced,
and the color of the upper layer near to the contact zone
changes gradually to blue through purple, and the entire acet-
ic acid layer shows a blue-green color through a deep blue
color.
Purity (1) Clarity and color of solution — Dissolve 20 mg
of Deslanoside in 1 mL of ethanol (95) and 3 mL of water by
warming, cool, and dilute to 100 mL with water: the solution
is clear and colorless.
(2) Related substances — Dissolve 10 mg of Deslanoside
in exactly 5 mL of methanol, and use this solution as the sam-
ple solution. Dissolve 1.0 mg of Deslanoside Reference Stan-
dard in exactly 5 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
/iL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of dichloromethane, methanol and water
(84:15:1) to a distance of about 13 cm, and air-dry the plate.
Spray evenly dilute sulfuric acid on the plate, and heat the
plate at 110°C for 10 minutes: the spots other than the prin-
cipal spot from the sample solution are not larger than and
not more intense than the spot from the standard solution.
Optical rotation <2.49> [a]™'- +6.5 - +8.5° (after drying,
0.5 g, anhydrous pyridine, 25 mL, 100 mm).
Loss on drying <2.41> Not more than 8.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Dissolve about 12 mg each of Deslanoside and Des-
lanoside Reference Standard, previously dried and accurately
weighed, in 20 mL each of methanol, add water to make ex-
actly 100 mL, and use these solutions as the sample solution
and the standard solution, respectively. Pipet 5 mL each of
these solutions, transfer to light-resistant, 25-mL volumetric
flasks, shake well with 5 mL each of 2,4,6-trinitrophenol TS
and 0.5 mL each of a solution of sodium hydroxide (1 in 10),
add diluted methanol (1 in 4) to make 25 mL, and allow to
stand at a temperature between 18°C and 22°C for 25
minutes. Determine the absorbances, A T and A s , of the sub-
sequent solutions of the sample solution and standard solu-
tion, respectively, at 485 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using a solution prepared
560
Deslanoside Injection / Official Monographs
JP XV
with 5 mL of diluted methanol (1 in 5) in the same manner as
the blank.
Amount (mg) of C47H 74 1 9
= W s x (A T /A S )
W s : Amount (mg) of Deslanoside Reference Standard
Containers and storage Containers — Tight containers.
Deslanoside Injection
tX7/-> Halt*
Deslanoside Injection is an aqueous solution for in-
jection.
It contains not less than 90% and not more than 110
% of the labeled amount of deslanoside
(C 47 H 74 19 : 943.08).
Method of preparation Dissolve Deslanoside in 10 vol%
ethanol and prepare as directed under Injections. It may con-
tain Glycerin. It may be prepared with a suitable amount of
Ethanol and Water for Injection.
Description Deslanoside Injection is a clear and colorless
liquid.
pH: 5.0-7.0
Identification (1) Place a volume of Deslanoside Injec-
tion, equivalent to 2 mg of Deslanoside according to the la-
beled amount, in a separator, add sodium chloride in the ra-
tio of 0.2 g to each mL of this solution, and extract with three
10-mL portions of chloroform. Combine the chloroform ex-
tracts, mix uniformly, pipet 15 mL of this solution, and
evaporate the chloroform under reduced pressure. Proceed
with the residue as directed in the Identification under Des-
lanoside.
(2) Place a volume of Deslanoside Injection, equivalent
to 1 mg of Deslanoside according to the labeled amount, in a
separator, add sodium chloride in the ratio of 0.2 g to each
mL of this solution, and extract with three 5-mL portions of
chloroform. Combine the chloroform extracts, evaporate the
chloroform under reduced pressure, dissolve the residue in 5
mL of methanol, and use this solution as the sample solution.
Separately, dissolve 1 mg of Deslanoside Reference Standard
in 5 mL of methanol, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 20 /xL each of these
solutions on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of
dichloromethane, methanol and water (84:15:1) to a distance
of about 13 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid upon the plate, and heat the plate at 110°C for 10
minutes: the spots from the sample solution and the standard
solution show a black color and have the same Rf value.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Deslanoside Injection,
equivalent to about 3 mg of deslanoside (C47H74O19). Add 5
mL of methanol and water to make 25 mL. Use this solution
as the sample solution, and proceed as directed in the Assay
under Deslanoside.
Amount (mg) of deslanoside (C4 7 H 7 40 19 )
= W s x (A T /A S ) x (1/4)
W s : Amount (mg) of Deslanoside Reference Standard
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Dexamethasone
C 22 H 29 F0 5 : 392.46
9-Fluoro- 11/3, 17,21 -trihydroxy-1 6a-methylpregna-l,4-diene-
3,20-dione [50-02-2]
Dexamethasone, when dried, contains not less than
97.0% and not more than 102.0% of C 22 H 29 F0 5 .
Description Dexamethasone occurs as white to pale yellow
crystals or crystalline powder.
It is sparingly soluble in methanol, in ethanol (95) and in
acetone, slightly soluble in acetonitrile, and practically in-
soluble in water.
Melting point: about 245°C (with decomposition).
Identification (1) Proceed with 10 mg of Dexamethasone
as directed under Oxygen Flask Combustion Method, using a
mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and
20 mL of water as the absorbing liquid: the solution obtained
responds to the Qualitative Tests <1.09> for fluoride
(2) Dissolve 1 mg of Dexamethasone in 10 mL of ethanol
(95). Mix 2 mL of the solution with 10 mL of phenylhydra-
zine hydrochloride TS, heat in a water bath at 60°C for 20
minutes, and cool the solution. Determine the absorption
spectrum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using as the blank the solution
prepared with 2 mL of ethanol (95) in the same manner as the
former solution, and compare the spectrum with the Refer-
ence Spectrum or the spectrum of a solution of Dexametha-
sone Reference Standard prepared in the same manner as the
sample solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Dex-
amethasone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Dexamethasone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve Dexamethasone and
Dexamethasone Reference Standard in acetone, respectively,
then evaporate the acetone to dryness, and repeat the test on
the residues.
Optical rotation <2.49> [ a ]g: +86
0.1 g, methanol, 10 mL, 100 mm).
■94° (after drying,
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
JPXV
Official Monographs / Dextran 40
561
Dexamethasone according to Method 2, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 30 ppm).
(2) Related substances — Dissolve 0.18 g of Dexametha-
sone in 100 mL of acetonitrile. To 33 mL of this solution add
a solution, prepared by dissolving 1.32 g of ammonium for-
mate in water to make 1000 mL and adjusted to pH 3.6 with
formic acid, to make 100 mL, and use this solution as the
sample solution. To exactly 1 mL of the sample solution add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/nL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the peak area other than
dexamethasone is not larger than the peak area of dex-
amethasone obtained from the standard solution, and the
total area of the peaks other than the peak of dexamethasone
from the sample solution is not larger than 2 times the peak
area of dexamethasone from the standard solution
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with phenylsilanized sili-
ca gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.32 g of ammonium formate in
1000 mL of water, and adjust the pH to 3.6 with formic acid.
To 670 mL of this solution add 330 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
dexamethasone is about 13 minutes.
Time span of measurement: About 4 times as long as the
retention time of dexamethasone beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 10 mL.
Confirm that the peak area of dexamethasone obtained with
10 /xL of this solution is equivalent to 8 to 12% of that with
10 /xL of the standard solution.
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of dexamethasone are not less than 5000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
dexamethasone is not more than 1.0%.
Loss on drying <2.41> Not more than 0.5% (0.2 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.2 g,
platinum crucible).
Assay Dissolve about 10 mg each of Dexamethasone and
Dexamethasone Reference Standard, previously dried and
accurately weighed, in 70 mL each of diluted methanol (1 in
2), add exactly 5 mL each of the internal standard solution,
then add diluted methanol (1 in 2) to make 100 mL, and use
these solutions as the sample solution and standard solution.
Perform the test with 10 /xL each of these solutions as direct-
ed under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, g T and Q s , of the
peak area of dexamethasone to that of the internal standard,
respectively.
Amount (mg) of C 22 H 29 F0 5 = W s x Q T /Q S
W s : Amount (mg) of Dexamethasone Reference Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in diluted methanol (1 in 2) (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (2:1).
Flow rate: Adjust the flow rate so that the retention time of
dexamethasone is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, dexamethasone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 6.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of dexamethasone to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dextran 40
t^'+X r-7>40
Dextran 40 is a product obtained by partial decom-
position of polysaccharide, which is produced by fer-
mentation of sucrose with Leuconostoc mesenteroides
van Tieghem (Lactobacillaceae), and the average
molecular mass is about 40,000.
When dried, it contains not less than 98.0% and not
more than 102.0% of dextran 40.
Description Dextran 40 occurs as a white, amorphous pow-
der. It is odorless and tasteless.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves gradually in water.
It is hygroscopic.
Identification To 1 mL of a solution of Dextran 40 (1 in
3000) add 2 mL of anthrone TS: a blue-green color develops
and turns gradually dark blue-green. Then to this solution
add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of acetic
acid (100): the solution does not change in color.
pH <2.54> Dissolve 1.0 g of Dextran 40 in 10 mL of water:
562
Dextran 40 Injection / Official Monographs
JP XV
the pH of this solution is between 5.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dextran 40 in 10 mL of water by warming: the solution is
clear and colorless.
(2) Chloride <1.03>— Perform the test with 2.0 g of
Dextran 40. Prepare the control solution with 1.0 mL of 0.01
mol/L hydrochloric acid (not more than 0.018%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dextran
40 according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.5 g
of Dextran 40 according to Method 1, and perform the test
(not more than 1.3 ppm).
(5) Nitrogen — Weigh accurately about 2 g of Dextran 40,
previously dried, and perform the test as directed under
Nitrogen Determination <1.08>, where 10 mL of sulfuric acid
is used for decomposition, and 45 mL of a solution of sodi-
um hydroxide (2 in 5) is added: the amount of nitrogen (N:
14.01) is not more than 0.010%.
(6) Reducing substances — Weigh exactly 3.00 g of Dex-
tran 40, previously dried, dissolve in water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh exactly 0.450 g of glucose, previously dried, dissolve in
water to make exactly 500 mL, and use this solution as the
control solution. Pipet 5 mL each of the sample solution and
the control solution, and add water to make exactly 50 mL,
respectively. Pipet 5 mL each of these solutions, add 5 mL of
alkaline copper TS, exactly measured, and heat for 15
minutes in a water bath. After cooling, add 1 mL of a solu-
tion of potassium iodine (1 in 40) and 1.5 mL of dilute sulfur-
ic acid, and titrate <2.50> with 0.005 mol/L sodium thiosul-
fate VS (indicator: 2 mL of starch TS).
The titrant consumed for the sample solution is not less
than that for the control solution.
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C,
6 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Viscosity <2.53> (1) Dextran 40 — Weigh accurately 0.2 to
0.5 g of Dextran 40, previously dried, dissolve in water to
make exactly 100 mL, and use this solution as the sample so-
lution. Perform the test with the sample solution and with
water as directed in Method 1 at 25 °C : the intrinsic viscosity
is between 0.16 and 0.19.
(2) High-molecular fraction — Weigh accurately about 6 g
of Dextran 40, previously dried, dissolve in water to make
exactly 100 mL, and transfer to a flask. Add slowly enough
methanol to get 7% to 10% of the precipitate (usually 80 to
90 mL) at 25 ± 1°C with stirring. Dissolve the precipitate at
35°C in a water bath with occasional shaking, and allow to
stand for more than 15 hours at 25 ± 1°C. Remove the su-
pernatant liquid by decantation, and heat the precipitate of
the lower layer to dryness on a water bath. Dry the residue,
and determine the intrinsic viscosity of the dried substance as
directed in (1): the value is not more than 0.27.
(3) Low-molecular fraction — Weigh accurately about 6 g
of Dextran 40, previously dried, dissolve in water to make
exactly 100 mL, and transfer to a flask. Add slowly enough
methanol to get 90% to 93% of the precipitate (usually 115 to
135 mL) at 25 ± 1°C with stirring, centrifuge at 25 °C, and
evaporate the supernatant liquid to dryness on a water bath.
Dry the residue, and determined the intrinsic viscosity of the
dried substance as directed in (1): the value is not less than
0.09.
Antigenicity Dissolve 10.0 g of Dextran 40 in isotonic sodi-
um chloride solution to make 100 mL, sterilize, and use this
solution as the sample solution. Inject 1.0 mL of the sample
solution on 3 occasions at intervals of 2 days into the
peritoneal cavity of each of 4 well-nourished, healthy guinea
pigs weighing 250 to 300 g. Inject 0.10 mL of horse serum
into the peritoneal cavity of each of 4 guinea pigs of another
group as a control. Inject 0.20 mL of the sample solution in-
travenously to each of 2 guinea pigs of the first group 14 days
after the first intraperitoneal injection and into each of the
remaining 2 guinea pigs 21 days after the injection, and inject
0.20 mL of horse serum intravenously in the same manner
into each guinea pig of the second group. Observe the signs
of respiratory distress, collapse or death of the animals for 30
minutes after each intravenous injection and 24 hours later:
the animals of the first group exhibit no signs mentioned
above.
All the animals of the second group exhibit symptoms of
respiratory distress or collapse and not less than 3 animals are
killed.
Pyrogen <4.04> Dissolve 10.0 g of Dextran 40 in isotonic so-
dium chloride solution to make 100 mL, and perform the
test: this solution meets the requirement.
Assay Weigh accurately about 3 g of Dextran 40, previous-
ly dried, dissolve in water to make exactly 50 mL, and use this
solution as the sample solution. Determine the optical rota-
tion a D with the sample solution as directed under Optical
Rotation Determination <2.49> in a 100-mL cell at 20 ± 1°C.
Amount (mg) of dextran 40 = a D x 253.8
Containers and storage Containers — Tight containers.
Dextran 40 Injection
r + X h7>40;±|t;R
Dextran 40 Injection is an aqueous solution for in-
jection.
It contains not less than 9.5 w/v% and not more
than 10.5 w/v% of dextran 40.
Method of preparation
Dextran 40
Isotonic Sodium Chloride
Solution
10 g
a sufficient quantity
To make
100 mL
Prepare as directed under Injections, with the above in-
gredients.
No preservative is added.
Description Dextran 40 Injection is a clear and colorless
liquid. It is slightly viscous.
Identification (1) Dilute 1 mL of Dextran 40 Injection
with water to 200 mL, and to 1 mL of the diluted solution
add 2 mL of anthrone TS: a blue-green color develops and
JPXV
Official Monographs / Dextran 70 563
turns gradually dark blue-green. Add 1 mL of diluted sulfur-
ic acid (1 in 2) or 1 mL of acetic acid (100) to this solution: the
solution does not change in color.
(2) Dextran 40 Injection responds to the Qualitative Tests
<1.09> for sodium salt and for chloride.
pH <2.54> 4.5 - 7.0
Bacterial endotoxins <4.01> Less than 0.50 EU/mL.
Extractable volume <6.05> It meets the requirement.
Viscosity <2.53> Measure exactly 2 to 5 mL of Dextran 40
Injection, add isotonic sodium, chloride solution to make ex-
actly 100 mL, and use this solution as the sample solution.
Perform the test with the sample solution and with isotonic
sodium chloride solution as directed in Method 1 at 25°C: the
intrinsic viscosity is between 0.16 and 0.19. Calculate the con-
centration of the sample solution (g/100 mL) as directed in
the Assay.
Assay To exactly 30 mL of Dextran 40 Injection add water
to make exactly 50 mL, and use this solution as the sample
solution. Determine the optical rotation a D with the sample
solution as directed under Optical Rotation Determination
<2.49> in a 100-mL cell at 20 ± 1°C.
Amount (mg) of dextran 40 in 100 mL of
Dextran 40 Injection
= a D x 846.0
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Storage — Avoid exposure to undue fluctuations in temper-
ature.
Dextran 70
r + 7. r-7> 70
Dextran 70 is a product obtained by partial decom-
position of polysaccharide, which is produced by fer-
mentation of sucrose with Leuconostoc mesenteroides
van Tieghem (Lactobacillaceae), and the average
molecular mass is about 70,000.
When dried, it contains not less than 98.0% and not
more than 102.0% of dextran 70.
Description Dextran 70 occurs as a white, amorphous pow-
der. It is odorless and tasteless.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves gradually in water.
It is hygroscopic.
Identification To 1 mL of a solution of Dextran 70 (1 in
3000) add 2 mL of anthrone TS: a blue-green color develops
and turns gradually dark blue-green. Then to this solution
add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of acetic
acid (100): the solution does not change in color.
pH <2.54> Dissolve 3.0 g of Dextran 70 in 50 mL of water:
the pH of this solution is between 5.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dextran 70 in 10 mL of water with warming: the solution is
clear and colorless.
(2) Chloride <1.03>— With 2.0 g of Dextran 70, perform
the test. Prepare the control solution with 1.0 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.018%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dextran
70 according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.5 g
of Dextran 70 according to Method 1, and perform the test
(not more than 1.3 ppm).
(5) Nitrogen — Weigh accurately about 2 g of Dextran 70,
previously dried, perform the test as directed under Nitrogen
Determination <1.08>, where 10 mL of sulfuric acid is used
for decomposition, and 45 mL of a solution of sodium
hydroxide (2 in 5) is added: the amount of nitrogen (N:
14.007) is not more than 0.010%.
(6) Reducing substances — Weigh exactly 3.00 g of Dex-
tran 70, previously dried, dissolve in water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh exactly 0.300 g of glucose, previously dried, dissolve in
water to make exactly 500 mL, and use this solution as the
control solution. Pipet 5 mL each of the sample solution and
the control solution, and add water to make exactly 50 mL,
respectively. Pipet 5 mL of these diluted solutions, add exact-
ly 5 mL of alkaline copper TS, and heat for 15 minutes in a
water bath. After cooling, add 1 mL of a solution of potassi-
um iodide (1 in 40) and 1.5 mL of dilute sulfuric acid, and ti-
trate <2.50> with 0.005 mol/L sodium thiosulfate VS (indica-
tor: 2 mL of starch TS).
The titrant consumed for the sample solution is not less
than that for the control solution.
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C,
6 hours).
Residue on ignition <2.44> Not more than 0.10% (1 g).
Viscosity <2.53> (1) Dextran 70 — Weigh accurately 0.2 to
0.5 g of Dextran 70, previously dried, dissolve in water to
make exactly 100 mL, and use this solution as the sample so-
lution. Perform the test with the sample solution and with
water as directed in Method 1 at 25 °C : the intrinsic viscosity
is between 0.21 and 0.26.
(2) High-molecular fraction — Weigh accurately about 6 g
of Dextran 70, previously dried, dissolve in water to make
exactly 100 mL, and transfer to a flask. Add slowly enough
methanol to get 7% to 10% of the precipitate (usually, 75 to
85 mL) at 25 ± 1°C with stirring. Dissolve the precipitate in
a water bath at 35°C with occasional shaking, and allow to
stand for more than 15 hours at 25 ± 1°C. Remove the
supernatant liquid by decantation, and heat the precipitate of
the lower layer on a water bath to dryness. Dry the residue,
and determine the intrinsic viscosity of the dried residue as
directed in (1): the value is not more than 0.35.
(3) Low-molecular fraction — Weigh accurately about 6 g
of Dextran 70, previously dried, dissolve in water to make
exactly 100 mL, and transfer to a flask. Add slowly enough
methanol to get 90% to 93% of the precipitate (usually 110 to
130 mL) at 25 ± 1 C C with stirring, centrifuge at 25°C, and
evaporate the supernatant liquid to dryness on a water bath.
Dry the residue, and determine the intrinsic viscosity of the
dried residue as directed in (1): the value is not less than 0.10.
Antigenicity Dissolve 6.0 g of Dextran 70 in isotonic sodi-
564 Dextran Sulfate Sodium Sulfur 5 / Official Monographs
JP XV
um chloride solution to make 100 mL, sterilize, and use this
solution as the sample solution. Inject 1.0 mL of the sample
solution on 3 occasions at intervals of 2 days into the
peritoneal cavity of each of 4 well-nourished, healthy guinea
pigs weighing 250 to 300 g. Separately, inject 0.10 mL of
horse serum into the peritoneal cavity of each of 4 guinea pigs
of another group as a control. Inject 0.20 mL of the sample
solution intravenously to each of 2 guinea pigs of the first
group 14 days after the first intraperitoneal injection and into
each of the remaining 2 guinea pigs 21 days after the injec-
tion, and inject 0.20 mL of horse serum intravenously in the
same manner into each guinea pigs of the second group. Ob-
serve the signs of respiratory distress, collapse or death of the
animals for 30 minutes after each intravenous injection and
24 hours later: the animals of the first group exhibit not signs.
All the animals of the second group exhibit symptoms of
respiratory distress or collapse and not less than 3 animals are
killed.
Pyrogen <4.04> Dissolve 6.0 g of Dextran 70 in isotonic so-
dium chloride solution to make 100 mL, and perform the
test: this solution meets the requirement.
Assay Weigh accurately about 3 g of Dextran 70, previous-
ly dried, dissolve in water to make exactly 50 mL, and use this
solution as the sample solution. Determine the optical rota-
tion « D as directed under Optical Rotation Determination
<2.49> in a 100-mL cell at 20 ± 1°C.
Amount (mg) of dextran 70 x a D = 253.8
Containers and storage Containers — Tight containers.
Dextran Sulfate Sodium Sulfur 5
Dextran Sulfate Sodium Sulfur 5 is a sodium salt of
sulfate ester obtained by sulfation of partial decompo-
sition products of dextran, which is produced by fer-
mentation of sucrose with Leuconostoc mesenteroides
Van Tieghem (Lactobacillaceae).
Description Dextran Sulfate Sodium Sulfur 5 occurs as a
white to light yellowish white powder. It is odorless, and has
a saline taste.
It is freely soluble in water and practically insoluble in
ethanol (95) and in diethyl ether.
It is hygroscopic.
Identification (1) To 10 mL of a solution of toluidine blue
(1 in 100,000) add 0.05 mL of a solution of Dextran Sulfate
Sodium Sulfur 5 (3 in 50) dropwise: a color of the solution
changes from blue to red-purple.
(2) To 1 mL of a solution of Dextran Sulfate Sodium Sul-
fur 5 (1 in 1500) add 2 mL of anthrone TS: a blue-green color
develops, which turns dark blue-green gradually. Then, add 1
mL of diluted sulfuric acid (1 in 2) or 1 mL of acetic acid
(100) to this solution: the solution remains dark blue-green.
(3) A solution of Dextran Sulfate Sodium Sulfur 5 (1 in
100) responds to the Qualitative Tests <1.09> (1) for sodium
salt.
Optical rotation <2.49> [ a £°: + 135.0 - + 155.0° (calculat-
ed on the dried basis, 1.5 g, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Dextran Sulfate Sodium Sulfur
5 in 20 mL of water: the pH of this solution is between 5.5
and 7.5.
Purity (1) Clarity of solution — Dissolve 2.5 g of Dextran
Sulfate Sodium Sulfur 5 in 50 mL of water: the solution is
clear. And, determine the absorbance of the solution at 420
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>: not more than 0.090.
(2) Chloride <1.03>— Perform the test with 0.10 g of Dex-
tran Sulfate Sodium Sulfur 5. Prepare the control solution
with 0.30 mL of 0.01 mol/L hydrochloric acid (not more
than 0.106%).
(3) Sulfate <1.14>— Dissolve 0.10 g of Dextran Sulfate
Sodium Sulfur 5 in 6 mL of water, add 0.6 mL of barium
chloride TS, and heat in a water bath for 4 minutes. After
cooling, add 1 mL of dilute hydrochloric acid and water to
make 50 mL, allow to stand for 10 minutes, and observe: the
turbidity of the solution is not more intense than that of the
control solution. Prepare the control solution as follows: to
0.50 mL of 0.005 mol/L sulfuric acid add 6 mL of water, and
proceed in the same manner (not more than 0.240%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Dextran
Sulfate Sodium Sulfur 5 according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Dextran Sulfate Sodium Sulfur 5 according to Method 3,
and perform the test (not more than 2 ppm).
Sulfur content Weigh accurately about 1.0 g of Dextran
Sulfate Sodium Sulfur 5, dissolve in 5 mL of water, add 1.5
mL of hydrochloric acid, and heat in a water bath for 1 hour.
After cooling, add water to make exactly 100 mL, and use
this solution as the sample solution. To exactly 10 mL of the
sample solution add exactly 20 mL of 0.02 mol/L barium
chloride VS, add 5 mL of methanol, and heat in a water bath
for 30 minutes. After cooling, neutralize with sodium
hydroxide TS, and add 70 mL of water, 10 mL of a solution
of zinc disodium ethylenediamine tetraacetate tetrahydrate (1
in 20), 3 mL of ammonium chloride TS and 7 mL of strong
ammonium water, and titrate <2.50> with 0.02 mol/L disodi-
um dihydrogen ethylenediamine tetraacetate VS until the
color of the solution changes from red to light blue (indica-
tor: 5 drops of eriochrome black T TS). Perform a blank de-
termination. Amount of sulfur (S: 32.07), calculated on the
dried basis, is between 3.0 and 6.0%.
Each mL of 0.02 mol/L barium chloride VS
= 0.6413 mg of S
Loss on drying <2.41> Not more than 10.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 4 hours).
Viscosity <2.53> Weigh accurately about 1.5 g of Dextran
Sulfate Sodium Sulfur 5, calculated on the dried basis, dis-
solve in a solution of sodium chloride (29 in 500) to make ex-
actly 100 mL, and use this solution as the sample solution.
Perform the test with the sample solution and a solution of
sodium chloride (29 in 500) at 25 ± 0.02°C as directed: the
intrinsic viscosity is between 0.030 and 0.040.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Dextrin 565
Dextran Sulfate Sodium Sulfur 18
x+7. h7>6S^iXxJU^-h U^a 4*^n
Dextran Sulfate Sodium Sulfur 18 is a sodium salt of
sulfate ester obtained by sulfation of partial decompo-
sition products of dextran, which is produced by fer-
mentation of sucrose with Leuconostoc mesenteroides
Van Tieghem (Lactobacillaceae).
Description Dextran Sulfate Sodium Sulfur 18 occurs as a
white to light yellowish white powder. It is odorless, and has
a saline taste.
It is freely soluble in water and practically insoluble in
ethanol (95) and in diethyl ether.
It is hygroscopic.
Identification (1) To 10 mL of a solution of toluidine blue
(1 in 100,000) add 0.05 mL of a solution of Dextran Sulfate
Sodium Sulfur 18 (3 in 50) dropwise: a color of the solution
changes from blue to red-purple.
(2) To 1 mL of a solution of Dextran Sulfate Sodium Sul-
fur 18 (1 in 1500) add 2 mL of anthrone TS: a blue-green
color develops, which turns dark blue-green gradually. Then,
add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of acetic
acid (100) to this solution: the solution remains dark blue-
green.
(3) A solution of Dextran Sulfate Sodium Sulfur 18 (1 in
100) responds to the Qualitative Tests <1.09> (1) for sodium
salt.
Optical rotation <2.49> [a]™: +90.0- +110.0° (calculated
on the dried basis, 1.5 g, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Dextran Sulfate Sodium Sulfur
18 in 20 mL of water: the pH of this solution is between 5.5
and 7.5.
Purity (1) Chloride <1.03>— Perform the test with 0.10 g
of Dextran Sulfate Sodium Sulfer 18. Prepare the control so-
lution with 0.30 mL of 0.01 mol/L hydrochloric acid (not
more than 0.106%).
(2) Sulfate <1.14>— Dissolve 0.10 g of Dextran Sulfate
Sodium Sulfur 18 in 6 mL of water, add 0.6 mL of barium
chloride TS, and heat in a water bath for 4 minutes. After
cooling, add 1 mL of dilute hydrochloric acid and water to
make 50 mL, allow to stand for 10 minutes, and observe: the
turbidity of the solution is not more intense than that of the
control solution. Prepare the control solution as follows: to
1.0 mL of 0.005 mol/L sulfuric acid add 6 mL of water, and
proceed in the same manner (not more than 0.480%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dextran
Sulfate Sodium Sulfur 18 according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Dextran Sulfate Sodium Sulfur 18 according to Method 3,
and perform the test (not more than 2 ppm).
Sulfur content Weigh accurately about 0.5 g of Dextran
Sulfate Sodium Sulfur 18, dissolve in 5 mL of water, add 1.5
mL of hydrochloric acid, and heat in a water bath for 1 hour.
After cooling, add water to make exactly 100 mL, and use
this solution as the sample solution. To exactly 10 mL of the
sample solution add exactly 20 mL of 0.02 mol/L barium
chloride VS, add 5 mL of methanol, and heat in a water bath
for 30 minutes. After cooling, neutralize with sodium
hydroxide TS, and add 70 mL of water, 10 mL of a solution
of zinc disodium ethylenediamine tetraacetate tetrahydrate (1
in 20), 3 mL of ammonium chloride TS and 7 mL of strong
ammonium water, and titrate <2.50> with 0.02 mol/L disodi-
um dihydrogen ethylenediamine tetraacetate VS until the
color of the solution changes from red to light blue (indica-
tor: 5 drops of eriochrome black T TS). Perform a blank de-
termination. Amount of sulfur (S: 32.07), calculated on the
dried basis, is between 15.0 and 20.0%.
Each mL of 0.02 mol/L barium chloride VS
= 0.6413 mg of S
Loss on drying <2.4I> Not more than 10.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 4 hours).
Viscosity <2.53> Weigh accurately about 1.5 g of Dextran
Sulfate Sodium Sulfur 18, calculated on the dried basis, dis-
solve in a solution of sodium chloride (29 in 500) to make ex-
actly 100 mL, and use this solution as the sample solution.
Perform the test with the sample solution and a solution of
sodium chloride (29 in 500) at 25 ± 0.02°C as directed: the
intrinsic viscosity is between 0.020 and 0.032.
Containers and storage Containers — Tight containers.
Dextrin
Description Dextrin occurs as a white or light yellow, amor-
phous powder or granules. It has a slight, characteristic odor
and a sweet taste. It does not irritate the tongue. Dextrin is
freely soluble in boiling water, soluble in water, and practi-
cally insoluble in ethanol (95) and in diethyl ether.
Identification To 0.1 g of Dextrin add 100 mL of water,
shake, and filter if necessary. To 5 mL of the filtrate add 1
drop of iodine TS: a light red-brown or light red-purple color
develops.
Purity (1) Clarity and color of solution — Take 2.0 g of
Dextrin in a Nessler tube, add 40 mL of water, dissolve by
heating, cool, and add water to make 50 mL: the solution is
colorless or light yellow. It is clear, and even if turbid, the
turbidity is not more than that of the following control solu-
tion.
Control solution: To 1.0 mL of 0.005 mol/L sulfuric acid
VS add 1 mL of dilute hydrochloric acid, 46 mL of water and
2 mL of barium chloride TS, allow to stand for 10 minutes,
and shake before use.
(2) Acidity — To 1.0 g of Dextrin add 5 mL of water, dis-
solve by heating, cool, and add 1 drop of phenolphthalein TS
and 0.50 mL of 0.1 mol/L sodium hydroxide VS: a red color
develops.
(3) Chloride <1.03>— To 2.0 g of Dextrin add 80 mL of
water, dissolve by heating, cool, add water to make 100 mL,
and filter. Take 40 mL of the filtrate, and add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more
566
Dextromethorphan Hydrobromide Hydrate / Official Monographs
JP XV
than 0.013%).
(4) Sulfate <1.14>— To 45 mL of the filtrate obtained in
(3) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 0.35 mL of 0.005
mol/L sulfuric acid VS (not more than 0.019%).
(5) Oxalate — To 1.0 g of Dextrin add 20 mL of water,
dissolve by heating, cool, add 1 mL of acetic acid (31), and
filter. To 5 mL of the filtrate add 5 drops of calcium chloride
TS: no turbidity is produced immediately.
(6) Calcium — To a 5-mL portion of the filtrate obtained
in (5) add 5 drops of ammonium oxalate TS: no turbidity is
immediately produced.
(7) Heavy metals <1.07> — Proceed with 0.5 g of Dextrin
according to Method 2, and perform the test. Prepare the
control solution with 2.5 mL of Standard Lead Solution (not
more than 50 ppm).
Loss on drying <2.41> Not more than 10% (0.5 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.5% (0.5 g).
Containers and storage Containers — Well-closed contain-
ers.
Dextromethorphan Hydrobromide
Hydrate
■ HBr * HjO
H3C-
C 18 H 25 NO.HBr.H 2 0: 370.32
(9 S, 1 3 S, 1 4 S)-3-Methoxy- 1 7-methylmorphinan
monohydrobromide monohydrate [6700-34-1]
Dextromethorphan Hydrobromide Hydrate contains
not less than 98.0% of dextromethorphan hydro-
bromide (C 18 H 25 NO.HBr: 352.31), calculated on the
anhydrous basis.
Description Dextromethorphan Hydrobromide Hydrate
occurs as white crystals or crystalline powder.
It is very soluble in methanol, freely soluble in ethanol (95)
and in acetic acid (100), and sparingly soluble in water.
Melting point: about 126°C (Insert the capillary tube into
the bath preheated to 116°C, and continue the heating so that
the temperature rises at a rate of about 3°C per minute.)
Identification (1) Determine the absorption spectrum of a
solution of Dextromethorphan Hydrobromide Hydrate (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Dex-
tromethorphan Hydrobromide Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) To 50 mL of a solution of Dextromethorphan
Hydrobromide Hydrate (1 in 100) add 2 drops of phenol-
phalein TS and sodium hydroxide TS until a red color de-
velops. Add 50 mL of chloroform, shake, and add 5 mL of
dilute nitric acid to 40 mL of the water layer. This solution
responds to the Qualitative Tests <1.09> for bromide.
Optical rotation <2.49> [a]™: + 26 - + 30° (0.34 g, calculat-
ed on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Dextromethorphan Hydro-
bromide Hydrate in 100 mL of water: the pH of this solution
is between 5.2 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Dextromethorphan Hydrobromide Hydrate in 20 mL of
water: the solution is clear and colorless.
(2) A r ,A r -dimethylaniline — To 0.50 g of Dextromethor-
phan Hydrobromide Hydrate add 20 mL of water, and dis-
solve by heating on a water bath. After cooling, add 2 mL of
dilute acetic acid, 1 mL of sodium nitrite TS and water to
make 25 mL: the solution has no more color than the follow-
ing control solution.
Control solution: Dissolve 0.10 g of 7V.,/V-dimethylaniline
in 400 mL of water by warming on a water bath, cool, and
add water to make 500 mL. Pipet 5 mL of this solution, and
add water to make 200 mL. To 1.0 mL of this solution add
2 mL of dilute acetic acid, 1 mL of sodium nitrite TS and
water to make 25 mL.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dex-
tromethorphan Hydrobromide Hydrate according to Method
4, and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(4) Phenolic compounds — Dissolve 5 mg of Dex-
tromethorphan Hydrobromide Hydrate in 1 drop of dilute
hydrochloric acid and 1 mL of water, add 2 drops of iron
(III) chloride TS and 2 drops of potassium hexacyanoferrate
(III) TS, shake, and allow to stand for 15 minutes: no blue-
green color develops.
(5) Related substances — Dissolve 0.25 g of Dex-
tromethorphan Hydrobromide Hydrate in 10 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add methanol to make exactly
200 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 juL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of toluene, ethyl acetate, methanol, dichloromethane and
13.5 mol/L ammonia TS (55:20:13:10:2) to a distance of
about 15 cm, and air-dry the plate. Spray evenly bismuth
potassium iodide TS on the plate, and then spray evenly
hydrogen peroxide TS on the plate: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Water <2.48> 4.0 - 5.5% (0.2 g, back titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Dextromethorphan
Hydrobromide Hydrate, dissolve in 10 mL of acetic acid
(100) and add 40 mL of acetic anhydride. Titrate <2.50> with
JPXV
Official Monographs / Diazepam 567
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 35.23 mg of C 18 H 25 NO.HBr
Containers and storage Containers — Well-closed contain-
ers.
Diastase
Diastase is an enzyme drug mainly prepared from
malt.
It has amylolytic activity.
It contains not less than 440 starch saccharifying ac-
tivity units per g.
It is usually diluted with suitable diluents.
Description Diastase occurs as a light yellow to light brown
powder.
It is hygroscopic.
Purity Rancidity — Diastase has no unpleasant or rancid
odor, and has no unpleasant or rancid taste.
Loss on drying <2.41> Not more than 4.0% (1 g, 105°C,
5 hours).
Assay (i) Substrate solution — Use potato starch TS for
amylolytic activity test.
(ii) Sample solution — Weigh accurately about 0.1 g of
Diastase, and dissolve in water to make exactly 100 mL.
(iii) Procedure — Proceed as directed in (i) Measurement
of starch saccharifying activity of (1) Assay for starch diges-
tive activity under Digestion Test <4.03>.
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
Diastase and Sodium Bicarbonate
Powder
Method of preparation
Diastase
Sodium Bicarbonate
Precipitated Calcium Carbonate
Magnesium Oxide
200 g
300 g
400 g
100 g
To make 1000 g
Prepare before use as directed under Powders, with the
above ingredients.
Description Diastase and Sodium Bicarbonate Powder oc-
curs as a light yellow powder. It has a characteristic, salty
taste.
Containers and storage Containers — Well-closed contain-
ers.
Compound Diastase and Sodium
Bicarbonate Powder
Method of preparation
Diastase
Sodium Bicarbonate
Magnesium Oxide
Powdered Gentian
200 g
600 g
150 g
50 g
To make 1000 g
Prepare before use as directed under Powders, with the
above ingredients.
Description Compound Diastase and Sodium Bicarbonate
Powder occurs as a slightly brownish, light yellow powder. It
has a characteristic odor and a bitter taste.
Containers and storage Containers — Well-closed contain-
ers.
Diazepam
v7-t£n°A
C 16 H 13 C1N 2 0: 284.74
7-Chloro- 1 -methyl-5-phenyl- 1 , 3-dihydro-2f/-l ,4-
benzodiazepin-2-one [439-14-5]
Diazepam, when dried, contains not less than 98.0%
of C 16 H 13 C1N 2 0.
Description Diazepam occurs as a white to light yellow,
crystalline powder. It is odorless, and has a slightly bitter
taste.
It is freely soluble in acetone, soluble in acetic anhydride
and in ethanol (95), sparingly soluble in diethyl ether, slightly
soluble in ethanol (99.5), and practically insoluble in water.
Identification (1) Dissolve 0.01 g of Diazepam in 3 mL of
sulfuric acid, and observe under ultraviolet light (main
wavelength: 365 nm): the solution shows a yellow-green
fluorescence.
(2) Dissolve 2 mg of Diazepam in 200 mL of a solution of
sulfuric acid in ethanol (99.5) (3 in 1000). Determine the
absorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Perform the test with Diazepam as directed under
Flame Coloration Test <1.04> (2): a blue to blue-green color
appears.
568
Dibasic Sodium Phosphate Hydrate / Official Monographs
JP XV
Absorbance <2.24> E\f m (285 nm): 425 - 445 [after drying,
2 mg, a solution of sulfuric acid in ethanol (99.5) (3 in 1000),
200 mL].
Melting point <2.60> 130 - 134°C
Purity (1) Clarity of solution — Dissolve 0.10 g of Diazep-
am in 20 mL of ethanol (95): the solution is clear.
(2) Chloride <1.03>— To 1.0 g of Diazepam add 50 mL of
water, allow to stand for 1 hour, with occasional shaking,
and filter. To 25 mL of the filtrate add 6 mL of dilute nitric
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution with
0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.014%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Diazep-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Related substances — Dissolve 1 .0 g of Diazepam in 10
mL of acetone, and use this solution as the sample solution.
Pipept 1 mL of the sample solution, and add acetone to make
exactly 100 mL. Pipet 1 mL of this solution, add acetone to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of ethyl acetate and hexane
(1:1) to a distance of about 12 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Diazepam, previ-
ously dried, dissolve in 60 mL of acetic anhydride, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.48 mg of C 16 H 13 C1N 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dibasic Sodium Phosphate Hydrate
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It effloresces in warm, dry air.
Identification A solution of Dibasic Sodium Phosphate Hy-
drate (1 in 10) responds to the Qualitative Tests <I.09> (1) and
(2) for sodium salt, and the Qualitative Tests <1.09> for phos-
phate.
pH <2.54> Dissolve 1 .0 g of Dibasic Sodium Phosphate Hy-
drate in 50 mL of water: the pH of this solution is between
9.0 and 9.4.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dibasic Sodium Phosphate Hydrate in 20 mL of water: the
solution is clear and colorless.
(2) Chloride <1.03>— Dissolve 1.0 g of Dibasic Sodium
Phosphate Hydrate in 7 mL of dilute nitric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.40 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.014%).
(3) Sulfate <1.14>— Dissolve 0.5 g of Dibasic Sodium
Phosphate Hydrate in 2 mL of dilute hydrochloric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 0.40 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.038%).
(4) Carbonate — To 2.0 g of Dibasic Sodium Phosphate
Hydrate add 5 mL of water, boil, and add 2 mL of
hydrochloric acid after cooling: the solution does not effer-
vesce.
(5) Heavy metals <1.07> — Dissolve 2.0 g of Dibasic Sodi-
um Phosphate Hydrate in 4 mL of acetic acid (31) and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 2.0 mL of
Standard Lead Solution by adding 2 mL of dilute acetic acid
and water to make 50 mL (not more than 10 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Dibasic Sodium Phosphate Hydrate according to Method
1, and perform the test (not more than 2 ppm).
Loss on drying <2.4I> 57.0-61.0% (10 g, at 40°C for
3 hours at first and then at 105°C for 5 hours).
Assay Dissolve about 3 g of Dibasic Sodium Phosphate Hy-
drate, previously dried and accurately weighed, in 50 mL of
water. Titrate <2.50> it with 0.5 mol/L sulfuric acid VS at 15°
C until the green color of the solution changes to dark-green-
ish red-purple (indicator: 3 to 4 drops of methyl orange-
xylenecyanol FF TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 142.0 mg of Na 2 HP0 4
Containers and storage Containers — Tight containers.
Na 2 HP0 4 .12H 2 0: 358.14
Dibasic Sodium Phosphate Hydrate, when dried,
contains not less than 98.0% of disodium hydrogen
phosphate (Na 2 HP0 4 : 141.96)
Description Dibasic Sodium Phosphate Hydrate occurs as
colorless or white crystals. It is odorless.
JPXV
Official Monographs / Dibucaine Hydrochloride
569
Dibekacin Sulfate
rl a N
• lHjSOi
C 18 H 37 N 5 8 .a:H 2 S04
3-Amino-3-deoxy-a-D-glucopyranosyl-(l->6)-[2,6-
diamino-2,3,4,6-tetradeoxy-a-D-ery?/!ro-hexopyranosyl-
(l->4)]-2-deoxy-D-streptamine sulfate [58580-55-5]
Dibekacin Sulfate is the sulfate of a derivative of be-
kanamycin.
It contains not less than 640 fig (potency) and not
more than 740 fig (potency) per mg, calculated on the
dried basis. The potency of Dibekacin Sulfate is ex-
pressed as mass (potency) of dibekacin (C 18 H 37 N 5 8 :
451.52).
Description Dibekacin Sulfate occurs as a white to yellow-
ish white powder.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 20 mg each of Dibekacin Sul-
fate and Dibekacin Sulfate Reference Standard in 1 mL of
water, and use these solutions as the sample solution and the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of ammonia solution (28) and methanol (1:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly 0.2% ninhydrin-water saturated 1-butanol TS, and
heat at 100°C for 10 minutes: the principal spots obtained
from the sample solution and standard solution show a pur-
ple-brown color and the same Rf value.
(2) To 5 mL of a solution of Dibekacin Sulfate (1 in 50)
add 1 drop of barium chloride TS: a white precipitate is pro-
duced.
Optical rotation <2.49> [a]™: + 96 - + 106° (0.25 g calculated
on the dried basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Dibekacin Sulfate in 20 mL of water is between 6.0 and
8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dibekacin Sulfate in 10 mL of water: the solution is clear and
colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Dibeka-
cin Sulfate according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
Loss on drying <2.41> Not more than 5.0% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer having pH 6.5 to 6.6 after sterilization.
(iii) Standard solutions - Weigh accurately an amount of
Dibekacin Sulfate Reference Standard, previously dried,
equivalent to about 20 mg (potency), dissolve in diluted phos-
phate buffer solution, pH 6.0 (1 in 2) to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5 to 15 °C and use within 30 days.
Take exactly a suitable amount of the standard stock solution
before use, add 0.1 mol/L phosphate buffer solution, pH 8.0
to make solutions so that each mL contains 20 fig (potency)
and 5 fig (potency), and use these solutions as the high con-
centration standard solution and low concentration standard
solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Dibekacin Sulfate, equivalent to about 20 mg (potency), and
dissolve in water to make exactly 50 mL. Take exactly a suita-
ble amount of this solution, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
20 fig (potency) and 5 fig (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Dibucaine Hydrochloride
Cinchocaine Hydrochloride
C 20 H 29 N 3 O 2 .HCl: 379.92
2-Butyloxy-A f -(2-diethylaminoethyl)-4-
quinolinecarboxamide monohydrochloride [61-12-1]
Dibucaine Hydrochloride, when dried, contains not
less than 98.0% of C 20 H 29 N 3 O 2 .HCl.
Description Dibucaine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is very soluble in water, in ethanol (95) and in acetic acid
(100), freely soluble in acetic anhydride, and practically in-
soluble in diethyl ether.
It is hygroscopic.
570
Diclofenac Sodium / Official Monographs
JP XV
Identification (1) Determine the absorption spectrum of a
solution of Dibucaine Hydrochloride in 1 mol/L hydrochlor-
ic acid TS (1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Dibucaine Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Dibucaine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 95-100°C Charge Dibucaine
Hydrochloride into a capillary tube for melting point deter-
mination, and dry in vacuum over phosphorus (V) oxide at
80°C for 5 hours. Seal immediately the open end of the tube,
and determine the melting point.
pH <2.54> Dissolve 1.0 g of Dibucaine Hydrochloride in 50
mL of water: the pH of this solution is between 5.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dibucaine Hydrochloride in 20 mL of water: the solution is
clear and colorless. Determine the absorbance of this solution
at 430 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using water as the blank: it is not more than
0.03.
(2) Sulfate <1.14>— Perform the test with 0.30 g of
Dibucaine Hydrochloride. Prepare the control solution with
0.35 mL of 0.005 mol/L sulfuric acid VS (not more than
0.056%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Dibucaine Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Related substances — Dissolve 0.20 g of Dibucaine
Hydrochloride in 5 mL of ethanol (95), and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add ethanol (95) to make exactly 20 mL, then pipet 2 mL of
this solution, add ethanol (95) to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 iXL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, water and acetic acid (100) (3:1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
phosphorus (V) oxide, 80°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Dibucaine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.00 mg of C 20 H 29 N 3 O 2 .HCl
Containers and storage Containers — Tight containers.
Diclofenac Sodium
oc
CO^Na
NH
C 14 H 10 Cl 2 NNaO 2 : 318.13
Monosodium 2-(2,6-dichlorophenylamino)phenylacetate
[15307-79-6]
Diclofenac Sodium, when dried, contains not less
than 98.5% of C 14 H 10 Cl 2 NNaO 2 .
Description Diclofenac Sodium occurs as white to pale yel-
lowish white crystals or crystalline powder.
It is freely soluble in methanol and in ethanol (95), sparing-
ly soluble in water and in acetic acid (100), and practically in-
soluble in diethyl ether.
It is hygroscopic.
Identification (1) To 1 mL of a solution of Diclofenac So-
dium in methanol (1 in 250) add 1 mL of nitric acid: a dark
red color develops.
(2) Perform the test with 5 mg of Diclofenac Sodium as
directed under Flame Coloration Test <1.04> (2): a light green
color appears.
(3) Determine the infrared absorption spectrum of
Diclofenac Sodium, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Diclofenac Sodium (1 in 100) responds
to the Qualitative Tests <1.09> for sodium salt.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Diclofenac Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Diclofenac Sodium according to Method 3, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.05 g of Diclofenac
Sodium in 50 mL of the mobile phase, and use this solution
as the sample solution. Pipet 2 mL of the sample solution,
and add the mobile phase to make exactly 50 mL. Pipet 5 mL
of this solution, add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /xh each of these solutions as directed
under Liquid Chromatography <2.01>. Determine each peak
area of these solutions by the automatic integration method:
the area of each peak other than the peak of diclofenac from
the sample solution is not larger than the peak area of the
standard solution.
JPXV
Official Monographs / Diclofenamide 571
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of methanol and diluted acetic
acid (100) (3 in 2500) (4:3).
Flow rate: Adjust the flow rate so that the retention time of
diclofenac is about 20 minutes.
Time span of measurement: About twice as long as the
retention time of diclofenac beginning after the solvent peak.
System suitability —
System performance: Dissolve 35 mg of ethyl parahydrox-
ybenzoate and 0.05 g of propyl parahydroxybenzoate in 100
mL of the mobile phase. To 1 mL of this solution add the
mobile phase to make 50 mL. When the procedure is run with
20 /xL of this solution under the above operating conditions,
ethyl parahydroxybenzoate and propyl parahydroxybenzoate
are eluted in this order with the resolution between these
peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of diclofenac is not more than 2.0%.
Loss on drying <2.41>
3 hours).
Not more than 0.5% (1 g, 105°C,
Assay Weigh accurately about 0.5 g of Diclofenac Sodium,
previously dried, dissolve with 40 mL of water in a separator,
add 2 mL of dilute hydrochloric acid, and extract the
precipitate formed with 50 mL of chloroform. Extract again
with two 20-mL portions of chloroform, and filter the extract
each time through a pledget of absorbent cotton moistened
with chloroform. Wash the tip of the separator and the ab-
sorbent cotton with 15 mL of chloroform, combine the wash-
ing with the extracts, add 10 mL of a solution of 1 mol/L
hydrochloric acid TS in ethanol (99.5) (1 in 100), and titrate
<2.50> with 0.1 mol/L potassium hydroxide-ethanol VS from
the first equivalent point to the second equivalent point
(potentiometric titration).
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 31.81 mg of C 14 H 10 Cl 2 NNaO 2
Containers and storage Containers — Tight containers.
Diclofenamide
Dichlorphenamide
* Nhfc
C 6 H 6 C1 2 N 2 4 S 2 : 305.16
4,5-Dichlorobenzene-l,3-disulfonamide
[120-97-8]
Diclofenamide, when dried, contains not less than
98.0% of C 6 H 6 C1 2 N 2 4 S 2 .
Description Diclofenamide occurs as a white, crystalline
powder.
It is very soluble in A^A^dimethylformamide, soluble in
ethanol (95), and very slightly soluble in water.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 0.01 g of Diclofenamide in 100
mL of 0.01 mol/L sodium hydroxide TS. To 10 mL of the
solution add 0.1 mL of hydrochloric acid. Determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Diclofenamide Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Diclofenamide, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Diclofenamide Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
Melting point <2.60> 237 - 240 C C
Purity (1) Chloride < 1.03 > —Dissolve 0.10 g of
Diclofenamide in 10 mL of A^TV-dimethylformamide, and
add 6 mL of dilute nitric acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 0.45 mL of 0.01 mol/L
hydrochloric acid VS add 10 mL of A^A'-dimethylfor-
mamide, 6 mL of dilute nitric acid and water to make 50 mL
(not more than 0.160%).
(2) Selenium — To 0.10 g of Diclofenamide add 0.5 mL of
a mixture of perchloric acid and sulfuric acid (1:1) and 2 mL
of nitric acid, and heat on a water bath until no more brown
gas evolves and the solution becomes to be a light yellow clear
solution. After cooling, add 4 mL of nitric acid to this solu-
tion, then add water to make exactly 50 mL, and use this so-
lution as the sample solution. Separately, pipet 3 mL of Stan-
dard Selenium Solution, add 0.5 mL of a mixture of per-
chloric acid and sulfuric acid (1:1) and 6 mL of nitric acid,
then add water to make exactly 50 mL, and use this solution
as the standard solution. Perform the test with the sample so-
lution and standard solution as directed under Atomic Ab-
572 Diclofenamide Tablets / Official Monographs
JP XV
sorption Spectrophotometry <2.23> according to the follow-
ing conditions, and determine constant absorbances, A T and
A s , obtained on a recorder after rapid increasing of the ab-
sorption: A-y is smaller than A s (not more than 30 ppm).
Perform the test by using a hydride generating system and
a thermal absorption cell.
Lamp: An selenium hollow cathode lamp
Wavelength: 196.0 nm
Temperature of sample atomizer: When an electric furnace
is used, about 1000°C.
Carrier gas: Nitrogen or Argon
(3) Heavy metals <1.07> — Proceed with 2.0 g of
Diclofenamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Related substances — Dissolve 0.10 g of Diclofena-
mide in 50 mL of the mobile phase, and use this solution as
the sample solution. Pipet 2 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peak of diclofenamide from the sample solu-
tion is not larger than the peak area of diclofenamide from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 5 times as long as the
retention time of diclofenamide.
System suitability —
Test for required detection: To exactly 5 mL of the stand-
ard solution add the mobile phase to make exactly 100 mL.
Confirm that the peak area of diclofenamide obtained from
10 /xL of this solution is equivalent to 3.5 to 6.5% of that of
diclofenamide obtained from 10 /xL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
diclofenamide is not more than 1.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 100°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Diclofena-
mide and Diclofenamide Reference Standard, previously
dried, and dissolve each in 30 mL of the mobile phase. To
each add exactly 10 mL of the internal standard solution and
the mobile phase to make 50 mL, and use these solutions as
the sample solution and the standard solution. Perform the
test with 10 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of diclofenamide to that of the
internal standard, respectively.
Amount (mg) of C 6 H 6 C1 2 N 2 4 S 2 = W s x (Q T /Q S )
W s : Amount (mg) of Diclofenamide Reference Standard
Internal standard solution — A solution of butyl parahydroxy
benzoate in the mobile phase (3 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of sodium phosphate TS and
acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
diclofenamide is about 7 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, diclofenamide and the internal standard are elut-
ed in this order with the resolution between these peaks being
not less than 9.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of diclofenamide to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Diclofenamide Tablets
Dichlorphenamide Tablets
Diclofenamide Tablets contain not less than 92%
and not more than 108% of the labeled amount of
diclofenamide (C 6 H 6 C1 2 N 2 4 S 2 : 305.16).
Method of preparation Prepare as directed under Tablets,
with Diclofenamide.
Identification To a quantity of powdered Diclofenamide
Tablets, equivalent to 0.2 g of Diclofenamide according to
the labeled amount, add 20 mL of methanol, shake, and
filter. Evaporate the filtrate on a water bath to dryness, and
dissolve 0.01 g of the residue in 100 mL of 0.01 mol/L sodi-
um hydroxide TS. To 10 mL of this solution add 0.1 mL of
hydrochloric acid TS, and determine the absorption spectrum
of this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 284 nm
and 288 nm, and between 293 nm and 297 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Diclofenamide Tablets at
50 revolutions per minute according to the Paddle method,
using 900 mL of water as the test solution. Take 20 mL or
more of the dissolved solution 60 minutes after starting the
test, and filter through a membrane filter with a pore size not
exceeding 0.8 fim. Discard the first 10 mL of the filtrate, and
use the subsequent as the sample solution. Separately, weigh
accurately about 55 mg of Diclofenamide Reference Stan-
JPXV
Official Monographs / Dicloxacillin Sodium Hydrate
573
dard, previously dried in vacuum at a pressure not exceeding
0.67 kPa at 100°C for 5 hours, dissolve in 10 mL of
methanol, and add water to make exactly 100 mL. Pipet 10
mL of this solution, add water to make exactly 100 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A T and A s , of the sample solution and the stan-
dard solution at 285 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate of
Diclofenamide Tablets in 60 minutes is not less than 70%.
Dissolution rate (%) with respect to
the labeled amount of diclofenamide (C 6 H 6 Cl2N 2 4 S2)
= W s x (A T /A S ) x (1/C) x 90
W s : Amount (mg) of Diclofenamide Reference Standard.
C: Labeled amount (mg) of diclofenamide
(C 6 H 6 C1 2 N 2 4 S 2 ) in 1 tablet.
Assay Weigh accurately, and powder not less than 20
tablets of Diclofenamide Tablets. Weigh accurately a portion
of the powder, equivalent to about 50 mg of diclofenamide
(C 6 H 6 C1 2 N 2 4 S 2 ), add exactly 25 mL of the mobile phase,
shake for 15 minutes, and centrifuge. Pipet 10 mL of the su-
pernatant liquid, add exactly 4 mL of the internal standard
solution and the mobile phase to make 20 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 50 mg of Diclofenamide Reference Standard, previ-
ously dried at 100°C in vacuum at a pressure not exceeding
0.67 kPa for 5 hours, dissolve in 30 mL of the mobile phase,
add exactly 10 mL of the internal standard solution and the
mobile phase to make 50 mL, and use this solution as the
standard solution. Proceed as directed in the Assay under
Diclofenamide.
Amount (mg) of diclofenamide (C 6 H 6 C1 2 N 2 4 S 2 )
= W s x (Qj/Q s )
W s : Amount (mg) of Diclofenamide Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in the mobile phase (3 in 5000).
Containers and storage Containers — Well-closed contain-
ers.
Dicloxacillin Sodium Hydrate
y <7 n *+*--> U >i- r- U O A7]ofnti
H
CI
.,o. CH o \S~
n V 3 ^r-N-"\ j
II H H
,COjNa
■CH 3
CH 3
•H;>0
C 19 H 16 Cl 2 N 3 Na0 5 S.H 2 0: 510.32
Monosodium (25 , ,5i?,6i?)-6-{[3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-
thia-l-azabicyclo[3.2.0]heptane-2-carboxylate
monohydrate [13412-64-1]
Dicloxacillin Sodium Hydrate contains not less than
910 fig (potency) and not more than 1020 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Dicloxacillin Sodium Hydrate is expressed as
mass (potency) of dicroxacillin (C^HnCy^OsS:
470.33).
Description Dicloxacillin Sodium Hydrate occurs as a white
to light yellowish white crystalline powder.
It is freely soluble in water and in methanol, and soluble in
ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Dicloxacillin Sodium Hydrate (1 in 2500) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Dicloxacillin Sodium Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of
Dicloxacillin Sodium Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Dicloxacillin Sodium Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) Dicloxacillin Sodium Hydrate responds to the Quan-
titative Tests <1.09> (1) for sodium salt.
Water <2.48> Not less than 3.0% and not more than 4.5%
(0.1 g, volumetric titration, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 6.5 to
6.6 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Dicloxacillin Sodium Reference Standard equivalent to about
50 mg (potency), dissolve in phosphate buffer solution, pH
6.0 to make exactly 50 mL, and use this solution as the stan-
dard stock solution. Keep the standard stock solution at 5°C
or below and use within 24 hours. Take exactly a suitable
amount of the standard stock solution before use, add phos-
phate buffer solution, pH 6.0 to make solutions so that each
mL contains 10 fig (potency) and 2.5 fig (potency), and use
these solutions as the high concentration standard solution
and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Dicloxacillin Sodium Hydrate equivalent to about 50 mg
(potency), dissolve in phosphate buffer solution, pH 6.0 to
make exactly 50 mL. Take exactly a suitable amount of the
solution, add phosphate buffer solution, pH 6.0 to make so-
lutions so that each mL contains 10 fig (potency) and 2.5 fig
(potency), and use these solutions as the high concentration
sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
574 Diethylcarbamazine Citrate / Official Monographs
JP XV
Diethylcarbamazine Citrate
H,C
CH 3
CH,
HO CO;H
C 10 H 21 N 3 O.C 6 H 8 O 7 : 391.42
7V,A^-Diethyl-4-methylpiperazine- 1 -carboxamide
monocitrate [1642-54-2]
Diethylcarbamazine Citrate, when dried,
not less than 98.0% of QoHziNjO.QHgCv
contains
Description Diethylcarbamazine Citrate occurs as a white,
crystalline powder. It is odorless, and has an acid and bitter
taste.
It is very soluble in water, soluble in ethanol (95), and prac-
tically insoluble in acetone, in chloroform and in diethyl
ether.
A solution of Diethylcarbamazine Citrate (1 in 20) is acid.
It is hygroscopic.
Identification (1) Dissolve 0.5 g of Diethylcarbamazine
Citrate in 2mL of water, add 10 mL of sodium hydroxide
TS, and extract with four 5-mL portions of chloroform.
Wash the combined chloroform extracts with 10 mL of
water, and evaporate the chloroform on a water bath. Add 1
mL of iodoethane to the residue, and boil gently under a
reflux condenser for 5 minutes. Evaporate the excess io-
doethane with the aid of a current of air, and dissolve the
residue in 4 mL of ethanol (95). Cool the ethanol solution in
an ice bath, with continuous stirring, add diethyl ether until
precipitates are formed, and stir until crystallization is
evident. Allow to stand in the ice bath for 30 minutes, and
collect the precipitate. Dissolve the precipitate in 4 mL of
ethanol (95), repeat the recrystallization in the same manner,
then dry at 105°C for 4 hours: the crystals so obtained melt
<2.60> between 151 °C and 155°C.
(2) Neutralize the remaining aqueous layer obtained in
(1) with dilute sulfuric acid: the solution responds to the
Qualitative Tests <1.09> (2) and (3) for citrate.
Melting point <2.60> 135.5 - 138. 5°C
Purity Heavy metals <1.07> — Proceed with 2.0 g of Diethyl-
carbamazine Citrate according to Method 4, and perform the
test. Prepare the control solution with 4.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41>
4 hours).
Not more than 1.0% (2 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.75 g of Diethylcarbama-
zine Citrate, previously dried, dissolve in 50 mL of acetic acid
(100) by warming, cool, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.14 mg of C 10 H 21 N 3 O.C 6 H 8 O 7
Containers and storage Containers — Tight containers.
Diethylcarbamazine Citrate Tablets
Diethylcarbamazine Citrate Tablets contain not less
than 95% and not more than 105% of the labeled
amount of diethylcarbamazine citrate (C 10 H 21 N 3 O.C 6
H 8 7 : 391.42).
Method of preparation Prepare as directed under Tablets,
with Diethylcarbamazine Citrate.
Identification (1) To a quantity of powdered Diethylcar-
bamazine Citrate Tablets, equivalent to 0.5 g of Diethylcar-
bamazine Citrate according to the labeled amount, add 10
mL of water, shake, and filter. Add 10 mL of sodium
hydroxide TS to the filtrate, and proceed as directed in the
Identification (1) under Diethylcarbamazine Citrate.
(2) To a quantity of powdered Diethylcarbamazine Ci-
trate Tablets, equivalent to 0.8 g of diethylcarbamazine ci-
trate according to the labeled amount, add 10 mL of water,
shake, centrifuge, and filter the supernatant liquid. To 5 mL
of the filtrate add 5 mL of sodium hydroxide TS, and extract
with two 20-mL portions of chloroform. Separate the aque-
ous layer, and neutralize with dilute hydrochloric acid: the
solution responds to the Qualitative Tests <1.09> (2) and (3)
for citrate.
Assay Weigh accurately and powder not less than 20
Diethylcarbamazine Citrate Tablets. Weigh accurately a por-
tion of the powder, equivalent to about 50 mg of diethylcar-
bamazine citrate (CioH 21 N 3 O.C 6 H 8 7 ), add 10 mL of water,
shake well, add 5 mL of sodium hydroxide TS, then add ex-
actly 20 mL of the internal standard solution, and shake
vigorously for 10 minutes. Centrifuge, discard the aqueous
layer, and use the chloroform layer as the sample solution.
Separately, weigh accurately about 50 mg of Diethylcar-
bamazine Citrate Reference Standard, previously dried at
105 °C for 4 hours, dissolve in 10 mL of water, add 5 mL of
sodium hydroxide TS, proceed in the same manner as the
preparation of the sample solution, and use the chloroform
layer as the standard solution. Perform the test with 2 /uL of
the sample solution and standard solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions, and calculate the ratios, g T and Q s , of the peak area
of diethylcarbamazine to that of the internal standard, re-
spectively.
Amount (mg) of diethylcarbamazine citrate
(C 10 H 21 N 3 O.C 6 H 8 O 7 )
= W s x (g T /Q s )
W s : Amount (mg) of Diethylcarbamazine Citrate Refer-
ence Standard
Internal standard solution — A solution of M-octadecane in
chloroform (1 in 1250).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass tube 3 mm in inside diameter and 1 m in
length, packed with silanized siliceous earth for gas chro-
JPXV
Official Monographs / Difenidol Hydrochloride
575
matography (180 to 250 //m in particle diameter) coated with
35% methylphenyldimethyl silicone polymer for gas chro-
matography in the ratio of 3%.
Column temperature: A constant temperature of about 145
°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
diethylcarbamazine is about 4 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, diethylcarbamazine and the internal standard are elut-
ed in this order with the resolution between these peaks being
not less than 5.
System repeatability: When the test is repeated 6 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of diethylcarbamazine to that of the internal stan-
dard is not more than 1.5%.
Containers and storage Containers — Well-closed contain-
ers.
Difenidol Hydrochloride
y7i- P-JUSMik
■HC1
C 21 H 27 NO.HCl: 345.91
1 , 1 -Diphenyl-4-piperidin- 1 -ylbutan- 1 -ol
monohydrochloride [3254-89-5]
Difenidol Hydrochloride, when dried, contains not
less than 98.5% of C 21 H 27 NO.HCl.
Description Difenidol Hydrochloride occurs as white crys-
tals or crystalline powder. It is odorless.
It is freely soluble in methanol, soluble in ethanol (95),
sparingly soluble in water and in acetic acid (100), and practi-
cally insoluble in diethyl ether.
Melting point: about 217°C (with decomposition).
Identification (1) Dissolve 0.01 g of Difenidol Hydrochlo-
ride in 1 mL of sulfuric acid: an orange-red color develops.
To this solution add carefully 3 drops of water: the solution
becomes yellowish brown, and colorless on the addition of 10
mL of water.
(2) To 5 mL of a solution of Difenidol Hydrochloride (1
in 100) add 2 mL of Reinecke salt TS: a light red precipitate is
formed.
(3) To 10 mL of a solution of Difenidol Hydrochloride (1
in 100) add 2 mL of sodium hydroxide TS, and extract with
two 15-mL portions of chloroform. Combine the extracts,
wash with three 10-mL portions of water, evaporate the chlo-
roform on a water bath, and dry the residue in a desiccator
(in vacuum, silica gel, 55 °C) for 5 hours: the residue melts
<2.60> between 103 °C and 106°C.
(4) A solution of Difenidol Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Difenidol Hydrochloride in 100
mL of freshly boiled and cooled water: the pH of this solu-
tion is between 4.7 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Difenidol Hydrochloride in 10 mL of methanol: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Difenidol
Hydrochloride according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Difenidol Hydrochloride according to Method 3, and per-
form the test (not more than 1 ppm).
(4) Related substances — Dissolve 0.10 g of Difenidol
Hydrochloride in methanol to make exactly 10 mL, and use
this solution as the sample solution. Separately, dissolve 10
mg of l,l-diphenyl-4-piperidino-l-butene hydrochloride for
thin-layer chromatography in methanol to make exactly 20
mL, pipet 1 mL of this solution, add methanol to make exact-
ly 10 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of toluene, methanol and acetic acid
(100) (10:2:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Difenidol
Hydrochloride, previously dried, dissolve in 30 mL of acetic
acid (100) by warming if necessary, cool, add 30 mL of acetic
anhydride, and titrate <2.50> with 0.05 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 17.30 mg of C 21 H 27 NO.HCl
Containers and storage Containers — Well-closed contain-
ers.
576 Digitoxin / Official Monographs
JP XV
Digitoxin
it^r r- +v>
C 41 H 64 13 : 764.94
3/?-[2,6-Dideoxy-/?-D-n'£>o-hexopyranosyl-(l->4)-
2,6-dideoxy-/?-D-nVjo-hexopyranosyl-(l->4)-2,6-dideoxy-
/?-D-n'£>o-hexopyranosyloxy]-14-hydroxy-5/?,14>S-card-
20(22)-enolide [71-63-6]
Digitoxin, when dried, contains not less than 90.0%
of C 41 H 64 Oi3.
Description Digitoxin occurs as a white to light yellowish
white, crystalline powder. It is odorless.
It is soluble in chloroform, sparingly soluble in methanol
and in ethanol (95), and practically insoluble in water and in
diethyl ether.
Identification (1) Transfer 1 mg of Digitoxin to a small
test tube about 10 mm in inside diameter, dissolve in 1 mL of
a solution of iron (III) chloride hexahydrate in acetic acid
(100) (1 in 10,000), and underlay gently with 1 mL of sulfuric
acid: at the zone of contact of the two liquids a brown ring
free from a reddish color is produced, and the color of the
upper layer near the contact zone changes to green through
purple. Finally the color of the entire acetic acid layer
changes to green through deep blue.
(2) To 2 mg of Digitoxin add 25 mL of a freshly prepared
solution of 1,3-dinitrobenzene in ethanol (95) (1 in 100), and
dissolve by shaking. Take 2 mL of this solution, add 2 mL of
a solution of tetramethylammonium hydroxide in ethanol
(95) (1 in 200), and mix: a red-purple color develops slowly,
and then fades.
(3) Dissolve 1 mg each of Digitoxin and Digitoxin Refer-
ence Standard in a mixture of chloroform and ethanol (95)
(1:1) to make 50 mL, and use these solutions as the sample
solution and the standard solution, respectively. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 20 /uL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of
dichloromethane, methanol and water (84:15:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid upon the plate, and heat at 110°C for 10 minutes:
the spot from the sample solution shows the same i?f value as
the spot from the standard solution.
Optical rotation <2.49> [a]™: + 16 - +18° (after drying. 0.5
g, chloroform, 20 mL, 200 mm).
Purity Digitonin — Dissolve 10 mg of Digitoxin in 2 mL of
ethanol (95) in a test tube, having the inner walls which are
free from scratches, add 2 mL of a solution of cholesterol in
ethanol (95) (1 in 200), mix gently, and allow to stand for 10
minutes: no turbidity is produced.
Loss on drying <2.41> Not more than 1.5% (0.5 g, in vacu-
um, 100°C, 2 hours).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Dissolve about 20 mg each of Digitoxin and Digitox-
in Reference Standard, previously dried and accurately
weighed, in methanol to make exactly 200 mL. Pipet 5 mL
each of these solutions, add exactly 10 mL of the internal
standard solution to each solution, add 12.5 mL of water,
then add methanol to make 50 mL, and use these solutions as
the sample solution and standard solution, respectively. Per-
form the test with 50 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g T and Q s , of the peak area of digitoxin to that of
the internal standard, respectively.
Amount (mg) of C 41 H 64 On
= W s x (Q T /Q S )
W s : Amount (mg) of Digitoxin Reference Standard
Internal standard solution — A solution of acenaphthene in
methanol (3 in 1,000,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 15 to 20 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /am in parti-
cle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of methanol and water (3:1).
Flow rate: Adjust the flow rate so that the retention time of
digitoxin is about 5 minutes.
Selection of column: Proceed with 50 fiL of the standard
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of digitoxin and
the internal standard in this order with the resolution between
these peaks being not less than 6.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Digitoxin Tablets
Digitoxin Tablets contain not less than 90% and not
more than 110% of the labeled amount of digitoxin
(C 41 H 64 13 : 764.94).
Method of preparation
with Digitoxin.
Prepare as directed under Tablets,
JPXV
Official Monographs / Digitoxin Tablets 577
Identification (1) Place a portion of powdered Digitoxin
Tablets, equivalent to 2 mg of digitoxin (C 41 H 64 I3 ) accord-
ing to the labeled amount, in a separator, shake with 30 mL
of water, and shake vigorously with 30 mL of chloroform.
Filter the chloroform extract with a funnel on which a small
amount of anhydrous sodium sulfate is placed, and transfer
to a round-bottomed flask connected by a universal joint.
Evaporate the solution to dryness by warming under reduced
pressure, and dissolve the residue in 10 mL of chloroform.
Transfer 5 mL of this solution to a small test tube about 10
mm in inside diameter, and evaporate to dryness on a water
bath with the aid of a current of air. Proceed with the residue
as directed in the Identification (1) under Digitoxin.
(2) Evaporate 4 mL of the chloroform solution obtained
in (1) to dryness, by warming under reduced pressure, add a
freshly prepared solution of 1,3-dinitrobenzene in ethanol
(95) (1 in 100) to the residue, and dissolve by shaking. Pro-
ceed with 2 mL of this solution as directed in the Identifica-
tion (2) under Digitoxin.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Transfer 1 tablet of Digitoxin Tablets to a 50-mL beaker,
add 0.5 mL of water to disintegrate the tablet, add 5 mL of
acetonitrile, and warm on a water bath for 5 minutes, cover-
ing the beaker with a watch glass. After cooling, transfer the
solution to separator A, rinse the beaker with 30 mL of chlo-
roform and then with 20 mL of water, transfer the rinsings to
separator A, and extract by vigorous shaking. Transfer the
chloroform extract to separator B containing 5 mL of a solu-
tion of sodium hydrogen carbonate (1 in 100), and shake to
wash. Filter the chloroform layer through a pledget of absor-
bent cotton, previously moistened with chloroform. Extract
the water layer in separator A with two 30-mL portions of
chloroform, wash the chloroform extract with a solution of
sodium hydrogen carbonate (1 in 100) in separator B, filter in
the same manner, and combine the filtrate with the first one.
Evaporate this filtrate to dryness under reduced pressure by
warming, add diluted ethanol (95) (4 in 5) to make exactly V
mL of a solution containing 5 fig of digitoxin (C 41 H 64 0i 3 ) per
ml. Shake vigorously for 20 minutes to dissolve, and use this
solution as the sample solution. Separately, weigh accurately
about 10 mg of Digitoxin Reference Standard, previously d-
ried at 100°C for 2 hours, and dissolve in diluted ethanol (95)
(4 in 5) to make exactly 100 mL. Pipet 5 mL of this solution,
add diluted ethanol (95) (4 in 5) to make exactly 100 mL, and
use this solution as the standard solution. Pipet 2 mL each of
the sample solution, the standard solution and diluted
ethanol (95) (4 in 5) into brown glass-stoppered test tubes T,
S and B. Add exactly 10 mL each of 0.02 w/v% L-ascorbic
acid-hydrochloric acid TS, shake well, and immediately add
exactly 1 mL each of dilute hydrogen peroxide TS. Shake
vigorously, and allow to stand at a constant temperature be-
tween 25 °C and 30°C for 45 minutes. Determine the fluores-
cence intensities, F T , F s and F B , of these solutions at 400 nm
of the excitation wavelength and at about 570 nm of the
fluorescence wavelength as directed under Fluorometry
<2.22>, respectively.
Amount (mg) of digitoxin (C 41 H 64 13 )
= W s x {(F T - F B )/(F S - F B )} x (F72000)
W s : Amount (mg) of Digitoxin Reference Standard
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Take 1 tablet of Digitoxin Tablets, and perform the test us-
ing 500 mL of diluted hydrochloric acid (3 in 500), degassed
by a suitable method, as the test solution at 100 revolutions
per minute as directed in the Basket method. At 30 minutes
after starting the test, take a + 15 mL of the dissolved solu-
tion, and immediately add the same volume of fresh test solu-
tion, previously warmed at 37 ± 0.5°C, to the vessel careful-
ly. Filter a + 15 mL of the dissolved solution through a
membrane filter (less than 0.8 //m in pore size). Discard the
first 10 mL of the filtrate, and use the subsequent filtrate as
the sample solution. Measure exactly a mL of the sample so-
lution, equivalent to about 2,Mg of digitoxin (C 41 H 64 0i 3 ) ac-
cording to the labeled amount, transfer to a glass-stoppered
centrifuge tube T 30 , and warm at 37 ± 0.5 °C for 30 minutes.
Further, at 60 minutes after starting the test, take a + 15 mL
of the dissolved solution, proceed in the same manner, meas-
ure exactly a mL of the sample solution, and transfer to a
glass-stoppered centrifuge tube T 60 . Separately, weigh ac-
curately 100 times the labeled amount of Digitoxin Reference
Standard, previously dried under reduced pressure at 100°C
for 2 hours, and dissolve in ethanol (95) to make exactly 100
mL. Measure exactly 1 mL of this solution, add the test solu-
tion to make exactly 500 mL, warm at 37 ± 0.5°C for 60
minutes, and filter through a membrane filter (less than 0.8
//m in pore size). Discard the first 10 mL of the filtrate, and
use the subsequent filtrate as the standard solution. Measure
exactly a mL each of the standard solution and the test solu-
tion, transfer to glass-stoppered centrifuge tubes T s and T B ,
respectively. Add exactly 7 mL of chloroform to each of the
glass-stoppered centrifuge tubes T 30 , T 60 , T s and T B , shake
vigorously for 10 minutes and centrifuge. Discard the aque-
ous layer, measure exactly 5 mL of the chloroform layer,
transfer to brown test tubes T 30 , T^o, T£ and T B , evaporate
the chloroform, add exactly 4 mL each of 0.05 w/v% L-as-
corbic acid-hydrochloric acid TS, shake well, and allow to
stand for 10 minutes. Then add exactly 0.5 mL each of dilute
hydrogen peroxide TS, shake well, and allow to stand at a
constant temperature between 25 °C and 30°C for 45
minutes. Determine the fluorescence intensities, F 30 , F m , F s
and F B , of these solutions at about 395 nm of the excitation
wavelength and at about 560 nm of the fluorescence
wavelength as directed under Fluorometry <2.22>, respective-
ly. Dissolution rates of Digitoxin Tablets after 30 minutes
and 60 minutes should be not less than 60% and 85%, respec-
tively.
No retest requirement is applied to Digitoxin Tablets.
Dissolution rate (%) with respect to the labeled
amount of digitoxin (C 41 H 64 13 ) for 30 minutes
= W s x {(F 30 - F B )/(F S - F B )} x (I/O
Dissolution rate (%) with respect to the labeled
amount of digitoxin (C 41 H 64 I3 ) for 60 minutes
= W, x
*h
F B
LA
F B
• + 15
500
W s : Amount (mg) of Digitoxin Reference Standard.
C: The labeled amount (mg) of digitoxin (C 14 H 64 Oi 3 ) in 1
tablet.
a + 15: Measured volume (mL) of dissolved solution at the
578 Digoxin / Official Monographs
JP XV
specified time.
Assay Weigh accurately and powder not less than 20
Digitoxin Tablets. Weigh accurately a portion of the powder,
equivalent to about 0.5 mg of digitoxin (C 4 iH 64 13 ), and
shake with 12.5 mL of water for 10 minutes. Add exactly 10
mL of the internal standard solution, shake for 20 minutes,
and add methanol to make 50 mL. Centrifuge this solution,
and use the supernatant liquid as the sample solution.
Separately, weigh accurately about 20 mg of Digitoxin Refer-
ence Standard, previously dried in vacuum at 100°C for 2
hours, dissolve in methanol to make exactly 200 mL. Pipet 5
mL of the solution, add exactly 10 mL of the internal stan-
dard solution, add 12.5 mL of water, then methanol to make
50 mL, and use this solution as the standard solution. Pro-
ceed as directed in the Assay under Digitoxin.
Amount (mg) of digitoxin (C 4I H 64 13 )
= Ws x (Qt/Qs) x 0.025
W s : Amount (mg) of Digitoxin Reference Standard
Internal standard solution — A solution of acenaphthene in
methanol (3 in 1,000,000).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Digoxin
CH,
OH
HO
"0"
£L oWh
OH
C 41 H 64 14 : 780.94
3/?-[2,6-Dideoxy-/?-D-n'£>o-hexopyranosyl-(l->4)-2,6-
dideoxy-/?-D-n7?o-hexopyranosyl-(l->4)-2,6-dideoxy-/?-D-
n'6o-hexopyranosyloxy]-12j8,14-dihydroxy-5j8,14/?-card-
20(22)-enolide [20830-75-5]
Digoxin, when dried, contains not less than 96.0%
and not more than 106.0% of C 41 H 64 14 .
Description Digoxin occurs as colorless or white crystals or
a white crystalline powder.
It is freely soluble in pyridine, slightly soluble in ethanol
(95), very slightly soluble in acetic acid (100), and practically
insoluble in water.
Identification (1) Transfer 1 mg of Digoxin to a small test
tube about 10 mm in inside diameter, dissolve in 1 mL of a
solution of iron (III) chloride hexahydrate in acetic acid (100)
(1 in 10,000), and underlay gently with 1 mL of sulfuric acid:
at the zone of contact of the two liquids a brown ring free
from a reddish color is produced, and the color of the upper
layer near the contact zone changes to green through purple.
Finally the entire acetic acid layer shows a green color
through a deep blue color.
(2) Determine the infrared absorption spectrum of
Digoxin, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]™: + 10.0 - + 13.0° (after drying,
0.20 g, dehydratead pyridine, 10 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Digoxin in 15 mL of diluted ethanol (95) (4 in 5) by warm-
ing: the solution is clear and colorless.
(2) Related substances — Dissolve exactly 25.0 mg of
Digoxin in 50 mL of warm ethanol (95), cool, and add
ethanol (95) to make exactly 100 mL. Pipet 10 mL of this
solution, add 10 mL of water and dilute ethanol to make 50
mL, and use this solution as the sample solution. Separately,
dissolve exactly 5.0 mg of Gitoxin Reference Standard, previ-
ously dried under reduced pressure at 105°C for 1 hour, in a
mixture of acetonitrile and water (7:3) to make exactly 200
mL. Pipet 2 mL of this solution, add dilute ethanol to make
50 mL, and use this solution as the standard solution. Per-
form the test with exactly 10 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of gitoxin: A T is
not larger than A s , and the total of the areas of the peaks
other than digitoxin and gitoxin, obtained by the area percen-
tage method, is not more than 3%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of digoxin beginning after the solvent peak.
System suitability —
Test for required detectability: Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the solution for system suitability test.
Pipet 1 mL of the solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of digoxin ob-
tained from 10 /uL of this solution is equivalent to 7 to 13%
of that from the solution for system suitability test.
System performance: Dissolve 25 mg of digoxin in 50 mL
of warm ethanol (95), cool, and add ethanol (95) to make
exactly 100 mL. Pipet 10 mL of this solution, add exactly
5 mL of a solution of propyl parahydroxybenzoate in ethanol
(95) (1 in 4000), 10 mL of water and dilute ethanol to make 50
mL. When the procedure is run with 10 ^L of this solution
under the above operating conditions, digoxin and propyl
parahydroxybenzoate are eluted in this order with the resolu-
tion between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of digoxin is not more than 1.0%.
JPXV
Official Monographs / Digoxin Injection
579
Loss on drying <2.41> Not more than 1.0% (0.5 g, in vacu-
um, 105°C, 1 hour).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Weigh accurately about 25 mg each of Digoxin and
Digoxin Reference Standard, previously dried, dissolve in
50 mL of warm ethanol (95), cool, and add ethanol (95) to
make exactly 100 mL. Pipet 10 mL of these solutions, add ex-
actly 5 mL of the internal standard solution, 10 mL of water
and dilute ethanol to make 50 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Qj and
Qs, of the peak area of digoxin to that of the internal stan-
dard.
Amount (mg) of C 4 iH 64 14 = W s x (Q T /Q S )
W s : Amount (mg) of Digoxin Reference Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in ethanol (95) (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
digoxin is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, digoxin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of digoxin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Digoxin Injection
Digoxin Injection is an aqueous solution for
injection.
It contains not less than 90.0% and not more than
105.0% of the labeled amount of digoxin (C 41 H 64 14 :
780.94).
Method of preparation Prepare as directed under Injec-
tions, with a solution of Digoxin in 5 to 50 vol% ethanol.
Description Digoxin Injection is a clear, colorless liquid.
Identification Dilute Digoxin Injection, if necessary, with
methanol so that each mL contains about 0.25 mg of Digoxin
according to the labeled amount, and use this solution as the
sample solution. In case where ingredients are suspected to
affect the test, remove them by means of a solid-phase extrac-
tion. Separately, dissolve 0.5 mg of Digoxin Reference Stan-
dard in 2 mL of methanol, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10,mL
each of the sample solution and standard solution on a plate
of octadecylsilanized silica gel for thin-layer chro-
matography. Develop the plate with a mixture of methanol
and water (7:3) to a distance of about 10 cm, and air-dry the
plate. Spray evenly a mixture of a solution of trichloroacetic
acid in ethanol (99.5) (1 in 4) and a freshly prepared solution
of sodium toluenesulfonchloramide trihydrate (3 in 100) (4:1)
on the plate, heat at 110°C for 10 minutes, and examine un-
der ultraviolet light (main wavelength: 366 nm): the Rf values
of the principal spots with the sample solution and the stan-
dard solution are not different each other.
Bacterial endotoxins <4.01> Less than 200 EU/mg.
Extractable volume <6.05> It meets the requirements.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay To an exact volume of Digoxin Injection, equivalent
to about 2.5 mg of digoxin (C 41 H 64 14 ), add exactly 5 mL of
the internal standard solution and dilute ethanol to make
50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 25 mg of Digoxin Refer-
ence Standard, previously dried under reduced pressure at
105°C for 1 hour, dissolve in 50 mL of warm ethanol (95),
cool, and add ethanol (95) to make exactly 100 mL. Pipet 10
mL of this solution, add exactly 5 mL of the internal stan-
dard solution, 10 mL of water and dilute ethanol to make 50
mL, and use this solution as the standard solution. Perform
the test with 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine the
ratios, Qt and Qs, of the peak area of digoxin to that of the
internal standard.
Amount (mg) of digoxin (C 41 H 64 14 )
= Ws x (St/Gs) x (1/10)
W s : Amount (mg) of Digoxin Reference Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in ethanol (95) (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
580
Digoxin Tablets / Official Monographs
JP XV
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
digoxin is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, digoxin and the internal standard are eluted in this ord-
er with the resolution between these peaks being not less than
5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of digoxin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Digoxin Tablets
Digoxin Tablets contain not less than 90.0% and not
more than 105.0% of the labeled amount of digoxin
(C 4 iH 64 14 : 780.94).
Method of preparation Prepare as directed under Tablets,
with Digoxin.
Identification To an amount of pulverized Digoxin Tablets,
equivalent to 0.5 mg of Digoxin according to the labeled
amount, add 2 mL of methanol, shake for 10 minutes, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 0.5 mg of Digoxin Reference Standard in 2 mL of
methanol, and use this as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /uL each of the sample solution
and standard solution on a plate of octadecylsilanized silica
gel for thin-layer chromatography. Develop the plate with a
mixture of methanol and water (7:3) to a distance of about 10
cm, and air-dry the plate. Spray evenly a mixture of a solu-
tion of trichloroacetic acid in ethanol (99.5) (1 in 4) and a
freshly prepared solution of sodium toluenesulfonchloramide
trihydrate (3 in 100) (4:1) on the plate, heat at 110°C for 10
minutes, and examine under ultraviolet light (main
wavelength: 366 nm): the Rf values of the principal spots
with the sample solution and the standard solution are not
different each other.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Digoxin Tablets add 0.5 mL of water to
disintegrate, then add exactly 0.5 mL of the internal standard
solution, and add KmL of dilute ethanol so that each mL
contains about 21 /ug of digoxin (C 41 H 64 0i 4 ). Exposure this
solution to ultrasonic waves for 20 minutes, shake for 5
minutes, filter, and use the filtrate as the sample solution.
Separately, weigh accurately about 25 mg of Digoxin Refer-
ence Standard, previously dried under reduced pressure at
105°C for 1 hour, dissolve in 50 mL of warm ethanol (95),
cool, and add ethanol (95) to make exactly 100 mL. Pipet
10 mL of this solution, and add ethanol (95) to make exactly
20 mL. Pipet 1 mL of this solution, add exactly 0.5 mL of the
internal standard solution, then add 1.5 mL of water and (V
- 2) mL of dilute ethanol, and use this as the standard
solution. Proceed with the sample solution and standard so-
lution as directed in the Assay.
Amount (mg) of digoxin (C 4 iH 64 14 )
= W s x (Q T /Q S ) x (1/200)
W s : Amount (mg) of Digoxin Reference Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in ethanol (95) (1 in 40,000/V).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Digoxin Tablets, using 500
mL of diluted hydrochloric acid (3 in 500) at 100 revolutions
per minute according to the Basket method as the dissolution
medium. Withdraw 30 mL or more of the dissolved solution
60 minutes after starting the test, and filter through a mem-
brane filter (less than 0.8 fim in pore size). Discard the first 10
mL of the filtrate, and use the subsequent filtrate as the sam-
ple solution. Separately, weigh accurately about 25 mg of
Digoxin Reference Standard, previously dried in vacuum at
105 °C for 1 hour, dissolve in a small portion of ethanol (95),
and add a mixture of ethanol (95) and water (4:1) to make ex-
actly 500 mL. Pipet 5 mL of this solution, add the dissolution
medium to make exactly 500 mL, and use this solution as the
standard solution. Pipet 2 mL each of the sample solution,
the standard solution and the dissolution medium, and trans-
fer to brown glass-stoppered test tubes. Add exactly 10 mL of
0.012 g/dL L-ascorbic acid-hydrochloric acid TS to these
tubes, and shake. Immediately add exactly 1 mL of dilute
hydrogen peroxide TS, shake well, and allow to stand at a
constant temperature between 25 °C and 30°C for 45
minutes. Determine the fluorescence intensities, F T , F$, and F
B , of these solutions at 360 nm of the excitation wavelength
and at 485 nm of the fluorescence wavelength as directed
under Fluorometry <2.22>, respectively: the dissolution rate
in 60 minutes is not less than 65%. No retest requirement is
applied to Digoxin Tablets.
Dissolution rate (%) with respect to the labeled amount of
digoxin (C 4 iH 64 14 )
= W s x {(F T - F B )/(F S - F B )} x (1/Q
W s : Amount (mg) of Digoxin Reference Standard
C: The labeled amount (mg) of digoxin (C 41 H 64 14 ) in 1
tablet
Assay Weigh accurately the mass of not less than 20 Digox-
in Tablets, and powder. Weigh accurately a portion of the
powder, equivalent to about 2.5 mg of digoxin (C 41 H 64 0, 4 ),
add 30 mL of dilute ethanol, exposure to ultrasonic waves for
20 minutes, and shake for 5 minutes. Add exactly 5 mL of the
internal standard solution and dilute ethanol to make 50 mL,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 25 mg of Digoxin
Reference Standard, previously dried under reduced pressure
at 105 C C for 1 hour , dissolve in 50 mL of warm ethanol (95),
cool, and add ethanol (95) to make exactly 100 mL. Pipet 10
mL of this solution, add exactly 5 mL of the internal stan-
dard solution, 10 mL of water and dilute ethanol to make 50
mL, and use this solution as the standard solution. Perform
JPXV
Official Monographs / Dihydrocodeine Phosphate
581
the test with 10 juL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qj and Q s , of the peak area of digoxin to that of the internal
standard.
Amount (mg) of digoxin (C 41 H 64 Oi 4 )
= W s x (Q T /Q S ) x (1/10)
W s : Amount (mg) of Digoxin Reference Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in ethanol (95) (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
digoxin is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, digoxin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of digoxin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dihydrocodeine Phosphate
■H HP=i^-f >U>i!±M
h 3 c-o
• H 3 P0 4
O' H H
C 18 N 23 N0 3 .H 3 P0 4 : 399.38
(5/?,6S)-4,5-Epoxy-3-methoxy-17-methylmorphinan-6-ol
monophosphate [24204-13-5]
Dihydrocodeine Phosphate contains not less than
98.0% of C 18 H 2 3N03.H 3 P04, calculated on the dried
basis.
Description Dihydrocodeine Phosphate occurs as a white to
yellowish white, crystalline powder.
It is freely soluble in water and in acetic acid (100), slightly
soluble in ethanol (95), and practically insoluble in diethyl
ether.
The pH of a solution of Dihydrocodeine Phosphate (1 in
10) is between 3.0 and 5.0.
It is affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Dihydrocodeine Phosphate (1 in 10,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared spectrum of Dihydrocodeine
Phosphate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Dihydrocodeine Phosphate (1 in 20)
responds to the Qualitative Tests <1.09> (1) for phosphate.
Purity (1) Chloride <7.0?>— Perform the test with 0.5 g of
Dihydrocodeine Phosphate. Prepare the control solution
with 0.30 mL of 0.01 mol/L hydrochloric acid (not more
than 0.021%).
(2) Sulfate <1.14>— Perform the test with 0.20 g of Di-
hydrocodeine Phosphate. Prepare the control solution with
1 .0 mL of 0.005 mol/L sulfuric acid (not more than 0.240%).
(3) Related substances — Dissolve 0.20 g of Dihydro-
codeine Phosphate in 10 mL of diluted ethanol (95) (1 in 2),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add diluted ethanol (95) (1 in 2) to make
exactly 50 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 jXL of the sam-
ple solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-chromatography. Develop the
plate with a mixture of ethanol (99.5), toluene, acetone and
ammonia solution (28) (14:14:7:1) to a distance of about 15
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41>
hours).
Not more than 1.0% (0.5 g, 105°C, 4
Assay Weigh accurately about 0.5 g of Dihydrocodeine
Phosphate, dissolve in 70 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to greenish
blue (indicator: 3 drops of crystal violet TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.94 mg of C 18 H 23 N0 3 .H 3 P0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
582
1% Dihydrocodeine Phosphate Powder / Official Monographs
JP XV
1% Dihydrocodeine Phosphate
Powder
1% Dihydrocodeine Phosphate Powder contains not
less than 0.90% and not more than 1.10% of di-
hydrocodeine phosphate (QgH^NOj.I^PCV 399.38).
Method of preparation
Dihydrocodeine Phosphate
Lactose Hydrate
10g
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification Determine the absorption spectrum of a solu-
tion of 1% Dihydrocodeine Phosphate Powder (1 in 100) as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 281 nm and 285 nm.
Assay Weigh accurately about 5g of 1% Dihydrocodeine
Phosphate Powder, dissolve in water to make exactly
100 mL, then pipet 10 mL of this solution, add exactly 10 mL
of the internal standard solution, and use this solution as the
sample solution. Separately, weigh accurately about 50 mg of
dihydrocodeine phosphate for assay (previously determine
the loss on drying <2.41> (105 °C, 4 hours)), dissolve in water
to make exactly 100 mL, then pipet 10 mL of this solution,
add exactly 10 mL of the internal standard solution, and use
this solution as the standard solution. Perform the test with
20 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Q T and Q s ,
of the peak area of dihydrocodeine to that of the internal
standard.
Amount (mg) of dihydrocodeine phosphate
(C 18 H 23 N03.H 3 P04)
= W s x (Q T /Q S )
W s : Amount (mg) of dihydrocodeine phosphate for assay,
calculated on the dried basis
Internal standard solution — A solution of ethylefurin
hydrochloride (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran.
Flow rate: Adjust the flow rate so that the retention time of
dihydrocodeine is about 9 minutes.
System suitability —
System performance: When the procedure is run with
20 /uL of the standard solution under the above operating
conditions, dihydrocodeine and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 4.
System repeatability: When the test is repeated 5 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of dihydrocodeine to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
10% Dihydrocodeine Phosphate
Powder
yth'naT-f>ij >m^m 10%
10% Dihydrocodeine Phosphate Powder contains
not less than 9.3% and not more than 10.7% of di-
hydrocodeine phosphate (QgHzsNOj.HjPO,,: 399.38).
Method of preparation
Dihydrocodeine Phosphate
Lactose Hydrate
100 g
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification Determine the absorption spectrum of a solu-
tion of 10% Dihydrocodeine Phosphate Powder (1 in 1000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 281 nm and 285 nm.
Assay Weigh accurately about 2.5 g of 10% Dihydro-
codeine Phosphate Powder, dissolve in water to make exactly
100 mL, then pipet 2 mL of this solution, add exactly 10 mL
of the internal standard solution and water to make 20 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 50 mg of dihydrocodeine phosphate
for assay, (previously determine the loss on drying <2.41>
(105°C, 4 hours)), dissolve in water to make exactly 100 mL,
then pipet 10 mL of this solution, add exactly 10 mL of the
internal standard solution, and use this solution as the stan-
dard solution. Perform the test with 20 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, g T and Q s , of the peak area of
dihydrocodeine to that of the internal standard.
Amount (mg) of dihydrocodeine phosphate
(C,
W s x (Qt/Qs) x 5
W s : Amount (mg) of dihydrocodeine phosphate for assay,
calculated on the dried basis
Internal standard solution — A solution of ethylefrine
hydrochloride (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
JPXV
Official Monographs / Dihydroergotamine Mesilate
583
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran.
Flow rate: Adjust the flow rate so that the retention time of
dihydrocodeine is about 9 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, dihydrocodeine and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 4.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of dihydrocodeine to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Tight containers.
Dihydroergotamine Mesilate
=>\i h*nuuzf?S >y vJL-^tg
H3C o
r^S H Y X^° * *
O H
3 C— SOgH
(5'S,10.R)-5'-Benzyl-12'-hydroxy-2'-
methyl-9, 10-dihydroergotaman-3' ,6' , 18-trione
monomethanesulfonate
[6190-39-2]
Dihydroergotamine Mesilate contains not less than
97.0% of CjsHsvNsOj.CH^S, calculated on the dried
basis.
Description Dihydroergotamine Mesilate occurs as a white
to yellowish white or grayish white to reddish white powder.
It is freely soluble in acetic acid (100), sparingly soluble in
methanol and in chloroform, slightly soluble in water and in
ethanol (95), and practically insoluble in acetic anhydride and
in diethyl ether.
It is gradually colored by light.
Melting point: about 214°C (with decomposition).
Identification (1) Dissolve 1 mg of Dihydroergotamine
Mesilate in 5 mL of a solution of L-tartaric acid (1 in 100). To
1 mL of this solution add 2 mL of 4-dimethylaminobenzalde-
hyde-ferric chloride TS, and shake: a blue color develops.
(2) To 0.1 g of Dihydroergotamine Mesilate add 0.4 g of
sodium hydroxide, stir well, and incinerate by gradual igni-
tion. After cooling, add 10 mL of water to the residue, heat
to boiling, cool, and filter. To the filtrate add 0.5 mL of
hydrochloric acid: the solution responds to the Qualitative
Tests <1.09> for sulfate. Separately, to 0.1 g of Dihydroer-
gotamine Mesilate add 5 mL of dilute hydrochloric acid,
shake for 5 minutes, filter, and to the filtrate add 1 mL of
barium chloride TS: the solution is clear.
(3) Determine the absorption spectrum of a solution of
Dihydroergotamine Mesilate in methanol (1 in 20,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of Di-
hydroergotamine Mesilate, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: - 16.7 - -22.7° [0.5 g, calcu-
lated on the dried basis, a mixture of ethanol (99.5), chlo-
roform and ammonia solution (28) (10:10:1), 20 mL, 100
mm].
pH <2.54> Dissolve 0.05 g of Dihydroergotamine Mesilate
in 50 mL of water: the pH of this solution is between 4.4 and
5.4.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Dihydroergotamine Mesilate in 0.1 mL of a solution of
methanesulfonic acid (7 in 100) and 50 mL of water: the solu-
tion is clear, and has no more color than the following con-
trol solutions [1] or [2].
Control solution [1]: Pipet 0.6 mL of Ferric Chloride Stock
CS and 0.15 mL of Cobaltous Chloride Stock CS, mix, and
add diluted hydrochloric acid (1 in 40) to make exactly 100
mL.
Control solution [2]: Pipet 0.6 mL of Ferric Chloride Stock
CS, 0.25 mL of Cobaltous Chloride Stock CS and 0.1 mL of
copper (II) sulfate CS, mix, and add diluted hydrochloric
acid (1 in 40) to make exactly 100 mL.
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Dihydroergotamine Mesilate in 5 mL of a mixture
of chloroform and methanol (9:1), and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add a
mixture of chloroform and methanol (9:1) to make exactly
200 mL, and use this solution as the standard solution (1).
Pipet 10 mL of the standard solution (1), add a mixture of
chloroform and methanol (9:1) to make exactly 25 mL, and
use this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solutions (1) and (2) on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of dichloromethane, ethyl acetate, methanol and ammonia
solution (28) (50:50:6:1) to a distance of about 15 cm, and
dry the plate with cold wind within 1 minute. Develop the
plate again immediately with a freshly prepared mixture of
dichloromethane, ethyl acetate, methanol and ammonia solu-
tion (28) (50:50:6:1) to a distance of about 15 cm, and air-dry
584
Dihydroergotoxine Mesilate / Official Monographs
JP XV
the plate. Spray evenly 4-dimethylaminobenzaldehyde TS for
spraying on the plate, and dry the plate with warm wind: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solution
(1), and the spots, which are more intense than the spot from
the standard solution (2), are not more than two.
Loss on drying <2.41> Not more than 4.0% (0.5 g, in vacu-
um at a pressure not exceeding 0.67 kPa, 100°C, 6 hours).
Assay Weigh accurately about 0.2 g of Dihydroergotamine
Mesilate, dissolve in 170 mL of a mixture of acetic anhydride
and acetic acid (100) (10:1), and titrate <2.50> with 0.02 mol/
L perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.02 mol/L perchloric acid VS
= 13.60 mg of CjjfL^NsOs.CH^S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dihydroergotoxine Mesilate
>\i h'nuuzf r-+->>y->;ng±g
• H 3 C-S0 3 H
Dihydroergocornine Mesilate : R =
Dihydro-a-ergocryptine Mesilate : R =
Dihydro-/$-ergocryptine Mesilate : R =±
Diriydroergocrrstine Mesilate : R =
Dihydroergocornine Mesilate
CjiH^NsOs.CH^S: 659.79
(5'S,10i?)-12'-Hydroxy-2',5'-bis(l-methylethyl)-
9, 10-dihydroergotaman-3' ,6' , 18-trione
monomethanesulfonate
Dihydro-a-ergocryptine Mesilate
C 3 2H43N50 5 .CH40 3 S: 673.82
(5'S,10i?)-12'-Hydroxy-2'-(l-methylethyl)-5'-(2-
methylpropyl)-9, 10-dihydroergotaman-3 ' ,6' , 1 8-trione
monomethanesulfonate
Dihydro-/3-ergocryptine Mesilate
C 32 H43N 5 05.CH 4 03S: 673.82
(5'S,10i?)-12'-Hydroxy-2'-(l-methylethyl)-5'-(l-
methylpropyl)-9, 10-dihydroergotaman-3 ' ,6' , 1 8-trione
monomethanesulfonate
Dihydroergocristine Mesilate
C 3 5H4,N50 5 .CH40 3 S: 707.84
(5'S,10i?)-5'-Benzyl-12'-hydroxy-2'-(l-methylethyl)-
9, 10-dihydroergotaman-3' ,6' , 18-trione
monomethanesulfonate
[8067-24-1, Dihydroergotoxine Mesilate]
Dihydroergotoxine Mesilate contains not less than
97.0% and not more than 103.0% of dihydroer-
gotoxine mesilate [as a mixture of dihydroergocornine
mesilate (CsirLuNsCVCrLtC^S), dihydro-a-ergocryp-
tine mesilate (C32H43N5O5.CH4O3S), dihydro-/?-er-
gocryptine mesilate (C32H43N5O5.CH4O3S) and di-
hydroergocristine mesilate (CssH^NsOs.CI^C^S)], cal-
culated on the anhydrous basis. The relative
contents of dihydroergocornine mesilate
(C3 1 H 41 N505.CH40 3 S), dihydroergocryptine mesilate
(C32H43N5O5.CH4O3S) and dihydroergocristine mesi-
late (C35H41N5O5.CH4O3S) are 30.3-36.3% each, and
the content ratio of dihydro-a-ergocryptine mesilate
and dihydro-/?-ergocryptine mesilate is 1.5-2.5:1.
Description Dihydroergotoxine Mesilate occurs as a white
to pale yellow powder.
It is soluble in methanol, sparingly soluble in ethanol (95),
slightly soluble in water, in acetonitrile and in chloroform,
and practically insoluble in diethyl ether.
Identification Determine the infrared absorption spectrum
of Dihydroergotoxine Mesilate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [«]£>: ■
lated on the anhydrous basis,
mm).
-11.0- + 15.0° (0.2 g, calcu-
dilute ethanol, 20 mL, 100
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Dihydroergotoxine Mesilate in 20 mL of water: the solu-
tion is clear and the color of the solution is not more intense
than that of the following control solution.
Control solution: To a mixture of 1.0 mL of Cobaltous
Chloride Stock CS, 0.4 mL of Cupric Sulfate Stock CS and
2.4 mL of Ferric Chloride Stock CS add diluted hydrochloric
acid (1 in 40) to make exactly 200 mL.
(2) Heavy metals <1. 07>— Proceed with 1.0 g of Di-
hydroergotoxine Mesilate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Weigh accurately 0.100 g of Di-
hydroergotoxine Mesilate, dissolve it in a mixture of chlo-
roform and methanol (9:1) to make exactly 5 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately 10 mg of dihydroergocristine mesilate for thin-layer
chromatography, and dissolve in a mixture of chloroform
and methanol (9:1) to make exactly 100 mL. Pipet 6mL, 4
mL and 2 mL of this solution, add a mixture of chloroform
and methanol (9:1) to make exactly 10 mL, respectively, and
use these solutions as the standard solutions (1), (2) and (3),
respectively. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03> without putting the
filter paper in the developing vessel. Spot 5 /uL each of the
sample solution and standard solutions (1), (2) and (3) on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of dichloromethane, ethyl acetate,
methanol and ammonia solution (28) (50:50:3:1) to a distance
of about 15 cm, and dry the plate with the aid of a cool air
JPXV
Official Monographs / Dihydroergotoxine Mesilate
585
stream. Immediately after that, develop the plate again with a
newly prepared mixture of dichloromethane, ethyl acetate,
methanol and ammonia solution (28) (50:50:3:1) to a distance
of about 15 cm, and dry the plate within 1 minute with the aid
of a cool air stream. Spray evenly />-dimethylaminobenzal-
dehyde-hydrochloric acid TS on the plate, dry the plate wi-
thin 2 minutes with the aid of a cool air stream, and heat it at
40°C for 15 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution (1), not more than 2 spots are
more intense than that from the standard solution (2), and
not more than 4 spots are more intense than that from the
standard solution (3).
Water <2.48> Not more than 5.0% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Dihydroergotoxine mesilate — Weigh accurately
about 30 mg each of Dihydroergotoxine Mesilate and Di-
hydroergotoxine Mesilate Reference Standard, and dissolve
them separately in a suitable amount of a mixture of water
and acetonitrile (3:1). To these solutions add exactly 10 mL
of the internal standard solution and an amount of a mixture
of water and acetonitrile (3:1) to make 50 mL, and use these
solutions as the sample solution and standard solution. Per-
form the test with 20 fiL of the sample solution and standard
solution as directed under Liquid Chromatography according
to the following conditions, and calculate the ratios of the
peak areas of dihydroergocornine, dihydro-a-ergocryptine,
dihydroergocristine and dihydro-/?-ergocryptine to the peak
area of the internal standard of these solutions.
Amount (mg) of dihydroergotoxine mesilate
= W s x {(M TA + M TB + M TC + M TD )/
(M s ,
M s
M s ,
M SD )}
W s : Amount (mg) of Dihydroergotoxine Mesilate Refer-
ence Standard, calculated on the anhydrous basis
M TA : Ratio of the peak area of dihydroergocornine to that
of the internal standard of the sample solution x
659.80
M TB : Ratio of the peak area of dihydro-a-ergocryptine to
that of the internal standard of the sample x 673.83
M TC : Ratio of the peak area of dihydroergocristine to that
of the internal standard of the sample solution x
707.85
M TD : Ratio of the peak area of dihydro-/?-ergocryptine to
that of the internal standard of the sample solution
x 673.83
M SA : Ratio of the peak area of dihydroergocornine to that
of the internal standard of the standard solution X
659.80
M SB : Ratio of the peak area of dihydro-a-ergocryptine to
that of the internal standard of the standard solution
x 673.83
M sc : Ratio of the peak area of dihydroergocristine to that
of the internal standard of the standard solution X
707.85
M SD : Ratio of the peak area of dihydro-/3-ergocryptine to
that of the internal standard of the standard solution
x 673.83
Internal standard solution — Dissolve 0.04 g of chloram-
phenicol in a mixture of water and acetonitrile (3:1) to make
250 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, acetonitrile and
triethylamine (30:10:1).
Flow rate: Adjust the flow rate so that the retention time of
chloramphenicol is about 5 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the standard solution under the above operating
conditions, the internal standard, dihydroergocornine, di-
hydro-a-ergocryptine, dihydroergocristine and dihydro-/3-er-
gocryptine are eluted in this order with the resolution
between the peaks of dihydro-a-ergocryptine and dihydroer-
gocristine being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of dihydroergocornine, dihydro-a-ergocryptine,
dihydroergocristine and dihydro-/?-ergocryptine to that of the
internal standard is not more than 0.5%.
(2) Relative contents of dihydroergocornine mesilate, di-
hydroergocryptine mesilate and dihydroergocristine mesilate
— Calculate the relative amounts of dihydroergocornine
mesilate, dihydroergocryptine mesilate (dihydro-a-ergocryp-
tine mesilate and dihydro-/?-ergocryptine mesilate) and di-
hydroergocristine mesilate from the chromatogram obtained
in Assay (1) for the sample solution using the following equa-
tions:
Relative amount (%) of dihydroergocornine mesilate
= {M TA /(M TA + M TB + M TC + M TD )} x 100
Relative amount (%) of dihydroergocryptine mesilate
= {(M TB + M TD )/(M TA + M TB + M TC + M TD )} x 100
Relative amount (%) of dihydroergocristine mesilate
= {M TC /(M TA + M TB + M TC + M TD )} x 100
(3) Ratio of the content of dihydro-a-ergocryptine mesi-
late to dihydro-/?-ergocryptine mesilate — Calculate the ratio
of the amount of dihydro-a-ergocryptine mesilate to di-
hydro-/?-ergocryptine mesilate from the chromatogram ob-
tained in the Assay (1) for the sample solution using the fol-
lowing equations:
Ratio of the content of dihydro-a-ergocryptine
mesilate to dihydro-/?-ergocryptine mesilate
= (M TB /M TD )
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
586
Dilazep Hydrochloride Hydrate / Official Monographs
JP XV
Dilazep Hydrochloride Hydrate
-/^-fef^iti^janti
C31H44N2do.2HCl.H2O: 695.63
3,3'-(l,4-Diazepane-l,4-diyl)dipropyl bis(3,4,5-
trimethoxybenzoate) dihydrochloride
monohydrate [20153-98-4, anhydride]
Dilazep Hydrochloride Hydrate contains not less
than 98.0% of dilazep hydrochloride (C31H44N2
O 10 .2HCl: 677.62), calculated on the dried basis.
Description Dilazep Hydrochloride Hydrate occurs as a
white, crystalline powder. It is odorless.
It is freely soluble in acetic acid (100) and in chloroform,
soluble in water, slightly soluble in ethanol (95) and in acetic
anhydride, and practically insoluble in diethyl ether.
Melting point: 200 - 204°C Immerse the sample in a bath
of 110°C, and raise the temperature at the rate of about 3°C
per minute from 140°C to 150°C, about 10°C per minute
from 160°Cto 195 °C and about 1°C per minute from 195°C.
Identification (1) To 1 mL of a solution of Dilazep
Hydrochloride Hydrate (1 in 100) add 0. 1 mL of a solution of
hydroxylammonium chloride (1 in 10) and 0.1 mL of 8 mol/
L potassium hydroxide TS, and warm in a water bath of 70°
C for 10 minutes. After cooling, add 0.5 mL of dilute
hydrochloric acid and 0.1 mL of iron (III) chloride TS: a pur-
ple color develops.
(2) To 5 mL of a solution of Dilazep Hydrochloride Hy-
drate (3 in 500) add 0.3 mL of Reinecke salt TS: a light red
precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Dilazep Hydrochloride Hydrate (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of
Dilazep Hydrochloride Hydrate as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> Dissolve 1 .0 g of Dilazep Hydrochloride Hydrate
in 100 mL of water: the pH of this solution is between 3.0 and
4.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dilazep Hydrochloride Hydrate in 20 mL of water: the solu-
tion is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.5 g of Dilazep
Hydrochloride Hydrate. Prepare the control solution with
0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Dilazep
Hydrochloride Hydrate according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than lOppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Dilazep Hydrochloride Hydrate according to Method 3,
and perform the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.40 g of Dilazep
Hydrochloride Hydrate in 10 mL of chloroform, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add chloroform to make exactly 200 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of methanol,
ethyl acetate, dichloromethane and hydrochloric acid
(500:200:100:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly Dragendorff's TS for spraying on the
plate: the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> 2.0 - 3.0% (1 g, 105°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Dilazep Hydrochlo-
ride Hydrate, dissolve in 40 mL of a mixture of acetic anhy-
dride and acetic acid (100) (7:3), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 33.88 mg of C3iH44N 2 1( ,.2HCl
Containers and storage Containers — Tight containers.
Diltiazem Hydrochloride
vJU-^T-tfA^I^
C22H2.sN2O4S.HCl: 450.98
(2S,3S>5-[2-(Dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-
oxo-2,3,4,5-tetrahydro-l,5-benzothiazepin-3-yl acetate
monohydrochloride [33286-22-5 ]
Diltiazem Hydrochloride, when dried, contains not
less than 98.5% of C 22 H 26 N 2 4 S.HC1.
Description Diltiazem Hydrochloride occurs as white crys-
tals or crystalline powder. It is odorless.
JPXV
Official Monographs / Diltiazem Hydrochloride
587
It is very soluble in formic acid, freely soluble in water, in
methanol and in chloroform, sparingly soluble in acetoni-
trile, slightly soluble in acetic anhydride and in ethanol
(99.5), and practically insoluble in diethyl ether.
Identification (1) Dissolve 0.05 g of Diltiazem Hydrochlo-
ride in 1 mL of 1 mol/L hydrochloric acid TS, add 2 mL of
ammonium thiocyanate-cobaltous nitrate TS and 5 mL of
chloroform, shake well, and allow to stand: a blue color de-
velops in the chloroform layer.
(2) Proceed as directed under Oxygen Flask Combustion
Method <1.06> with 0.03 g of Diltiazem Hydrochloride, using
20 mL of water as the absorbing liquid, and prepare the test
solution: the test solution responds to the Qualitative Tests
<1.09> (1) for sulfate.
(3) Dissolve 0.01 g of Diltiazem Hydrchloride in 0.01
mol/L hydrochloric acid TS to make 100 mL. To 2 mL of the
solution add 0.01 mol/L hydrochloric acid TS to make 20
mL. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of Diltia-
zem Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 1741 cm -1 , 1678 cm -1 , 1252 cm -1 and
1025 cm" 1 .
(5) A solution of Diltiazem Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]™: + 115 - + 120° (after drying,
0.20 g, water, 20 mL, 100 mm).
Melting point <2.60> 210 - 215 °C (with decomposition).
pH <2.54> Dissolve 1 .0 g of Diltiazem Hydrochloride in 100
mL of water: the pH of this solution is between 4.3 and 5.3.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Diltiazem Hydrochloride in 20 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test with 1.0 g of Diltia-
zem Hydrochloride. Prepare the control solution with 0.50
mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Diltia-
zem Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Place 1.0 g of Diltiazem Hydrochlo-
ride in a decomposition flask, add 5 mL of nitric acid and 2
mL of sulfuric acid, put a small funnel on the neck of the
flask, and heat cautiously until white fumes are evolved. Af-
ter cooling, add 2 mL of nitric acid, heat, and repeat this
procedure twice, add several 2-mL portions of hydrogen
peroxide (30), and heat until the solution becomes colorless
to pale yellow. After cooling, add 2 mL of saturated solution
of ammonium oxalate monohydrate, and heat again until
white fumes are evolved. After cooling, add water to make 5
mL, use this solution as the test solution, and perform the
test: the test solution has no more color than the following
control solution (not more than 2 ppm).
Control solution: Proceed in the same manner as the test
solution without Diltiazem Hydrochloride, add 2.0 mL of
Standard Arsenic Solution and water to make 5 mL, and pro-
ceed in the same manner as the test solution.
(5) Related substances — Dissolve 50 mg of Diltiazem
Hydrochloride in 50 mL of diluted ethanol (99.5) (4 in 5), and
use this solution as the sample solution. Measure exactly
1 mL of the sample solution, add diluted ethanol (99.5) (4 in
5) to make exactly 200 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 20 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions. Determine each peak area of both solutions by auto-
matic integration method: the total peak area of peaks other
than the peak of diltiazem obtained from the sample solution
is not more than 3/5 times the peak area of diltiazem
obtained from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 240 nm).
Column: A stainless steel column 4.6 mm in inside diame-
ter and 15 cm in length, packed with octadecylsilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 8 g of sodium acetate trihydrate
and 1.5 g of tf-camphorsulfonic acid in 500 mL of water, and
filter using a membrane filter (0.4 fim in pore size). Add 250
mL each of acetonitrile and methanol to the filtrate, and ad-
just the solution to a pH of 6.6 by adding sodium acetate tri-
hydrate.
Flow rate: Adjust the flow rate so that the retention time of
diltiazem is about 9 minutes.
Time span of measurement: About twice as long as the
retention time of diltiazem beginning after the solvent peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add diluted ethanol (99.5) (4 in 5) to make
exactly 10 mL. Confirm that the peak area of diltiazem
obtained from 20 /xL of this solution is equivalent to 15 to
25% of that of diltiazem obtained from 20 /uL of the stan-
dard solution.
System performance: Dissolve 0.03 g of Diltiazem
Hydrochloride, 0.02 g of d-3-hydroxy-cis-2,3-dihydro-5-[2-
(dimethylamino)ethyl]-2-(4-methoxyphenyl)-l , 5-benzothia-
zepin-4-(5//)-one hydrochloride and 0.02 g of phenylbenzo-
ate in 160 mL of ethanol (99.5), and add water to make 200
mL. Perform the test with 20 /xL of this solution as directed
under Liquid Chromatography under the above operating
conditions: rf-3-hydroxy-c/s-2,3-dihydro-5-[2-(dimethyl-
amino)ethyl] - 2 - (4 - methoxyphenyl) - 1, 5 - benzothiazepin-
4(5//)-one, diltiazem and phenyl benzoate are eluted in this
order with the resolutions between the peaks of rf-3-hydroxy-
cis - 2, 3 - dihydro - 5 - [2 - (dimethylamino)ethyl] - 2 - (4 -
methoxyphenyl)-l,5-benzothiazepin-4(5//)-one and diltia-
zem and between the peaks of diltiazem and phenyl benzoate
being not less than 2.5, respectively.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
diltiazem is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
588
Dimemorfan Phosphate / Official Monographs
JP XV
Assay Weigh accurately about 0.7 g of Diltiazem
Hydrochloride, previously dried, dissolve in 2.0 mL of for-
mic acid, add 60 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 45.10 mg of C 22 H 26 N 2 4 S.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dimemorfan Phosphate
; y z EJL-7 7>U>lg±M
HjPOj
C 18 H 25 N.H 3 P04: 353.39
(95, 135',14S)-3,17-Dimethylmorphinan monophosphate
[36304-84-4]
Dimemorfan Phosphate, when dried, contains not
less than 98.5% of C 18 H 25 N.H 3 P04.
Description Dimemorfan Phosphate occurs as white to pale
yellowish white crystals or crystalline powder.
It is freely soluble in acetic acid (100), sparingly soluble in
water and in methanol, slightly soluble in ethanol (95), and
practically insoluble in diethyl ether.
Melting point: about 265 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Dimemorfan Phosphate (1 in 5000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelenghs.
(2) Determine the infrared absorption spectrum of
Dimemorfan Phosphate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibits similar intensities
of absorption at the same wave numbers.
(3) To 2 mL of a solution of Dimemorfan Phosphate (1
in 100) add 2 to 3 drops of silver nitrate TS: a yellow
precipitate is formed, and it dissolves on the addition of di-
lute nitric acid.
Optical rotation <2.49> [a]™: +25 - +27° (after drying, 1
g, methanol, 100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Dimemorfan Phosphate in 100
mL of water: the pH of this solution is between 4.0 and 5.0.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Dimemorfan Phosphate according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.U> — Prepare the test solution with 1.0 g
of Dimemorfan Phosphate according to Method 3, and per-
form the test. Use 10 mL of a solution of magnesium nitrate
hexahydrate in ethanol (95) (1 in 10) (not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Dimemorfan
Phosphate in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of methanol, chloroform and ammonia
solution (28) (150:150:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly the plate with Dragendorff's
TS for spraying: the spots other than the principal spot from
the sample solution are not more intense than the spot from
the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Assay Weigh accurately about 0.6 g of Dimemorfan Phos-
phate, previously dried, dissolve in 100 mL of acetic acid
(100), and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 35.34 mg of C 18 H 25 N.H 3 P04
Containers and storage Containers — Tight containers.
Dimenhydrinate
V
->*>\1 KU^-r-
CH 3
I
CH,
C 17 H 21 N0.C 7 H 7 C1N 4 2 : 469.96
2-(Diphenylmethoxy)-A r ,Af-dimethylethylamine —
8-chloro-l,3-dimethyl-l//-purine-2,6(3//,7//)-dione
(1/1) [525-57-5]
Dimenhydrinate, when dried, contains not less
than 53.0% and not more than 55.5% of diphenhydra-
mine (C 17 H 2 iNO: 255.36), and not less than 44.0% and
not more than 47.0% of 8-chlorotheophylline
(C 7 H 7 C1N 4 2 : 214.61).
Description Dimenhydrinate occurs as a white, crystalline
powder. It is odorless, and has a bitter taste.
It is very soluble in chloroform, freely soluble in ethanol
(95), and slightly soluble in water and in diethyl ether.
Identification (1) Dissolve 0.5 g of Dimenhydrinate in 30
mL of dilute ethanol, add 30 mL of water, and use this solu-
tion as the sample solution. Transfer 30 mL of the sample so-
lution to a separator, and add 2 mL of ammonia solution
(28). Extract with two 10-mL portions of diethyl ether, com-
JPXV
Official Monographs / Dimenhydrinate Tablets
589
bine the diethyl ether extracts, wash the combined extracts
with 5 mL of water, and then extract the combined extracts
with 15 mL of diluted hydrochloric acid (1 in 100). With this
acid extract perform the following tests.
(i) To 5 mL of this acid extract add 5 drops of Reinecke
salt TS: a light red precipitate is produced.
(ii) To 10 mL of this acid extract add 10 mL of 2,4,6-
trinitrophenol TS dropwise, and allow to stand for 30
minutes. Collect the precipitate by filtrating, recrystallize
from dilute ethanol, and dry at 105°C for 30 minutes: the
crystals melt <2.60> between 128°C and 133°C.
(2) To 30 mL of the sample solution obtained in the Iden-
tification (1) add 2 mL of dilute sulfuric acid, and cool for 30
minutes. Scratch the inside wall of the container frequently to
facilitate crystallization. Filter, and wash the white crystals
with a small amount of ice-cooled water. Dry the crystals for
1 hour at 105°C: the crystals melt <2.60> between 300°C and
305 °C with decomposition.
(3) To 0.01 g of the crystals obtained in the Identification
(2) add 10 drops of hydrogen peroxide TS and 1 drop of
hydrochloric acid, and evaporate on a water bath to dryness:
the residue shows a yellow-red color. When the dish contain-
ing the residue is held over a vessel containing 2 to 3 drops of
ammonia TS, the color changes to red-purple, which is dis-
charged on the addition of 2 to 3 drops of sodium hydroxide
TS.
(4) Mix well 0.05 g of the crystals obtained in the Identifi-
cation (2) with 0.5 g of sodium peroxide in a nickel crucible,
and heat until the mass melts. Cool, dissolve the melted mass
in 20 mL of water, and acidify with dilute nitric acid: the
solution responds to the Qualitative Tests for chloride <1.09>.
Melting point <2.60> 102 - 107 °C
Purity (1) Chloride <1.03>— Transfer 50 mL of the filtrate
obtained in the Assay (2) to a Nessler tube, add 1 mL of nitric
acid, and allow to stand for 5 minutes: the turbidity of the
solution is not greater than that of the following control solu-
tion.
Control solution: Dilute 0.25 mL of O.Olmol/L
hydrochloric acid VS with 6 mL of dilute nitric acid and with
water to make 50 mL, add 1 mL of silver nitrate TS, and al-
low to stand for 5 minutes (not more than 0.044%).
(2) Bromide and iodide — Place 0. 10 g of Dimenhydrinate
in a glass-stoppered test tube, and add 0.05 g of sodium ni-
trite, 10 mL of chloroform and 10 mL of dilute hydrochloric
acid. Stopper, shake well, and allow to stand: the chloroform
layer remains colorless.
Loss on drying <2.41> Not more than 0.5% (3 g, in vacuum,
phosphorus (V) oxide, 24 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay (1) Diphenhydramine — Weigh accurately about 0.5
g of Dimenhydrinate, previously dried, transfer to a 250-mL
separator, and add 50 mL of water, 3 mL of ammonia TS
and 10 g of sodium chloride. Extract with six 15-mL portions
of diethyl ether with shaking, combine the diethyl ether ex-
tracts, and wash the combined diethyl ether extracts with
three 50-mL portions of water. To the diethyl ether extracts
add exactly 25 mL of 0.05 mol/L sulfuric acid VS, and add
25 mL of water. Shake thoroughly, and evaporate the diethyl
ether gently. Cool, and titrate the excess sulfuric acid with 0. 1
mol/L sodium hydroxide VS (indicator: 3 drops of methyl
red TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.05 mol/L sulfuric acid VS
= 25.54 mg C 17 H 21 NO
(2) 8-Chlorotheophylline — Weigh accurately about 0.8 g
of Dimenhydrinate, previously dried, transfer to a 200-mL
volumetric flask, add 50 mL of water, 3 mL of ammonia TS
and 6 mL of a solution of ammonium nitrate (1 in 10), and
heat on a water bath for 5 minutes. Add exactly 25 mL of 0.1
mol/L silver nitrate VS, heat on a water bath for 15 minutes
with occasional shaking, cool, and add water to make exactly
200 mL. Allow to stand overnight to settle the precipitate,
and filter through a dry filter paper, discarding the first 20 mL
of the filtrate. Measure exactly 100 mL of the subsequent
filtrate, acidify with nitric acid, add 3 mL of nitric acid, and
titrate the excess silver nitrate with 0.1 mol/L ammonium
thiocyanate VS (indicator: 2 mL of ammonium iron (III) sul-
fate TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 21.46 mg of C 7 H 7 C1N 4 2
Containers and storage Containers — Well-closed contain-
ers.
Dimenhydrinate Tablets
Dimenhydrinate Tablets contain not less than 95%
and not more than 105% of the labeled amount of
dimenhydrinate (C 17 H 21 N0.C 7 H 7 C1N 4 2 : 469.96).
Method of preparation Prepare as directed under Tablets,
with Dimenhydrinate.
Identification (1) Triturate a quanity of powdered Dimen-
hydrinate Tablets, equivalent to 0.5 g of Dimenhydrinate ac-
cording to the labeled amount, with 25 mL of warm ethanol
(95), and filter. Dilute the filtrate with 40 mL of water, and
filter again. Use the filtrate as the sample solution. Transfer
30 mL of the sample solution to a separator, and proceed as
directed in the Identification (1) under Dimenhydrinate.
(2) With 30 mL of the sample solution obtained in (1),
proceed as directed in the Identification (2), (3) and (4) under
Dimenhydrinate.
Assay Weigh accurately, and powder not less than 20
Dimenhydrinate Tablets. Weigh accurately a portion of the
powder, equivalent to about 0.5 g of dimenhydrinate
(C 17 H 21 N0.C 7 H 7 C1N 4 2 ), transfer to a flask, add 40 mL of
ethanol (95), and heat with swirling on a water bath until the
solution just boils. Continue to heat for 30 seconds, and filter
through a glass filter (G4). Wash the filter with warm ethanol
(95), transfer the filtrate and washings to a flask, and
evaporate the ethanol on a water bath to make 5 mL. Add 50
mL of water, 3 mL of ammonia TS and 6 mL of a solution of
ammonium nitrate (1 in 10), heat the mixture on a water bath
for 5 minutes, add exactly 25 mL of 0.1 mol/L silver nitrate
VS, and heat on a water bath for 15 minutes with occasional
shaking. Transfer the mixture to a 200-mL volumetric flask,
590
Dimercaprol / Official Monographs
JP XV
using water to rinse the flask, cool, add water to make exactly
200 mL, and proceed as directed in the Assay (2) under
Dimenhydrinate .
Each mL of 0.1 mol/L silver nitrate VS
= 47.00 mg of C 17 H 21 N0.C 7 H 7 C1N 4 2
Containers and storage Containers — Well-closed contain-
ers.
Dimercaprol
and enantiomer
C 3 H 8 OS 2 : 124.23
(2flS>2,3-Disulfanylpropan-l-ol [59-52-9]
Dimercaprol contains not less than 98.5% and not
more than 101.5% of C 3 H 8 OS 2 .
Description Dimercaprol is a colorless or pale yellow liquid.
It has a mercaptan-like, disagreeable odor.
It is miscible with methanol and with ethanol (99.5)
It is soluble in peanut oil, and sparingly soluble in water.
It shows no optical rotation.
Identification (1) Add 1 drop of Dimercaprol to a mixture
of 1 drop of a solution of cobalt (II) chloride hexahydrate (1
in 200) and 5 mL of water: a yellow-brown color develops.
(2) Determine the infrared absorption spectrum of
Dimercaprol as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Specific gravity <2.56> df : 1.238 - 1.248
Purity (1) Clarity and color of solution — Dissolve 1.0 mL
of Dimercaprol in 20 mL of peanut oil: the solution is clear
and colorless to pale yellow.
(2) Bromide — To 2.0 g of Dimercaprol add 25 mL of di-
lute potassium hydroxide-ethanol TS, and heat in a water
bath under a reflux condenser for 2 hours. Evaporate the
ethanol in a current of warm air, add 20 mL of water, and
cool. Add a mixture of 10 mL of strong hydrogen peroxide
and 40 mL of water, boil gently under a reflux condenser for
10 minutes, and filter rapidly after cooling. Wash the residue
with two 10-mL portions of water, combine the washings
with the filtrate, add 10 mL of dilute nitric acid and exactly 5
mL of 0.1 mol/L silver nitrate VS, and titrate <2.50> the ex-
cess silver nitrate with 0.1 mol/L ammonium thiocyanate VS
(indicator: 2 mL of ammonium iron (III) sulfate TS). Per-
form a blank determination: not more than 1.0 mL of 0.1
mol/L silver nitrate VS is consumed.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dimer-
caprol according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
Assay Weigh accurately about 0.15 g of Dimercaprol into a
glass-stoppered flask, dissolve in 10 mL of methanol, and ti-
trate <2.50> immediately with 0.05 mol/L iodine VS until a
pale yellow color is produced. Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.05 mol/L iodine VS = 6.211 mg of C 3 H 8 OS 2
Containers and storage Containers — Tight containers.
Storage — Not exceeding 5°C.
Dimercaprol Injection
Dimercaprol Injection is an oily solution for injec-
tion.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of dimercaprol
(C 3 H 8 OS 2 : 124.23).
Method of preparation Prepare as directed under Injec-
tions, with Dimercaprol. Benzyl Benzoate or Benzyl Alcohol
may be added to increase the solubility.
Description Dimercaprol Injection is a clear, colorless or
light yellow liquid. It has an unpleasant odor.
Identification Measure a volume of Dimercaprol Injection,
equivalent to 30 mg of Dimercaprol according to the labeled
amount, and proceed as directed in the Identification (1).
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 2: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Pipet a volume of Dimercaprol Injection, equivalent
to about 0.1 g of dimercaprol (C 3 H 8 OS 2 ), into a flask, and
rinse the pipet several times with a mixture of methanol and
diethyl ether (3:1), adding the rinsings to the flask. Add the
mixture of methanol and diethyl ether (3:1) to make 50 mL,
and titrate <2.50> with 0.05 mol/L iodine VS until a yellow
color persists. Perform a blank determination, and make any
necessary correction.
Each mL of 0.05 mol/L iodine VS
= 6.211 mg of C 3 H 8 OS 2
Containers and storage Containers — Hermetic containers.
Storage — In a cold place.
JPXV
Official Monographs / Dimorpholamine Injection 591
Dimorpholamine
i>=£)l*s7Z. >
o
Y
A.
CHj
KJ
C 2 „H 38 N 4 4 : 398.54
A f ,A^'-Ethylenebis(A f -butylmorpholine-4-carboxamide)
[119-48-2]
Dimorpholamine, when dried, contains not less than
98.0% and not more than 101.0% of C 20 H 38 N 4 O 4 .
Description Dimorpholamine is a white to light yellow,
crystalline powder, masses or syrupy liquid.
It is very soluble in ethanol (99.5) and in acetic anhydride,
and soluble in water.
The pH of a solution prepared by dissolving 1.0 g of
Dimorpholamine in 10 mL of water is between 6.0 and 7.0.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Dimorpholamine (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Dimorpholamine, previously dried, as directed in the potassi-
um bromide disk method under the Infrared Spectrophoto-
metry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dimorpholamine in 50 mL of water: the solution is clear and
colorless to pale yellow.
(2) Chloride <1.03> — To 20 mL of the solution obtained
in (1) add 6 mL of dilute nitric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 0.40 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(3) Sulfate <1.14> — To 10 mL of the solution obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.096%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Dimor-
pholamine according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(5) Related substances — Dissolve 0.20 g of Dimorphola-
mine in 10 mL of ethanol (99.5), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
ethanol (99.5) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethanol (99.5) and water
(4:1) to a distance of about 10 cm, and air-dry the plate. Al-
low the plate to stand in iodine vapor for 10 minutes: the spot
other than the principal spot from the sample solution is not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 8 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Dimorpholamine,
previously dried, dissolve in 50 mL of acetic anhydride, and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.85 mg of C 2 oH3 8 N 4 04
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dimorpholamine Injection
-x^EJUTfcvS >%Mfe
Dimorpholamine Injection is an aqueous solution
for injection.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of dimorpholamine
(C 2 oH 38 N 4 4 : 398.54).
Method of preparation Prepare as directed under Injec-
tions, with Dimorpholamine.
Description Dimorpholamine Injection is a clear, colorless
liquid.
pH: 3.0-5.5
Identification (1) To a volume of Dimorpholamine Injec-
tion, equivalent to 0.1 g of Dimorpholamine according to the
labeled amount, add 3 drops of Dragendorff's TS: an orange
color develops.
(2) To a volume of Dimorpholamine Injection, equiva-
lent to 50 mg of Dimorpholamine according to the labeled
amount, add 1 mL of dilute hydrochloric acid, and evaporate
on a water bath to dryness. Dissolve the residue in 2 mL of
hydrochloric acid, boil for 10 minutes under a reflux con-
denser, and evaporate to dryness on a water bath. Dissolve
the residue with 1 mL of water, neurtralize with sodium
hydroxide TS, and add 0.2 mL of a solution of acetaldehyde
(1 in 20), 0.1 mL of sodium pentacyanonitrosyl ferrate (III)
TS and 0.5 mL of sodium carbonate TS: a blue color de-
velops.
Bacterial endotoxins <4.01> Less than 5.0 EU/mg. Perform
the test with the sample diluted to 0.15 w/v% with water for
bacterial endotoxins test.
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
592 Dinoprost / Official Monographs
JP XV
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Measure exactly a volume of Dimorpholamine
Injection, equivalent to about 30 mg of dimorpholamine
(C 2 oH3 8 N 4 04), and add water to make exactly 200 mL. Pipet
1 mL of this solution, shake with exactly 4 mL of the inernal
standard solution for 5 minutes, and use this solution as the
sample solution. Separately, weigh accurately about 0.15 g of
dimorpholamine for assay, previously dried in a desiccator
(in vacuum, phosphorus (V) oxide) for 8 hours, and dissolve
in water to make exactly 1000 mL. Pipet 1 mL of this
solutionn, shake with exactly 4 mL of the inernal standard
solution for 5 minutes, and use this solution as the standard
solution. Perform the test with 10 /xh each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
dimorpholamine to that of the inernal standard.
Amount (mg) of dimorpholamine (C20H38N4O4)
= W s x (Qj/Qs) x (1/5)
W s : Amount (mg) of dimorpholamine for assay
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 25,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 216 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
dimorpholamine is about 4 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, dimorpholamine and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of dimorpholamine to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Dinoprost
Prostaglandin F 2a
HO. f >'
C20H34O5: 354.48
(5Z)-7-{(17?,27?,37?,5S)-3,5-Dihydroxy-2-[(17},3S)-3-
hydroxyoct-l-en-l-yl]cyclopentyl}hept-5-enoic acid
[557-77-7]
Dinoprost contains not less than 98.5% of C20H34O5,
calculated on the anhydrous basis.
Description Dinoprost occurs as white, waxy masses or
powder, or a clear, colorless to light yellow and viscous liq-
uid. It is odorless.
It is very soluble in 7V,7V-dimethylformamide, freely solu-
ble in methanol, in ethanol (99.5) and in diethyl ether, and
very slightly soluble in water.
Identification (1) To 5 mg of Dinoprost add 2 mL of sul-
furic acid, and dissolve by shaking for 5 minutes: a dark red
color develops. To this solution add 30 mL of sulfuric acid:
an orange color develops with a green fluorescence.
(2) Dissolve 1 mg of Dinoprost in 50 mL of diluted sul-
furic acid (7 in 10), and warm in a water bath heated at 50°C
for 40 minutes. After cooling, determine the absorption spec-
trum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(3) Warm Dinoprost at 40°C to effect a liquid, and deter-
mine the infrared absorption spectrum of the liquid as direct-
ed in the liquid film method under Infrared Spectrophoto-
metry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibits similar intensities of absorp-
tion at the same wave numbers.
Optical rotation <2.49> [a]™:
(99.5), 10 mL, 100 mm).
+ 24- +31° (0.2 g, ethanol
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Dinoprost in 5 mL of ethanol (99.5): the solution is clear
and colorless to pale yellow.
(2) Related substances — Dissolve 10 mg of Dinoprost in 2
mL of methanol, add water to make 10 mL, and use this
solution as the sample solution. Pipet 3 mL of the sample so-
lution, add diluted methanol (1 in 5) to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine each
peak area of these solutions by the automatic integration
method: the total area of the peaks other than the peak of
dinoprost from the sample solution is not larger than the
peak area of dinoprost from the standard solution.
JPXV
Official Monographs / Diphenhydramine and Bromovalerylurea Powder
593
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 205 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.02 mol/L potassium di-
hydrogenphosphate TS and acetonitrile (5:2).
Flow rate: Adjust the flow rate so that the retention time of
dinoprost is about 20 minutes.
Selection of column: Dissolve 0.01 g each of isopropyl
parahydroxybenzoate and propyl parahydroxybenzoate in 2
mL of methanol, and add water to make 10 mL. To 1 mL of
this solution add diluted methanol (1 in 5) to make 30 mL,
proceed with 10 /uL of this solution under the above operating
conditions, and calculate the resolution. Use a column giving
elution of isopropyl parahydroxybenzoate and propyl para-
hydroxybenzoate in this order with the resolution between
these peaks being not less than 2.5.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of dinoprost from the standard solution
composes 5% to 15% of the full scale.
Time span of measurement: About 1.5 times as long as the
retention time of dinoprost beginning after the solvent peak.
Water <2.48> Not more than 0.5% (0.3 g, direct titration).
Assay Weigh accurately about 50 mg of Dinoprost, dissolve
in 30 mL of /V,7V-dimethylformamide, and titrate <2.50> with
0.02 mol/L tetramethylammonium hydroxide VS under a
stream of nitrogen (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.02 mol/L tetramethylammonium
hydroxide VS
= 7.090 mg of C 2 oH 3 40 5
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a place not exceeding
5°C.
Diphenhydramine
/7i>t K7
C 17 H 21 NO: 255.35
2-(Diphenylmethoxy)-/V,A r -dimethylethylamine [58-73-1]
It is miscible with acetic anhydride, with acetic acid (100),
with ethanol (95) and with diethyl ether.
It is very slightly soluble in water.
Boiling point: about 162°C (in vacuum, 0.67 kPa).
Refractive index n 2 °: about 1.55
It is gradually affected by light.
Identification (1) To 0.05 g of Diphenhydramine add 2
mL of sulfuric acid: an orange-red precipitate is produced
immediately, and its color changes to red-brown on standing.
Add carefully 2 mL of water to this solution: the intensity of
the color changes, but the color tone does not change.
(2) Dissolve 0.1 g of Diphenhydramine in 1 mL of dilute
ethanol, add an excess of a saturated solution of 2,4,6-
trinitrophenol in dilute ethanol with stirring, and cool in ice.
Collect the produced crystals, recrystallize from dilute
ethanol, and dry at 105 °C for 30 minutes: the crystals melt
between 128°C and 133°C.
Specific gravity <2.56> df : 1.013 - 1.020
Purity (1) /?-Dimethylaminoethanol — Dissolve 1.0 g of
Diphenhydramine in 20 mL of diethyl ether, and extract with
two 10-mL portions of water with thorough shaking. Com-
bine the water extracts, and add 2 drops of phenolphthalein
TS and 1.0 mL of 0.05 mol/L sulfuric acid VS: no red color
develops.
(2) Benzohydrol — Transfer 1.0 g of Diphenhydramine to
a separator, dissolve in 20 mL of diethyl ether, and extract
with two 25-mL portions of diluted hydrochloric acid (1 in
15) with thorough shaking. Separate the diethyl ether layer,
evaporate slowly on a water bath, and dry in a desiccator (in
vacuum, silica gel) for 2 hours: the mass of the residue is not
more than 20 mg.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Diphen-
hydramine according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Diphenhydramine,
dissolve in 50 mL of a mixture of acetic anhydride and acetic
acid (100) (7:3), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 25.54 mg of C 17 H 21 NO
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled.
Diphenhydramine and
Bromovalerylurea Powder
Diphenhydramine contains not less than 96.0% of
C 17 H 21 NO.
Description Diphenhydramine is a clear, light yellow to yel-
low liquid. It has a characteristic odor, and has a burning
taste at first, followed by a slight sensation of numbness on
the tongue.
Method of preparation
Diphenhydramine Tannate
Bromovalerylurea
Starch, Lactose Hydrate, or
their mixture
90 g
500 g
a sufficient quantity
594
Diphenhydramine Hydrochloride / Official Monographs
JP XV
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Diphenhydramine and Bromovalerylurea Pow-
der occurs as a slightly grayish white powder.
Identification (1) To 0.1 g of Diphenhydramine and
Bromovalerylurea Powder add 5 mL of dilute hydrochloric
acid, 1 mL of ethanol (95) and 10 mL of water, shake, and
filter. To the filtrate add 10 mL of sodium hydroxide TS, and
extract with 10 mL of chloroform. Separate the chloroform
layer, add 1 mL of bromophenol blue TS, and shake: a yel-
low color develops in the chloroform layer (diphenhydramine
tannate).
(2) Shake 0.02 g of Diphenhydramine and Bromo-
valerylurea Powder with 10 mL of diethyl ether, filter, and
evaporate the filtrate on a water bath. Dissolve the residue in
2 mL of sodium hydroxide TS, and add 5 mL of dimethyl-
glyoxime-thiosemicarbazide TS, and heat on a water bath for
30 minutes: a red color develops (bromovalerylurea).
(3) Shake 0.3 g of Diphenhydramine and
Bromovalerylurea with 5 mL of methanol, filter, and use the
filtrate as the sample solution. Dissolve 0.15 of
bromovalerylurea and 0.03 g of diphenhydramine tannate in
5 mL each of methanol, and use the solutions as standard so-
lution (1) and standard solution (2). Perform the test as
directed under Thin-layer Chromatography <2.03> with these
solutions. Spot 5 /uL each of the sample solution and stan-
dard solutions (1) and (2) on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate in a mixture of ethyl acetate, ethanol (99.5) and ammo-
nia solution (28) (50:5:1) to a distance of about 10 cm. Air-
dry the plate, and examine under ultraviolet light (main
wavelength: 254 nm): 3 spots from the sample solution and
the corresponding spot from standard solutions (1) and (2)
show the same Rf value. Spray Dragendorff's TS for spraying
evenly on the plate: the spot from the standard solution (2)
and the corresponding spot from the sample solution reveal
an orange color.
Containers and storage Containers — Well-closed contain-
ers.
Diphenhydramine Hydrochloride
'7x>t: h'7
-m.mik
C 17 H 21 NO.HCl: 291.82
2-(Diphenylmethoxy)-Af,iV-dimethylethylamine
monohydrochloride [147-24-0]
Diphenhydramine Hydrochloride, when dried, con-
tains not less than 98.0% of C 17 H 21 NO.HCl.
Description Diphenhydramine Hydrochloride occurs as
white crystals or crystalline powder. It is odorless, and has a
bitter taste, followed by a sensation of numbness on the ton-
gue.
It is very soluble in methanol and in acetic acid (100), freely
soluble in water and in ethanol (95), sparingly soluble in acet-
ic anhydride, and practically insoluble in diethyl ether.
It is gradually affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Diphenhydramine Hydrochloride in methanol (1
in 2000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Dipenhydramine Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Diphenhydramine Hydrochloride (1 in
50) responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Diphenhydramine Hydrochlo-
ride in 10 mL of water: the pH of this solution is between 4.0
and 5.0.
Melting point <2.60> 166 - 170°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Diphenhydramine Hydrochloride in 10 mL of water: the so-
lution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Diphen-
hydramine Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Diphenhydra-
mine Hydrochloride in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add methanol to make exactly 200 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of hexane,
ethyl acetate, methanol and ammonia solution (28) (10:4:2:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly iodine TS on the plate: the spots other than the prin-
cipal spot from the sample solution and the spot on the origi-
nal point are not more intense than the spot from the stan-
dard solution.
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Diphenhydramine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3). Titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.18 mg of C 17 H 21 NO.HCl
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Diphenhydramine Tannate
595
Storage — Light-resistant.
Diphenhydramine, Phenol and
Zinc Oxide Liniment
•/7i>tp7; >-7i/-JU-
Method of preparation
Diphenhydramine
Phenol and Zinc Oxide Liniment
•U-/>r-
20 j
980;
To make 1000 g
Dissolve and mix the above ingredients.
Description Diphenhydramine, Phenol and Zinc Oxide
Liniment is a white to whitish, pasty mass. It has a slight odor
of phenol.
Identification (1) To 3 g of Diphenhydramine, Phenol
and Zinc Oxide Liniment add 20 mL of hexane, shake well,
and separate the hexane layer. Shake thoroughly the hexane
solution with 10 mL of 0.2 mol/L hydrochloric acid.
Separate the aqueous layer, and adjust with sodium
hydroxide TS to a pH of 4.6. Add 1 mL of bromophenol
blue-potassium biphthalate TS and 10 mL of chloroform,
and shake: a yellow color develops in the chloroform layer
(diphenhydramine) .
(2) Place 1 g of Diphenhydramine, Phenol and Zinc
Oxide Liniment in a porcelain crucible, gradually raise the
temperature by heating until the mass is charred, and ignite
strongly: a yellow color is produced, and disappears on cool-
ing. To the residue add 10 mL of water and 5 mL of dilute
hydrochloric acid, shake well, and filter. Add 2 to 3 drops of
potassium hexacyanoferrate (II) TS to the filtrate: a white
precipitate is produced (zinc oxide).
(3) Shake 0.5 g of Diphenhydramine, Phenol and Zinc
Oxide Liniment with 1 mL of water and 5 mL of chloroform,
filter, and use the filtrate as the sample solution. Dissolve 0.01
g each of diphenhydramine and phenol in 5 mL each of chlo-
roform, and use these solutions as standard solution (1) and
standard solution (2). Perform the test as directed under
Thin-layer Chromatography <2.03> with the sample solution
and the standard solutions. Spot 5 /uL each of the sample so-
lution and standard solutions (1) and (2) on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethyl acetate, ethanol (99.5) and ammonia solu-
tion (28) (50:5:1) to a distance of about 10 cm, and air-dry the
plate. Allow the plate to stand in iodine vapor: two spots
from the sample solution and each spot from standard solu-
tion (1) and standard solution (2) show the same Rf value.
Sublime iodine, and spray Dragendorff's TS evenly upon the
plate: the spot from standard solution (1) and the cor-
responding spot from the sample solution reveal an orange
color.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Diphenhydramine Tannate
Diphenhydramine Tannate is a compound of
diphenhydramine and tannic acid, and contains not
less than 25.0% and not more than 35.0% of diphen-
hydramine (C 17 H 21 NO: 255.35).
Description Diphenhydramine Tannate occurs as a grayish
white to light brown powder. It is odorless or has a slight,
characteristic odor. It is tasteless.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water and in diethyl ether.
Identification (1) To 1 g of Diphenhydramine Tannate
add 15 mL of water and 0.3 mL of dilute hydrochloric acid,
shake thoroughly for 1 minute, filter, and use this filtrate as
the sample solution. Transfer 10 mL of the sample solution
to a separator, extract with two 20-mL portions of chlo-
roform, combine the chloroform extracts, and evaporate on
a water bath to dryness. To 5 mL of a solution of the residue
(1 in 100) add 5 drops of Reinecke salt TS: a light red
precipitate is produced.
(2) To 10 mL of a solution of the residue obtained in (1)
(1 in 100) add 10 mL of 2,4,6-trinitrophenol TS dropwise,
and allow to stand for 30 minutes. Collect the precipitate by
filtration, recrystallize from dilute ethanol, and dry at 105°C
for 30 minutes: the crystals melt <2.60> between 128°C and
133°C.
(3) To 1 mL of the sample solution obtained in (1) add 1
drop of iron (III) chloride TS: a dark blue-purple color de-
velops.
Purity Heavy metals <1.07> — Proceed with 1 .0 g of Diphen-
hydramine Tannate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41>
5 hours).
Not more than 7.0% (1 g, 105°C,
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Transfer about 1.7 g of Diphenhydramine Tannate,
accurately weighed, to a separator, dissolve in 20 mL of
water and 3.0 mL of dilute hydrochloric acid with thorough
shaking, add 20 mL of a solution of sodium hydroxide (1 in
10) and exactly 25 mL of isooctane, shake vigorously for 5
minutes, dissolve 2 g of sodium chloride with shaking, and
allow to stand. To 20 mL of the isooctane layer add exactly
80 mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 25.54 mg of Ci 7 H 21 NO
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
596
Freeze-dried Diphtheria Antitoxin, Equine / Official Monographs
JP XV
Freeze-dried Diphtheria Antitoxin,
Equine
Freeze-dried Diphtheria Antitoxin, Equine, is a
preparation for injection which is dissolved before use.
It contains diphtheria antitoxin in immunoglobulin
of horse origin.
It conforms to the requirements of Freeze-dried
Diphtheria Antitoxin, Equine, in the Minimum Re-
quirements for Biological Products.
Description Freeze-dried Diphtheria Antitoxin, Equine,
becomes a colorless or light yellow-brown, clear liquid or a
slightly whitish turbid liquid on addition of solvent.
Diphtheria Toxoid
^fuyh+v-f k
Diphtheria Toxoid is a liquid for injection contain-
ing diphtheria toxoid prepared by treating diphtheria
toxin with formaldehyde by a method involving no ap-
preciable loss of the immunogenicity.
It conforms to the requirements of Diphtheria Tox-
oid in the Minimum Requirements for Biological
Products.
Description Diphtheria Toxoid is a clear, colorless to light
yellow-brown liquid.
Adsorbed Diphtheria Toxoid for
Adult Use
Adsorbed Diphtheria Toxoid for Adult Use is a liq-
uid for injection containing diphtheria toxoid prepared
by treating diphtheria toxin with formaldehyde by a
method involving no appreciable loss of the immuno-
genicity and very few antigenic substances other than
toxoid, and rendered insoluble with aluminum salt.
It conforms to the requirements of Adsorbed
Diphtheria Toxoid for Adult Use in the Minimum Re-
quirements of Biological Products.
Description Adsorbed Diphtheria Toxoid for Adult Use
becomes a homogeneous, whitish turbid liquid on shaking.
Adsorbed Diphtheria-Purified
Pertussis-Tetanus Combined
Vaccine
Adsorbed Diphtheria-Purified Pertussis-Tetanus
Combined Vaccine is a liquid for injection consisting
of a liquid containing the protective antigen of Bor-
detella pertussis, Diphtheria Toxoid and a liquid con-
taining tetanus toxoid obtained by detoxifying the teta-
nus toxin with formaldehyde solution without impair-
ing its immunogenicity, to which aluminum is added to
make the antigen and the toxoids insoluble.
It conforms to the requirements of Adsorbed
Diphtheria-Purified Pertussis-Tetanus Combined Vac-
cine in the Minimum Requirements for Biological
Products.
Description Adsorbed Diphtheria-Purified Pertussis-Teta-
nus Combined Vaccine becomes a homogeneous, white tur-
bid liquid on shaking.
Diphtheria-Tetanus Combined
Toxoid
'7t'J 7««Ja££ h * V 4 K
v
Diphtheria-Tetanus Combined Toxoid is a liquid for
injection containing diphtheria toxoid and tetanus tox-
oid which are prepared by treating diphtheria toxin and
tetanus toxin, respectively, with formaldehyde by a
method involving no appreciable loss of the immuno-
genicity.
It conforms to the requirements of Diphtheria-Teta-
nus Combined Toxoid in the Minimum Requirements
of Biological Products.
Description Diphtheria-Tetanus Combined Toxoid is a
colorless or light yellow-brown, clear liquid.
Adsorbed Diphtheria-Tetanus
Combined Toxoid
y7fU 7«fca££ r- * V -i K
Adsorbed Diphtheria-Tetanus Combined Toxoid is a
liquid for injection containing diphtheria toxoid and
tetanus toxoid which are prepared by treating diphther-
ia toxin and tetanus toxin, respectively, with formalde-
hyde by a method involving no appreciable loss of the
immunogenicity and rendered insoluble by adding alu-
minum salt.
It conforms to the requirements of Adsorbed
JPXV
Official Monographs / Dipyridamole 597
Diphtheria-Tetanus Combined Toxoid in the
mum Requirements for Biological Products.
Mini-
Description Adsorbed Diphtheria-Tetanus Combined Tox-
oid becomes a homogeneous, whitish turbid liquid on shak-
ing.
Dipyridamole
C24H 4 oN 8 4 : 504.63
2,2',2",2'"-{[4,8-Di(piperidin-l-yl)pyrimido[5,4-rf]
pyrimidine-2,6-diyl]dinitrilo}tetraethanol [58-32-2]
Dipyridamole, when dried,
98.5% of C^H^NsO,
contains not less than
'24 n 40 i>l 8 , -'4-
Description Dipyridamole occurs as yellow crystals or crys-
talline powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in chloroform, sparingly soluble in
methanol and in ethanol (99.5), and practically insoluble in
water and in diethyl ether.
Identification (1) Dissolve 5 mg of Dipyridamole in 2 mL
of sulfuric acid, add 2 drops of nitric acid, and shake: a deep
purple color develops.
(2) Determine the absorption spectrum of a solution of
Dipyridamole in a mixture of methanol and hydrochloric
acid (99:1) (1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Dipyridamole, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 165 - 169 °C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Dipyridamole in 10 mL of chloroform: the solution is clear,
and shows a yellow color.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Dipyridamole according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Dipyridamole according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Dipyridamole
in 50 mL of the mobile phase, and use this solution as the
sample solution. Pipet 0.5 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly
20 [iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peak of dipyridamole from the sample solu-
tion is not larger than the peak area of dipyridamole from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octylsilanized silica gel for
liquid chromatography (5 /tm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 0.2 g of potassium dihydrogen
phosphate in 200 mL of water, and add 800 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
dipyridamole is about 4 minutes.
Time span of measurement: About 5 times as long as the
retention time of dipyridamole.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add the mobile phase to make exactly 25 mL.
Confirm that the peak area of dipyridamole obtained from 20
/iL of this solution is equivalent to 15 to 25% of that of
dipyridamole obtained from 20 /xL of the standard solution.
System performance: Dissolve 7 mg of Dipyridamole and 3
mg of terphenyl in 50 mL of methanol. When the procedure
is run with 20 /iL of this solution under the above operating
conditions, dipyridamole and terphenyl are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
20 /iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
dipyridamole is not more than 1.0%.
Loss on drying <2.41> Not more than 0.2% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Dipyridamole,
previously dried, dissolve in 70 mL of methanol, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 50.46 mg of C 24 H 40 N 8 O4
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
598
Disodium Edetate Hydrate / Official Monographs
JP XV
Disodium Edetate Hydrate
EDTA Sodium Hydrate
NaOsC
HO;?C
•2H z O
C 10 H 14 N 2 Na 2 O 8 .2H 2 O: 372.24
Disodium dihydrogen ethylenediaminetetraacetate
dihydrate [6381-92-6]
Disodium Edetate Hydrate contains not less than
99.0% of C 10 H 14 N 2 Na 2 O 8 .2H 2 O.
Description Disodium Edetate Hydrate occurs as white
crystals or crystalline powder. It is odorless and has a slight,
acid taste.
It is soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
Identification (1) Dissolve 0.01 g of Disodium Edetate
Hydrate in 5 mL of water, add 2 mL of a solution of potassi-
um chromate (1 in 200) and 2 mL of arsenic (III) trioxide TS,
and heat in a water bath for 2 minutes: a purple color de-
velops.
(2) Dissolve 0.5 g of Disodium Edetate Hydrate in 20 mL
of water, and add 1 mL of dilute hydrochloric acid: a white
precipitate is produced. Collect the precipitate, wash with 50
mL of water, and dry at 105 C C for 1 hour: the precipitate
melts <2.60> between 240°C and 244°C (with decomposition).
(3) A solution of Disodium Edetate Hydrate (1 in 20)
responds to the Qualitative Tests <1.09> (1) for sodium salt.
pH <2.54> Dissolve 1 g of Disodium Edetate Hydrate in 100
mL of water: the pH of this solution is between 4.3 and 4.7.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Disodium Edetate Hydrate in 50 mL of water: the solution is
clear and colorless.
(2) Cyanide — Transfer 1.0 g of Disodium Edetate Hy-
drate to a round-bottomed flask, dissolve in 100 mL of water,
add 10 mL of phosphoric acid, and distil. Place 15 mL of 0.5
mol/L sodium hydroxide VS in a 100-mL measuring cylin-
der, which is used as a receiver, and immerse the bottom end
of the condenser into the solution. Distil the mixture until the
distillate measures 100 mL, and use this solution as the sam-
ple solution. Transfer 20 mL of the sample solution to a
glass-stoppered test tube, add 1 drop of phenolphthalein TS,
neutralize with dilute acetic acid, and add 5 mL of phosphate
buffer solution, pH 6.8, and 1.0 mL of diluted sodium tol-
uensulfonchloramide TS (1 in 5). Immediately stopper the
tube, mix gently, and allow to stand for a few minutes. Mix
well with 5 mL of pyridine-pyrazolone TS, and allow to stand
between 20°C and 30°C for 50 minutes: the solution has no
more color than the following control solution.
Control solution: Pipet 1.0 mL of Standard Cyanide Solu-
tion, add 15 mL of 0.5 mol/L sodium hydroxide VS and
water to make exactly 1000 mL, transfer 20 mL of this solu-
tion to a glass-stoppered test tube, and proceed as directed
for the sample solution.
(3) Heavy metals <1.07> — Proceed with 2.0 g of Disodi-
um Edetate Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Disodium Edetate Hydrate according to Method 1, and
perform the test (not more than 2 ppm).
Residue on ignition <2.44> 37.0 - 39.0% (1 g).
Assay Weigh accurately about 1 g of Disodium Edetate Hy-
drate, dissolve in 50 mL of water, add 2 mL of ammonia-am-
monium chloride buffer solution, pH 10.7, and 0.04 g of
eriochrome black T-sodium chloride indicator, and titrate
<2.50> with 0.1 mol/L zinc VS until the color of the solution
changes from blue to red.
Each mL of 0.1 mol/L zinc VS
= 37.22 mg of C 10 H 14 N 2 Na 2 O 8 .2H 2 O
Containers and storage Containers — Well-closed contain-
ers.
Disopyramide
5?Vtf75 K
and enantiomer
C 2I H 29 N 3 0: 339.47
(2i?5')-4-Bis(l-methylethyl)amino-2-phenyl-2-(pyridin-2-
yl)butanamide [3737-09-5]
Disopyramide contains not less than 98.5% of
C21H29N3O, calculated on the dried basis.
Description Disopyramide occurs as white crystals or crys-
talline powder.
It is very soluble in methanol and in ethanol (95), freely
soluble in acetic anhydride, in acetic acid (100) and in diethyl
ether, and slightly soluble in water.
Identification (1) To 1 mL of a solution of Disopyramide
in ethanol (95) (1 in 20) add 10 mL of 2,4,6-trinitrophenol
TS, and warm: a yellow precipitate is formed. Filter this
precipitate, wash with water, and dry at 105°C for 1 hour:
the residue melts <2.60> between 172°C and 176°C.
(2) Determine the absorption spectrum of a solution of
Disopyramide in 0.05 mol/L sulfuric acid-methanol TS (1 in
25,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Determine the infrared absorption spectrum of Dis-
opyramide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
JPXV
Official Monographs / Distigmine Bromide
599
Absorbance <2.24>
(269 nm): 194-205 (10 mg, 0.05
mol/L sulfuric acid-methanol TS, 500 mL).
Purity (1) Heavy metals <1.07> — Dissolve 1.0 g of Dis-
opyramide in 10 mL of ethanol (95), and add 2 mL of dilute
acetic acid (31) and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control so-
lution as follows: to 2.0 mL of Standard Lead Solution add
10 mL of ethanol (95), 2 mL of dilute acetic acid and water to
make 50 mL (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Disopyramide according to Method 3, and perform the
test (not more than 2 ppm).
(3) Related substances — Dissolve 0.40 g of Disopyramide
in 10 mL of methanol, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add methanol to
make exactly 400 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /iL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and ammonia solution (28) (45:4:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, 80°C, 2 hours).
Residue on ignition <2.44> Not more than 0.20% (1 g).
Assay Weigh accurately about 0.25 g of Disopyramide,
dissolve in 30 mL of acetic acid (100), and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 16.97 mg of C 2I H 29 N 3
Containers and storage Containers — Tight containers.
Distigmine Bromide
CH 3
k
a°i
CH 3
N
I
CH
IX)
2Br
V
I
CH;J
C 22 H 32 Br 2 N 4 4 : 576.32
3,3'-[Hexamethylenebis(methyliminocarbonyloxy)]bis(l-
methylpyridinium) dibromide [15876-67-2]
Distigmine Bromide contains not less than 98.5% of
C 2 2H32Br2N 4 04, calculated on the anhydrous basis.
Description Distigmine Bromide occurs as a white, crystal-
line powder.
It is very soluble in water, freely soluble in methanol, in
ethanol (95) and in acetic acid (100), and slightly soluble in a-
cetic anhydride.
The pH of a solution of Distigmine Bromide (1 in 100) is
between 5.0 and 5.5.
It is slightly hygroscopic.
It is gradually colored by light.
Melting point: about 150°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Distigmine Bromide (1 in 25,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Distigmine Bromide as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) To 5 mL of a solution of Distigmine Bromide (1 in 10)
add 2 mL of dilute nitric acid: the solution responds to the
Qualitative Tests <1.09> (1) for bromide.
Purity (1) Clarity and color of solution — Dissolve 0.25 g
of Distigmine Bromide in 5 mL of water: the solution is clear
and colorless.
(2) Sulfate <1.14>— Perform the test with 0.40 g of Distig-
mine Bromide. Prepare the control solution with 0.40 mL of
0.005 mol/L sulfuric acid VS (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Distig-
mine Bromide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Related substances — Dissolve 40 mg of Distigmine
Bromide in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of this solution, add methanol
to make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solution on a plate of
cellulose with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water, ethanol (99.5) and acetic acid (100) (8:3:2:1) to a dis-
tance of about 13 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution. Spray even-
ly Dragendorff's TS for spraying on the plate: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Water <2.48> Not more than 1.0% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Distigmine
Bromide, dissolve in 60 mL of a mixture of acetic anhydride
and acetic acid (100) (8:1), and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration with platinum
electrode). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.82 mg of C 22 H 32 Br 2 N 4 4
Containers and storage Containers — Tight containers.
600
Distigmine Bromide Tablets / Official Monographs
JP XV
Storage — Light-resistant.
Distigmine Bromide Tablets
Distigmine Bromide Tablets contain not less than
95% and not more than 105% of the labeled amount of
distigmine bromide (C 2 2H32Br2N 4 04: 576.32).
Method of preparation Prepare as directed under Tablets,
with Distigmine Bromide.
Identification Determine the absorption spectrum of the so-
lution obtained in the Assay, as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 268 nm and 272 nm, and a minimum between 239 nm
and 243 nm.
Assay Weigh accurately and powder not less than 20 tablets
of Distigmine Bromide Tablets. Weigh accurately a portion
of the powder, equivalent to about 15 mg of Distigmine
Bromide (Q^Br^O,,), add 30 mL of 0.1 mol/L
hydrochloric acid TS, shake for 1 hour, add 0.1 mol/L
hydrochloric acid TS to make exactly 50 mL, and filter. Dis-
card the first 20 mL of the filtrate, pipet 10 mL of the subse-
quent filtrate, add 0.1 mol/L hydrochloric acid TS to make
exactly 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 30 mg of distigmine
bromide for assay (previously determine the water), and dis-
solve in 0.1 mol/L hydrochloric acid TS to make exactly 100
mL. Pipet 10 mL of this solution, add 0.1 mol/L hydrochlor-
ic acid TS to make exactly 100 mL, and use this solution as
the standard solution. Determine the absorbances of the
sample solution and standard solution, A T2 and A S2 , at 270
nm and, A TI and A sl , at 241 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, respectively.
Amount (mg) of distigmine bromide (C22H32Br 2 N 4 04)
= W s x {(A T2 - A T1 )/(.A S2 - A S1 )} x d/2)
W s : Amount (mg) of distigmine bromide for assay, calcu-
lated on the anhydrous basis
Containers and storage Containers — Tight containers.
Disulfiram
; XJl»7-f 7A
,CH S
H 3 G
C10H20N2S4: 296.54
Tetraethylthiuram disulfide [97-77-8]
Disulfiram, when dried, contains not less than
99.0% of C 10 H 20 N 2 S4.
Description Disulfiram occurs as white to yellowish white
crystals or crystalline powder.
It is freely soluble in acetone and in toluene, sparingly solu-
ble in methanol and in ethanol (95), and practically insoluble
in water.
Identification (1) Determine the absorption spectrum of a
solution of Disulfiram in ethanol (95) (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Disul-
firam, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Melting point <2.60> 70 - 73 °C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Disulfiram according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Disulfiram according to Method 4, and perform the test
(not more than 2 ppm).
(3) Diethyldithiocarbamic acid — Dissolve 0.10 g of Dis-
ulfiram in 10 mL of toluene, and shake with 10 mL of diluted
sodium carbonate TS (1 in 20). Discard the toluene layer,
wash the water layer with 10 mL of toluene, shake with 5
drops of a solution of cupric sulfate (1 in 250) and 2 mL of
toluene, and allow to stand: no light yellow color develops in
the toluene layer.
(4) Related substances — Dissolve 50 mg of Disulfiram in
40 mL of methanol, add water to make 50 mL, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add the mobile phase to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /uL each of the sample solution and the standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of both solutions by the automatic integration method:
the total area of the peaks other than the peak of disulfiram
from the sample solution is not larger than the peak area of
disulfiram from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (7:3).
Flow rate: Adjust the flow rate so that the retention time of
disulfiram is about 8 minutes.
Selection of column: Dissolve 50 mg of Disulfiram and 50
mg of benzophenone in 40 mL of methanol, and add water to
make 50 mL. To 1 mL of this solution add the mobile phase
to make 200 mL. Proceed with 10 fiL of this solution under
the above operating conditions, and calculate the resolution.
Use a column giving elution of benzophenone and disulfiram
in this order with the resolution between these peaks being
JP XV
Official Monographs / Dobutamine Hydrochloride
601
not less than 4.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of disulfiram obtained from 10 /uL of the
standard solution is 15-30 mm.
Time span of measurement: About 3.5 times of the reten-
tion time of disulfiram.
Loss on drying <2.41>
24 hours).
Not more than 0.20% (2 g, silica gel,
Residue on ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately about 0.2 g of Disulfiram, previ-
ously dried, in an iodine bottle, dissolve in 20 mL of acetone,
add 1.5 mL of water and 1.0 g of potassium iodide, and dis-
solve by shaking thoroughly. To this solution add 3.0 mL of
hydrochloric acid, stopper the bottle tightly, shake, and al-
low to stand in a dark place for 3 minutes. Add 70 mL of
water, and titrate <2.50> with 0.1 mol/L sodium thiosulfate
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 14.83 mg of C 10 H 20 N 2 S 4
Containers and storage Containers — Tight containers.
Dobutamine Hydrochloride
H CH 3
C 18 H 23 N0 3 .HC1: 337.84
4-{2-[(li?S)-3-(4-Hydroxyphenyl)-
1 -methylpropylamino]ethyl} benzene- 1 ,2-diol
monohydrochloride [49745-95-1 ]
Dobutamine Hydrochloride, when dried,
not less than 98.0% of QgH^NOj.HCl.
contains
Description Dobutamine Hydrochloride occurs as white to
very pale orange crystalline powder or grains.
It is freely soluble in methanol, sparingly soluble in water
and in ethanol (95), and practically insoluble in diethyl ether.
A solution of Dobutamine Hydrochloride (1 in 100) shows
no optical rotation.
Identification (1) Determine the infrared absorption spec-
tra of Dobutamine Hydrochloride, previously dried, as
directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum or the spectrum of dried
Dobutamine Hydrochloride Reference Standard: both spec-
tra exhibit similar intensities of absorption at the same wave
numbers.
(2) A solution of Dobutamine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Dobutamine Hydrochloride in
100 mL of water: the pH of this solution is between 4.0 and
5.5.
Melting point <2.60> 188-191°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Dobutamine Hydrochloride in 30 mL of water: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Dissolve 1.0 g of Dobutamine
Hydrochloride in 40 mL of water by warming, cool, and add
2 mL of dilute acetic acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 2.0 mL of Standard Lead So-
lution add water to make 50 mL (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Dobutamine
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol and formic
acid (78:22:5) to a distance of about 12 cm, and air-dry the
plate. Allow the plate to stand for 5 minutes in iodine vapor:
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41>
3 hours).
Not more than 0.30% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g each of Dobutamine
Hydrochloride and Dobutamine Hydrochloride Reference
Standard, each previously dried, dissolve separately in ex-
actly 10 mL of the internal standard solution, add diluted
methanol (1 in 2) to make 50 mL, and use these solutions as
the sample solution and standard solution respectively. Per-
form the test with 5 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g T and Q s , of the peak area of dobutamine to that
of the internal standard, respectively.
Amount (mg) of C, 8 H 23 N0 3 .HC1 = W s x (Q T /Q S )
W s : Amount (mg) of Dobutamine Hydrochloride Refer-
ence Standard
Internal standard solution — A solution of salicylamide in
diluted methanol (1 in 2) (1 in 125).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of tartrate buffer solution, pH 3.0
and methanol (7:3).
Flow rate: Adjust the flow rate so that the retention time of
dobutamine is about 7 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, dobutamine and internal standard are eluted in this
602
Dopamine Hydrochloride / Official Monographs
JP XV
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of dobutamine to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Dopamine Hydrochloride
.OH
■ HCI
OH
C 8 H n N0 2 .HCl: 189.64
4-(2-Aminoethyl)benzene-l ,2-diol monohydrochloride
[62-31-7]
Dopamine Hydrochloride, when dried, contains not
less than 98.5% of C 8 H„N0 2 .HC1.
Description Dopamine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in water and in formic acid, and sparing-
ly soluble in ethanol (95).
Melting point: about 248°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Dopamine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 25,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Dopa-
mine Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) A solution of Dopamine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (1) for chloride.
pH <2.54> Dissolve 1.0 g of Dopamine Hydrochloride in 50
mL of water: the pH of this solution is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dopamine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.8 g of Dopa-
mine Hydrochloride. Prepare the control solution with 0.35
mL of 0.005 mol/L sulfuric acid VS (not more than 0.021%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dopa-
mine Hydrochloride according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Dopamine Hydrochloride according to Method 1 , and per-
form the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Dopamine
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 250 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of cellulose with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-propanol,
water and acetic acid (100) (16:8:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly a solution of nin-
hydrin in acetone (1 in 50) on the plate, and heat at 90°C for
10 minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Dopamine
Hydrochloride, previously dried, dissolve in 5 mL of formic
acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS, and
heat on a water bath for 15 minutes. After cooling, add 50
mL of acetic acid (100), and titrate <2.50> the excess perchlor-
ic acid with 0.1 mol/L sodium acetate VS (potentiometric
titration). Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 18.96 mg of C 8 H„N0 2 . HCI
Containers and storage Containers — Tight containers.
Dopamine Hydrochloride Injection
i*7f$>£K£a*t«
Dopamine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 97% and not more than 103
% of the labeled amount of dopamine hydrochloride
(C 8 H„N0 2 .HC1: 189.64).
Method of preparation Prepare as directed under Injec-
tions, with Dopamine Hydrochloride.
Description Dopamine Hydrochloride Injection occurs as a
clear, colorless liquid.
Identification To a volume of Dopamine Hydrochloride In-
jection, equivalent to 0.04 g of Dopamine Hydrochloride ac-
cording to the labeled amount, add 0.1 mol/L hydrochloric
acid TS to make 100 mL. To 5 mL of this solution add 0.1
mol/L hydrochloric acid TS to make 50 mL. Determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 278 nm and 282 nm.
pH <2.54> 3.0-5.0
Bacterial endotoxins <4.01> Less than 4.2 EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay To an exact volume of Dopamine Hydrochloride
Injection, equivalent to about 30 mg of dopamine hydrochlo-
ride (C 8 H U N0 2 .HC1), add the mobile phase to make exactly
50 mL. Pipet 2.5 mL of this solution, add exactly 2.5 mL of
JPXV
Official Monographs / Doxapram Hydrochloride Hydrate
603
the internal standard solution and the mobile phase to make
20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 30 mg of dopamine
hydrochloride for assay, previously dried at 105°C for 3
hours, dissolve in the mobile phase to make exactly 50 mL.
Pipet 2.5 mL of this solution, add exactly 2.5 mL of the
internal standard solution and the mobile phase to make
20 mL, and use this solution as the standard solution. Per-
form the test with 10 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g T and Q s , of the peak area of dopamine to that of
the internal standard.
Amount (mg) of dopamine hydrochloride (C 8 H U N0 2 .HC1)
= W s x (Qj/Qs)
W s : Amount (mg) of dopamine hydrochloride for assay
Internal standard solution — A solution of uracil in the
mobile phase (3 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Disodium hydrogen phosphate-citric acid
buffer solution, pH 3.0
Flow rate: Adjust the flow rate so that the retention time of
dopamine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the internal standard and dopamine are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of
peak area of dopamine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Doxapram Hydrochloride Hydrate
H^+f^A^gti^kftt)
r
■ hci • H 2
and enantiomer
C24H30N2O2.HCl.H2O: 432.98
(4i?S)-i-Ethyl-4-[2-(morpholin-4-yl)ethyl]-3,3-
diphenylpyrrolidin-2-one monohydrochloride
monohydrate [7081-53-0]
Doxapram Hydrochloride Hydrate contains not less
than 98.0% of doxapram hydrochloride (C24H 30 N 2
2 .HC1: 414.97), calculated on the anhydrous basis.
Description Doxapram Hydrochloride Hydrate occurs as
white crystals or crystalline powder.
It is freely soluble in methanol and in acetic acid (100),
sparingly soluble in water, in ethanol (95) and in acetic anhy
dride, and practically insoluble in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Doxapram Hydrochloride Hydrate (1 in 2500) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Dox-
apram Hydrochloride Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Doxapram Hydrochloride Hydrate (1 in
50) responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Doxapram Hydrochloride Hy-
drate in 50 mL of water: the pH of this solution is between
3.5 and 5.0.
Melting point <2.60> 218 - 222 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Doxapram Hydrochloride Hydrate in 50 mL of water: the so-
lution is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 1.0 g of Dox-
apram Hydrochloride Hydrate. Prepare the control solution
with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.024%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Dox-
apram Hydrochloride Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Doxapram Hydrochloride Hydrate according to Method
3, and perform the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.5 g of Doxapram
Hydrochloride Hydrate in 10 mL of methanol, and use this
solution as the sample solution. Pipet 3 mL of the sample so-
lution, and add methanol to make exactly 100 mL. Pipet 5
mL of this solution, add methanol to make exactly 50 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 6//L each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, formic acid, ethyl formate and methanol (8:3:3:2) to
a distance of about 10 cm, and air-dry the plate. Allow the
plate to stand in iodine vapor: the spots other than the prin-
cipal spot from the sample solution are not more intense than
the spot from the standard solution.
Water <2.48> 3.5 - 4.5% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.3% (1 g).
604
Doxifluridine / Official Monographs
JP XV
Assay Weigh accurately about 0.8 g of Doxapram
Hydrochloride, dissolve in 50 mL of a mixture of acetic anhy-
dride and acetic acid (100) (7:3), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 41.50 mg of C 24 H 30 N 2 O 2 .HCl
Containers and storage Containers — Tight containers.
Doxifluridine
H+->7JU'J >■:
A'
HO OH
C 9 H u FN 2 5 : 246.19
5 ' -Deoxy-5-fluorouridine
[3094-09-5]
Doxifluridine, when dried, contains not less than
98.5% and not more than 101.0% of C 9 H n FN 2 5 .
Description Doxifluridine occurs as a white crystalline pow-
der.
It is freely soluble in 7V,7V-dimethylformamide, soluble in
water and in methanol, and slightly soluble in ethanol (99.5).
It dissolves in 0.1 mol/L hydrochloric acid TS and in 0.01
mol/L sodium hydroxide TS.
Melting point: about 191 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Doxifluridine in 0.1 mol/L hydrochloric acid TS
(1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Dox-
ifluridine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
20-mL volumetric flask, add 5.0 mL of diluted 0.01 mol/L
sodium hydroxide TS (1 in 20) and 5 mL of the mixture of
acetone and alizarin complexone TS (2:1), then proceed in
the same manner as for preparation of the sample solution,
and use the solution so obtained as the standard solution. De-
termine the absorbances, A T and A s , of the sample solution
and standard solution at 620 nm as directed under Ultrav-
iolet-visible Spectrophotometory <2.24>, using a solution ob-
tained in the same way with 5.0 mL of diluted 0.01 mol/L so-
dium hydroxide TS (1 in 20) as a blank: A T is not larger than
A s .
(2) Chloride <1.03>— Perform the test with 0.30 g of Dox-
ifluridine. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.035%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Dox-
ifluridine according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(4) Related substances — Dissolve 20 mg of Doxifluridine
in 2 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add methanol to
make exactly 25 mL. Pipet 5 mL of this solution, add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, acetic acid (100) and water (17:2:1) to a distance of
about 12 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the number of the spot
other than the principal spot is not more than three, and they
are not more intense than the spot with the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible)
Assay Weigh accurately about 0.25 g of Doxifluridine,
previously dried, dissolve in 50 mL of A^TV-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L tetramethylam-
monium hydroxide VS (potentiometric titration). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L tetramethylammonium hydroxide VS
= 24.62 mg of C 9 H„FN 2 5
Containers and storage Containers — Tight containers.
Optical rotation <2.49>
0.1 g, water, 10 mL, 100 mm)
[«]£: +160
+ 174° (after drying, Doxifluridine Capsules
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Doxifluridine in 10 mL of water is between 4.2 and 5.2.
Purity (1) Fluoride — Dissolve 0.10 g of Doxifluridine in
10.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in
20). Transfer 5.0 mL of this solution into a 20-mL volumetric
flask, add 5 mL of a mixture of acetone and lanthanum-aliza-
rin complexone TS (2:1) and water to make 20 mL, allow to
stand for 1 hour, and use this solution as the sample solution.
Separately, put 1.0 mL of Standard Fluorine Solution in a
K + WJL-Uv^^-feJU
Doxifluridine Capsules contain not less than 95.0%
and not more than 105.0% of doxifluridine
(C 9 H„FN 2 5 : 246.19).
Method of preparation
with Doxifluridine.
Prepare as directed under Capsules,
Identification (1) Dissolve an amount of the contents of
Doxifluridine Capsules, equivalent to 20 mg of Doxifluridine
JP XV
Official Monographs / Doxorubicin Hydrochloride
605
according to the labeled amount, in 0.1 mol/L hydrochloric
acid TS to make 100 mL, and filter. To 1 mL of the filtrate
add 0.1 mol/L hydrochloric acid TS to make 20 mL, and de-
termine the absorption spectrum of this solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using
0.1 mol/L hydrochloric acid TS as the blank: it exhibits a
maximum between 267 nm and 271 nm.
(2) To an amount of powdered contents of Doxifluridine
Capsules, equivalent to 20 mg of Doxifluridine according to
the labeled amount, add 2 mL of methanol, shake, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 20 mg of doxifluridine in 2 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, acetic acid (100) and
water (17:2:1) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the principal spot with the sample solution and the spot
with the standard solution show a dark purple color and these
Rf values are the same.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with one capsule of Doxifluridine Cap-
sules at 50 revolutions per minute according to the Paddle
method, using 900 mL of water as the dissolution medium,
using the sinker. Withdraw 20 mL or more of the dissolution
medium 30 minutes after starting the test, and filter through a
membrane filter with pore size of not more than 0.45 ^m.
Discard the first 10 mL of the filtrate, pipet the subsequent V
mL, add water to make exactly V mL so that each mL con-
tains about 13 n% of doxifluridine (C9H U FN 2 5 ) according to
the labeled amount, and use this solution as the sample solu-
tion. Separately, weigh accurately about 26 mg of doxifluri-
dine for assay, previously dried at 105°C for 4 hours, and dis-
solve in water to make exactly 100 mL. Pipet 5 mL of this so-
lution, add water to make exactly 100 mL, and use this solu-
tion as the standard solution. Determine the absorbances, A T
and A s , of these solutions at 269 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>. The dissolution rate
in 30 minutes is not less than 85%.
Dissolution rate (%) with respect to the labeled amount of
doxifluridine (C 9 H u FN 2 5 )
= W s x (A T /A S ) x (V/V) x (1/QX9
W s : Amount (mg) of doxifluridine for assay
C: Labeled amount (mg) of doxifluridine (C9H U FN 2 5 ) in
1 capsule
Assay Weigh accurately the mass and powder the contents
of not less than 20 Doxifluridine Capsules. Weigh accurately
a portion of the powder, equivalent to about 50 mg of dox-
ifluridine (C9H U FN 2 5 ) according to the labeled amount, add
40 mL of water, shake for about 10 minutes, add water to
make exactly 50 mL, and filter. Discard the first 10 mL of the
filtrate, pipet 5 mL of the subsequent filtrate, add exactly 10
mL of the internal standard solution, then add a mixture of
water and methanol (5:3) to make 100 mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 50 mg of doxifluridine for assay, previously dried at
105 °C for 4 hours, and dissolve in water to make exactly 50
mL. Pipet 5 mL of this solution, add exactly 10 mL of the in-
ternal standard solution, then add the mixture of water and
methanol (5:3) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with 10 /xL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the ratios, g T and Q s , of the peak
height of doxifluridine to that of the internal standard.
Amount (mg) of doxifluridine (C9H U FN 2 5 )
= W s x(Q T /Q s )
W s : Amount (mg) of doxifluridine for assay
Internal standard solution — A solution of anhydrous caffeine
(1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and methanol (13:7).
Flow rate: Adjust the flow rate so that the retention time of
doxifluridine is about 2.5 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, doxifluridine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak height of doxifluridine to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Doxorubicin Hydrochloride
H+yjutf
HCI
C 27 H 29 N0 U -HC1: 579.98
(2S,4S)-4-(3-Amino-2,3,6-trideoxy-a-L-/yAro-
hexopyranosyloxy)-2,5,12-trihydroxy-2-hydroxyacetyl-7-
methoxy-l,2,3,4-tetrahydrotetracene-6,ll-dione
monohydrochloride [25316-40-9]
Doxorubicin Hydrochloride is the hydrochloride of
606
Doxycycline Hydrochloride Hydrate / Official Monographs
JP XV
a derivative of daunorubicin.
It contains not less than 980 fig (potency) and not
more than 1080 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Doxorubicin
Hydrochloride is expressed as mass (potency) of dox-
orubicin hydrochloride (C27H 2 9NO n .HCl).
Description Doxorubicin Hydrochloride occurs as a red-
orange crystalline powder.
It is sparingly soluble in water, slightly soluble in
methanol, very slightly soluble in ethanol (99.5), and practi-
cally insoluble in acetonitrile.
Identification (1) Determine the absorption spectrum of a
solution of Doxorubicin Hydrochloride in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Doxorubicin
Hydrochloride Reference Standard prepared in the same
manner as the sample solution: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Dox-
orubicin Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Doxorubicin Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Doxorubicin Hydrochloride (1 in 200)
responds to the Qualitative Tests <1.09> (1) for chloride.
Optical rotation <2.49> [a]™: +240 - +290° (20 mg calculat-
ed on the anhydrous basis, methanol, 20 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 50
mg of Doxorubicin Hydrochloride in 10 mL of water is be-
tween 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 50 mg
of Doxorubicin Hydrochloride in 10 mL of water: the solu-
tion is clear and red.
(2) Related substances — Dissolve 25 mg of Doxorubicin
Hydrochloride in 100 mL of the mobile phase, and use this
solution as the sample solution. Pipet 2 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 20 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the area of
the peak other than doxorubicin obtained from the sample
solution is not more than 1/4 times the peak area of dox-
orubicin from the standard solution, and the total area of the
peaks other than doxorubicin is not more than the peak area
of doxorubicin from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 3 g of sodium lauryl sulfate in 1000
mL of diluted phosphoric acid (7 in 5000), and add 1000 mL
of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
doxorubicin is about 8 minutes.
Time span of measurement: About 3 times as long as the
retention time of doxorubicin.
System suitability —
Test for required detectability: Measure 1 mL of the stan-
dard solution, and add the mobile phase to make exactly
20 mL. Confirm that the peak area of doxorubicin obtained
from 20 fiL of this solution is equivalent to 3.5 to 6.5% of
that from 20 fiL of the standard solution.
System performance: Dissolve 5 mg of Doxorubicin
Hydrochloride in 20 mL of water, add 1.5 mL of phosphoric
acid, and allow to stand at room temperature for 30 minutes.
Adjust the pH of this solution to 2.5 with 2 mol/L sodium
hydroxide TS. When the procedure is run with 20 fiL of this
solution under the above operating conditions, doxorubici-
none, having the relative retention time of about 0.6 with
respect to doxorubicin, and doxorubicin are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
doxorubicin is not more than 2.0%.
Water <2.48> Not more than 3.0% (0.3 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Doxorubicin
Hydrochloride and Doxorubicin Hydrochloride Reference
Standard, equivalent to about 10 mg (potency), dissolve each
in water to make exactly 25 mL. Pipet 5 mL each of these
solutions, add water to make exactly 100 mL, and use these
solutions as the sample solution and the standard solution,
respectively. Determine the absorbances at 495 nm, A T and
j4 s , of the sample solution and standard solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>.
Amount [Mg (potency)] of C27H 29 NO u .HCl
= W s x (Aj/A s ) x 1000
W s : Amount [mg (potency)] of Doxorubicin Hydrochlo-
ride Reference Standard
Containers and storage Containers — Tight containers.
Doxycycline Hydrochloride Hydrate
■HCI
, L H 3 C
'OH
JHjO
HOH HH N-
H 3 C
C 22 H 24 N 2 8 .HCl.i/ 2 C 2 H 6 0.i/ 2 H 2 0: 512.94
(4S,4ai?,5S,5a^,6/?,12a5)-4-Dimethylamino-
3,5,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-
1 ,4, 4a, 5, 5a, 6, 1 1 , 1 2a-octahydrotetracene-
2-carboxamide monohydrochloride
hemiethanolate hemihydrate [564-25-0, Doxycycline]
JPXV
Official Monographs / Doxycycline Hydrochloride Hydrate
607
Doxycycline Hydrochloride Hydrate is the
hydrochloride of a derivative of oxy tetracycline.
It contains not less than 880 fig (potency) and not
more than 943 fig (potency) per mg, calculated on the
anhydrous basis and corrected by the amount of
ethanol. The potency of Doxycycline Hydrochloride
Hydrate is expressed as mass (potency) of doxycycline
(C 22 H 24 N 2 8 : 444.43).
Description Doxycycline Hydrochloride Hydrate occurs as
yellow to dark yellow, crystals or crystalline powder.
It is freely soluble in water and in methanol, and slightly
soluble in ethanol (99.5).
Identification (1) Determine the infrared absorption spec-
trum of Doxycycline Hydrochloride Hydrate as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Doxycycline
Hydrochloride Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(2) Dissolve 10 mg of Doxycycline Hydrochloride Hy-
drate in 10 mL of water, and add silver nitrate TS: a white
turbidity is produced.
Absorbance <2.24> E\°f m (349 nm): 285-315 (10 mg, 0.01
mol/L hydrochloric acid-methanol TS, 500 mL).
Optical rotation <2.49> [a]™: - 105 - - 120° (0.25 g calcu-
lated on the anhydrous basis and correcred by the amount of
ethanol, 0.01 mol/L hydrochloric acid-methanol TS, 25 mL,
100 mm). Determine within 5 minutes after the sample solu-
tion is prepared.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Doxycycline Hydrochloride Hydrate according to Method 2,
and perform the test. Prepare the control solution with 5.0
mL of Standard Lead Solution (not more than 50ppm).
(2) Related substance — Dissolve 20 mg of Doxycycline
Hydrochloride Hydrate in 0.01 mol/L hydrochloric acid TS
to make exactly 25 mL, and use this solution as the sample
solution. Separately, dissolve 20 mg of 6-epidoxycycline
hydrochloride in 0.01 mol/L hydrochloric acid TS to make
exactly 25 mL, and use this solution as 6-epidoxycycline
hydrochloride stock solution. Separately, dissolve 20 mg of
metacycline hydrochloride in 0.01 mol/L hydrochloric acid
TS to make exactly 25 mL, and use this solution as metacy-
cline hydrochloride stock solution. Pipet 2 mL each of 6-
epidoxycycline hydrochloride stock solution and metacycline
hydrochloride stock solution, add 0.01 mol/L hydrochloric
acid TS to make exatly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 fiL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the peak areas of metacycline and
6-epidoxycycline obtained from the sample solution are not
more than the peak areas of them obtained from the standard
solution, respectively, and the areas of the two peaks, ap-
peared between the solvent peak and metacycline and behind
of doxycycline, obtained from the sample solution are not
more than 1/4 of the peak area of 6-epidoxycycline from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with styrene-divinylben-
zene copolymer for liquid chromatography (8 ftm in particle
diameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase: Mix 125 mL of 0.2 mol/L potassium
dihydrogen phosphate TS, 117 mL of 0.2 mol/L sodium
hydroxide TS, and add water to make 500 mL. To 400 mL of
this solution add 50 mL of a solution of tetrabutylammoni-
um hydrogensulfate (1 in 100), 10 mL of a solution of disodi-
um dihydrogen ethylenediamine tetraacetate dihydrate (1 in
25), 60 g of ^-butanol and 200 mL of water, adjust the pH to
8.0 with 2 mol/L sodium hydroxide TS, and add water to
make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
doxycycline is about 19 minutes.
Time span of measurement: About 2.4 times as long as the
retention time of doxycycline beginning after the solvent
peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add 0.01 mol/L hydrochloric acid
TS to make exactly 20 mL. Confirm that the peak areas of 6-
epidoxycycline and metacycline obtained from 20 fiL of this
solution are equivalent to 3.5 to 6.5% of them obtained from
20 fiL of the standard solution, respectively.
System performance: To 8 mL of the sample solution,
3 mL of 6-epidoxycycline hydrochloride stock solution and
2 mL of metacycline hydrochloride stock solution add
0.01 mol/L hydrochloric acid TS to make 50 mL. When the
procedure is run with 20 fiL of this solution under the above
operating conditions, metacycline, 6-epidoxycycline and dox-
ycycline are eluted in this order with the resolutions between
the peaks, metacycline and 6-epidoxycycline, and 6-epidox-
ycycline and doxycycline, being not less than 1.3 and not less
than 2.0, respectively, and the symmetry factor of the peak of
doxycycline is not more than 1.3.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviations of the peak area
of metacycline and 6-epidoxycycline are not more than 3.0%
and not more than 2.0%, respectively.
Ethanol Weigh accurately about 0.1 g of Doxycycline
Hydrochloride Hydrate, dissolve in the internal standard so-
lution to make exactly 10 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.4 g of
ethanol (99.5), and add the internal standard solution to
make exactly 100 mL. Pipet 1 mL of this solution, add the in-
ternal standard solution to make exactly 10 mL, and use this
solution as the standard solution. Perform the test with 1 fiL
each of the sample solution and standard solution as directed
under Gas Chromatography <2.02> according to the follow-
ing conditions, and determine the ratios, Qj and Q s , of the
peak area of ethanol to that of the internal standard: the
amount of ethanol is not less than 4.3% and not more than
6.0%.
Amount (%) of ethanol = (W S /W T ) x (g T /Q s )
W s : Amount (mg) of ethanol (99.5)
608
Droperidol / Official Monographs
JP XV
W T : Amount (mg) of the sample
Internal standard solution — A solution of 1-propanol (1 in
2000).
Operating conditions —
Detector: An hydrogen fiame-ionization detector.
Column: A glass column 3.2 mm in inside diameter
and 1.5 m in length, packed with porous ethylvinylbenzene-
divinylbenzene copolymer for gas chromatography
(0.0075 //m in average pore size, 500 - 600 m 2 /g in specific
surface area) (150 - 180 /xm in particle diameter).
Column temperature: A constant temperature of about
135°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethanol is about 5 minutes.
System suitability —
System performance: When the procedure is run with 1 /xL
of the standard solution under the above operating condi-
tions, ethanol and the internal standard are eluted in this ord-
er with the resolution between these peaks being not less than
2.0.
System repeatability: When the test is repeated 5 times with
1 /xL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of ethanol to that of the internal standard is not
more than 2.0%.
Water <2.48> Not less than 1.4% and not more than 2.8%
(0.6 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately an amount of Doxycycline
Hydrochloride Hydrate and Doxycycline Hydrochloride
Reference Standard, equivalent to about 50 mg (potency),
dissolve each in water to make exactly 50 mL, and use these
solutions as the sample solution and standard solution. Per-
form the test with exactly 10 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of doxycycline.
Amount Lug (potency)] of doxycycline (C22H 2 4N 2 8 )
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Doxycycline Hydrochlo-
ride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 7.0 g of sodium dihydrogen phos-
phate dihydrate in 450 mL of water, add 553 mL of a mixture
of methanol and N, TV-dim ethyl-M-octylamine (550:3), and
adjust the pH to 8.0 with a solution of sodium hydroxide (43
in 200).
Flow rate: Adjust the flow rate so that the retention time of
doxycycline is about 6 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the theoretical plates and the symmetry factor of
the peak of doxycycline are not less than 1000 and not more
than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
doxycycline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Droperidol
(ip^lj \!-)\,
H
H
C 22 H 22 FN 3 2 : 379.43
l-{l-[4-(4-Fluorophenyl)-4-oxobutyl]-l,2,3,6-
tetrahydropyridin-4-yl}-l,3-dihydro-2//-benzoimidazol-
2-one [548-73-2]
Droperidol, when dried, contains not less than
98.0% of C 22 H 22 FN 3 2 .
Description Droperidol occurs as a white to light yellow
powder.
It is freely soluble in acetic acid (100), soluble in
dichloromethane, slightly soluble in ethanol (99.5), and prac-
tically insoluble in water.
It is gradually colored by light.
Identification (1) Put 30 mg of Droperidol in a brown
volumetric flask, and dissolve in 10 mL of 0.1 mol/L
hydrochloric acid TS and ethanol (95) to make 100 mL.
Transfer 5 mL of the solution to a brown volumetric flask,
and add 10 mL of 0.1 mol/L hydrochloric acid TS and
ethanol (95) to make 100 mL. Determine the absorption spec-
trum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Droperidol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, dissolve Droperidol in acetone, evaporate the
acetone, dry the residue in a desiccator (in vacuum, silica gel,
70°C) for 4 hours, and perform the test with the residue.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Droperidol in a platinum crucible according to Method 2,
and perform the test. Prepare the control solution with 2.0
JPXV
Official Monographs / Dydrogesterone
609
mL of Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve 50
mg of Droperidol in 5 mL of dichloromethane, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add dichloromethane to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, chloroform, methanol and a-
cetic acid-sodium acetate buffer solution, pH 4.7,
(54:23:18:5) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 3.0% (0.5 g, in vacu-
um, silica gel, 70°C, 4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.5 g of Droperidol, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 37.94 mg of C 22 H 22 FN 3 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dydrogesterone
■> Hn^Xxn
C 21 H 28 2 : 312.45
9/?,10a-Pregna-4,6-diene-3,20-dione [152-62-5]
Dydrogesterone, when dried, contains not less than
98.0% and not more than 102.0% of C 21 H 28 2 .
Description Dydrogesterone occurs as white to light yellow-
ish white crystals or crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in acetonitrile,
sparingly soluble in methanol and in ethanol (95), slightly
soluble in diethyl ether, and practically insoluble in water.
Identification (1) To 5 mg of Dydrogesterone add 5 mL of
4-methoxybenzaldehyde-acetic acid TS and 2 to 3 drops of
sulfuric acid, and heat in a water bath for 2 minutes: an oran-
ge-red color develops.
(2) Determine the absorption spectrum of a solution of
Dydrogesterone in methanol (1 in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Dydrogesterone, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]p : -470-
0.1 g, chloroform, 10 mL, 100 mm).
Melting point <2.60> 167 - 171 °C
■500° (after drying,
Purity (1) Heavy metals <l.07> — Proceed with 1.0 g of
Dydrogesterone according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 10 mg of Dydrogester-
one in 200 mL of the mobile phase, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area of these
solutions by the automatic integration method: the total area
of peaks other than the peak of dydrogesterone from the
sample solution is not larger than the peak area of
dydrogesterone from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (3 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, ethanol (95) and
acetonitrile (53:26:21).
Flow rate: Adjust the flow rate so that the retention time of
dydrogesterone is about 12 minutes.
Selection of column: Dissolve 1 mg each of Dydrogester-
one and progesterone in 20 mL of the mobile phase. Proceed
with 10 /xL each of these solutions under the above operating
conditions, and calculate the resolution. Use a column giving
elution of dydrogesterone and progesterone in this order with
the resolution between these peaks being not less than 8.
Wavelength is 265 nm.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of dydrogesterone obtained from 10 /uL
of the standard solution is between 5 mm and 10 mm.
Time span of measurement: About twice as long as the
retention time of dydrogesterone beginning after the solvent
peak.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg of Dydrogesterone,
previously dried, and dissolve in methanol to make exactly
610
Dydrogesterone Tablets / Official Monographs
JP XV
100 mL. Pipet 1 mL of this solution, and add methanol to
make exactly 100 mL. Determine the absorbance A of this so-
lution at the wavelength of maximum absorption at about
286 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>.
Amount (mg) of C 21 H 28 2 = (A/ $45) x 100,000
Containers and storage Containers — Tight containers.
Dydrogesterone Tablets
Dydrogesterone Tablets contain not less than 95%
and not more than 105% of the labeled amount of
dydrogesterone (C2iH 2 80 2 : 312.45).
Method of preparation
with Dydrogesterone.
Prepare as directed under Tablets,
Identification (1) To a quantity of powdered Dydrogester-
one Tablets, equivalent to 0.05 g of Dydrogesterone accord-
ing to the labeled amount, add 50 mL of methanol, shake
well, and filter. Evaporate 5 mL of the filtrate on a water bath
to dryness. Proceed with the residue as directed in the Iden-
tification (1) under Dydrogesterone.
(2) To 1 mL of the filtrate obtained in (1) add methanol
to make 200 mL. Determine the absorption spectrum of this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 284 nm and 288
nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Dydrogesterone Tablets at
50 revolutions per minute according to the Paddle method,
using 900 mL of water as the dissolution medium. Take 20
mL or more of the dissolved solution 30 minutes after start-
ing the test, and filter. Discard the first 10 mL of the filtrate,
and use the subsequent as the sample solution. Separately,
weigh accurately about 50 mg of dydrogesterone for assay,
previously dried in a desiccator (in vacuum, phosphorus (V)
oxide) for 24 hours, and dissolve in methanol to make exactly
100 mL. Pipet 1 mL of this solution, add water to make ex-
actly 100 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and standard solution at 296 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Dydrogesterone Tablets in 30
minutes is not less than 80%.
Dissolution rate (%) with respect to
the labeled amount of dydrogesterone (C 2 iH 28 02)
= W s x (Aj/As) x (1/C) x 9
W s : Amount (mg) of dydrogesterone for assay
C: Labeled amount (mg) of dydrogesterone (C 21 H 28 2 ) in 1
tablet.
Assay Weigh accurately and powder not less than 20
Dydrogesterone Tablets. Weigh accurately a portion of the
powder, equivalent to about 10 mg of dydrogesterone
(C 2I H 28 2 ), shake well with 50 mL of methanol, and add
methanol to make exactly 100 mL. Filter this solution, dis-
card the first 20 mL of the filtrate, pipet the subsequent 5 mL,
add methanol to make exactly 100 mL, and use this solution
as the sample solution. Separately, weigh accurately about
0.01 g of dydrogesterone for assay, previously dried in a
desiccator (in vacuum, phosphorus (V) oxide) for 24 hours,
proceed in the same manner as the preparation of the sample
solution, and use the solution as the standard solution. Deter-
mine the absorbances, A T and A s , of the sample solution and
standard solution at 286 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>.
Amount (mg) of dydrogesterone (C 2 iH 28 2 )
= W s x (A T /A S )
W s : Amount (mg) of dydrogesterone for assay
Containers and storage Containers — Tight containers.
Ecothiopate Iodide
H 3 C V
-o' s CH: '
C 9 H 23 IN0 3 PS: 383.23
2-(Diethoxyphosphorylsulfanyl)-./V,A r ,./V-
trimethylethylaminium iodide [513-10-0]
Ecothiopate Iodide contains not less than 95.0%
C9H23INO3PS, calculated on the dried basis.
of
Description Ecothiopate Iodide occurs as white crystals or
crystalline powder.
It is very soluble in water, freely soluble in methanol,
slightly soluble in ethanol (95), and practically insoluble in
diethyl ether.
Identification (1) Dissolve 0.1 g of Ecothiopate Iodide in
2 mL of water, and add 1 mL of nitric acid: a brown
precipitate is formed. To 1 drop of the turbid solution con-
taining this precipitate add 1 mL of hexane, and shake: a
light red color develops in the hexane layer.
(2) Heat the suspension of the precipitate obtained in (1)
until it becomes colorless, cool, add 10 mL of water, and use
this solution as the sample solution. Two mL of the sample
solution responds to the Qualitative Tests <1.09> (2) for phos-
phate.
(3) Two mL of the sample solution obtained in (2)
responds to the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 0. 1 g of Ecothiopate Iodide in 40 mL of
water: the pH of this solution is between 3.0 and 5.0.
Melting point <2.60> 116 - 122°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Ecothiopate Iodide in 5 mL of water: the solution is clear and
colorless.
(2) Heavy metals <1.07> — To 1.0 g of Ecothiopate Iodide
in a Kjeldahl flask add 5 mL of nitric acid and 2 mL of sulfur-
ic acid, put a small funnel on the mouth of the flask, and heat
carefully until white fumes are evolved. After cooling, add 2
mL of nitric acid, and heat. Repeat this procedure twice, add
several 2-mL portions of hydrogen peroxide (30), and heat
JPXV
Official Monographs / Edrophonium Chloride 611
until the solution becomes colorless, and white fumes are
evolved. After cooling, transfer the solution together with a
small quantity of water to a Nessler tube, and add water to
make about 20 mL. Adjust the solution with ammonia solu-
tion (28) and ammonia TS to a pH between 3.0 and 3.5, add
water to make 50 mL, and use this solution as the test solu-
tion. Prepare the control solution as follows: proceed in the
same manner as the preparation of the test solution, and add
2.0 mL of Standard Lead Solution and water to make 50 mL
(not more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Ecothiopate
Iodide in 10 mL of methanol, and use this solution as the
sample solution. Pipet 3 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of cellulose for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, water and acetic acid (100)
(4:2:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly Dragendorff's TS for spraying on the plate: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
phosphorus (V) oxide, 50°C, 3 hours).
Assay Weigh accurately about 0.125 g of Ecothiopate
Iodide, and dissolve in water to make exactly 100 mL. Pipet
10 mL of of this solution, add 30 mL of water, then add
exactly 10 mL of phosphate buffer solution, pH 12, stopper
the container, and allow to stand at 25 ± 3°C for 20 minutes.
To this solution add quickly 2 mL of acetic acid (100), and ti-
trate <2.50> with 0.002 mol/L iodine VS (potentiometric
titration). Perform the test in the same manner without phos-
phate buffer solution, pH 12, and make any necessary correc-
tion.
Each mL of 0.002 mol/L iodine VS
= 1.533 mg of C 9 H 23 IN0 3 PS
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 0°C.
Edrophonium Chloride
ih-p*z^AMi
C 10 H 16 C1NO: 201.69
A r -Ethyl-3-hydroxy-A^,A r -dimethylanilinium chloride
[116-38-1]
Edrophonium Chloride, when dried, contains not
less than 98.0% of C 10 H 16 C1NO.
Description Edrophonium Chloride occurs as white crystals
or crystalline powder. It is odorless.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), and practically insoluble in acetic anhy-
dride and in diethyl ether.
It is hygroscopic.
It is gradually colored by light.
Identification (1) To 5 mL of a solution of Edrophonium
Chloride (1 in 100) add 1 drop of iron (III) chloride TS: a
light red-purple color develops.
(2) Determine the absorption spectrum of a solution of
Edrophonium Chloride in 0.1 mol/L hydrochloric acid TS (1
in 20,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Edrophonium
Chloride Reference Standard prepared in the same manner as
the sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(3) A solution of Edrophonium Chloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Edrophonium Chloride in 10
mL of water: the pH of this solution is between 3.5 and 5.0.
Melting point <2.60> 166 - 171 °C (with decomposition).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Edrophonium Chloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Edrophonium Chloride according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Edrophonium Chloride according to Method 1, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.50 g of Edrophonium
Chloride in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
ethanol (95) to make exactly 100 mL. Pipet 3 mL of this solu-
tion, add ethanol (95) to make exactly 10 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of methanol, chloroform and ammonia solution (28) (16:4:1)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, in vacu-
um, phosphorus (V) oxide, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Edrophonium Chlo-
ride, previously dried, and dissolve in 100 mL of a mixture of
acetic anhydride and acetic acid (100) (7:3). Titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 20.17 mg of C 10 H 16 C1NO
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
612 Edrophonium Chloride Injection / Official Monographs
JP XV
Edrophonium Chloride Injection
i K n^-^A^btiaW*
Edrophonium Chloride Injection is an aqueous solu-
tion for injection. It contains not less than 95% and
not more than 105% of the labeled amount of
edrophonium chloride (C 10 H 16 C1NO: 201.69).
Method of preparation Prepare as directed under Injec-
tions, with Edrophonium Chloride.
Description Edrophonium Chloride Injection is a clear and
colorless liquid.
Identification (1) To a volume of Edrophonium Chloride
Injection, equivalent to 0.04 g of Edrophonium Chloride ac-
cording to the labeled amount, add 4 mL of barium nitrate
TS, shake, and filter. Proceed with the filtrate as directed in
the Identification (1) under Edrophonium Chloride.
(2) Determine the absorption spectrum of the sample
solution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 272 nm and 276 nm.
pH <2.54> 6.5 - 8.0
Extractable volume <6.05> It meets the requirement.
Assay Conduct this procedure without exposure to day-
light, using light-resistant containers. Measure exactly a
volume of Edrophonium Chloride Injection, equivalent to
about 50 mg of edrophonium chloride (Ci H 16 ClNO), place
in a chromatographic column prepared by pouring 10 mL of
weakly basic DEAE-bridged dextran anion exchanger (CI
type) (50 to 150//m in particle diameter) into a chromato-
graphic tube about 2 cm in inside diameter and about 10 cm
in length, add 25 mL of water, and elute at the flow rate of 1
to 2 mL per minute. Wash the column with two 25-mL por-
tions of water at the flow rate of 1 to 2 mL per minute. Com-
bine the washings with above effluent solutions, and add
water to make exactly 100 mL. Measure exactly 10 mL of this
solution, and add 10 mL of phosphate buffer solution, pH
8.0, and 5 g of sodium chloride. Wash this solution with four
20-mL portions of a mixture of diethyl ether and hexane
(1:1), collect the water layer, add 0.1 mol/L hydrochloric
acid TS to make exactly 100 mL, and use this solution as the
sample solution. Separately, weigh accurately about 50 mg of
Edrophonium Chloride Reference Standard, previously dried
in a desiccator (in vacuum, phosphorus (V) oxide) for 3
hours, and dissolve in water to make exactly 100 mL. Meas-
ure exactly 10 mL of this solution, and prepare the standard
solution in the same manner as the sample solution. Deter-
mine the absorbances, A T and A s , of the sample solution and
standard solution at 273 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>.
Amount (mg) of edrophonium chloride (C 10 H 16 C1NO)
= W s x (A T /As)
W s : Amount (mg) of Edrophonium Chloride Reference
Standard
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Elcatonin
Va I ■ Leu ■ Gly- Lys-Leu-Ser-G I n-G In- Leu-
His- Lys-Leu-G I n-Thr-Tyr- Pro- Arg-Th r-Asp- Val-G ly-Ala-Gly-Thr-P ro-N H 5
C 148 H 2 4 4 N 42 04 7 : 3363.77
[60731-46-6]
Elcatonin contains not less than 5000 Elcatonin
Units and not more than 7000 Elcatonin Units per 1 mg
of peptide, calculated on the dehydrated and de-acetic
acid basis.
Description Elcatonin is a white powder.
It is very soluble in water, freely soluble in ethanol (95),
and practically insoluble in acetonitrile.
It is hygroscopic.
The pH of its solution (1 in 500) is between 4.5 and 7.0.
Identification Dissolve 5 mg of Elcatonin in 5 mL of water.
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
Constituent amino acids Put about 1 mg of Elcatonin in a
test tube for hydrolysis, add phenol-hydrochloric acid TS to
dissolve, replace the air inside with Nitrogen, seal the tube
under reduced pressure, and heat at 110 ± 2°C for 24 hours.
After cooling, open the tube, evaporate the hydrolyzate to
dryness under reduced pressure, dissolve the residue in 1 mL
of 0.02 mol/L hydrochloric acid TS, and use this solution as
the sample solution. Separately, weigh exactly 1.33 mg of
L-aspartic acid, 1.19 mg of L-threonine, 1.05 mg of L-serine,
1.47 mg of L-glutamic acid, 1.15 mg of L-proline, 0.75 mg of
glycine, 0.89 mg of L-alanine, 1.17 mg of L-valine, 1 .89 mg of
L-2-aminosuberic acid, 1.31 mg of L-leucine, 1.81 mg of
L-tyrosine, 1.83 mg of L-lysine hydrochloride, 2.10 mg of
L-histidine hydrochloride monohydrate and 2.11 mg of
L-arginine hydrochloride, dissolve them in 0.02 mol/L
hydrochloric acid TS to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with exaxtly
10 /iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions: 14 peaks of amino acids appear on
the chromatogram obtained from the sample solution, and
their respective molar ratios against alanine are 1.7 - 2.2 for
aspartic acid, 3.5 - 4.2 for threonine, 2.4 - 3.0 for serine, 2.7
-3.2 for glutamic acid, 1.7-2.2 for proline, 2.7-3.2 for
glycine, 1.6-2.2 for valine, 0.8-1.2 for 2-aminosuberic
acid, 4.5 - 5.2 for leucine, 0.7 - 1.2 for tyrosine, 1.7 - 2.2 for
lysine, 0.8 - 1.2 for histidine and 0.7 - 1.2 for arginine.
Operating conditions —
Detector: A visible spectrophotometer (wavelength: 440
JPXV
Official Monographs / Elcatonin 613
nm and 570 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 8 cm in length, packed with strongly acidic
ion-exchange resin for liquid chromatography composed
with a sulfonated styrene-divinylbenzene copolymer (3 fim in
particle diameter).
Column temperature: Varied between 50°C and 65°C.
Chemical reaction vessel temperature: A constant tempera-
ture of about 130°C.
Color developing time: About 1 minute.
Mobile phase: Buffer solutions A, B, C and D, with sodium
ion concentrations of O.lOmol/L, 0.135 mol/L, 1.26mol/L
and 0.20 mol/L, respectively. The ion concentration of the
mobile phase is changed stepwise from 0.10 mol/L to 1.26
mol/L by using these buffer solutions.
Components of buffer solutions
Buffer solution:
A
B
C
D
Citric acid
8.85 g
7.72 g
6.10g
—
Sodium
citrate
3.87 g
10.05 g
26.67 g
—
Sodium
hydroxide
—
—
2.50 g
8.00 g
Sodium
chloride
3.54 g
1.87 g
54.35 g
—
Ethanol
60.0 mL
—
—
60.0 mL
Thiodiglycol
5.0 mL
5.0 mL
—
—
Purified
a sufficient
a sufficient
a sufficient
a sufficient
water
amount
amount
amount
amount
Total amount 1000 mL 1000 mL 1000 mL 1000 mL
Reaction reagent: Mix 407 g of lithium acetate dihydrate,
245 mL of acetic acid (100) and 801 mL of l-methoxy-2-
propanol, add water to make 2000 mL, stir for about 20
minutes while passing Nitrogen, and use this solution as solu-
tion A. Separately, to 1957 mL of l-methoxy-2-propanol add
77 g of ninhydrin and 0.134 g of sodium borohydride, stir for
about 20 minutes while passing Nitrogen, and use this solu-
tion as solution B. Mix solution A and solution B before use.
Flow rate of mobile phase: Adjust the flow rate so that the
retention time of arginine is about 75 minutes.
Flow rate of reaction reagent: About 0.2 mL per minute.
Selection of column: Proceed with 10 iiL of the standard
solution under the above operating conditions. Use a column
from which aspartic acid, threonine, serine, glutamic acid,
proline, glycine, alanine, valine, 2-aminosuberic acid, leu-
cine, tyrosine, lysine, histidine and arginine are eluted in this
order, with complete separation of each peak.
Purity (1) Acetic acid — Weigh accurately 3-6 mg of
Elcatonin quickly under conditions of 25 ± 2°C and 50 ±
5% relative humidity, add exactly 1 mL of the internal stan-
dard solution to dissolve it, and use this solution as the sam-
ple solution. Separately, weigh accurately about 0.5 g of acet-
ic acid (100), and add the internal standard solution to make
exactly 100 mL. Pipet 5 mL of this solution, add the internal
standard solution to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with 20 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of acetic acid to that of the internal standard: the
amount of acetic acid is not more than 7.0%.
Amount (%) of acetic acid (CH 3 COOH)
= (W SJ /W SA ) x (Qt/Gs) x 50
W ST : Amount (g) of acetic acid (100)
W SA : Amount (mg) of sample
Internal standard solution — A solution of citric acid (1 in
4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 13.2 g of diammonium hydrogen-
phosphate in 900 mL of water, add phosphoric acid to adjust
the pH to 2.5, and add water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
acetic acid is about 4 minutes.
Selection of column: Proceed with 20 fxL of the standard
solution under the above operating conditions, and calculate
the resolution. Use a column from which acetic acid and
citric acid are eluted in this order with the resolution between
their peaks being not less than 2.0.
(2) Related substances — Dissolve 1.0 mg of Elcatonin in
1 mL of a mixture of trifluoroacetic acid TS and acetonitrile
(2:1), and use this solution as the sample solution. Take
exactly 0.3 mL of the sample solution, add a mixture of
trifluoroacetic acid TS and acetonitrile (2:1) to make exactly
10 mL, and use this solution as the standard solution. Per-
form the test with exactly 10,mL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the total of the peak areas other than the peak of el-
catonin of the sample solution is not larger than the peak area
of elcatonin of the standard solution, and each peak area
other than the peak of elcatonin of the sample solution is not
larger than 1/3 of the peak area of elcatonin of the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of trifluoroacetic acid TS and
acetonitrile (change the ratio linearly from 85:15 to 55:45 in
30 minutes).
Flow rate: Adjust the flow rate so that the retention time of
elcatonin is about 25 minutes.
Selection of column: Dissolve 2 mg of Elcatonin in 200 fiL
of trypsin TS for test of elcatonin, warm at 37°C for 1 hour,
then add 1 drop of acetic acid (100), and heat at 95 °C for 1
minute. To 10,uL of this solution add 50,mL of the sample
solution, and mix. Proceed with 10 iiL of this solution under
the above operating conditions, and calculate the resolution.
Use a column such that the resolution between the peak of
elcatonin and the peak which appears immediately before the
614
Elcatonin / Official Monographs
JP XV
peak of elcatonin is not less than 2.0, and the retention time
of elcatonin is about 25 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of elcatonin from 10 /xL of the standard
solution is between 50 mm and 200 mm.
Time span of measurement: Continue measurement until
the regularly changing base-line of the chromatogram disap-
pears, beginning after the solvent peak.
Water <2.48> Weigh accurately 1-3 mg of Elcatonin quick-
ly under conditions of 25 ± 2°C and 50 ± 5% relative hu-
midity, and perform the test as directed in 2. Coulometric
titration: not more than 8.0%.
Nitrogen content Weigh accurately 0.015- 0.02 g of El-
catonin quickly under conditions of 25 ± 2°C and 50 ± 5%
relative humidity, and perform the test as directed under
Nitrogen Determination <1.08>: it contains not less than
16.1% and not more than 18.7% of nitrogen (N: 14.01) in the
peptide, calculated on the dehydrated and de-acetic acid ba-
sis.
Assay (i) Animals: Select healthy male Sprague-Dawley rats
each weighing between 90 g and 110 g. Keep the rats for not
less than 3 days before use, providing an appropriate uniform
diet and water.
(ii) Diluent for elcatonin: Dissolve 2.72 g of sodium acetate
in water to make 200 mL, add 0.2 g of bovine serum albu-
min, and adjust the pH to 6.0 with acetic acid (100). Prepare
before use.
(iii) Standard solution: Dissolve Elcatonin Reference Stan-
dard in the diluent for elcatonin to make two standard solu-
tions, one to contain exactly 0.075 Unit in each mL which is
designated as the high-dose standard solution, S H , and the
other to contain exactly 0.0375 Unit in each mL which is
designated as the low-dose standard solution, S L .
(iv) Sample solution: Weigh accurately 0.5 - 2.0 mg of El-
catonin quickly under conditions of 25 ± 2°C and 50 ± 5%
relative humidity, and dissolve in the diluent for elcatonin to
make two sample solutions, the high-dose sample solution,
T H , which contains the Units per mL equivalent to S H and the
low-dose sample solution, T L , which contains the Units per
mL equivalent to S L .
(v) Deproteinizing solution for elcatonin: Dissolve 160 g of
trichloroacetic acid and 30.6 g of strontium chloride in water
to make 3600 mL.
(vi) Procedure: Divide the animals into 4 equal groups of
not less than 10 animals each. Withhold all food, but not
water, for 18 to 24 hours before the injections, and withhold
water during the assay until the final blood sample is taken.
Handle the animals with care in order to avoid undue excite-
ment.
Inject exactly 0.2 mL each of the standard solutions and
the sample solutions into the tail vein of each animal as indi-
cated in the following design:
First group S H Third group T H
Second group S L Fourth group T L
At 1 hour after the injection, take a sufficient blood sample
to perform the test from the carotid artery and vein of each
animal under ether anesthesia, centrifuge the blood samples
to separate serum, and determine the serum calcium accord-
ing to the following (vii).
(vii) Serum calcium determination: Take exactly 0.3 mL of
the serum, add the deproteinizing solution for elcatonin to
make exactly 3 mL, mix well, centrifuge, and use the super-
natant liquid as the sample solution for calcium determina-
tion. Separately, pipet 1 mL of Standard Calcium Solution
for Atomic Absorption Spectrophotometry <2.23>, and add a
solution of sodium chloride (17 in 2000) to make exactly 10
mL. Pipet 5 mL of this solution, add the deproteinizing solu-
tion for elcatonin to make exactly 50 mL, and use this solu-
tion as the standard solution for calcium determination. De-
termine the absorbances, A T and A s , of the sample solution
and the standard solution as directed under Atomic Absorp-
tion Spectrophotometry <2.23> according to the following
conditions. Determine the absorbance, A , of a solution ob-
tained in the same manner used for preparation of the stan-
dard solution, but with 1 mL of water instead of the standard
solution.
Amount (mg) of calcium (Ca) in 100 mL of the serum
= 0.01 x {(A T -A )/(A S -A )} x 10 x 100
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Calcium hollow-cathode lamp
Wavelength: 422.7 nm
(viii) Calculation: Amounts of calcium in 100 mL of the se-
rum obtained with S H , S L , T H and T L in (vii) are symbolized
as y>\, y 2 , )>i and y 4 , respectively. Sum up individual y lt y 2 , y^
and y 4 to obtain Y lt Y 2 , T 3 and Y 4 , respectively.
Units per mg of peptide, calculated on the dehydrated
and de-acetic acid basis
= antilog M x (units per mL of S H x (b/a)
M = 0.3010 x (YJY b )
y. = - y, - y, + y, + y 4
y b = y - y 2 + y 3 - y 4
a: Amount (mg) of the sample
x {100 - [water content (%) + acetic acid content (%)]/100}
b: Total volume (mL) of the high-dose sample solution pre-
pared by dissolving the sample with diluent for elcato-
F' computed by the following equation should be smaller
than F shown in the table against n with which s 2 is calculat-
ed. Calculate L (P = 0.95) by use of the following equation: L
should be not more than 0.20. Iff exceeds F, or if L exceeds
0.20, repeat the test, increasing the number of animals or ar-
ranging the assay conditions so thatF' is not more than F and
L is not more than 0.20.
F = (- y, + y 2 + y, - y 4 ) 2 /4/^ 2
/: Number of the animals of each group.
s 2 = {T.y 2 - (Y/f)}/n
£y 2 : The sum of squares of y lt y 2 , y^ and y 4 in each
group.
y = y 2 + y 2 2 + y, 2 + y, 2
n = 4 (/ - 1)
L = 2^(C - 1XCM 2 + 0.09062)
t 2 : Value shown in the following table against n used to cal-
culate s 2 .
JPXV
Official Monographs / Enflurane 615
n
P = F
n
t 2 = F
n
t 2 = F
1
161.45
13
AMI
25
4.242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
oo
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Containers and storage Containers-
Storage — Not exceeding 8°C.
Enflurane
i>7JL-^:
-Tight containers.
R F F F
xv
and enantiomer
C 3 H 2 C1F 5 0: 184.49
(2/?S)-2-Chloro-l-(difluoromethoxy)-l,l,2-trifluoroethane
[13838-16-9]
Description Enflurane is a clear, colorless liquid.
It is slightly soluble in water.
It is miscible with ethanol (95) and with diethyl ether.
It is a volatile, and not an inflammable.
It shows no optical rotation.
Boiling point: 54 - 57°C
Identification (1) Take 50 /uL of Enflurane, and prepare
the test solution as directed to the Oxygen Flask Combus-
tion Method <1.06> using 40 mL of water as the absorbing
liquid. The test solution responds to the Qualitative Tests
<1.09> for chloride and fluoride.
(2) Determine the infrared absorption spectrum of Enflu-
rane as directed in the liquid film method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
Refractive index <2.45> n 2 °: 1.302 - 1.304
Specific gravity <2.56> d™: 1.520 - 1.540
Purity (1) Acidity or alkalinity — To 60 mL of Enflurane
add 60 mL of freshly boiled and cooled water, shake for 3
minutes, separate the water later, and use the layer as the
sample solution. To 20 mL of the sample solution add one
drop of bromocresol purple TS and 0.10 mL of 0.01 mol/L
sodium hydroxide TS: the color of the solution is purple. To
20 mL of the sample solution add one drop of bromocresol
purple TS and 0.06 mL of 0.01 mol/L hydrochloric acid TS:
the color of the solution is yellow.
(2) Chloride <1.03>— To 20 g of Enflurane add 20 mL of
water, shake well, and separate the water layer. Take 10 mL
of the water layer add 6 mL of dilute nitric acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid (not more than 0.001%).
(3) Nonvolatile residue — Evaporate exactly 65 mL of En-
flurane on a water bath to dryness, and dry the residue at
105 °C for 1 hour: the of the residue is not more than 1.0 mg.
(4) Related substances — Proceed the test with 5 /uL of En-
flurane as directed under Gas chromatography <2.02> accord-
ing to the following conditions. Determine each peak area
other than the peak of air which appears soon after injection
of the sample by the automatic integration method, and cal-
culate the amount of each peak by the area percentage
method: the amount of the substances other than enflurane is
not more than 0.10%.
Operating conditions —
Detector: A thermal conductivity detector.
Column: A column 3 mm in inside diameter and 3 m in
length, packed with siliceous earth for gas chromatography,
180 to 250 /um in particle diameter, coated with diethylene
glycol succinate ester for gas chromatography in the ratio of
20%.
Column temperature: A constant temperature of about
80°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
enflurane is about 3 minutes.
Time span of measurement: About three times as long as
the retention time of enflurane.
System suitability —
Test for required detection: Pipet exactly 1 mL of enflu-
rane add 2-propanol to make exactly 100 mL. To exactly
2 mL of this solution add 2-propanol to make exactly 10 mL,
and use this solution as the solution for system suitability
test. Pipet 1 mL of the solution, and add 2-propanol to make
exactly 10 mL. Confirm that the peak area of enflurane ob-
tained from 5 /uL of this solution is equivalent to 7 to 13% of
that of enflurane obtained from 5 jXL of the solution for sys-
tem suitability test.
System performance: Mix 5 mL of Enflurane and 5 mL of
2-propanol. When the procedure is run with 5 /uL of this mix-
ture under the above operating conditions, enflurane and 2-
propanol are eluted in this order with the resolution between
these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of enflurane is not more than 2.0%.
Water <2.48> Not more than 0.10% (10 g, direct titration).
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
616
Enoxacin Hydrate / Official Monographs
JP XV
Enoxacin Hydrate
iy*t-»*iffl»
HN
\^- n vx n sj- n
r
•i-j H20
i ^
CO;H
C 15 H 17 FN 4 03.1 1 /2H20: 347.34
l-Ethyl-6-fluoro-4-oxo-7-(piperazin-l-yl)-l,4-dihydro-l,8-
naphthyridine-3-carboxylic acid sesquihydrate
[84294-96-2]
Enoxacin Hydrate, when dried, contains not less
than 98.5% of enoxacin (Ci 5 H 17 FN 4 3 : 320.32).
Description Enoxacin Hydrate occurs as white to pale yel-
low-brown crystals or crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
methanol, very slightly soluble in chloroform, and practically
insoluble in water, in ethanol (95) and in diethyl ether.
It dissolves in dilute sodium hydroxide TS.
It is gradually colored by light.
Identification (1) Place 0.02 g of Enoxacin Hydrate and
0.05 g of sodium in a test tube, and heat gradually to ignition
with precaution. After cooling, add 0.5 mL of methanol and
then 5 mL of water, and heat to boiling. To this solution add
2 mL of dilute acetic acid, and filter: the filtrate responds to
the Qualitative Tests <1.09> (2) for fluoride.
(2) Dissolve 0.05 g of Enoxacin Hydrate in dilute sodium
hydroxide TS to make 100 mL. To 1 mL of the solution add
water to make 100 mL. Determine the absorption spectrum
of the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Enox-
acin Hydrate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 225 - 229°C (after drying).
Purity (1) Sulfate <1.14> — Dissolve 1.0 g of Enoxacin Hy-
drate in 50 mL of dilute sodium hydroxide TS, shake with 10
mL of dilute hydrochloric acid, and centrifuge. Filter the su-
pernatant liquid, and to 30 mL of the filtrate add water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution as follows: to 0.50 mL
of 0.005 mol/L sulfuric acid VS add 25 mL of dilute sodium
hydroxide TS, 5 mL of dilute hydrochloric acid TS and water
to make 50 mL (not more than 0.048%).
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Enoxacin
Hydrate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Enoxacin Hydrate according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Enoxacin Hy-
drate in 25 mL of a mixture of chloroform and methanol
(7:3), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add a mixture of chloroform and
methanol (7:3) to make exactly 200 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 [iL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (3:1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.4I> 7.0 - 9.0% (1 g, 105 °C, 3 hours).
Residue on ignition <2.44>
num crucible).
Not more than 0.1% (1 g, plati-
Assay Weigh accurately about 0.3 g of Enoxacin Hydrate,
previously dried, dissolve in 30 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.03 mg of C 15 H 17 FN 4 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Enviomycin Sulfate
i>b^"7>f->>8fE8J£
°H,N H
m /
HO
s~ . . I,NH HN^^-0
HN'^O HN
•1^-H 2 S0 4
O J> NH
HN
H S N-^C
Tuberactinomycin N : R = OH
Tuberactinomycin O : R = H
Tuberactinomycin N Sulfate
C 25 H43N 1 30 1 „.lyH 2 S0 4 : 832.81
Tuberactinomycin O Sulfate
C 25 H43N 13 9 .lyH 2 S04: 816.81
Tuberactinomycin N Sulfate
(3i?,4i?)-Aq(3S,9S,12S,15S)-9,12-Bis(hydroxymethyl)-3-
[(4R )-2-iminohexahydropyrimidin-4-yl] -2,5,8,11,14-
pentaoxo-6-(Z)-ureidomethylene-l,4,7,10,13-
pentaazacyclohexadec-15-yl]-3,6-diamino-4-
hydroxyhexanamide sesquisulfate
JPXV
Official Monographs / Enviomycin Sulfate 617
[33103-22-9, Tuberactinomycin N]
Tuberactinomycin O Sulfate
(3S>iV-[(3S,9S,12S,15S)-9,12-Bis(hydroxymethyl)-3-[(4fl)-
2-iminohexahydropyrimidin-4-yl]-2,5,8, 1 1 , 14-pentaoxo-
6-(Z)-ureidomethylene-l,4,7,10,13-pentaazacyclohexadec-
15-yl]-3,6-diaminohexanamide sesquisulfate
[33137-73-4, Tuberactinomycin O]
Enviomycin Sulfate is the sulfate of a mixture of
peptide substances having antibacterial activity pro-
duced by the growth of Streptomyces griseoverticilla-
tus var. tuberacticus.
It contains not less than 770 iig (potency) per mg,
calculated on the dried basis. The potency of Enviomy-
cin Sulfate is expressed as mass (potency) of tuberac-
tinomycin N (C 2 5H43N 13 1 o: 685.69).
Description Enviomycin Sulfate occurs as a white powder.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) To 5 mL of a solution of Enviomycin
Sulfate (1 in 200) add 1.5 mL of sodium hydroxide TS, and
add 1 drop of a mixture of 0.01 mol/L citric acid TS and cop-
per (II) sulfate TS (97:3) : a blue-purple color develops.
(2) Determine the absorption spectrum of a solution of
Enviomycin Sulfate (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) To 2 mL of a solution of Enviomycin Sulfate (1 in 20)
add 1 drop of barium chloride TS: a white precipitate is pro-
duced.
Optical rotation <2.49> [a]™: -16- -22° (0.5 g calculated
on the dried basis, water, 50 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 2.0 g
of Enviomycin Sulfate in 20 mL of water is between 5.5 and
7.5.
Content ratio of the active principle Dissolve 0.1 g of En-
viomycin Sulfate in water to make 100 mL, and use this solu-
tion as the sample solution. Perform the test with 3 /xL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A Tl and A T2 , of tuberactinomycin N and
tuberactinomycin O, having the relative retention time, 1.4
± 0.4, with respect to tuberactinomycin N, by the automatic
integration method: A T2 /(A Tl + A T2 ) is between 0.090 and
0.150.
Operating conditions-
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with silica gel for liquid
chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of ammonium acetate TS, 1,4-di-
oxane, tetrahydrofuran, water and ammonia solution (28)
(100:75:50:23:2).
Flow rate: Adjust the flow rate so that the retention time of
tuberactinomycin N is about 9 minutes.
System suitability —
System performance: When the procedure is run with 3 /uL
of the sample solution under the above operating conditions,
tuberactinomycin N and tuberactinomycin O are eluted in
this order with the resolution between these peaks being not
less than 1.5.
System repeatability: When the test is repeated 6 times with
3 /uL of the sample solution under the above operating condi-
tions, the relative standard deviation of the peak area of
tuberactinomycin N is not more than 2.0%.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Enviomycin Sulfate in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of En-
viomycin Sulfate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Enviomycin Sulfate according to Method 1 , and perform
the test (not more than 1 ppm).
Loss on drying <2.41> Not more than 4.0% (0.2 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillius subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er.
(iii) Standard solutions — Weigh accurately an amount of
Enviomycin Sulfate Reference Standard, equivalent to about
20 mg (potency), dissolve in water to make exactly 20 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at a temperature not exceeding 5°C
and use within 10 days. Take exactly a suitable amount of the
standard stock solution before use, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 400 /ug (potency) and 100 /ug (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Enviomycin Sulfate, equivalent to about 20 mg (potency),
and dissolve in water to make exactly 20 mL. Take exactly a
suitable amount of this solution, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 400 /ug (potency) and 100 /ug (potency), and use these
solutions as the high concentration sample solution and low
concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
618
Eperisone Hydrochloride / Official Monographs
JP XV
Eperisone Hydrochloride
i-<U7">±£i^
• HCI
and enantiomer
C 17 H 25 NO.HCl: 295.85
(2RS)- 1 -(4-Ethylphenyl)-2-methyl-3-piperidin- 1 -ylpropan-1 -
one monohydrochloride [56839-43-1]
Eperisone Hydrochloride contains not less than
98.5% and not more than 101.0% of C 17 H 25 NO.HCl,
calculated on the anhydrous basis.
Description Eperisone Hydrochloride occurs as a white
crystalline powder.
It is freely soluble in water, in methanol and in acetic acid
(100), and soluble in ethanol (99.5).
Melting point: about 167°C (with decomposition).
A solution of Eperisone Hydrochloride in methanol (1 in
100) shows no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Eperisone Hydrochloride in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Eperi-
sone Hydrochloride as directed in the potassium chloride disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) A solution of Eperisone Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Eperisone Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Piperidine hydrochloride — Dissolve 1.0 g of Eperi-
sone Hydrochloride in 20 mL of water, add 2.0 mL of diluted
hydrochloric acid (1 in 2), 2.0 mL of a solution of copper (II)
sulfate pentahydrate (1 in 20) and 1.5 mL of ammonia solu-
tion (28), and use this solution as the sample solution.
Separately, to 2.0 mL of a solution of piperidine hydrochlo-
ride (1 in 1000) add 18 mL of water, 2.0 mL of diluted
hydrochloric acid (1 in 2), 2.0 mL of a solution of copper (II)
sulfate pentahydrate (1 in 20) and 1.5 mL of ammonia solu-
tion (28), and use this solution as the standard solution. To
each of the sample solution and standard solution add 10 mL
of a mixture of isopropylether and carbon disulfide (3:1),
shake for 30 seconds, allow them to stand for 2 minutes, and
compare the color of the upper layer: the color obtained from
the sample solution is not more darker than that from the
standard solution.
(3) Related substances — Dissolve 0.1 g of Eperisone
Hydrochloride in 100 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 liL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the total area of
the peaks other than the peak of eperisone is not more than
1/5 of the peak area of eperisone from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 iim in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of methanol, 0.0375 mol/L sodi-
um 1-decanesulfonate TS and perchloric acid (600:400:1).
Flow rate: Adjust the flow rate so that the retention time of
eperisone is about 17 minutes.
Time span of measurement: About 2 times as long as the
retention time of eperisone.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add the mobile phase to make exactly 10 mL.
Confirm that the peak area of eperisone obtained from 10 iiL
of this solution is equivalent to 7 to 13% of that obtained
from 10 tiL of the standard solution.
System performance: When the procedure is run with 10
iiL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of eperisone are not less than 4000 steps
and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
eperisone is not more than 3.0%.
Water <2.48> Not more than 0.2% (0. 1 g, coulometric titra-
tion).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.6 g of Eperisone
Hydrochloride, dissolve in 20 mL of acetic acid (100), add
80 mL of acetic anhydride, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.58 mg of C I7 H 25 NO.HCl
Containers and storage Containers — Well-closed contain-
JPXV
Official Monographs / Ephedrine Hydrochloride Injection
619
Ephedrine Hydrochloride
I7i KU>£8£
H OH
CM,
■HCI
Ci H 15 NO.HCl: 201.69
( 1 R ,2 S)-2-Methylamino- 1 -phenylpropan-1 -ol
monohydrochloride [50-98-6]
Ephedrine Hydrochloride, when dried, contains not
less than 99.0% of C 10 H 15 NO.HC1.
Description Ephedrine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in water, soluble in ethanol (95), slightly
soluble in acetic acid (100), and practically insoluble in
acetonitrile and in acetic anhydride.
Identification (1) Determine the absorption spectrum of a
solution of Ephedrine Hydrochloride (1 in 2000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Ephe-
drine Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Ephedrine Hydrochloride (1 in 15)
responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [a]™: —33.0 ■
drying, 1 g, water, 20 mL, 100 mm).
■36.0° (after
pH <2.54> Dissolve 1.0 g of Ephedrine Hydrochloride in
20 mL of water: the pH of this solution is between 4.5 and
6.5.
Melting point <2.60> 218 - 222°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Ephedrine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate < 1. 14>— Dissolve 0.05 g of Ephedrine
Hydrochloride in 40 mL of water, add 1 mL of dilute
hydrochloric acid and 1 mL of barium chloride TS, and allow
to stand for 10 minutes: no turbidity is produced.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Ephe-
drine Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 1.0 mL of Standard
Lead Solution (not more than 10 ppm).
(4) Related substances — Dissolve 0.05 g of Ephedrine
Hydrochloride in 50 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample
solution, add the mobile phase to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 /uL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the areas
of each peak by the automatic integration method: the total
area of the peaks other than ephedrine from the sample solu-
tion is not larger than the peak area of ephedrine from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
45°C.
Mobile phase: A mixture of a solution of sodium lauryl
sulfate (1 in 128), acetonitrile and phosphoric acid
(640:360:1).
Flow rate: Adjust the flow rate so that the retention time of
ephedrine is about 14 minutes.
Time span of measurement: About 3 times as long as the
retention time of ephedrine beginning after the solvent peak.
System suitability-
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of ephedrine obtained from 10 /uL
of this solution is equivalent to 4 to 6% of that from the stan-
dard solution.
System performance: Dissolve 1 mg of ephedrine
hydrochloride for assay and 4 mg of atropine sulfate in 100
mL of diluted methanol (1 in 2). When the procedure is run
with 10 /iL of this solution under the above operating condi-
tions, ephedrine and atropine are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of ephedrine is not more than 2.0%.
Loss on drying <2.41>
3 hours).
Not more than 0.5% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Ephedrine
Hydrochloride, previously dried, and dissolve in 50 mL of a
mixture of acetic anhydride and acetic acid (100) (7:3) by
warming. Cool, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 20.17 mg of C 10 H 15 NO.HC1
Containers and storage Containers — Well-closed contain-
ers.
Ephedrine Hydrochloride Injection
i7i Ku>*«*aw«
Ephedrine Hydrochloride Injection is aqueous solu-
tion for injection. It contains not less than 95% and
not more than 105% of the labeled amount of ephe-
620 10% Ephedrine Hydrochloride Powder / Official Monographs
JP XV
drine hydrochloride (C 10 H 15 NO.HC1: 201.69).
Method of preparation Prepare as directed under Injec-
tions, with Ephedrine Hydrochloride.
Description Ephedrine Hydrochloride Injection is a clear,
colorless liquid.
pH: 4.5-6.5
Identification To a volume of Ephedrine Hydrochloride
Injection, equivalent to 0.05 g of Ephedrine Hydrochloride
according to the labeled amount, add water to make 100 mL,
and determine the absorption spectrum of this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 249 nm and 253 nm, between 255
nm and 259 nm, and between 261 nm and 265 nm.
Bacterial endotoxins <4.01> Less than 7.5 EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay To an exact volume of Ephedrine Hydrochloride
Injection, equivalent to about 40 mg of ephedrine hydrochlo-
ride (C 10 H 15 NO.HC1) according to the labeled amount, add
exactly 10 mL of the internal standard solution and water to
make 200 mL, and use this solution as the sample solution.
Separately, weigh accurately about 40 mg of ephedrine
hydrochloride for assay, previously dried at 105°C for 3
hours, add exactly 10 mL of the internal standard solution to
dissolve, add water to make 200 mL, and use this solution as
the standard solution. Perform the test with 10 iiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of ephedrine to that of the internal standard of each solution.
Amount (mg) of ephedrine hydrochloride (C 10 H 15 NO.HC1)
= W s x (Gt/Gs)
W s : Amount (mg) of ephedrine hydrochloride for assay
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (4) under Ephedrine Hydrochloride.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the internal standard and ephedrine are eluted in this
order with the resolution between these peaks being not less
than 15.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ephedrine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
10% Ephedrine Hydrochloride
Powder
Ephedrine Hydrochloride Powder
17 x K U >£B8£tt 10%
10% Ephedrine Hydrochloride Powder contains not
less than 9.3% and not more than 10.7% of ephedrine
hydrochloride (C 10 H 15 NO.HC1: 201.69).
Method of preparation
Ephedrine Hydrochloride
Starch, Lactose Hydrate or
their mixture
100;
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification To 0.5 g of 10% Ephedrine Hydrochloride
Powder add 100 mL of water, shake for 20 minutes, and
filter. Determine the absorption spectrum of the filtrate as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 249 nm and 253 nm, between 255
nm and 259 nm, and between 261 nm and 265 nm.
Assay Weigh accurately about 0.4 g of 10% Ephedrine
Hydrochloride Powder, add 150 mL of water, and extract
with the aid of ultrasonicator for 10 minutes with occasional
shaking. Shake more for 10 minutes, then add exactly 10 mL
of the internal standard solution and water to make 200 mL,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 40 mg of ephedrine
hydrochloride for assay, previously dried at 105°C for 3
hours, add exactly 10 mL of the internal standard solution to
dissolve, add water to make 200 mL, and use this solution as
the standard solution. Perform the test with 10 iiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of ephedrine to that of the internal standard of each solution.
Amount (mg) of ephedrine hydrochloride (Ci Hi 5 NO.HCl)
= W s x (Gt/Gs)
W s : Amount (mg) of ephedrine hydrochloride for assay
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Operating conditions —
Detector, column, column temperature, mobile phase and
flow rate: Perform as directed in the operating conditions in
the Purity (4) under Ephedrine Hydrochloride.
System suitability —
System performance: When the procedure is run with 10
iiL of the standard solution under the above operating condi-
tions, the internal standard and ephedrine are eluted in this
order with the resolution between these peaks being not less
than 15.
System repeatability: When the test is repeated 6 times with
JP XV
Official Monographs / Epirizole
621
10 fih of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ephedrine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Ephedrine Hydrochloride Tablets
i7i KU>£K£S£
Ephedrine Hydrochloride Tablets contain not less
than 93% and not more than 107% of the labeled
amount of ephedrine hydrochloride (C 10 H 15 NO.HC1:
201.69).
Method of preparation Prepare as directed under Tablets,
with Ephedrine Hydrochloride.
Identification To an amount of powdered Ephedrine
Hydrochloride Tablets, equivalent to 0.05 g of Ephedrine
Hydrochloride, add 100 mL of water, shake for 20 minutes,
and filter. Determine the absorption spectrum of the filtrate
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits maxima between 249 nm and 253 nm, be-
tween 255 nm and 259 nm, and between 261 nm and 265 nm.
Assay Weigh accurately not less than 20 tablets of Ephe-
drine Hydrochloride Tablets, and powder. Weigh accurately
an amount of the powder, equivalent to about 40 mg of ephe-
drine hydrochloride (C 10 H 15 NO.HC1), add 150 mL of water,
and extract with the aid of ultrasonicator for 10 minutes with
occasional shaking. Shake more for 10 minutes, then add ex-
actly 10 mL of the internal standard solution and water to
make 200 mL, centrifuge, and use the supernatant liquid as
the sample solution. Separately, weigh accurately about 40
mg of ephedrine hydrochloride for assay, previously dried at
105 °C for 3 hours, add exactly 10 mL of the internal stan-
dard solution to dissolve, add water to make 200 mL, and use
this solution as the standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Qt and Qs,
of the peak area of ephedrine to that of the internal standard
of each solution.
Amount (mg) of ephedrine hydrochloride (Ci H 15 NO.HCl)
= W s x (Q T /Q S )
W s : Amount (mg) of ephedrine hydrochloride for assay
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Operating conditions—
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (4) under Ephedrine Hydrochloride.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the internal standard and ephedrine are eluted in this
order with the resolution between these peaks being not less
than 15.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ephedrine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Epirizole
Mepirizole
itr u7-;u
Y f
*v \\ /
N
CH 3
C„H 14 N40 2 : 234.25
4-Methoxy-2-(5-methoxy-3-methyl-l//-pyrazol-l-yl)-6-
methylpyrimidine [18694-40-1 ]
Epirizole, when dried, contains not less than 99.0%
of C n HuN 4 02.
Description Epirizole occurs as white crystals or crystalline
powder. It is odorless, and has a bitter taste.
It is very soluble in methanol and in acetic acid (100), freely
soluble in ethanol (95), and sparingly soluble in water and in
diethyl ether.
It dissolves in dilute hydrochloric acid and in sulfuric acid.
The pH of a solution of Epirizole (1 in 100) is between 6.0
and 7.0.
Identification (1) To 0.1 g of Epirizole add 0.1 g of vanil-
lin, 5 mL of water and 2 mL of sulfuric acid, and mix with
shaking for a while: a yellow precipitate is formed.
(2) Dissolve 0.1 g of Epirizole in 10 mL of water, and add
10 mL of 2,4,6-trinitrophenol TS: a yellow precipitate is
produced. Collect the precipitate by filtration, wash with 50
mL of water, and dry at 105°C for 1 hour: it melts <2.60> be-
tween 163°C and 169°C.
(3) Determine the absorption spectrum of a solution of
Epirizole in 0.1 mol/L hydrochloric acid TS (1 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
Melting point <2.60>
91°C
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Epirizole in 20 mL of water: the solution is clear and color-
less.
(2) Chloride <1.03> — Add 0.5 g of Epirizole to a ground
mixture of 0.7 g of potassium nitrate and 1.2 g of anhydrous
sodium carbonate, mix well, transfer little by little to a plati-
num crucible, previously heated, and heat until the reaction is
completed. After cooling, add 15 mL of dilute sulfuric acid
and 5 mL of water to the residue, boil for 5 minutes, filter,
wash the insoluble matter with 10 mL of water, and add 6 mL
622
Epirubicin Hydrochloride / Official Monographs
JP XV
of dilute nitric acid and water to the combined filtrate and
washings to make 50 mL. Perform the test with this solution
as the test solution. Prepare the control solution as follows:
proceed with the same quantities of the same reagents as
directed for the preparation of the test solution, and add 0.25
mL of 0.01 mol/L hydrochloric acid VS and water to make
50 mL (not more than 0.018%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Epirizole
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Epirizole according to Method 3, and perform the test (not
more than 2 ppm).
(5) Related substances — Dissolve 1.0 g of Epirizole in 10
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add methanol to
make exactly 50 mL. Pipet 1 mL of this solution, add
methanol to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2//L
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of isopropyl
diethyl ether, ethanol (95) and water (23:10:2) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution. Place this plate in a
chamber filled with iodine vapor: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
(6) Readily carbonizable substances <1.15> — Perform the
test with 0.10 g of Epirizole: the solution has no more color
than Matching Fluid A.
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Epirizole, previous-
ly dried, dissolve in 40 mL of acetic acid (100) and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 2 drops
of crystal violet TS) until the color of the solution changes
from purple through blue-green to green.
Perform a blank determination, and make any necessary
correction.
Each mL of 0.1 mol/L perchloric acid VS
= 23.43 mg of C„H 14 NA
Containers and storage Containers — Well-closed contain-
ers.
Epirubicin Hydrochloride
xb°)ut:~v>±iig±s
H : ,C
(2S,4S)-4-(3-Amino-2,3,6-trideoxy-a-L-ara6wo-
hexopyranosyloxy)-2,5,12-trihydroxy-2-hydroxyacetyl-
7-methoxy-l,2,3,4-tetrahydrotetracene-6,ll-dione
monohydrochloride [56390-09-1]
Epirubicin Hydrochloride is the hydrochloride of a
derivative of daunorubicin.
It contains not less than 970 /ug (potency) and not
more than 1020 fig (potency) per mg, calculated on the
anhydrous basis and corrected by the amount of the
residual solvent. The potency of Epirubicin
Hydrochloride is expressed as mass (potency) of
epirubicin hydrochloride (^T^gNOn-HCl).
Description Epirubicin Hydrochloride occurs as a pale
yellowish red to brownish red powder.
It is soluble in water and in methanol, slightly soluble in
ethanol (95), and practically insoluble in acetonitrile.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Epirubicin Hydrochloride in methanol (3 in
200,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Epirubicin Hydrochloride and Epirubicin Hydrochloride
Reference Standard as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare these spectra: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: +310 - +340° (10 mg calcu-
lated on the anhydrous basis and collected by the amount of
the residual solvent, methanol, 20 mL, 100 mm).
pH <2.54> Dissolve 10 mg of Epirubicin Hydrochloride in 2
mL of water: the pH of the solution is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 50 mg
of Epirubicin Hydrochloride in 5 mL of water: the solution is
clear and dark red.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Epirubi-
cin Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Perform the test with 10,uL of
JPXV
Official Monographs / Epirubicin Hydrochloride
623
the sample solution obtained in the Assay as directed under
Liquid Chromatography <2.01> according to the following
conditions, determine each peak area by the automatic in-
tegration method, and calculate the sum amount of the peaks
other than epirubicin and 2-naphthalenesulfonic acid by the
area percentage method: not more than 5.0%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of epirubicin beginning after the solvent peak.
System suitability —
Test for required detectability: To 1 mL of the sample
solution add the mobile phase to make 100 mL, and use this
solution as the solution for system suitability test. Pipet 1 mL
of the solution for system suitability test, and add the mobile
phase to make exactly 10 mL. Confirm that the peak area of
epirubicin obtained from 10 iiL of this solution is equivalent
to 7 to 13% of that obtained from 10 /uL of the solution for
system suitability test.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
(4) Residual solvents <2.46> — Weigh accurately about 0.3
g of Epirubicin Hydrochloride, add exactly 0.6 mL of the in-
ternal standard solution, add 7V,,/V-dimethylformamide to
make 6mL, and use this solution as the sample solution.
Separately, pipet 1 mL of methanol, add TV.JV-dimethylfor-
mamide to make exactly 25 mL, and use this solution as
methanol standard stock solution. Take exactly 125 ^L of
acetone, 30 /uL of ethanol (99.5), 32 /uL of 1-propanol and
17 /uL of the methanol standard stock solution, add exactly
10 mL of the internal standard solution and N, iV-dimethyl-
formamide to make 100 mL, and use this solution as the stan-
dard solution. Perform the test with 1 /xL each of the sample
solution and standard solution as directed under Gas Chro-
matography <2.02> according to the following condition, and
determine the ratios of the peak areas of acetone, ethanol, 1-
propanol and methanol to that of the internal standard, Q TA
and Q SA , Q TB and Q SB , Q TC and Q sc , and g TD and g SD , re-
spectively. Calculate the amounts of acetone, ethanol, 1-
propanol and methanol by the following equations: the
amounts of acetone, ethanol, 1-propanol and methanol are
not more than 1.5%, not more than 0.5%, not more than 0.5
% and not more than 0.1%, respectively.
Amount (%) of acetone
= (1/Wt) x (Sta/Gsa) x 593
Amount (%) of ethanol
= (1/Wt) x (Stb/Ssb) x 142
Amount (%) of 1-propanol
= (1/Wt) x (Qjc/Qsc) x 154
Amount (%) of methanol
= (l/W T ) x (Q 1D /Q SD ) x 2.23
W T : Amount (mg) of Epirubicin Hydrochloride
Internal standard solution — A solution of 1,4-dioxane in
A^Af-dimethylformamide (1 in 100).
Operating conditions —
Detector: Hydrogen flame-ionization detector.
Column: A fused silica column 0.53 mm in inside diameter
and 30 m in length, coated with polyethylene glycol for gas-
chromatography 1 fim in thickness.
Column temperature: 40°C for 11 minutes after injection
of the sample, then rise to 90°C at a rate of 10°C per minute.
If necessary, rise to 130°C at a rate of 50°C per minute and
maintain the temperature for 30 minutes.
Injection port temperature: A constant temperature of
about 120°C.
Detector temperature: A constant temperature of about
150°C.
Carrier gas: Herium
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 8 minutes.
Split ratio: 1:15
System suitability —
System performance: When the procedure is run with 1 /uL
of the standard solution under the above operating condi-
tions, acetone, methanol, ethanol, 1-propanol and the inter-
nal standard are eluted in this order with the resolution be-
tween the peaks of acetone and the internal standard being
not less than 30.
System repeatability: When the test is repeated 6 times with
1 /uL of the standard solution under the above operating con-
ditions, the relative standard deviations of the peak areas of
acetone, methanol, ethanol and 1-propanol are not more
than 4.0%, respectively.
Water Not more than 8.0% (0.1 g, volumetric titration,
direct titration).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Weigh accurately an amount of Epirubicin Hydro-
chloride and Epirubicin Hydrochloride Reference Standard,
equivalent to about 50 mg (potency), dissolve each in the
internal standard solution to make exactly 50 mL, and use
these solutions as the sample solution and standard solution,
respectively. Perform the test with 10 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Q T and Q s , of the peak area
of epirubicin to that of the internal standard.
Amount [wg (potency)] of C 2 7H 2 9NOn.HCl
= W s x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Epirubicin Hydrochloride
Reference Standard
Internal standard solution — A solution of sodium 2-naphtha-
lene sulfonate in a mixture of water, acetonitrile, methanol
and phosphoric acid (540:290:170:1) (1 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with trimethylsilanized silica gel
for liquid chromatography (6 fim in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 2 g of sodium lauryl sulfate in a
mixture of water, acetonitrile, methanol and phosphoric acid
(540:290:170:1) to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
epirubicin is about 9.5 minutes.
System suitability —
624 Ergocalciferol / Official Monographs
JP XV
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the internal standard and epirubicin are eluted in
this order with the resolution between these peaks being not
less than 20.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of epirubicin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — At a temperature between 0°C and 5°C.
Ergocalciferol
Calciferol
Vitamin D 2
H CH 3
CH 3
C 28 H 4 40: 396.65
(3S , ,5Z,7£,22£')-9,10-Secoergosta-5,7,10(19),22-tetraen-3-ol
[50-14-6]
Ergocalciferol contains not less than 97.0% and not
more than 103.0%, of Q-gl^O.
Description Ergocalciferol occurs as white crystals. It is
odorless, or has a faint, characteristic odor.
It is freely soluble in ethanol (95), in diethyl ether and in
chloroform, sparingly soluble in isooctane, and practically
insoluble in water.
It is affected by air and by light.
Melting point: 115-118°C Transfer Ergocalciferol to
a capillary tube, and dry for 3 hours in a desiccator (in vacu-
um at a pressure not exceeding 2.67 kPa). Immediately fire-
seal the capillary tube, put it in a bath fluid, previously heated
to a temperature about 10°C below the expected melting
point, and heat at a rate of rise of about 3°C per minute, and
read the melting point.
Identification (1) Dissolve 0.5 mg of Ergocalciferol in 5
mL of chloroform, add 0.3 mL of acetic anhydride and 0.1
mL of sulfuric acid, and shake: a red color is produced, and
rapidly changes through purple and blue to green.
(2) Determine the infrared absorption spectrum of Er-
gocalciferol as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Ergocalciferol Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
Absorbance <2.24> E\* m (265 nm): 445-485 (0.01 g,
ethanol (95), 100 mL).
Optical rotation <2.49> [a]™: + 102 - + 107° (0.3 g, ethanol
(95), 20 mL, 100 mm). Prepare the solution of Ergocalciferol
within 30 minutes after the container has been opened, and
determine the rotation within 30 minutes after the solution
has been prepared.
Purity Ergosterol — Dissolve 10 mg of Ergocalciferol in 2.0
mL of diluted ethanol (95) (9 in 10), add a solution of 20 mg
of digitonin in 2.0 mL of diluted ethanol (95) (9 in 10), and
allow the mixture to stand for 18 hours: no precipitate is
formed.
Assay Weigh accurately about 30 mg each of Ergocalciferol
and Ergocalciferol Reference Standard, and dissolve each in
isooctane to make exactly 50 mL. Pipet 10 mL each of these
solutions, add exactly 3 mL each of the internal standard
solution, then add the mobile phase to make 50 mL, and use
these solutions as the sample solution and the standard solu-
tion, respectively. Perform the test with 10 to 20 /xL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Q T and Q s , of the peak
area of ergocalciferol to that of the internal standard. Per-
form the procedure rapidly avoiding contact with air or other
oxidizing agents and using light-resistant containers.
Amount (mg) of C 28 H 44 = W s x (Q T /Q S )
W s : Amount (mg) of Ergocalciferol Reference Standard
Internal standard solution — A solution of dimethyl phthalate
in isooctane (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with a silica gel for liq-
uid chromatography (10 /xm particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of hexane and n-amylalcohol
(997:3).
Flow rate: Adjust the flow rate so that the retention time of
ergocalciferol is about 25 minutes.
System suitability —
System performance: Dissolve 15 mg of Ergocalciferol
Reference Standard in 25 mL of isooctane. Transfer this
solution to a flask, heat in an oil bath under a reflux condens-
er for 2 hours, and cool immediately to room temperature.
Transfer the solution to a quartz test tube, and irradiate with
a short-wave lamp (main wavelength: 254 nm) and a long-
wave lamp (main wavelength: 365 nm) for 3 hours. To 10 mL
of this solution add the mobile phase to make 50 mL. When
the procedure is run with 10 /xL of this solution under the
above operating conditions. Use a column with the ratios of
the retention time of previtamin D 2 , trans-vitamin D 2 and
tachysterol 2 to that of ergocalciferol being about 0.5, about
0.6 and about 1.1, respectively, and with resolution between
previtamin D 2 and trans-vitamin D 2 being not less than 0.7,
and that between ergocalciferol and tachysterol 2 being not
less than 1.0.
System repeatability: When the test is repeated 6 times with
JP XV
Official Monographs / Ergometrine Maleate Injection
625
10 fih of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ergocalciferol to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, under Nitrogen atmosphere, and
in a cold place.
Ergometrine Maleate
C 19 H 2 3N30 2 .C4H 4 04: 441.48
(8 S )-N-[( 1 S )-2-Hydroxy- 1 -methylethyl] -6-methyl-
9, 10-didehydroergoline-8-carboxamide monomaleate
[129-51-1]
Ergometrine Maleate, when dried, contains not less
than 98.0% of Q^NjCVC^CV
Description Ergometrine Maleate occurs as a white to pale
yellow, crystalline powder. It is odorless.
It is sparingly soluble in water, slightly soluble in methanol
and in ethanol (95), and practically insoluble in diethyl ether.
Melting point: about 185°C (with decomposition).
It gradually changes to yellow in color on exposure to light.
Identification (1) Prepare a solution of Ergometrine
Maleate (1 in 50): the solution shows a blue fluorescence.
(2) Dissolve 1 mg of Ergometrine Maleate in 5 mL of
water. To 1 mL of this solution add 2 mL of 4-
dimethylaminobenzaldehyde-ferric chloride TS, shake, and
allow to stand for 5 to 10 minutes: a deep blue color de-
velops.
(3) To 5 mL of a solution of Ergometrine Maleate (1 in
500) add 1 drop of potassium permanganate TS: the red color
of the solution disappears immediately.
Optical rotation <2.49> [a]™
0.25 g, water, 25 mL, 100 mm).
+ 48- +57° (after drying
pH <2.54> Dissolve 0.10 g of Ergometrine Maleate in 10 mL
of water. The pH of the solution is between 3.0 and 5.0.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Ergometrine Maleate in 10 mL of water: the solution is
clear and colorless to light yellow.
(2) Ergotamine and ergotoxine — To 0.02 g of Ergome-
trine Maleate add 2 mL of a solution of sodium hydroxide (1
in 10), and heat to boiling: the gas evolved does not change
moistened red litmus paper to blue.
(3) Related substances — Dissolve 5 mg each of Ergome-
trine Maleate and Ergometrine Maleate Reference Standard
in 1.0 mL of methanol, and use these solutions as the sample
solution and standard solution, respectively. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate, prepared with silica gel for
thin-layer chromatography and dilute sodium hydroxide TS.
Develop the plate with a mixture of chloroform and
methanol (4:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly 4-dimethylaminobenzaldehyde TS on the
plate: the spots obtained from the sample solution and the
standard solution show a red-purple color and the same Rf
value, and any spot from the sample solution other than that
corresponding to the spot from the standard solution does
not appear.
Loss on drying <2.41>
4 hours).
Not more than 2.0% (0.2 g, silica gel,
Assay Weigh accurately about 10 mg each of Ergometrine
Maleate and Ergometrine Maleate Reference Standard,
previously dried in a desiccator (silica gel) for 4 hours, dis-
solve in water to make exactly 250 mL, and use these solu-
tions as the sample solution and the standard solution,
respectively. Pipet 2 mL of each solution into a separate
brown glass-stoppered tube. To each tube add 4 mL of 4-
dimethylaminobenzaldehyde-iron (III) chloride TS, exactly
measured, while cooling in an ice bath, then warm at 45°C
for 10 minutes. Allow to stand at room temperature for 20
minutes, and perform the test with these solutions as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using a
solution, prepared with 2 mL of water in the same manner, as
the blank. Determine the absorbances, A T and A s , of the sub-
sequent solutions of the sample solution and the standard so-
lution at 550 nm, respectively.
Amount (mg) of Q9H23N3O2.C4H4O4
= W s x (A T /A S )
W s : Amount (mg) of Ergometrine Maleate Reference Stan-
dard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ergometrine Maleate Injection
iJUaV h- U >7N >m&%tt%L
Ergometrine Maleate Injection is an aqueous solu-
tion for injection.
It contains not less than 90% and not more than 110
% of the labeled amount of ergometrine maleate (C 19 H
23 N30 2 .C 4 H 4 4 : 441.48).
Method of preparation Prepare as directed under Injec-
tions, with Ergometrine Maleate.
Description Ergometrine Maleate Injection is a clear, color-
less to pale yellow liquid.
pH: 2.7-3.5
Identification (1) Measure a volume of Ergometrine Male-
ate Injection, equivalent to 3 mg of Ergometrine Maleate ac-
cording to the labeled amount, if necessary, dilute with water
or evaporate on a water bath to make 15 mL, and use this
solution as the sample solution. The sample solution shows a
blue fluorescence.
(2) To 1 mL of the sample solution obtained in (1) add 1
626
Ergometrine Maleate Tablets / Official Monographs
JP XV
mL of ammonia TS, and extract with 20 mL of diethyl ether.
To the diethyl ether extract add 1 mL of dilute sulfuric acid,
shake, and warm to remove diethyl ether in a water bath.
Cool, to the residue obtained add 2 mL of 4-
dimethylaminobenzaldehyde-iron (III) chloride TS, and al-
low to stand for 5 to 10 minutes: a deep blue color develops.
(3) To 5 mL of the sample solution obtained in (1) add 1
drop of potassium permanganate TS: a red color disappears
immediately.
Extractable volume <6.05> It meets the requirement.
Assay Transfer an exactly measured volume of Ergome-
trine Maleate Injection, equivalent to about 2 mg of ergome-
trine maleate (C19H23N3O2.C4H4O4), and add sodium chlo-
ride in a ratio of 0.3 g to 1 mL of the solution. To this mix-
ture add 20 mL of diethyl ether and 2 mL of ammonia TS,
shake, and extract. Further, extract with three 15-mL por-
tions of diethyl ether, combine all the extracts, add 5 g of an-
hydrous sodium sulfate, filter through a pledget of absorbent
cotton, and wash with three 5-mL portions of diethyl ether.
Add the washings to the filtrate, shake with 5 mL of dilute
sulfuric acid, evaporate the diethyl ether by warming in a cur-
rent of nitrogen, to the remaining solution add water to make
exactly 50 mL, and use this solution as the sample solution.
Weigh accurately about 2 mg of Ergometrine Maleate Refer-
ence Standard, previously dried in a desiccator (silica gel) for
4 hours, add water to make exactly 50 mL, and use this solu-
tion as the standard solution. Transfer 2 mL each of the sam-
ple solution and standard solution, accurately measured, to
separate glass-stoppered test tubes, and proceed as directed in
the Assay under Ergometrine Maleate.
Amount (mg) of ergometrine maleate (Q9H23N3O2.C4H4O4)
= W s x (A T /A S )
W s : Amount (mg) of Ergometrine Maleate Reference Stan-
dard
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant, and in a cold place.
Ergometrine Maleate Tablets
Ergometrine Maleate Tablets contain not less than
90% and not more than 1 10% of the labeled amount of
ergometrine maleate (Q9H23N3O2.C4H4O4: 441.48).
Method of preparation Prepare as directed under Tablets,
with Ergometrine Maleate.
Identification To a quantity of powdered Ergometrine
Maleate Tablets, equivalent to 3 mg of Ergometrine Maleate
according to the labeled amount, add 15 mL of warm water,
shake, and filter: the filtrate shows a blue fluorescence. Pro-
ceed with this solution as directed in the Identification (2) and
(3) under Ergometrine Maleate.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Transfer 1 tablet of Ergometrine Maleate Tablets to a
glass-stoppered centrifuge tube, and add a solution of L-tar-
taric acid (1 in 100) to make exactly KmL of a solution con-
taining about 40 fig of ergometrine maleate
(Q9H23N3O2.C4H4O4) per ml. Stopper the tube, shake for 30
minutes vigorously, centrifuge, and use the supernatant liq-
uid as the sample solution. Separately, weigh accurately
about 4 mg of Ergometrine Maleate Reference Standard,
previously dried in a desiccator (silica gel) for 4 hours, dis-
solve in water to make exactly 100 mL, and use this solution
as the standard solution. Pipet 4 mL each of the sample solu-
tion and standard solution into separate brown glass-stop-
pered test tubes, add exactly 8 mL each of 4-
dimethylaminobenzaldehyde-iron (III) chloride TS while
cooling in an ice bath, after shaking, and allow to stand for 1
hour at ordinary temperature. Perform the test with these so-
lutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using a solution, prepared with 4 mL of water
in the same manner, as the blank. Determine the absor-
bances, A T and A s , of the subsequent solutions of the sample
solution and the standard solution at 550 nm, respectively.
Amount (mg) of ergometrine maleate(Ci9H 2 3N202.C4H40 4 )
= W s x (A T /A S ) x (K/100)
W s : Amount (mg) of Ergometrine Maleate Reference Stan-
dard
Assay Weigh accurately, and powder not less the 20 Er-
gometrine Maleate Tablets. Weigh accurately a portion of the
powder, equivalent to about 2 mg of ergometrine maleate
(C 19 H23N302.C4H 4 04), transfer to a glass filter (G4), add 10
mL of a solution of L-tartaric acid (1 in 100), and filter with
thorough shaking. Repeat the procedures 3 times, combine
the filtrates, add a solution of L-tartaric acid (1 in 100) to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 2 mg of Ergome-
trine Maleate Reference Standard, previously dried in a desic-
cator (silica gel) for 4 hours, dissolve in a solution of L-tartar-
ic acid (1 in 100) to make exactly 50 mL, and use this solution
as the standard solution. Pipet 2 mL each of the sample solu-
tion and standard solution, and proceed as directed in the As-
say under Ergometrine Maleate.
Amount (mg) of ergometrine maleate (C19H23N3O2.C4H4O4)
= W s x (A T /A S )
W s : Amount (mg) of Ergometrine Maleate Reference Stan-
dard
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
JPXV
Official Monographs / Erythromycin 627
Ergotamine Tartrate
■ ' L '- °vt N ^
(>
HOjC
CO>H
OH
(C33H 35 N 5 5 ) 2 .C 4 H 6 6 : 1313.41
(5'S)-5'-Benzyl-12'-hydroxy-2'-methylergotaman-3',6',18-
trione hemitartrate [379-79-3]
Ergotamine Tartrate contains not less than 98.0% of
(€331^35^05)2. C 4 H 6 6 , calculated on the dried basis.
Description Ergotamine Tartrate occurs as colorless crys-
tals, or a white to pale yellowish white or grayish white, crys-
talline powder.
It is slightly soluble in water and in ethanol (95).
Melting point: about 180°C (with decomposition).
Identification (1) Dissolve 1 mg of Ergotamine Tartrate in
10 mL of a mixture of acetic acid (100) and ethyl acetate
(1:1). To 0.5 mL of this solution add slowly 0.5 mL of sulfur-
ic acid, with shaking in cold water, and allow to stand: a pur-
ple color develops. To this solution add 0.1 mL of diluted
iron (III) chloride TS (1 in 12): the color of the solution
changes to blue to blue-purple.
(2) Dissolve 1 mg of Ergotamine Tartrate in 5 mL of a so-
lution of L-tartaric acid (1 in 100). To 1 mL of this solution
add 2 mL of 4-dimethylaminobenzaldehyde-iron (III) chlo-
ride TS, and shake: a blue color develops.
Optical rotation <2.49> Ergotamine base [a\o- — 155— -
165°. Dissolve 0.35 g of Ergotamine Tartrate in 25 mL of a
solution of L-tartaric acid (1 in 100), add 0.5 g of sodium
hydrogen carbonate, shake gently and sufficiently, and ex-
tract with four 10-mL portions of ethanol-free chloroform.
Filter the extracts successively through a small filter paper,
moistened with ethanol-free chloroform, into a 50-mL volu-
metric flask. Allow the flask to stand in a water bath at 20°C
for 10 minutes, and determine the optical rotation in a
100-mm cell. Separately, pipet 25 mL of this solution,
evaporate to dryness under reduced pressure at a temperature
not higher than 45°C, dissolve the residue in 25 mL of acetic
acid (100), and titrate <2.50> with 0.05 mol/L perchloric acid
VS (indicator: 1 drop of crystal violet TS). Perform a blank
determination, and make any necessary correction. Calculate
the specific rotation of the ergotamine base from the con-
sumed volume of 0.05 mol/L perchloric acid VS and the opti-
cal rotation.
Each mL of 0.05 mol/L perchloric acid VS
= 29.08 mg of C33H35N5O5
Purity Related substances — Conduct this procedure
without exposure to daylight, using light-resistant vessels. To
40 mg of Ergotamine Tartrate add 10 mL of a solution of L-
tartaric acid in diluted methanol (1 in 2) (1 in 1000), dissolve
with thorough shaking, and use this solution as the sample
solution. Pipet 1 mL of this solution, add a solution of L-tar-
taric acid in diluted methanol (1 in 2) (1 in 1000) to make ex-
actly 50 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of chloroform and methanol (9:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly 4-dimethylaminoben-
zaldehyde TS on the plate: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 5.0% (0.1 g, in vacu-
um, 60°C, 4 hours).
Assay Weigh accurately about 0.2 g of Ergotamine Tar-
trate, dissolve in 15 mL of a mixture of acetic acid (100) and
acetic anhydride (50:3), and titrate <2.50> with 0.05 mol/L
perchloric acid VS (indicator: 1 drop of crystal violet TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.05 mol/L perchloric acid VS
= 32.84 mg of (C33H 35 N 5 5 ) 2 .C 4 H 6 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled, or under
nitrogen atmosphere, and not exceeding 5°C.
Erythromycin
C 37 H 67 N0 1 3: 733.93
(2R,3S,4S,5R,6R,8R,10RMR,l2S,l3R)-5-(3A,6-
Trideoxy-3-dimethylamino-/?-D-xy/o-hexopyranosyloxy)-
3-(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-n7jo-
hexopyranosyloxy)-6, 1 1 , 12-trihydroxy-
2,4,6,8,10,12-hexamethyl-9-oxopentadecan-13-olide
[114-07-8]
Erythromycin is a macrolide substance having an-
tibacterial activity produced by the growth of Sac-
charopolyspora erythraea.
It contains not less than 930 fig (potency) and not
more than 1020 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Erythromycin is
expressed as mass (potency) of erythromycin
(C 37 H 67 N0 13 ).
Description Erythromycin occurs as a white to light yellow-
628
Erythromycin / Official Monographs
JP XV
ish white powder.
It is very soluble in 7V,iV-dimethylformamide, freely solu-
ble in methanol and in ethanol (95), and very slightly soluble
in water.
Identification (1) Determine the infrared absorption spec-
trum of Erythromycin as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Erythromycin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(2) Dissolve 10 mg each of Erythromycin and
Erythromycin Reference Standard in 1 mL of methanol, and
use these solutions as the sample solution and standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of methanol and ammonia solution (28) (50:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
methoxybenzaldehyde-sulfuric acid TS on the plate, and heat
at 100°C for 15 minutes: the principal spot from the sample
solution and the spot from the standard solution are dark
purple in color, and their i?f values are the same.
Optical rotation <2.49> [ a ]^°: -71 - -78° (1 g calculated
on the anhydrous basis, ethanol (95), 50 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Erythromycin according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Erythromycin according to Method 5 using hydrochloric
acid instead of diluted hydrochloric acid (1 in 2), and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 40 mg of Erythromycin
in 2 mL of methanol, add a mixture of phosphate buffer solu-
tion, pH 7 . and methanol ( 1 5 : 1 ) to make exactly 1 mL , and
use this solution as the sample solution. Separately, dissolve
16 mg of Erythromycin Reference Standard in 2 mL of
methanol, add a mixture of phosphate buffer solution, pH
7.0 and methanol (15:1) to make exactly 10 mL, and use this
solution as the standard stock solution. Dissolve 5 mg each of
erythromycin B and erythromycin C in 2 mL of methanol,
add exactly 2 mL of the standard stock solution, add a mix-
ture of phosphate buffer solution, pH 7.0 and methanol
(15:1) to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with exactly 100 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the peak areas of erythromycin B and
erythromycin C from the sample solution are not more than
those of erythromycin B and erythromycin C from the stan-
dard solution, respectively, and each area of the peaks other
than erythromycin, erythromycin B and erythromycin C is
not more than the area of the peak of erythromycin from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 215 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with styrene-divinylben-
zene copolymer for liquid chromatography (8 //m in particle
diameter).
Column temperature: A constant temperature of about
70°C.
Mobile phase: Dissolve 3.5 g of dipotassium hydrogen
phosphate in water to make 100 mL, and adjust the pH to 9.0
with diluted phosphoric acid (1 in 10). To 50 mL of this solu-
tion add 190 mL of ?-butanol, 30 mL of acetonitrile and
water to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
erythromycin is about 20 minutes.
Time span of measurement: About 4 times as long as the
retention time of erythromycin beginning after the solvent
peak.
System suitability —
System performance: Dissolve 2 mg of Af-demethyl-
erythromycin in 10 mL of the standard solution. When the
procedure is run with 100 fiL of this solution under the above
operating conditions, A^-demethylerythromycin, erythromy-
cin C, erythromycin and erythromycin B are eluted in this
order, with the resolution between the peaks of N-
demethylerythromycin and erythromycin C being not less
than 0.8, and with the resolution between the peaks of N-
demethylerythromycin and erythromycin being not less than
5.5.
System repeatability: When the test is repeated 3 times with
100 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
erythromycin is not more than 3.0%.
Water <2.48> Not more than 10.0% (0.2 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Erythromycin Reference Standard, equivalent to about
25 mg (potency), dissolve in 25 mL of methanol, add
0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly
100 mL, and use this solution as the standard stock solution.
Keep the standard stock solution at 5 C C or below, and use
within 7 days. Take exactly a suitable amount of the standard
stock solution before use, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
20 /ug (potency) and 5 /xg (potency), and use these solutions as
the high concentration standard solution and low concentra-
tion standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Erythromycin, equivalent to about 25 mg (potency), dissolve
in 25 mL of methanol, and add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make exactly 100 mL. Take exactly a suit-
able amount of this solution, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 20 /ug (potency) and 5 /ug (potency), and use these
solutions as the high concentration sample solution and low
JPXV
Official Monographs / Erythromycin Lactobionate
629
concentration sample solution, respectively.
Containers and storage Containers — Well-closed contain-
ers.
Erythromycin Ethylsuccinate
H 3 C
H CHi
H H ' X °
h g- CH V
C 4 3H 75 N0 16 : 862.05
(2R,3S,4S,5R,6R,8R,l0R,llR,l2S,l3R)-5-
[3,4,6-Trideoxy-2-0-(3-ethoxycarbonylpropanoyl)-
3-dimethylamino-/?-D-xy/o-hexopyranosyloxy]-3-
(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-n&o-
hexopyranosyloxy)-6 ,11,1 2-trihy droxy-
2,4,6,8,10,12-hexamethyl-9-oxopentadecan-13-olide
[41342-53-4]
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Erythromycin Reference Standard equivalent to about 50 mg
(potency), dissolve in 50 mL of methanol, add 0.1 mol/L
phosphate buffer solution, pH 8.0 to make exactly 100 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5°C or below and use within 7
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solu-
tion, pH 8.0 to make solutions so that each mL contains 20
fig (potency) and 5 fig (potency), and use these solutions as
the high concentration standard solution and low concentra-
tion standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Erythromycin Ethylsuccinate equivalent to about 50 mg
(potency), dissolve in 50 mL of methanol, and add 0.1
mol/L phosphate buffer solution, pH 8.0 to make exactly 100
mL. Take exactly a suitable amount of the solution, add 0.1
mol/L phosphate buffer solution, pH 8.0 to make solutions
so that each mL contains 20 fig (potency) and 5 fig (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Erythromycin Lactobionate
I'jXPT'fy/?^ ht:*7|->^
Erythromycin Ethylsuccinate is a derivative of
erythromycin.
It contains not less than 780 fig (potency) and not
more than 900 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Erythromycin Ethyl-
succinate is expressed as mass (potency) of erythromy-
cin (C 37 H 67 N0 13 : 733.93).
Description Erythromycin Ethylsuccinate occurs as a white
powder.
It is freely soluble in methanol and in acetone, soluble in
ethanol (95), and practically insoluble in water.
Identification (1) Dissolve 3 mg of Erythromycin Ethyl-
succinate in 2 mL of acetone, and add 2 mL of hydrochloric
acid: an orange color develops and is immediately changed to
red to deep purple.
(2) Determine the infrared absorption spectrum of
Erythromycin Ethylsuccinate, previously dried in a desicca-
tor (reduced pressure, silica gel) for 24 hours, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Water <2.48> Not more than 5.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
C.H-,
HO H H OH
HO -^X^X^oc I
HO
H OH
T— ^fH H O\ °
OH HO j O i H CHj
L/ch, \i
CH 3
C 37 H 67 N0 13 .C 12 H 22 12 : 1092.22
(2i?,3S,4S,5fl,6i?,8i?,10fl,llfl,12S,13fl)-5-(3,4,6-Trideoxy-3-
dimethylamino-/?-D-x>'/o-hexopyranosyloxy)-3-(2,6-dideoxy-
3-C-methyl-3-0-methyl-a-L-n'£>o-hexopyranosyloxy)-
6,ll,12-trihydroxy-2,4,6,8,10,12-hexamethyl-9-
oxopentadecan-13-olide mono(4-0-/?-D-galactopyranosyl-
D-gluconate) [3847-29-8]
Erythromycin Lactobionate is the lactobionate of
erythromycin.
It contains not less than 590 ^g (potency) and not
more than 700 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Erythromycin Lac-
tobionate is expressed as mass (potency) of erythromy-
cin (C 37 H 67 N0 13 : 733.93).
Description Erythromycin Lactobionate occurs as a white
powder.
It is freely soluble in water, in methanol and in ethanol
(99.5), and very slightly soluble in acetone.
Identification (1) To 3 mg of Erythromycin Lactobionate
add 2 mL of acetone, and add 2 mL of hydrochloric acid: an
orange color is produced, and it changes immediately to red
630
Erythromycin Stearate / Official Monographs
JP XV
to deep purple.
(2) Transfer about 0.3 g of Erythromycin Lactobionate
to a separator, add 15 mL of ammonia TS and 15 mL of
chloroform, shake, and take the separated aqueous layer.
Wash the aqueous layer with three 15-mL portions of chlo-
roform, and evaporate the aqueous liquid on a water bath to
dryness. Dissolve the residue in 10 mL of a mixture of
methanol and water (3:2), and use this solution as the sample
solution. Separately, dissolve 0.10 g of lactobionic acid in
10 mL of a mixture of methanol and water (3 :2), and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 fiL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chromatograph.
Develop the plate with the upper layer obtained from a mix-
ture of water , 1 -butanol and acetic acid ( 1 00) (3 : 3 : 1 ) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly di-
lute sulfuric acid, and heat at 105°C for 20 minutes: the prin-
cipal spot obtained from the sample solution shows a deep
brown and the Ri value which are the same as those of the
principal spot from the standard solution.
pH <2.54> The pH of a solution obtained by dissolving 0.5 g
of Erythromycin Lactobionate in 10 mL of water is between
5.0 and 7.5.
Water <2.48> Not more than 5.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount
of Erythromycin Reference Standard, equivalent to about
50 mg (potency), dissolve in 50 mL of methanol, add
0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly
100 mL, and use this solution as the standard stock solution.
Keep the standard stock solution at not exceeding 5°C and
use within 7days. Take exactly a suitable amount of the stan-
dard stock solution before use, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 20 fig (potency) and 5 fig (potency), and use these so-
lutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Erythromycin Lactobionate, equivalent to about 50 mg
(potency), dissolve in 50 mL of methanol, and add 0.1 mol/L
phosphate buffer solution, pH 8.0 to make exactly 100 mL.
Take exactly a suitable amount of this solution, add 0.1
mol/L phosphate buffer solution, pH 8.0 to make solutions
so that each mL contains 20 fig (potency) and 5 fig (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Erythromycin Stearate
o
C, 7 H 67 N0 13 .C 18 H 36 2 : 1018.40
(2i?,35,45,5i?,6i?,8i?,10/?,ll J R,12S,13i?)-5-(3,4,6-
Trideoxy-3-dimethylamino-yS-D-xy/o-hexopyranosyloxy)-
3-(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-n7jo-
hexopyranosyloxy)-6,ll,12-trihydroxy-2,4,6,8,10,12-
hexamethyl-9-oxopentadecan-13-olide monostearate
[643-22-1]
Erythromycin Stearate is the stearate of erythromy-
cin.
It contains not less than 600 fig (potency) and not
more than 720 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Erythromycin
Stearate is expressed as mass (potency) of erythromy-
cin (C 3 7H 67 N0 13 : 733.93).
Description Erythromycin Stearate occurs as a white pow-
der.
It is freely soluble in ethanol (95) and in acetone, soluble in
methanol, and practically insoluble in water.
Identification (1) Dissolve 3 mg of Erythromycin Stearate
in 2 mL of acetone, and add 2 mL of hydrochloric acid: an
orange color develops and is immediately changed to red to
deep purple.
(2) Determine the infrared absorption spectrum of
Erythromycin Stearate, previously dried in a desiccator
(reduced pressure, silica gel) for 24 hours, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Water <2.48> Not more than 5.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Erythromycin Reference Standard equivalent to about 50 mg
JPXV
Official Monographs / Estradiol Benzoate
631
(potency), dissolve in 50 mL of methanol, add 0.1 mol/L
phosphate buffer solution, pH 8.0 to make exactly 100 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5°C or below and use within 7
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solu-
tion, pH 8.0 to make solutions so that each mL contains 20
fig (potency) and 5 ng (potency), and use these solutions as
the high concentration standard solution and low concentra-
tion standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Erythromycin Stearate equivalent to about 50 mg (potency),
dissolve in 50 mL of methanol, and add 0.1
mol/L phosphate buffer solution, pH 8.0 to make exactly 100
mL. Take exactly a suitable amount of the solution, add 0.1
mol/L phosphate buffer solution, pH 8.0 to make solutions
so that each mL contains 20 /ug (potency) and 5 fig (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Estazolam
ix^'M
294.74
8-Chloro-6-phenyl-4//-[l,2,4]triazolo[4,3-
a][l, ^benzodiazepine [29975-16-4]
Estazolam, when dried, contains not less than 98.5%
of C 16 H„C1N 4 .
Description Estazolam occurs as white to pale yellowish
white crystals or crystalline powder. It is odorless, and has a
bitter taste.
It is soluble in methanol and in acetic anhydride, sparingly
soluble in ethanol (95), and practically insoluble in water and
in diethyl ether.
Identification (1) Dissolve 0.01 g of Estazolam in 3 mL of
sulfuric acid: the solution shows a yellow-green fluorescence
under ultraviolet light (main wavelength: 365 nm).
(2) Determine the absorption spectrum of a solution of
Estazolam in 1 mol/L hydrochloric acid TS (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Perform the test with Estazolam as directed under
Flame Coloration Test (2) <1.04>: a green color appears.
Melting point <2.60> 229 - 233°C
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Estazolam in 10 mL of ethanol (95): the solution is clear
and colorless.
(2) Chloride <1.03>— Dissolve 1.0 g of Estazolam in 10
mL of ethanol (95) by heating, add 40 mL of water, cool with
shaking in ice water, allow to stand to attain ordinary tem-
perature, and filter. To 30 mL of the filtrate add 6 mL of di-
lute nitric acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.25 mL of 0.01 mol/L hydrochloric acid
VS and 6 mL of ethanol (95) (not more than 0.015%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Estazol-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Estazolam according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.20 g of Estazolam in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 fih each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of hexane, chloroform and
methanol (5:3:1) to a distance of about 10 cm, and air-dry the
plate. Examine the plate under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the prin-
cipal spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately about 0.25 g of Estazolam, previ-
ously dried, dissolve in 100 mL of acetic anhydride, and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (potentiomet-
ric titration), until the solution changes to the second equiva-
lence point. Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 14.74 mg of C 16 H U C1N 4
Containers and storage Containers — Well-closed contain-
ers.
Estradiol Benzoate
C 25 H 28 3 : 376.49
Estra-l,3,5(10)-triene-3,17/?-diol 3-benzoate [50-50-0]
Estradiol Benzoate, when dried, contains not less
632
Estradiol Benzoate Injection / Official Monographs
JP XV
than 97.0% of C 25 H 28 3 .
Description Estradiol Benzoate occurs as a white, crystal-
line powder. It is odorless.
It is sparingly soluble in acetone, slightly soluble in
methanol, in ethanol (95) and in diethyl ether, and practically
insoluble in water.
Identification (1) To 2 mg of Estradiol Benzoate add 2
mL of sulfuric acid: a yellowish green color with a blue
fluorescence is produced, and the color of the solution
changes to light orange on the careful addition of 2 mL of
water.
(2) Determine the infrared absorption spectrum of Es-
tradiol Benzoate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of dried Estradiol Benzoate Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Optical rotation <2.49> [ a £°: +54- +58° (after drying,
0.1 g, acetone, 10 mL, 100 mm).
Melting point <2.60> 191 - 198°C
Purity (1) 3,17a-Estradiol — Dissolve 5.0 mg each of Es-
tradiol Benzoate and Estradiol Benzoate Reference Standard
in acetone to make exactly 100 mL, and use these solutions as
the sample solution and standard solution, respectively.
Place exactly 2 mL each of the sample solution and standard
solution in separate glass-stoppered test tube, add boiling
stones, evaporate the acetone by heating in a water bath, and
dry the residue in a desiccator (in vacuum, phosphorus (V)
oxide) for 1 hour. Add 1.0 mL of dilute iron-phenol TS to
each test tube. Stopper the test tubes loosely, heat for 30 se-
conds in a water bath, shake in a water bath for several se-
conds, and heat for 2 minutes. Cool the solutions in ice for 2
minutes, add 4.0 mL of diluted sulfuric acid (7 in 20), and
mix well: the solution obtained from the sample solution has
no more color than that from the standard solution.
(2) Related substances — Dissolve 40 mg of Estradiol Ben-
zoate in 2 mL of acetone, and use this solution as the sample
solution. Pipet 1 mL of this solution, add acetone to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and diethyla-
mine (19:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.2% (0.1 g).
Assay Weigh accurately about 10 mg each of Estradiol Ben-
zoate and Estradiol Benzoate Reference Standard, previously
dried, and dissolve each in methanol to make exactly 20 mL.
Pipet 5 mL each of these solutions, add 5 mL of the internal
standard solution, then add methanol to make 20 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 5 /uL of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, g T and Q s , of the peak area of
estradiol benzoate to that of the internal standard.
Amount (mg) of C 25 H 28 3 = W s x (Q T /Q S )
W s : Amount (mg) of Estradiol Benzoate Reference Stand-
ard
Internal standard solution — A solution of progesterone in
methanol (13 in 80,000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of acetonitrile and water (7:3).
Flow rate: Adjust the flow rate so that the retention time of
estradiol benzoate is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, the internal standard and estradiol benzoate are eluted
in this order with the resolution between these peaks being
not less than 9.
System repeatability: When the test is repeated 6 times with
5 iXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of estradiol benzoate to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Estradiol Benzoate Injection
xx h : 7^7l--;ug?,&S^i7.-TJuaw>ft
Estradiol Benzoate Injection is an oily solution for
injection.
It contains not less than 90% and not more than 110
% of the labeled amount of estradiol benzoate (C 25 H 2 g
3 : 376.49).
Method of preparation Prepare as directed under Injec-
tions, with Estradiol Benzoate.
Description Estradiol Benzoate Injection is a clear, oily liq-
uid.
Identification To a volume of Estradiol Benzoate Injection,
equivalent to 1 mg of Estradiol Benzoate according to the la-
beled amount, add chloroform to make 5 mL, and use this
solution as the sample solution. Separately dissolve 1 mg of
Estradiol Benzoate Reference Standard in 5 mL of chlo-
roform, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 50 /uL each of the sample so-
JPXV
Official Monographs / Estriol 633
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with dichloromethane to a distance of about 15 cm,
and air-dry the plate. Then develop the plate with a mixture
of chloroform and methanol (99:1) to a distance of about 15
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the principal spot obtained from
the sample solution and the spot obtained from the standard
solution show the same Rf value.
Extractable volume <6.05> It meets the requirement.
Assay Transfer an exactly measured volume of Estradiol
Benzoate Injection, equivalent to about 10 mg of estradiol
benzoate (C25H28O3), to a separator, add 30 mL of hexane
saturated with diluted methanol (9 in 10), and extract with
five 15-mL portions of dilute methanol (9 in 10) saturated
with hexane. Filter the extract through a filter paper washed
with 10 mL of diluted methanol (9 in 10), to the filtrate add
methanol to make exactly 200 mL, and use this solution as
the sample solution. Separately, weigh accurately about 25
mg of Estradiol Benzoate Reference Standard, previously d-
ried in a desiccator (in vacuum, phosphorus (V) oxide) for 4
hours, and dissolve in methanol to make exactly 100 mL.
Pipet 10 mL of this solution, add methanol to make exactly
50 mL, and use this solution as the standard solution. Trans-
fer 2 mL each of the sample solution and standard solution,
exactly measured, to light-resistant 20-mL volumetric flasks,
and evaporate to dryness on a water bath with the aid of a
current of air. Dissolve the residue in 1 mL of methanol, add
10 mL of boric acid-methanol buffer solution, shake, and
boil under a reflux condenser for 30 minutes. Cool, add 5 mL
of boric acid-methanol buffer solution, shake, and cool with
ice. To each solution add 2 mL of ice-cold diazo TS quickly,
shake vigorously, add 2 mL of sodium hydroxide TS, then
add water to make 20 mL, and filter after shaking. Discard
the first 3 mL of the filtrate, and perform the test with the
subsequent filtrate as directed under Ultraviolet-visible Spec-
trophotometry <2.24> using a solution, prepared with 2 mL
of methanol in the same manner, as the blank. Determine the
absorbances, A T and A s , of the subsequent solutions ob-
tained from the sample solution and standard solution in a
4-cm cell at 490 nm, respectively.
Amount (mg) of estradiol benzoate (C25H28O3)
= W s x (A T /A S ) x (2/5)
W s : Amount (mg) of Estradiol Benzoate Reference Stan-
dard
Containers and storage Containers — Hermetic containers.
with Estradiol Benzoate.
Description Estradiol Benzoate Injection (Aqueous Suspen-
sion) produces a white turbidity on shaking.
Identification Extract a volume of Estradiol Benzoate In-
jection (Aqueous Suspension), equivalent to 1 mg of Es-
tradiol Benzoate according to the labeled amount, with 5 mL
of chloroform, and use this extract as the sample solution.
Separately, dissolve 1 mg of Estradiol Benzoate Reference
Standard in 5 mL of chloroform, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 50
/uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and methanol (99:1) to a distance of about 15 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the principal spot obtained from the
sample solution and the spot obtained from the standard so-
lution show the same Rf value.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of well-mixed Estradiol
Benzoate Injection (Aqueous Suspension), equivalent to
about 2 mg of estradiol benzoate (C25H 28 3 ), dissolve the
crystals with an appropriate quantity of methanol, and add
methanol to make exactly 20 mL. Pipet 10 mL of this solu-
tion, add exactly 10 mL of the internal standard solution,
add methanol to make 100 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Estradiol Benzoate Reference Standard, previously dried in
desiccator (reduced pressure, phosphorus (V) oxide) for 4
hours, and dissolve in methanol to make exactly 100 mL.
Pipet 10 mL of this solution, add exactly 10 mL of the inter-
nal standard solution and methanol to make 100 mL, and use
this solution as the standard solution. Proceed with these so-
lutions as directed in the Assay under Estradiol Benzoate.
Amount (mg) of estradiol benzoate (C25H28O3)
= W s x (St/Gs) x (1/5)
W s : Amount (mg) of Estradiol Benzoate Reference Stan-
dard
Internal standard solution — A solution of progesterone in
methanol (13 in 100,000).
Containers and storage Containers — Hermetic containers.
Estriol
Estradiol Benzoate Injection
(Aqueous Suspension)
Estradiol Benzoate Injection (Aqueous Suspension)
is an aqueous suspension for injection. It contains not
less than 90% and not more than 110% of the labeled
amount of estradiol benzoate (C25H28O3: 376.49).
Method of preparation Prepare as directed under Injection,
IX r- U7T--JU
C 18 H 2 40 3 : 288.38
Estra-l,3,5(10)-triene-3,16a,17/?-triol [50-27-1]
Estriol, when dried, contains not less than 97.0%
and not more than 102.0% of C lg H 2 40 3 .
634
Estriol Injection (Aqueous Suspension) / Official Monographs
JP XV
Description Estriol occurs as a white, crystalline powder. It
is odorless.
It is sparingly soluble in methanol, slightly soluble in
ethanol (95) and in 1,4-dioxane, and practically insoluble in
water and in diethyl ether.
Identification (1) Dissolve 0.01 g of Estriol in 100 mL of
ethanol (95) by warming, and use this solution as the sample
solution. Evaporate 1 mL of this solution on a water bath to
dryness, add 5 mL of a solution of sodium p-phenolsulf onate
in diluted phosphoric acid (1 in 50), heat at 150°C for 10
minutes, and cool: a red-purple color develops.
(2) Determine the absorption spectrum of the sample so-
lution obtained in (1) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of Es-
triol Reference Standard prepared in the same manner as the
sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Es-
triol, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of previously dried Estriol Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Optical rotation <2.49> [a]™: + 54 - + 62° (after drying, 40
mg, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 281 - 286°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of Es-
triol according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(2) Related substances — Dissolve 40 mg of Estriol in 10
mL of ethanol (95) by warming, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
ethanol (95) to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
[iL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol, acetone and
acetic acid (100) (18:1:1:1) to a distance of about 15 cm, and
air-dry the plate. Spray evenly diluted sulfuric acid (1 in 2) on
the plate, and heat at 105°C for 15 minutes: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 25 mg each of Estriol and
Estriol Reference Standard, previously dried, and dissolve
each in methanol to make exactly 50 mL. Pipet 10 mL each
of these solutions, add exactly 5 mL of the internal standard
solution, add methanol to make 100 mL, and use these solu-
tions as the sample solution and the standard solution,
respectively. Perform the test with 10 /iL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
estriol to that of the internal standard, respectively.
Amount (mg) of C 18 H 24 3 = W s x (Q T /Q S )
W s : Amount (mg) of Estriol Reference Standard
Internal standard solution — A solution of methyl benzoate
for estriol limit test in methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /tm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and methanol (51:49).
Flow rate: Adjust the flow rate so that the retention time of
estriol is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, estriol and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of estriol to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Estriol Injection
(Aqueous Suspension)
iX h U :t-JU7kte!8>S&*f*
Estriol Injection (Aqueous Suspension) is an aque-
ous suspension for injection. It contains not less than
90% and not more than 1 10% of the labeled amount of
estriol (C 18 H 2 40 3 : 288.38).
Method of preparation Prepare as directed under Injec-
tions, with Estriol.
Description Shake Estriol Injection (Aqueous Suspension):
a white turbidity is produced.
Identification (1) Shake well, take a volume of Estriol In-
jection (Aqueous Suspension), equivalent to 2 mg of Estriol
according to the labeled amount, add ethanol (95) to make 20
mL, and use this solution as the sample solution. Proceed
with the sample solution as directed in the Identification (1)
under Estriol.
(2) Determine the absorption spectrum of the sample so-
lution obrtained in (1) as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
279 nm and 283 nm.
Extractable volume <6.05> It meets the requirement.
Assay Shake well, pipet a volume of Estriol Injection
(Aqueous Suspension), equivalent to about 5 mg of estriol
JPXV
Official Monographs / Estriol Tablets 635
(C18H24O3), and dissolve in methanol to make exactly 20 mL.
Pipet 4 mL of this solution, add exactly 5 mL of the internal
standard solution, then add methanol to make 50 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Estriol Reference Standard, previ-
ously dried at 105°C for 3 hours, and dissolve in methanol to
make exactly 100 mL. Pipet 4 mL of this solution, add exact-
ly 5 mL of the internal standard solution, then add methanol
to make 50 mL, and use this solution as the standard solu-
tion. Proceed as directed in the Assay under Estriol.
Amount (mg) of estriol (C18H24O3)
= W s x (Q T /Q S ) x (1/5)
W s : Amount (mg) of Estriol Reference Standard
Internal standard solution — A solution of methyl benzoate
for estriol limit test in ethanol (95) (1 in 5000).
Containers and storage Containers — Hermetic containers.
Estriol Tablets
IX r- U*-JH5e
Estriol Tablets contain not less than 90% and not
more than 110% of the labeled amount of estriol
(C 18 H 24 3 : 288.38).
Method of preparation Prepare as directed under Tablets,
with Estriol.
Identification (1) Weigh a portion of powdered Estriol
Tablets, equivalent to 2 mg of Estriol according to the labeled
amount, add ethanol (95) to make 20 mL, shake for 10
minutes, centrifuge, and use the supernatant liquid as the
sample solution. Proceed with the sample solution as directed
in the Identification (1) under Estriol.
(2) Determine the absorption spectrum of the sample so-
lution obtained in (1) as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
279 nm and 283 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Estriol Tablets add exactly 5 mL of water,
disperse the fine particles with ultrasonic wave, add exactly 15
mL of methanol, and shake for 15 minutes. Centrifuge this
solution for 10 minutes, pipet a definite amount of the super-
natant liquid, and add methanol to make exactly a definite
amount of solution so that each ml of the solution contains
about 5 fig of estriol (C 18 H 24 3 ). Pipet 5 mL of this solution,
add exactly 1 mL of the internal standard solution, and use
this solution as the sample solution. Proceed with 20 /uL of
the sample solution as directed in the Assay under Estriol.
Use a solution of methyl benzoate in methanol (1 in 40,000)
as the internal standard solution. Calculate the mean value
from each ratio of peak areas of 10 samples: the samples con-
form to the requirements if the deviation (%) of the mean
value and each ratio of peak areas is within 15%. If the devia-
tion (%) exceeds 15%, and 1 sample shows deviation within
25%, repeat the test with 20 samples. Calculate the deviation
(%) of the mean value from each ratio of peak areas of the 30
samples used in the 2 tests and each ratio of peak areas: the
samples conform to the requirements if the deviation exceeds
15%, not more than 1 sample shows deviation within 25%,
and no sample shows deviation exceeding 25%.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Estriol Tablets at 50 revo-
lutions per minute according to the Paddle method, using 900
mL of water as the dissolution medium. Take 20 mL or more
of the dissolved solution 30 minutes after starting the test,
and filter through a membrane filter with pore size of not
more than 0.8 ^m. Discard the first 10 mL of the filtrate,
pipet the subsequent KmL, add water to make exactly V mL
so that each mL contains about 0.1 /ug of estriol (C18H24O3)
according to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Estriol Reference Standard, previously dried at 105°C for 3
hours, dissolve in methanol to make exactly 100 mL, then
pipet 5 mL of this solution, and add water to make exactly
100 mL. Pipet 2 mL of this solution, add water to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 100 /xL each of the sample solu-
tion and standard solution according to the operating condi-
tions as directed in the Assay under Estriol, and determine
the peak areas of estriol, A T and A s , from these solutions.
The dissolution rate of Estriol Tablets in 30 minutes is not
less than 80%.
Dissolution rate (%) with respect to
the labeled amount of estriol (C 18 H 24 3 )
= W s x (A T /A S ) x (V'/V) x (1/Q x (9/10)
W s : Amount (mg) of Estriol Reference Standard
C: Labeled amount (mg) of estriol (C 18 H 24 3 ) in 1 tablet
Assay Weigh accurately and powder not less than 20 Estriol
Tablets. Weigh accurately a portion of the powder, equiva-
lent to about 1 mg of estriol (C I8 H 24 3 ), add exactly 5 mL of
water, disperse the fine particles with ultrasonic wave, shake
with 25 mL of methanol for 10 minutes, centrifuge, and take
the supernatant liquid. Add 25 mL of methanol, repeat the
above procedure twice, combine the supernatant liquid, add
exactly 5 mL of the internal standard solution, then add
methanol to make 100 mL, and use this solution as the sam-
ple solution. Separately, weigh accurately about 25 mg of Es-
triol Reference Standard, previously dried at 105 °C for 3
hours, and dissolve in methanol to make exactly 100 mL.
Pipet 4 mL of this solution, add exactly 5 mL of the internal
standard solution, then add methanol to make 100 mL, and
use this solution as the standard solution. Proceed with 20 /uL
each of the sample solution and standard solution as directed
in the Assay under Estriol.
Amount (mg) of estriol (C 18 H 24 3 )
= W s x (Qj/Qs) x (1/25)
W s : Amount (mg) of Estriol Reference Standard
Internal standard solution — A solution of methyl benzoate
for estriol limit test in methanol (1 in 5000).
Containers and storage Containers — Tight containers.
636
Etacrynic Acid / Official Monographs
JP XV
Etacrynic Acid
i??y >K
COjH
C 13 H 12 C1 2 4 : 303.14
[2,3-Dichloro-4-(2-ethylacryloyl)phenoxy]acetic acid
[58-54-8]
Etacrynic Acid, when dried, contains not less than
98.0% of C 13 H 12 C1 2 4 .
Description Etacrynic Acid occurs as a white, crystalline
powder. It is odorless, and has a slightly bitter taste.
It is very soluble in methanol, freely soluble in ethanol
(95), in acetic acid (100) and in diethyl ether, and very slightly
soluble in water.
Identification (1) Dissolve 0.2 g of Etacrynic Acid in 10
mL of acetic acid (100), and to 5 mL of this solution add 0.1
mL of bromine TS: the color of the test solution disappears.
To the remaining 5 mL of the solution add 0. 1 mL of potassi-
um permanganate TS: the color of the test solution changes
to light orange immediately.
(2) To 0.01 g of Etacrynic Acid add 1 mL of sodium
hydroxide TS, and heat in a water bath for 3 minutes. After
cooling, add 1 mL of disodium chlomotropate TS, and heat
in a water bath for 10 minutes: a deep purple color develops.
(3) Determine the absorption spectrum of a solution of
Etacrynic Acid in methanol (1 in 20,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Perform the test with Etacrynic Acid as directed un-
der Flame Coloration Test (2) <1.04>: a green color appears.
Melting point <2.60> 121 - 125°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Etacrynic Acid in 10 mL of methanol: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Etacrynic
Acid according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Etacrynic Acid according to Method 3, and perform the
test. Add 10 mL of a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 50), then add 1.5 mL of hydrogen
peroxide (30), and fire to burn (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Etacrynic
Acid in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 3 mL of the sample solution, add
ethanol (95) to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, ethyl acetate and acetic acid (100) (6:5:2) to a dis-
tance of about 15 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.25% (1 g, in vacu-
um, 60 C C, 2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of Etacrynic Acid,
previously dried, place in an iodine bottle, dissolve in 20 mL
of acetic acid (100), and add exactly 20 mL of 0.05 mol/L
bromine VS. To this solution add 3 mL of hydrochloric acid,
stopper tightly at once, shake, and allow to stand in a dark
place for 60 minutes. Add carefully 50 mL of water and 15
mL of potassium iodide TS, stopper tightly at once, shake
well, and titrate <2.50> the liberated iodine with 0.1 mol/L
sodium thiosulfate VS (indicator: 1 mL of starch TS). Per-
form a blank determination.
Each mL of 0.05 mol/L bromine VS
= 15.16 mg of C 13 Hi 2 Cl 2 4
Containers and storage Containers — Well-closed contain-
ers.
Etacrynic Acid Tablets
Etacrynic Acid Tablets contain not less than 90%
and not more than 110% of the labeled amount of
etacrynic acid (C13H12CI2O4: 303.14).
Method of preparation
with Etacrynic Acid.
Prepare as directed under Tablets,
Identification (1) Weigh a quantity of powdered Etacrynic
Acid Tablets, equivalent to 0.3 g of Etacrynic Acid according
to the labeled amount, add 25 mL of 0.1 mol/L hydrochloric
acid TS, and extract with 50 mL of Dichloromethane. Filter
the dichloromethane extract, and evaporate the filtrate on a
water bath to dryness. Proceed with the residue as directed in
the Identification (1), (2) and (4) under Etacrynic Acid.
(2) Prepare a solution of the residue obtained in (1),
equivalent to a solution of Etacrynic Acid in methanol (1 in
20,000), and determine the absorption spectrum as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 268 nm and 272 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Etacrynic Acid Tablets at
50 revolutions per minute according to the Paddle method,
using 900 mL of water as the test solution. Take 20 mL or
more of the dissolved solution 45 minutes after starting the
test, and filter through a membrane filter with pore size of not
more than 0.8 fim. Discard the first 10 mL of the filtrate, and
use the subsequent as the sample solution. Separately, weigh
accurately about 55 mg of etacrynic acid for assay, previous-
ly dried at 60°C for 2 hours, dissolve in 10 mL of methanol,
and add water to make exactly 100 mL. Pipet 5 mL of this so-
JPXV
Official Monographs / Ethambutol Hydrochloride 637
lution, add water to make exactly 100 mL, and use this solu-
tion as the standard solution. Determine the absorbances, A T
and A s , of the sample solution and standard solution at 277
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>.
The dissolution rate of Etacrynic Acid Tablets in 45
minutes is not less than 70%.
Dissolution rate (%) with respect to
the labeled amount of etacrynic acid (Ci 3 Hi 2 Cl 2 4 )
= W s x (A T /A S ) X (I/O X 45
W s : Amount (mg) of etacrynic acid for assay
C: Labeled amount (mg) of etacrynic acid (CnHi 2 Cl 2 4 ) in
1 tablet
Assay Weigh accurately and powder not less than 20
Etacrynic Acid Tablets. Weigh accurately a portion of the
powder, equivalent to about 0.1 g of etacrynic acid
(Ci3H 12 Cl 2 4 ), add 25 mL of 0.1 mol/L hydrochloric acid
TS, and extract with three 30-mL portions of
dichloromethane. Filter the dichloromethane extracts
through a pledget of absorbent cotton into an iodine bottle.
Wash the pledget of absorbent cotton with a small amount of
dichloromethane, and combine the washing with the extracts.
Evaporate this solution on a water bath to dryness in a cur-
rent of air, to the residue add 20 mL of acetic acid (100), and
proceed as directed in the Assay under Etacrynic Acid.
Each mL of 0.05 mol/L bromine VS
= 15.16 mg of C 13 H 12 C1 2 4
Containers and storage Containers — Well-closed contain-
ers.
Ethambutol Hydrochloride
i*>7r--,/U£&£
OH
i
n
X^CH 3
■2HCI
OH
C 10 H 24 N 2 O 2 .2HCl: 277.23
2,2 ' -(Ethylenediimino)bis [(2 <S)-butan- 1 -ol] dihydrochloride
[1070-11-7]
Ethambutol Hydrochloride, when dried, contains
not less than 98.5% of C 10 H 24 N 2 O 2 .2HCl.
Description Ethambutol Hydrochloride occurs as white
crystals or crystalline powder. It is odorless, and has a bitter
taste.
It is very soluble in water, soluble in methanol and in
ethanol (95), and practically insoluble in diethyl ether.
The pH of a solution of Ethambutol Hydrochloride (1 in
20) is between 3.4 and 4.0.
Identification (1) To 10 mL of a solution of Ethambutol
Hydrochloride (1 in 100) add 0.5 mL of copper (II) sulfate TS
and 2 mL of sodium hydroxide TS: a deep blue color is
produced.
(2) Dissolve 0. 1 g of Ethambutol Hydrochloride in 40 mL
of water, add 20 mL of 2,4,6-trinitrophenol TS, and allow to
stand for 1 hour. Collect the precipitate, wash with 50 mL of
water, and dry at 105°C for 2 hours: the precipitate melts
<2.60> between 193°C and 197°C.
(3) A solution of Ethambutol Hydrochloride (1 in 30)
responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [a]™: +5.5 - +6.1° (after drying,
5 g, water, 50 mL, 200 mm).
Melting point <2.60> 200 - 204°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ethambutol Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g Ethambutol
Hydrochloride according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ethambutol Hydrochloride according to Method 1, and
perform the test (not more than 2 ppm).
(4) 2-Aminobutanol — Dissolve 5.0 g of Ethambutol
Hydrochloride in methanol to make exactly 100 mL, and use
this solution as the sample solution. Dissolve 0.05 g of 2-
amino-1-butanol in methanol to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 2//L each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, acetic acid (100), hydrochloric acid and water
(11:7:1:1) to a distance of about 10 cm, air-dry the plate, and
heat at 105°C for 5 minutes. Cool, spray evenly ninhydrin-L-
ascorbic acid TS upon the plate, air-dry the plate, and heat at
105 °C for 5 minutes: the spot from the sample solution, cor-
responding to that from the standard solution, has no more
color than that from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Ethambutol
Hydrochloride, previously dried, dissolve in 20 mL of water,
and add 1 . 8 mL of copper (II) sulfate TS . To the solution add
7 mL of sodium hydroxide TS with shaking, add water to
make exactly 50 mL, and centrifuge. Pipet 10 mL of the su-
pernatant liquid, add 10 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.0 and 100 mL of water, and titrate
<2.50> with 0.01 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS until the color of the solution changes
from blue-purple through light red to light yellow (indicator:
0.15 mL of Cu-PAN TS). Perform a blank determination,
and make any necessary correction.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.772 mg of Ci H 24 N 2 O 2 .2HCl
Containers and storage Containers — Tight containers.
638
Ethanol / Official Monographs
JP XV
Ethanol
Alcohol
H 3 C
on
C 2 H 6 0: 46.07
Ethanol [64-17-5]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Ethanol contains not less than 95.1 vol% and not
more than 96.9 vol% (by specific gravity) of C 2 H 6 at
15°C.
♦Description Ethanol is a clear, colorless liquid.
It is miscible with water.
It is flammable and burns with a light blue flame on igni-
tion.
It is volatile. »
Identification (1) Determine the infrared absorption spec-
trum of Ethanol as directed in the liquid film method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
Specific gravity <2.56> d\\: 0.809 - 0.816
Purity (1) Clarity and color of solution — Ethanol is clear
and colorless. To 1.0 mL of Ethanol add water to make 20
mL, and allow to stand for 5 minutes: the resulting liquid is
clear. Control solution: water.
(2) Acidity or alkalinity— To 20 mL of Ethanol add 20
mL of freshly boiled and cooled water and 0.1 mL of a solu-
tion prepared by addition of 7.0 mL of ethanol (95) and 2.0
mL of water to 1.0 mL of phenolphthalein TS : no color de-
velops. Add 1.0 mL of 0.01 mol/L sodium hydroxide VS to
this solution: a light red color develops.
(3) Volatile impurities — Pipet 500 mL of Ethanol, add
150 /uL of 4-methylpentan-2-ol, and use this solution as the
sample solution. Separately, to 100 /xL of anhydrous
methanol add Ethanol to make exactly 50 mL. Pipet 5 mL of
this solution, add Ethanol to make exactly 50 mL, and use
this solution as the standard solution (1). Separately, to ex-
actly 50 fiL each of anhydrous methanol and acetaldehyde
add Ethanol to make exactly 50 mL. To exactly lOO^L of
this solution add Ethanol to make exactly 10 mL, and use this
solution as the standard solution (2). Separately, to exactly
150 /uL of acetal add Ethanol to make exactly 50 mL. To ex-
actly 100 jXL of this solution add Ethanol to make exactly 10
mL, and use this solution as the standard solution (3).
Separately, to exactly 100 /uL of benzene add Ethanol to
make exactly 100 mL. To exactly 100 fiL of this solution add
Ethanol to make exactly 50 mL, and use this solution as the
standard solution (4). Perform the test with exactly 1 /uL each
of Ethanol, the sample solution and standard solutions (1),
(2), (3) and (4) as directed under Gas Chromatography <2.02>
according to the following conditions, and determine the
peak areas of acetaldehyde, A E , benzene, B E and acetal, C E
obtained with Ethanol, and the peak area of methanol with
the standard solution (1), the peak area of acetaldehyde, A T
with the standard solution (2), the peak area of acetal, C T
with the standard solution (3) and the peak area of benzene,
B T : the peak area of methanol is not more than 1/2 times the
peak area of methanol with the standard solution (1). When
calculate the amounts of the volatile impurities by the follow-
ing equation, the total amount of acetaldehyde and acetal is
not more than 10 vol ppm as acetaldehyde, and the amount
of benzene is not more than 2 vol ppm. The total area of the
peaks other than the peak mentioned above and the peak
having the area not more than 3% that of 4-methylpentan-2-
ol is not larger than the peak area of 4-methylpentan-2-ol.
Total amount (vol ppm) of acetaldehyde and acetal
= {(10xA e )/(A t -A e )} + {(30XC E )/(C T -C E )}
Amount (vol ppm) of benzene = 2B E /(B T — B E )
If necessary, identify the peak of benzene by using a different
stationary liquid phase and suitable chromatographic condi-
tions.
Operating conditions —
Detector: A hydrogen flame-ionization detector
Column: A fused silica tube 0.32 mm in inside diameter
and 30 m in length, coated with 6% cyanopropyl phenyl-94%
dimethyl silicone polymer for gas chromatography in 1 .8 /um
thickness.
Column temperature: Inject at a constant temperature of
about 40°C, maintain the temperature for 12 minutes, then
rise up to 240°C at the rate of 10°C per minute, and maintain
at a constant temperature of about 240°C for 10 minutes.
Carrier gas: Helium
Flow rate: 35 cm/min.
Split ratio: 1: 20
System suitability —
System performance: When the procedure is run with 1 fiL of
the standard solution (2) under the above operating condi-
tions, acetaldehyde and methanol are eluted in this order with
the resolution between these peaks being not less than 1.5.
(4) Other impurities (absorbance) — Perform the test with
Ethanol as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: the absorbances at 240 nm, between 250 nm
and 260 nm and between 270 nm and 340 nm are not more
than 0.40, 0.30, and 0.10, respectively. The absorption spec-
trum determined in a 5-cm cell using water as a blank shows a
flat absorption curve between 235 nm and 340 nm.
(5) Residue on evaporation — Evaporate 100 mL of
Ethanol, exactly measured, in a tared dish on a water bath,
and dry for 1 hour at 105°C: the mass of the residue does not
exceed 2.5 mg.
Containers and storage *Containers — Tight containers. ♦
Storage — Without exposure to light.
JPXV
Official Monographs / Anhydrous Ethanol
639
Anhydrous Ethanol
Dehydrated Alcohol
H 3 C-
"OH
C 2 H 6 0: 46.07
Ethanol [64-17-5]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Anhydrous Ethanol contains not less than 99.5 vol%
(by specific gravity) of C 2 H 6 at 15°C.
♦Description Anhydrous Ethanol is a clear, colorless liq-
uid.
It is miscible with water.
It is flammable and burns with a light blue flame on igni-
tion.
It is volatile.
Boiling point: 78 - 79°C»
Identification (1) Determine the infrared absorption spec-
trum of Anhydrous Ethanol as directed in the solution
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Specific gravity <2.56>
d\\: 0.794-
0.797
Purity (1) Clarity and color of solution — Anhydrouse
Ethanol is clear and colorless. To 1.0 mL of Anhydrous
Ethanol add water to make 20 mL, and allow to stand for 5
minutes: the resulting liquid is clear. Control solution: water
(2) Acidity or alkalinity — To 20 mL of Anhydrous
Ethanol add 20 mL of freshly boiled and cooled water and
0.1 mL of a solution obtained by addition of 7.0 mL of
ethanol (95) and 2.0 mL of water to 1.0 mL of
phenolphthalein TS: no color develops. Add 1.0 mL of 0.01
mol/L sodium hydroxide VS to this solution: a light red color
develops.
(3) Volatile impurities — Pipet 500 mL of Anhydrous
Ethanol, add 150 /xL of 4-methylpentan-2-ol, and use this so-
lution as the sample solution. Separately, to 100^L of anhy-
drous ethanol add Anhydrous Ethanol to make exactly 50
mL. Pipet 5 mL of this solution, add Anhydrous Ethanol to
make exactly 50 mL, and use this solution as the standard so-
lution (1). Separately, to exactly 50 /uL each of anhydrous
methanol and acetaldehyde add Anhydrous Ethanol to make
exactly 50 mL. To exactly 100 /xh of this solution add Anhy-
drous Ethanol to make exactly 10 mL, and use this solution
as the standard solution (2). Separately, to exactly 150/^L of
acetal add Anhydrous Ethanol to make exactly 50 mL. To ex-
actly 100 fiL of this solution add Anhydrous Ethanol to make
exactly 10 mL, and use this solution as the standard solution
(3). Separately, to exactly 100 /uL of benzene add Anhydrous
Ethanol to make exactly 100 mL. To exactly 100 /xh of this
solution add Anhydrous Ethanol to make exactly 50 mL, and
use this solution as the standard solution (4). Perform the test
with exactly 1 /xL each of Anhydrous Ethanol, the sample so-
lution and standard solutions (1), (2), (3) and (4) as directed
under Gas Chromatography <2.02> according to the follow-
ing conditions, and determine the peak areas of acetalde-
hyde, A E , benzene, B E and acetal, C E obtained with Anhy-
drous Ethanol, and the peak area of methanol with the stan-
dard solution (1), the peak area of acetaldehyde, A T with the
standard solution (2), the peak area of acetal, C T with the
standard solution (3) and the peak area of benzene, B T : the
peak area of methanol is not more than 1/2 times the peak
area of methanol with the standard solution (1). When calcu-
late the amounts of the volatile impurities by the following e-
quation, the total amount of acetaldehyde and acetal is not
more than 10 vol ppm as acetaldehyde, and the amount of
benzene is not more than 2 vol ppm. The total area of the
peaks other than the peak mentioned above and the peak
having the area not more than 3% that of 4-methylpentan-2-
ol is not larger than the peak area of 4-methylpentan-2-ol.
Total amount (vol ppm) of acetaldehyde and acetal
= {(10x^l E )/(y4 T -^ E )} + {(30XC E )/(C T -C E )}
Amount (vol ppm) of benzene = 2B E /(Bj—B E )
If necessary, identify the peak of benzene by using a different
stationary liquid phase and suitable chromatographic condi-
tions.
Operating conditions —
Detector: A hydrogen flame-ionization detector
Column: A fused silica tube 0.32 mm in inside diameter
and 30 m in length, coated with 6% cyanopropyl phenyl-94%
dimethyl silicone polymer for gas chromatography in 1 .8 fim
thickness.
Column temperature: Inject at a constant temperature of
about 40°C, maintain the temperature for 12 minutes, then
rise up to 240°C at the rate of 10°C per minute, and maintain
at a constant temperature of about 240°C for 10 minutes.
Carrier gas: Helium
Flow rate: 35 cm/min.
Split ratio: 1: 20
System suitability —
System performance: When the procedure is run with 1 /xh
of the standard solution (2) under the above operating condi-
tions, acetaldehyde and methanol are eluted in this order with
the resolution between these peaks being not less than 1.5.
(4) Other impurities (absorbance) — Perform the test with
Ethanol as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: the absorbances at 240 nm, between 250 nm
and 260 nm and between 270 nm and 340 nm are not more
than 0.40, 0.30, and 0.10, respectively. The absorption spec-
trum determined in a 5-cm cell using water as a blank shows a
flat absorption curve between 235 nm and 340 nm.
(5) Residue on evaporation — Evaporate 100 mL of De-
hydrated Ethanol, exactly measured, in a tared dish on a
water bath, and dry for 1 hour at 105°C: the mass of the
residue does not exceed 2.5 mg.
Containers and storage *Containers — Tight containers. ♦
Storage — Without exposure to light.
640
Ethanol for Disinfection / Official Monographs
JP XV
Ethanol for Disinfection
Alcohol for Disinfection
Ethanol for Disinfection contains not less than 76.9
vol% and not more than 81.4 vol% (by specific gravity)
of ethanol (C 2 H 6 0: 46.07) at 15°C.
Method of preparation
Ethanol
Purified Water
830mL
a sufficient quantity
To make 1000 mL
Prepare by mixing the above ingredients.
Description Ethanol for Disinfection is a colorless, clear
liquid.
It is miscible with water.
It burns with a light blue flame on ignition.
It is volatile.
Identification (1) To 1 mL of Ethanol for Disinfection
add 2 mL of iodine TS and 1 mL of sodium hydroxide TS,
and mix: light yellow precipitates appear.
(2) To 1 mL of Ethanol for Disinfection add 1 mL of
acetic acid (100) and 3 drops of sulfuric acid, and heat: the
odor of ethyl acetate is produced.
Specific gravity <2.56> d\\: 0.860 - 0.873
Purity Proceed as directed in the Purity under Ethanol.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ethenzamide
Ethoxybenzamide
O CH 3
C 9 H„N0 2 : 165.19
2-Ethoxybenzamide [938-73-8]
Ethenzamide, when dried, contains not less than
98.0% of C 9 H„N0 2 .
Description Ethenzamide occurs as white crystals or crys-
talline powder. It is odorless and tasteless.
It is soluble in ethanol (95) and in acetone, slightly soluble
in diethyl ether, and practically insoluble in water.
Its saturated solution is neutral.
It begins to sublime slightly at about 105°C.
Identification (1) To 0.5 g of Ethenzamide add 5 mL of
sodium hydroxide TS, and heat the mixture gently: the gas
evolved turns moistened red litmus paper to blue.
(2) To 0.2 g of Ethenzamide add 10 mL of hydrobromic
acid, and boil the mixture gently for 1 hour under a reflux
condenser. Cool in ice water, collect the separated crystalline
precipitate, wash with three 5-mL portions of ice water, and
dry in a desiccator (in vacuum, silica gel) for 2 hours: the
precipitate melts <2.60> between 158°C and 161 °C.
Melting point <2.60> 131 - 134°C
Purity (1) Chloride <1.03>— Dissolve 0.5 g of Ethen-
zamide in 30 mL of acetone, add 6 mL of dilute nitric acid,
and dilute with water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 0.7 mL of 0.01 mol/L hydrochloric acid VS
add 30 mL of acetone and 6 mL of dilute nitric acid, and di-
lute with water to make 50 mL (not more than 0.050%).
(2) Sulfate <1.14>— Dissolve 0.5 g of Ethenzamide in 30
mL of acetone, add 1 mL of dilute hydrochloric acid, and di-
lute with water to 50 mL. Perform the test using this solution
as the test solution. Prepare the control solution as follows:
to 0.50 mL of 0.005 mol/L sulfuric acid VS add 30 mL of
acetone and 1 mL of dilute hydrochloric acid, and dilute with
water to 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Ethen-
zamide according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(4) Arsenic <1.11>— To 0.40 g of Ethenzamide add 0.3 g
of potassium nitrate and 0.5 g of anhydrous sodium car-
bonate, mix thoroughly, ignite the mixture gradually, and
cool. Dissolve the residue in 10 mL of dilute sulfuric acid,
and heat the solution until white fumes begin to evolve. After
cooling, add water carefully to make 5 mL, use this solution
as the test solution, and perform the test (not more than 5
ppm).
(5) Salicylamide — Dissolve 0.20 g of Ethenzamide in 15
mL of diluted ethanol (95) (2 in 3), and add 2 to 3 drops of
dilute iron (III) chloride TS: no purple color develops.
Loss on drying <2.41>
3 hours).
Not more than 1.0% (1 g, silica gel,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 20 mg each of Ethenzamide
and Ethenzamide Reference Standard, previously dried, and
dissolve each in 70 mL of ethanol (95) by warming, and after
cooling, add ethanol (95) to make exactly 100 mL. Pipet 5
mL each of these solutions, add ethanol (95) to make exactly
50 mL, and use these solutions as the sample solution and
standard solution, respectively. Determine the absorbances,
A T and A s , of the sample solution and standard solution at
290 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using ethanol (95) as the blank.
Amount (mg) of C 9 H n N0 2
= W s x (A T /A S )
W s : Amount (mg) of Ethenzamide Reference Standard
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Anesthetic Ether
641
Ether
i-xJU
Anesthetic Ether
H 3 C O CH 3
C 4 H 10 O: 74.12
Diethyl ether [60-29-7]
Ether contains not less than 96% and not more than
98% (by specific gravity) of C 4 H 10 O.
It contains a small quantity of ethanol and water. It
cannot be used for anesthesia.
Description Ether is a colorless, clear, mobile liquid, having
a characteristic odor.
It is miscible with ethanol (95).
It is soluble in water.
It is highly volatile and flammable.
It is slowly oxidized by the action of air and light, with the
formation of peroxides.
Its vapor, when mixed with air and ignited, may explode
violently.
Boiling point: 35 - 37°C
Specific gravity <2.56> df : 0.718
0.721
Purity (1) Foreign odor — Place 10 mL of Ether in an
evaporating dish, and allow it to evaporate spontaneously to
a volume of about 1 mL: no foreign odor is perceptible. Drop
this residue onto a piece of clean, odorless filter paper to
evaporate the ether: no foreign odor is perceptible.
(2) Acidity— Place 10 mL of diluted ethanol (95) (4 in 5)
and 0.5 mL of phenolphthalein TS in a 50-mL glass-stop-
pered flask, and add 0.2mol/L sodium hydroxide dropwise
to produce a red color which persists after shaking for 30 se-
conds. Add 25 mL of Ether, stopper the flask, shake gently,
and add 0.40 mL of 0.02 mol/L sodium hydroxide VS with
shaking: a red color develops.
(3) Aldehyde — Place 10 mL of Ether in a Nessler tube,
add 1 mL of potassium hydroxide TS, and allow the mixture
to stand for 2 hours, protecting from light, with occasional
shaking: no color is produced in the ether layer and the aque-
ous layer.
(4) Peroxide — Place 10 mL of Ether in a Nessler tube,
add 1 mL of a freshly prepared solution of potassium iodide
(1 in 10), shake for 1 minute, then add 1 mL of starch TS,
and shake well: no color is produced in the ether layer and in
the aqueous layer.
(5) Residue on evaporation — Evaporate 140 mL of
Ether, and dry the residue at 105 °C for 1 hour: the mass of
the residue does not more than 1.0 mg.
Containers and storage Containers — Tight containers.
Storage — Without fill up, light-resistant, remote from fire,
and not exceeding 25 °C.
HjC O CH3
C 4 H 10 O: 74.12
Diethyl ether [60-29-7]
Anesthetic Ether contains not less than 96% and not
more than 98% (by specific gravity) of C 4 H 10 O.
It contains small quantities of ethanol and water.
Suitable stabilizers may be added.
It is not to be used for anesthesia if it has been re-
moved from the original container for more than 24
hours.
Description Anesthetic Ether occurs as a colorless, clear,
mobile liquid, having a characteristic odor.
It is miscible with ethanol (95).
It is soluble in water.
It is highly volatile and flammable.
It is slowly oxidized by the action of air and light, with the
formation of peroxides.
Its vapor, when mixed with air and ignited, may explode
violently.
Boiling point: 35 - 37°C
Specific gravity <2.56> df : 0.718 - 0.721
Purity (1) Foreign odor — Place 10 mL of Anesthetic
Ether in an evaporating dish, and allow it to evaporate spon-
taneously to a volume of about 1 mL: no foreign odor is per-
ceptible. Drop this residue onto a piece of clean, odorless
filter paper to evaporate the ether: no foreign odor is percep-
tible.
(2) Acidity— Place 10 mL of diluted ethanol (95) (4 in 5)
and 0.5 mL of phenolphthalein TS in a 50-mL glass-stop-
pered flask, and add 0.2 mol/L sodium hydroxide dropwise
to produce a red color which persists after shaking for 30 se-
conds. Add 25 mL of Anesthetic Ether, stopper the flask,
shake gently, and add 0.40 mL of 0.02 mol/L sodium
hydroxide VS with shaking: a red color develops.
(3) Aldehyde — To 100 mL of water in a 200-mL glass-
stoppered flask add 10 mL of Anesthetic Ether and 1 mL of a
solution of sodium hydrogen sulfite (1 in 1000), stopper tight-
ly, shake vigorously for 10 seconds, and allow the mixture to
stand in a cool place for 30 minutes, protected from light.
Add 2 mL of starch TS, and add dropwise 0.01 mol/L iodine
VS until a pale blue color develops. Shake with about 2 g of
sodium hydrogen carbonate to decolorize the solution, and
add 1 mL of diluted 0.01 mol/L iodine VS (9 in 40): a blue
color develops. Keep the temperature of the solution below
18 C C during the procedure.
(4) Peroxide — Place 10 mL of Anesthetic Ether in a Ness-
ler tube, add 1 mL of a freshly prepared solution of potassi-
um iodide (1 in 10), shake occasionally for 1 hour, protecting
from light, then add 1 mL of starch TS, and shake well: no
color is produced and in the aqueous layer and in the ether
layer.
(5) Residue on evaporation — Evaporate 50 mL of
Anesthetic Ether, and dry the residue at 105°C for 1 hour:
642 Ethinylestradiol / Official Monographs
JP XV
the mass of the residue is not more than 1.0 mg.
Containers and storage Containers — Tight containers.
Storage — Without fill up, light-resistant, remote from fire,
and not exceeding 25 °C.
Ethinylestradiol
= 29.64 mg of C 20 H 24 O 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ethinylestradiol Tablets
if-JHX r- V-/7|--JL-ffi
^CH
C 20 H 24 O 2 : 296.40
17a-Ethynylestra-l,3,5(10)-triene-3,17/?-diol [57-63-6]
Ethinylestradiol, when dried, contains not less than
98.0% of C 20 H 24 O 2 .
Description Ethinylestradiol occurs as white to pale yellow
crystals or crystalline powder. It is odorless.
It is freely soluble in pyridine and in tetrahydrofuran, solu-
ble in ethanol (95) and in diethyl ether, and practically insolu-
ble in water.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 2 mg of Ethinylestradiol in 1
mL of a mixture of ethanol (95) and sulfuric acid (1:1): a pur-
plish red color develops with a yellow-green fluorescence.
Add carefully 2 mL of water to this solution: the color of the
solution changes to red-purple.
(2) Transfer 0.02 g of Ethinylestradiol to a glass-stop-
pered test tube, dissolve in 10 mL of a solution of potassium
hydroxide (1 in 20), add 0.1 g of benzoyl chloride, and shake.
Collect the resulting precipitate, recrystallize from methanol,
and dry in a desiccator (in vacuum, phosphorus (V) oxide):
the precipitate melts <2.60> between 200°C and 202°C.
Optical rotation <2.49> [a]™'- -26- -31° (after drying, 0.1
g, pyridine, 25 mL, 200 mm).
Melting point <2.60> 180 - 186°C or 142 - 146°C
Purity Estrone — Dissolve 5 mg of Ethinylestradiol in 0.5
mL of ethanol (95), and add 0.05 g of 1,3-dinitrobenzene.
Add 0.5 mL of freshly prepared dilute potassium hydroxide-
ethanol TS, allow to stand in a dark place for 1 hour, and add
10 mL of ethanol (95): the solution has no more color than
the following control solution.
Control solution: Proceed in the same manner as men-
tioned above, omitting Ethinylestradiol.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.2 g of Ethinylestradiol,
previously dried, and dissolve in 40 mL of tetrahydrofuran.
Add 10 mL of a solution of silver nitrate (1 in 20), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (potentiometric
titration).
Each mL of 0.1 mol/L sodium hydroxide VS
Ethinylestradiol Tablets contain not less than 90%
and not more than 110% of the labeled amount of
ethinylestradiol (C 20 H 24 O 2 : 296.40).
Method of preparation Prepare as directed under Tablets,
with Ethinylestradiol.
Identification (1) Evaporate to dryness 5 mL of the sam-
ple solution obtained in Assay, and add 2 mL of a mixture of
sulfuric acid and ethanol (95) (2:1) to the residue: a light red
color with a yellow fluorescence develops. To the solution
add carefully 4 mL of water: the color of the solution changes
to red-purple.
(2) Evaporate to dryness 10 mL of the sample solution
obtained in Assay, add 0.2 mL of acetic acid (31) and 2 mL
of phosphoric acid to the residue, and heat on a water bath
for 5 minutes: a red color with a yellow-green fluorescence
develops.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Place 1 tablet of Ethinylestradiol Tablets in a separator,
add 10 mL of 2nd fluid for disintegration test, and shake un-
til the tablet is disintegrated. Add 10 mL of dilute sulfuric
acid and 20 mL of chloroform, shake vigorously for 5
minutes, and filter the chloroform layer into a conical flask
through filter paper on which 5 g of anhydrous sodium sul-
fate is placed. Extract the aqueous layer with two 20-mL por-
tions of chloroform, proceed with the extracts in the same
manner as before, and combine the filtrates with the previous
one. Evaporate gently the combined filtrate on a water bath
with the aid of a current of nitrogen, dissolve the residue in
exactly 100 mL of methanol, and centrifuge, if necessary.
Pipet x mL of the supernatant liquid, add methanol to make
exactly FmL of a solution containing about 0.04 /ug of
ethinylestradiol (C 20 H 24 O 2 ) per mL, and use this solution as
the sample solution. Separately, weigh accurately about 10
mg of Ethinylestradiol Reference Standard, previously dried
in a desiccator (in vacuum, phosphorus (V) oxide) for 4
hours, dissolve in methanol, dilute to a volume containing
about 0.04 //g of ethinylestradiol (C 20 H 24 O 2 ) per mL, and use
this solution as the standard solution. Pipet 4 mL each of sul-
furic acid-methanol TS into three glass-stoppered test tubes,
T, S and B, cool in ice, to each tube add exactly 1 mL each of
the sample solution, the standard solution and methanol,
shake immediately, and allow to stand in a water bath at
30°C for 40 minutes, then allow to stand in a water bath at
20°C for 5 minutes. Perform the test with these solutions as
directed under Fluorometry <2.22>. Determine the fluores-
cence intensities, F T , F s and F B , of these solutions using the
fluorophotometer, at about 460 nm of the excitation and at
about 493 nm of the fluorescence.
Amount (mg) of ethinylestradiol (C 20 H 24 O 2 )
JPXV
Official Monographs / Ethionamide
643
= W s x {(F T - F B )/(F S - F B )} x (F/2500) X (1/x)
W s : Amount (mg) of Ethinylestradiol Reference Standard
Assay (i) Chromatographic tube: Pack a pledget of glass
wool in the bottom of a tube 25 mm in inside diameter and
300 mm in length, and place 5 g of anhydrous sodium sulfate
on the glass wool.
(ii) Chromatographic column: Place 5 g of siliceous earth
for chromatography in a 200-mL beaker, soak well in 4 mL
of 1 mol/L hydrochloric acid TS, and mix uniformly. Put the
siliceous earth into the chromatographic tube in small por-
tions to make 60 to 80 mm in height in proper hardness with a
tamping rod.
(iii) Standard solution: Weigh accurately about 10 mg of
Ethinylestradiol Reference Standard, previously dried in a
desiccator (in vacuum, phosphorus (V) oxide) for 4 hours,
and dissolve in chloroform to make exactly 100 mL. Pipet 5
mL of this solution, and add chloroform to make exactly 100
mL.
(iv) Sample: Weigh accurately not less than 20 Ethinyles-
tradiol Tablets, and powder. Weigh accurately a portion of
the powder, equivalent to about 0.5 mg of ethinylestradiol
(C20H24O2), place in a 50-mL beaker, add 2 mL of water,
shake well, add 3 mL of chloroform, and shake well again.
Add 4 g of siliceous earth for chromatography, mix well until
the contents do not stick to the inner wall of the beaker, and
use the substance as the sample.
(v) Procedure: To the chromatographic column add the
sample with a funnel, and pack in proper hardness. Mix well
the sample sticking to the beaker with 0.5 g of siliceous earth
for chromatography, and place in the chromatographic tube.
Wipe off the sample solution sticking to the beaker and the
tamping rod with glass wool, and place it in the chromato-
graphic tube. Push down the sample, and press lightly on the
chromatographic column to make the height of the column
110 mm to 130 mm. Take 70 mL of chloroform, rinse the in-
ner wall of the chromatographic tube with a portion of the
chloroform, and transfer the remaining portion to the chro-
matographic tube. Collect the effluent solution at a flow rate
not more than 0.8 mL per minute. After completing the elu-
tion, rinse the lower end of the chromatographic tube with a
small quantity of chloroform, add chloroform to make exact-
ly 100 mL, and use this solution as the sample solution.
Transfer 6 mL each of the sample solution and standard solu-
tion to each separators, and add 20 mL each of isooctane.
Add exactly 10 mL of a mixture of sulfuric acid and
methanol (7:3), shake vigorously for 5 minutes, allow to
stand in a dark place for 15 minutes, and centrifuge. Perform
the test with the resulting color solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using a solu-
tion, prepared with 6 mL of chloroform in the same manner,
as the blank. Determine the absorbances, A T and A s , of the
subsequent solutions obtained from the sample solution and
standard solution at 540 nm, respectively.
Amount (mg) of ethinylestradiol (C20H24O2)
= W s X (Aj/A s ) x (1/20)
W s : Amount (mg) of Ethinylestradiol Reference Standard
Containers and storage Coniners — Well-closed containers.
Ethionamide
C 8 H l0 N 2 S: 166.24
2-Ethylpyridine-4-carbothioamide
[536-33-4]
Ethionamide, when dried, contains not less than
98.5% and not more than 101.0% of C 8 H 10 N 2 S.
Description Ethionamide occurs as yellow crystals or crys-
talline powder, having a characteristic odor.
It is soluble in methanol and in acetic acid (100), sparingly
soluble in ethanol (99.5) and in acetone, and practically in-
soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Ethionamide in methanol (3 in 160,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Ethionamide as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 161 - 165°C
Purity (1) Acidity — Dissolve 3.0 g of Ethionamide in 30
mL of methanol by warming, add 90 mL of water, allow to
stand in ice water for 1 hour, and filter. To 80 mL of the
filtrate add 0.8 mL of cresol red TS and 0.20 mL of 0.1
mol/L sodium hydroxide VS: a red color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Ethionamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ethionamide according to Method 3. Add 10 mL of a so-
lution of magnesium nitrate hexahydrate in ethanol (95) (1 in
50), then add 1.5 mL of hydrogen peroxide (30), and fire to
burn (not more than 2 ppm).
(4) Related substances — Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve 0.20 g
of Ethionamide in 10 mL of acetone, and use this solution as
the sample solution. Pipet 0.5 mL of the sample solution,
add acetone to make exactly 100 mL, and use this solution as
the standard solution (1). Separately, pipet exactly 0.2 mL of
the sample solution, add acetone to make exactly 100 mL,
and use this solution as the standard solution (2). Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 10 /xh each of the sample solu-
tion and standard solutions (1) and (2) on a plate of silica gel
with fluorescent indicator for thin-layer chromatography, de-
644
Ethosuximide / Official Monographs
JP XV
velop with a mixture of ethyl acetate, hexane and methanol
(6:2:1) to a distance of about 15 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spot other than the principal spot obtained with the sam-
ple solution is not more intense than the spot with the stan-
dard solution (1), and number of the spot other than the prin-
cipal spot obtained with the sample solution which is more in-
tense than the spot with the standard solution (2) is not more
than one.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Ethionamide, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from orange-red to dark orange-brown
(indicator: 2 mL of p-naphtholbenzein TS). Perform a blank
determination in the same manner, and make any necessary
correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.62 mg of C 8 H 10 N 2 S
Containers and storage Containers — Well-closed contain-
ers.
Ethosuximide
H
CH 3 and enantiomer
C 7 H u N0 2 : 141.17
(2.RS>2-Ethyl-2-methylsuccinimide [ 77-67-8]
Ethosuximide contains not less than 98.5% of
C 7 H n N0 2 calculated on the anhydrous basis.
Description Ethosuximide occurs as a white, paraffin-like
solid or powder. It is odorless or has a slight, characteristic
odor.
It is very soluble in methanol, in ethanol (95), in diethyl
ether, and in A^^-dimethylformamide, and freely soluble in
water.
Melting point: about 48°C
Identification (1) To 0.2 g of Ethosuximide add 10 mL of
sodium hydroxide TS, and boil: the gas evolved turns a
moistened red litmus paper blue.
(2) Dissolve 0.05 g of Ethosuximide in 1 mL of ethanol
(95), add 3 drops of a solution of copper (II) acetate monohy-
drate (1 in 100), warm slightly, and add 1 to 2 drops of sodi-
um hydroxide TS: a purple color is produced.
(3) Determine the absorption spectrum of a solution of
Ethosuximide in ethanol (95) (1 in 2000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ethosuximide in 10 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— With 1.0 g of Ethosuximide, per-
form the test. Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.011%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Ethosux-
imide according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ethosuximide, according to Method 1, and perform the
test (not more than 2 ppm).
(5) Acid anhydride — Dissolve 0.50 g of Ethosuximide in
1 mL of ethanol (95), add 1 mL of hydroxylammonium chlo-
ride-iron (III) chloride TS, and allow to stand for 5 minutes.
Add 3 mL of water, mix, and allow to stand for 5 minutes:
the red to red-purple color of this solution is not more intense
than that of the following control solution.
Control solution: Dissolve 0.070 g of succinic anhydride in
ethanol (95) to make exactly 100 mL. To 1.0 mL of this solu-
tion add 1 mL of hydroxylammonium chloride-iron (III)
chloride TS, and proceed in the same manner.
(6) Cyanide — Dissolve 1 .0 g of Ethosuximide in 10 mL of
ethanol (95), and add 3 drops of iron (II) sulfate TS, 1 mL of
sodium hydroxide TS and 2 to 3 drops of iron (III) chloride
TS. Warm gently, and acidify with dilute sulfuric acid: not a
blue precipitate and a blue color are produced within 15
minutes.
Water <2.48> Not more than 0.5% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Ethosuximide, dis-
solve in 20 mL of iV,./V-dimethylformamide, and titrate <2.50>
with 0.1 mol/L tetramethylammonium hydroxide VS (poten-
tiometric titration). Perform a blank determination.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 14.12 mg of C 7 H„N0 2
Containers and storage Containers — Tight containers.
Ethyl Aminobenzoate
Anesthamine
Benzocaine
"CHa
C 9 H„N0 2 : 165.19
Ethyl 4-aminobenzoate [94-09-7]
Ethyl Aminobenzoate, when dried, contains not less
than 99.0% of C 9 H„N0 2 .
Description Ethyl Aminobenzoate occurs as white crystals
or crystalline powder. It is odorless. It has a slightly bitter
JPXV
Official Monographs / Ethyl L-Cysteine Hydrochloride
645
taste, numbing the tongue.
It is freely soluble in ethanol (95) and in diethyl ether, and
very slightly soluble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Dissolve 0.01 g of Ethyl Aminobenzoate
in 1 mL of dilute hydrochloric acid and 4 mL of water. This
solution responds to the Qualitative Tests <1.09> for primary
aromatic amines.
(2) Dissolve 0.1 g of Ethyl Aminobenzoate in 5 mL of
water with the aid of dilute hydrochloric acid added drop-
wise, and add iodine TS dropwise: a brown precipitate is
produced.
(3) Warm 0.05 g of Ethyl Aminobenzoate with 2 drops of
acetic acid (31) and 5 drops of sulfuric acid: the odor of ethyl
acetate is perceptible.
Melting point <2.60> 89 - 91 C C
Purity (1) Acidity — Dissolve 1.0 g of Ethyl Aminobenzo-
ate in 10 mL of neutralized ethanol, and add 10 mL of water,
2 drops of phenolphthalein TS and 0.50 mL of 0.01 mol/L
sodium hydroxide VS: a red color is produced.
(2) Chloride — Dissolve 0.20 g of Ethyl Aminobenzoate in
5 mL of ethanol (95), add 2 to 3 drops each of dilute nitric
acid and of silver nitrate TS: no change occurs immediately.
(3) Heavy metals <1.07>— Dissolve 2.0 g of Ethyl
Aminobenzoate in 20 mL of ethanol (95), add 2 mL of dilute
acetic acid and ethanol (95) to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 2 mL of dilute acetic acid and sufficient ethanol (95) to
make 50 mL (not more than 10 ppm).
(4) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Ethyl Aminobenzoate: the solution has no
more color than Matching Fluid A.
Loss on drying <2.41> Not more than 1.0% (1 g, silica gel, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Ethyl Aminoben-
zoate, previously dried, dissolve in 10 mL of hydrochloric
acid and 70 mL of water, add 10 mL of a solution of potassi-
um bromide (3 in 10), and cool to a temperature below 15°C.
Then titrate <2.50> with 0.1 mol/L sodium nitrite VS by the
potentiometric titration or the amperometric titration.
Each mL of 0.1 mol/L sodium nitrite VS
= 16.52 mg of C 9 H„N0 2
Containers and storage Containers — Well-closed contain-
ers.
Ethyl L-Cysteine Hydrochloride
Ethyl Cysteine Hydrochloride
L-ifJlyXj'f >M&k
H NH 2
C 5 H„N0 2 S.HC1: 185.67
Ethyl (2R )-2-amino-3 -sulf anylpropanoate
monohydrochloride
[868-59-7]
Ethyl L-Cysteine Hydrochloride, when dried, con-
tains not less than 98.5% of CjHuNOjS.HCl.
Description Ethyl L-Cysteine Hydrochloride occurs as
white crystals or crystalline powder. It has a characteristic
odor, and has a bitter taste at first with a burning aftertaste.
It is very soluble in water, and freely soluble in ethanol
(95).
Melting point: about 126°C (with decomposition).
Identification (1) Determine the infrared absorption spec-
trum of Ethyl L-Cysteine Hydrochloride as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(2) A solution of Ethyl L-Cysteine Hydrochloride (1 in
20) responds to the Qualitative Tests <1.09> (1) for chloride.
Optical rotation <2.49> [a\o- -10.0 --13.0° (after
drying, 2.0 g, 1 mol/L hydrochloric acid TS, 25 mL, 100
mm).
Purity (1) Sulfate <I.14>— Perform the test with 0.6 g of
Ethyl L-Cysteine Hydrochloride. Prepare the the control so-
lution with 0.35 mL of 0.005 mol/L sulfuric acid (not more
than 0.028%).
(2) Heavy metals <1.07>— Proceed with 1.0 g of Ethyl L-
Cysteine Hydrochloride according to Method 1, and perform
the test. Prepare the control solution with 1.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Related substances — Conduct this procedure rapidly.
Dissolve 0.05 g each of Ethyl L-Cysteine Hydrochloride and
Af-ethylmaleimide in 5 mL of mobile phase, allow to stand
for 30 minutes, and use this solution as the sample solution.
Pipet 3 mL of the sample solution, add the mobile phase to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with exactly 2//L each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of these solutions by the
automatic integration method: a peak area from the sample
solution with the ratio of the retention time to ethyl L-
cysteine-7V-ethylmaleimide complex from the standard solu-
tion being about 0.7 is not larger than the peak area of ethyl
L-cysteine-iV-ethylmaleimide complex from the standard so-
lution. Each area of all peaks other than the peaks of ethyl l-
cysteine-iV-ethylmaleimide complex and TV-ethylmaleimide
from the sample solution is not larger than 1/3 of the peak
area of ethyl L-cysteine JV-ethylmaleimide complex from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 250 nm).
Column: A stainless steel column about 6 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.02 mol/L monobasic potas-
646
Ethyl Icosapentate / Official Monographs
JP XV
sium phosphate TS and acetonitrile (2:1).
Flow rate: Adjust the flow rate so that the retention time of
ethyl L-cysteine-TV-ethylmaleimide complex is about 4
minutes.
Selection of column: Dissolve 0.05 g of Ethyl L-Cysteine
Hydrochloride, 0.01 g of L-cysteine hydrochloride and 0.05 g
of 7V-ethylmaleimide in 25 mL of the mobile phase, and allow
to stand for 30 minutes. Proceed with 2//L of this solution
under the above conditions, and calculate the resolution. Use
a column giving elution of L-cysteine-/V-ethylmaleimide com-
plex, ethyl L-cysteine-/V-ethylmaleimide complex and N-
ethylmaleimide in this order, complete resolution of each
component, and the resolution of the peaks of L-cysteine-7V-
ethylmaleimide complex and ethyl L-cysteine-7V-
ethylmaleimide complex being not less than 3.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of ethyl L-cysteine-TV-ethylmaleimide
complex obtained from 2 /uL of the standard solution is be-
tween 10 mm and 20 mm.
Time span of measurement: About 3 times as long as the
retention time of ethyl L-cysteine-/V-ethylmaleimide complex.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus oxide (V), 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Ethyl L-Cysteine
Hydrochloride, previously dried, transfer into a glass-stop-
pered flask, and dissolve in 10 mL of water previously freshly
boiled and cooled to a temperature not exceeding 5°C in a
stream of nitrogen. Add exactly 20 mL of 0.05 mol/L iodine
VS, previously cooled to a temperature not exceeding 5°C,
and allow to stand for 30 seconds, then titrate <2.50> with 0.1
mol/L sodium thiosulfate VS, on cooling below 5°C (indica-
tor: 1 mL of starch TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.05 mol/L iodine VS
= 18.57 mg of C5H„N0 2 SHC1
Containers and storage Containers — Tight containers.
Ethyl Icosapentate
„(X XHa
C 22 H 34 2 : 330.50
Ethyl (5Z, 8Z, 1 1 Z, 1 4Z, 1 7Z)-icosa-5 ,8,11,14,17-
pentaenoate
[86227-47-6]
Ethyl Icosapentate contains not less than 96.5% and
not more than 101.0% of C22H34O2.
It may contain a suitable antioxidant.
Description Icosapentate is a colorless or pale yellow, clear
liquid. It has a faint, characteristic odor.
It is miscible with ethanol (99.5), with acetic acid (100) and
with hexane. It is practically insoluble in water and in ethy-
lene glycol.
Identification (1) To 20 mg of Ethyl Icosapentate add 3
mL of a solution of potassium hydroxide in ethylene glycol
(21 in 100), stopper tightly while passing a current of nitro-
gen, and heat at 180°C for 15 minutes. After cooling, add
methanol to make 100 mL. To 4 mL of this solution add
methanol to make 100 mL, and use this solution as the sam-
ple solution. Determine the absorption spectrum of the sam-
ple solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using a solution, prepared in the same
manner as the sample solution with 3 mL of the solution of
potassium hydroxide in ethylene glycol (21 in 100), as a con-
trol, and compare the spectrum with the Reference Spectrum
or the spectrum of a solution of Ethyl Icosapentate Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Ethyl
Icosapentate as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum or the spectrum of Ethyl
Icosapentate Reference Standard: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
Refractive index <2.45> «g>: 1.481 - 1.491
Specific gravity <2.56> df : 0.905 - 0.915
Acid value <1.13> Not more than 0.5
Saponification value <1.13> 165 - 175
Iodine value <1.13> 365 - 395. Perform the test with 20 mg
of Ethyl Icosapentate.
Purity (1) Heavy metals <1.07>— Mix 1.0 g of Ethyl
Icosapentate with ethanol (99.5), and add 2 mL of dilute a-
cetic acid and ethanol (99.5) to make 50 mL. Perform the test
with this solution as the test solution. Control solution: To
1.0 mL of Standard Lead Solution add 2 mL of dilute acetic
acid and ethanol (99.5) to make 50 mL (not more than 10
ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ethyl Icosapentate according to Method 3, and perform
the test (not more than 2 ppm).
(3) Related substances — To 0.40 g of Ethyl Icosapentate
add hexane to make 50 mL, and use this solution as the sam-
ple solution. Perform the test with 1.5 fiL of the sample solu-
tion as directed under Gas Chromatography <2.02> according
to the following conditions. Determine each peak area by the
automatic integration method, and calculate the amounts of
these peaks by the area percentage method: the area of the
peak, having the relative retention time of about 0.53 with
respect to ethyl icosapentate, is not more than 0.5%, the
areas of the peaks other than the principal peak and other
than the peak mentioned above are not more than 1.0%, and
the total amount of these peaks other than the principal peak
is not more than 3.5%.
(the rest is omitted)
(4) Peroxide — Weigh accurately about 1 g of Ethyl
Icosapentate, put in a 200-mL glass-stoppered conical flask,
add 25 mL of a mixture of acetic acid (100) and chloroform
(3:2), and dissolve by gentle shaking. Add 1 mL of saturated
potassium iodide solution, immediately stopper tightly,
shake gently, and allow to stand in a dark place for 10
minutes. Then add 30 mL of water, shake vigorously for 5 to
10 seconds, and titrate <2.50> with 0.01 mol/L sodium
thiosulfate VS until the blue color of the solution disappears
JPXV
Official Monographs / Ethyl Parahydroxybenzoate
647
after addition of 1 mL of starch TS. Calculate the amount of
peroxide by the following equation: not more than 2 meq/kg.
Amount (meq/kg) of peroxide = (V/W) X 10
V: Volume (mL) of 0.01 mol/L sodium thiosulfate VS
consumed
W: Amount (g) of the sample
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Ethyl Icosapentate,
and add hexane to make exactly 50 mL. Pipet 2 mL of this
solution, add exactly 2 mL of the internal standard solution,
and use this solution as the sample solution. Separately,
weigh accurately about 80 mg of Ethyl Icosapentate Refer-
ence Standard, and add hexane to make exactly 10 mL. Pipet
2 mL of this solution, add exactly 2 mL of the internal stan-
dard solution, and use this solution as the standard solution.
Perform the test with 3 /uL each of the sample solution and
standard solution as directed under Gas Chromatography
<2.02> according to the following conditions, and determine
the ratios, Qt and Q s , of the peak area of ethyl icosapentate
to that of the internal standard.
Amount (mg) of ethyl icosapentate (C22H34O2)
= W s x (Q T /Q S ) x 5
W s : Amount (mg) of Ethyl Icosapentate Reference Stan-
dard
Internal standard solution — A solution of methyl docosanate
in hexane (1 in 125).
Operating conditions —
Detector: A hydrogen-flame ionization detector
Column: A glass column in 4 mm inside diameter and 1.8
m in length, packed with siliceous earth for gas chro-
matography (175 to 246 /um in particle diameter), coated with
diethylene glycol succinate polyester for gas chromatography
in the ratio of 25%.
Column temperature: A constant temperature of about
190°C
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethyl icosapentate is about 30 minutes.
System suitability —
System performance: When the procedure is run with 3 fiL
of the standard solution under the above operating condi-
tions, the internal standard and ethyl icosapentate are eluted
in this order with the resolution between these peaks being
not less than 3.
System repeatability: When the test is repeated 6 times with
3 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of ethyl icosapentate to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Being fully filled, or replacing the air with Nitro-
gen.
Ethyl Parahydroxybenzoate
HO
A ^.
C 9 H 10 O 3 : 166.17
Ethyl 4-hydroxybenzoate [120-47-8]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Ethyl Parahydroxybenzoate contains not less than
98.0% and not more than 102.0%, of C 9 H 10 O 3 .
♦Description Ethyl Parahydroxybenzoate occurs as color-
less crystals or a white, crystalline powder.
It is freely soluble in ethanol (95) and in acetone, and very
slightly soluble in water. ♦
Identification (1) The melting point <2.60> of Ethyl Para-
hydroxybenzoate is between 115 C C and 118°C.
*(2) Determine the infrared absorption spectrum of
Ethyl Parahydroxybenzoate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.*
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ethyl Parahydroxybenzoate in 10 mL of ethanol (95): the so-
lution is clear and not more intensely colored than the follow-
ing control solution.
Control solution: To 5.0 mL of cobalt (II) chloride colori-
metric stock solution, 12.0 mL of iron (III) chloride colori-
metric stock solution and 2.0 mL of cupper (II) sulfate colori-
metric stock solution add water to make 1000 mL.
(2) Acidity — Dissolve 0.20 g of Ethyl Parahydroxybenzo-
ate in 5 mL of ethanol (95), add 5 mL of freshly boiled and
cooled water and 0.1 mL of bromocresol green-sodium
hydroxide-ethanol TS, then add 0. 1 mL of 0. 1 mol/L sodium
hydroxide VS: the solution shows a blue color.
♦(3) Heavy metals <1. 07>— Dissolve 1.0 g of Ethyl Para-
hydroxybenzoate in 25 mL of acetone, add 2 mL of dilute
acetic acid and water to make 50 mL, and perform the test
using this solution as the test solution. Prepare the control
solution as follows: to 2.0 mL of Standard Lead Solution add
25 mL of acetone, 2 mL of dilute acetic acid, and water to
make 50 mL (not more than 20 ppm).»
(4) Related substances — Dissolve 0.10 g of Ethyl Para-
hydroxybenzoate in 10 mL of acetone, and use this solution
as the sample solution. Pipet 0.5 mL of the sample solution,
add acetone to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
//L each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
methanol, water and acetic acid (100) (70:30:1) to a distance
648
Ethylenediamine / Official Monographs
JP XV
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot other than the
principal spot is not more intense than the spot obtained with
the standard solution.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1.0 g of Ethyl Parahydrox-
ybenzoate, add exactly 20 mL of 1 mol/L sodium hydroxide
VS, heat at about 70°C for 1 hour, and immediately cool in
ice. Titrate <2.50> the excess sodium hydroxide with 0.5
mol/L sulfuric acid VS up to the second equivalent point,
(potentiometric titration). Perform a blank determination.
Each mL of 1 mol/L sodium hydroxide VS
= 166.2 mg of C 9 H 10 O 3
♦Containers and storage Containers — Well-closed contain-
ers.*
Ethylenediamine
ifl^>y7; >
HjN
C 2 H 8 N 2 : 60.10
Ethane- 1 ,2-diamine
[107-15-3]
Ethylenediamine contains not less than 97.0%
C 2 H 8 N 2 .
of
Description Ethylenediamine is a clear, colorless to pale
yellow liquid. It has an ammonia-like odor.
It is miscible with water, with ethanol (95) and with diethyl
ether.
It has a caustic nature and an irritating property.
It is gradually affected by air.
Specific gravity df : about 0.898
Identification (1) A solution of Ethylenediamine (1 in 500)
is alkaline.
(2) To 2 mL of copper (II) sulfate TS add 2 drops of
Ethylenediamine: a blue-purple color develops.
(3) To 0.04 g of Ethylenediamine add 6 drops of benzoyl
chloride and 2 mL of a solution of sodium hydroxide (1 in
10), warm for 2 to 3 minutes with occasional shaking, collect
the white precipitate formed, and wash with water. Dissolve
the precipitate in 8 mL of ethanol (95) by warming, promptly
add 8 mL of water, cool, filter the crystals, wash with water,
and dry at 105°C for 1 hour: it melts <2.60> between 247°C
and251°C.
Purity (1) Heavy metals < 1. 07>— Place 1.0 g of
Ethylenediamine in a porcelain crucible, evaporate to dryness
on a water bath, cover loosely, ignite at a low temperature
until charred, proceed according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Residue on evaporation — Pipet 5 mL of Ethylenedia-
mine, heat on a water bath to dryness, and dry to constant
mass at 105°C: the mass of the residue does not exceed 3.0
mg.
Assay Weigh accurately about 0.7 g of Ethylenediamine in
a glass-stoppered conical flask, add 50 mL of water, and ti-
trate <2.50> with 1 mol/L hydrochloric acid VS (indicator: 3
drops of bromophenol blue TS).
Each mL of 1 mol/L hydrochloric acid VS
= 30.05 mg of C 2 H 8 N 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled.
Ethylmorphine Hydrochloride
Hydrate
Dionin
• HCI • 2H;0
C 19 H 23 N0 3 .HC1.2H 2 0: 385.88
(5i?,6S)-4,5-Epoxy-3-ethoxy-17-methyl-
7,8-didehydromorphinan-6-ol monohydrochloride dihydrate
[125-30-4, anhydride]
Ethylmorphine Hydrochloride Hydrate contains not
less than 98.0% of ethylmorphine hydrochloride
(C 19 H 2 3N0 3 .HC1: 349.86), calculated on the anhydrous
basis.
Description Ethylmorphine Hydrochloride Hydrate occurs
as white to pale yellow crystals or crystalline powder.
It is very soluble in methanol and in acetic acid (100), freely
soluble in water, soluble in ethanol (95), sparingly soluble in
acetic anhydride, and practically insoluble in diethyl ether.
It is affected by light.
Melting point: about 123°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ethylmorphine Hydrochloride Hydrate (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Ethylmorphine Hydrochloride Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Ethylmorphine Hydrochloride Hydrate
(1 in 50) responds to the Qualitative Tests <1.09> (2) for chlo-
ride.
Optical rotation <2.49> [a]™: -103 - -106° (0.4 g calcu-
lated on the anhydrous basis, water, 20 mL, 100 mm).
Distilling range <2.57> 1 14 - 119°C, not less than 95 vol%. pH <254> Disso i ve .10 g of Ethylmorphine Hydrochloride
JPXV
Official Monographs / Etidronate Disodium
649
Hydrate in 10 mL of water: the pH of this solution is between
4.0 and 6.0.
Purity Related substances — Dissolve 0.20 g of Ethylmor-
phine Hydrochloride Hydrate in 10 mL of diluted ethanol
(95) (1 in 2), and use this solution as the sample solution.
Pipet 0.5 mL of the sample solution, add diluted ethanol (95)
(1 in 2) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
jXL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ethanol
(99.5), toluene, acetone and ammonia solution (28)
(14:14:7:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Water <2.48> 8.0 - 10.0% (0.25 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.5 g of Ethylmorphine
Hydrochloride Hydrate, and dissolve in 50 mL of a mixture
of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.99 mg of C 19 H 23 N03.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Etidronate Disodium
if K a >m.—-f V U ^A
H 3 C POjHNa
HO''^POjHNa
C 2 H 6 Na 2 7 P 2 : 249.99
Disodium dihydrogen (l-hydroxyethylidene)diphosphonate
[7414-83-7]
Etidronate Disodium, when dried, contains not less
than 98.0% and not more than 101.0% of
C 2 H 6 Na 2 7 P 2 .
Description Etidronate Disodium occurs as a white powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
The pH of a solution prepared by dissolving 0.10 g of
Etidronate Disodium in 10 mL of water is between 4.4 and
5.4.
It is hygroscopic.
Identification (1) To 5 mL of a solution of Etidronate
Disodium (1 in 100) add 1 mL of copper (II) sulfate TS, and
mix for 10 minutes: a blue precipitate is formed.
(2) Determine the infrared absorption spectrum of
Etidronate Disodium, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Etidronate Disodium (1 in 100)
responds to the Qualitative Tests <1.09> for sodium salt.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Etidronate Disodium according to Method 4, and perform
the test using the supernatant liquid obtained by centrifuging
after addition of 2 mL of dilute acetic acid. Prepare the con-
trol solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Etidronate Disodium according to Method 1 , and perform
the test (not more than 2 ppm).
(3) Phosphite — Weigh accurately about 3.5 g of
Etidronate Disodium, dissolve in 100 mL of 0.1 mol/L sodi-
um dihydrogen phosphate TS adjusted the pH to 8.0 with so-
dium hydroxide TS, add exactly 20 mL of 0.05 mol/L iodine
VS, and immediately stopper tightly. Allow to stand in a dark
place for 30 minutes, add 1 mL of acetic acid (100), and ti-
trate <2.50> the excess of iodine with 0.1 mol/L sodium
thiosulfate VS (indicator: 1 mL of starch TS). Perform a
blank determination in the same manner, and make any
necessary correction. The amount of phosphite (NaH 2 P0 3 ) is
not more than 1.0%.
Each mL of 0.05 mol/L iodine VS = 5.199mg of NaH 2 P0 3
(4) Methanol — Weigh accurately about 0.5 g of
Etidronate Disodium, dissolve in water to make exactly 5
mL, and use this solution as the sample solution. Separately,
pipet 1 mL of methanol, and add water to make exactly 100
mL. Pipet 1 mL of this solution, add water to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 1 /xL each of the sample solution
and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and determine the peak areas of methanol, A T and A s , and
determine the amount of methanol (CH 4 0) by the following
equation: not more than 0.1%.
Amount (%) of methanol (CH 4 0)
= (\/W) x (A T /A S ) x (1/20) x 0.79
W; Amount (g) of sample
0.79: Density (g/mL) of methanol
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with porous copolymer beads for gas chro-
matography (180 - 250 [im in particle diameter).
Column temperature: A constant temperature of about
130°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
methanol is about 2 minutes.
System suitability —
System performance: To 1 mL of methanol and 1 mL of
ethanol (99.5) add water to make 100 mL. To 1 mL of this so-
lution add water to make 100 mL. When the procedure is run
with 1 /uL of this solution under the above operating condi-
tions, methanol and ethanol are eluted in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
650
Etidronate Disodium Tablets / Official Monographs
JP XV
1 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
methanol is not more than 5.0%.
Loss on drying <2.41> Not more than 5.0% (0.5 g, 210°C, 2
hours).
Assay Weigh accurately about 0.5 g of Etidronate Disodi-
um, previously dried, and dissolve in water to make exactly
50 mL. Transfer exactly 15 mL of this solution to a chro-
matographic column of 10 mm in internal diameter contain-
ing 5 mL of strongly acidic ion exchange resin for column
chromatography (H type), allow to flow at a flow rate of
about 1.5 mL per minute, and wash the column with two
25-mL portions of water. Combine the eluate and the wash-
ings, and titrate <2.50> with 0.1 mol/L sodium hydroxide VS
(potentiometric titration). Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.50 mg of C 2 H 6 Na 2 7 P 2
Containers and storage Containers — Tight containers.
Etidronate Disodium Tablets
if K n >M-i- r- U OA&
Etidronate Disodium Tablets contains not less than
93.0% and not more than 107.0% of the labeled
amount of etidronate disodium (C2H 6 Na 2 7 P2:
249.99).
Method of preparation Prepare as directed under Tablets,
with Etidronate Disodium.
Identification (1) Shake an amount of pulverized
Etidronate Disodium Tablets, equivalent to 0.2 g of
Etidronate Disodium according to the labeled amount, with
20 mL of water, and filter. Proceed with the filtrate as direct-
ed in the Identification (1) under Etidronate Disodium.
(2) Shake an amount of pulverized Etidronate Disodium
Tablets, equivalent to 0.4 g of Etidronate Disodium accord-
ing to the labeled amount, with 10 mL of water, and filter.
Evaporate total amount of the filtrate to dryness under
reduced pressure, shake the residue with 15 mL of ethanol
(99.5), centrifuge, and dry the precipitate at 150°C for 4
hours. Determine the infrared absorption spectrum of the
precipitate as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 1170 cm -1 , 1056 cm -1 ,
916 cm -1 and 811 cm -1 .
Uniformity of dosage unit <6.02>
of the Mass variation test.
It meets the requirement
Dissolution <6.I0> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Etidronate Disodium
Tablets at 50 revolutions per minute according to the Paddle
method, using 900 mL of water as the dissolution medium.
Withdraw not less than 20 mL of the dissolution medium 60
minutes after start of the test, and filter through a membrane
filter with pore size of not more than 0.45 fim. Discard the
first 10 mL of the filtrate, take exactly FmL of the subse-
quent filtrate, add water to make exactly V mL so that each
mL contains about 0.22 mg of etidronate disodium (C 2 H 6 Na 2
7 P 2 ) according to the labeled amount, and use this solution
as the sample solution. Separately, weigh accurately about 30
mg of etidronate disodium for assay, previously dried at 210°
C for 2 hours, and dissolve in water to make exactly 100 mL.
Dilute exactly a suitable amount of this solution with water to
make solutions so that each mL contains about 0.12 mg,
about 0.21 mg and about 0.24 mg of etidronate disodium (C 2
H 6 Na 2 7 P 2 ), and use these solutions as the standard solu-
tions. Pipet 2 mL each of the sample solution and standard
solutions, add exactly 2 mL of a solution of copper (II) sul-
fate (7 in 10,000) and water to make exactly 10 mL. Deter-
mine the absorbances of these solutions at 233 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24> using a
solution prepared by diluting exactly 2 mL of the solution of
copper (II) sulfate (7 in 10,000) with water to make exactly 10
mL as the control. From the calibration curve obtained with
the standard solutions determine the concentration of
etidronate disodium, C T , in the sample solution. The dissolu-
tion rate of Etidronate Disodium Tablets in 60 minutes is not
less than 85%.
Dissolution rate (%) with respect to the labeled amount of
etidronate disodium (C 2 H 6 Na 2 7 P 2 )
= C T x (V'/V) x (l/Q x 90
C T : Concentration (Mg/mL) of etidronate disodium
(C 2 H 6 Na 2 7 P 2 ) in the sample solution
C: Labeled amount (mg) of etidronate disodium
(C 2 H 6 Na 2 7 P 2 ) in 1 tablet
Assay Weigh accurately, and powder not less than 20
Etidronate Disodium Tablets. Weigh accurately a portion of
the powder, equivalent to about 0.5 g of etidronate disodium
(C 2 H 6 Na 2 7 P 2 ), add 30 mL of water, shake vigorously for 10
minutes, add water to make exactly 50 mL, and filter. Pro-
ceed with the filtrate as directed in the Assay under
Etidronate Disodium.
Containers and storage Containers — Tight containers.
Etilefrine Hydrochloride
i^7U>£K£
H OH
and enantiomer
C 10 H 15 NO 2 .HCl: 217.69
(li?5)-2-Ethylamino-l-(3-hydroxyphenyl)ethanol
monohydrochloride [943-17-9]
Etilefrine Hydrochloride, when dried, contains not
less than 98.0%> and not more than 101.0% of
Ci H 15 NO 2 .HCl.
Description Etilefrine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is very soluble in water, freely soluble in ethanol (99.5),
and sparingly soluble in acetic acid (100).
It is gradually colored to yellow-brown by light.
JPXV
Official Monographs / Etilefrine Hydrochloride Tablets 651
A solution of Etilefrine Hydrochloride (1 in 20) shows no
optical rotation.
Identification (1) Dissolve 5 mg of Etilefrine Hydrochlo-
ride in 100 mL of diluted hydrochloric acid (1 in 1000). Deter-
mine the absorption spectrum of the solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Etilefrine Hydrochloride as directed in the potassium chlo-
ride disk method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) A solution of Etilefrine Hydrochloride (1 in 1000)
responds to the Qualitative Tests <1.09> (2) for chloride.
Melting point <2.60> 1 18 - 122°C
Purity (1) Acidity or alkalinity — To 10 mL of a solution
of Etilefrine Hydrochloride (1 in 50) add 0.1 mL of methyl
red TS for acid or alkali test and 0.2 mL of 0.01 mol/L sodi-
um hydroxide VS: a yellow color develops, and the necessary
volume of 0.01 mol/L hydrochloric acid VS to change the
color to red is not more than 0.4 mL.
(2) Clarity and color of solution — Dissolve 0.5 g of
Etilefrine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(3) Sulfate <1.14>— Perform the test with 0.85 g of
Etilefrine Hydrochloride. Prepare the control solution with
0.35 mL of 0.005 mol/L sulfuric acid VS (not more than
0.020%).
(4) Heavy metals <1.07> — Dissolve 1.0 g of Etilefrine
Hydrochloride in 30 mL of water and 2 mL of acetic acid
(100), adjust with sodium hydroxide TS to a pH of 3.3, add
water to make 50 mL, and perform the test. Prepare the con-
trol solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.15 g of Etilefrine
Hydrochloride, previously dried, dissolve in 20 mL of acetic
acid (100), add 50 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rectoin.
Each mL of 0.1 mol/L perchloric acid VS
= 21.77 mg of C 10 Hi 5 NO 2 .HCl
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Etilefrine Hydrochloride Tablets
ifU7'J>Mili
Etilefrine Hydrochloride Tablets contain not less
than 93.0% and not more than 107. 0%> of the labeled
amount of etilefrine hydrochloride (C 10 H 15 NO 2 .HCl:
217.69).
Method of preparation Prepare as directed under Tablets,
with Etilefrine Hydrochloride.
Identification To a quantity of powdered Etilefrine
Hydrochloride Tablets, equivalent to 5 mg of Etilefrine
Hydrochloride according to the labeled amount, add 60 mL
of diluted hydrochloric acid (1 in 1000), shake well, add
40 mL of diluted hydrochloric acid (1 in 1000), and filter. De-
termine the absorption spectrum of the filtrate as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, using dilut-
ed hydrochloric acid (1 in 1000) as the blank: it exhibits a
maximum between 271 nm and 275 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Etilefrine Hydrochloride Tablets add 60 mL
of diluted hydrochloric acid (1 in 1000), and proceed as
directed in the Assay.
Amount (mg) of etilefrine hydrochloride (C 10 H I5 NO 2 .HCl)
= W s x (A T /A S ) x (1/10)
W s : Amount (mg) of etilefrine hydrochloride for assay
Assay Weigh accurately the mass of not less than 20
Etilefrine Hydrochloride Tablets, and powder. Weigh ac-
curately a portion of the powder, equivalent to about 5 mg of
etilefrine hydrochloride (C 10 H 15 NO 2 .HCl), add 60 mL of
diluted hydrochloric acid (1 in 1000), shake for 10 minutes,
add diluted hydrochloric acid (1 in 1000) to make exactly 100
mL, and filter. Discard the first 20 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Separately,
weigh accurately about 50 mg of etilefrine hydrochloride for
assay, previously dried at 105°C for 4 hours, and dissolve in
diluted hydrochloric acid (1 in 1000) to make exactly 100 mL.
Pipet 10 mL of this solution, add diluted hydrochloric acid (1
in 1000) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 20 /xL each
of the sample solution and standard solution as direct under
Liquid Chromatography <2.01> according to the following
conditions, and determine the peak areas, A T and A s , of
etilefrine.
Amount (mg) of etilefrine hydrochloride (CioH 15 N0 2 .HCl)
= W s x (A T /A S ) x (1/10)
W s : Amount (mg) of etilefrine hydrochloride for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 5 g of sodium lauryl sulfate in
940 mL of water and 500 mL of acetonitrile, and adjust the
pH to 2.3 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
etilefrine is about 6 minutes.
System suitability —
System performance: Dissolve 4 mg of bamethan sulfate
652 Etizolam / Official Monographs
JP XV
and 4 mg of etilefrine hydrochloride in the mobile phase to
make 50 mL. When the procedure is run with 20 ^L of this
solution under the above operating conditions, etilefrine and
bamethan are eluted in this order with the resolution between
these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
etilefrine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Etizolam
lf'/7A
C 17 H 15 C1N 4 S: 342.85
4-(2-Chlorophenyl)-2-ethyl-9-methyl-6//-
thieno[3,2-/][l,2,4]triazolo[4,3-a][l,4]diazepine
[40054-69-1]
Etizolam contains not less than 98.5% and not more
than 101.0% of etizolam (C 17 H 15 C1N 4 S).
Description Etizolam occurs as a white to pale yellowish
white crystalline powder.
It is soluble in ethanol (99.5), sparingly soluble in aceto-
nitrile and in acetic anhydride, and practically insoluble in
water.
Identification (1) Determine the absorption spectrum of a
solution of Etizolam in ethanol (99.5) (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Etizolam as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Melting point <2.60> 146 - 149°C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Etizolam according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve 20 mg of Etizolam in 50
mL of acetonitrile, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, and add acetoni-
trile to make exactly 20 mL. Pipet 1 mL of this solution, add
acetonitrile to make exactly 50 mL, and use this solution as
the standard solution. Perform the test with exactly 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
etizolam obtained from the sample solution is not more than
the peak area of etizolam from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 1.36 g of potassium dihydrogen
phosphate in water to make 1000 mL, and adjust the pH to
3.5 with diluted phosphoric acid (1 in 10). To 550 mL of this
solution add 450 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
etizolam is about 6 minutes.
Time span of measurement: About 5 times as long as the
retention time of etizolam beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add acetonitrile to make exactly 20
mL. Confirm that the peak area of etizolam obtained from
10 /uL of this solution is equivalent to 8 to 12% of that from
10 [iL of the standard solution.
System performance: Dissolve 0.02 g each of Etizolam and
ethyl parahydroxybenzoate in the mobile phase to make 50
mL. To 1 mL of this solution add the mobile phase to make
50 mL. When the procedure is run with 10 /xL of this solution
under the above operating conditions, ethyl parahydroxyben-
zoate and etizolam are eluted in this order with the resolution
between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
etizolam is not more than 2%.
Loss on drying <2.41>
hours).
Not more than 0.5% (1 g, 105 °C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Etizolam, previous-
ly dried, dissolve in 70 mL of a mixture of acetic anhydride
and acetic acid (100) (7:3), and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). The end point is
the second equivalent point. Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 17.14 mg of C 17 H 15 C1N 4 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Etoposide 653
Etodolac
IH*=7?
CH 3
C0 2 H
and enanliomer
C 17 H 21 N0 3 : 287.35
2-[(li?S)-l,8-Diethyl-l,3,4,9-tetrahydropyrano[3,4-d]indol-
1-yl] acetic acid
[41340-25-4]
Etodolac, when dried, contains not less than 98.5%
and not more than 101.0% of C 17 H 21 N0 3 .
Description Etodolac occurs as white to pale yellow, crys-
tals or crystalline powder.
It is freely soluble in methanol and in ethanol (99.5), and
practically insoluble in water.
A solution of Etodolac in methanol (1 in 50) shows no opti-
cal rotation.
Melting point: about 147°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Etodolac in ethanol (99.5) (3 in 200,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Etodolac as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Etodolac according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve 0.5 g of Etodolac in 10
mL of methanol, and use this solution as the sample solution.
Pipet 2 mL of the sample solution, and add methanol to
make exactly 100 mL. Pipet 5 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the
standard solution (1). Pipet 4 mL of the standard solution
(1), add methanol to make exactly 10 mL, and use this solu-
tion as the standard solution (2). Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Previously develop a plate of silica gel with fluores-
cent indicator for thin-layer chromatography in a developing
container containing 2 cm depth of a solution of L-ascorbic
acid in a mixture of methanol and water (4: 1) (1 in 200 mL) to
the distance of 3 cm, and air-dry for 30 minutes. Spot 10 /uL
each of the sample solution and standard solutions (1) and (2)
on the plate 2.5 cm away from the bottom of the plate, then
immediately develop with a mixture of toluene, ethanol (95)
and acetic acid (100) (140:60:1) to a distance of about 15 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): any spot other than the principal spot
obtained with the sample solution is not more intense than
the spot with the standard solution (1), and the number of
spots which are more intense than the spot with the standard
solution (2) is not more than 2.
Loss on drying <2.41>
60°C, 4 hours).
Not more than 0.5% (1 g, in vacuum,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Etodolac, previous-
ly dried, dissolve in 50 mL of ethanol (99.5), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 28.74 mg of C 17 H 21 N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Etoposide
h*°->H
(5i?,5afl,8afl,9S)-9-{[4,6-0-(U?)-Ethylidene-
/?-D-glucopyranosyl]oxy}5-(4-hydroxy-
3,5-dimethoxyphenyl)-5,8,8a,9-
tetrahydrofuro[3' ,4' :6,7]naphtho[2,3-rf]-l ,3-dioxol-
6(5a//)-one [33419-42-0]
Etoposide contains not less than 98.0% and not
more than 102.0% of C29H 3 20 13 , calculated on the
anhydrous basis.
Description Etoposide occurs as white crystals or crystalline
powder.
It is sparingly soluble in methanol, slightly soluble in
ethanol (99.5), and very slightly soluble in water.
Melting point: about 260°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Etoposide in methanol (1 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Etoposide Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Etoposide as directed in the potassium bromide disk method
654 Eucalyptus Oil / Official Monographs
JP XV
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Etoposide Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]g>: - 100 - - 105° (0.1 g calculated
on the anhydrous basis, methanol, 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Etoposide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Dissolve 50 mg of Etoposide in
10 mL of methanol, add the mobile phase to make 50 mL,
and use this solution as the sample solution. Pipet 2 mL of
the sample solution, add the mobile phase to make exactly
200 mL, and use this solution as the standard solution. Per-
form the test with exactly 50 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the area of the peak other than etoposide is not larg-
er than 1/5 times the peak area of etoposide with the stan-
dard solution, and the total area of the peaks other than the
peak of etoposide with the sample solution is not larger than
1/2 times the peak area of etoposide with the standard solu-
tion.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of etoposide beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make
exactly 10 mL. Confirm that the peak area of etoposide
obtained with 50 /xL of this solution is equivalent to 7 to 13%
of that with 50 /xL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
etoposide is not more than 2.0%.
Water <2.48> Not more than 4.0% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 25 mg each of Etoposide and
Etoposide Reference Standard (previously determined the
water <2.48> in the same manner as Etoposide) dissolve
separately in methanol to make exactly 25 mL. Pipet 10 mL
each of these solutions, add exactly 5 mL of the internal stan-
dard solution and the mobile phase to make 50 mL, and use
these solutions as the sample solution and standard solution.
Perform the test with 50 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, g T and Q s , of the peak area of etoposide to that of
the internal standard.
Amount (mg) of C 29 H 32 13 = W s x (Q T /Q S )
W s : Amount (mg) of Etoposide Reference Standard,
calculated on the anhydrous basis
Internal standard solution — A solution of 2,6-dichloro-
phenol in methanol (3 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 290 nm).
Column: A stainless steel column 3.9 mm in inside
diameter and 30 cm in length, packed with phenylsilanized
silica gel for liquid chromatography (10 /um in particle
diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 6.44 g of sodium sulfate decahy-
drate in diluted acetic acid (100) (1 in 100) to make 1000 mL,
and add 250 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
etoposide is about 20 minutes.
System suitability —
System performance: Dissolve 10 mg of Etoposide in 2 mL
of methanol, add 8 mL of the mobile phase, and mix well.
Add 0.1 mL of diluted acetic acid (100) (1 in 25) and 0.1 mL
of phenolphthalein TS, and add sodium hydroxide TS until
the color of the solution changes to faintly red. After
allowing to stand for 15 minutes, add 0.1 mL of diluted acetic
acid (100) (1 in 25). When the procedure is run with 10 fiL of
this solution under the above operating conditions, the reso-
lution between the peak of etoposide and the peak having the
relative retention time of about 1.3 with respect to etoposide
is not less than 3.
System repeatability: When the test is repeated 6 times with
50 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of etoposide to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Eucalyptus Oil
Oleum Eucalypti
Eucalyptus Oil is the essential oil distilled with steam
from the leaves of Eucalyptus globulus Labillardiere or
allied plants (Myrtaceae).
It contains not less than 70.0% of cineol (C 10 H 18 O:
154.25).
Description Eucalyptus Oil is a clear, colorless or pale yel-
low liquid. It has a characteristic, aromatic odor and a pun-
gent taste.
It is neutral.
Identification Shake 1 mL of Eucalyptus Oil vigorously
with 1 mL of phosphoric acid, and allow to stand: the solu-
tion congeals within 30 minutes.
Refractive index <2.45> n™: 1.458 - 1.470
Specific gravity <7.75> df : 0.907 - 0.927
Purity (1) Clarity of solution — Mix 1.0 mL of Eucalyptus
JPXV
Official Monographs / Famotidine 655
Oil with 5 mL of diluted ethanol (7 in 10): the solution is
clear.
(2) Heavy metals <1.07> — Proceed with 1.0 mL of Eu-
calyptus Oil according to Method 2, and perform the test.
Prepare the control solution with 4.0 mL of Standard Lead
Solution (not more than 40 ppm).
Assay Weigh accurately about 0.1 g of Eucalyptus Oil, and
dissolve in hexane to make exactly 25 mL. Pipet 5 mL of this
solution, add exactly 5 mL of the internal standard solution,
then add hexane to make 100 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.1 g of
cineol for assay, proceed as directed in the sample solution,
and use this solution as the standard solution. Perform the
test with 2 /uL each of the sample solution and standard solu-
tion as directed under Gas Chromatography <2.02> according
to the following conditions. Calculate the ratios, Q T and Q s ,
of the peak area of cineol to that of the internal standard of
each solutions, respectively.
Amount (mg) of cineol (C 10 H 18 O)
= W s x (Q T /Q S )
W s : Amount (mg) of cineol for assay
Internal standard solution — A solution of anisol in hexane (1
in 250).
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 5 m in length, having alkylene glycol phthalate es-
ter for gas chromatography coated at the ratio of 10% on
silanized siliceous earth for gas chromatography (150 to 180
/um in particle diameter).
Column temperature: A constant temperature of about
120°C.
Carrier gas: Nitrogen.
Flow rate: Adjust the flow rate so that the retention time of
cineol is about 11 minutes.
Selection of column: Dissolve 0.1 g each of cineol and
limonene in 25 mL of hexane. To 1 mL of this solution add
hexane to make 20 mL. Proceed with about 2 fiL of this solu-
tion under the above operating conditions, and calculate the
resolution. Use a column giving elution of limonene and
cineol in this order with the resolution between these peaks
being not less than 1.5.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Famotidine
7/ : Efy>
C 8 H 15 N 7 2 S 3 : 337.45
A r -Aminosulfonyl-3-{[2-(diaminomethyleneamino)-l,3-
thiazol-4-yl]methylsulfanyl}propanimidamide
[76824-35-6]
Famotidine, when dried, contains not less than
98.5% of C 8 H 15 N 7 2 S 3 .
Description Famotidine occurs as white to yellowish white
crystals.
It is freely soluble in acetic acid (100), slightly soluble in
ethanol (95), and very slightly soluble in water.
It dissolves in 0.5 mol/L hydrochloric acid TS.
It is gradually colored by light.
Melting point: about 164°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Famotidine in 0.05 mol/L potassium dihydrogen-
phosphate TS (1 in 50,000) as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Famotidine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Famotidine in 10 mL of 0.5 mol/L hydrochloric acid TS: the
solution is clear and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Famoti-
dine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Related substances — Dissolve 0.20 g of Famotidine in
10 mL of acetic acid (100), and use this solution as the sample
solution. Pipet 1 mL of the sample solution, and add acetic
acid (100) to make exactly 100 mL. Pipet 1 mL, 2 mL and 3
mL of this solution, add acetic acid (100) to make exactly 10
mL, respectively, and use these solutions as the standard so-
lution (1), the standard solution (2) and the standard solution
(3). Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solutions (1), (2) and (3) on a
plate of silica gel (5 to 7 /xm) with fluorescent indicator for
thin-layer chromatography, and dry in a stream of nitrogen.
Develop the plate with a mixture of ethyl acetate,
methanol, toluene and ammonia solution (28) (40:25:20:2) to
a distance of about 8 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot and other than the spot of the
starting point from the sample solution are not more intense
than the spot from the standard solution (3). Total intensity
of the spots other than the principal spot and other than the
spot of the starting point from the sample solution is not
more than 0.5% calculated on the basis of intensities of the
spots from the standard solution (1) and the standard solu-
tion (2) (each spot is equivalent to 0.1% and 0.2%, respec-
tively).
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 80°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Famotidine, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
656
Famotidine for Injection / Official Monographs
JP XV
Each mL of 0.1 mol/L perchloric acid VS
= 16.87 mg of C 8 H 15 N 7 2 S 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Famotidine for Injection
Famotidine for Injection is a preparation for injec-
tion which is dissolved before use.
It contains not less than 94.0% and not more than
106.0% of the labeled amount of famotidine (C 8 H 15 N 7
2 S 3 : 337.45).
Method of preparation Prepare as directed under Injection,
with Famotidine.
Description Famotidine for Injection occurs as white
porous masses or powder.
Identification Dissolve an amount of Famotidine for Injec-
tion, equivalent to 0.01 g of Famotidine according to the la-
beled amount, in 50 mL of 0.05 mol/L potassium di-
hydrogenphosphate TS. To 5 mL of this solution add 0.05
mol/L potassium dihydrogenphosphate TS to make 50 mL,
and determine the absorption spectrum of this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 263 nm and 267 nm.
pH <2.54> Dissolve an amount of Famotidine for Injection,
equivalent to 0.02 g of Famotidine according to the labeled
amount, in 1 mL of water: the pH of this solution is between
4.9 and 5.5.
Purity (1) Clarity and color of solution — Dissolve an
amount of Famotidine for Injection, equivalent to 0.02 g of
Famotidine according to the labeled amount, in 1 mL of
water: the solution is clear and colorless.
(2) Related substances — Take a number of Famotidine
for Injection, equivalent to about 0.1 g of famotidine
(C 8 H 15 N 7 2 S3), dissolve each content in water, wash the
inside of the container with water, combine the solutions of
the contents with the washings, add water to the combined
solution to make exactly 100 mL, and use this solution as the
sample solition. Pipet 1 mL of the sample solution, add water
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 5 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area of these solutions by the
automatic integration method: the total area of the peaks
other than peak of famotidine from the sample solution is
not larger than peak area of famotidine from the standard so-
lution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of famotidine beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the water to make exactly 20 mL. Confirm
that the peak area of famotidine obtained from 5 /uL of this
solution is equivalent to 8 to 12% of that of famotidine
obtained from 5 /uL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
famotidine is not more than 2.0%.
Water <2.48> Not more than 1.5% (0.1 g, coulometric titra-
tion).
Bacterial endotoxins <4.01> Not more than 15 EU/mg.
Assay Take a number of Famotidine for Injection, equiva-
lent to about 0.1 g of famotidine (C 8 H 15 N 7 2 S3), dissolve
each content in water, wash the inside of each container with
water, combine the solutions of the contents with the wash-
ings, add water to the combined solution to make exactly 100
mL, and use this solution as the sample stock solution. Pipet
5 mL of this solution, add exactly 5 mL of the internal stan-
dard solution, add the mobile phase to make exactly 50 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 50 mg of famotidine for assay, previ-
ously dried in vacuum with phosphorus (V) oxide at 80°C for
4 hours, dissolve in the mobile phase to make exactly 50 mL.
Pipet 5 mL of this solution, add 5 mL of the internal stan-
dard solution, add the mobile phase to make exactly 50 mL,
and use this solution as the standard solution. Perform the
test with 5 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qt and g s , of the peak area of famotidine to that of the in-
ternal standard.
Amount (mg) of famotidine (C 8 H 15 N 7 2 S3)
= W s x (Q T /Q S ) x 2
W s : Amount (mg) of famotidine for assay
Internal standard solution — To 5 mL of a solution of methyl
parahydroxybenzoate in methanol (1 in 500) add water to
make 50 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2 g of sodium 1-heptane sulfonate
in 900 mL of water, adjust to pH 3.0 with acetic acid (100),
and add water to make 1000 mL. To this solution add 240
mL of acetonitrile and 40 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
famotidine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
JPXV
Official Monographs / Famotidine Tablets 657
tions, famotidine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 11.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of famotidine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Famotidine Powder
Famotidine Powder contains not less than 94.0%
and not more than 106.0% of the labeled amount of
famotidine (C 8 H 15 N 7 2 S 3 : 337.45).
Method of preparation Prepare as directed under Powder,
with Famotidine.
Identification Weigh a portion of Famotidine Powder,
equivalent to 0.01 g of Famotidine according to the labeled
amount, add 50 mL of 0.05 mol/L potassium dihydrogen-
phosphate TS, shake well, and centrifuge. To 5 mL of the su-
pernatant liquid add 0.05 mol/L potassium dihydrogen-
phosphate TS to make 50 mL, and determine the absorption
spectrum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
263 nm and 267 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Weigh accurately an amount of Famotidine Powder,
equivalent to about 20 mg of famotidine (C8FL5N7O2S3) ac-
cording to the labeled amount, and perform the test at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of 0.05 mol/L acetic acid-sodium acetate buffer
solution, pH 4.0 as the dissolution medium. Withdraw not
less than 20 mL of the dissolution medium 15 minutes after
starting the test, and filter through a membrane filter with
pore size of not more than 0.5 [im. Discard the first 10 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. Separately, weigh accurately about 40 mg of famotidine
for assay, previously dried in vacuum on phosphorus (V)
oxide at 80°C for 4 hours, dissolve in 0.05 mol/L acetic acid-
sodium acetate buffer solution, pH 4.0 to make exactly 100
mL. Pipet 5 mL of this solution, add the same buffer solution
to make exactly 100 mL, and use this solution as the standard
solution. Determine the absorbances, A T and A s , of the sam-
ple solution and standard solution at 266 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>. The disso-
lution rate of a 20-mg/g powder and a 100-mg/g powder in
15 minutes of Famotidine Powder are not less than 80% and
not less than 85%, respectively.
Dissolution rate (%) with respect to the labeled amount of
famotidine (C 8 H 15 N 7 2 S3)
= (W S / Wj) x (A T /A S ) x (1/Q x 45
W s : Amount (mg) of famotidine for assay
Wj\ Amount (mg) of the sample.
C: Labeled amount (mg) of famotidine (C 8 H 15 N 7 2 S3) in
lg
Assay Weigh accurately a portion of Famotidine Powder,
equivalent to about 20 mg of famotidine (C8FL5N7O2S3), add
20 mL of water, and shake well. Add 20 mL of methanol,
then shake well, add methanol to make exactly 50 mL, and
centrifuge. Pipet 5 mL of the supernatant liquid, add exactly
2 mL of the internal standard solution, add the mobile phase
to make 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.1 g of famotidine for
assay, previously dried in vacuum with phosphorus (V) oxide
at 80°C for 4 hours, dissolve in methanol to make exactly 50
mL. Pipet 10 mL of this solution, and add methanol to make
exactly 50 mL. Pipet 5 mL of this solution, add exactly 2 mL
of the internal standard solution, add the mobile phase to
make 20 mL, and use this solution as the standard solution.
Perform the test with 5 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of famotidine to that
of the internal standard.
Amount (mg) of famotidine (C 8 H 15 N 7 2 S3)
= W s x (Q T /Q S ) x (1/5)
W s : Amount (mg) of famotidine for assay
Internal standard solution — To 5 mL of a solution of methyl
parahydroxybenzoate in methanol (1 in 500) add water to
make 50 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2 g of sodium 1-heptane sulfonate
in 900 mL of water, adjust to pH 3.0 with acetic acid (100),
and add water to make 1000 mL. To this solution add 240
mL of acetonitrile and 40 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
famotidine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, famotidine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 11.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of famotidine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Famotidine Tablets
7r : Efy>fi
Famotidine Tablets contain not less than 94.0% and
658
Faropenem Sodium Hydrate / Official Monographs
JP XV
not more than 106.0% of the labeled amount of
famotidine (C 8 H 15 N 7 2 S 3 : 337.45).
Method of preparation Prepare as directed under Tablets,
with Famotidine.
Identification Weigh a portion of powdered Famotidine
Tablets, equivalent to 0.01 g of Famotidine according to the
labeled amount, add 50 mL of 0.05 mol/L potassium di-
hydrogenphosphate TS, shake well, and centrifuge. To 5 mL
of the supernatant liquid add 0.05 mol/L potassium di-
hydrogenphosphate TS to make 50 mL, and determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 263 nm and 267 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Famotidine Tablets add 2 mL of water,
shake to disintegrate, then add a suitable amount of
methanol, and shake well. Add methanol to make exactly V
mL of a solution containing about 0.2 mg of famotidine (C 8
H 15 N 7 2 S 3 ) per mL, and centrifuge. Pipet 10 mL of the su-
pernatant liquid, add exactly 2 mL of the internal standard
solution, add the mobile phase to make 20 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 0.1 g of famotidine for assay, previously dried in vacu-
um with phosphorus (V) oxide at 80°C for 4 hours, dissolve
in methanol to make exactly 100 mL. Pipet 10 mL of this so-
lution, and add methanol to make exactly 50 mL. Pipet 10
mL of this solution, add exactly 2 mL of the internal stan-
dard solution, add the mobile phase to make 20 mL, and use
this solution as the standard solution. Perform the test with 5
fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the operating conditions described in the Assay, and calculate
the ratios, Q T and Q s , of the peak area of famotidine to that
of the internal standard.
Amount (mg) of famotidine (CgH^NvC^Sj)
= W s x (Q T /Q S ) x (K/500)
W s : Amount (mg) of famotidine for assay
Internal standard solution — To 5 mL of a solution of methyl
paraphydroxybenzoate in methanol (1 in 500) add water to
make 50 mL.
Dissolution Being specified separately.
Assay Take a number of Famotidine Tablets, equivalent to
0.2 g of famotidine (CgH^NvO^), add 50 mL of water, and
disintegrate by shaking well. Add 100 mL of methanol, then
shake well, add methanol to make exactly 200 mL, and cen-
trifuge. Pipet 5 mL of the supernatant liquid, add exactly 5
mL of the internal standard solution, add the mobile phase to
make 50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.1 g of famotidine for
assay, previously dried in vacuum with phosphorus (V) oxide
at 80°C for 4 hours, dissolve in methanol to make exactly 100
mL. Pipet 5 mL of this solution, add exactly 5 mL of the in-
ternal standard solution, add the mobile phase to make 50
mL, and use this solution as the standard solution. Perform
the test with 5 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qt and Q s , of the peak area of famotidine to that of the in-
ternal standard.
Amount (mg) of famotidine (C 8 H 15 N 7 02S3)
= Ws x (Qt/Qs) x 2
W s : Amount (mg) of famotidine for assay
Internal standard solution — To 5 mL of a solution of methyl
parahydroxybenzoate in methanol (1 in 500) add water to
make 50 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2 g of sodium 1-heptane sulfonate
in 900 mL of water, adjust to pH 3.0 with acetic acid (100),
and add water to make 1000 mL. To this solution add 240
mL of acetonitrile and 40 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
famotidine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, famotidine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 11.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of famotidine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Faropenem Sodium Hydrate
7/n <*Ai- h U ^ A7Kftti
o
H V-«
COjNa
H 3 C H H
.1.
■2"2 H 2°
H O'
C 12 H 14 NNa0 5 S.2-H 2 0: 352.34
Monosodium (5i?,6S)-6-[(l/?)-l-hydroxyethyl]-7-oxo-
3-[(2/?)-tetrahydrofuran-2-yl]-4-thia-l-
azabicyclo[3.2.0]hept-2-ene-2-carboxylate
hemipentahydrate [122547-49-3, anhydride]
Faropenem Sodium Hydrate contains not less than
870 fig (potency) and not more than 943 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Faropenem Sodium Hydrate is expressed as mass
(potency) of faropenem (C 12 H 15 N0 5 S: 285.32).
Description Faropenem Sodium Hydrate occurs as white to
light yellow, crystals or crystalline powder.
It is freely soluble in water and in methanol, slightly solu-
JPXV
Official Monographs / Faropenem Sodium for Syrup
659
ble in ethanol (95), and practically insoluble in diethyl ether.
Identification (1) Dissolve 5 mg of Faropenem Sodium
Hydrate in 1 mL of hydroxylammonium chloride-ethanol
TS, allow to stand for 3 minutes, add 1 mL of acidic ammo-
nium iron (III) sulfate TS, and shake: a red-brown to brown
color develops.
(2) Determine the absorption spectra of solutions of
Faropenem Sodium Hydrate and Faropenem Sodium Refer-
ence Standard (1 in 20,000) as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, and compare the spectra: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectra of
Faropenem Sodium Hydrate and Faropenem Sodium Refer-
ence Standard as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectra: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Optical rotation <2.49> [a\^: +145- +150° (0.5 g calcu-
lated as the anhydrous basis, water, 50 mL, 100 mm).
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Faropen-
em Sodium Hydrate according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Related substances — Being specified separately.
Water <2.48> Not less than 12.6% and not more than
13.1% (20 mg, coulometric titration).
Assay Weigh accurately an amount of Faropenem Sodium
Hydrate and Faropenem Sodium Reference Standard, e-
quivalent to about 25 mg (potency), each of these, add ex-
actly 10 mL each of the internal standard solution, add water
to make 50 mL, and use these solutions as the sample solu-
tion and the standard solution, respectively. Perform the test
with 20 iuL of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Q T and Q s ,
of the peak area of faropenem to that of the internal stan-
dard.
Amount [Mg (potency)] of faropenem (C I2 H 15 N0 5 S)
= W s x (Qj/Q s ) x 1000
W s : amount (mg) of Faropenem Sodium Reference Stan-
dard
Internal standard solution — Dissolve 0.5 g of m-hydroxya-
cetophenone in 20 mL of acetonitrile, and add water to make
200 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 305 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 4.8 g of potassium dihydrogen-
phosphate, 5.4 g of disodium hydrogen phosphate dodecahy-
drate and 1.0 g of tetra n-butyl ammonium bromide in water
to make 1000 mL. To 870 mL of this solution add 130 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
faropenem is about 11 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, the internal standard and faropenem are eluted in this
order with the resolution between these peaks being not less
than 1.5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of faropenem to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Faropenem Sodium for Syrup
Faropenem Sodium for Syrup is a preparation for
syrup, which is dissolved before use. It contains not
less than 93.0% and not more than 106.0% of the la-
beled amount of faropenem (C 12 H 15 N0 5 S: 285.32).
Method of preparation Prepare as directed under Syrups,
with Faropenem Sodium Hydrate.
Identification Dissolve an amount of pulverized Faropenem
Sodium for Syrup, equivalent to 25 mg (potency) of Faropen-
em Sodium Hydrate according to the labeled amount, in 50
mL of water. To 5 mL of this solution add water to make 50
mL, centrifuge, if necessary, and determine the absorption
spectrum of the solution so obtained as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 254 nm and 258 nm, and between 304 nm and
308 nm.
Water <2.48> Not less than 1.5% and not more than 2.1%
(80 mg, coulometric titration).
Uniformity of dosage units <6.02> Faropenem Sodium for
Syrup in single-unit container meets the requirement of the
Mass variation test.
Assay Powder, if necessary, and weigh accurately an
amount of Faropenem Sodium for Syrup, equivalent to
about 25 mg (potency) of faropenem (C 12 H 15 N0 5 S), add ex-
actly 10 mL of the internal standard solution and a suitable
amount of water, shake well, and add water to make 50 mL.
Filter, discard the first 10 mL of the filtrate, and use the sub-
sequent filtrate as the sample solution. Separately, weigh
accurately about 25 mg (potency) of Faropenem Sodium
Reference Standard, add exactly 10 mL of the internal stan-
dard solution and water to make 50 mL, and use this solution
as the standard solution. Preceed as directed in the Assay un-
der Faropenem Sodium Hydrate.
Amount [mg (potency)] of faropenem (C 12 H I5 N0 5 S)
= W s x (Q^/Qs)
W s : Amount [mg (potency)] of Faropenem Sodium Refer-
ence Standard
660
Faropenem Sodium Tablets / Official Monographs
JP XV
Internal standard solution — Dissolve 0.5 g of m-hydroxya-
cetophenone in 20 mL of acetonitrile, and add water to make
200 mL.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Faropenem Sodium Tablets
Faropenem Sodium Tablets contain not less than
94.0% and not more than 106.0% of the labeled
amount of faropenem (C 12 H 15 N0 5 S: 285.32).
Method of preparation Prepare as directed under Tablets,
with Faropenem Sodium Hydrate.
Identification To pulverized Faropenem Sodium Tablets,
equivalent to 70 mg (potency) of Faropenem Sodium Hy-
drate according to the labeled amount, add water to make
100 mL. To 5 mL of this solution add water to make 100 mL,
filter, if necessary, and determine the absorption spectrum of
this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 254 nm
and 258 nm and between 304 nm and 308 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Faropenem Sodium Tablets add 180 mL of
water, shake vigorously until the tablets are disintegrated,
and add water to make exactly FmL so that each mL con-
tains about 1 mg (potency) of Faropenem Sodium Hydrate.
Pipet 5 mL of this solution, add water to make exactly 100
mL, and filter. Discard the first 10 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Separately,
weigh accurately about 25 mg (potency) of Faropenem Sodi-
um Reference Standard, and dissolve in water to make ex-
actly 50 mL. Pipet 10 mL of this solution, add water to make
exactly 100 mL, and use this solution as the standard solu-
tion. Determine the absorbances, A T2 js, ^4t305, A t}54 , A S2 i5,
A SW5 and ^4 S 354> of the sample solution and standard solution
at 275 nm, 305 nm and 354 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and calculate A T and A s ,
using the following equations.
A T = A TW s ~ (49 XA T275 + 30 Xy4 T 3 54 )/79
^s=^s30 5 -(49x^ S275 + 30x^ S354 )/79
Amount [mg (potency)] of faropenem (C 12 H 15 N0 5 S)
= W s x(A 1 /A s )x(V/25)
W s : Amount [mg (potency)] of Faropenem Sodium Refer-
ence Standard
Assay Weigh accurately the mass of not less than 5
Faropenem Sodium Tablets, and powder. Weigh accurately a
portion of the powder, equivalent to about 25 mg (potency)
of faropenem (C^H^NC^S), add exactly 10 mL of the inter-
nal standard solution, shake well, and add water to make ex-
actly 50 mL. Filter, discard the first 10 mL of the filtrate, and
use the subsequent filtrate as the sample solution. Separately,
weigh accurately about 25 mg (potency) of Faropenem Sodi-
um Hydrate Reference Standard, add exactly 10 mL of the
internal standarad solution and water to make exactly 50 mL,
and use this solution as the standard solution. Proceed as
directed in the Assay under Faropenem Sodium Hydrate.
Amount [mg (potency)] of faropenem (C^H^NC^S)
= ^ s x(g T /g s )
W s : Amount [mg (potency)] of Faropenem Sodium Hy-
drate Reference Standard
Internal standard solution — Dissolve 0.5 g of m-hydorxya-
cetophenone in 20 mL of acetonitrile, and add water to make
200 mL.
Containers and storage Containers — Tight containers.
Fenbufen
7 i >7"7i
COjH
C 16 H 14 3 : 254.28
4-(Biphenyl-4-yl)-4-oxobutanoic acid [36330-85-5]
Fenbufen, when dried, contains not less than 98.0%
of C 16 H 14 3 .
Description Fenbufen occurs as a white crystalline powder.
It has a bitter taste.
It is sparingly soluble in acetone, slightly soluble in
methanol, in ethanol (95) and in diethyl ether, and practically
insoluble in water.
Melting point: about 188°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Fenbufen in ethanol (95) (1 in 200,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Fen-
bufen, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Purity (1) Heavy metals <1.07> — Take 2.0 g of Fenbufen,
add 2 mL of sulfuric acid, and carbonize by gentle heating,
proceed according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Fenbufen according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.1 g of Fenbufen in 20
mL of acetone, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add acetone to make ex-
actly 100 mL, and use this solution as the standard solution.
JPXV
Official Monographs / Ferrous Sulfate Hydrate
661
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of dichloromethane, methanol and
water (80:20:3) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.3% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Fenbufen, previous-
ly dried, dissolve in 100 mL of ethanol (99.5), and titrate
<2.50> with 0.1 mol/L potassium hydroxide-ethanol VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 25.43 mg of C 16 H 14 3
Containers and storage Containers — Tight containers.
Fentanyl Citrate
CH,
HO CO?H
C 22 H 28 N 2 O.C 6 H 8 7 : 528.59
A f -(l-Phenethylpiperidin-4-yl)-/V-phenylpropanamide
monocitrate [990-73-8]
Fentanyl Citrate contains not less than 98.0%
C 2 2H2 8 N 2 O.C 6 H g 07, calculated on the dried basis.
of
Description Fentanyl Citrate occurs as white crystals or
crystalline powder.
It is freely soluble in methanol and in acetic acid (100),
sparingly soluble in water and in ethanol (95), and very slight-
ly soluble in diethyl ether.
Identification (1) Dissolve 0.05 g of Fentanyl Citrate in 10
mL of 0.1 mol/L hydrochloric acid TS and ethanol (95) to
make 100 mL. Determine the absorption spectrum of the so-
lution as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Fen-
tanyl Citrate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Fentanyl Citrate (1 in 100) responds to
the Qualitative Tests <1.09> (1) for citrate.
pH <2.54> Dissolve 0.10 g of Fentanyl Citrate in 10 mL of
water: the pH of this solution is between 3.0 and 5.0.
Melting point <2.60> 150 - 154°C
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Fentanyl Citrate according to Method 2, and perform the
test. Prepare the control solution with 1.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.10 g of Fentanyl Ci-
trate in 5 mL of methanol, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-butanol, water and acetic acid (100) (3:1:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
Dragendorff's TS for spraying on the plate: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.2 g, in vacu-
um, silica gel, 60°C, 2 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 0.075 g of Fentanyl Citrate,
dissolve in 50 mL of acetic acid (100), and titrate <2.50> with
0.02 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.02 mol/L perchloric acid VS
= 10.57 mg of C 22 H 28 N 2 .C 6 H 8 7
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ferrous Sulfate Hydrate
BSKiMcWl*i
FeS0 4 .7H 2 0: 278.01
Ferrous Sulfate Hydrate contains not less than 98.0
% and not more than 104.0%, of FeS0 4 .7H 2 0.
Description Ferrous Sulfate Hydrate occurs as pale green
crystals or crystalline powder. It is odorless, and has an as-
tringent taste.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It is efflorescent in dry air, and its surface becomes yellow-
ish brown in moist air.
Identification A solution of Ferrous Sulfate Hydrate (1 in
10) responds to the Qualitative Tests <1.09> for ferrous salt
and for sulfate.
Purity (1) Clarity of solution — Dissolve 1.0 g of Ferrous
Sulfate Hydrate in 20 mL of water and 1 mL of dilute sulfur-
ic acid: the solution is clear.
662
Flavin Adenine Dinucleotide Sodium / Official Monographs
JP XV
(2) Acidity — To 5.0 g of powdered Ferrous Sulfate Hy-
drate add 50 mL of ethanol (95), shake well for 2 minutes,
and filter the mixture. To 25 mL of the filtrate add 50 mL of
water, 3 drops of bromothymol blue TS and 0.5 mL of dilute
sodium hydroxide TS: a blue color develops.
(3) Heavy metals <1.07> — Take 1.0 g of Ferrous Sulfate
Hydrate in a porcelain dish, add 3 mL of aqua regia, and dis-
solve. Then evaporate on a water bath to dryness. To the
residue add 5 mL of 6 mol/L hydrochloric acid TS, and dis-
solve. Transfer this solution to a separator. Wash the por-
celain dish with two 5-mL portions of 6 mol/L hydrochloric
acid TS, and combine the washings and the solution in the
separator. Pour two 40-mL portions and one 20-mL portion
of diethyl ether in the separator, shaking each time to mix.
Allow to stand, and discard each separated diethyl ether lay-
er. To the aqueous layer add 0.05 g of hydroxylammonium
chloride, dissolve, and heat on a water bath for 10 minutes.
Cool, adjust the solution to a pH of 3 to 4 by dropping strong
ammonia solution, add water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution as follows: take 2.5 mL of Standard Lead
Solution in a porcelain dish, add 3 mL of aqua regia, and
proceed as directed for the preparation of the test solution
(not more than 25 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Ferrous Sulfate Hydrate according to Method 1, and per-
form the test (not more than 2 ppm).
Assay Dissolve about 0.7 g of Ferrous Sulfate Hydrate, ac-
curately weighed, in a mixture of 20 mL of water and 20 mL
of dilute sulfuric acid, add 2 mL of phosphoric acid, and im-
mediately titrate <2.50> with 0.02 mol/L potassium perman-
ganate VS.
Each mL of 0.02 mol/L potassium permanganate VS
= 27.80 mg of FeS0 4 .7H 2
Containers and storage Containers — Tight containers.
Flavin Adenine Dinucleotide
Sodium
NH;
M
N' M
O^ ,.0,. „cl
p p ^.
AS A |A
H OH
3Xrt
OH OH
C 27 H 3l N 9 Na 2 O l5 P 2 : 829.51
Disodium adenosine 5'-[(2/?,35',4S)-5-(7,8-dimethyl-
2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2//)-yl)-2,3,4-
trihydroxypentyl diphosphate] [84366-81-4]
Flavin Adenine Dinucleotide Sodium contains not
less than 93.0% of C^F^NsNajO^P;,, calculated on
the anhydrous basis.
Description Flavin Adenine Dinucleotide Sodium occurs as
an orange-yellow to light yellow-brown powder. It is odorless
or has a slight, characteristic odor, and has a slightly bitter
taste.
It is freely soluble in water, and practically insoluble, in
methanol, in ethanol (95), in ethyleneglycol and in diethyl
ether.
It is hygroscopic.
It is decomposed by light.
Identification (1) A solution of Flavin Adenine Dinucleo-
tide Sodium (1 in 100,000) is light yellow-green in color, and
shows a strong yellow-green fluorescence. To 5 mL of the so-
lution add 0.02 g of hydrosulfite sodium: the color and the
fluorescence of the solution disappear, and gradually reap-
pear when the solution is shaken in air. Add dilute
hydrochloric acid or sodium hydroxide TS dropwise: the
fluorescence of the solution disappears.
(2) Determine the infrared absorption spectrum of Flavin
Adenine Dinucleotide Sodium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) To 0.1 g of Flavin Adenine Dinucleotide Sodium add
10 mL of nitric acid, evaporate on a water bath to dryness,
and ignite. To the residue add 10 mL of diluted nitric acid (1
in 50), boil for 5 minutes, and after cooling, neutralize with
ammonia TS , then filter the solution if necessary: the solution
responds to the Qualitative Tests <1.09> for sodium salt and
the Qualitative Tests <1.09> (1) and (3) for phosphate.
Optical rotation <2.49> [a]?, : -21.0 - -25.5° (0.3 g, calcu-
lated on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Flavin Adenine Dinucleotide
Sodium in 100 mL of water: the pH of this solution is be-
tween 5.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Flavin Adenine Dinucleotide Sodium in 10 mL of water:
the solution is clear and orange-yellow in color.
(2) Free phosphoric acid — Weigh accurately about 0.02 g
of Flavin Adenine Dinucleotide Sodium, dissolve in 10 mL of
water, and use this solution as the sample solution. Separate-
ly, measure exactly 2 mL of Standard Phosphoric Acid Solu-
tion, add 10 mL of water, and use this solution as the stan-
dard solution. To each of the sample solution and standard
solution add 2 mL of diluted perchloric acid (100 in 117),
then add 1 mL of hexaammonium heptamolybdate TS and 2
mL of 2,4-diaminophenol hydrochloride TS, respectively,
shake, add water to make exactly 25 mL, and allow to stand
at 20 ± 1°C for 30 minutes. Perform the test with these solu-
tions as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using a solution prepared in the same manner with 2
mL of water, as the blank, and determine the absorbances, A
T and A s , of the subsequent solutions of the sample solution
and standard solution at 730 nm, respectively: the amount of
free phosphoric acid is less than 0.25%.
Amount (%) of free phosphoric acid (H 3 P0 4 )
= (At/ A s ) x (\/W) x 5.16
W: Amount (mg) of flavin adenine dinucleotide sodium,
JPXV
Official Monographs / Flavin Adenine Dinucleotide Sodium
663
calculated on the anhydrous basis.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Flavin
Adenine Dinucleotide Sodium according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Flavin Adenine Dinucleotide Sodium according to Method
3, and perform the test (not more than 1 ppm).
(5) Related substances — Dissolve 0.10 g of Flavin Ade-
nine Dinucleotide Sodium in 200 mL of the mobile phase,
and use this solution as the sample solution. Perform the test
with 20 fiL of the sample solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine the peak area, A, of flavin adenine
dinucleotide and the total area, S, of peaks other than the
peak of flavin adenine dinucleotide by the automatic integra-
tion method: S/(A +5) is not more than 0.10.
Operating conditions —
Column, column temperature, mobile phase, flow rate,
and time span of measurement: Proceed as directed in the
operating conditions in the Procedure (ii) under Assay (1).
Detector: An ultraviolet absorption photometer
(wavelength: 260 nm).
System suitability —
Test for required detection: To exactly 2 mL of the sample
solution add the mobile phase to make exactly 20 mL, and
use this solution as the solution for system suitability test.
Confirm that the peak area of flavin adenine dinucleotide ob-
tained from 20 fiL of this solution is equivalent to 8 to 12%
of that of flavin adenine dinucleotide obtained from 20 fiL of
the sample solution.
System performance: Proceed as directed in the system
suitability in the Procedure (ii) under Assay (1).
System repeatability: When the test is repeated 6 times with
20 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of flavin adenine dinucleotide is not more
than 1.0%.
Water <2.48> Take 50 mL of a mixture of methanol for Karl
Fischer method and ethyleneglycol for Karl Fischer method
(1:1) into a dry titration flask, and titrate with Karl Fischer
TS until end point. Weigh accurately about 0.1 g of Flavin
Adenine Dinucleotide Sodium, transfer quickly to the titra-
tion flask, add an excess and constant volume of Karl Fischer
TS, dissolve by stirring for 10 minutes, and perform the test:
the water content is not more than 10.0%.
Assay (1) Procedure (i) Total flavin content — Conduct
this procedure without exposure to daylight, using light-
resistant vessels. Weigh accurately about 0.1 g of Flavin Ade-
nine Dinucleotide Sodium, and dissolve in water to make ex-
actly 200 mL. Pipet 5 mL of this solution, add 5 mL of zinc
chloride TS, and heat in a water bath for 30 minutes. After
cooling, add water to make exactly 100 mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 50 mg of Riboflavin Reference Standard, previously
dried at 105°C for 2 hours, dissolve in 200 mL of diluted a-
cetic acid (100) (1 in 100) by warming, cool, add water to
make exactly 500 mL. Pipet 10 mL of this solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and^4 s , of
the sample solution and standard solution at 450 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using water as the blank.
Total amout (mg) of flavin
= W s x (A T /A S ) x (4/5)
W s : Amount (mg) of Riboflavin Reference Standard
(ii) Peak area ratio of flavin adenine dinucleotide — Dis-
solve 0.1 g of Flavin Adenine Dinucleotide Sodium in 200 mL
of water, and use this solution as the sample solution. Per-
form the test with 5 fiL of this solution as directed under the
Liquid Chromatography <2.01> according to the following
conditions. Determine the peak area, A of flavin adenine
dinucleotide, and the total area, S, of the peaks other than
flavin adenine dinucleotide by the automatic integration
method.
Peak area ratio of flavin adenine dinucleotide
= 1.08/4/(1.08/1 + S)
Operating conditions —
Detector: A visible spectrophotometer (wavelength: 450
nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ftm in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of a solution of potassium di-
hydrogen phosphate (1 in 500) and methanol (4:1).
Flow rate: Adjust the flow rate so that the retention time of
flavin adenine dinucleotide is about 10 minutes.
Time span of measurement: About 4.5 times as long as the
retention time of flavin adenine dinucleotide.
System suitability —
Test for required detection: To exactly 2 mL of the sample
solution add water to make exactly 20 mL, and use this solu-
tion as the solution for system suitability test. Pipet 2 mL of
the solution, and add water to make exactly 20 mL. Confirm
that the peak area of flavin adenine dinucleotide obtained
from 5 fiL of this solution is equivalent to 8 to 1 2% of that of
flavin adenine dinucleotide obtained from 5 fiL of the
solution for system suitability test.
System performance: Dissolve 20 mg each of Flavin
Adenine Dinucleotide Sodium and riboflavin sodium phos-
phate in 100 mL of water. When the procedure is run with
5 fiL of this solution under the above operating conditions,
flavin adenine dinucleotide and riboflavin phosphate are elut-
ed in this order with the resolution between these peaks being
not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of flavin adenine dinucleotide is not more
than 1.0%.
(2) Calculation
Amount (mg) of C27H 31 N9Na20i5P2
=/ T x/rX 2.2040
/ T : Total amount (mg) of flavin in Flavin Adenine
Dinucleotide Sodium obtained from the procedure (i).
/ R : Peak area ratio of flavin adenine dinucleotide in Flavin
Adenine Dinucleotide Sodium obtained from the proce-
dure (ii).
664
Flavoxate Hydrochloride / Official Monographs
JP XV
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Flavoxate Hydrochloride
7=7**-y— r-£K£
>HCI
C 24 H 25 N04.HC1: 427.92
2-(Piperidin-l-yl)ethyl 3-methyl-4-oxo-2-phenyl-4//-
chromene-8-carboxylate monohydrochloride
[3717-88-2]
Flavoxate Hydrochloride, when dried, contains not
less than 99.0% of C 24 H 25 N0 4 .HC1.
Description Flavoxate Hydrochloride occurs as white crys-
tals or crystalline powder.
It is sparingly soluble in acetic acid (100) and in chlo-
roform, slightly soluble in water and in ethanol (95), and
practically insoluble in acetonitrile and in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Flavoxate Hydrochloride in 0.01 mol/L
hydrochloric acid TS (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Flavoxate Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Flavoxate Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Flavoxate Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Flavoxate Hydrochloride according to Method 4, and per-
form the test (not more than 1 ppm).
(3) Related substances — Dissolve 80 mg of Flavoxate
Hydrochloride in 10 mL of chloroform, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add chloroform to make exactly 20 mL, then pipet 1 mL of
this solution, add chloroform to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 fiL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of 1-butanol, water and acetic acid (100)
(3:1:1) to a distance of about 12 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 1.0% (1 g, reduced
pressure, silica gel, 2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Flavoxate
Hydrochloride, previously dried, add 10 mL of acetic acid
(100) and 40 mL of acetonitrile to dissolve, add 50 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 42.79 mg of C 24 H 25 N04.HC1
Containers and storage Containers — Tight containers.
Flomoxef Sodium
7 P^E =Hz7^ h U7A
..OH
COjNa N v\
VnA
^^r-k
Or
O H
CH,
C 15 H 17 F 2 N 6 Na0 7 S 2 : 518.45
Monosodium (6R,1R)-1-
{[(diifuoromethylsulfanyl)acetyl]amino}-
3-[ 1 -(2-hydroxyethyl)- l//-tetrazol-5-ylsulf anylmethyl] -
7-methoxy-8-oxo-5-oxa-l-azabicyclo[4.2.0]oct-2-ene-
2-carboxylate [92823-03-5]
Flomoxef Sodium contains not less than 870 fig
(potency) and not more than 985 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Flomoxef Sodium is expressed as mass (potency) of
flomoxef (C 15 H 18 F 2 N 6 7 S 2 : 496.47).
Description Flomoxef Sodium occurs as white to light yel-
lowish white, powder or masses.
It is very soluble in water, freely soluble in methanol, and
sparingly soluble in ethanol (99.5).
Identification (1) Decompose 0.01 g of Flomoxef Sodium
as directed under Oxygen Flask Combustion Method <1.06>,
using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide
TS and 20 mL of water as the absorbing liquid. To 2 mL of
the test solution so obtained add 1 .5 mL of a mixture of aliza-
rin complexone TS, acetic acid-potassium acetate buffer solu-
tion, pH 4.3 and cerium (III) nitrate TS (1:1:1): blue-purple
color develops.
(2) Determine the absorption spectrum of a solution of
Flomoxef Sodium (3 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Flomoxef Sodium as directed in the potassium bromide disk
JPXV
Official Monographs / Flomoxef Sodium
665
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(4) Determine the spectrum of a solution of Flomoxef So-
dium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a single signal A at around <5 3.5 ppm, a single signal
or a sharp multiple signal B at around <53.7 ppm, and a single
signal C at around 8 5. 2 ppm. The ratio of the integrated in-
tensity of these signals, A:B:C, is about 3:2:1.
(5) Flomoxef Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: -8 - -13° (1 g calculated on
the anhydrous basis, a mixture of water and ethanol (99.5)
(4:1), 50 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.5 g
of Flomoxef Sodium in 5 mL of water is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Flomoxef Sodium in 10 mL of water: the solution is clear and
pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Flomox-
ef Sodium in a quartz crucible according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — To 1.0 g of Flomoxef Sodium 5 mL
of sulfuric acid and 5 mL of nitric acid, heat carefully until
the solution changes to colorless to light yellow with oc-
casional addition of 2 mL of nitric acid. After cooling, add
10 mL of ammonium oxalate TS, heat until white fumes
evolve, and concentrate to 2 to 3 mL. After cooling, add
water to make 10 mL, and perform the test using this solution
as the test solution: the color is not darker than that of the
control solution.
Control solution: Proceed to prepare a solution in the same
manner as the test solution without Flomoxef Sodium, and
transfer 10 mL of the solution so obtained to the generator
bottle, add exactly 2 mL of Standard Arsenic Solution, and
proceed in the same manner as the test solution (not more
than 2 ppm).
(4) l-(2-Hydroxyethyl)-li/-tetrazol-5-thiol— Weigh ac-
curately about 20 mg of l-(2-hydroxyethyl)-l//-tetrazol-5-
thiol, and dissolve in water to make exactly 100 mL. Pipet
5 mL of this solution, add exactly 25 mL of the internal stan-
dard solution and water to make 50 mL, and use this solution
as the standard solution. Perform the test with 5 /xL each of
the sample solution obtained in the Assay and standard solu-
tion as directed under Liquid Chromatography <2.0J> ac-
cording to the following conditions, and determine the ratios,
Q T and Q s , of the peak area of l-(2-hydroxyethyl)-l//-
tetrazol-5-thiol to that of the internal standard: the amount
of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol is not more than
1.0% of the amount of Flomoxef Sodium calculated on the
anhydrous basis.
Amount (mg) of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol
(C 3 H 6 N 4 OS)
= W s x (<2 T /0s) x (1/10)
thiol
Internal standard solution — A solution of m-cresol (3 in
1000)
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
Proceed as directed in the system suitability in the Assay.
Water <2.48> Not more than 1.5% (0.5 g, volumetric titra-
tion, back titration).
Assay Weigh accurately an amount of Flomoxef Sodium
and Flomoxef Triethylammonium Reference Standard,
equivalent to about 50 mg (potency), and dissolve each in ex-
actly 50 mL of the internal standard solution, add water to
make 100 mL, and use these solutions as the sample solution
and standard solution. Perform the test with 5 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak area
of flomoxef to that of the internal standard.
Amount L"g (potency)] of flomoxef (C 15 H 18 F 2 N 6 07S2)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Flomoxef Triethylammo-
nium Reference Standard
Internal standard solution — A solution of m-cresol (3 in
1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 246 nm).
Column: A stainless steel column 4 mm in inside diameter
and 20 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 - 10,Mm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.94 g of potassium dihydrogen
phosphate, 3.22 g of disodium hydrogen phosphate dodeca-
hydrate and 1.60 g of tetra-M-butylammonium bromide in
water to make 1000 mL. To 750 mL of this solution add 250
mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
flomoxef is about 9 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, flomoxef and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 3 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of flomoxef to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage— At 5°C or below.
W s : Amount (mg) of l-(2-hydroxyethyl)-l//-tetrazol-5-
666
Flomoxef Sodium for Injection / Official Monographs
JP XV
Flomoxef Sodium for Injection
Flomoxef Sodium for Injection is a preparation for
injection which is dissolved before use.
It contains not less than 90.0% and not more
than 110.0% of the labeled amount of flomoxef
(C 15 H 18 F 2 N 6 7 S 2 : 496.47).
Method of preparation Prepare as directed under Injec-
tions, with Flomoxef Sodium.
Description Flomoxef Sodium for Injection occurs as white
to light yellowish white, friable masses or powder.
Identification Proceed as directed in the Identification (3)
under Flomoxef Sodium.
pH <2.54> The pH of a solution obtained by dissolving an
amount of Flomoxef Sodium for Injection, equivalent to 0.5
g (potency) of Flomoxef Sodium according to the labeled
amount, in 5 mL of water is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve an
amount of Flomoxef Sodium for Injection, equivalent to 1.0
g (potency) of Flomoxef Sodium according to the labeled
amount, in 10 mL of water: the solution is clear and colorless
or pale yellow.
(2) l-(2-Hydroxyethyl)-l//-tetrazol-5-thiol— Use the
sample solution obtained in the Assay as the sample solution.
Weigh accurately about 20 mg of l-(2-hydroxy ethyl)- IH-
tetrazol-5-thiol, and dissolve in water to make exactly 100
mL. Pipet 5 mL of this solution, add exactly 25 mL of the in-
ternal standard solution and water to make 50 mL, and use
this solution as the standard solution. Perform the test with 5
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Qj and
Qs, of the peak area of l-(2-hydroxyethyl)-l//-tetrazol-5-
thiol to that of the internal standard. Calculate the amount of
l-(2-hydroxyethyl)-l//-tetrazol-5-thiol per 1 g (potency) of
Flomoxef Sodium for Injection by the following formula:
not more than 10 mg.
Amount (mg) of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol
(C 3 H 6 N 4 OS)
= W s x (Q T /Q S ) x (1/10)
W s : Amount (mg) of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol
Operating conditions —
Proceed as directed in the Assay.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add water to make exactly 20 mL. Confirm that
the peak area of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol ob-
tained from 5 nL of this solution is equivalent to 3.5 - 6.5%
of that obtained from 5 /uL of the standard solution.
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, l-(2-hydroxyethyl)-l//-tetrazol-5-thiol and the internal
standard are eluted in this order with the resolution between
these peaks being not less than 20.
System repeatability: When the test is repeated 3 times with
5 ,uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of l-(2-hydroxyethyl)-l//-tetrazol-5-thiol to that of
the internal standard is not more than 1.0%.
Water <2.48> Not more than 1.5% (0.5 g, volumetric titra-
tion, back titration).
Bacterial endotoxins <4.01> Less than 0.025 EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Flomoxef Sodium for Injection, and calculate the
average mass of the content. Spread out thinly about 1 g of
the content in a petri dish, allow the dish to stand in a desic-
cator containing a saturated solution of magnesium bromide
without light exposure to equilibrate the sample to constant
water content. Determine the water content, separately, with
about 0.1 g of the sample according to the method described
in Water. Weigh accurately an amount of the sample, equiva-
lent to about 50 mg (potency) of Flomoxef Sodium according
to the labeled amount, add exactly 50 mL of the internal stan-
dard solution to dissolve, add water to make 100 mL, and use
this solution as the sample solution. Separately weigh ac-
curately about 50 mg (potency) of Flomoxef Triethylammo-
nium Reference Standard, add exactly 50 mL of the internal
standard solution to dissolve, add water to make 100 mL,
and use this solution as the standard solution. Proceed as
directed in the Assay under Flomoxef Sodium.
Amount L"g (potency)] of flomoxef (C 15 H 18 F 2 N 6 07S2)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Flomoxef Triethylammoni-
um Reference Standard
Internal standard solution — A solution of m-cresol (3 in
1000).
Containers and storage Containers — Hermetic containers.
Polyethylene or polypropylene containers for aqueous
injection may be used.
JPXV
Official Monographs / Flopropione Capsules
667
Flopropione
H 3 C
noAA
C 9 H 10 O 4 : 182.17
1 -(2,4,6-Trihydroxyphenyl)propan- 1 -one [2295-58-1 ]
Flopropione contains not less than 98.0%
C 9 H 10 O 4 , calculated on the anhydrous basis.
of
Description Flopropione occurs as a white to pale yellow-
brown, crystalline powder. It is odorless, and has a bitter
taste.
It is very soluble in AfAf-dimethylformamide, freely solu-
ble in methanol, in ethanol (99.5) and in diethyl ether, and
practically insoluble in water.
Identification (1) To 1 mL of a solution of Flopropione in
ethanol (99.5) (1 in 200) add 4 mL of water and 1 mL of iron
(III) nitrate TS: a red-purple color develops.
(2) Determine the absorption spectrum of a solution of
Flopropione in ethanol (99.5) (1 in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same wave-
lengths.
Melting point <2. 60> 1 77 - 1 8 1 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Flopropione in 10 mL of ethanol (99.5): the solution is clear,
and has no more color than Matching Fluid H.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Flopropione according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Flopropione according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Flopropione
in 10 mL of ethanol (99.5), and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add ethanol
(99.5) to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of hexane, ethanol (99.5) and acetic acid
(100) (40:20:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly />-nitrobenzenediazonium TS for spraying
on the plate, and dry in cold wind for about 5 minutes: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Water <2.48> Not more than 4.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Flopropione, dis-
solve in 30 mL of 7V,./V-dimethylformamide, and titrate <2.50>
with 0.1 mol/L tetramethylammonium hydroxide VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 18.22 mg of C 9 H 10 O 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Flopropione Capsules
Flopropione Capsules contain not less than 93.0%
and not more than 107.0% of the labeled amount of
flopropione (C 9 H 10 O 4 : 182.17).
Method of preparation Prepare as directed under the
Capsules, with Flopropione.
Identification (1) Powder the contents of Flopropione
Capsules. To a portion of the powder, equivalent to 60 mg of
Flopropione according to the labeled amount, add 40 mL of
water, shake well, and filter. To 5 mL of the filtrate add 1 mL
of iron (III) nitrate TS: a red-purple color appears.
(2) Powder the contents of Flopropione Capsules. To a
portion of the powder, equivalent to 90 mg of Flopropione
according to the labeled amount, add 100 mL of ethanol
(99.5), shake well, and filter. To 5 mL of the filtrate add
ethanol (99.5) to make 50 mL. To 5 mL of this solution add
ethanol (99.5) to make 100 mL, and use this solution as the
sample solution. Determine the absorption spectrum of the
sample solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 283
nm and 287 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 capsule of Flopropione Capsules add 43 mL of a mix-
ture of water and phosphoric acid (86:1), and disintegrate the
capsule in a water bath at 50°C. After cooling, add a suitable
amount of acetonitrile to make exactly FmL of a solution
containing about 0.4 mg of flopropione (C 9 H 10 O 4 ) per mL.
Stir the solution for 10 minutes, centrifuge a part of the solu-
tion at 3000 rpm for 5 minutes, and use the supernatant liq-
uid as the sample solution. Proceed as directed in the Assay.
Amount (mg) of flopropione (C 9 H 10 O 4 )
= W s x(A T /A s )x(V/l00)
W s : Amount (mg) of flopropione for assay, calculated on
the anhydrous basis
Assay Take out the contents of not less than 20
Flopropione Capsules, weigh accurately the mass of the con-
tents, and power. Weigh accurately a part of the powder,
equivalent to about 40 mg of flopropione (C 9 H 10 O 4 ), and add
the mobile phase to make exactly 100 mL. Stir the solution
for 10 minutes, centrifuge a part of this solution for 5
minutes at 3000 rpm, and use the supernatant liquid as the
sample solution. Separately, weigh accurately about 40 mg of
668
Flucytosine / Official Monographs
JP XV
flopropione for assay (previously determine the water <2.48>
in the same manner as Flopropione), add 70 mL of the mo-
bile phase, and dissolve by exposure for 10 minutes to ultra-
sonic vibration. Add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 5 /uL each of the sample solution and
standard solution as directed under Liquid chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of flopropione.
Amount (mg) of flopropione (C 9 H 10 O 4 ) = W s x (A T /A S )
fV s : Amount (mg) of flopropione for assay, calculated on
the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 267 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of acetonitrile, water and phos-
phoric acid (114:86:1)
Flow rate: Adjust the flow rate so that the retention time of
flopropione is about 3 minutes.
System suitability —
System performance: Dissolve 50 mg of flopropione in 50
mL of the mobile phase. To 20 mL of the solution add 25 mL
of a solution prepared by dissolving 25 mg of ethyl para-
hydroxybenzoate in 30 mL of acetonitrile and add water to
make 50 mL, and then add the mobile phase to make 50 mL.
When the procedure is run with 5 /uL of this solution under
the above operating conditions, Flopropione and ethyl para-
hydroxybenzoate are eluted in this order with the resolution
between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
flopropione is not more than 1.0%.
Containers and storage Containers — Tight containers.
Flucytosine
7JUv r-V>
H
NH,
C4H4FN3O: 129.09
5-Fluorocytosine [2022-85-7]
Flucytosine, when dried, contains not less than
98.5% of C4H4FN3O, and not less than 14.0% and not
more than 15.5% of fluorine (F: 19.00).
Description Flucytosine occurs as a white, crystalline pow-
der.
It is odorless.
It is sparingly soluble in water, slightly soluble in
methanol, in ethanol (95), in acetic anhydride and in acetic
acid (100), and practically insoluble in diethyl ether.
It dissolves in 0.1 mol/L hydrochloric acid TS.
The pH of a solution of Flucytosine (1 in 100) is between
5.5 and 7.5.
It is slightly hygroscopic.
Melting point: about 295°C (with decomposition).
Identification (1) Add 0.2 mL of bromine TS to 5 mL of a
solution of Flucytosine (1 in 500): a yellow-brown color of
bromine TS is immediately discharged. Further add 2 mL of
barium hydroxide TS: a purple precipitate is formed.
(2) Proceed with 0.1 g of Flucytosine as directed under
Oxygen Flask Combustion Method <1.06>, using a mixture of
0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of
water as the absorbing liquid. The solution responds to the
Qualitative Tests <I.09> (2) for fluoride.
(3) Determine the absorption spectrum of a solution of
Flucytosine in 0.1 mol/L hydrochloric acid TS (1 in 125,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Flucytosine in 100 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Dissolve 1.0 g of Flucytosine in 80
mL of water by heating on a water bath. After cooling, to 40
mL of this solution add 6 mL of dilute nitric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 0.20 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.014%).
(3) Fluoride — Dissolve 0.10 g of Flucytosine in 10.0 mL
of diluted 0.01 mol/L sodium hydroxide TS (1 in 20). Trans-
fer 5.0 mL of this solution to a 20-mL volumetric flask, add
10 mL of a mixture of alizarin complexone TS, acetic acid-
potassium acetate buffer solution, pH 4.3, and cerrous nitrate
TS (1:1:1), and add water to make 20 mL. Allow the mixture
to stand for 1 hour, and use this solution as the sample solu-
tion. Separately, transfer 4.0 mL of Standard Fluorine Solu-
tion to a 20-mL volumetric flask, add 5.0 mL of diluted 0.01
mol/L sodium hydroxide TS (1 in 20), add 10 mL of a mix-
ture of alizarin complexone TS, acetic acid-potassium acetate
buffer solution, pH 4.3, and cerrous nitrate TS (1:1:1). Pro-
ceed in the same manner as directed in the preparation of the
sample solution, and use this solution as the standard solu-
tion. Transfer 5.0 mL of diluted 0.01 mol/L sodium
hydroxide TS (1 in 20) to a 20-mL volumetric flask, proceed
in the same manner as directed in the preparation of the stan-
dard solution, and use this solution as the blank solution. De-
termine the absorbances, A T and A s , of the sample solution
and standard solution at 600 nm, using the blank solution as
the control as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: A T is not larger than A s (not more
than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Flucyto-
sine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Flucytosine according to Method 2, and perform the test
(not more than 2 ppm).
JPXV
Official Monographs / Fludiazepam
669
(6) Related substances — Dissolve 50 mg of Flucytosine in
5 mL of diluted methanol (1 in 2), and use this solution as the
sample solution. Measure accurately 1 mL of this solution,
add diluted methanol (1 in 2) to make exactly 25 mL. Meas-
ure accurately 1 mL of this solution, add diluted methanol (1
in 2) to make exactly 20 mL, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 20 /xL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the chromatogram with a mixture of
ethyl acetate, methanol and water (5:3:2) to a distance of
about 12 cm, air-dry the plate, and observe the spots under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.4l> Not more than 1.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Flucytosine — Weigh accurately about 0.2 g of
Flucytosine, previously dried, dissolve in 40 mL of acetic acid
(100), add 100 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 12.91 mg of C4H4FN3O
(2) Fluorine — Weigh accurately about 0.01 g of Flucyto-
sine, previously dried, and proceed as directed in the determi-
nation of fluorine under Oxygen Flask Combustion Method
<1.06>, using a mixture of 0.5 mL of 0.01 mol/L sodium
hydroxide VS and 20 mL of water as the absorbing liquid.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Fludiazepam
7;u-x7-£a°a
C 16 H 12 C1FN 2 0: 302.73
7-Chloro-5-(2-fluorophenyl)- 1 -methyl- 1 , 3-dihydro-2//- 1 ,4-
benzodiazepin-2-one [3900-31-0]
Fludiazepam, when dried, contains not less than
99.0% of C 16 H 12 C1FN 2 0.
Description Fludiazepam occurs as white to light yellow
crystals or crystalline powder.
It is very soluble in chloroform, freely soluble in methanol,
in ethanol (95), in acetic acid (100) and in diethyl ether, and
practically insoluble in water.
Identification (1) Prepare the test solution with 0.01 g of
Fludiazepam as directed under Oxygen Flask Combustion
Method <1.06>, using a mixture of 0.5 mL of 0.01 mol/L so-
dium hydroxide TS and 20 mL of water as the absorbing liq-
uid: the test solution responds to the Qualitative Tests <1.09>
(2) for fluoride.
(2) Determine the absorption spectrum of a solution of
Fludiazepam in methanol (1 in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum 1 : both spectra ex-
hibit similar intensities of absorption at the same wave-
lengths. Separately, determine the absorption spectrum of a
solution of Fludiazepam in methanol (1 in 20,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum 2: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Fludiazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) Perform the test with Fludiazepam as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 91 - 94°C
Purity (1) Chloride <1.03>— Dissolve 1.0 g of Fludiazep-
am in 50 mL of diethyl ether, add 50 mL of water, and shake.
Separate the water layer, wash it with two 20-mL portions of
diethyl ether, and filter the water layer. To 20 mL of the
filtrate add 6 mL of dilute nitric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 0.40 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Fludia-
zepam according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Related substances — Dissolve 0.10 g of Fludiazepam
in 20 mL of chloroform, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, and add chlo-
roform to make exactly 50 mL. Pipet 2 mL of this solution,
add chloroform to make exactly 20 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and ethyl acetate (10:7) to a distance of about 12 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, in vacu-
um, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.5 g of Fludiazepam, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
670
Flu nitrazepam / Official Monographs
JP XV
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.27 mg of C 16 H 12 C1FN 2
Containers and storage Containers — Tight containers.
Flunitrazepam
313.28
5-(2-Fluorophenyl)-l-methyl-7-nitro-l,3-dihydro-2.H-l,4-
benzodiazepin-2-one [1622-62-4]
Flunitrazepam, when dried, contains not less than
99.0% of C 16 H 12 FN 3 3 .
Description Flunitrazepam occurs as a white to pale yellow
crystalline powder.
It is freely soluble in acetic acid (100), soluble in acetic
anhydride and in acetone, slightly soluble in ethanol (99.5)
and in diethyl ether, and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Flunitrazepam in ethanol (99.5) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Flunitrazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 168 - 172°C
Purity (1) Chloride <1.03>— To 1.0 g of Flunitrazepam
add 50 mL of water, allow to stand for 1 hour with oc-
casional stirring, and filter. To 20 mL of the filtrate add 6 mL
of dilute nitric acid and water to make 50 mL, and perform
the test with this solution. Prepare the control solution with
0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.022%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Flunitrazepam according to Method 4 using a platinum cruci-
ble, and perform the test. Prepare the control solution with
2.0 mL of Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 50 mg of Flunitrazep-
am in 10 mL of acetone, and use this solution as the sample
solution. Pipet 2 mL of the sample solution, and add acetone
to make exactly 20 mL. Pipet 1 mL of this solution, add
acetone to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 1,2-
dichloroethane, diethyl ether and ammonia solution (28)
(200:100:3) to a distance of about 12 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): number of the spots other than the principal spot from
the sample solution is not more than 2, and they are not more
intense than the spot from the standard solution.
Loss on drying <2.41>
4 hours).
Not more than 0.5% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g,
platinum crucible).
Assay Weigh accurately about 0.5 g of Flunitrazepam,
previously dried, dissolve in 20 mL of acetic acid (100), add
50 mL of acetic anhydride, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.33 mg of C I6 H 12 FN 3 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Fluocinolone Acetonide
7;U7t-->/ n>7-te h-K
H F
C 24 H 30 F 2 O 6 : 452.49
6a,9-Difluoro-ll/?,21-dihydroxy-16a,17-
(1 -methylethylidenedioxy)pregna-l ,4-diene-3 ,20-dione
[67-73-2]
Fluocinolone Acetonide, when dried, contains not
less than 97.0% and not more than 102.0% of
C24H 3 oF 2 6 .
Description Fluocinolone Acetonide occurs as white crys-
tals or crystalline powder. It is odorless.
It is freely soluble in acetic acid (100) and in acetone,
soluble in ethanol (95) and ethanol (99.5), sparingly soluble
in methanol and in chloroform, slightly soluble in acetoni-
trile, very slightly soluble in diethyl ether, and practically
insoluble in water.
Melting point: 266 - 274°C (with decomposition).
Identification (1) To 2 mg of Fluocinolone Acetonide add
2 mL of sulfuric acid: a yellow color is produced.
(2) Dissolve 0.01 g of Fluocinolone Acetonide in 1 mL of
methanol, add 1 mL of Fehling's TS, and heat: a red
precipitate is produced.
(3) Proceed as directed under Oxygen Flask Combustion
Method <1.06> with 0.01 g of Fluocinolone Acetonide, using
JP XV
Official Monographs / Fluocinolone Acetonide 671
a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and
20 mL of water as the absorbing liquid. When the process is
completed, shake well, and force the combustion gas into the
absorbing liquid: this liquid responds to the Qualitative Tests
<l.09> for fluoride.
(4) Determine the infrared absorption spectrum of
Fluocinolone Acetonide, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of previously dried
Fluocinolone Acetonide Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers. If any difference appears between the spectra,
dissolve Fluocinolone Acetonide and Fluocinolone Acetonide
Reference Standard in acetone, respectively, then evaporate
the acetone to dryness, and repeat the test on the residues.
Optical rotation <2.49> [a]™: +9S
0.1 g, methanol, 10 mL, 100 mm).
108° (after drying,
Purity Related substances — Dissolve 15 mg of Fluocino-
lone Acetonide in 25 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 2 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of each solution by the automatic integration method:
the total area of the peaks other than the peak of fluocinolone
acetonide from the sample solution is not larger than the
peak area of fluocinolone acetonide from the standard solu-
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with silica gel (5 /xm in
particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water-saturated chloroform,
methanol and acetic acid (100) (200:3:2).
Flow rate: Adjust the flow rate so that the retention time of
fluocinolone acetonide is about 12 minutes.
Time span of measurement: About twice as long as the
retention time of fluocinolone acetonide beginning after the
solvent peak.
System suitability —
Test for required detection: To exactly 5 mL of the
standard solution add the mobile phase to make exactly 100
mL. Confirm that the peak area of fluocinolone acetonide
obtained from 20 /xh of this solution is equivalent to 4 to 6%
of that of fluocinolone acetonide obtained from 20 fiL of the
standard solution.
System performance: Dissolve 15 mg each of Fluocinolone
Acetonide and triamcinolone acetonide in 25 mL of the mo-
bile phase. To 5 mL of this solution add the mobile phase to
make 20 mL. When the procedure is run with 20,mL of this
solution under the above operating conditions, triamcinolone
acetonide and fluocinolone acetonide are eluted in this order
with the resolution between these peaks being not less than
1.9.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of fluocinolone acetonide is not more than 1.0%.
Loss on drying <2.41> Not more than 1.0% (0.2 g, in vacu-
um, 105°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.2 g,
platinum crucible).
Assay Dissolve about 20 mg each of Fluocinolone
Acetonide and Fluocinolone Acetonide Reference Standard,
previously dried and accurately weighed, in 40 mL each of
methanol, add exactly 10 mL each of the internal standard
solution, then add water to make 100 mL, and use these solu-
tions as the sample solution and standard solution. Perform
the test with 20 juL each of these solutions as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Qt and g s , of the peak
area of fluocinolone acetonide to that of the internal stan-
dard, respectively.
Amount (mg) of fluocinolone acetonide (C24H 3 oF 2 6 )
= W s x (Qj/Qs)
W s : Amount (mg) of Fluocinolone Acetonide Reference
Standard
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel (5 /um in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
fluocinolone acetonide is about 20 minutes.
System suitability —
System performance: Dissolve 5 mg each of isopropyl
parahydroxybenzoate and propyl parahydroxybenzoate in 50
mL of acetonitrile, and add water to make 100 mL. When the
procedure is run with 20 /xh of this solution under the above
operating conditions, isopropyl parahydroxybenzoate and
propyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 1.9.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of fluocinolone acetonide to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
672 Fluocinonide / Official Monographs
JP XV
Fluocinonide
7ju^-->/- k
CH 3
0^
H H3C
J CH 3
r.°-/-cH 3
HiC T H 1
\--0
■/ H
C 26 H 32 F 2 7 : 494.52
6a,9-Difluoro-ll/?,21-dihydroxy-16a,17-
( 1 -methylethylidenedioxy)pregna-l ,4-diene-3 ,20-dione 21-
acetate [356-12-7]
Fluocinonide, when dried, contains not less than
97.0% and not more than 103.0% of C 26 H 32 F 2 7 .
Description Fluocinonide occurs as white crystals or crys-
talline powder.
It is sparingly soluble in chloroform, slightly soluble in
acetonitrile, in methanol, in ethanol (95) and in ethyl acetate,
very slightly soluble in diethyl ether, and practically insoluble
in water.
Identification (1) To 0.01 g of Fluocinonide add 4 mL of
water and 1 mL of Fehling's TS, and heat: a red precipitate is
formed.
(2) Prepare the test solution with 0.01 g of Fluocinonide
as directed under Oxygen Flask Combustion Method <1.06>,
using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide
TS and 20 mL of water as an absorbing liquid: the test solu-
tion responds to the Qualitative Tests <1.09> for fluoride.
(3) Determine the absorption spectrum of a solution of
Fluocinonide in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Fluocinonide Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectra of
Fluocinonide and Fluocinonide Reference Standard, previ-
ously dried, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare both spectra: both the sample and the Reference Stan-
dard exhibit similar intensities of absorption at the same
wave numbers. If any difference appears in the absorption
spectra, dissolve the sample and the Reference Standard in
ethyl acetate, respectively, evaporate the ethyl acetate, and
perform the test with the residue in the same manner.
Optical rotation <2.49> [a]™: + 81 - + 89° (after drying, 0.2
g, chloroform, 20 mL, 100 mm).
Purity Related substances — Dissolve 10 mg of
Fluocinonide in 2 mL of chloroform, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
chloroform to make exactly 100, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
11L each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform and methanol (97:3) to a
distance of about 12 cm, and air-dry the plate. Spray evenly
alkaline blue tetrazolium TS on the plate: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 1.0% (0.5 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (0.5 g,
platinum crucible).
Assay Weigh accurately about 20 mg of Fluocinonide and
Fluocinonide Reference Standard, previously dried, dissolve
each in 50 mL of acetonitrile, to each add exactly 8 mL of the
internal standard solution and water to make 100 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 20 tiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of fluocinonide to that of the internal standard, respectively.
Amount (mg) of fluocinonide (C 26 H 32 F 2 7 )
= W s x (Q T /Q S )
W s : Amount (mg) of Fluocinonide Reference Standard
Internal standard solution — A solution of propyl benzoate in
acetonitrile (1 in 100).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of acetonitrile and water (1:1).
Flow rate: Adjust the flow rate so that the retention time of
fluocinonide is about 8 minutes.
System suitability —
System performance: When the procedure is run with 20
11L of the standard solution under the above operating condi-
tions, fluocinonide and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of fluocinonide to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Fluorescein Sodium 673
Fluorescein Sodium
NaO„ j*^ _d
C 2 „H 10 Na 2 O 5 : 376.27
Disodium 2-(6-oxido-3-oxo-3//-xanthen-9-yl)benzoate
[518-47-8]
Fluorescein Sodium contains not less than 98.5% of
C 2 oH 10 Na 2 05, calculated on the dried basis.
Description Fluorescein Sodium occurs as an orange pow-
der. It is odorless, and tasteless.
It is freely soluble in water, in methanol and in ethanol
(95), and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) To a solution of Fluorescein Sodium (1
in 100) having a strong green fluorescence, add a large quanti-
ty of water: the fluorescence remains. Acidify the solution
with hydrochloric acid: the fluorescence disappears. Then
render the solution alkaline with sodium hydroxide TS: the
fluorescence reappears.
(2) Place 1 drop of a solution of Fluorescein Sodium (1 in
2000) on a piece of filter paper: a yellow spot develops. Ex-
pose the spot, while moist, to the vapor of bromine for 1
minute and then to ammonia vapor: the yellow color of the
spot changes to red.
(3) Char 0.5 g of Fluorescein Sodium by ignition, cool,
mix the residue with 20 mL of water, and filter: the filtrate
responds to the Qualitative Tests <1.09> for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 g of
Fluorescein Sodium in 10 mL of water: the solution is clear,
and shows a red color.
(2) Chloride <1.03> — Dissolve 0.15 g of Fluorescein Sodi-
um in 20 mL of water, add 6 mL of dilute nitric acid and
water to make 30 mL, and filter. To 20 mL of the filtrate add
2 mL of dilute nitric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 1.0 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.355%).
(3) Sulfate <1.14>— Dissolve 0.20 g of Fluorescein Sodi-
um in 30 mL of water, add 2.5 mL of dilute hydrochloric acid
and water to make 40 mL, and filter. To 20 mL of the filtrate
add water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with 1 .0
mL of 0.005 mol/L sulfuric acid VS (not more than 0.480%).
(4) Zinc — Dissolve 0.10 g of Fluorescein Sodium in 10
mL of water, add 2 mL of hydrochloric acid, and filter. To
the filtrate add 0.1 mL of potassium hexacyanoferrate (II)
TS: no turbidity is produced immediately.
(5) Related substances — Dissolve 0.20 g of Fluorescein
Sodium in 10 mL of methanol, and use this solution as the
sample solution. Perform the test with this solution as direct-
ed under Thin-layer Chromatography <2.03>. Spot 5 juL of
the sample solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chlo-
roform, methanol and ammonia solution (28) (30:15:1) to a
distance of about 10 cm, and air-dry the plate: any colored
spot other than the principal spot does not appear.
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C,
constant mass).
Assay Transfer about 0.5 g of Fluorescein Sodium, ac-
curately weighed, to a separator. Dissolve in 20 mL of water,
add 5 mL of dilute hydrochloric acid, and extract the solution
with four 20-mL portions of a mixture of 2-methyl-l-
propanol and chloroform (1:1). Wash each extract succes-
sively with the same 10 mL of water. Evaporate the combined
extracts on a water bath with the aid of a current of air.
Dissolve the residue in 10 mL of ethanol (99.5), evaporate the
solution on a water bath to dryness, dry the residue at 105 °C
for 1 hour, and weigh as fluorescein (C 20 H 12 O 5 : 332.31).
Amount (mg) of C 20 H 10 Na 2 O 5
= amount (mg) of fluorescein (C 20 H 12 O 5 ) x 1.1323
Containers and storage Containers — Tight containers.
Fluorometholone
7;u7t-ny hn>
O. XH 3
H CH 3
C 22 H 29 F0 4 : 376.46
9-Fluoro- 11/6,1 7-dihydroxy-6a-methylpregna-l,4-diene-3, 20-
dione [426-13-1 ]
Fluorometholone, when dried, contains not less than
97.0% and not more than 103.0% of C 22 H 29 F0 4 .
Description Fluorometholone occurs as a white to light
yellowish white, odorless, crystalline powder.
It is freely soluble in pyridine, slightly soluble in methanol,
in ethanol (99.5) and in tetrahydrofuran, and practically in-
soluble in water and in diethyl ether.
Identification (1) Proceed with 7 mg of Fluorometholone
as directed under Oxygen Flask Combustion Method <1.06>,
using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide
TS and 20 mL of water as an absorbing liquid: the liquid
responds to the Qualitative Tests <1.09> (2) for fluoride.
(2) Determine the absorption spectrum of a solution of
Fluorometholone in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Fluorometholone Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the infrared absorption spectrum of
Fluorometholone, previously dried, as directed in the potassi-
674
Fluorouracil / Official Monographs
JP XV
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Fluorometholone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: +52- +60° (after drying,
0.1 g, pyridine, 10 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Fluorometholone according to Method 3, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 20 mg of Fluorometho-
lone in 10 mL of tetrahydrofuran, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
tetrahydrofuran to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with these
solutions as directed under Thin-layer chromatography
<2.03>. Spot 25 nL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of dichloromethane, acetone and methanol (45:5:1) to a dis-
tance of about 12 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (0.2 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2 % (0.2 g,
platinum crucible).
Assay Weigh accurately about 0.1 g each of Fluorometho-
lone and Fluorometholone Reference Standard, previously
dried, and dissolve each in methanol to make exactly 100 mL.
Pipet 5 mL each of these solutions, and add diluted methanol
(7 in 10) to make exactly 50 mL. Pipet 10 mL each of these
solutions, add exactly 10 mL of the internal standard solu-
tion and diluted methanol (7 in 10) to make 100 mL, and use
these solutions as the sample solution and standard solution,
respectively. Perform the test with 20 /xh each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions and determine the ratios, Q T and Q s , of the peak area of
fluorometholone to that of the internal standard.
Amount (mg) of fluorometholone (C22H29FO4)
= W s x (Q T /Q S )
W s : Amount (mg) of Fluorometholone Reference Stan-
dard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 25 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Diluted methanol (7 in 10).
Flow rate: Adjust the flow rate so that the retention time of
fluorometholone is about 8 minutes.
Selection of column: Proceed with 20 [iL of the standard
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of fluorometho-
lone and the internal standard in this order with the resolu-
tion between these peaks being not less than 4.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Fluorouracil
H
V
NH
C 4 H 3 FN 2 2 : 130.08
5-Fluorouracil [51-21-8]
Fluorouracil, when dried, contains not less than
98.5% of C 4 H 3 FN 2 2 , and not less than 13.1% and not
more than 16.1% of fluorine (F: 19.00).
Description Fluorouracil occurs as white crystals or crystal-
line powder. It is odorless.
It is freely soluble in A^A^-dimethylformamide, sparingly
soluble in water, slightly soluble in ethanol (95), and practi-
cally insoluble in diethyl ether.
Melting point: about 282°C (with decomposition).
Identification (1) Add 0.2 mL of bromine TS to 5 mL of a
solution of Fluorouracil (1 in 500): the color of bromine TS is
discharged. Further add 2 mL of barium hydroxide TS: a
purple precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Fluorouracil in 0.1 mol/L hydrochloric acid TS (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Proceed with 0.01 g of Fluorouracil as directed under
Oxygen Flask Combustion Method <1.06>, using a mixture of
0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of
water as the absorbing liquid. After combustion is complet-
ed, shake well to absorb the gas evolved: the solution
responds to the Qualitative Tests <1.09> for fluoride.
Purity (1) Clarity and color of solution — Add 20 mL of
water to 0.20 g of Fluorouracil, and dissolve by warming: the
solution is clear and colorless.
(2) Fluoride — Dissolve 0.10 g of Fluorouracil in 10.0 mL
of diluted 0.01 mol/L sodium hydroxide TS (1 in 20). Trans-
fer 5.0 mL of this solution to a 20-mL volumetric flask, add
10 mL of a mixture of alizarin complexone TS, acetic acid-
potassium acetate buffer solution, pH4.3, and cerium (III)
nitrate TS (1:1:1), and add water to make 20 mL. Allow to
stand for 1 hour, and use this solution as the sample solution.
Separately, transfer 1 .0 mL of Standard Fluorine Solution to
a 20-mL volumetric flask, add 5.0 mL of diluted 0.01 mol/L
sodium hydroxide TS (1 in 20), and add 10 mL of a mixture
JPXV
Official Monographs / Fluoxymesterone 675
of alizarin complexone TS, acetic acid-potassium acetate
buffer solution, pH4.3, and cerium (III) nitrate TS (1:1:1).
Proceed in the same manner as directed for the preparation
of the sample solution, and use this solution as the standard
solution. Perform the test as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, using a solution, prepared
with 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in
20) in the same manner, as the blank: the absorbance of the
sample solution at 600 nm is not larger than that of the stan-
dard solution (not more than 0.012%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Fluorouracil according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — To 1.0 g of Fluorouracil in a crucible
add 10 mL of a solution of magnesium nitrate hexahydrate in
ethanol (95) (1 in 10), ignite the ethanol to burn, and inciner-
ate by strong heating at 750°C to 850°C. If a carbonized sub-
stance remains in this method, moisten with a small amount
of nitric acid, and incinerate by strong heating. Cool, add 10
mL of dilute hydrochloric acid to the residue, dissolve it by
warming on a water bath, use this solution as the test solu-
tion, and perform the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Fluorouracil
in 10 mL of water, and use this solution as the sample solu-
tion. Measure exactly 1 mL of this solution, add water to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, acetone and water (7:4:1) to a distance of about 12
cm, air-dry the plate, and examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
80°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Fluorouracil — Weigh accurately about 0.2 g of
Fluorouracil, previously dried, dissolve in 20 mL of N,N-
dimethylformamide, and titrate <2.50> with 0.1 mol/L tetra-
methylammonium hydroxide VS until the color of the solu-
tion changes from yellow through blue-green to blue (indica-
tor: 3 drops of thymol blue-dimethylformamide TS). Per-
form a blank determination.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 13.01 mg of C 4 H 3 FN 2 2
(2) Fluorine — Weigh accurately about 4 mg of
Fluorouracil, previously dried, and proceed as directed in the
determination of fluorine under Oxygen Flask Combustion
Method <1.06>, using a mixture of 0.5 mL of 0.01 mol/L so-
dium hydroxide TS and 20 mL of water as the absorbing liq-
uid.
Containers and storage Containers — Tight containers.
Fluoxymesterone
C 20 H 29 FO 3 : 336.44
9-Fluoro-ll/?,17/?-dihydroxy-17-methylandrost-4-en-3-one
[76-43-7]
Fluoxymesterone, when dried, contains not less than
97.0% and not more than 102.0% of C 2 oH 29 F0 3 .
Description Fluoxymesterone occurs as white crystals or
crystalline powder. It is odorless.
It is sparingly soluble in methanol, slightly soluble in
ethanol (95) and in chloroform, very slightly soluble in
diethyl ether, and practically insoluble in water.
Identification (1) Dissolve 5 mg of Fluoxymesterone in 2
mL of sulfuric acid: a yellow color develops.
(2) Prepare the test solution with 0.01 g of Fluoxymester-
one as directed under Oxygen Flask Combustion Method
<1.06>, using a mixture of 0.5 mL of 0.01 mol/L sodium
hydroxide TS and 20 mL of water as an absorbing liquid: the
test solution responds to the Qualitative Tests <1.09> (2) for
fluoride.
(3) Determine the absorption spectrum of a solution of
Fluoxymesterone in ethanol (95) (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Fluoxymesterone Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of Fluox-
ymesterone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Fluoxymesterone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve Fluoxymesterone and
Fluoxymesterone Reference Standard in ethanol (99.5),
respectively, then evaporate the ethanol to dryness, and
repeat the test on the residues.
Optical rotation <2.49> [a]™'- + 104
0.1 g, ethanol (95), 10 mL, 100 mm).
112° (after drying,
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Fluoxymesterone according to Method 2, and perform the
test. Prepare the control solution with 1.5 mL of Standard
Lead Solution (not more than 30 ppm).
(2) Related substances — Dissolve 0.03 g of Fluoxymester-
one in 10 mL of methanol, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
676
Fluphenazine Enanthate / Official Monographs
JP XV
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of toluene,
ethanol (95) and ethyl acetate (3 : 1 : 1) to a distance of about 12
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g,
platinum crucible).
Assay Weigh accurately about 25 mg each of Fluoxymester-
one and Fluoxymesterone Reference Standard, previously d-
ried, dissolve each in the internal standard solution to make
exactly 100 mL, and use these solutions as the sample solu-
tion and standard solution, respectively. Perform the test
with 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Qs, of the peak area of fluoxymesterone to that of the inter-
nal standard, respectively.
Amount (mg) of C20H29FO3
= W s x (Q T /Q S )
W s : Amount (mg) of Fluoxymesterone Reference Standard
Internal standard solution — A solution of methylpredniso-
lone in a mixture of chloroform and methanol (19:1) (1 in
5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 30 cm in length, packed with silica gel for liquid
chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of n-butyl chloride, water-satu-
rated M-butyl chloride, tetrahydrofuran, methanol and acetic
acid (100) (95:95:14:7:6).
Flow rate: Adjust the flow rate so that the retention time of
fluoxymesterone is about 9 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, fluoxymesterone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 6.
System repeatability: When the test is repeated 6 times with
10 liL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of fluoxymesterone to that of the internal standard
is not more than 1.5%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Fluphenazine Enanthate
7JI,7ity>it> HiXxJl
.0...
C 29 H 38 F 3 N 3 2 S: 549.69
2-(4- {3 - [2-(Trifluoromethyl)- 1 0//-phenothiazin- 1 0-
yl]propyl}piperazin-l-yl)ethyl heptanoate [2746-81-8]
Fluphenazine Enanthate, when dried, contains not
less than 98.5% of C 29 H3 8 F 3 N 3 2 S.
Description Fluphenazine Enanthate is a light yellow to yel-
lowish orange viscous liquid. It is generally clear, and can be
opaque by producing crystals.
It is freely soluble in methanol and in diethyl ether, soluble
in acetic acid (100) and in ethanol (95), and practically insolu-
ble in water.
Identification (1) Prepare the test solution with 0.01 g of
Fluphenazine Enanthate as directed under Oxygen Flask
Combustion Method <1.06>, using a mixture of 0.5 mL of
0.01 mol/L sodium hydroxide TS and 20 mL of water as the
absorbing liquid: the test solution responds to the Qualitative
Tests <1.09> for fluoride.
(2) Dissolve 2 mg of Fluphenazine Enanthate in 200 mL
of a solution of hydrochloric acid in methanol (17 in 2000).
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Fluphenazine Enanthate as directed in the liquid firm method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Fluphenazine Enanthate according to Method 2, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 30 ppm).
(2) Related substances — Dissolve 0.25 g of Fluphenazine
Enanthate in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
20 /uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ace-
tone, hexane and ammonia solution (28) (16:6:1) to a dis-
tance of about 15 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution. Then spray
evenly diluted sulfuric acid (1 in 2) on the plate: the spots
JPXV
Official Monographs / Flurazepam
677
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Fluphenazine Enan-
thate, previously dried, dissolve in 50 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (indica-
tor: 2 drops of crystal violet TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 27.49 mg of C 29 H 38 F 3 N 3 2 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Flurazepam
C 2 iH 23 ClFN 3 0: 387.88
7-Chloro-l-[2-(diethylamino)ethyl]-5-(2-fluorophenyl)-l,3-
dihydro-2//-l,4-benzodiazepin-2-one [17617-23-1]
Flurazepam, when dried, contains not less than
99.0% of C 21 H 23 C1FN 3 0.
Description Flurazepam occurs as white to light yellow
crystals or crystalline powder.
It is very soluble in chloroform, freely soluble in methanol,
in ethanol (95), in acetic anhydride and in diethyl ether, and
practically insoluble in water.
Identification (1) Dissolve 0.01 g of Flurazepam in 3 mL
of sulfuric acid: the solution shows a greenish yellow fluores-
cence under ultraviolet light (main wavelength: 365 nm).
(2) Dissolve 0.01 g of Flurazepam in 3 mL of citric acid-
acetic acid TS, and heat in a water bath for 4 minutes: a dark
red color develops.
(3) Prepare the test solution with 0.01 g of Flurazepam as
directed under Oxygen Flask Combustion Method <1.06>, us-
ing a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS
and 20 mL of water as an absorbing liquid: the test solution
responds to the Qualitative Tests <1.09> (2) for fluoride.
(4) Determine the absorption spectrum of a solution of
Flurazepam in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum 1 : both spectra ex-
hibit similar intensities of absorption at the same
wavelengths. Separately, determine the absorption spectrum
of a solution of Flurazepam in methanol (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 2:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(5) Perform the test with Flurazepam as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 79 - 83 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Flurazepam in 10 mL of ethanol (95): the solution is clear and
colorless to light yellow.
(2) Chloride <1.03> — Dissolve 1.0 g of Flurazepam in 50
mL of diethyl ether, add 46 mL of water and 4 mL of sodium
carbonate TS, shake, separate the water layer, wash with two
20-mL portions of diethyl ether, and filter the water layer.
Neutralize 20 mL of the filtrate with dilute nitric acid, and
add 6 mL of dilute nitric acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution with 0.40 mL of 0.01 mol/L hydrochlor-
ic acid VS (not more than 0.036%).
(3) Sulfate <1.14>— Neutralize 20 mL of the filtrate ob-
tained in (2) with dilute hydrochloric acid, and add 1 mL of
dilute hydrochloric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 0.40 mL of 0.005 mol/L sulfuric acid
VS (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Flurazep-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Flurazepam according to Method 3, and perform the test
(not more than 2 ppm).
(6) Related substances — Dissolve 0.20 g of Flurazepam in
20 mL of chloroform, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, and add chlo-
roform to make exactly 20 mL. Pipet 3 mL of this solution,
add chloroform to make exactly 50 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of cyclo-
hexane, acetone and ammonia solution (28) (60:40: 1) to a dis-
tance of about 12 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, in vacu-
um, 60°C, 2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.3 g of Flurazepam, previ-
ously dried, dissolve in 50 mL of acetic anhydride, and titrate
<2.50> with 0.1 mol/L perchloric acid VS to the second e-
quivalence point (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.39 mg of C 21 H 23 C1FN 3
Containers and storage Containers — Well-closed contain-
ers.
678
Flurazepam Capsules / Official Monographs
JP XV
Storage — Light-resistant.
Flurazepam Capsules
Flurazepam Capsules contain not less than 93% and
not more than 107% of the labeled amount of flurazep-
am (C 21 H 23 C1FN 3 0: 387.88).
Flurazepam Hydrochloride
Method of preparation
with Flurazepam.
Prepare as directed under Capsules,
Identification (1) Powder the contents of Flurazepam
Capsules. To a quantity of the powder, equivalent to 0.1 g of
Flurazepam according to the labeled amount, add 100 mL of
0.1 mol/L hydrochloric acid TS, stir, and filter. To 40 mL of
the filtrate add 80 mL of a solution of sodium hydroxide (1 in
250) and 100 mL of hexane, extract by shaking well, and use
the hexane layer as the sample solution. Evaporate 25 mL of
the sample solution on a water bath to dryness. Dissolve the
residue in 3 mL of sulfuric acid: the solution shows a greenish
yellow fluorescence under ultraviolet light.
(2) Evaporate 25 mL of the sample solution obtained in
(1) on a water bath to dryness. Dissolve the residue in 3 mL of
citric acid-acetic acid TS, and heat in a water bath for 4
minutes: a dark red color develops.
(3) Determine the absorption spectrum of the sample so-
lution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 315 nm and 319 nm, and a minimum between 297 nm
and 301 nm.
Assay Weigh accurately the contents of not less than 20
Flurazepam Capsules, and powder the combined contents.
Weigh accurately a portion of the powder, equivalent to
about 0.05 g of flurazepam (C 21 H 23 C1FN 3 0), add 30 mL of
methanol, stir well for 10 minutes, and add methanol to
make exactly 50 mL. Filter this solution, discard the first 20
mL of the filtrate, pipet 6 mL of the subsequent filtrate, add
methanol to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.05 g of
flurazepam for assay, previously dried in vacuum at 60°C for
2 hours, and dissolve in methanol to make exactly 50 mL.
Pipet 6 mL of this solution, add methanol to make exactly 50
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution at 317 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>.
Amount (mg) of flurazepam (C21H23CIFN3O)
= W s x (A T /A S )
W s : Amount (mg) of flurazepam for assay
Containers and storage Containers — Tight containers.
7JUvHfA°A±^ig±g
C 2 iH 23 C1FN 3 O.HC1: 424.34
7-Chloro- 1 - [2-(diethylamino)ethyl] -5-(2-fluorophenyl)-
l,3-dihydro-2//-l,4-benzodiazepin-2-one
monohydrochloride [36105-20-1]
Flurazepam Hydrochloride, when dried, contains
not less than 99.0% of C 21 H 23 ClFN 3 O.HCl.
Description Flurazepam Hydrochloride occurs as white to
yellowish white crystals or crystalline powder.
It is freely soluble in water, in ethanol (95), in ethanol
(99.5) and in acetic acid (100).
Melting point: about 197°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Flurazepam Hydrochloride in sulfuric acid-
ethanol TS (1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Flurazepam Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Flurazepam Hydrochloride (1 in 20)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Flurazepam Hydrochloride in
20 mL of water: the pH of this solution is between 5.0 and
6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Flurazepam Hydrochloride in 10 mL of water: the solution is
clear and colorless to pale yellow.
(2) Sulfate <1.14>— Perform the test with 1.5 g of
Flurazepam Hydrochloride. Prepare the control solution
with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than
0.011%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Flurazep-
am Hydrochloride in a platinum crucible according to
Method 2, and perform the test. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 20
ppm).
(4) Related substances — Dissolve 0.05 g of Flurazepam
Hydrochloride in 5 mL of ethanol (95), and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add ethanol (95) to make exactly 50 mL. Pipet 1 mL of this
solution, add ethanol (95) to make exactly 10 mL, and use
JPXV
Official Monographs / Flurbiprofen 679
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 20 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Place the plate in a chamber
filled with ammonia vapor, allow to stand for about 15
minutes, and immediately develop the plate with a mixture of
diethyl ether and diethylamine (39:1) to a distance of about
12 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): not more than 3 spots other than
the principal spot and the spot on the starting point from the
sample solution appear, and are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Flurazepam
Hydrochloride, previously dried, dissolve in 10 mL of acetic
acid (100), add 40 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 21.22 mg of C 21 H 23 ClFN 3 O.HCl
Containers and storage Containers — Tight containers.
Flurbiprofen
and enantiomer
C 15 H 13 F0 2 : 244.26
(2/?5')-2-(2-Fluorobiphenyl-4-yl)propanoic acid
[5104-49-4]
Flurbiprofen, when dried, contains not less than
98.0% of C 15 H 13 F0 2 .
Description Flurbiprofen occurs as a white, crystalline
powder. It has a slightly irritating odor.
It is freely soluble in methanol, in ethanol (95), in acetone
and in diethyl ether, soluble in acetonitrile, and practically
insoluble in water.
A solution of Flurbiprofen in ethanol (95) (1 in 50) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Flurbiprofen in methanol (1 in 200,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Flur-
biprofen, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 114 - 117°C
Purity (1) Chloride <1.03>— Dissolve 0.6 g of Flur-
biprofen in 40 mL of acetone, and add 6 mL of dilute nitric
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS add 40
mL of acetone, 6 mL of dilute nitric acid and water to make
50 mL (not more than 0.015%).
(2) Heavy metals <1.07> — Dissolve 2.0 g of Flurbiprofen
in 30 mL of acetone, and add 2 mL of dilute acetic acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
2.0 mL of Standard Lead Solution add 30 mL of acetone, 2
mL of dilute acetic acid and water to make 50 mL (not more
than 10 ppm).
(3) Related substances — Dissolve 20 mg of Flurbiprofen
in 10 mL of a mixture of water and acetonitrile (11:9), and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, and add a mixture of water and acetonitrile
(11:9) to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with exactly 20 //L each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area of both solutions
by the automatic integration method: each area of the peaks
other than the peak of flurbiprofen from the sample solution
is not larger than the peak area of flurbiprofen from the stan-
dard solution, and the total area of these peaks is not larger
than twice the peak area of flurbiprofen from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (12:7:1).
Flow rate: Adjust the flow rate so that the retention time of
flurbiprofen is about 20 minutes.
Time span of measurement: About twice as long as the
retention time of flurbiprofen beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add a mixture of water and acetonitrile (11:9)
to make exactly 25 mL. Confirm that the peak area of flur-
biprofen obtained from 20 /uL of this solution is equivalent to
16 to 24% of that of flurbiprofen obtained from 20 fiL of the
standard solution.
System performance: Dissolve 0.04 g of flurbiprofen and
0.02 g of butyl parahydroxybenzoate in 100 mL of a mixture
of water and acetonitrile (1 1 :9). To 5 mL of this solution add
a mixture of water and acetonitrile (11:9) to make 50 mL.
When the procedure is run with 20 fiL of this solution under
the above operating conditions, butyl parahydroxybenzoate
680
Folic Acid / Official Monographs
JP XV
and flurbiprofen are eluted in this order with the resolution
between these peaks being not less than 12.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
flurbiprofen is not more than 2.0%.
Loss on drying <2.41> Not more than 0.10% (1 g, in vacu-
um at a pressure not exceeding 0.67 kPa, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.6 g of Flurbiprofen, previ-
ously dried, dissolve in 50 mL of ethanol (95), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 24.43 mg of C 15 H 13 F0 2
Containers and storage Containers — Well-closed contain-
ers.
Folic Acid
f? H CO;H
CO^H
C 19 H 19 N 7 6 : 441.40
N- {4- [(2-Amino-4-hydroxypteridin-
6-ylmethyl)amino]benzoylj-L-glutamic acid [59-30-3]
Folic Acid contains not less than 98.0% and not
more than 102.0% of C 19 H 19 N 7 6 , calculated on the
anhydrous basis.
Description Folic Acid occurs as a yellow to orange-yellow,
crystalline powder. It is odorless.
It is practically insoluble in water, in methanol, in ethanol
(95), in pyridine and in diethyl ether.
It dissolves in hydrochloric acid, in sulfuric acid, in dilute
sodium hydroxide TS and in a solution of sodium carbonate
decahydrate (1 in 100), and these solutions are yellow in
color.
It is slowly affected by light.
Identification (1) Dissolve 1.5 mg of Folic Acid in dilute
sodium hydroxide TS to make 100 mL. Determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Folic Acid Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) To 10 mL of the solution obtained in (1) add 1 drop of
potassium permanganate TS, and mix well until the color
changes to blue, and immediately observe under ultraviolet
light (main wavelength: 365 nm): a blue fluorescence is
produced.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Folic Acid in 10 mL of dilute sodium hydroxide TS : the
solution is clear and yellow in color.
(2) Free amines — Pipet 30 mL of the sample solution ob-
tained in the Assay, add 20 mL of dilute hydrochloric acid
and water to make exactly 100 mL, and use this solution as
the sample solution. Weigh accurately about 50 mg of
jP-Aminobenzoylglutamic Acid Reference Standard, previ-
ously dried in a desiccator (in vacuum, silica gel) for 4 hours,
dissolve in diluted ethanol (95) (2 in 5) to make exactly 100
mL. Pipet 3 mL of this solution, add water to make exactly
1000 mL, and use this solution as the standard solution.
Pipet 4 mL each of the sample solution and the standard
solution, proceed as directed in the Assay, and perform the
test as directed under Ultraviolet-visible Spectrophotometry
<2.24>. Determine the absorbances, A T and A s , of subsequent
solutions of the sample solution and the standard solution at
550 nm: the content of free amines is not more than 1.0%.
Content (%) of free amines = (A T /A S ) x (W'/W)
W : Amount (mg) of Folic Acid, calculated on the anhy-
drous basis
W: Amount (mg) of p-Aminobenzoylglutamic Acid
Reference Standard
Water <2.48> Not more than 8.5% (10 mg, coulometric
titration).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately about 50 mg each of Folic Acid
and Folic Acid Reference Standard. To each add 50 mL of
dilute sodium hydroxide TS, mix well to dissolve, add dilute
sodium hydroxide TS to make exactly 100 mL, and use these
solutions as the sample solution and standard solution. To 30
mL each of these solutions, accurately measured, add 20 mL
of dilute hydrochloric acid and water to make exactly 100
mL. To 60 mL each of these solutions add 0.5 g of zinc pow-
der, and allow to stand with frequent shaking for 20 minutes.
Filter each mixture through a dry filter paper, and discard the
first 10 mL of the filtrate. Pipet 10 mL each of the subsequent
filtrate, and add water to make exactly 100 mL. To 4 mL each
of solutions, accurately measured, add 1 mL of water, 1 mL
of dilute hydrochloric acid and 1 mL of a solution of sodium
nitrite (1 in 1000), mix well, and allow to stand for 2 minutes.
To each solution add 1 mL of a solution of ammonium
amidosulfate (1 in 200), mix thoroughly, and allow to stand
for 2 minutes. To each of these solutions, add 1 mL of a solu-
tion of 7V-(l-naphthyl)-/V'-diethylethylenediamine oxalate (1
in 1000), shake, allow to stand for 10 minutes, and add water
to make exactly 20 mL. Separately, to 30 mL of the sample
solution, accurately measured, add 20 mL of dilute
hydrochloric acid and water to make exactly 100 mL. Pipet
10 mL of this solution, add 18 mL of dilute hydrochloric acid
and water to make exactly 100 mL. Pipet 4 mL of this solu-
tion, and prepare the blank solution in the same manner as
the sample solution. Perform the test with these solutions as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution prepared with 4 mL of water in the same
manner as a blank. Determine the absorbances, A T , A s and A
c, of the subsequent solution of the sample solution, the stan-
dard solution and the blank solution at 550 nm.
Amount (mg) of Ci 9 H 19 N 7 6
JPXV
Official Monographs / Formalin
681
= W s x {(A T - A c )/A s }
W s : Amount (mg) of Folic Acid Reference Standard, cal-
culated on the anyhdrous basis
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Folic Acid Tablets
m$&
Folic Acid Injection
EB&I4*
Folic Acid Injection is an aqueous solution for injec-
tion.
It contains not less than 95% and not more than 115
% of the labeled amount of folic acid (C 19 H 19 N 7 6 :
441.40).
Method of preparation Dissolve Folic Acid in water with
the aid of Sodium Hydroxide or Sodium Carbonate, and pre-
pare as directed under Injections.
Description Folic Acid Injection is a yellow to orange-yel-
low, clear liquid.
pH: 8.0- 11.0
Identification (1) To a volume of Folic Acid Injection,
equivalent to 1.5 mg of Folic Acid according to the labeled
amount, add dilute sodium hydroxide TS to make 100 mL.
Proceed as directed in the Identification (2) under Folic Acid,
using this solution as the sample solution.
(2) Determine the absorption spectrum of the sample so-
lution obtained in (1) as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 255
nm and 257 nm, between 281 nm and 285 nm and between
361 nm and 369 nm. Separately, determine the maximal ab-
sorbances of the sample solution, A { and A 2 , between 255 nm
and 257 nm and between 361 nm and 369 nm, respectively:
the ratio of AJA 2 is between 2.80 and 3.00.
(3) Folic Acid Injection responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Extractable volume <6.05> It meets the requirement.
Assay To an exactly measured volume of Folic Acid Injec-
tion, equivalent to about 50 mg of folic acid (C 19 H 19 N 7 6 )
add dilute sodium hydroxide TS to make exactly 100 mL, and
use this solution as the sample solution. Separately, weigh
accurately about 50 mg of Folic Acid Reference Standard,
dissolve in dilute sodium hydroxide TS to make exactly 100
mL, and use this solution as the standard solution. Proceed
with 30 mL each of the sample solution and standard solu-
tion, exactly measured, as directed in the Assay under Folic
Acid.
Amount (mg) of folic acid (C 19 H 19 N 7 6 )
= W s x {{A-, - A c )/A s }
W s : Amount (mg) of Folic Acid Reference Standard, cal-
culated on the anhydrous basis
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Folic Acid Tablets contain not less than 90% and not
more than 115% of the labeled amount of folic acid
(C 19 H 19 N 7 6 : 441.40).
Method of preparation
with Folic Acid.
Prepare as directed under Tablets,
Identification (1) Take a quantity of powdered Folic Acid
Tablets, equivalent to 1.5 mg of Folic Acid according to the
labeled amount, add 100 mL of dilute sodium hydroxide TS,
shake, and filter. Discard the first 10 mL of the filtrate, use
the subsequent filtrate as the sample solution, and proceed as
directed in the Identification (2) under Folic Acid.
(2) Determine the absorption spectrum of the filtrate ob-
tained in (1) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 255 nm
and 257 nm, between 281 nm and 285 nm and between 361
nm and 369 nm. Separately, determine the maximal absor-
bances of the filtrate, A x andy4 2 , between 255 nm and 257 nm
and between 361 nm and 369 nm, respectively: the ratio of A l
/A 2 is between 2.80 and 3.00.
Assay Weigh accurately and powder not less than 20 Folic
Acid Tablets. Weigh accurately a portion of the powder,
equivalent to about 50 mg of folic acid (C 19 H 19 N 7 6 ). Add 50
mL of dilute sodium hydroxide TS, shake frequently, then
filter into a 100-mL volumetric flask, and wash with dilute so-
dium hydroxide TS. To the combined filtrate and washings
add dilute sodium hydroxide TS to make exactly 100 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of Folic Acid Reference Standard, dis-
solve in dilute sodium hydroxide TS to make exactly 100 mL,
and use this solution as the standard solution. Take 30 mL
each of the sample solution and standard solution, exactly
measured, and proceed as directed in the Assay under Folic
Acid.
Amount (mg) of folic acid (C 19 H 19 N 7 6 )
= W s x {(A T - A c )/A s ]
W s : Amount (mg) of Folic Acid Reference Standard, cal-
culated on the anhydrous basis
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Formalin
7k)l -7 U >
Formalin contains not less than 35.0% and not more
than 38.0% of formaldehyde (CH 2 Or 30.03.)
It contains 5% to 13% of methanol to prevent poly-
merization.
Description Formalin is a clear, colorless liquid. Its vapor is
682
Formalin Water / Official Monographs
JP XV
irritating to the mucous membrane.
It is miscible with water and with ethanol (95).
When stored for a long time, especially in a cold place, it
may become cloudy.
Identification (1) Dilute 2 mL of Formalin with 10 mL of
water in a test tube, and add 1 mL of silver nitrate-ammonia
TS: a gray precipitate is produced, or a silver mirror is
formed on the wall of the test tube.
(2) To 5 mL of sulfuric acid in which 0.1 g of salicylic
acid has been dissolved add 2 drops of Formalin, and warm
the solution: a persistent, dark red color develops.
Purity Acidity — Dilute 20 mL of Formalin with 20 mL of
water, and add 5.0 mL of 0.1 mol/L sodium hydroxide VS
and 2 drops of bromothymol blue TS: a blue color develops.
Residue on ignition <2.44>
mL, after evaporation).
Not more than 0.06 w/v% (5
Assay Weigh accurately a weighing bottle containing 5 mL
of water, add about 1 g of Formalin, and weigh accurately
again. Add water to make exactly 100 mL. Pipet 10 mL of
this solution, add exactly 50 mL of 0.05 mol/L iodine VS and
20 mL of potassium hydroxide TS, and allow to stand for 15
minutes at an ordinary temperature. To this mixture add 15
mL of dilute sulfuric acid, and titrate <2.50> the excess iodine
with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of
starch TS). Perform a blank determination.
Each mL of 0.05 mol/L iodine VS = 1 .501 mg of CH 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Formalin Water
Formalin Water contains not less than 0.9 w/v%
and not more than 1.1 w/v% of formaldehyde
(CH 2 Q: 30.03).
Method of preparation
Formalin
Water or Purified Water
30 mL
a sufficient quantity
To make 1000 mL
Prepare by mixing the above ingredients.
Description Formalin Water is a clear, colorless liquid. It
has a slight odor of formaldehyde.
It is almost neutral.
Assay Transfer 20 mL of Formalin Water, measured exact-
ly, to a 100-mL volumetric flask containing 2.5 mL of 1 mol/
L sodium hydroxide VS, and add water to make 100 mL.
Pipet 10 mL of this solution, and proceed as directed in the
Assay under Formalin.
Each mL of 0.05 mol/L iodine VS = 1 .501 mg of CH 2
Containers and storage Containers — Tight containers.
Formoterol Fumarate Hydrate
H OH
CHO
HOjC
^^-C02H , 2H20
and enanliomer
(C 19 H 2 4N 2 04)2.C4H 4 04.2H 2 0: 840.91
iV-(2-Hydroxy-5- {(1 RS )- 1 -hydroxy-
2-[(l.RS)-2-(4-methoxyphenyl)-
1 -methylethylamino] ethyl} phenyl)f ormamide
hemifumarate monohydrate [43229-80-7, anhydride]
Formoterol Fumarate Hydrate contains not less
than 98.5% of formoterol fumarate [(QsF^^C^.
C 4 H 4 4 : 804.88], calculated on the anhydrous basis.
Description Formoterol Fumarate Hydrate occurs as a
white to yellowish white, crystalline powder.
It is freely soluble in acetic acid (100), soluble in methanol,
very slightly soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
A solution of Formoterol Fumarate in methanol (1 in 100)
shows no optical rotation.
Melting point: about 138°C (with decomposition).
Identification (1) Dissolve 0.5 g of Formoterol Fumarate
Hydrate in 20 mL of 0.5 mol/L sulfuric acid TS, and extract
with three 25-mL portions of diethyl ether. Wash the com-
bined diethyl ether extracts with 10 mL of 0.5 mol/L sulfuric
acid TS, and evaporate the ether layer under reduced pres-
sure, and dry the residue at 105°C for 3 hours: the residue
melts <2.60> at about 290°C (with decomposition, in a sealed
tube).
(2) Determine the absorption spectrum of a solution of
Formoterol Fumarate Hydrate in methanol (1 in 40,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the infrared absorption spectrum of For-
moterol Fumarate Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry <
2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Formoterol Fumarate Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related Substances — Dissolve 0.20 g of Formoterol
Fumarate Hydrate in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add methanol to make exactly 200 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
JPXV
Official Monographs / Fosfestrol
683
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chlo-
roform, 1,4-dioxane, ethanol (99.5) and ammonia solution
(28) (20:20:10:3) to a distance of about 12 cm, and air-dry the
plate. Allow the plate to stand for 5 minutes in iodine vapor:
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Water <2.48> 4.0 - 5.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Formoterol
Fumarate Hydrate, dissolve in 50 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 40.24 mg of (C 19 H 2 4N 2 04)2-C4H 4 04
Containers and storage Containers — Tight containers.
Fosfestrol
Diethylstilbestrol Diphosphate
OP0 3 H?
HiO^PO
C 18 H 22 8 P 2 : 428.31
(£')-4,4'-(Hex-3-ene-3,4-diyl)bis(phenyl dihydrogen
phosphate) [522-40-7]
Fosfestrol, when dried, contains not less than 98.5%
of C 18 H 2 20 8 P 2 .
Description Fosfestrol occurs as a white, crystalline pow-
der. It is odorless.
It is freely soluble in ethanol (95), soluble in formamide,
slightly soluble in water, and practically insoluble in acetoni-
trile and in diethyl ether.
It dissolves in sodium hydroxide TS.
Melting point: about 234°C (with decomposition).
Identification (1) Dissolve 15 mg of Fosfestrol in 1 mL of
sulfuric acid: a yellow to orange color develops. To this solu-
tion add 10 mL of water: the color of the solution disappears.
(2) Determine the infrared absorption spectrum of Fos-
festrol as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or with the spectrum
of Fosfestrol Reference Standard: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
(3) Place 0.4 g of Fosfestrol in a crucible, wet by adding
0.1 mL of sulfuric acid, and heat to carbonize. Add 10 mL of
water to the residue, stir well, and filter. Add 0.1 mL of nitric
acid to the filtrate, and heat in a water bath for 15 minutes:
this solution responds to the Qualitative Tests <1.09> for
phosphate.
pH <2.54> Dissolve 0.10 g of Fosfestrol in 30 mL of water:
the pH of this solution is between 1.0 and 2.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Fosfestrol in 15 mL of sodium hydroxide TS: the solution is
clear and colorless.
(2) Chloride <1.03>— Dissolve 0.10 g of Fosfestrol in 30
mL of ethanol (95), add 6 mL of dilute nitric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution as follows: to 0.70
mL of 0.01 mol/L hydrochloric acid VS add 6 mL of dilute
nitric acid, 30 mL of ethanol (95) and water to make 50 mL
(not more than 0.248%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Fosfes-
trol according to Method 4, and perform the test. Use a solu-
tion of magnesium nitrate hexahydrate in ethanol (95) (1 in
5). Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Fosfestrol according to Method 3, and perform the test.
Use a solution of magnesium nitrate hexahydrate in ethanol
(95) (1 in 5) (not more than 2 ppm).
(5) Free phosphoric acid — Weigh accurately about 0.4 g
of Fosfestrol, dissolve in a mixture of water and formamide
(1:1) to make exactly 200 mL, and use this solution as the
sample solution. Separately, weigh accurately 0. 1 12 g of
monobasic potassium phosphate, previously dried in a desic-
cator (silica gel) to constant mass, dissolve in 10 mL of dilut-
ed sulfuric acid (1 in 10) and water to make exactly 1000 mL.
Measure exactly 10 mL of this solution, add 100 mL of for-
mamide and water to make exactly 200 mL, and use this solu-
tion as the standard solution. Measure exactly 10 mL each of
the sample solution and standard solution, and place in a
25-mL volumetric flask, respectively. To each of these solu-
tions add 2.5 mL of hexaammonium heptamolybdate-sulfur-
ic acid TS and 1 mL of l-amino-2-naphthol-4-sulfonic acid
TS, shake, add water to make 25 mL, and allow to stand at
20 ± 1°C for 30 minutes. Perform the test with these solu-
tions, using a solution obtained in the same manner with 10
mL of a mixture of water and formamide (1:1) as the blank,
as directed under Ultraviolet-visible Spectrophotometry
<2.24>. Determine the absorbances, A T and A s , of the solu-
tions obtained from the sample solution and standard solu-
tion at 740 nm: the amount of free phosphoric acid is not
more than 0.2%.
Amount (%) of free phosphoric acid (H 3 P0 4 )
= (Aj/As) x (1/ W) x 80.65
W; Amount (mg) of Fosfestrol
(6) Related substances — Dissolve 20 mg of Fosfestrol in
100 mL of the mobile phase, and use this solution as the sam-
ple solution. Measure exactly 5 mL of this solution, and add
the mobile phase to make exactly 50 mL. Pipet 3 mL of this
solution, add the mobile phase to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of these solutions by the automatic integration method:
the total area of the peaks other than the peak of fosfestrol
from the sample solution is not larger than the peak area of
fosfestrol from the standard solution.
684
Fosfestrol Tablets / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of potassium di-
hydrogen phosphate (1 in 500), acetonitrile and tetrabutylam-
monium hydroxide TS (70:30:1).
Flow rate: Adjust the flow rate so that the retention time of
fosfestrol is about 8 minutes.
Selection of column: Dissolve 0.02 g of Fosfestrol and 8
mg of methyl parahydroxybenzoate in 100 mL of the mobile
phase. Proceed with lO^L of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of methyl parahydroxybenzoate and
fosfestrol in this order with the resolution between these
peaks being not less than 3.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of fosfestrol obtained from 10 /xL of the
standard solution is between 5 mm and 15 mm.
Time span of measurement: Three times as long as the
retention time of fosfestrol.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
4 hours).
Assay Weigh accurately about 0.2 g of Fosfestrol, previ-
ously dried, dissolve in 60 mL of water, and titrate <2.50>
with 0.1 mol/L sodium hydroxide VS (potentiometric titra-
tion). The end point is the second equivalent point. Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 10.71 mg of C 18 H 22 8 P 2
Containers and storage Containers — Tight containers.
Fosfestrol Tablets
Diethylstilbestrol Diphosphate Tablets
*X7iX r- n-juffi
Fosfestrol Tablets contain not less than 93% and not
more than 107% of the labeled amount of fosfestrol
(C 18 H 22 8 P 2 : 428.31).
Method of preparation Prepare as directed under Tablets,
with Fosfestrol.
Identification (1) To a quantity of powdered Fosfestrol
Tablets, equivalent to 0.5 g of Fosfestrol according to the
labeled amount, add 50 mL of 0.1 mol/L hydrochloric acid
TS, shake well, and filter. To the filtrate add 100 mL of
diethyl ether, extract, and evaporate carefully the diethyl
ether extract on a water bath to dryness. Proceed with 0.015 g
of the residue as directed in the Identification (1) under Fos-
festrol.
(2) Dry 0.01 g of the residue obtained in (1) at 105 °C for
4 hours, and determine the infrared absorption spectrum as
directed in the potassium bromide disk method under
Infrared Spectrometry <2.25>: it exhibits absorption at the
wave numbers of about 2970 cm -1 , 1605 cm -1 , 1505 cm -1 ,
1207 cm" 1 and 1006 cm" 1 .
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Fosfestrol Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of water. Take 20 mL or more of the dissolved
solution 20 minutes after starting the test, and filter through a
membrane filter with pore size of not more than 0.8 ftm. Dis-
card the first 10 mL of the filtrate, pipet 2 mL of the subse-
quent, add a solution of sodium hydroxide (1 in 250) to make
exactly 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 50 mg of Fosfestrol
Reference Standard, previously dried at 105°C for 4 hours,
and dissolve in a solution of sodium hydroxide (1 in 250) to
make exactly 100 mL. Pipet 2 mL of this solution, add a solu-
tion of sodium hydroxide (1 in 250) to make exactly 100 mL,
and use this solution as the standard solution. Determine the
absorbances, A T and A s , of the sample solution and standard
solution at 242 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
The dissolution rate of Fosfestrol Tablets in 20 minutes is
not less than 80%.
Dissolution rate (%) with respect to the labeled amount
of fosfestrol (C 18 H 22 O s P 2 )
= W s x (A T /A S ) x (1/C) x 180
W s : Amount (mg) of Fosfestrol Reference Standard
C: Labeled amount (mg) of fosfestrol (C 18 H 22 8 P 2 ) in 1
tablet
Assay Weigh accurately not less than 20 Fosfestrol Tablets,
and powder. Weigh accurately a quantity of the powder,
equivalent to about 1 g of fosfestrol (C 18 H 22 8 P 2 ) according
to the labeled amount, add 100 mL of a solution of sodium
hydroxide (1 in 125), shake well, add water to make exactly
500 mL. Filter this solution, discard the first 30 mL of the
filtrate, pipet the subsequent 2 mL of the filtrate, add 30 mL
of a solution of sodium hydroxide (1 in 125) and water to
make exactly 250 mL, and use this solution as the sample
solution. Separately, weigh accurately about 80 mg of Fos-
festrol Reference Standard, previously dried at 105°C for 4
hours, and dissolve in a solution of sodium hydroxide (1 in
125) to make exactly 50 mL. Pipet 1 mL of this solution, add
10 mL of a solution of sodium hydroxide (1 in 125) and water
to make exactly 100 mL, and use this solution as the standard
solution. Determine the absorbances, A T and A s , of the sam-
ple solution and standard solution at 242 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of fosfestrol (Ci 8 H 22 8 P 2 )
= W s x (Aj/A s ) x (25/2)
W s : Amount (mg) of Fosfestrol Reference Standard
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Fosfomycin Calcium Hydrate
685
Fosfomycin Calcium Hydrate
h 3 c. p, .P0 3 ca
'H;.0
C 3 H 5 Ca0 4 P.H 2 0: 194.14
Monocalcium (2/?,35')-3-methyloxiran-2-ylphosphonate
monohydrate [26016-98-8]
Fosfomycin Calcium Hydrate is the calcium salt of a
substance having antibacterial actively produced by the
growth of Streptomyces fradiae or by the chemical syn-
thesis.
It contains not less than 725 fig (potency) and not
more than 805 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Fosfomycin Calcium
Hydrate is expressed as mass (potency) of fosfomycin
(C 3 H 7 4 P: 138.06).
Description Fosfomycin Calcium Hydrate occurs as a white
crystalline powder.
It is slightly soluble in water, and practically insoluble in
methanol and in ethanol (99.5).
Identification (1) Determine the infrared absorption spec-
trum of Fosfomycin Calcium Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(2) Determine the spectrum of a solution of Fosfomycin
Calcium Hydrate in heavy water for nuclear magnetic
resonance spectroscopy (1 in 300), using sodium 3-trimethyl-
silylpropanesulfonate for nuclear magnetic resonance spec-
troscopy as an internal reference compound, as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
('H): it exhibits a double signal at around d 1.5 ppm, a duple
double signal at around 5 2.9 ppm, a multiple signal at
around 5 3.3 ppm, and no signal at around 5 1.4 ppm.
(3) A solution of Fosfomycin Calcium Hydrate (1 in 500)
responds to the Qualitative Tests <1.09> (3) for calcium salt.
Optical rotation <2.49> [a]™: -2.5 - -5.4° (0.5 g calculat-
ed on the anhydrous bases, 0.4mol/L disodium dihydrogen
ethylenediamine tetraacetate TS, pH 8.5, 10 mL, 100 mm).
Phosphorus Content Weigh accurately about 0.1 g of Fos-
fomycin Calcium Hydrate, add 40 mL of sodium periodate
(107 in 10,000) and 2 mL of perchloric acid, and heat in a
water bath for 1 hour. After cooling, add water to make ex-
actly 200 mL. Pipet 10 mL of this solution, and add 1 mL of
potassium iodide TS. To this solution add sodium thiosulfate
TS until the solution is colorless, add water to make exactly
100 mL, and use this solution as the sample stock solution.
Separately, weigh accurately about 70 mg of potassium di-
hydrogenphosphate, proceed with this solution in the same
manner as directed for the preparation of the sample stock
solution, and use the solution so obtained as the standard
stock solution. Proceed and prepare a solution in the same
manner for the preparation of the sample stock solution
without using Fosfomycin Calcium, and use the solution so
obtained as the blank stock solution. Pipet 5 mL each of the
sample stock solution, the standard stock solution, and the
blank stock solution, add 2.5 mL of ammonium molybdate-
sulfuric acid TS and 1 mL of l-amino-2-naphthol-4-sulfonic
acid TS, mix, and add water to make exactly 25 mL, and use
these solutions as the sample solution, the standard solution,
and the blank solution, respectively. After allowing these so-
lutions to stand for 30 minutes at 20 ± 1°C, perform the test
with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using water as a blank, and deter-
mine the absorbances at 740 nm, A T , A s and A B , of the sam-
ple solution, the standard solution and the blank solution:
the content of phosphorus is 15.2 - 16.7%.
Amount (mg) of phosphorus (P)
= W s x {(A T - A B )/(A S - A B )} x 0.22760
W s : Amount (mg) of potassium dihydrogenphosphate
Calcium Content Weigh accurately about 0.2 g of Fos-
fomycin Calcium Hydrate, add 4 mL of lmol/L
Hydrochloric acid TS, and shake well until the sample is
completely dissolved. To this solution add 100 mL of water,
9 mL of sodium hydroxide TS and 0.1 g of methylthymol
blue-sodium chloride indicator, and titrate <2.50> with 0.05
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
until the color of the solution changes from clear blue to gray
or gray-purple: calcium content is 19.6-21.7%. Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS = 2.004 mg of Ca
Purity (1) Heavy metals <1.07> — To 1.0 g of Fosfomycin
Calcium Hydrate add 40 mL of 0.25 mol/L acetic acid TS
and water to make 50 mL. Proceed with this solution accord-
ing to Method 1, and perform the test. Prepare the control
solution with 2.0 mL of Standard Lead Solution (not more
than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Fosfomycin Calcium Hydrate according to Method 3, and
perform the test (not more than 2 ppm).
Water <2.48> Not more than 12.0% (0.1 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1)
instead of methanol for water determination).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Proteus sp. (MB838)
(ii) Culture medium — Dissolve 5.0 g of peptone, 3.0 g of
meat extract, 2.0 g of yeast extract, and 15 g of agar in 1000
mL of water, sterilize, and use as the agar media for base
layer and seed layer with the pH of between 6.5 and 6.6 after
sterilization.
(iii) Seeded agar layer — Incubate the test organism on the
slant of the agar medium for transferring test organisms at
37°C for 40-48 hours. Subcultures at least three times.
Inoculate the grown organisms onto the surface of 300 mL of
the agar medium for transferring test organisms in a Roux
bottle, incubate at 37 °C for 40 - 48 hours, and suspend the
grown organisms in about 30 mL of water. To the suspension
add water, and use this as the stock suspension of test organ-
686
Fosfomycin Sodium / Official Monographs
JP XV
ism. The amount of the water to be added is adjust so that the
percent transmission at 560 nm of the suspension diluted ten
times with water is 17%. Keep the stock suspension at 10°C
or below and use within 7 days. Add 1.0- 2.0 mL of the
stock suspension of test organism to 100 mL of the agar
medium for seed layer previously kept at 48 °C, mix
thoroughly, and use this as the deeded agar layer.
(iv) Standard solutions — Weigh accurately an amount of
Fosfomycin Phenethylammonium Reference Standard
equivalent to about 20 mg (potency), dissolve in 0.05
mol/L Tris buffer solution, pH 7.0 to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5°C or below and use within 7
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.05 mol/L Tris buffer solution, pH
7.0 to make solutions so that each mL contains 10 fig (poten-
cy) and 5 fig (potency), and use these solutions as the high
concentration standard solution and low concentration stan-
dard solution, respectively.
(v) Sample solutions — Weigh accurately an amount of
Fosfomycin Calcium Hydrate equivalent to about 20 mg
(potency), and dissolve in 0.05 mol/L Tris buffer solution,
pH 7.0 to make exactly 50 mL. To exactly a suitable amount
of this solution add 0.05 mol/L Tris buffer solution, pH 7.0
to make solutions so that each mL contains 10 fig (potency)
and 5 fig (potency), and use these solutions as the high con-
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Tight containers.
Fosfomycin Sodium
H 3 C. P ,P0 3 Na 2
t/\
C 3 H 5 Na 2 4 P: 182.02
Disodium (2R, 3 5)-3-methyloxiran-2-ylphosphonate
[26016-99-9]
Fosfomycin Sodium is the sodium salt of a substance
having antibacterial activity produced by the growth of
Streptomyces fradiae or by the chemical synthesis.
It contains not less than 725 fig (potency) and not
more than 11 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Fosfomycin Sodium
is expressed as mass (potency) of fosfomycin
(C 3 H 7 4 P: 138.06).
Description Fosfomycin Sodium occurs as a white crystal-
line powder.
It is very soluble in water, sparingly soluble in methanol,
and practically insoluble in ethanol (99.5).
Identification (1) Determine the infrared absorption spec-
trum of Fosfomycin Sodium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) Determine the spectrum of a solution of Fosfomycin
Sodium in heavy water for nuclear magnetic resonance
spectroscopy (1 in 300), using sodium 3-trimethylsilyl-
propanesulfonate for nuclear magnetic resonance spec-
troscopy as an internal reference compound, as directed
under Nuclear Magnetic Resonance Spectroscopy <2.21>
OH): it exhibits a double signal at around 3 1.5 ppm, a duple
double signal at around d 2.8 ppm, a multiple signal at
around d 3.3 ppm, and no signal at around <5 1.3 ppm.
(3) A solution of Fosfomycin Sodium (1 in 500) responds
to the Quantitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [a]^°: - 3.5 - - 5.5° (0.5 g calculat-
ed on the anhydrous bases, water, 10 mL, 100 mm).
pH <2.54> Dissolve 0.70 g of Fosfomycin Sodium in 10 mL
of water: the pH of the solution is between 8.5 and 10.5.
Phosphorus Content Weigh accurately about 0.1 g of Fos-
fomycin Sodium, add 40 mL of a solution of sodium perio-
date (107 in 10,000) and 2 mL of perchloric acid, and heat in
a water bath for 1 hour. After cooling, add water to make
exactly 200 mL. Pipet 10 mL of this solution, and add 1 mL
of potassium iodide TS. To this solution add sodium thiosul-
fate TS until the solution is colorless, add water to make
exactly 100 mL, and use this solution as the sample stock so-
lution. Separately, weigh accurately about 70 mg of potassi-
um dihydrogenphosphate, proceed with this solution in the
same manner as directed for the preparation of the sample
stock solution, and use the solution so obtained as the stan-
dard stock solution. Proceed and prepare a solution in the
same manner for the preparation of the sample stock solution
without using Fosfomycin Sodium, and use the solution so
obtained as the blank stock solution. Pipet 5 mL each of the
sample stock solution, the standard stock solution, and the
blank stock solution, add 2.5 mL of ammonium molybdate-
sulfuric acid TS and 1 mL of l-amino-2-naphthol-4-sulfonic
acid TS, mix, and add water to make exactly 25 mL, and use
these solutions as the sample solution, the standard solution,
and the blank solution, respectively. After allowing these so-
lutions to stand for 30 minutes at 20 ± 1°C, perform the test
with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using water as a blank, and deter-
mine the absorbances at 740 nm, A T , A s and^4 B , of the sam-
ple solution, the standard solution and the blank solution:
the content of phosphorus is 16.2 - 17.9%.
Amount (mg) of phosphorus (P)
= W x {(A T - A B )/(A S - A B )} x 0.22760
W: Amount (mg) of potassium dihydrogenphosphate
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Fosfomycin Sodium in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Fos-
fomycin Sodium according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Fosfomycin Sodium accordiong to Method 3, and perform
the test (not more than 2 ppm).
Water <2.48> Not more than 3.0% (0.2 g, volumetric titra-
JPXV
Official Monographs / Fosfomycin Sodium for Injection
687
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism— Proteus sp. (MB838)
(ii) Culture medium — Mix 5 .0 g of peptone, 3 .0 g of meat
extract, 2.0 g of yeast extract, and 15 g of agar in 1000 mL of
water, sterilize, and use as the agar media for base layer and
seed layer with the pH of between 6.5 and 6.6 after steriliza-
tion.
(iii) Seeded agar layer — Incubate the test organism on the
slant of the agar medium for transferring test organisms at 37
°C for 40 - 48 hours. Subcultures at least three times. Inocu-
late the grown organisms onto the surface of 300 mL of the
agar medium for transferring test organisms in a Roux bottle,
incubate at 37 °C for 40 - 48 hours, and suspend the grown
organisms in about 30 mL of water. To the suspension add
water, and use this as the stock suspension of test organism.
The amount of the water to be added is adjust so that the per-
cent transmission at 560 nm of the suspension diluted ten
times with water is 17%. Keep the stock suspension at 10°C
or below and use within 7 days. Add 1.0- 2.0 mL of the
stock suspension of test organism to 100 mL of the agar
medium for seed layer previously kept at 48°C, mix
thoroughly, and use this as the deeded agar layer.
(iv) Standard solutions — Weigh accurately an amount of
Fosfomycin Phenethylammonium Reference Standard
equivalent to about 20 mg (potency), dissolve in 0.05
mol/L Tris buffer solution, pH 7.0 to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5°C or below and use within 7
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.05 mol/L Tris buffer solution, pH
7.0 to make solutions so that each mL contains 10 /ug (poten-
cy) and 5 /ug (potency), and use these solutions as the high
concentration standard solution and low concentration stan-
dard solution, respectively.
(v) Sample solutions — Weigh accurately an amount of
Fosfomycin Sodium equivalent to about 20 mg (potency),
and dissolve in 0.05 mol/L Tris buffer solution, pH 7.0 to
make exactly 50 mL. To exactly a suitable amount of this so-
lution add 0.05 mol/L Tris buffer solution, pH 7.0 to make
solutions so that each mL contains 10 fig (potency) and 5 /ug
(potency), and use these solutions as the high concentration
sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Hermetic containers.
Fosfomycin Sodium for Injection
Fosfomycin Sodium for Injection is a preparation
for injection which is dissolved before use. It contains
not less than 90.0% and not more than 1 10.0% of the
labeled amount of fosfomycin (C 3 H 7 4 P: 138.06).
Method of preparation Prepare as directed under Injec-
tions, with Fosfomycin Sodium.
Description Fosfomycin Sodium for Injection occurs as a
white crystalline powder.
Identification (1) Dissolve about 0.1 g of Fosfomycin So-
dium for Injection in 3 mL of a solution of perchloric acid (1
in 4), add 1 mL of 0.1 mol/L sodium periodate solution, and
heat in a water bath at 60°C for 30 minutes. After cooling,
add 50 mL of water, neutralize with saturated sodium hydro-
gen carbonate solution, and add 1 mL of potassium iodide
TS; the solution does not reveal a red color, while the blank
solution reveals a red color.
(2) To 2 mL of a solution of Fosfomycin Sodium for In-
jection (1 in 250) add 1 mL of perchloric acid and 2 mL of 0. 1
mol/L sodium periodate solution, and heat in a water bath
for 10 minutes. After cooling, add 1 mL of ammonium
molybdate-sulfuric acid TS and 1 mL of l-amino-2-naphtol-
4-sulfonic acid TS, and allow to stand for 30 minutes: a blue
color develops.
(3) Dissolve an amount of Fosfomycin Sodium for Injec-
tion, equivalent to 0.1 g (potency) of Fosfomycin Sodium, in
50 mL of water. Perform the test with this solution as direct-
ed in the Identification (3) under Fosfomycin Sodium.
pH <2.54> The pH of a solution prepared by dissolving an
amount of Fosfomycin Sodium for Injection, equivalent to
1.0 g (potency) of Fosfomycin Sodium according to the la-
beled amount, in 20 mL of water is between 6.5 and 8.5.
Purity Clarity and color of solution — Dissolve an amount
of Fosfomycin Sodium for Injection, equivalent to 1.0 g
(potency) of Fosfomycin Sodium according to the labeled
amount, in 10 mL of water: the solution is clear and color-
less.
Water <2.48> Not more than 4.0% (25 mg, volumetric titra-
tion, direct titration).
Bacterial endotoxins <4.01> Less than 0.025 EU/mg (poten-
cy).
Uniformity of dosage unit <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, seeded agar layer,
and standard solutions — Proceed as directed in the Assay un-
der Fosfomycin Sodium.
(ii) Sample solutions — Weigh accurately the mass of the
contents of not less than 10 Fosfomycin Sodium for Injec-
tion. Weigh accurately an amount of the content, equivalent
to about 20 mg (potency) of Fosfomycin Sodium according
to the labeled amount, and dissolve in 0.05 mol/L tris buffer
solution, pH 7.0 to make exactly 50 mL. Take exactly a suita-
ble amount of this solution, add 0.05 mol/L tris buffer solu-
tion, pH 7.0 to make solutions so that each mL contains 10
fig (potency) and 5 /ug (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
688
Fradiomycin Sulfate / Official Monographs
JP XV
Containers and storage Containers — Hermetic containers.
Polyethylene or polypropylene containers for aqueous injec-
tions may be used.
Fradiomycin Sulfate
Neomycin Sulfate
■ 3H2SO4
H NH 2
Fradiomycin B:R'=H R 2 =CH 2 NH 2
Fradiomycin C^'sCHsNHs R^H
C23H 46 N 6 13 .3H 2 S04: 908.88
Fradiomycin Sulfate B
2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(l -> 4)-
[2,6-diamino-2,6-dideoxy-/?-L-idopyranosyl-(l->3)-/?-D-
ribofuranosyl-(l ->5)]-2-deoxy-D-streptamine trisulfate
[119-04-0, Neomycin B]
Fradiomycin Sulfate C
2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(l -» 4)-
[2,6-diamino-2,6-dideoxy-a-D-glucopyranosyl-(l->3)-/?-D-
ribofuranosyl-(l ->5)]-2-deoxy-D-streptamine trisulfate
[66-86-4, Neomycin C] [1405-10-3, Neomycin Sulfate]
Fradiomycin Sulfate is the sulfate of a mixture of
aminoglycoside substances having antibacterial activity
produced by the growth of Streptomyces fradiae.
It, when dried, contains not less than 623 ng (poten-
cy) and not more than 740 fig (potency) per mg. The
potency of Fradiomycin Sulfate is expressed as mass
(potency) of fradiomycin (C 2 3H 46 N 6 13 : 614.64).
Description Fradiomycin Sulfate occurs as a white to light
yellow powder.
It is freely soluble in water, and practically insoluble in
ethanol (95).
It is hygroscopic.
Identification (1) Dissolve 50 mg each of Fradiomycin
Sulfate and Fradiomycin Sulfate Reference Standard in 1 mL
of water, and use these solutions as the sample solution and
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of methanol, ammonia solution (28) and
dichloromethane (3:2:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly a solution of ninhydrin in ace-
tone (1 in 50) on the plate, and heat at 1 10°C for 15 minutes:
the Rf values of the principal spots from the sample solution
and the standard solution are not different each other.
(2) A solution of Fradiomycin Sulfate (1 in 20) responds
to the Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [ a } 2 °: +53.5 - +59.0° (1 g calculated
on the dried basis, water, 10 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Fradiomycin Sulfate in 10 mL of water is between 5.0 and
7.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Fradiomycin Sulfate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Fradiomycin Sulfate according to Method 1, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.63 g of Fradiomycin
Sulfate in 5 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 50 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 1 fiL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of methanol, ammonia solution (28) and
dichloromethane (3:2:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly a solution of ninhydrin in ace-
tone (1 in 50) on the plate, and heat at 1 10°C for 15 minutes:
the spot at around R f 0.4 from the sample solution is not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 8.0% (0.2 g, in vac-
cum, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Agar medium for seed and base layer
Glucose 1.0 g
Peptone 6.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Sodium chloride 2.5 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients and sterilize. Adjust the pH after
sterilization to 7.8 - 8.0 with sodium hydroxide TS.
(iii) Standard solutions - Weigh accurately an amount of
Fradiomycin Sulfate Reference Standard, previously dried,
equivalent to about 50 mg (potency), dissolve in 0.1 mol/L
phosphate buffer solution for antibiotics, pH 8.0 to make
exactly 50 mL, and use this solution as the standard stock so-
lution. Keep the standard stock solution at 5°C or below and
use within 14 days. Take exactly a suitable amount of the
standard stock solution before use, add 0.1 mol/L phosphate
buffer solution for antibiotics, pH 8.0 to make solutions so
JPXV
Official Monographs / Fructose Injection
689
that each mL contains 80 fig (potency) and 20 fig (potency),
and use these solutions as the high concentration standard
solution and low concentration standard solution, respec-
tively.
(iv) Sample solutions — Weigh accurately an amount of
Fradiomycin Sulfate, previously dried, equivalent to about
50 mg (potency), dissolve in 0.1 mol/L phosphate buffer
solution for antibiotics, pH 8.0 to make exactly 50 mL. Take
exactly a suitable amount of this solution, add 0.1 mol/L
phosphate buffer solution for antibiotics, pH 8.0 to make so-
lutions so that each mL contains 80 fig (potency) and 20 fig
(potency), and use these solutions as the high concentration
sample solution and low concentration sample solution, re-
spectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Fructose
Hr- OOH
hot — r k.
OH H
C 6 H 12 6 : 180.16
/?-D-Fructopyranose [57-48-7]
Fructose,
of C 6 H 12 6
when dried, contains not less than 98.0%
Description Fructose occurs as colorless to white crystals or
crystalline powder. It is odorless and has a sweet taste.
It is very soluble in water, sparingly soluble in ethanol (95)
and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) Add 2 to 3 drops of a solution of Fruc-
tose (1 in 20) to 5 mL of boiling Fehling's TS: a red
precipitate is produced.
(2) Determine the infrared absorption spectrum of Fruc-
tose as directed in the paste method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
pH <2.54> Dissolve 4.0 g of Fructose in 20 mL of water: the
pH of the solution is between 4.0 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 25.0 g
of Fructose in 50 mL of water: the solution is clear and has
no more color than the following control solution.
Control solution: To a mixture of 1.0 mL of Cobaltous
Chloride Stock CS, 3.0 mL of Ferric Chloride Stock CS and
2.0 mL of Cupric Sulfate Stock CS, and add water to make
10.0 mL. To 3.0 mL of the solution add water to make 50
mL.
(2) Acidity — Dissolve 5.0 g of Fructose in 50 mL of
freshly boiled and cooled water, and add 3 drops of
phenolphthalein TS and 0.60 mL of 0.01 mol/L sodium
hydroxide VS: a red color develops.
(3) Chloride <1.03>— Perform the test with 2.0 g of Fruc-
tose. Prepare the control solution with 1.0 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.018%).
(4) Sulfate <1.14>— Perform the test with 2.0 g of Fruc-
tose. Prepare the control solution with 1.0 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.024%).
(5) Sulfite — Dissolve 0.5 g of Fructose in 5 mL of water,
and add 0.25 mL of 0.02 mol/L iodine: the color of the solu-
tion is yellow.
(6) Heavy metals <1.07> — Proceed with 5.0 g of Fructose
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 4 ppm).
(7) Calcium — Dissolve 0.5 g of Fructose in 5 mL of
water, add 2 to 3 drops of ammonia TS and 1 mL of ammoni-
um oxalate TS, and allow to stand for 1 minute: the solution
is clear.
(8) Arsenic <1.11> — Dissolve 1.5 g of Fructose in 5 mL of
water, heat with 5 mL of dilute sulfuric acid and 1 mL of bro-
mine TS on a water bath for 5 minutes, concentrate to 5 mL,
and cool. Perform the test with this solution as the test solu-
tion (not more than 1.3 ppm).
(9) 5-Hydroxymethylfurfurals — Dissolve 5.0 g of Fruc-
tose in 100 mL of water, and read the absorbance at 284 nm
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: the absorbance is not more than 0.32.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 4 g of Fructose, previously
dried, dissolve in 0.2 mL of ammonia TS and 80 mL of
water, and after standing for 30 minutes add water to make
exactly 100 mL, and determine the optical rotation, a D , in a
100-mm cell at 20 ± 1°C as directed under Optical Rotation
Determination <2.49>.
Amount (mg) of C 6 H 12 6 = |« D | x 1087.0
Containers and storage Containers — Tight containers.
Fructose Injection
JMt<tfc
Fructose Injection is an aqueous solution for injec-
tion.
It contains not less than 95% and not more than 105
% of the labeled amount of fructose (C 6 H 12 6 :
180.16).
Method of preparation Prepare as directed under Injec-
tions, with Fructose. No preservative is added.
Description Fructose Injection is a colorless to pale yellow,
clear liquid. It has a sweet taste.
Identification (1) Take a volume of Fructose Injection,
equivalent to 1 g of Fructose according to the labeled
amount, dilute with water or concentrate on a water bath to
20 mL, if necessary, and use this solution as the sample solu-
tion. Add 2 to 3 drops of the sample solution to 5 mL of boil-
ing Fehling's TS: a red precipitate is produced.
(2) To 10 mL of the sample solution obtained in (1) add
0.1 g of resorcinol and 1 mL of hydrochloric acid, and warm
690
Furosemide / Official Monographs
JP XV
in a water bath for 3 minutes: a red color develops.
pH <2.54> 3.0 - 6.5 In the case where the labeled concentra-
tion
of the injection exceeds 5%, dilute to 5% with water before
the test.
Purity (1) Heavy metals <1.07> — Take a volume of Fruc-
tose Injection, equivalent to 5.0 g of Fructose, according to
the labeled amount, and evaporate on a water bath to dry-
ness. With the residue, proceed according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution.
(2) Arsenic <1.11> — Take a volume of Fructose Injection,
equivalent to 1.5 g of Fructose, according to the labeled
amount, dilute with water or concentrate on a water bath to 5
mL, if necessary, and add 5 mL of dilute sulfuric acid and 1
mL of bromine TS. Proceed as directed in the purity (8)
under Fructose.
Residue on ignition <2.44> Measure exactly a volume of
Fructose Injection, equivalent to 2 g of Fructose according to
the labeled amount, evaporate on a water bath to dryness,
and perform the test: the residue weighs not more than 2 mg.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Perform the test with Fructose Injection
stored in a container in a volume exceeding 10 mL: it meets
the requirement.
Assay Measure exactly a volume of Fructose Injection
equivalent to about 4 g of fructose (C 6 H 12 6 ), add 0.2 mL of
ammonia TS, dilute with water to make exactly 100 mL,
shake well, and after allowing to stand for 30 minutes, deter-
mine the optical rotation, a D , in a 100-mm cell at 20 ± 1°C
as directed under Optical Rotation Determination <2.49>.
Amount (mg) of fructose (QH 12 6 ) = |« D | x 1087.0
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Furosemide
"7n-tz5 K
C 12 H U C1N 2 5 S: 330.74
4-Chloro-2-[(furan-2-ylmethyl)amino]-5-sulfamoylbenzoic
acid [54-31-9]
Furosemide, when dried, contains not less than
98.0% and not more than 101.0% of C 12 H n ClN 2 5 S.
Description Furosemide occurs as white, crystals or crystal-
line powder.
It is freely soluble in 7V,/V-dimethylformamide, soluble in
methanol, sparingly soluble in ethanol (99.5), slightly soluble
in acetonitrile and in acetic acid (100), and practically
insoluble in water.
It dissolves in dilute sodium hydroxide TS.
It is gradually colored by light.
Melting point: about 205°C (with decomposition).
Identification (1) Dissolve 25 mg of Furosemide in 10 mL
of methanol. To 1 mL of this solution add 10 mL of 2 mol/L
hydrochloric acid TS. Heat the solution under a reflux con-
denser on a water bath for 15 minutes, cool, and add 18 mL
of sodium hydroxide TS to make weakly acidic: the solution
responds to the Qualitative Tests <1.09> for primary aromatic
amines, producing a red to red-purple color.
(2) Determine the absorption spectrum of a solution of
Furosemide in dilute sodium hydroxide TS (1 in 125,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Furosemide Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Furosemide as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the
spectrum of Furosemide Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Furosemide in 10 mL of a solution of sodium hydroxide (1 in
50): the solution is clear and colorless.
(2) Chloride <1.03> — Dissolve 2.6 g of Furosemide in 90
mL of dilute sodium hydroxide TS, add 2 mL of nitric acid,
and filter. To 25 mL of the filtrate add 6 mL of dilute nitric
acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: To 0.40 mL of 0.01 mol/L hydrochloric acid VS
add 6 mL of dilute nitric acid and water to make 50 mL (not
more than 0.020%).
(3) Sulfate <1.14>— To 20 mL of the filtrate obtained in
(2) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution as follows: To 0.35 mL
of 0.005 mol/L sulfuric acid VS add 1 mL of dilute
hydrochloric acid and water to make 50 mL (not more than
0.030%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of
Furosemide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Related substances — Dissolve 25 mg of Furosemide in
25 mL of the dissolving solution, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add the
dissolving solution to make exactly 200 mL, and use this so-
lution as the standard solution. Perform the test with exactly
20 [iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of each peak
appeared ahead of the peak of furosemide is not more than
2/5 times the peak area of furosemide from the standard so-
lution, the area of each peak appeared behind the peak of
furosemide is not more than 1/4 times the peak area of
furosemide from the standard solution, and the total area of
these peaks is not more than 2 times the peak area of
furosemide from the standard solution.
JPXV
Official Monographs / Furosemide Tablets
691
Dissolving solution — To 22 mL of acetic acid (100) add a
mixture of water and acetonitrile (1:1) to make 1000 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 272 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, tetrahydrofuran and
acetic acid (100) (70:30:1).
Flow rate: Adjust the flow rate so that the retention time of
furosemide is about 18 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of furosemide beginning after the solvent
peak.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the dissolving solution to
make exactly 50 mL. Confirm that the peak area of
furosemide obtained from 20 /xh of this solution is equivalent
to 3.2 to 4.8% of that obtained from 20 /xL of the standard
solution.
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of furosemide is not less than 7000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
furosemide is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Furosemide, previ-
ously dried, dissolve in 50 mL of 7V,iV-dimethylformamide,
and titrate <2.50> with 0.1 mol/L sodium hydroxide VS until
the color of the solution changes from yellow to blue (indica-
tor: 3 drops of bromothymol blue TS). Perform a blank de-
termination with a mixture of 50 mL of TV.Af-dimethylfor-
mamide and 15 mL of water, and make any necessary correc-
tion
Each mL of 0.1 mol/L sodium hydroxide VS
= 33.07 mg of C 12 H„C1N 2 5 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Furosemide Tablets
Furosemide Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of
furosemide (C 12 H n ClN 2 5 S: 330.74).
Method of preparation Prepare as directed under Tablets,
with Furosemide.
Identification (1) Shake well a quantity of powdered
Furosemide Tablets, equivalent to 0.2 g of Furosemide
according to the labeled amount, with 40 mL of acetone, and
filter. To 0.5 mL of the filtrate add 10 mL of 2 mol/L
hydrochloric acid TS, and heat under a reflux condenser on a
water bath for 15 minutes. After cooling, add 18 mL of
sodium hydroxide TS to make the solution slightly acetic: the
solution responds to the Qualitative Tests <1.09> for primary
aromatic amines, producing a red to red-purple color.
(2) Determine the absorption spectrum of the sample
solution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits maxima between
227 nm and 231 nm, between 269 nm and 273 nm, and
between 330 nm and 336 nm.
Purity To a quantity of powdered Furosemide Tablets,
equivalent to 40 mg of Furosemide according to the labeled
amount, add about 30 mL of acetone, shake well, and add
acetone to make exactly 50 mL. Centrifuge the solution, add
3.0 mL of water to 1.0 mL of the supernatant liquid, cool in
ice, add 3.0 mL of dilute hydrochloric acid and 0.15 mL of
sodium nitrite TS, shake, and allow to stand for 1 minute.
Add 1.0 mL of ammonium amidosulfate TS, shake well,
allow to stand for 3 minutes, add 1.0 mL of A^TV-diethyl-TV'-
1-naphthylethylenediamine oxalate TS, shake well, and allow
to stand for 5 minutes. Perform the test with this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution prepared in the same manner with 1.0 mL of
acetone as the blank: the absorbance at 530 nm is not more
than 0.10.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Furosemide Tablets add a suitable amount
of 0.05 mol/L sodium hydroxide TS, shake to disintegrate,
then add 0.05 mol/L sodium hydroxide TS to make exactly
FmL so that each mL contains about 0.4 mg of furosemide
(C 12 H U C1N 2 5 S). Filter the solution, discard the first 10 mL
of the filtrate, pipet the subsequent 2 mL of the filtrate, add
0.05 mol/L sodium hydroxide TS to make exactly 100 mL,
and use this solution as the sample solution. Proceed as
directed in the Assay.
Amount (mg) of furosemide (C 12 H U C1N 2 5 S)
= W s x (Aj/As) x (F/50)
W s : Amount (mg) of Furosemide Reference Standard
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Furosemide Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of 2nd fluid for dissolution test as the dissolution
medium. Withdraw 20 mL or more of the dissolution medi-
um 15 minutes after starting the test for a 20-mg tablet or 30
minutes after for a 40-mg tablet, and filter through a mem-
brane filter with pore size of not more than 0.45 fim. Discard
the first 10 mL of the filtrate, pipet KmL of the subsequent
filtrate, add 2nd fluid for dissolution test to make exactly V
mL so that each mL contains about 10 /ug of furosemide (C 12
H U C1N 2 5 S) according to the labeled amount, and use this
solution as the sample solution. Separately, weigh accurately
about 20 mg of Furosemide Reference Standard, previously
692
Fursultiamine Hydrochloride / Official Monographs
JP XV
dried at 105 °C for 4 hours, and dissolve in 5 mL of
methanol, and add 2nd fluid for dissolution test to make ex-
actly 100 mL. Pipet 5 mL of this solution, add 2nd fluid for
dissolution test to make exactly 100 mL, and use this solution
as the standard solution. Determine the absorbances, A T and
^4 S > of the sample solution and the standard solution at 277
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>: the dissolution rates for a 20-mg tablet in 15 minutes
and for a 40-mg tablet in 30 minutes are not less than 80%,
respectively.
Dissolution rate (%) with respect to the labeled amount of
furosemide (C 12 H n ClN 2 5 S)
= W s x (Aj/A s ) x (V'/V) x (1/Q x 45
W s : Amount (mg) of Furosemide Reference Standard
C: Labeled amount (mg) of furosemide (C 12 H U C1N 2 05S)
in 1 tablet
Assay Weigh accurately the mass of not less than 20
Furosemide Tablets, and powder. Weigh accurately a portion
of the powder, equivalent to about 40 mg of furosemide
(C 12 H„C1N 2 5 S), add about 70 mL of 0.05 mol/L sodium
hydroxide TS, shake well, and add 0.05 mol/L sodium
hydroxide TS to make exactly 100 mL. Filter, discard the first
10 mL of the filtrate, pipet 2 mL of the subsequent filtrate,
add 0.05 mol/L sodium hydroxide TS to make exactly 100
mL, and use this solution as the sample solution. Separately,
weigh accurately about 20 mg of Furosemide Reference Stan-
dard, previously dried at 105 °C for 4 hours, and dissolve in
0.05 mol/L sodium hydroxide TS to make exactly 50 mL.
Pipet 2 mL of this solution, add 0.05 mol/L sodium
hydroxide TS to make exactly 100 mL, and use this solution
as the standard solution. Determine the absorbances, A T and
A s , of the sample solution and standard solution at 271 nm as
directed under the Ultraviolet-visible Spectrophotometry
<2.24>.
Amount (mg) of furosemide (C I2 H H C1N 2 5 S)
= Ws X (,4 T A4 S ) x 2
W s : Amount (mg) of Furosemide Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Fursultiamine Hydrochloride
HCI
C 17 H 26 N 4 3 S 2 .HC1: 435.00
Af-(4-Amino-2-methylpyrimidin-5-ylmethyl)-A r -{(lZ)-4-
hydroxy-l-methyl-2-[(2/?5')-tetrahydrofuran-2-ylmethyl-
disulfanyl]but-l-en-l-yl} formamide monohydrochloride
[804-30-8, Fursultiamine]
Fursultiamine Hydrochloride contains not less than
98.5% of C 17 H 26 N 4 3 S 2 .HC1, calculated on the dried
basis.
Description Fursultiamine Hydrochloride occurs as white
crystals or crystalline powder. It is odorless or has a charac-
teristic odor, and has a bitter taste.
It is freely soluble in water, in methanol and in ethanol
(95), and practically insoluble in diethyl ether.
Identification (1) Dissolve 5 mg of Fursultiamine
Hydrochloride in 6 mL of 0.1 mol/L hydrochloric acid TS,
add 0.1 g of zinc powder, allow to stand for several minutes,
and filter. To 3 mL of the filtrate, add 3 mL of sodium
hydroxide TS and 0.5 mL of potassium hexacyanoferrate
(III) TS, then add 5 mL of 2-methyl-l-propanol, shake
vigorously for 2 minutes, allow to stand to separate the 2-
methyl-1-propanol layer, and examine under ultraviolet light
(main wavelength: 365 nm): the 2-methyl-l-propanol layer
shows a blue-purple fluorescence. The fluorescence disap-
pears by acidifying, and appears again by alkalifying.
(2) Determine the infrared absorption spectrum of a solu-
tion of Fursultiamine Hydrochloride, previously dried in a
desiccator (in vacuum, phosphorus (V) oxide) for 24 hours,
as directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum, or with the spectrum of Fursul-
tiamine Hydrochloride Reference Standard, previously dried
in a desiccator (in vacuum, phosphorus (V) oxide) for 24
hours: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any differences appear, dissolve
the Fursultiamine Hydrochloride in water, evaporate the
water, and dry the residue in a desiccator (in vacuum, phos-
phorus (V) oxide) for 24 hours, and repeat the test.
(3) A solution of Fursultiamine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
Purity (1) Clarity of solution — Dissolve 1.0 g of Fursul-
tiamine Hydrochloride in 20 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14> — Proceed with 1.5 g of Fursultiamine
Hydrochloride, and perform the test. Prepare the control so-
lution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not
more than 0.011%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Fursul-
tiamine Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(4) Related substances — Dissolve 0.10 g of Fursultiamine
Hydrochloride in 100 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following condition. Determine each peak area
of each solution by the automatic integration method: the
total area of the peaks other than the peak of fursultiamine
from the sample solution is not larger than the peak area of
fursultiamine from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed as directed in the
operating conditions in the Assay.
Detector sensitivity: Adjust the detection sensitivity so that
JPXV
Official Monographs / Gabexate Mesilate
693
the peak height of fursultiamine from 10 juL of the standard
solution is between 20 mm and 30 mm.
Time span of measurement: About 3 times as long as the
retention time of fursultiamine.
Water <2.48> Not more than 5.0% (0.3 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 55 mg each of Fursultiamine
Hydrochloride and Fursultiamine Hydrochloride Reference
Standard (previously determined the water <2.48> in the same
manner as Fursultiamine Hydrochiloride) and dissolve each
in 50 mL of water, and add exactly 10 mL each of the internal
standard solution, then add water to make exactly 100 mL.
To 8 mL each of the solution add water to make 50 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with 10 ^L each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Q s , of the peak area
of fursultiamine to that of the internal standard, respectively.
Amount (mg) of C 17 H 26 N 4 3 S 2 .HC1
= W s x (Q T /Q S )
W s : Amount (mg) of Fursultiamine Hydrochloride Refer-
ence Standard, calculated on the anhydrous basis
Internal standard solution — A solution of isopropyl 4-
aminobenzoate in ethanol (95) (3 in 400).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 1.01 g of sodium 1-heptane sul-
fonate in 1000 mL of diluted acetic acid (100) (1 in 100). To
675 mL of this solution add 325 mL of a mixture of methanol
and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
Fursultiamine is about 9 minutes.
Selection of column: Proceed with 10 /xL of the standard
solution under the above operating conditions and calculate
the resolution. Use a column giving elution of fursultiamine
and the internal standard in this order with the resolution be-
tween these peaks being not less than 10.
Containers and storage Containers — Tight containers.
Gabexate Mesilate
. H^-SQjH
C 16 H 23 N304.CH 4 03S: 417.48
Ethyl 4-(6-guanidinohexanoyloxy)benzoate
monomethanesulfonate [56974-61-9]
Gabexate Mesilate, when dried, contains not less
than 98.5% of QgH^CVCrLAS.
Description Gabexate Mesilate occurs as white crystals or
crystalline powder.
It is very soluble in water, freely soluble in ethanol (95),
and practically insoluble in diethyl ether.
Identification (1) To 4 mL of a solution of Gabexate
Mesilate (1 in 2000) add 2 mL of 1-naphthol TS and 1 mL of
diacetyl TS, and allow to stand for 10 minutes: a red color
develops.
(2) Dissolve 1 g of Gabexate Mesilate in 5 mL of water,
add 2 mL of sodium hydroxide TS, and heat in a water bath
for 5 minutes. After cooling, add 2 mL of dilute nitric acid
and 5 mL of ethanol (95), shake, add 5 drops of iron (III)
chloride TS, and shake: a purple color develops.
(3) Determine the absorption spectrum of a solution of
Gabexate Mesilate (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Gabexate Mesilate Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) To 0.1 g of Gabexate Mesilate add 0.2 g of sodium
hydroxide, fuse by heating gently, and continue the heating
for 20 to 30 seconds. After cooling, add 0.5 mL of water and
3 mL of dilute hydrochloric acid, and warm: the gas evolved
changes a potassium iodate-starch paper to blue.
pH <2.54> Dissolve 1.0 g of Gabexate Mesilate in 10 mL of
water: the pH of the solution is between 4.5 and 5.5.
Melting point <2.60> 90 - 93 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Gabexate Mesilate in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Gabexate
Mesilate according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11> — Dissolve 2.0 g of Gabexate Mesilate
in 20 mL of 1 mol/L hydrochloric acid TS by heating in a
water bath, and continue the heating for 20 minutes. After
cooling, centrifuge, and use 10 mL of the supernatant liquid
as the test solution. Perform the test (not more than 2 ppm).
(4) Ethyl parahydroxybenzoate — Weigh 50 mg of
Gabexate Mesilate, previously dried, and dissolve in dilute
ethanol to make exactly 100 mL. Pipet 5 mL of this solution,
add exactly 5 mL of the internal standard solution, and use
this solution as the sample solution. Separately, dissolve 5.0
mg of ethyl parahydroxybenzoate in dilute ethanol to make
exactly 100 mL. Pipet 1 mL of this solution, and add dilute
ethanol to make exactly 20 mL. To exactly 5 mL of this solu-
tion add exactly 5 mL of the internal standard solution, and
use this solution as the standard solution. Perform the test
with 3 //L each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Qs, of the peak area of ethyl parahydroxybenzoate to that of
the internal standard: Q T is not larger than Qs-
694 jS-Galactosidase (Aspergillus) / Official Monographs
JP XV
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in dilute ethanol (1 in 5000).
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
Proceed as directed in the system suitability in the Assay.
(5) Related substances — Dissolve 0.20 g of Gabexate
Mesilate in 5 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
ethanol (95) to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
jXL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, water and acetic acid
(100) (3:1:1) to a distance of about 10 cm, and air-dry the
plate until it has no acetic odor. Spray evenly a solution of 8-
quinolinol in acetone (1 in 1000) on the plate, and after air-
drying, spray evenly bromine-sodium hydroxide TS: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.30% (1 g, in vacu-
um, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Gabexate
Mesilate and Gabexate Mesilate Reference Standard, previ-
ously dried, and dissolve each in dilute ethanol to make ex-
actly 100 mL. Pipet 5 mL each of these solutions, add exactly
5 mL each of the internal standard solution, and use these so-
lutions as the sample solution and standard solution, respec-
tively. Perform the test with 3 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
gabexate to that of the internal standard.
Amount (mg) of C^H^CvCH^S
= W s x (Qt/Qs)
W s : Amount (mg) of Gabexate Mesilate Reference Stan-
dard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in dilute ethanol (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 245 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol, a solution of sodi-
um lauryl sulfate (1 in 1000), a solution of sodium 1-heptane
sulfonate (1 in 200) and acetic acid (100) (540:200:20:1).
Flow rate: Adjust the flow rate so that the retention time of
gabexate is about 13 minutes.
System suitability —
System performance: When the procedure is run with 3 fiL
of the standard solution under the above operating condi-
tions, the internal standard and gabexate are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
3 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of gabexate to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
/?-Galactosidase (Aspergillus)
[9031-11-2]
/?-Galactosidase (Aspergillus) contains an enzyme
produced by Aspergillus oryzae. It is an enzyme drug
having lactose decomposition activity.
It contains 8000 to 12000 units per g.
Usually, it is diluted with a mixture of Maltose Hy-
drate and Dextrin, Maltose Hydrate and D-Mannitol,
or Maltose Hydrate, Dextrin and D-Mannitol.
Description /?-Galactosidase (Aspergillus) occurs as a white
to light yellow powder.
It is slightly soluble in water with a turbidity, and practical-
ly insoluble in ethanol (95) and in diethyl ether.
Identification (1) Dissolve 25 mg of /?-Galactosidase
(Aspergillus) in 100 mL of water, then to 1 mL of this solu-
tion add 9 mL of lactose substrate TS, and stand at 30°C for
10 minutes. To 1 mL of this solution add 6 mL of glucose de-
tection TS, and stand at 30°C for 10 minutes: a red to red-
purple color develops.
(2) Dissolve 0.1 g of /?-Galactosidase (Aspergillus) in 100
mL of water, and filter the solution if necessary. Determine
the absorption spectrum of the solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
Purity (1) Odor — /?-Galactosidase (Aspergillus) has no
any rancid odor.
(2) Heavy metals <1. 07>— Proceed with 1 .0 g of /?-Galac-
tosidase (Aspergillus) according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of /?-Galactosidase (Aspergillus) according to Method 3, and
perform the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 9.0% (0.5 g, in vacu-
um, 80°C, 4 hours).
Residue on ignition <2.44> Not more than 3% (0.5 g).
Nitrogen content Weigh accurately about 70 mg of /?-
Galactosidase (Aspergillus), and perform the test as directed
under Nitrogen Determination <1.08>: the amount of nitro-
gen (N: 14.01) is between 0.5% and 5.0%, calculated on the
dried basis.
JPXV
Official Monographs / /?-Galactosidase (Penicillium)
695
Assay (i) Substrate solution: Dissolve 0.172 g of 2-
nitrophenyl-/M>galactopyranoside in disodium hydrogen-
phosphate-citric acid buffer solution, pH 4.5 to make 100
mL.
(ii) Procedure: Weigh accurately about 25 mg of /?-
Galactosidase (Aspergillus), dissolve in water to make exactly
100 mL, then pipet 2 mL of this solution, add water to make
exactly 50 mL, and use this solution as the sample solution.
Take exactly 3.5 mL of the substrate solution, stand at 30 ±
0.1 °C for 5 minutes, add exactly 0.5 mL of the sample solu-
tion, immediately mix, and stand at 30 ± 0.1 °C for exactly
10 minutes, then add exactly 1 mL of sodium carbonate TS
and mix immediately. Perfoum the test as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and determine
the absorbance, Ay, of this solution at 420 nm using water as
the control. Separately, take exactly 3.5 mL of the substrate
solution, add exactly 1 mL of sodium carbonate TS and mix,
then add exactly 0.5 mL of the sample solution and mix. De-
termine the absorbance, A 2 , of this solution in the same man-
ner as above.
Units per g of /3-Galactosidase (Aspergillus)
= (1/W)x {(Ay-A 2 )/0.9\1\ x (1/0.5) X (1/10)
0.917: Absorbance of 1 ^mol/5 mL of o-nitrophenol
W: Amount (g) of the sample in the sample solution per
mL
Unit: One unit indicates an amount of the enzyme which
decomposes 1 //mol of 2-nitrophenyl-/?-D-galac-
topyranoside in 1 minute under the above conditions.
Containers and storage Containers — Tight containers.
Storage — In a cold place.
/?-Galactosidase (Penicillium)
[9031-11-2]
/?-Galactosidase (Penicillium) contains an enzyme,
having lactose decomposition activity, produced by
Penicillium multicolor.
It contains not less than 8500 units and not more
than 11,500 units in each g.
Usually, it is diluted with D-mannitol.
Description /?-Galactosidase (Penicillium) occurs as a white
to pale yellowish white, crystalline powder or powder.
It is soluble in water with a turbidity, and practically in-
soluble in ethanol (95).
It is hygroscopic.
Identification (1) Dissolve 0.05 g of /?-Galactosidase
(Penicillium) in 100 mL of water, then to 0.2 mL of this solu-
tion add 0.2 mL of lactose substrate TS, and allow to stand at
30°C for 10 minutes. To this solution add 3 mL of glucose
detection TS, and allow to stand at 30°C for 10 minutes: a
red to red-purple color develops.
(2) Dissolve 0.15 g of /?-Galactosidase (Penicillium) in
100 mL of water, filter if necessary, and determine the ab-
sorption spectrum of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 278 nm and 282 nm.
Purity (1) Odor — /?-Galactosidase (Penicillium) has no
any rancid odor.
(2) Heavy metals <1. 07>— Proceed with 1 .0 g of /?-Galac-
tosidase (Penicillium) according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of /?-Galactosidase (Penicillium) according to Method 3, and
perform the test (not more than 2 ppm).
(4) Nitrogen — Weigh accurately about 0.1 g of /?-Galac-
tosidase (Penicillium), and perform the test as directed under
Nitrogen Determination <1.08>: not more than 3 mg of nitro-
gen (N: 14.01) is found for each labeled 1000 Units.
(5) Protein contaminants — Dissolve 0.15 g of /?-Galac-
tosidase (Penicillium) in 4 mL of water, and use this solution
as the sample solution. Perform the test with 15 liL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
each peak area by the automatic integration method: the total
area of the peaks other than the peak having retention time of
about 19 minutes is not more than 75% of the total area of all
peaks, and the areas of peaks other than the peaks having
retention times of about 3, 16 and 19 minutes are not more
than 15% of the total area of all peaks.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column about 7.5 mm in inside
diameter and about 75 mm in length, packed with strongly
acidic ion-exchange resin for liquid chromatography of sul-
fopropyl group-binding hydrophilic polymer (10 fim in parti-
cle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A solution obtained by dissolving 2.83 g of
sodium acetate in 1000 mL of water, and adjusting to pH 4.5
with acetic acid (100) (mobile phase A), and a solution ob-
tained by dissolving 29.2 g of sodium chloride in 1000 mL of
mobile phase A (mobile phase B).
Flow system: Adjust a linear concentration gradient from
the mobile phase A to the mobile phase B immediately after
injection of the sample so that the retention times of non-
retaining protein and the enzyme protein are about 3 minutes
and 19 minutes, respectively, when the flow runs 0.8 mL per
minute, and then continue the running of the mobile phase B.
Selection of column: Dissolve 15 mg of /Mactoglobulin in
4.5 mL of water, add 0.5 mL of a solution of cytosine (1 in
5000), and use this solution as the column-selecting solution.
Proceed with 15 LiL of the column-selecting solution under
the above operating conditions, and calculate the resolution.
Use a column giving elution of cytosine and /Mactoglobulin
in this order with the resolution between these peaks being
not less than 4.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of /Mactoglobulin from 15 liL of the
column-selecting solution is between 5 cm and 14 cm.
Time span of measurement: About 1.4 times as long as the
retention time of /Mactoglobulin.
Loss on drying <2.41> Not more than 5.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 2% (1 g).
696
Gallium ( 67 Ga) Citrate Injection / Official Monographs
JP XV
Assay (i) Substrate solution — Dissolve 0.603 g of 2-
nitrophenyl-/?-D-galactopyranoside in disodium hydrogen
phosphate-citric acid buffer solution, pH 4.5 to make 100
mL.
(ii) Procedure — Weigh accurately about 0.15 g of /?-
Galactosidase (Penicillium), dissolve in water with thorough
shaking to make exactly 100 mL, and allow to stand at room
temperature for an hour. Pipet 2 mL of this solution, add
disodium hydrogen phosphate-citric acid buffer solution, pH
4.5 to make exactly 100 mL, and use this solution as the sam-
ple solution. Transfer exactly 0.5 mL of the sample solution
to a test tube, stand at 30 ± 0.1 °C for 10 minutes, add exact-
ly 0.5 mL of the substrate solution previously kept at 30 ±
0.1 °C, then mix immediately, and stand at 30 ± 0.1 °C for
exactly 10 minutes. Then add exactly 1 mL of sodium car-
bonate TS, mix immediately to stop the reaction. To this so-
lution add exactly 8 mL of water, mix, and use as the colored
sample solution. Separately, pipet 0.5 mL of disodium
hydrogen phosphate-citric acid buffer solution, pH 4.5, then
proceed in the same manner as the sample solution, and use
the solution so obtained as the colored blank solution. Per-
form the test with the colored sample solution and the
colored blank solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using water as the blank, and de-
termine the absorbances, A T and A B , at 420 nm.
Units per g of y5-Galactosidase (Penicillium)
= (l/W)x {(A T -A B )/0A59} x(l/10)
0.459: Absorbance of 1 ^mol/10mL of o-nitrophenol
W: Amount (g) of the sample in 0.5 mL of the sample solu-
tion
Unit: One unit indicates an amount of the enzyme which
decomposes 1 ^mol of 2-nitrophenyl-/?-D-galactopyra-
noside in 1 minute under the above conditions.
Containers and storage Containers — Tight containers.
Gallium ( 67 Ga) Citrate Injection
?i >mii U ^ A ( 67 Ga) £#};«
Gallium ( 67 Ga) Citrate Injection is an aqueous solu-
tion for injection containing gallium-67 ( 67 Ga) in the
form of gallium citrate.
It conforms to the requirements of Gallium ( 67 Ga)
Citrate Injection in the Minimum Requirements for
Radiopharmaceuticals .
Test for Extractable volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Gallium ( 67 Ga) Citrate Injection is a clear,
colorless or light red liquid.
Gas Gangrene Antitoxin, Equine
Gas Gangrene Antitoxin, Equine, is a liquid for in-
jection containing Clostridium perfringens (C. welchii)
Type A antitoxin, Clostridium septicum (Vibrion sep-
tique) antitoxin and Clostridium oedematiens (C.
novyi) antitoxin in immunoglobulin of horse origin.
It may contain also Clostridium histolyticum an-
titoxin.
It conforms to the requirements of Gas Gangrene
Antitoxin, Equine, in the Minimum Requirements for
Biological Products.
Description Gas Gangrene Antitoxin, Equine, is a colorless
to light yellow-brown, clear liquid or a slightly whitish turbid
liquid.
Gelatin
■If7f>
Gelatin is a product prepared from aqueous extract
of raw collagen by heating. The raw collagen is ob-
tained by acid or alkali treatment of the bone, skin,
ligament or tendon of animals.
Description Gelatin occurs as colorless or white to light yel-
low-brown sheets, shreds, granules or powder. It is odorless
and tasteless.
Gelatin is very soluble in hot water, and practically insolu-
ble in ethanol (95) and in diethyl ether.
Gelatin does not dissolve in water, but slowly swells and
softens when immersed in it, gradually absorbing water 5 to
10 times its own mass.
Gelatin derived from an acid-treated collagen exhibits an
isoelectric point between pH 7.0 and 9.0, and Gelatin derived
from an alkali-treated collagen exhibits an isoelectric point
between pH 4.5 and 5.0.
Identification (1) To 5 mL of a solution of Gelatin (1 in
100) add chromium (VI) oxide TS or 2,4,6-trinitrophenol TS
dropwise: a precipitate is formed.
(2) To 5 mL of a solution of Gelatin (1 in 5000) add tan-
nic acid TS dropwise: the solution becomes turbid.
Purity (1) Foreign odor and water-insoluble substan-
ces — Dissolve 1.0 g of Gelatin in 40 mL of water by heating:
the solution has no disagreeable odor. It is clear, or only
slightly opalescent. The solution has no more color than
Matching Fluid A.
(2) Sulfite — Take 20.0 g of Gelatin in a round-bottomed
flask, dissolve in 150 mL of hot water, and add 3 to 5 drops
of silicone resin, 5 mL of phosphoric acid and 1 g of sodium
hydrogen carbonate. Attach a condenser, immediately distil
the solution, immersing the end of the condenser into a
receiver containing 50 mL of iodine TS, and continue the dis-
tillation until 50 mL of distillate is obtained. Acidify the dis-
tillate with 2 to 3 drops of hydrochloric acid, add 2 mL of
barium chloride TS, and heat on a water bath until the color
of iodine TS is discharged. Collect the precipitates, wash with
water, and ignite: the mass of the residue is not more than 4.5
mg, but the mass of the residue obtained from Gelatin for use
in the preparation of capsules and tablets is not more than 75
mg. Perform a blank determination, and make any necessary
correction.
(3) Heavy metals <1.07> — Proceed with 0.5 g of Gelatin
JPXV
Official Monographs / Purified Gelatin 697
according to Method 2, and perform the test. Prepare the
control solution with 2.5 mL of Standard Lead Solution (not
more than 50 ppm).
(4) Arsenic <1.11> — Take 15.0 g of Gelatin in a flask, add
60 mL of diluted hydrochloric acid (1 in 5), and heat until so-
lution is effected. Add 15 mL of bromine TS, heat until the
excess of bromine is expelled, neutralize with ammonia TS,
add 1.5 g of disodium hydrogen phosphate dodecahydrate,
and allow to cool. To this solution add 30 mL of magnesia
TS, allow to stand for 1 hour, and collect the precipitates.
Wash the precipitates with five 10-mL portions of diluted am-
monia TS (1 in 4), and dissolve in diluted hydrochloric acid (1
in 4) to make exactly 50 mL. Perform the test with 5 mL of
this solution: the solution has no more color than the follow-
ing standard stain.
Standard stain: Proceed with 15 mL of Standard Arsenic
Solution, instead of Gelatin, in the same manner (not more
than 1 ppm).
(5) Mercury — Place 2.0 g of Gelatin in a decomposition
flask, add 20 mL of diluted sulfuric acid (1 in 2) and 100 mL
of a solution of potassium permanganate (3 in 50), heat gent-
ly under a reflux condenser, and boil for 2 hours. If the solu-
tion becomes clear during boiling, reduce the temperature of
the solution to about 60°C, add further 5 mL of a solution of
potassium permanganate (3 in 50), boil again, and repeat the
above-mentioned procedure until the precipitate of man-
ganese dioxide remains for about 20 minutes. Cool, add a so-
lution of hydroxylammonium chloride (1 in 5) until the
precipitate of manganese dioxide disappears, add water to
make exactly 150 mL, and use the solution as the sample so-
lution. Perform the test as directed under Atomic Absorption
Spectrophotometry <2.23> (Cold vapor type) using the sample
solution. Place the sample solution in a sample water bottle
of the atomic absorption spectrophotometer, add 10 mL of
tin (II) chloride-sulfuric acid TS, connect the bottle immedi-
ately to the atomic absorption spectrophotometer, and circu-
late air. Determine the absorbance A T of the sample solution
at 253.7 nm when the indication of the recorder has risen
rapidly and become constant. On the other hand, place 2.0
mL of Standard Mercury Solution in a decomposition flask,
add 20 mL of diluted sulfuric acid (1 in 2) and and 100 mL of
a solution of potassium permanganate (3 in 50), and proceed
in the same manner as for the sample solution. Determine the
absorbance A s of the standard solution: A T is not more than
A s (not more than 0.1 ppm).
Loss on drying Not more than 15.0%. Take about 1 g of
Gelatin, accurately weighed, in a tared 200-mL beaker con-
taining 10 g of sea sand (No. 1) previously dried at 110°C for
3 hours. Add 20 mL of water, allow to stand for 30 minutes
with occasional shaking, evaporate to dryness on a water
bath with occasional shaking, and dry the residue at 110°C
for 3 hours.
Residue on ignition <2.44> Not more than 2.0% (0.5 g).
Containers and storage Containers — Tight containers.
Purified Gelatin
Purified Gelatin is a product prepared from aqueous
extract of raw collagen by heating. The raw collagen is
obtained by acid or alkali treatment of the bone, skin,
ligament, or tendon of animals.
Description Purified Gelatin occurs as colorless to light yel-
low sheets, shreds, pellets or powder. It is odorless and taste-
less.
It is very soluble in hot water, and practically insoluble in
ethanol (95) and in diethyl ether.
Purified Gelatin does not dissolve in water. It slowly swells
and softens when immersed in water, and absorbs water 5 to
10 times its own mass.
Purified Gelatin derived from an acid-treated collagen has
an isoelectric point at pH 7.0 to 9.0, and Purified Gelatin der-
ived from an alkali-treated collagen has an isoelectric point at
pH 4.5 to 5.0.
Identification (1) To 5 mL of a solution of Purified Gela-
tin (1 in 100) add chromium (VI) oxide TS or 2,4,6-
trinitrophenol TS dropwise: a precipitate is formed.
(2) To 5 mL of a solution of Purified Gelatin (1 in 5000)
add tannic acid TS dropwise: the solution becomes turbid.
Purity (1) Foreign odor and water-insoluble substan-
ces — Dissolve 1.0 g of Purified Gelatin in 40 mL of water by
heating: the solution is clear, colorless and free from any dis-
agreeable odor when the layer of the solution is 20 mm in
depth.
(2) Sulfite — Take 20.0 g of Purified Gelatin in a round-
bottomed flask, dissolve in 150 mL of hot water, and add 3 to
5 drops of silicone resin, 5 mL of phosphoric acid and 1 g of
sodium hydrogen carbonate. Attach a condenser, immediate-
ly distil the solution, immersing the end of the condenser into
a receiver containing 50 mL of iodine TS, and continue the
distillation until 50 mL of distillate is obtained. Acidify the
distillate by dropwise addition of hydrochloric acid, add 2
mL of barium chloride TS, and heat on a water bath until the
color of iodine TS is discharged. Collect the precipitates,
wash with water, and ignite: the mass of the residue is not
more than 1.5 mg. Perform a blank determination, and make
any necessary correction.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Purified
Gelatin according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(4) Arsenic <1.11> — Place 15.0 g of Purified Gelatin in a
flask, add 60 mL of diluted hydrochloric acid (1 in 5), and
heat until solution is effected. Add 15 mL of bromine TS,
heat until the excess of bromine is expelled, neutralize with
ammonia TS, add 1.5 g of disodium hydrogen phosphate
dodecahydrate, and allow to cool. To this solution add 30
mL of magnesia TS, allow to stand for 1 hour, and collect the
precipitates. Wash the precipitates with five 10-mL portions
of diluted ammonia TS (1 in 4), and dissolve in diluted
hydrochloric acid (1 in 4) to make exactly 50 mL. Perform
the test with 5 mL of this solution: the solution has no more
color than the following standard solution.
Standard solution: Proceed with 15 mL of Standard Ar-
senic Solution, instead of Purified Gelatin, in the same man-
ner (not more than 1 ppm).
(5) Mercury — Place 2.0 g of Purified Gelatin in a decom-
position flask, add 20 mL of diluted sulfuric acid (1 in 2) and
100 mL of a solution of potassium permanganate (3 in 50),
heat gently under a reflux condenser, and boil for 2 hours. If
698
Gentamicin Sulfate / Official Monographs
JP XV
the solution becomes clear during boiling, reduce the temper-
ature of the solution to about 60°C, add further 5 mL of a so-
lution of potassium permanganate (3 in 50), boil again, and
repeat the above-mentioned procedure until the precipitate of
manganese dioxide remains for about 20 minutes. Cool, add
a solution of hydroxylammonium chloride (1 in 5) until the
precipitate of manganese dioxide disappears, add water to
make exactly 150 mL, and use the solution as the sample so-
lution. Perform the test as directed under Atomic Absorption
Spectrophotometry <2.23> (Cold vapor type) using the sample
solution. Place the sample solution in a sample water bottle
of the atomic absorption spectrophotometer, add 10 mL of
tin (II) chloride-sulfuric acid TS, connect the bottle immedi-
ately to the atomic absorption spectrophotometer, and circu-
late air. Determine the absorbance A T of the sample solution
at 253.7 nm when the indication of the recorder has risen
rapidly and become constant. On the other hand, place 2.0
mL of Standard Mercury Solution in a decomposition flask,
add 20 mL of diluted sulfuric acid (1 in 2) and 100 mL of a
solution of potassium permanganate (3 in 50), and proceed in
the same manner as for the sample solution. Determine the
absorbance AS of the standard solution: A T is not more than
A s (not more than 0.1 ppm).
Loss on drying Not more than 15.0%. Take about 1 g of
Purified Gelatin, accurately weighed, in a tared 200-mL beak-
er containing 10 g of sea sand (No. 1), previously dried at 110
°C for 3 hours. Add 20 mL of water, allow to stand for 30
minutes with occasional shaking, evaporate on a water bath
to dryness with occasional shaking, and dry the residue at 1 10
°C for 3 hours.
Residue on ignition <2.44> Not more than 2.0% (0.5 g).
Containers and storage Containers — Tight containers.
Gentamicin Sulfate
?>* Vf <>>«»»
iHjSOj
NH 2
Gentamicin Sulfate Ci : R' = CH 3 R z = NHCH3
Gentamicin Sulfate C? : Ft' = CH 3 ft a = NH £
Gentamicin Sultate C,„ ; R 1 = H R 2 = NH 2
Gentamicin Sulfate Q:
(6/?)-2-Amino-2,3,4,6-tetradeoxy-6-methylamino-
6-methyl-a-D-ery?/!ro-hexopyranosyl-(l -» 4)-[3-deoxy-
4-C-methyl-3-methylamino-/?-L-arabinopyranosyl-(l->6)]-
2-deoxy-D-streptamine sulfate
Gentamicin Sulfate C 2 :
(6i?)-2,6-Diamino-2,3,4,6-tetradeoxy-6-methyl-a-D-ery?/!ro-
hexopyranosyl-(l- > 4)-[3-deoxy-4-C-methyl-
3-methylamino-/?-L-arabinopyranosyl-(l -> 6)]-2-deoxy-
D-streptamine sulfate
Gentamicin Sulfate C la :
2,6-Diamino-2,3,4,6-tetradeoxy-a-D-ery?/!ro-
hexopyranosyl-(l- > 4)-[3-deoxy-4-C-methyl-3-
methylamino-/?-L-arabinopyranosyl-(l -> 6)]-2-deoxy-
D-streptamine sulfate
[1405-41-0, Gentamicin Sulfate]
Gentamicin Sulfate is the sulfate of a mixture of
aminoglycoside substances having antibacterial activity
produced by the growth of Micromonospora purpurea
or Micromonospora echinospora.
It contains not less than 590 /ug (potency) and not
more than 775 fig (potency) per mg, calculated on the
dried basis. The potency of Gentamicin Sulfate is
expressed as mass (potency) of gentamicin Ci
(C 21 H4 3 N 5 7 : 477.60).
Description Gentamicin Sulfate occurs as a white to light
yellowish white powder.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
It is hygroscopic.
Identification (1) Dissolve 50 mg of Gentamicin Sulfate in
1 mL of water, and add 2 drops of a solution of 1-naphthol in
ethanol (95) (1 in 500). Gently superimpose this solution on 1
mL of sulfuric acid: a blue-purple color develops at the zone
of contact.
(2) Dissolve 50 mg each of Gentamicin Sulfate and Gen-
tamicin Sulfate Reference Standard in 10 mL of water, and
use these solutions as the sample solution and standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 20 fiL of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Separately, shake a mixture of
chloroform, ammonia solution (28) and methanol (2:1:1) in a
separator, and allow the mixture to stand for more than 1
hour. To 20 mL of the lower layer so obtained add 0.5 mL of
methanol, and use this as the developing solvent. Develop the
plate with the developing solvent to a distance of about 17 cm
in a developing container with a cover, having an opening of
about 20 mm 2 , and without putting a filter paper in the con-
tainer, and air-dry the plate. Allow the plate to stand in io-
dine vapors: three principal spots obtained from the sample
solution are the same with the corresponding spots from the
standard solution in color tone and the Rf value, respectively.
(3) Dissolve 50 mg of Gentamicin Sulfate in 5 mL of
water, and add 0.5 mL of barium chloride TS: a white
precipitate is formed.
Optical rotation <2.49> [a] 2 /: + 107 - + 121° (0.25 g calculat-
ed on the dried basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.20
g of Gentamicin Sulfate in 5 mL of water is between 3.5 and
5.5.
Content ratio of the active principle Dissolve 50 mg of Gen-
tamicin Sulfate in water to make 10 mL, and use this solution
as the sample solution. Perform the test with this solution as
directed under Thin-layer Chromatography <2.03>. Spot 20
JP XV
Official Monographs / Glibenclamide
699
/uh of the sample solution on a plate of silica gel for thin-layer
chromatography. Separately, shake a mixture of chloroform,
ammonia solution (28) and methanol (2:1:1) in a separator,
and allow the mixture to stand for more than 1 hour. To 20
mL of the lower layer so obtained add 0.5 mL of methanol,
and use this as the developing solvent. Develop the plate with
the developing solvent to a distance of about 17 cm in a de-
veloping container with a cover, having an opening of about
20 mm 2 , without putting a filter paper in the container, and
air-dry the plate. Allow the plate to stand in iodine vapor.
Determine the integral absorbances, A^, A b and A c , of the
colored spots of gentamicin Ci (Rf value: about 0.3), gen-
tamicin C 2 (Rf value: about 0.2) and gentamicin C u (Rf
value: about 0.1), respectively, by a densitometer (wave-
length: 450 nm) while covering the plate with a glass plate,
and calculate these amounts by the following formulae: gen-
tamicin C] is between 25% and 55%, gentamicin C 2 is be-
tween 25% and 50%, and gentamicin C la is between 5% and
30%.
Amount (%) of gentamicin Ci
= U a /(/l a + 1.35A + A c )} x 100
Amount (%) of gentamicin C 2
= {1.35A/(A + 1.35,4 b + A c )} x 100
Amount (%) of gentamicin C la
= U c /G4 a + l.3SA b + A c )} x 100
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Gentamicin Sulfate in 10 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Gentami-
cin Sulfate according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Dissolve 50 mg of Gentamicin
Sulfate in water to make 10 mL, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
/uL of the sample solution and standard solution on a plate of
silica gel for thin-layer chromatography. Separately, shake a
mixture of chloroform, ammonia solution (28) and methanol
(2:1:1) in a separator, and allow the mixture to stand for
more than 1 hour. To 20 mL of the lower layer so obtained
add 0.5 mL of methanol, and use this as the developing sol-
vent. Develop the plate with the developing solvent to a dis-
tance of about 17 cm in a developing container with a cover,
having an opening of about 20 mm 2 , without putting a filter
paper in the container, and air-dry the plate. Allow the plate
to stand in iodine vapor, and compare the colored spots while
covering with a glass plate: the spots other than the spots of
gentamicin Q (Rf value: about 0.3), gentamicin C 2 (Rf
value: about 0.2) and gentamicin C la (Rf value: about 0.1)
obtained from the sample solution are not more intense than
the spot of gentamicin C 2 from the standard solution.
Loss on drying <2.4I> Not more than 18.0% (0.15 g,
reduced pressure not exceeding 0.67 kPa, 110°C, 3 hours).
Handle the sample avoiding absorption of moisture.
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus epidermidis ATCC
12228
(ii) Agar media for seed and base layer —
Glucose 1.0 g
Peptone 6.0 g
Meat extract 1.5 g
Yeast extract 3.0 g
Sodium chloride 10.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 7.8 to 8.0 after sterilization.
(iii) Agar medium for transferring test organisms — Use
the medium ii in 2) Medium for other organisms under (2)
Agar media for transferring test organisms.
(iv) Standard solutions — Weigh accurately an amount of
Gentamicin Sulfate Reference Standard, equivalent to about
25 mg (potency), dissolve in 0.1 mol/L phosphate buffer
solution, pH 8.0 to make exactly 25 mL, and use this solution
as the standard stock solution. Keep the standard stock solu-
tion at 15°C or lower, and use within 30 days. Take exactly a
suitable amount of the standard stock solution before use,
add 0.1 mol/L phosphate buffer solution, pH 8.0 to make
solutions so that each mL contains 4 /ug (potency) and 1 /ug
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(v) Sample solutions — Weigh accurately an amount of
Gentamicin Sulfate, equivalent to about 25 mg (potency),
and dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0
to make exactly 25 mL. Take exactly a suitable amount of
this solution, add 0.1 mol/L phosphate buffer solution, pH
8.0 to make solutions so that each mL contains 4//g (potency)
and 1 /ug (potency), and use these solutions as the high con-
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Tight containers.
Glibenclamide
^'J-<>?v$ K
-::;..
C
I
CH;
o o o f'S
'n^>i'
C 23 H 28 C1N 3 5 S: 494.00
4-[2-(5-Chloro-2-methoxybenzoylamino)ethyl]-
Af-(cyclohexylcarbamoyl)benzenesulfonamide [10238-21-8]
Glibenclamide, when dried, contains not less than
98.5% of C 23 H 28 C1N30 5 S.
Description Glibenclamide occurs as white to pale yellowish
white crystals or crystalline powder.
It is freely soluble in dimethylformamide, sparingly soluble
700
Glucose / Official Monographs
JP XV
in chloroform, slightly soluble in methanol and in ethanol
(95), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Glinbenclamide in methanol (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Glibenclamide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Perform the test with Glibenclamide as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 169 - 174°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Glibenclamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.20 g of Gliben-
clamide in 20 mL of chloroform, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
chloroform to make exactly 20 mL. Pipet 1 mL of this solu-
tion, add chloroform to make exactly 10 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of 1-propanol, chloroform and diluted ammonia TS (4 in 5)
(11:7:2) to a distance of about 12 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Assay Weigh accurately about 0.9 g of Glibenclamide,
previously dried, dissolve in 50 mL of Af/Y-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (indicator: 3 drops of phenolphthalein TS). Perform a
blank determination with a solution prepared by adding 18
mL of water to 50 mL of /V,7V-dimethylformamide, and
make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 49.40 mg of C 2 3H 28 C1N 3 5 S
Containers and storage Containers — Tight containers.
Glucose
a -0- Gluoopyranose : R'=H. R ! =OH
/?-D-Glucopyrarcose : R 1 =OH, R Z =H
C 6 H 12 6 : 180.16
D-Glucopyranose
[50-99-7]
Glucose is a-D-glucopyranose, /?-D-glucopyranose,
or a mixture of them, and when dried, it contains not
less than 99.5% of C 6 H 12 6 .
Description Glucose occurs as white crystals or crystalline
powder. It is odorless, and has a sweet taste.
It is freely soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
Identification Add 2 to 3 drops of a solution of Glucose (1
in 20) to 5 mL of boiling Fehling's TS: a red precipitate is
produced.
Purity (1) Clarity and color of solution — Add 25 g of
Glucose to 30 mL of water in a Nessler tube, warm at 60°C in
a water bath until solution is effected, cool, and add water to
make 50 mL: the solution is clear and has no more color than
the following control solution.
Control solution: To a mixture of 1.0 mL of Cobaltous
Chloride Stock CS, 3.0 mL of Ferric Chloride stock CS, and
2.0 mL of Cupric Sulfate Stock CS, add water to make 10.0
mL. To 3.0 mL of this solution add water to make 50 mL.
(2) Acidity — Dissolve 5.0 g of Glucose in 50 mL of fresh-
ly boiled and cooled water, and add 3 drops of
phenolphthalein TS and 0.60 mL of 0.01 mol/L sodium
hydroxide VS: a red color develops.
(3) Chloride <1.03>— Perform the test with 2.0 g of Glu-
cose. Prepare the control solution with 1 .0 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.018%).
(4) Sulfate <1.14>— Perform the test with 2.0 g of Glu-
cose. Prepare the control solution with 1.0 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.024%).
(5) Heavy metals <1.07> — Proceed with 5.0 g of Glucose
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 4 ppm).
(6) Arsenic <1.11> — Dissolve 1.5 g of Glucose in 5 mL of
water, add 5 mL of dilute sulfuric acid and 1 mL of bromine
TS, heat on a water bath for 5 minutes, and concentrate to 5
mL. After cooling, perform the test with this solution as the
test solution (not more than 1.3 ppm).
(7) Dextrin— To 1.0 g of Glucose add 20 mL of ethanol
(95), and boil under a reflux condenser: the solution is clear.
(8) Soluble starch and sulfite — Dissolve 1.0 g of Glucose
in 10 mL of water, and add 1 drop of iodine TS: a yellow
color develops.
Loss on drying <2.4I> Not more than 1.0% (1 g, 105 °C,
JPXV
Official Monographs / Glutathione
701
6 hours).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately about 10 g of Glucose, previously
dried, dissolve in 0.2 mL of ammonia TS and water to make
exactly 100 mL, allow to stand for 30 minutes, and determine
the optical rotation, a D , of this solution at 20 ± 1°C in a
100-mm cell as directed under Optical Rotation Determina-
tion <2.49>.
Amount (mg) of C 6 H 12 6 =a D x 1895.4
Containers and storage Containers — Tight containers.
Glucose Injection
7 K ^iiilt*
Glucose Injection is an aqueous solution for injec-
tion. It contains not less than 95% and not more than
105% of the labeled amount of glucose (C 6 H 12 6 :
180.16).
Method of preparation Prepare as directed under Injec-
tions, with Glucose. No preservative is added.
Description Glucose Injection is a clear, colorless liquid. It
has a sweet taste. It occurs as a colorless to pale yellow, clear
liquid when its labeled concentration exceeds 40%.
Identification Measure a volume of Glucose Injection,
equivalent to 0.1 g of Glucose according to the labeled
amount, and, if necessary, add water or evaporate on a water
bath to a volume of 2 mL. Add 2 to 3 drops of the solution to
5 mL of boiling Fehling's TS: a red precipitate is produced.
pH <2.54> 3.5 - 6.5 In the case where the labeled concentra-
tion
of the injection exceeds 5%, dilute to 5% with water before
the test.
Purity 5-Hydroxymethylfurfural and related substan-
ces — Measure exactly a volume of Glucose Injection, equiva-
lent to 2.5 g of Glucose according to the labeled amount, and
add water to make exactly 100 mL. Determine the absor-
bance of this solution at 284 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it is not more than
0.80.
Bacterial endotoxins <4.01> Less than 0.50 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Measure accurately a volume of Glucose Injection,
equivalent to about 4 g of glucose (C 6 H 12 6 ), and add 0.2 mL
of ammonia TS and water to make exactly 100 mL. Shake the
solution well, allow to stand for 30 minutes, and determine
the optical rotation, aD, at 20 ± 1°C in a 100-mm cell as
directed under Optical Rotation Determination <2.49>.
Amount (mg) of glucose (C 6 H 12 6 ) = a D x 1895.4
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Glutathione
1 H O
HOjC\
- N yS
C0 2 H
H NHj
C 10 H 17 N 3 O 6 S: 307.32
(2S)-2-Amino-4-[l-(carboxymethyl)carbamoyl-(2i?)-2-
sulfanylethylcarbamoyljbutanoic acid [70-18-8]
Glutathione, when dried, contains not less than
98.0% and not more than 101.0% of C 10 H 17 N 3 O 6 S.
Description Glutathione occurs as a white crystalline
powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Melting point: about 185°C (with decomposition).
Identification Determine the infrared absorption spectrum
of Glutathione, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a] D °: -
drying, 2 g, water, 50 mL, 100 mm).
15.5- -17.5° (after
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Glutathione in 10 mL of water: the solution is clear and
colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Glutathione according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Glutathione according to Method 1, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 50 mg of Glutathione
in 100 mL of the mobile phase, and use this solution as the
sample solution. Pipet 2 mL of the sample solution, add the
mobile phase to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 10 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak having the rel-
ative retention time of about 4 with respect to glutathione is
not more than 3/4 times the peak area of glutathione from
the standard solution, and the total area of the peaks other
than the peak of glutathione is not more than the peak area
of glutathione from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
702
Glycerin / Official Monographs
JP XV
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen
phosphate and 2.02 g of sodium 1-heptane sulfonate in 1000
mL of water, and adjust the pH to 3.0 with phosphoric acid.
To 970 mL of this solution add 30 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
glutathione is about 5 minutes.
Time span of measurement: About 6 times as long as the
retention time of glutathione beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 10 mL of the standard
solution, and add the mobile phase to make exactly 100 mL.
Confirm that the peak area of glutathione obtained from 10
[iL of this solution is equivalent to 8 to 12% of that obtained
from 10 /uL of the standard solution.
System performance: Dissolve 50 mg of glutathione, 10 mg
of D-phenylglycine and 50 mg of ascorbic acid in 100 mL of
water. When the procedure is run with 10 tiL of this solution
under the above operating conditions, ascorbic acid,
glutathione and D-phenylglycine are eluted in this order, and
the resolutions between the peaks of ascorbic acid and
glutathione and between the peaks of glutathione and
D-phenylglycine are not less than 5, respectively.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glutathione is not more than 1.5%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Glutathione,
previously dried, dissolve in 50 mL of a solution of
metaphosphoric acid (1 in 50), and titrate <2.50> with 0.05
mol/L iodine VS (indicator: 1 mL of starch TS). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.05 mol/L iodine VS
= 30.73 mg of C 10 H 17 N 3 O 6 S
Containers and storage Containers — Tight containers.
Glycerin
Glycerol
C 3 H 8 3 : 92.09
Glycerin contains not less than 84.0% and not more
than 87.0% of C 3 H 8 3 .
Description Glycerin is a clear, colorless, viscous liquid. It
has a sweet taste.
It is miscible with water and with ethanol (99.5).
It is hygroscopic.
Identification Determine the infrared absorption spectrum
of Glycerin as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Refractive index <2.45> n™: 1.449 - 1.454
Specific gravity <2.56> df : 1.221 - 1.230
Purity (1) Color — Place 50 mL of Glycerin in a Nessler
tube, and observe downward: the solution has no more color
than the following control solution. Control solution: Place
0.40 mL of Ferric Chloride Colorimetric Stock Solution in a
Nessler tube, and add water to make 50 mL.
(2) Acidity or alkalinity — To 2 mL of Glycerin add 8 mL
of water and mix: the solution is neutral.
(3) Chloride <7.ft?>— Take 10.0 g of Glycerin, and per-
form the test: Prepare the control solution with 0.30 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.001%).
(4) Sulfate <1.14>— Take 10.0 g of Glycerin, and perform
the test. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.002%).
(5) Ammonium — To 5 mL of Glycerin add 5 mL of a
solution of sodium hydroxide (1 in 10), and boil: the gas
evolved does not change moistened red litmus paper to blue.
(6) Heavy metals <1.07> — Proceed with 5.0 g of Glycerin
according to Method 1, and perform the test: Prepare the
control solution with 2.5 mL of Standard Lead Solution (not
more than 5 ppm).
(7) Calcium — To 5 mL of the solution obtained in (2) add
3 drops of ammonium oxalate TS: the solution remains un-
changed.
(8) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Glycerin according to Method 1, and perform the test (not
more than 2 ppm).
(9) Acrolein, glucose, and other reducing substances — To
1.0 g of Glycerin add 1 mL of ammonia TS, mix, and warm
in a water bath at 60 °C for 5 minutes: no yellow color is
produced. Take the solution out of the water bath, add 3
drops of silver nitrate TS immediately, and allow to stand in
a dark place for 5 minutes: the color of the solution does not
change, and no turbidity is produced.
(10) Fatty acids and esters — Mix 50 g of Glycerin with 50
mL of freshly boiled and cooled water, add exactly 10 mL of
0.1 mol/L sodium hydroxide VS, boil the mixture for 15
minutes, cool, and titrate the excess sodium hydroxide with
0.1 mol/L hydrochloric acid VS: 0.1 mol/L sodium
hydroxide VS consumed is not more than 3.0 mL (indicator:
3 drops of phenolphthalein TS). Perform a blank determina-
tion.
(11) Readily carbonizable substances — To 5 mL of
Glycerin add carefully 5 mL of sulfuric acid for readily car-
bonizable substances, mix gently at a temperature between
18°C and 20°C, and allow to stand for 1 hour between 15°C
and 25°C: the solution has not more color than Matching
Fluid H.
Water <2.48> 13 - 17% (0.1 g, volumetric titration, direct
titration).
Residue on ignition <2.44> Weigh accurately about 10 g of
Glycerin in a tared crucible, heat to boiling, and fire to burn
immediately. After cooling, moisten the residue with 1 to 2
drops of sulfuric acid, and ignite cautiously to constant mass:
the mass of the residue is not more than 0.01%.
JPXV
Official Monographs / Concentrated Glycerin
703
Assay Weigh accurately about 0.2 g of Glycerin, transfer
into a glass-stoppered flask, add 50 mL of water, mix, add ex-
actly 50 mL of sodium periodate TS, shake, and allow to
stand in a dark place at a room temperature for about 30
minutes. Add 10 mL of a mixture of water and ethylene
glycol (1:1), allow to stand for about 20 minutes, add 100 mL
of water, and titrate <2.50> with 0. 1 mol/L sodium hydroxide
VS (indicator: 2 drops of phenolphthalein TS). Perform a
blank determination, and make the necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 9.209 mg of C 3 H 8 3
Containers and storage Containers — Tight containers.
Concentrated Glycerin
Concentrated Glycerol
C 3 H 8 3 : 92.09
Propane-1 ,2,3-triol
[56-81-5]
Concentrated Glycerin contains not less than 98.0%
and not more than 101.0% of glycerin (C 3 H 8 3 ), calcu-
lated of the anhydrous basis.
Description Concentrated Glycerin is a clear, colorless and
viscous liquid. It has a sweet taste.
It is miscible with water and with ethanol (99.5).
It is hygroscopic.
Identification Determine the infrared absorption spectrum
of Concentrated Glycerin as directed in the liquid film
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Refractive index <2.45> n 2 °: Not less than 1.470.
Specific gravity <2.56> df : Not less than 1.258.
Purity (1) Color — Place 50 mL of Concentrated Glycerin
in a Nessler tube, and observe downward: the solution has no
more color than the following control solution.
Control solution: Pipet 0.40 mL of Ferric Chloride Colori-
metric Stock Solution into a Nessler tube, and add water to
make 50 mL.
(2) Acidity or alkalinity — To 2 mL of Concentrated
Glycerin add 8 mL of water and mix: the solution is neutral.
(3) Chloride <1.03>— Take 10.0 g of Concentrated Glyce-
rin, and perform the test. Prepare the control solution with
0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.001%).
(4) Sulfate <1.14>— Take 10.0 g of Concentrated Glyce-
rin, and perform the test. Prepare the control solution with
0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.002%).
(5) Ammonium — To 5 mL of Concentrated Glycerin add
5 mL of a solution of sodium hydroxide (1 in 10), and boil:
the gas evolved does not change moistened red litmus paper
to blue.
(6) Heavy metals <1.07> — Proceed with 5.0 g of Concen-
trated Glycerin according to Method 1, and perform the test.
Prepare the control solution with 2.5 mL of Standard Lead
Solution (not more than 5 ppm).
(7) Calcium — To 5 mL of the solution obtained in (2) add
3 drops of ammonium oxalate TS: the solution remains un-
changed.
(8) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Concentrated Glycerin according to Method 1, and per-
form the test (not more than 2 ppm).
(9) Acrolein, glucose, or other reducing substances — To
1.0 g of Concentrated Glycerin add 1 mL of ammonia TS,
mix, and warm in a water bath at 60°C for 5 minutes: no yel-
low color is produced. Take the solution out of the water
bath, add 3 drops of silver nitrate TS immediately, and allow
to stand in a dark place for 5 minutes: the color of the solu-
tion does not change, and no turbidity is produced.
(10) Fatty acids and esters — Mix 50 g of Concentrated
Glycerin with 50 mL of freshly boiled and cooled water, add
10 mL of 0.1 mol/L sodium hydroxide VS, accurately meas-
ured, boil the mixture for 15 minutes, cool, and titrate the ex-
cess sodium hydroxide with 0.1 mol/L hydrochloric acid VS:
not more than 3.0 mL of 0.1 mol/L sodium hydroxide VS is
consumed (indicator: 3 drops of phenolphthalein TS). Per-
form a blank determination.
(11) Readily carbonizable substances — To 5 mL of Con-
centrated Glycerin add carefully 5 mL of sulfuric acid for
readily carbonizable substances, mix gently at a temperature
between 18°C and 20°C, and allow to stand for 1 hour be-
tween 15°C and 25°C: the solution has no more color than
Matching Fluid H.
Water <2.48> Not more than 2.0% (6 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Weigh accurately about 10 g of
Concentrated Glycerin in a tared crucible, heat to boiling,
and fire to burn immediately. Cool, moisten the residue with
1 to 2 drops of sulfuric acid, and ignite cautiously to constant
mass: the mass of the residue is not more than 0.01%.
Assay Weigh accurately about 0.2 g of Concentrated Glyc-
erin, transfer into a glass-stoppered flask, add 50 mL of
water, mix, add exactly 50 mL of sodium periodate TS,
shake, and allow to stand in a dark place at a room tempera-
ture for about 30 minutes. Add 10 mL of a mixture of water
and ethylene glycol (1:1), allow to stand for about 20
minutes, add 100 mL of water, and titrate <2.50> with 0.1
mol/L sodium hydroxide VS (indicator: 2 drops of
phenolphthalein TS). Perform a blank determination, and
make the necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 9.209 mg of C 3 H 8 3
Containers and storage Containers — Tight containers.
704
Glycerin and Potash Solution / Official Monographs
JP XV
Glycerin and Potash Solution
yy-tzu>ay«
Method of preparation
Potassium Hydroxide
Glycerin
Ethanol
Aromatic substance
Water or Purified Water
3g
200 mL
250 mL
a suitable quantity
a sufficient quantity
To make 1000 mL
Dissolve Potassium Hydroxide in a portion of Water or
Purified Water, add Glycerin, Ethanol, a suitable quantity of
aromatic substance and another portion of Water or Purified
Water to volume, and filter. Concentrated Glycerin may be
used in place of Glycerin.
Description Glycerin and Potash Solution is a clear, color-
less liquid, having an aromatic odor.
The pH of a solution of Glycerin and Potash Solution (1 in
5) is about 12.
Specific gravity df : about 1.02
Identification (1) A solution of Glycerin and Potash Solu-
tion (1 in 2) is alkaline (potassium hydroxide).
(2) Place 10 mL of a solution of Glycerin and Potash So-
lution (1 in 10) in a glass-stoppered test tube, add 2 mL of so-
dium hydroxide TS and 1 mL of copper (II) sulfate TS, and
shake: a blue color is produced (glycerin).
(3) Glycerin and Potash Solution responds to the
Qualitative Tests <1.09> for potassium salt.
Containers and storage Containers — Tight containers.
Glyceryl Monostearate
=E/Xx7'J>g^Uir'J>
Glyceryl Monostearate is a mixture of a- and /?-
glyceryl monostearate and other fatty acid esters of
glycerin.
Description Glyceryl Monostearate occurs as white to light
yellow, waxy masses, thin flakes, or granules. It has a charac-
teristic odor and taste.
It is very soluble in hot ethanol (95), soluble in chloroform,
sparingly soluble in diethyl ether, and practically insoluble in
water and in ethanol (95).
It is slowly affected by light.
Identification (1) Heat 0.2 g of Glyceryl Monostearate
with 0.5 g of potassium hydrogen sulfate until thoroughly
charred: the irritative odor of acrolein is perceptible.
(2) Dissolve 0.1 g of Glyceryl Monostearate in 2 mL of
ethanol (95) by warming, heat with 5 mL of dilute sulfuric
acid in a water bath for 30 minutes, and cool: a white to yel-
low solid is produced. This separated solid dissolves when
shaken with 3 mL of diethyl ether.
Melting point <7.73> Not below 55°C.
Acid value <I.I3> Not more than 15.
Saponification value <1.13> 157 - 170
Iodine value <1.13> Not more than 3.0. Use chloroform in-
stead of cyclohexane.
Purity <1.13> Acidity or alkalinity — To 1.0 g of Glyceryl
Monostearate add 20 mL of boiling water, and cool with
swirling: the solution is neutral.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Glycine
Aminoacetic Acid
H £ N COjH
C 2 H 5 N0 2 : 75.07
Aminoacetic acid
[56-40-6]
Glycine, when dried, contains not less than 98.5% of
C 2 H 5 N0 2 .
Description Glycine occurs as white crystals or crystalline
powder. It is odorless. It has a sweet taste.
It is freely soluble in water and in formic acid, and practi-
cally insoluble in ethanol (95).
Identification Determine the infrared absorption spectrum
of Glycine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, dissolve Glycine in water, evaporate the water to
dryness, and repeat the test with the residue.
pH <2.54> Dissolve 1.0 g of Glycine in 20 mL of water: the
pH of the solution is between 5.6 and 6.6.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Glycine in 10 mL of water: the solution is clear and colorless.
(2) Chloride <1.03>— Perform the test with 0.5 g of Gly-
cine. Prepare the control solution with 0.30 mL of 0.01 mol/
L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of Gly-
cine. Prepare the control solution with 0.35 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.028%).
(4) Ammonium<7.ft2> — Perform the test using 0.25 g of
Glycine. Prepare the control solution with 5.0 mL of Stan-
dard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of Glycine
according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Glycine according to Method 1, and perform the test (not
more than 2 ppm).
(7) Related substences — Dissolve 0.10 g of Glycine in 25
JP XV
Official Monographs / Gonadorelin Acetate
705
mL of water and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add water to make exactly
50 mL. Pipet 5 mL of this solution, add water to make exact-
ly 20 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 [iL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (3:1:1) to a distance of
about 10 cm, and dry the plate at 80°C for 30 minutes. Spray
evenly a solution of ninhydrin in acetone (1 in 50), and heat
at 80°C for 5 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 80 mg of Glycine, previously
dried, dissolve in 3 mL of formic acid, add 50 mL of acetic
acid (100), and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 7.507 mg of C 2 H 5 N0 2
Containers and storage Containers — Well-closed contain-
ers.
Gonadorelin Acetate
V-
°<T H
His-Trp-Ser-Tyr-Gly-Leu-Arg-Pra-Gly-NH., . gHjC-COjH
C 55 H 75 N 17 13 .2C 2 H40 2 : 1302.39
5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-
glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide
diacetate [34973-08-5]
Gonadorelin Acetate contains not less than 96.0%
and not more than 102.0% of C 5 5H 7 5N 17 13 .2C 2 H40 2 ,
calculated on the anhydrous basis.
Description Gonadorelin Acetate occurs as a white to pale
yellow powder. It is odorless or has a slight, acetic odor.
It is freely soluble in water, in methanol and in acetic acid
(100), and sparingly soluble in ethanol (95).
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Gonadorelin Acetate in methanol (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Gonadorelin Acetate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Gonadorelin Acetate Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Dissolve 20 mg of Gonadorelin Acetate in 0.5 mL of
ethanol (99.5), add 1 mL of sulfuric acid, and heat: the odor
of ethyl acetate is perceptible.
Optical rotation <2.49>: [a]™: -53.0- -57.0° (0.10 g cal-
culated on the anhydrous basis, diluted acetic acid (100) (1 in
100), 10 mL, 100 mm).
pH <2.54> Dissolve 0.10 g of Gonadorelin Acetate in 10 mL
of water: the pH of this solution is between 4.8 and 5.8.
Constituent amino acids Put 10 mg of Gonadorelin Acetate
in a test tube for hydrolysis, add 0.5 mL of hydrochloric acid
and 0.5 mL of a solution of mercaptoacetic acid (2 in 25), seal
the tube under reduced pressure, and heat at 110°C for 5
hours. After cooling, open the tube, transfer the hydrolyzate
into a beaker, and evaporate to dryness on a water bath. Add
exactly 100 mL of 0.02 mol/L hydrochloric acid TS to dis-
solve the residue, and use this solution as the sample solution.
Separately, weigh exactly 0.105 g of L-serine, 0.147 g of L-
glutamic acid, 0.115 g of L-proline, 75 mg of glycine, 0.131 g
of L-leucine, 0.181 g of L-tyrosine, 0.210 g of L-histidine
hydrochloride monohydrate, 0.204 g of L-tryptophan and
0.211 g of L-arginine hydrochloride, which are all previously
dried at 105°C for 3 hours, add 50 mL of 1 mol/L
hydrochloric acid TS to dissolve them, and add water to
make exactly 1000 mL. Pipet 10 mL of this solution, add 0.02
mol/L hydrochloric acid TS to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
50 /iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions: the peaks of nine constituent amino
acids are observed on the chromatogram, and their respective
molar ratios with respect to arginine are 0.7 - 1.0 for serine
and tryptophan, 0.8 - 1.2 for proline, 0.9 - 1.1 for glutamic
acid, leucine, tyrosine and histidine, respectively, and 1.8 -
2.2 for glycine.
Operating conditions —
Detector: A visible spectrophotometer (wavelength: 440
nm for proline and 570 nm for others).
Column: A stainless steel column 4 mm in inside diameter
and 8 cm in length, packed with strongly acidic ion-exchange
resin for liquid chromatography composed with a sulfonated
polystyrene copolymer (5 ^m in particle diameter).
Column temperature: A constant temperature of about
57°C.
Chemical reaction bath temperature: A constant tempera-
ture of about 130°C.
Mobile phase: Prepare the mobile phases A, B, C and D
according to the following table.
706
Gonadorelin Acetate / Official Monographs
JP XV
Mobile phase
A
B
C
D
Trisodium citrate di-
hydrate
6.19g
7.74 g
26.67 g
-
Sodium hydroxide
—
—
—
8.00 g
Sodium chloride
5.66 g
7.07 g
54.35 g
—
Citric acid monohydrate
19.80 g
22.00 g
6.10g
—
Ethanol (99.5)
130 mL
20 mL
—
100 mL
Benzyl alcohol
—
—
5mL
—
Thiodiglycol
5mL
5mL
—
—
Lauromacrogol solution
in diethyl ether (1 in 4)
4mL
4mL
4mL
4mL
Caprylic acid
0.1 mL
0.1 mL
0.1 mL
0.1 mL
a sufficient
a sufficient
a sufficient
a sufficient
amount
amount
amount
amount
Total volume
1000 mL
1000 mL
1000 mL
1000 mL
Flowing of mobile phase: Control the gradient by mixing
the mobile phases A, B, C and D as directed in the following
table.
Time after
injection of
sample (min)
Mobile
phase A
(vol%)
Mobile
phase B
(vol%)
Mobile
phase C
(vol%)
Mobile
phase D
(vol%)
0-9
100
9-25
100
25-61
100^0
0^100
61-76
100
76-96
100
Reaction reagent: Dissolve 204 g of lithium acetate dihy-
drate in 336 mL of water, add 123 mL of acetic acid (100) and
401 mL of l-methoxy-2-propanol, and use as Solution A.
Separately, dissolve 39 g of ninhydrin and 81 mg of sodium
borohydride in 979 mL of l-methoxy-2-propanol, and use as
Solution B. Mix the same volume of Solution A and Soluiton
B before use.
Flow rate of mobile phase: 0.25 mL per minute.
Flow rate of reaction reagent: 0.3 mL per minute.
System suitability —
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
tions, serine, glutamic acid, proline, glycine, leucine, tyro-
sine, histidine, tryptophan and arginine are eluted in this ord-
er with enough separation between these peaks.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 0.10 g of Gonadorelin Acetate in 10 mL
of water is clear, and the absorbance of this solution at 350
nm determined as directed under Ultraviolet-visible Spec-
trophotometry <2.24> is not more than 0.10.
(2) Related substances — Dissolve 50 mg of Gonadorelin
Acetate in 100 mL of the mobile phase A, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 20 mL, and use
this solution as the standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the peak area other than
gonadorelin from the sample solution is not more than 1/5
times the peak area of gonadorelin from the standard solu-
tion, and the total area of the peaks other than the peak of
gonadorelin is not more than 3/5 of the peak area of
gonadorelin from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2.5 times as long as the
retention time of gonadorelin beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 100 mL.
Confirm that the peak area of gonadorelin obtained from 10
/xL of this solution is equivalent to 1 to 3% of that obtained
from 10 /xL of the standard solution.
System performance: Dissolve 4 mg of Gonadorelin
Acetate in a suitable amount of the mobile phase, add 5 mL
of a solution of phenacetin in acetonitrile (1 in 1000) and the
mobile phase to make 50 mL. When the procedure is run with
10,mL of this solution under the above operating conditions,
gonadorelin and phenacetin are eluted in this order with the
resolution between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
gonadorelin is not more than 5%.
Water <2.48> Not more than 8.0% (0.15 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (0.1 g).
Assay Weigh accurately about 40 mg of Gonadorelin
Acetate and Gonadorelin Acetate Reference Standard
(separately determine the water <2.48> in the same manner as
Gonadorelin Acetate) and dissolve in diluted acetic acid (100)
(1 in 1000) to make exactly 25 mL each. Pipet 5 mL each of
these solutions, add exactly 5 mL of the internal standard so-
lution and water to make 25 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with 10 [iL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Qj and
Qs, of the peak area of gonadorelin to that of the internal
standard.
Amount (mg) of C 55 H 75 N 17 13 .2C 2 H 4 2 = W s x(Q T /Q s )
W s : Amount (mg) of Gonadorelin Acetate Reference
Standard, calculated on the anhydrous basis
Internal standard solution — A solution of phenacetin in a
mixture of water and acetonitrile (3:2) (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0 and acetonitrile (90:17).
Flow rate: Adjust the flow rate so that the retention time of
gonadorelin is about 13 minutes.
System suitability —
JPXV
Official Monographs / Human Chorionic Gonadotropin
707
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, gonadorelin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of gonadorelin to that of the internal standard is
not more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Human Chorionic Gonadotropin
Chorionic Gonadotrophin
Human Chorionic Gonadotrophin is a dried preparation
of gonad-stimulating hormone obtained from the urine of
healthy pregnant women after the manufacturing process to
remove or inactivate the virus.
It contains not less than 2500 human chorionic gonadotro-
phin Units per mg, and contains not less than 3000 chorionic
gonadotrophin Units per mg protein.
It contains not less than 80% and not more than 125% of
the labeled human chorionic gonadotrophin Units.
Description Human Chorionic Gonadotrophin occurs as a
white to light yellow-brown powder.
It is freely soluble in water.
Identification Calculate b by the following equation, using
F 3 and Y 4 obtained in the Assay: b is not less than 120.
b = (E/I)
E=(Y 3 -Y 4 )/f
f: Number of test animals per group.
7=log(T H /T L )
Purity (1) Clarity and color of solution — Dissolve 0.05 g
of Human Chorionic Gonadotrophin in 5 mL of isotonic so-
dium chloride solution: the solution is clear and colorless or
light yellow.
(2) Estrogen — Inject subcutaneously into each of three
female albino rats or albino mice ovariectomized at least two
weeks before the test, single dose of 100 units according to
the labeled Units dissolved in 0.5 mL of isotonic sodium
chloride solution. Take vaginal smear twice daily, on the
third, fourth and fifth day. Place the smear thinly on a slide
glass, dry, stain with Giemsa's TS, wash with water, and
again dry: no estrus figure is shown microscopically <5.01>.
Loss on drying <2.41> Not more than 5.0% (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.03 EU/unit.
Abnormal toxicity Dilute Human Chorionic Gonadotro-
phin with isotonic sodium chloride solution so that each mL
of the solution contains 120 Units, and use this solution as
the sample solution. Inject 5.0 mL of the sample solution
into the peritoneal cavity of each of 2 or more of well-
nourished, healthy guinea pigs weighing about 350 g, and ob-
serve the conditions of the animals for more than 7 days: all
the animals exhibit no abnormalities.
Specific activity When calculate from the results obtained
by the Assay and the following test, the specific activity is not
less than 3000 human chorionic gonadotrophin Units per mg
protein.
(i) Sample solution — To an exactly amount of Chorionic
Gonadotrophin add water to make a solution so that each
mL contains about 500 Units of human chorionic gonadotro-
phin according to the labeled amount.
(ii) Standard solution — Weigh accurately about 10 mg of
bovine serum albumin, and dissolve in water to make exactly
20 mL. To a suitable volume of this solution add water to
make four solutions containing exactly 300, 200, 100 and 50
//g of the albumin per mL, respectively.
(hi) Procedure — Pipet 0.5 mL each of the sample solu-
tion and the standard solutions, put them in glass test tubes
about 18 mm in inside diameter and about 130 mm in length,
add exactly 5 mL of alkaline copper TS, mix, and allow the
tubes to stand in a water bath at 30°C for 10 minutes. Then
add exactly 0.5 mL of diluted Folin's TS (1 in 2), mix, and
warm in a water bath at 30°C for 20 minutes. Determine the
absorbances of these solutions at 750 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24> using a solution
obtained in the same manner with 0.5 mL of water as the
blank.
Plot the absorbances of the standard solutions on the
vertical axis and their protein concentrations on the horizon-
tal axis to prepare a calibration curve, and determine the pro-
tein content of the sample solution from its absorbance by us-
ing this curve. Then calculate the amount of the protein in the
sample.
Assay (i) Test animals — Select healthy female albino rats
weighing about 45 to 65 g.
(ii) Standard solution — Dissolve a quantity of Human
Chorionic Gonadotrophin Reference Standard in bovine se-
rum albumin-isotonic sodium chloride solution to prepare
four kinds of solutions, having 7.5, 15, 30 and 60 Units per
2.5 mL, respectively. Inject these solutions into four groups
consisting of five test animals each, and weigh their ovaries,
as directed in procedure of (iv). Inject bovine serum albumin-
isotonic sodium chloride solution to another group, and use
this group as the control group. According to the result of
this test, designate the concentration of the reference stan-
dard which will increase the masses of the ovaries about 2.5
times the mass of the ovaries of the control group as a low-
dose concentration of the standard solution, and the concen-
tration 1.5 to 2.0 times the low-dose concentration as a high-
dose concentration. Dissolve a quantity of Human Chorionic
Gonadotrophin Reference Standard, in bovine serum al-
bumin-isotonic sodium chloride solution, and prepare a high-
dose standard solution S H and a low-dose standard solution
S L whose concentrations are equal to those determined by the
above test.
(iii) Sample solution — According to the labeled units,
weigh accurately a suitable quantity of Human Chorionic
Gonadotrophin, dissolve in bovine serum albumin-isotonic
sodium chloride solution, and prepare a high-dose sample so-
lution T H and a low-dose sample solution T L having Units e-
qual to the standard solutions in equal volumes, respectively.
708
Human Chorionic Gonadotrophin for Injection / Official Monographs
JP XV
(iv) Procedure — Divide the test animals at random into 4
groups, A, B, C and D, with not less than 10 animals and
equal numbers in each group. Inject subcutaneously 0.5 mL
of S H , S L , T H and T L in each group for 5 days. On the sixth
day, excise the ovaries, remove the fat and other unwonted
tissues attached to the ovaries, and remove the adhering
water by lightly pressing between filter paper, and immediate-
ly weigh the ovaries.
(v) Calculation — Designate the mass of ovaries by S H , S L ,
T H and T L as y lt y 2 , y-$ and y 4 , respectively. Sum up y lt y 2 , yi
and y 4 on each set to obtain Y,, Y 2 , Y 3 and Y 4 .
Units per mg of Human Chorionic Gonadotrophin
= antilog Mx (units per mL of S H ) x (b/a)
M=IYJY b
/=log(S H /S L ) = log(T H /T L )
Y i =-Y 1 -Y 1 +Y 3 +Y A
Y b =Y l -Y 2 +Y i -Y 4
a: Mass (mg) of sample.
b: Total volume (mL) of the high dose of the test solution
prepared by diluting with bovine serum albumin-isotonic
sodium chloride solution.
F' computed by the following equation should be smaller
than Fi against n when s 2 is calculated. And compute L (P =
0.95) by the following equation: L should be not more than
0.3. If F' exceeds F it or if L exceeds 0.3, repeat the test in-
creasing the number of the test animals or arranging the assay
method in a better way until F' is smaller than Fj or L is not
more than 0.3.
F'=(Y 1 -Y 2 -Y 3 +Y 4 )V4/5 2
/: Number of test animals per group.
s 2 = {Y.y 2 -(Y/f)}/n
Ey 2 : The sum of the squares of each y lt y 2 , yi and y 4 .
Y=Y i 2 +Y 2 2 +Y i 2 +Y 4 2
n = 4(/-l)
L = 2J(C-1)(CM2 + P)
C=Y b 2 /(Y b 2 -4fs 2 f)
t 2 : Value shown in the following table against n used to
calculate s 2 .
n
f = F i
n
t 2 = F l
n
t 2 = F l
1
161.45
13
AMI
25
4.242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
oo
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Human Chorionic Gonadotrophin
for Injection
Chorionic Gonadotrophin for Injection
Human Chorionic Gonadotrophin for Injection is a
preparation for injection which is dissolved before use.
It contains not less than 80% and not more than
125% of the labeled human chorionic gonadotrophin
Units.
Method of preparation Prepare as directed under Injections
with Human Chorionic Gonadotrophin.
Description Human Chorionic Gonadotrophin for Injec-
tion occurs as a white to light yellow-brown powder or mass-
es.
Identification Proceed as directed in the Identification un-
der Human Chorionic Gonadotrophin.
pH <2.54> Prepare a solution so that each mL of isotonic
sodium chloride solution contains 2 mg of Human Chorionic
Gonadotorophin for Injection: the pH of this solution is be-
tween 5.0 and 7.0.
Loss on drying <2.41> Not more than 5.0% (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.03 EU/unit.
Uniformity of dosage units <6.02> When calculate the ac-
ceptance value using the mean of estimated contents of the u-
nits tested as M, it meets the requirements of the Mass varia-
tion test.
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Proceed as directed in the Assay under Human
Chorionic Gonadotrophin. The ratio of the assayed Units to
the labeled Units should be calculated by the following equa-
tion.
The ratio of the assayed Units to the labeled Units
= antilog M
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, and in a cold place.
Human Menopausal Gonadotrophin
t: hTSftttttJ»»J»*;u ; E>
Human Menopausal Gonadotrophin is a dried
preparation of gonad-stimulating hormone obtained
JPXV
Official Monographs / Human Menopausal Gonadotropin
709
from the urine of postmenopausal healthy women,
after processing for virus removal or inactivation. It
has follicle-stimulating hormonal action and luteiniz-
ing hormonal action.
It contains not less than 40 follicle-stimulating hor-
mone Units per mg.
Description Human Menopausal Gonadotrophin occurs as
a white to pale yellow powder.
It is soluble in water.
Purity Interstitial cell-stimulating hormone — Perform the
test according to the following method: the ratio of the unit
of interstitial cell-stimulating hormone (luteinizing hormone)
to that of follicle-stimulating hormone is not more than 1 .
(i) Test animals — Select healthy male albino rats weigh-
ing about 45 to 65 g.
(ii) Standard solutions — Weigh accurately a suitable
amount of Human Menopausal Gonadotrophin Reference
Standard, and dissolve in bovine serum albumin-sodium
chloride-phosphate buffer solution, pH 7.2, to prepare three
kinds of solutions, containing 10, 20 and 40 interstitial cell-
stimulating hormone (luteinizing hormone) units per 1.0 mL,
respectively. Inject these solutions into three groups consist-
ing of five test animals each, and weigh their seminal vesicles
as directed in (iv). According to the result of the test, desig-
nate the concentration of the reference standard, which will
make the mass of the seminal vesicle 20 to 35 mg, as the high-
dose standard solution, S H . Dilute the S H to 1.5 to 2.0 times
the initial volume with the bovine serum albumin-sodium
chloride-phosphate buffer solution, pH 7.2, and designate
this solution as the low-dose standard solution, S L . Store
these solutions at 2 - 8°C.
(iii) Sample solution — According to the labeled units,
weigh accurately a suitable amount of Human Menopausal
Gonadotrophin, and dissolve in the bovine serum albumin-
sodium chloride-phosphate buffer solution, pH 7.2, to pre-
pare the high-dose sample solution, T H and the low-dose
sample solution, T L , so that their concentrations are similar
to those of the corresponding standard solutions, respec-
tively. Store these solutions at 2 - 8°C.
(iv) Procedure — Divide the test animals at random into 4
groups, A, B, C and D, with not less than 10 animals and
equal numbers in each group. Inject subcutaneously once
every day 0.2 mL each of S H , S L , T H and T L into the animals
in each group. On the sixth day, excise the seminal vesicles,
remove extraneous tissue, remove fluid adhering to the vesi-
cles and the contents of the vesicles by lightly pressing be-
tween filter papers, and weigh the vesicles.
(v) Calculation — Proceed as directed in (v) in the Assay.
Bacterial endotoxins <4.01> Dissolve Human Menopausal
Gonadotrophin in water for bacterial endotoxins test to pre-
pare a solution containing 75 follicle-stimulating hormone
Units per mL, and perform the test: less than 0.66 EU/ fol-
licle-stimulating hormone Unit.
Water <2.48> Not more than 5.0% (0.2 g, volumetric titra-
tion, direct titration).
Specific activity Perform the test with Human Menopausal
Gonadotrophin according to the following method, and cal-
culate the specific activity using the amount (Unit) obtained
in the Assay: it is not less than 50 follicle-stimulating hor-
mone Units per 1 mg of protein.
(i) Sample solution — Weigh accurately about 10 mg of
Human Menopausal Gonadotrophin, dissolve in water so
that each mL contains exactly 200 /ug, and use this solution as
the sample solution.
(ii) Standard solutions — Weigh accurately about 10 mg
of bovine serum albumin, and dissolve in water to make ex-
actly 20 mL. To this solution add water to make four solu-
tions containing exactly 300 ^g, 200 //g, 100 /ug and 50 /ug of
the albumin per mL, respectively, and use these solutions as
the standard solutions.
(iii) Procedure — To glass test tubes, about 18 mm in in-
side diameter and about 130 mm in height, add separately ex-
actly 0.5 mL each of the sample solution and the standard so-
lutions. To these tubes add exactly 5 mL of alkaline copper
TS, warm in a water bath at 30°C for 10 minutes, then add
exactly 0.5 mL of diluted Folin's TS (1 in 2), and warm in a
water bath at 30 °C for 20 minutes. Perform the test with
these solutions as directed under Ultraviolet-visible Spectro-
metry <2.24>, and determine the absorbances at 750 nm, us-
ing a liquid obtained with 0.5 mL of water in the same man-
ner as above as a blank. Prepare a calibration curve from the
absorbances of the standard solutions, with absorbance on
the vertical axis and concentration on the horizontal axis.
Determine the amount of protein in the sample solution from
the absorbance of the sample solution using the curve, and
calculate the protein content of the sample.
Assay
(i) Test animals — Select healthy female albino rats weigh-
ing about 45 to 65 g.
(ii) Standard solutions — Weigh accurately a suitable
amount of Human Menopausal Gonadotrophin Reference
Standard, dissolve in human chorionic gonadotrophin TS to
make three solutions which contain 0.75, 1.5 and 3.0 follicle-
stimulating hormone Units per 1.0 mL, respectively. Inject
these solutions into three groups consisting of five test
animals each, and weigh their ovaries, as directed in (iv). Ac-
cording to the result of the test, designate the concentration
of the reference standard, which will make the mass of the
ovary about 120 to 160 mg, as the high-dose standard solu-
tion, S H . Dilute the S H to 1.5 to 2.0 times the initial volume
with the human chorionic gonadotrophin TS, and designate
the solution as the low-dose standard solution, S L .
(iii) Sample solutions — According to the labeled units,
weigh accurately a suitable amount of Human Menopausal
Gonadotrophin, dissolve in human chorionic gonadotrophin
TS, and prepare the high-dose sample solution, T H , and the
low-dose sample solution, T L , which have similar numbers of
units to those of corresponding standard solutions in equal
volume, respectively.
(iv) Procedure — Divide the test animals at random into 4
groups, A, B, C and D, with not less than 10 animals and
equal numbers in each group. Inject subcutaneously 0.2 mL
each of S H , S L , T H and T L into the animals in each group,
once in the afternoon on the first day, three times in the
morning, noon and afternoon on the second day, and two
times in the morning and afternoon on the third day. On the
fifth day, excise the ovaries, remove the fat and extraneous
tissue, remove fluid adhering to the ovaries by lightly pressing
between filter papers, and immediately weigh the ovaries.
(v) Calculation — Designate the mass of ovaries by S H , S L ,
T H and T L as y lt y 2 , v 3 and y 4 , respectively. Sum up y lt v 2 , v 3
and v 4 on each set to obtain Y x , Y 2 , Y" 3 and Y 4 .
710
Serum Gonadotrophin / Official Monographs
JP XV
Units per mg of Human Menopausal Gonadotrophin
= antilog Mx (units per mL of S H ) x (b/a)
M = IY a /Y b
7=log(S H /S L ) = log(T H /T L )
y a =-y,-y 2 +y 3 +y 4
Y b =Y 1 -Y 2 +Y 1 -Y 4
a: Mass (mg) of sample.
b: Total volume (mL) of the high dose of the test solution
prepared by diluting with bovine serum albumin-isotonic
sodium chloride solution.
F' computed by the following equation should be smaller
than F\ against n when s 2 is calculated. And compute L (P =
0.95) by the following equation: L should be not more than
0.3. If F' exceeds F lt or if L exceeds 0.3, repeat the test in-
creasing the number of the test animals or arranging the assay
method in a better way until F' is smaller than Fj or L is not
more than 0.3.
F'=(Y l -Y 2 -Y 3 -Y 4 ) 2 /(4fs 2 )
f: Number of test animals per group.
s 2 = {ly 2 -(Y/f)}/n
Yy 2 : The sum of the squares of each y u y 2 , j 3 and y A .
Y= — Y\2 — Y 22 + Y}2 + Y 42
k = 4(/-1)
L = 2V(C-1)(CM 2 + / 2 )
C=Y b 2 /(Y b 2 -4fs 2 t 2 )
t 2 : Value shown in the following table against n used to
calculate s 2 .
n
t 2 = F x
n
fl = F 1
n
fl = F 1
1
161.45
13
AMI
25
4. 242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
CO
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Serum Gonadotrophin
Serum Gonadotrophin is a dried preparation of
gonad-stimulating hormone, obtained from pregnant
mares' serum which has adequately inspected viruses,
and subjected to a suitable process for removal or inac-
tivation of viruses.
It contains not less than 2000 serum gonadotrophin
Units per mg.
It contains not less than 80% and not more than
125% of the labeled serum gonadotrophin Units.
Description Serum Gonadotrophin occurs as a white
powder.
It is freely soluble in water.
Identification Calculate b by the following equation, using
Y 3 and Y 4 obtained in the Assay: b is not less than 120.
b=E/I
E=(Y i -Y A )/f
f: Number of test animals per group.
7=log(T„/T L )
Purity Clarity and color of solution — Dissolve Serum
Gonadotrophin in isotonic sodium chloride solution to pre-
pare a solution containing 9000 units per mL according to the
labeled Units: the solution is clear and colorless.
Loss on drying <2.41> Not more than 8.0% (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.1 EU/unit.
Specific activity When calculated from the results obtained
by the Assay and the following test, Serum Gonadotrophin
containes not less than 3000 serum gonadotrophin Units per
mg of protein.
(i) Standard solutions — Dissolve about 3 mg of bovine
serum albumin in water to make a solution containing 500 /xg
of the albumin in each mL. To this solution add water to
make four standard solutions so that each mL contains
exactly 200 /xg, 150 /ug, 100,Mg and 50 /xg of the albumin,
respectively.
(ii) Sample solution — Dissolve about 1 mg of Serum
Gonadotrophin in water to make a solution containing
exactly ISO /ug in each mL.
(iii) Sodium carbonate solution — Dissolve 2 g of sodium
carbonate (standard reagent) in 0.1 mol/L sodium hydroxide
TS to make 100 mL.
(iv) Sodium tartrate solution — Dissolve about 1 g of
sodium tartrate dihydrate in water to make 100 mL.
(v) Copper (II) sulfate solution — Dissolve 0.5 g of copper
(II) sulfate pentahydrate in the sodium tartrate solution to
make 100 mL.
(vi) Alkaline copper solution — Mix 50 mL of the sodium
carbonate solution and 1 mL of the copper (II) sulfate
solution. Prepare before use. Use within the day of prepara-
tion.
(vii) Procedure — Pipet 0.5 mL each of the standard
solutions and the sample solution in small test tubes, add
3 mL of the alkaline copper solution to them, and mix. Allow
them to stand at the room temperature for not less than 10
minutes, add 0.3 mL of diluted Folin's TS (1 in 2), mix imme-
diately, and allow to stand for not less than 30 minutes. De-
termine the absorbances of these solutions so obtained at 750
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24> using a solution, prepared in the same manner with 0.5
mL of water, as the blank. Plot the calibration curve from
the absorbances obtained with the standard solutions, and
determine the amount of protein in the sample solution from
JPXV
Official Monographs / Serum Gonadotrophin 711
this curve.
Specific activity (unit/mg protein)
= [(units per mg, obtained in the Assay)/
(amount (%) of protein in the sample)] X 100
Abnormal toxicity Dissolve Serum Gonadotrophin in
isotonic sodium chloride solution so that each 5 mL of the so-
lution contains 4000 Units, and use this solution as the sam-
ple solution. Inject 5.0 mL of the sample solution into the
peritoneal cavity of each of 2 or more of well-nourished,
healthy guinea pigs weighing about 350 g, and inject 0.5 mL
of the sample solution into the peritoneal cavity of each of 2
or more of well-nourished, healthy mice aged about 5 weeks.
Observe the conditions of the animals for more than 7 days:
all the animals exhibit no abnormalities.
Assay
(i) Test animals — Select healthy female albino rats weigh-
ing about 45 g.
(ii) Standard solution — Dissolve a quantity of Serum
Gonadotrophin Reference Standard in bovine serum al-
bumin-isotonic sodium chloride solution to prepare four
kinds of solutions, containing 10, 20, 40 and 80 Units per 0.5
mL, respectively. Inject these solutions into four groups con-
sisting of five test animals each, and weigh their ovaries, as
directed in procedure of (iv). Inject bovine serum albumin-
isotonic sodium chloride solution to another group, and use
this group as the control group. According to the result of
this test, designate the concentration of the reference stan-
dard which will increase the masses of the ovaries about 3
times the mass of the ovaries of the control group as a low-
dose concentration of the standard solution, and the concen-
tration 1.5 to 2.0 times the low-dose concentration as a high-
dose concentration. Weigh accurately a suitable quantity of
Serum Gonadotrophin Reference Standard, dissolve in bo-
vine serum albumin-isotonic sodium chloride solution, and
prepare a high-dose standard solution S H and a low-dose
standard solution S L whose concentrations are equal to those
determined by the above test.
(iii) Sample solution — According to the labeled units,
weigh accurately a suitable quantity of Serum Gonadotro-
phin, dissolve in bovine serum albumin-isotonic sodium chlo-
ride solution, and prepare a high-dose sample solution T H
and a low-dose sample solution T L having Units equal to the
standard solutions in equal volumes, respectively.
(iv) Procedure — Divide the test animals at random into 4
groups, A, B, C and D, with not less than 10 animals and
equal numbers in each group. Inject once subcutaneously 0.5
mL of S H , S L , T H and T L in each group. On the sixth day, ex-
cise the ovaries, remove the fat and other unwonted tissues
attached to the ovaries, and remove the adhering water by
lightly pressing between filter paper, and immediately weigh
the ovaries.
(v) Calculation — Designate the mass of ovaries by S H , S L ,
T H and T L as y x , y 2 , y 3 and y 4 , respectively. Sum up y x , y 2 , y-j
and y 4 on each set to obtain Y lt Y 2 , Y } and Y 4 .
Units per mg of Serum Gonadotrophin
= antilog MX (units per mL of SH) x (b/a)
M=IYJY b
/=log(S H /S L ) = log(T H /T L )
Y a =-Y t -Y 2 +Y,+ Y 4
Y^Yi-Yt+Yi-Yt
a: Mass (mg) of sample.
b: Total volume (mL) of the high dose of the test solution
prepared by diluting with bovine serum albumin-isotonic
sodium chloride solution.
F' computed by the following equation should be smaller
than Fj against n when s 2 is calculated. And compute L (P =
0.95) by the following equation: L should be not more than
0.3. If F' exceeds F u or if L exceeds 0.3, repeat the test in-
creasing the number of the test animals or arranging the assay
method in a better way until F' is smaller than F-y or L is not
more than 0.3.
F'={Y X -Y 2 -Yi+Y 4 y/Afs 2
f: Number of test animals per group.
s 2 = {T.y 2 -{Y/f)}/n
Ey 2 : The sum of the squares of each y u y 2 , y^ and y 4 .
Y=Y l 2 +Y 2 2 +Y i 2 +Y 4 2
« = 4(/-l)
£ = 2V(C-l)(CM2 + /2)
C=Y b 2 /(Y b 2 -4fs 2 t 2 )
t 2 : Value shown in the following table against n used to
calculate s 2 .
n
fi = F,
n
fi=F t
n
fi=F t
1
161.45
13
4.667
25
4.242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
oo
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Serum Gonadotrophin for Injection
Serum Gonadotrophin for Injection is a preparation
for injection which is dissolved before use.
It contains not less than 80% and not more than
125% of the labeled Serum Gonadotrophin Units.
Method of preparation Prepare as directed under Injections
with Serum Gonadotrophin.
Description Serum Gonadotrophin for Injection occurs as
white powder or masses.
Identification Proceed as directed in the Identification un-
der Serum Gonadotrophin.
pH <2.54> Dissolve 30 mg of Serum Gonadotrophin for In-
712 Gramicidin / Official Monographs
JP XV
jection in 20 mL of isotonic sodium chloride solution: the pH
of this solution is between 5.0 and 7.0.
Loss on drying <2.41> Not more than 5.0% (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.1 EU/unit.
Assay Proceed as directed in the Assay under Serum
Gonadotrophin. The ratio of the Units assayed to the labeled
Units should be calculated by the following equation.
The ratio of the assayed Units to the labeled Units
= antilog M
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, and in a cold place.
Gramicidin
[1405-97-6]
Gramicidin is a mixture of peptide substances having
antibacterial activity produced by the growth of Bacil-
lus brevis Dubos.
It contains not less than 900 fig (potency) per mg,
calculated on the dried basis. The potency of
Gramicidin is expressed as mass (potency) of
gramicidin.
Description Gramicidin occurs as a white to light yellowish
white crystalline powder.
It is freely soluble in methanol, soluble in ethanol (99.5),
and practically insoluble in water.
Identification (1) To 10 mg of Gramicidin add 2 mL of
6 mol/L hydrochloric acid TS, and heat in a water bath for
30 minutes with occasional stirring. After cooling, neutralize
with 6 mol/L sodium hydroxide TS, add 1 mL of ninhydrin
TS and 0.5 mL of pyridine, and heat for 2 minutes: a blue-
purple to red-purple color develops.
(2) Determine the absorption spectrum of a solution of
Gramicidin in ethanol (95) (1 in 20,000), as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Gramicidin Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Loss on drying <2.41> Not more than 3.0% (0.1 g, in vacu-
um, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Turbidimetric
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Enterococcus hirae ATCC 10541
(ii) Agar medium for transferring test organism
Glucose 10.0 g
Casein peptone 5.0 g
Yeast extract 20.0 g
Potassium dihydrogen phosphate 2.0 g
Polysorbate 80 0.1 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.7 to 6.8 after sterilization.
(iii) Liquid medium for suspending test organism — Use
the culture medium (2).
(iv) Preparation of the test organism suspension — Pun-
cture the test organism in the medium, prepared by dispens-
ing 10 mL of the agar medium for transferring test organism
in a test tube about 16 mm in inside diameter, incubate at
36.5 to 37.5°C for 20 to 24 hours. After sub-culturing at least
three times, keep between 1 to 5°C. Transfer the organism so
obtained in 10 mL of the liquid medium for suspending test
organism, incubate at 36.5 to 37.5°C for 20 to 24 hours, and
use this medium as the test organism stock suspension. Be-
fore use, add the test organism stock suspension to the liquid
medium for suspending test organism so that the transmit-
tance at 580 nm is 50 to 60%. Mix one volume of this suspen-
sion and 200 volume of the liquid medium for suspending test
organism, and use this as the test organism suspension.
(v) Standard solution — Weigh accurately an amount of
Gramicidin Reference Standard, previously dried under
reduced pressure not exceeding 0.67 kPa at 60°C for 3 hours,
equivalent to about 10 mg (potency), dissolve in ethanol
(99.5) to make exactly 100 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
not exceeding 5°C and use within 7 days. Take exactly a suit-
able amount of the standard stock solution before use, add
the following diluting solution to make a solution so that
each mL contains 0.02 /ug (potency), and use this solution as
the standard solution.
Diluting solution: To 390 mL of propylene glycol add 210
mL of a mixture of ethanol (99.5) and acetone (9:1) and
Sterile Purified Water to make 1000 mL.
(vi) Sample solution — Weigh accurately an amount of
Gramicidin, equivalent to about 10 mg (potency), and dis-
solve in ethanol (99.5) to make exactly 100 mL. Take exactly
a suitable amount of this solution, add the diluting solution
obtained in (v) to make a solution so that each mL contains
0.02 n% (potency), and use this solution as the sample solu-
tion.
(vii) Procedure— Transfer 0.155 mL, 0.125 mL, 0.100
mL, 0.080 mL and 0.065 mL each of the standard solution,
0.100 mL of the sample solution and 0.100 mL of the diluting
solution, separately, in test tubes about 14 mm in inside di-
ameter and about 15 cm in length, and make three sets for
each. To each of the test tube add 10 mL of the test organism
suspension, stopper the tube, incubate in a water bath at 36.5
to 37.5°C for 180 to 270 minutes, add 0.5 mL of a solution of
formaldehyde (1 in 3), and determine their transmittances at
580 nm.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Griseofulvin 713
Griseofulvin
^U-tz7|-7JHf>
H 3 C
H;,C
C 17 H 17 C10 6 : 352.77
(2S,6'i?)-7-Chloro-2',4,6-trimethoxy-6'-
methylspiro [benzo [b] furan-2(3 H) , 1 ' -(cyclohex-2' -ene)] -
3,4'-dione [126-07-8]
Griseofulvin is a substance having antifungal activity
produced by the growth of Penicillium griseofulvum or
Penicillium janczewskii.
It contains not less than 960 fig (potency) and not
more than 1020 fig (potency) per mg, calculated on the
dried basis. The potency of Griseofulvin is expressed as
mass (potency) of griseofulvin (C 17 H 17 C10 6 ).
Description Griseofulvin occurs as white, crystals or crys-
talline powder.
It is soluble in _/V,7V-dimethylformamide, sparingly soluble
in acetone, slightly soluble in methanol and in ethanol (95),
and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Griseofulvin in ethanol (95) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Griseofulvin Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Griseofulvin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the
spectrum of Griseofulvin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™: + 350 - + 364° (0.25 g calcu-
lated on the dried basis, ./V,iV-dimethylformamide, 25 mL,
100 mm).
Melting point <2.60> 218 - 222 °C
Purity (1) Acidity — Dissolve 0.25 g of Griseofulvin in
20 mL of neutralized ethanol, and add 2 drops of
phenolphthalein TS and 1.0 mL of 0.02 mol/L sodium
hydroxide VS: the color of the solution is red.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Griseofulvin according to Method 2, and perform the test.
Prepare the control solution with 2.5 mL of Standard Lead
Solution (not more than 25 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Griseofulvin according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — To 0.10 g of Griseofulvin add
exactly 1 mL of the internal standard solution and acetone to
make 10 mL, and use this solution as the sample solution.
Separately, to 5.0 mg of Griseofulvin Reference Standard
add exactly 1 mL of the internal standard solution and ace-
tone to make 10 mL, and use this solution as the standard so-
lution. Perform the test with 2 fiL each of the sample solution
and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
determine each peak area by the automatic integration
method, and calculate the ratio, Q lt of the peak area of
dechlorogriseofulvin, having the relative retention time of
about 0.6 with respect to griseofulvin, to that of the internal
standard obtained from the sample solution, the ratio, Q 2 , of
the peak area of dehydrogriseofulvin, having the relative
retention time of about 1.2 with respect to griseofulvin, to
that of the internal standard obtained from the sample solu-
tion and the ratio, Q s , of the peak area of griseofulvin to that
of the internal standard obtained from the standard solution:
Qi/Qs is not more than 0.6, and Q 2 /Qs is not more than 0.15.
Internal standard solution — A solution of 9,10-
diphenylanthracene in acetone (1 in 500).
Operating conditions —
Detector: An hydrogen flame-ionization detector.
Column: A glass column 4 mm in inside diameter and 1 m
in length, packed with siliceous earth for gas chro-
matography coated with 25% phenyl-25% cyanopropyl-
methylsilicone polymer for gas chromatography in the ratio
of 1% (150 - 180 //m in particle diameter).
Column temperature: A constant temperature of about
250°C.
Temperature of injection port: A constant temperature of
about 270°C.
Temperature of detector: A constant temperature of about
300°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
griseofulvin is about 10 minutes.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the internal standard solution
diluted with acetone (1 in 10) to make exactly 10 mL. Con-
firm that the ratio of the peak area of griseofulvin to that of
the internal standard obtained from 2 fiL of this solution is
equivalent to 7 to 13% of that obtained from 2 fiL of the
standard solution.
System performance: When the procedure is run with 2 fiL
of the standard solution under the above operating condi-
tions, the internal standard and griseofulvin are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of griseofulvin to that of the internal standard is
not more than 5.0%.
(5) Petroleum ether soluble substances — To 1.0 g of
Griseofulvin add 20 mL of petroleum ether, shake, and boil
for 10 minutes under a reflux condenser. After cooling, filter
through a dried filter paper, wash the filter paper with two
15-mL portions of petroleum ether, combine the washings to
the filtrate, evaporate the petroleum ether on a water bath,
and dry the residue at 105°C for 1 hour: the amount of the
714 Guaifenesin / Official Monographs
JP XV
residue is not more than 0.2%.
Loss on drying <2.41> Not more than 1.0% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately an amount of Griseofulvin and
Griseofulvin Reference Standard, equivalent to about 50 mg
(potency), dissolve each in 50 mL of iV,./V-dimethylform-
amide, add exactly 20 mL of the internal standard solution
and water to make 250 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Qj and
Qs, of the peak area of griseofulvin to that of the internal
standard.
Amount [Mg (potency)] of Ci 7 H 17 C10 6
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Griseofulvin Reference
Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 400).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
griseofulvin is about 6 minutes.
System suitability —
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, griseofulvin and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 4.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of griseofulvin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Guaifenesin
Guaiacol Glyceryl Ether
and enantiomer
C 10 H 14 O 4 : 198.22
(2i?5')-3-(2-Methoxyphenoxy)propane-l,2-diol
[93-14-1]
Guaifenesin, when dried, contains not less then
98.0% and not more than 102.0%, of C 10 H 14 O 4 .
Description Guaifenesin occurs as a white crystals or crys-
talline powder.
It is freely soluble in ethanol (95), and sparingly soluble in
water.
A solution of ethanol (95) (1 in 20) shows no optical rota-
tion.
Identification (1) Determine the absorption spectrum of a
solution of Guaifenesin (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Guaifenesin Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Guaifenesin, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Guaifenesin Refer-
ence Standard: both spectra exhibit similar intensities of
absorption at the same wave numbers.
Melting point <2.60> 80 - 83 °C
pH <2.54> Dissolve 1.0 g of Guaifenesin in 100 mL of
water: the pH of the solution is between 5.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Guaifenesin in 10 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Dissolve 0.7 g of Guaifenesin in 25
mL of water by warming. Cool, add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution with
0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.020%).
(3) Heavy metals <1.07> — Dissolve 2.0 g of Guaifenesin
in 25 mL of water by warming. Cool, add 2 mL of dilute
acetic acid and water to make 50 mL, and perform the test
using this solution as the test solution. Prepare the control
solution with 2.0 mL of Standard Lead Solution (not more
than 10 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Guaifenesin according to Method 3, and perform the test
(not more than 2 ppm).
(5) Free guaiacol — To 1.0 g of Guaifenesin add exactly
25 mL of water, dissolve by warming, cool, and use this solu-
JPXV
Official Monographs / Guanabenz Acetate 715
tion as the sample solution. Separately, dissolve 0.100 g of
guaiacol in water to make exactly 1000 mL. Pipet 3 mL of
this solution, add exactly 22 mL of water, and use this solu-
tion as the standard solution. To each of the sample solution
and the standard solution add 1.0 mL of potassium hexac-
yanoferrate (III) TS and 5.0 mL of a solution of 4-aminoan-
tipyrine (1 in 200), and immediately after shaking for exactly
5 seconds add a solution of sodium hydrogen carbonate (1 in
1200) to make exactly 100 mL. Determine the absorbances of
these solutions at 500 nm exactly 15 minutes after the addi-
tion of the 4-aminoantipyrine solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using a solu-
tion, prepared in the same manner with 25 mL of water, as
the blank: the absorbance of the solution obtained from the
sample solution is not greater than that from the standard so-
lution.
(6) Related substances — Dissolve 1.0 g of Guaifenesin in
100 mL of ethanol (95), and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /iL each
of the sample solution and standard solution on a plate of sil-
ica gel for thin-layer chromatography. Develop the plate with
a mixture of diethyl ether, ethanol (95), and ammonia solu-
tion (28) (40:10:1) to a distance of about 10 cm, and air-dry
the plate. Spray evenly 4-dimethylaminobenzaldehyde TS for
spraying on the plate, and heat at 110°C for 10 minutes: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 60 mg of Guaifenesin and
Guaifenesin Reference Standard, previously dried, and dis-
solve each then in water to make exactly 100 mL. Pipet 5 mL
of these solutions, and add water to make exactly 100 mL,
and use these solutions as the sample solution and standard
solution. Determine the absorbances, A T and A s , of the sam-
ple solution and standard solution at 273 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 10 H 14 O 4
= W s x (At/ A s )
W s : Amount (mg) of Guaifenesin Reference Standard
Containers and storage Containers — Tight containers.
Guanabenz Acetate
H,N^ „N
CI
HjC-CCVt
0!
C 8 H 8 C1 2 N 4 .C 2 H 4 2 : 291.13
(£')-l-(2,6-Dichlorobenzylideneamino)guanidine
monoacetate [23256-50-0]
Guanabenz Acetate, when dried, contains not less
than 98.5% of C 8 H 8 C1 2 N 4 .C 2 H 4 2 .
Description Guanabenz Acetate occurs as white crystals or
crystalline powder.
It is freely soluble in acetic acid (100), soluble in methanol
and in ethanol (95), slightly soluble in water, and practically
insoluble in diethyl ether.
It is gradually affected by light.
Melting point: about 190°C (with decomposition).
Identification (1) To 5 mL of a solution of Guanabenz
Acetate (1 in 1000) add 0.5 mL of a diluted ethanol (95) (5 in
6) which contains 16 g of urea and 0.2 g of 1-naphthol in 100
mL, and add 1 mL of iV-bromosuccinimide TS: a purple
color develops.
(2) Determine the absorption spectrum of a solution of
Guanabenz Acetate in methanol (1 in 100,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Guanabenz Acetate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) To 0.1 g of Guanabenz Acetate add 5 mL of water
and 1 mL of ammonia TS, shake, filter, and neutralize the
filtrate with dilute hydrochloric acid: the solution responds to
the Qualitative Tests <1.09> (3) for acetate.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Guanabenz Acetate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than lOppm).
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.05 g of Guanabenz Acetate in 5 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 10 mL, then pipet 1
mL of this solution, add methanol to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, methanol and ammonia solu-
tion (28) (80:20:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution. Place the plate in a chamber filled with io-
dine vapor for 10 minutes: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 50°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.25 g of Guanabenz
Acetate, previously dried, dissolve in 50 mL of acetic acid
716 Guanethidine Sulfate / Official Monographs
JP XV
(100), and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.11 mg of C 8 H 8 C1 2 N 4 .C 2 H 4 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Guanethidine Sulfate
C 10 H 22 N 4 .H 2 SO 4 : 296.39
1 - [2-(Hexahydroazocin- 1 (2/f)-yl)ethyl]guanidine
monosulfate [645-43-2]
Guanethidine Sulfate, when dried, contains not less
than 98.5% of C 10 H 22 N 4 .H 2 SO4.
Description Guanethidine Sulfate occurs as white crystals
or crystalline powder. It is odorless or has a slight, charac-
teristic odor and a bitter taste.
It is very soluble in formic acid, freely soluble in water, and
practically insoluble in ethanol (95) and in diethyl ether.
Melting point: 251 -256°C (an evacuated sealed capillary
tube, with decomposition).
Identification (1) To 4 mL of a solution of Guanethidine
Sulfate (1 in 4000) add 2 mL of 1-naphthol TS, 1 mL of
diacetyl TS and 15 mL of water, and allow to stand for 30
minutes: a red color develops.
(2) Determine the infrared absorption spectrum of
Guanethidine Sulfate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Guanethidine Sulfate (1 in 10) responds
to the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 1.0 g of Guanethidine Sulfate in 50 mL
of water: the pH of the solution is between 4.7 and 5.7.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Guanethidine Sulfate in 50 mL of water: the solution is clear
and colorless.
(2) Methylisothiourea sulfate — Dissolve 2.0 g of
Guanethidine Sulfate in 80 mL of sodium hydroxide TS, and
allow to stand for 10 minutes. Add 60 mL of hydrochloric
acid, 2 g of sodium bromide and water to make 200 mL.
Then, to this solution add 0.70 mL of 1/60 mol/L potassium
bromate VS and 2 mL of zinc iodide-starch paste TS: a blue
color develops.
(3) Heavy metals <1.07> — Proceed with 2.0 g of
Guanethidine Sulfate according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Guanethidine Sul-
fate, previously dried, dissolve in 2 mL of formic acid, add 70
mL of a mixture of acetic anhydride and acetic acid (100)
(6:1), and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.64 mg of C, H 22 N 4 .H 2 SO 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Adsorbed Habu-venom Toxoid
>*»(*-$: r-*7^ K
Adsorbed Habu-venom Toxoid is a liquid for injec-
tion containing habu toxoid prepared by treating toxic
substances produced by habu (Trimeresurus flavoviri-
dis) with formaldehyde by a method involving no ap-
preciable loss of the immunogenicity and rendered in-
soluble by the addition of aluminum salt.
It conforms to the requirements of Adsorbed Habu-
venom Toxoid in the Minimum Requirements for Bio-
logical Products.
Description Adsorbed Habu-venom Toxoid becomes a
uniform whitish turbid liquid on shaking.
Freeze-dried Habu Antivenom,
Equine
Freeze-dried Habu Antivenom, Equine, is a prepara-
tion for injection which is dissolved before use.
It contains Trimeresurus flavoviridis antivenom in
immunoglobulin of horse origin.
It conforms to the requirements of Freeze-dried
Habu Anti-venom, Equine, in the Minimum Require-
ments for Biological Products.
Description Freeze-dried Habu Antivenom, Equine,
becomes colorless or light yellow-brown, clear liquid or a
slightly whitish turbid liquid on addition of solvent.
Adsorbed Hepatitis B Vaccine
Adsorbed Hepatitis B Vaccine is a liquid for injec-
tion prepared by adding an aluminum salt to a liquid
containing a surface antigen of hepatitis B virus to
make the HBs antigen insoluble.
JPXV
Official Monographs / Ilaloperidol 717
It conforms to the requirements of Adsorbed Hepa-
titis B Vaccine in the Minimum Requirements for Bio-
logical Products.
Description Adsorbed Hepatitis B Vaccine becomes a
homogeneous, whitish turbid liquid on shaking.
Haloperidol
CI
II J OH
C 2 iH 23 ClFN0 2 : 375.86
4-[4-(4-Chlorophenyl)-4-hydroxypiperidin-l-yl]-l-(4-
fluorophenyl)butan-l-one [52-86-8]
Haloperidol, when dried, contains not less than
99.0% and not more than 101.0% of C 21 H 23 C1FN0 2 .
Description Haloperidol occurs as white to pale yellow
crystals or powder.
It is freely soluble in acetic acid (100), sparingly soluble in
methanol, slightly soluble in 2-propanol and in ethanol
(99.5), and practically insoluble in water.
Identification (1) Dissolve 30 mg of Haloperidol in 100
mL of 2-propanol. To 5 mL of the solution add 10 mL of 0. 1
mol/L hydrochloric acid TS and 2-propanol to make 100
mL. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Haloperidol as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 149 - 153°C
Purity (1) Sulfate </./4>— To 1.0 g of Haloperidol add 50
mL of water, shake, and filter. To 25 mL of the filtrate add 1
mL of dilute hydrochloric acid and water to make 50 mL,
and perform the test using this solution as the test solution.
Prepare the control solution with 0.50 mL of 0.005 mol/L
sulfuric acid VS (not more than 0.048%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Haloperidol according to method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 25 mg of Haloperidol
in 50 mL of the mobile phase, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add the
mobile phase to make exactly 200 mL, and use this solution
as the standard solution. Perform the test with exactly 10 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
haloperidol is not larger than the peak area of haroperidol
from the standard solution, and the total area of these peaks
is not larger than 2 times the peak area of haloperidol from
the standard solution. For this calculation, use the peak areas
for the related substances, having the relative retention time
of about 0.5, about 1.2 and about 2.6 with respect to
haloperidol, after multiplying by their relative response fac-
tors, 0.75, 1.47 and 0.76, respectively.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.95 g of trisodium citrate dihy-
drate in 900 mL of water, adjust to pH 3.5 with dilute
hydrochloric acid, and add water to make 1000 mL. To 300
mL of this solution add 700 mL of methanol and 1 .0 g of so-
dium lauryl sulfate.
Flow rate: Adjust the flow rate so that the retention time of
haloperidol is about 9 minutes.
Time span of measurement: About 2 times as long as the
retention time of haloperidol beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add the mobile phase to make exactly 25 mL.
Confirm that the peak area of haloperidol obtained with 10
fiL of this solution is equivalent to 15 to 25% of that with 10
fiL of the standard solution.
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of haloperidol are not less than 4000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
haloperidol is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, phosphorus (V) oxide, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of haloperidol, previ-
ously dried, and dissolve in 40 mL of acetic acid (100), and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (indicator: 1
drop of crystal violet TS). Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 37.59 mg of C 21 H 23 C1FN0 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
718
Ilaloperidol Tablets / Official Monographs
JP XV
Haloperidol Tablets
Haloperidol Tablets contain not less than 93.0% and
not more than 107.0% of the labeled amount of
haloperidol (C21H23CIFNO2: 375.86).
Method of preparation Prepare as directed under Tablets,
with Haloperidol.
Identification To pulverized Haloperidol Tablets, equiva-
lent to 6 mg of Haloperidol according to the labeled amount,
add 70 mL of 2-propanol, and heat on a water bath until to
boiling while shaking. After cooling, add 2-propanol to make
100 mL, and centrifuge. To 5 mL of the supernatant liquid
add 2 mL of 0.1 mol/L hydrochloric acid TS and 2-propanol
to make 20 mL. Determine the absorption spectrum of the
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits maxima between 219 nm and 223 nm
and between 243 nm and 247 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Haloperidol Tablets add 5 mL of the mobile
phase, disperse the particle with the aid of ultrasonic waves,
add 30 mL of the mobile phase, and extract for 30 minutes
with the aid of ultrasonic waves with occasional shaking.
Shake for more 30 minutes, and add the mobile phase to
make exactly 50 mL. Centrifuge the solution, pipet FmL of
the supernatant liquid, equivalent to about 0.3 mg of
haloperidol (C 21 H 23 C1FN0 2 ), add exactly 2 mL of the inter-
nal standard solution and the mobile phase to make 25 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 20 mg of haloperidol for assay, previ-
ously dried in vacuum at 60°C for 3 hours on phosphorus (V)
oxide, and dissolve in the mobile phase to make exactly 100
mL. Pipet 15 mL of this solution, and add the mobile phase
to make exactly 50 mL. Pipet 5 mL of this solution, add ex-
actly 2 mL of the internal standard solution and the mobile
phase to make 25 mL, and use this solution as the standard
solution. Perform the test with 10 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according the following conditions, and
determine the ratios, Q T and Q s , of the peak area of
haloperidol to that of the internal standard.
Amount (mg) of haloperidol (C 21 H 23 C1FN0 2 )
= W s x (Q T /Q S ) X (\/V) X (3/4)
fV s : Amount (mg) of haloperidol for assay
Internal standard solution — A solution of diphenyl in the
mobile phase (1 in 6700).
Operating conditions —
Proceed as detected in the operating condition in the
Assay.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, haloperidol and diphenyl are eluted in this order with
the resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of haloperidol to that of the internal standard is
not more than 1.0%.
Dissolution Being specified separately.
Assay Weigh accurately, and powder not less than 20
Haloperidol Tablets. Weigh accurately a portion of the
powder, equivalent to about 10 mg of haloperidol
(C 21 H 23 C1FN0 2 ), add 10 mL of water, disperse the particle
with the aid of ultrasonic waves, add exactly 20 mL of the in-
ternal standard solution, extract for 30 minutes with the aid
of ultrasonic waves with occasional shaking, and add the mo-
bile phase to make 100 mL. Centrifuge after shaking for
more 30 minutes, and use the supernatant liquid as the sam-
ple solution. Separately, weigh accurately about 25 mg of
haloperidol for assay, previously dried in vacuum at 60°C for
3 hours on phosphorus (V) oxide, and dissolve in methanol to
make exactly 25 mL. Pipet 10 mL of this solution, add ex-
actly 20 mL of the internal standard solution and the mobile
phase to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with 10 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Qs, of the peak area
of haloperidol to that of the internal standard.
Amount (mg) of haloperidol (C 21 H 23 C1FN0 2 )
= W s x (Qj/Q s ) x (2/5)
W s : Amount (mg) of haloperidol for assay
Internal standard solution — A solution of diphenyl in
methanol (1 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.95 g of trisodium citrate dihy-
drate in 900 mL of water, adjust to pH 3.5 with dilute
hydrochloric acid, and add water to make 1000 mL. To 250
mL of this solution add 750 mL of methanol and 1 .0 g of so-
dium lauryl sulfate, and mix to dissolve.
Flow rate: Adjust the flow rate so that the retention time of
haloperidol is about 9 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, haloperidol and diphenyl are eluted in this order with
the resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of haloperidol to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant for the tablets without coating.
JPXV
Official Monographs / Haloxazolam 719
Halothane
J\n9>
F, F
Br CI
and enantiomer
C 2 HBrClF 3 : 197.38
(2/?5')-2-Bromo-2-chloro-l , 1 , 1 -trifluoroethane
[151-67-7]
Halothane contains not less than 0.008% and not
more than 0.012% of Thymol as a stabilizer.
Description Halothane is a clear, colorless, and mobile liq-
uid.
It is miscible with ethanol (95), with diethyl ether and with
isooctane.
It is slightly soluble in water.
It is a volatile, nonflammable liquid, and setting fire to its
heated vapor does not support combustion.
It is affected by light.
Refractive index n™\ 1.369 - 1.371
Identification Transfer about 3 /uL of Halothane to a gas
cell having light path 10 cm in length, and determine the in-
frared absorption spectrum as directed in the gas sampling
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Specific gravity <2.56>
d 2 2 ° : 1.872-1.877
Purity (1) Acidity or alkalinity — Shake 60 mL of
Halothane with 60 mL of freshly boiled and cooled water
vigorously for 3 minutes. Separate the water layer, and use
this as the sample solution. To 20 mL of the sample solution
add 1 drop of bromocresol purple TS and 0.10 mL of 0.01
mol/L sodium hydroxide VS: a red-purple color develops. To
20 mL of the sample solution add 1 drop of bromocresol pur-
ple TS and 0.6 mL of 0.01 mol/L hydrochloric acid VS: a yel-
low color is produced.
(2) Halide and halogen — To 5 mL of the sample solution
obtained in (1) add 1 drop of nitric acid and 0.20 mL of silver
nitrate TS: no turbidity is produced. To 10 mL of the sample
solution obtained in (1) add 1 mL of potassium iodide TS and
2 drops of starch TS, and allow to stand for 5 minutes: no
blue color develops.
(3) Phosgene — Transfer 50 mL of Halothane to a dried
300-mL conical flask, suspend a strip of phosgene test paper
vertically inside the flask with the lower end about 10 mm
above the surface of the liquid, insert the stopper, and allow
to stand at a dark place for 20 to 24 hours: the test paper
shows no yellow color.
(4) Residue on evaporation — Pipet 50 mL of Halothane,
evaporate on a water bath, and dry the residue at 105°C for 2
hours: the mass of the residue is not more than 1.0 mg.
(5) Volatile related substances — To 100 mL of Halothane
add exactly 5.0 /uL of the internal standard, and use this solu-
tion as the sample solution. Perform the test with 5 /iL of the
sample solution as directed under Gas Chromatography
<2.02>, and determine each peak area by the automatic in-
tegration method: the total area of the peaks other than those
of halothane and the internal standard is not larger than the
peak area of the internal standard.
Internal standard — l,l,2-Trichloro-l,2,2-trifluoroethane.
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A column about 3 mm in inside diameter and 3 m
in length, at the first 2 m from the injection port, having mac-
rogol 400 coated in the ratio of 30% on siliceous earth for gas
chromatography (180 to 250 fim in particle diameter), and at
the remaining 1 m, having dinonyl phthalate coated in the ra-
tio of 30% on siliceous earth for gas chromatography (180 to
250 //m in particle diameter).
Column temperature: A constant temperature of about
50°C
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is 2 to 3 minutes.
Selection of column: Mix 3 mL of Halothane and 1 mL of
the internal standard. Proceed with 1 fiL of this solution un-
der the above operating conditions, and calculate the resolu-
tion. Use a column giving elution of the internal standard and
halothane in this order with the resolution between these
peaks being not less than 10.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of the internal standard obtained from 5
fiL of the sample solution composes 30 to 70% of the full
scale.
Time span of measurement: About 3 times as long as the
retention time of halothane.
Distilling range <2.57> Not less than 95 vol distils within a 1
°C range between 49°C and 51 °C.
Thymol To 0.50 mL of Halothane add 5.0 mL of isooctane
and 5.0 mL of titanium (IV) oxide TS, shake vigorously for
30 seconds, and allow to stand: the separated upper layer has
more color than the following control solution A, and has no
more color than the following control solution B.
Control solution: Dissolve 0.225 g of thymol for assay in
isooctane to make exactly 100 mL. To 10 mL each of this so-
lution, accurately measured, add isooctane to make exactly
150 mL and 100 mL, respectively. Proceed with 0.50 mL
each of these solutions in the same manner as Halothane, and
use the separated upper layers so obtained as the control solu-
tion A and B, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 30°C.
Haloxazolam
Ant*-/7A
and enantiomer
C 17 H 14 BrFN 2 2 : 377.21
(llbi?S)-10-Bromo-llb-(2-fluorophenyl)-2, 3,7,1 lb-
tetrahydro [ 1 , 3] oxazolo [3 ,2-d] [ 1 ,4]benzodiazepin-6(5//)-one
[59128-97-1]
720
Haloxazolam / Official Monographs
JP XV
Haloxazolam, when dried, contains not less than
99.0% of C 17 H 14 BrFN 2 2 .
Description Haloxazolam occurs as white crystals or crys-
talline powder. It is odorless and tasteless.
It is freely soluble in acetic acid (100), sparingly soluble in
acetonitrile, in methanol and in ethanol (99.5), slightly solu-
ble in diethyl ether, and practically insoluble in water.
Melting point: about 183°C (with decomposition).
Identification (1) Dissolve 0.01 g of Haloxazolam in 10
mL of methanol, add 1 drop of hydrochloric acid: the solu-
tion shows a yellow-green fluorescence under ultraviolet light
(main wavelength: 365 nm). To this solution add 1 mL of so-
dium hydroxide TS: the fluorescence disappears immediately.
(2) Prepare the test solution with 0.05 g of Haloxazolam
as directed under Oxygen Flask Combustion Method <1.06>,
using a mixture of 20 mL of dilute sodium hydroxide TS and
1 mL of hydrogen peroxide (30) as an absorbing liquid: the
test solution responds to the Qualitative Tests <1.09> for
bromide and for fluoride.
(3) Determine the absorption spectrum of a solution of
Haloxazolam in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of
Haloxazolam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry <
2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Absorbance <2.24> E\°^ (247 nm): 390 - 410 (10 mg,
methanol, 1000 mL).
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Haloxazolam in 20 mL of ethanol (99.5): the solution is
clear and colorless.
(2) Soluble halides — To 1 .0 g of Haloxazolam add 50 mL
of water, allow to stand for 1 hour with occasional shaking,
and filter. To 25 mL of the filtrate add 6 mL of dilute nitric
acid and water to make 50 mL. Perform the test with this so-
lution as directed under Chloride Limit Test <1.03>. Prepare
the control solution with 0.10 mL of 0.01 mol/L hydrochlor-
ic acid VS.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Halox-
azolam according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(4) Arsenic <1.11> — To 1 .0 g of Haloxazolam in a decom-
position flask add 5 mL of nitric acid and 2 mL of sulfuric
acid, place a small funnel on the mouth of the flask, and heat
carefully until white fumes are evolved. After cooling, add 2
mL of nitric acid, heat, repeat this procedure twice, add
several 2-mL portions of hydrogen peroxide (30), and heat
until the solution is colorless to pale yellow. After cooling,
add 2 mL of a saturated solution of ammonium oxalate
monohydrate, and heat until white fumes are evolved. After
cooling, add water to make 5 mL, and perform the test with
this solution: the solution has no more color than the follow-
ing control solution (not more than 2 ppm).
Control solution: Proceed in the same manner as above
without using Haloxazolam, add 2.0 mL of Standard Arsenic
Solution and water to make 5 mL, and proceed in the same
manner as the test solution.
(5) Related substances — Dissolve 0.10 g of Haloxazolam
in 100 mL of acetonitrile, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add acetonitrile
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of both solutions by the
automatic integration method: the total area of all peaks
other than the area of the haloxazolam from the sample
solution is not larger than the peak area of the haloxazolam
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 250 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.2 g of boric acid and 7.5 g of
potassium chloride in 900 mL of water, adjust the pH with
triethylamine to 8.5, and add water to make 1000 mL. To 300
mL of this solution add 200 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
haloxazolam is about 10 minutes.
Time span of measurement: About 3 times as long as the
retention time of haloxazolam beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add acetonitrile to make exactly 50 mL. Con-
firm that the peak area of haloxazolam obtained from 10 /iL
of this solution is equivalent to 8 to 12% of that of haloxazol-
am obtained from 10 [iL of the standard solution.
System performance: Dissolve 10 mg each of Haloxazolam
and cloxazolam in 200 mL of acetonitrile. When the proce-
dure is run with 10 /iL of this solution under the above oper-
ating conditions, haloxazolam and cloxazolam are eluted in
this order with the resolution between these peaks being not
less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
haloxazolam is not more than 1.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.5 g of Haloxazolam, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 37.72 mg of C 17 H 14 BrFN 2 2
Containers and storage Containers — Tight containers.
JPXV
Storage — Light-resistant.
Heparin Sodium
"\a°U >i-V U^A
Heparin Sodium is obtained from the livers, the
lungs and the intestinal mucosa of healthy edible
animals, and prolongs the clotting time of blood.
Heparin Sodium obtained from the livers and the lungs
contains not less than 110 Heparin Units per mg, and
that obtained from the intestinal mucosa contains not
less than 130 Heparin Units per mg.
Heparin Sodium, calculated on the dried basis, con-
tains not less than 90% and not more than 110% of the
labeled Units.
Label the name of the organ used as the starting
material.
Description Heparin Sodium occurs as a white to grayish
brown powder or grains. It is odorless.
It is soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
It is hygroscopic.
pH <2.54> The pH of a solution of 1 .0 g of Heparin Sodium
in 100 mL of water is between 6.0 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Heparin Sodium in 20 mL of water: the solution is clear and
colorless to light yellow.
(2) Barium — Dissolve 0.03 g of Heparin Sodium in 3.0
mL of water, and use this solution as the sample solution. To
1.0 mL of the sample solution add 3 drops of dilute sulfuric
acid, and allow to stand for 10 minutes: no turbidity is
produced.
(3) Total nitrogen — Weigh accurately about 0.1 g of
Heparin Sodium, previously dried at 60°C for 3 hours under
reduced pressure, and perform the test as directed under
Nitrogen Determination <1.08>: the amount of nitrogen (N:
14.01) is not more than 3.0%.
(4) Protein — To 1.0 mL of the sample solution obtained
in (2) add 5 drops of a solution of trichloroacetic acid (1 in 5):
neither a precipitate nor turbidity is produced.
Loss on drying <2.41> Not more than 10% (20 mg, in vacu-
um, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 40% (after
drying, 20 mg).
Pyrogen <4.04> Dissolve Heparin Sodium in isotonic sodi-
um chloride solution so as to contain 1000 Units per mL ac-
cording to the labeled Units. Inject into rabbits 2 mL of this
solution per kg: it meets the requirement.
Assay (i) Substrate solution: Dissolve 15 mg of N-ben-
zoyl-L-isoleucyl-L-glutamyl(y-OR)-glycyl-L-arginyl-j9-
nitroanilide hydrochloride in 20 mL of water.
(ii) Antithrombin III solution: Dissolve human an-
tithrombin III in water to make a solution containing 1 Unit
per mL.
(iii) Activated blood coagulation factor X solution: Dis-
solve bovine activated blood coagulation factor X in water to
Official Monographs / Heparin Sodium 721
make a solution containing 0.426 Units per mL.
(iv) Buffer solution: Dissolve 6.06 g of 2-amino-2-
hydroxymethyl-l,3-propanediol in 750 mL of water, adjust
the pH to 8.4 with 1 mol/L hydrochloric acid TS, and add
water to make 1000 mL.
(v) Reaction stop solution: To 20 mL of acetic acid (100)
add water to make 40 mL.
(vi) Heparin standard solutions: Dissolve Heparin Sodi-
um Reference Standard in isotonic sodium chloride solution
to make a solution containing 10 Units per mL, and use as the
standard stock solution. To 100 /xh of the standard stock so-
lution add the buffer solution to make exactly 5 mL, and use
this solution as the standard solution. Prepare the heparin
standard solutions (1), (2), (3), (4) and (5) by addition of an-
tithrombin III solution, human normal plasma and the buffer
solution to the standard solution as directed in the following
table.
Heparin standard
solution
No.
solution „ „ , ,.., , . Human
Buffer Antithrombin ,
IT ■ solution III solution ,
Heparin , , . , , . plasma
concentration ^ L; ^ L; (juL)
(Unit/mL)
(1)
800
100
100
(2)
0.02
700
100
100
100
(3)
0.04
600
100
100
200
(4)
0.06
500
100
100
300
(5)
0.08
400
100
100
400
Standard
solution
(vii) Sample solution: Weigh accurately an adequate
amount of Heparin Sodium, dissolve in isotonic sodium chlo-
ride solution so that each mL contains about 0.5 Units ac-
cording to the labeled amount. To 100 /xL of this solution
add 100 fiL of antithrombin III solution, 100 //L of human
normal plasma and 700 iiL of the buffer solution, and use this
solution as the sample solution.
(viii) Procedure: Transfer 400 /xL of the sample solution
to a test tube, and warm at 37°C for 4 minutes. Add 200 /iL
of the activated blood coagulation factor X solution, mix
well, warm at 37°C for exactly 30 seconds, add 400 /uL of the
substrate solution, previously warmed at 37 °C, and mix well.
Allow the tube to stand at 37°C for exactly 3 minutes, add
600 //L of the reaction stop solution, mix immediately, and
determine the absorbance at 405 nm, using the blank solution
prepared by addition of 600 /uL of the reaction stop solution
and 600 fiL of water to 400 /uL of the sample solution. Pro-
ceed the same way with the heparin standard solution (1), the
heparin standard solution (2), the heparin standard solution
(3), the heparin standard solution (4) and the heparin stan-
dard solution (5), and determine their absorbances.
(ix) Calculation: Plot the absorbances of the standard so-
lutions on the vertical axis and their heparin concentrations
on the horizontal axis to prepare a calibration curve. Deter-
mine the heparin concentration, C, of the sample solution
from its absorbance by using the curve, and calculate heparin
Units per mg of Heparin Sodium from the following formu-
la.
Units per mg of Heparin Sodium = C x 10 x (b/a)
722 Heparin Sodium Injection / Official Monographs
JP XV
a: Amount of sample (mg)
b: Total volume (mL) of isotonic sodium chloride solution
used to dissolve the sample to make the solution
containing about 0.5 Units per mL
Containers and storage Containers — Tight containers.
Heparin Sodium Injection
"V?U>-J-r- u^Aaw*
Heparin Sodium Injection is an aqueous solution for
injection. It contains not less than 90% and not more
than 110% of the labeled heparin Units.
Label the name of organ used as the starting material
of Heparin Sodium supplied for preparing Heparin
Sodium Injection.
Method of preparation Dissolve Heparin Sodium in Isoton-
ic Sodium Chloride Solution and prepare as directed under
Injections.
Description Heparin Sodium Injection is a clear, colorless
to light yellow liquid.
pH <2.54> 5.5-8.0
Purity (1) Barium — Measure exactly a portion of Heparin
Sodium Injection, equivalent to 3000 Units of Heparin Sodi-
um according to the labeled Unit. Add water to make 3.0 mL
and use this solution as the sample solution. To 1.0 mL of the
sample solution add 3 drops of dilute sulfuric acid, and allow
to stand for 10 minutes: no turbidity is produced.
(2) Protein — Proceed as directed in the Purity (4) under
Heparin Sodium.
Bacterial endotoxins <4.01> Less than 0.0030 EU/unit.
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter Perform the test according to
Method 1: it meets the requirement.
Insoluble particulate matter Perform the test according to
Method 1: it meets the requirement.
Sterility Perform the test according to the Membrane filtra-
tion method: it meets the requirement.
Assay Proceed as directed in the Assay under Heparin
Sodium, replacing the sample solution indicated in (vii) and
the calculation in (ix) with the following.
Sample solution: Measure exactly an adequate portion of
Heparin Sodium Injection according to the labeled Units,
dilute it with isotonic sodium chloride solution so that each
mL contains about 0.5 Units. To 100 /xh of this solution add
100 fiL of antithrombin III solution, 100 fiL of human nor-
mal plasma and 700 /uL of the buffer solution, and use this so-
lution as the sample solution.
Calculation: Plot the absorbances of the standard solu-
tions on the vertical axis and their heparin concentrations on
the horizontal axis to prepare a calibration curve. Determine
the heparin concentration, C, of the sample solution from its
absorbance by using the curve, and calculate heparin Units
per mL of Heparin Sodium Injection from the following for-
mula.
Units per mL of Heparin Sodium Injection
= C x 10 x (b/a)
a: Amount of sample (mL)
b: Total volume (mL) of isotonic sodium chloride solution
used to dilute the sample to make the solution contain-
ing about 0.5 Units per mL
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Homatropine Hydrobromide
*-7r-ntf>JMbk*#*
H
C 16 H 2I N0 3 .HBr: 356.25
(li?,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl [(2RS)-2-
hydroxy-2-phenyl]acetate monohydrobromide [51-56-9]
Homatropine Hydrobromide contains not less than
99.0% of C 16 H 21 N0 3 .HBr, calculated on the dried
basis.
Description Homatropine Hydrobromide occurs as white
crystals or crystalline powder. It is odorless.
It is freely soluble in water, sparingly soluble in ethanol
(95), slightly soluble in acetic acid (100), very slightly soluble
in acetic anhydride, and practically insoluble in diethyl ether.
It is affected by light.
Melting point: about 214°C (with decomposition).
Identification (1) To 5 mL of a solution of Homatropine
Hydrobromide (1 in 20) add 2 to 3 drops of iodine TS: a
brown precipitate is produced.
(2) Dissolve 0.05 g of Homatropine Hydrobromide in 5
mL of water, and add 3 mL of 2,4,6-trinitrophenol TS: a yel-
low precipitate is produced. Filter the precipitate, wash with
five 10-mL portions of water, and dry at 105 °C for 2 hours: it
melts <2.60> between 184°C and 187°C.
(3) A solution of Homatropine Hydrobromide (1 in 20)
responds to the Qualitative Tests <1.09> for bromide.
Purity (1) Acidity — Dissolve 1.0 g of Homatropine
Hydrobromide in 20 mL of water, and add 0.40 mL of 0.01
mol/L sodium hydroxide VS and 1 drop of methyl red-
methylene blue TS: a green color develops.
(2) Atropine, hyoscyamine and scopolamine — To 10 mg
of Homatropine Hydrobromide add 5 drops of nitric acid,
evaporate on a water bath to dryness, and cool. Dissolve the
residue in 1 mL of 7V,A r -dimethylformamide, and add 5 to 6
drops of tetraethylammonium hydroxide TS: no red-purple
color is produced.
(3) Related substances — Dissolve 0.15 g of Homatropine
Hydrobromide in 3 mL of water, and use this solution as the
sample solution.
(i) To 1 mL of the sample solution add 2 to 3 drops of
tannic acid TS: no precipitate is produced.
(ii) To 1 mL of the sample solution add 2 to 3 drops each
JPXV
Official Monographs / Homochlorcyclizine Hydrochloride
723
of dilute hydrochloric acid and platinic chloride TS: no
precipitate is produced.
Loss on drying <2.41> Not more than 1.5% (0.5 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (0.2 g).
Assay Dissolve by warming about 0.4 g of Homatropine
Hydrobromide in 60 mL of a mixture of acetic anhydride and
acetic acid (100) (7:3). Cool, and titrate <2.50> with 0.1 mol/
L perchloric acid VS (potentiometric titration). Perform a
blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 35.63 mg of C 16 H 21 N0 3 .HBr
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Homochlorcyclizine Hydrochloride
•2HCI
H I N-
and enantiomer
C 19 H 23 C1N 2 .2HC1: 387.77
l-[(i?S)-(4-Chlorophenyl)(phenyl)methyl]-
4-methylhexahydro-l//-l ,4-diazepine dihydrochloride
[1982-36-1]
Homochlorcyclizine Hydrochloride, when dried,
contains not less than 98.0% of C 19 H 23 C1N 2 .2HC1.
Description Homochlorcyclizine Hydrochloride occurs as
white to pale brown, crystals or powder.
It is very soluble in water, freely soluble in acetic acid
(100), slightly soluble in ethanol (99.5), and very slightly solu-
ble in acetonitrile and in acetic anhydride.
It dissolves in 0.1 mol/L hydrochloric acid TS.
It is hygroscopic.
It is colored slightly by light.
A solution of Homochlorcyclizine Hydrochloride (1 in 10)
shows no optical rotation.
Melting point: about 227°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Homochlorcyclizine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Homochlorcyclizine Hydrochloride, previously dried, as
directed in the potassium chloride disk method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
(3) A solution of Homochlorcyclizine Hydrochloride (1
in 100) responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Homochlorcyclizine Hydrochloride according to Method 2,
and perform the test. Prepare the control solution with
2.0 mL of Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.10 g of Homochlor-
cyclizine Hydrochloride in 100 mL of the mobile phase, and
use this solution as the sample solution. Measure exactly
1 mL of this solution, add the mobile phase to make exactly
100 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas by the automatic integration
method: the areas of the peaks other than homochlorcycli-
zine obtained from the sample solution are not more than 1/2
times the peak area of homochlorcyclizine from the standard
solution, and the total area of the peaks other than
homochlorcyclizine from the sample solution is not more
than the peak area of homochlorcyclizine from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 223 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile and per-
chloric acid (134:66:1).
Flow rate: Adjust the flow rate so that the retention time of
homochlorcyclizine is about 10 minutes.
Time span of measurement: About 2 times as long as the
retention time of homochlorcyclizine.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add the mobile phase to make exactly 50 mL.
Confirm that the peak area of homochlorcyclizine obtained
from 10 /uL of this solution is equivalent to 7 to 13% of that
of homochlorcyclizine obtained from lO/iL of the standard
solution.
System performance: Dissolve 5 mg each of Homochlorcy-
clizine Hydrochloride and methyl parahydroxybenzoic acid
in 100 mL of the mobile phase. When the procedure is run
with 10 juL of this solution under the above operating condi-
tions, methyl parahydroxybenzoic acid and homo-
chlorcyclizine are eluted in this order with the resolution
between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10//L of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
homochlorcyclizine is not more than 1.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, 110°C,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Homochlorcyclizine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
724
Human Normal Immunoglobulin / Official Monographs
JP XV
= 19.39 mg of C 19 H 23 C1N 2 .2HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Human Normal Immunoglobulin
Human Normal Immunoglobulin is a liquid for in-
jection containing immunoglobulin G in serum globu-
lins of humans.
It conforms to the requirements of Human Normal
Immunoglobulin in the Minimum Requirements for
Biological Products.
Description Human Normal Immunoglobulin is a clear,
colorless or yellow-brown liquid.
Hydralazine Hydrochloride
t^77y>«S
,,-NH;,
■HCI
C 8 H 8 N 4 .HC1: 196.64
Phthalazin-1 -ylhydrazine monohydrochloride
[304-20-1]
Hydralazine Hydrochloride, when dried, contains
not less than 98.0% of C 8 H 8 N 4 .HC1.
Description Hydralazine Hydrochloride occurs as a white,
crystalline powder. It is odorless, and has a bitter taste.
It is soluble in water, slightly soluble in ethanol (95), and
practically insoluble in diethyl ether.
Melting point: about 275 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Hydralazine Hydrochloride (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Hydralazine Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Hydralazine Hydrochloride (1 in 4000)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Hydralazine Hydrochloride in
50 mL of water: the pH of the solution is between 3.5 and
4.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Hydralazine Hydrochloride in 50 mL of water: the solution is
clear, and colorless or pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Hydrala-
zine Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 8 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.15 g of Hydralazine
Hydrochloride, previously dried, transfer it to a glass-stop-
pered flask, dissolve in 25 mL of water, add 25 mL of
hydrochloric acid, cool to room temperature, add 5 mL of
chloroform, and titrate <2.50> with 0.05 mol/L potassium io-
date VS while shaking until the purple color of the chlo-
roform layer disappears. The end point is reached when the
red-purple color no more reappears in the chloroform layer
within 5 minutes after the layer has been decolorized.
Each mL of 0.05 mol/L potassium iodate VS
= 9.832 mg of C 8 H 8 N 4 .HC1
Containers and storage Containers — Tight containers.
Hydralazine Hydrochloride for
Injection
Mfflt: P77y>
Hydralazine Hydrochloride for Injection is a prepa-
ration for injection which is dissolved before use. It
contains not less than 99% and not more than 113% of
the labeled amount of hydralazine hydrochloride
(QH 8 N 4 .HC1: 196.64).
Method of preparation Prepare as directed under Injec-
tions, with Hydralazine Hydrochloride.
Description Hydralazine Hydrochloride for Injection oc-
curs as a white to pale yellow powder or mass. It is odorless,
and has a bitter taste.
Identification Determine the absorption spectrum of a solu-
tion of Hydralazine Hydrochloride for Injection (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits maxima between 238 nm and 242
nm, between 258 nm and 262 nm, between 301 nm and 305
nm, and between 313 nm and 317 nm.
pH <2.54> Dissolve 1.0 g of Hydralazine Hydrochloride for
Injection in 50 mL of water: the pH of this solution is be-
tween 3.5 and 4.5.
Assay Weigh accurately the contents of not less than 10
samples of Hydralazine Hydrochloride for Injection. Weigh
accurately about 0.15 g of the contents, transfer it to a glass-
stoppered flask, dissolve in 25 mL of water, add 25 mL of
hydrochloric acid, cool to room temperature, and proceed as
directed in the Assay under Hydralazine Hydrochloride.
Each mL of 0.05 mol/L potassium iodate VS
= 9.832 mg of C 8 H 8 N 4 .HC1
Containers and storage Containers — Hermetic containers.
JPXV
Official Monographs / Hydrochloric Acid
725
Hydralazine Hydrochloride Powder
t^77y>MM
Hydralazine Hydrochloride Powder contains not
less than 95% and not more than 105% of the labeled
amount of hydralazine hydrochloride (QH 8 N 4 .HC1:
196.64).
Method of preparation Prepare as directed under Powder,
with Hydralazine Hydrochloride.
Identification Weigh a portion of Hydralazine Hydrochlo-
ride Powder, equivalent to 25 mg of Hydralazine Hydrochlo-
ride according to the labeled amount, add 100 mL of water,
shake well, and filter, if necessary. Add water to 2 mL of the
filtrate to make 50 mL and determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 238
nm and 242 nm, between 258 nm and 262 nm, between 301
nm and 305 nm, and between 313 nm and 317 nm.
Assay Weigh accurately a portion of Hydralazine
Hydrochloride Powder, equivalent to about 0.15 g of
Hydralazine Hydrochloride, transfer it to a glass-stoppered
flask, add 25 mL of water, shake well, add 25 mL of
hydrochloric acid, cool to room temperature, and proceed as
directed in the Assay under Hydralazine Hydrochloride.
Each mL of 0.05 mol/L potassium iodate VS
= 9.832 mg of C 8 H 8 N 4 .HC1
Containers and storage Containers — Tight containers.
ter start of the dissolution test, and filter through a mem-
brane filter with pore size of not more than 0.8 fim. Discard
the first 10 mL of the filtrate, pipet the subsequent KmL, add
water to make exactly V mL so that each mL contains about
11 /ug of hydralazine hydrochloride (C 8 H 8 N 4 .HC1) according
to the labeled amount, and use this solution as the sample so-
lution. Separately, weigh accurately about 0.05 g of hydrala-
zine hydrochloride for assay, previously dried at 105°C for 3
hours, and dissolve in water to make exactly 50 mL. Pipet 1
mL of this solution, add water to make exactly 100 mL, and
use this solution as the standard solution. Determine the
absorbances, A T and A s , of the sample solution and the stan-
dard solution at 260 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate of Hydrala-
zine Hydrochloride Tablets in 45 minutes is not less than
80%.
Dissolution rate (%) with respect to the labeled
amount of hydralazine hydrochloride (C 8 H 8 N 4 .HC1)
= W s x (A T /A S ) x (V'/V) x (l/C) x 18
W s : Amount (mg) of hydralazine hydrochloride for assay.
C: Labeled amount (mg) of hydralazine hydrochloride
(C 8 H 8 N 4 .HC1) in 1 tablet.
Assay Weigh accurately not less than 20 Hydralazine
Hydrochloride Tablets, and powder. Weigh accurately a por-
tion of the powder, equivalent to about 0.15 g of hydralazine
hydrochloride (C 8 H 8 N 4 .HC1), transfer it to a glass-stoppered
flask, and proceed as directed in the Assay under Hydralazine
Hydrochloride.
Each mL of 0.05 mol/L potassium iodate VS
= 9.832 mg of C 8 H 8 N 4 .HC1
Containers and storage Containers — Tight containers.
Hydralazine Hydrochloride Tablets
tF77y>M^i
Hydrochloric Acid
Hydralazine Hydrochloride Tablets contain not less
than 95% and not more than 105% of the labeled
amount of hydralazine hydrochloride (C 8 H 8 N 4 .HC1:
196.64).
Method of preparation Prepare as directed under Tablets,
with Hydralazine Hydrochloride.
Identification Weigh a quantity of powdered Hydralazine
Hydrochloride Tablets, equivalent to 25 mg of Hydralazine
Hydrochloride according to the labeled amount, add 100 mL
of water, mix well, and filter if necessary. To 2 mL of this so-
lution add water to make 50 mL, and determine the absorp-
tion spectrum of this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits maxima between
238 nm and 242 nm, between 258 nm and 262 nm, between
301 nm and 305 nm and between 313 nm and 317 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Hydralazine Hydrochlo-
ride Tablets at 50 revolutions per minute according to the
Paddle method, using 900 mL of water as the test solution.
Take 30 mL or more of the dissolved solution 45 minutes af-
Hydrochloric Acid contains not less than 35.0% and
not more than 38.0% of hydrogen chloride (HC1:
36.46).
Description Hydrochloric Acid is a colorless liquid having a
pungent odor.
It is fuming but ceases to fume when it is diluted with 2
volumes of water.
Specific gravity d 2 2 ° : about 1.18
Identification (1) Allow a glass stick wet with ammonia
TS to come near the surface of Hydrochloric Acid: a remark-
able white smoke evolves.
(2) A solution of Hydrochloric Acid (1 in 100) changes
blue litmus paper to red, and responds to the Qualitative
Tests <1.09> for chloride.
Purity (1) Sulfate <1.14>— To 15 mL of Hydrochloric
Acid add water to make 50 mL, and use this solution as the
sample solution. To 3.0 mL of the sample solution add 5 mL
of water and 5 drops of barium chloride TS, and allow to
stand for 1 hour: no turbidity is produced.
(2) Sulfite — To 3.0 mL of the sample solution obtained in
(1) add 5 ml of water and 1 drop of iodine TS: the color of io-
726 Dilute Hydrochloric Acid / Official Monographs
JP XV
dine TS does not disappear.
(3) Bromide or iodide — Place 10 mL of the sample solu-
tion obtained in (1) in a glass-stoppered test tube, add 1 mL
of chloroform and 1 drop of 0.002 mol/L potassium perman-
ganate VS, and shake well: the chloroform layer remains
colorless.
(4) Bromine or chlorine — Place 10 mL of the sample so-
lution obtained in (1) in a glass-stoppered test tube, add 5
drops of potassium iodide TS and 1 mL of chloroform, and
shake for 1 minute: the chloroform layer remains free from a
purple color.
(5) Heavy metals < 1.07 > — Evaporate 5 mL of
Hydrochloric Acid on a water bath to dryness, and add 2 mL
of dilute acetic acid and water to the residue to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution as follons: to 3.0 mL of Standard
Lead Solution add 2 mL of dilute acetic acid and water to
make 50 mL (not more than 5 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.7 mL
of Hydrochloric Acid according to Method 1, and perform
the test (not more than 1 ppm).
(7) Mercury — Dilute 20 mL of Hydrochloric Acid with
water to make exactly 100 mL, and use the solution as the
sample solution. Perform the test with this sample solution as
directed under Atomic Absorption Spectrophotometry <2.23>
(cold vapor type). Place the sample solution in a sample
bottle of the atomic absorption spectrophotometer, add 10
mL of tin (II) chloride-sulfuric acid TS, connect the bottle
immediately to the spectrophotometer, circulate air, and de-
termine the absorbance A T of the sample solution after the
recorder reading has risen rapidly, and becomes constant at a
wavelength of 253.7 nm. On the other hand, to 8 mL of Stan-
dard Mercury Solution add water to make exactly 100 mL,
and determine the absorbance A s of the solution obtained by
the same procedure as used for the sample solution: A T is
smaller than A s (not more than 0.04 ppm).
Residue on ignition <2.44> Pipet 10 mL of Hydrochloric
Acid, add 2 drops of sulfuric acid, evaporate to dryness, and
ignite: not more than 1.0 mg of residue remains.
Assay Weigh accurately a glass-stoppered flask containing
20 mL of water, add about 3 mL of Hydrochloric Acid, and
weigh accurately again. Dilute with 25 mL of water, and ti-
trate <2.50> with 1 mol/L sodium hydroxide VS (indicator: 2
to 3 drops of methyl red TS).
Each mL of 1 mol/L sodium hydroxide VS
= 36.46 mg of HC1
Containers and storage Containers — Tight containers.
Dilute Hydrochloric Acid
^fJ*min&
Dilute Hydrochloric Acid contains not less than 9.5
w/v% and not more than 10.5 w/v% of hydrogen
chloride (HC1: 36.46).
Description Dilute Hydrochloric Acid is a colorless liquid.
It is odorless and has a strong acid taste.
Specific gravity d^JJ: about 1.05
Identification A solution of Dilute Hydrochloric Acid (1 in
30) changes blue litmus paper to red and responds to the
Qualitative Tests <1.09> for chloride.
Purity (1) Sulfate — To 3.0 mL of Dilute Hydrochloric
Acid add 5 mL of water and 5 drops of barium chloride TS,
and allow to stand for 1 hour: no turbidity is produced.
(2) Sulfite— To 3.0 mL of Dilute Hydrochloric Acid add
5 mL of water and 1 drop of iodine TS: the color of iodine TS
does not disappear.
(3) Bromide or iodide — Place 10 mL of Dilute
Hydrochloric Acid in a glass-stoppered test tube, add 1 mL
of chloroform and 1 drop of 0.002 mol/L potassium perman-
ganate VS, and shake well: the chloroform layer remains
colorless.
(4) Bromine or chlorine — Place 10 mL of Dilute
Hydrochloric Acid in a glass-stoppered test tube, add 5 drops
of potassium iodide TS and 1 mL of chloroform, and shake
for 1 minute: the chloroform layer remains free from a pur-
ple color.
(5) Heavy metals <1.07> — Evaporate 9.5 mL of Dilute
Hydrochloric Acid on a water bath to dryness, add 2 mL of
dilute acetic acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 3.0 mL of Standard Lead Solution
add 2 mL of dilute acetic acid and water to make 50 mL (not
more than 3 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 4.0 mL
of Dilute Hydrochloric Acid according to Method 1 , and per-
form the test (not more than 0.5 ppm).
(7) Mercury — Dilute 80 mL of Dilute Hydrochloric Acid
with water to make exactly 100 mL, and use this solution as
the sample solution. Perform the test with this solution ac-
cording to the Atomic Absorption Spectrophotometry <2.23>
(cold vapor type). Place the sample solution in a sample bot-
tle of the atomic absorption spectrophotometer, add 10 mL
of tin (II) chloride-sulfuric acid TS, connect the bottle im-
mediately to the spectrophotometer, circulate air, and read
the absorbance A T of the sample solution after the recorder
reading has risen rapidly and become constant at a
wavelength of 253.7 nm. On the other hand, to 8 mL of Stan-
dard Mercury Solution add water to make exactly 100 mL,
and read the absorbance A s of the solution obtained by the
same procedure as used for the sample solution: A T is smaller
than A s (not more than 0.01 ppm).
Residue on ignition <2.44> Pipet 10 mL of Dilute
Hydrochloric Acid, add 2 drops of sulfuric acid, evaporate to
dryness, and ignite: the mass of the residue is not more than
l.Omg.
Assay Measure exactly 10 mL of Dilute Hydrochloric Acid,
and dilute with 20 mL of water. Titrate <2.50> with 1 mol/L
sodium hydroxide VS (indicator: 2 to 3 drops of methyl red
TS).
Each mL of 1 mol/L sodium hydroxide VS
= 36.46 mg of HC1
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Hydrochlorothiazide
727
Hydrochloric Acid Lemonade
£»'J=E-*-
Method of preparation
Dilute Hydrochloric Acid
Simple Syrup
Purified Water
5mL
80 mL
a sufficient quantity
To make 1000 mL
Prepare before use as directed under Lemonades, with the
above ingredients.
Description Hydrochloric Acid Lemonade is a clear, color-
less liquid. It has a sweet, cool, acid taste.
Containers and storage Containers — Tight containers.
Hydrochlorothiazide
t Hn^nnfTv K
a p o, p
s s
l^^h,^
C 7 H 8 C1N 3 4 S 2 : 297.74
6-Chloro-3,4-dihydro-2//-l,2,4-benzothiadiazine-7-
sulfonamide 1,1 -dioxide [58-93-5]
Hydrochlorothiazide, when dried, contains not less
than 99.0% of C 7 H 8 C1N 3 4 S 2 .
Description Hydrochlorothiazide occurs as white crystals
or crystalline powder. It is odorless, and has a slightly bitter
taste.
It is freely soluble in acetone, sparingly soluble in acetoni-
trile, very slightly soluble in water and in ethanol (95), and
practically insoluble in diethyl ether.
It dissolves in sodium hydroxide TS.
Melting point: about 267°C (with decomposition).
Identification (1) To 5 mg of Hydrochlorothiazide add 5
mL of disodium chlomotropate TS, and allow to stand for 5
minutes: a purple color develops.
(2) Fuse a mixture of 0.1 g of Hydrochlorothiazide and
0.5 g of sodium carbonate decahydrate cautiously: the gas
evolved changes moistened red litmus paper to blue. After
cooling, crush with a glass rod, add 10 mL of water, stir, and
filter. To 4 mL of the filtrate add 2 drops of hydrogen
peroxide (30), 5 mL of diluted hydrochloric acid (1 in 5) and 2
to 3 drops of barium chloride TS: a white precipitate is
produced.
(3) To 4 mL of the filtrate obtained in (2) add 5 mL of di-
lute nitric acid and 3 drops of silver nitrate TS: a white
precipitate is produced.
(4) Dissolve 12 mg of Hydrochlorothiazide in 100 mL of
sodium hydroxide TS. Dilute 10 mL of the solution with
water to make 100 mL. Determine the absorption spectrum
of the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum or the spectrum of a solution of
Hydrochlorothiazide Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Purity (1) Chloride <1.03> — Dissolve 1.0 g of Hydrochloro-
thiazide in 30 mL of acetone, add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 1.0 mL of 0.01 mol/L hydrochloric acid VS add 30
mL of acetone, 6 mL of dilute nitric acid and water to make
50 mL (not more than 0.036%).
(2) Sulfate <1.14>— Dissolve 1.0 g of Hydrochloro-
thiazide in 30 mL of acetone, add 1 mL of dilute hydrochlor-
ic acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 1.0 mL of 0.005 mol/L sulfuric acid VS add 30
mL of acetone, 1 mL of dilute hydrochloric acid and water to
make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Hydrochlo-
rothiazide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(4) Primary aromatic amines — Dissolve 80 mg of
Hydrochlorothiazide in acetone to make exactly 100 mL.
Measure exactly 1 mL of the solution, add 3.0 mL of dilute
hydrochloric acid, 3.0 mL of water and 0.15 mL of sodium
nitrite TS, shake, and allow to stand for 1 minute. Shake this
solution with 1.0 mL of ammonium amidosulfate TS, allow
to stand for 3 minutes, then add 1.0 mL of /V-(l-naphthyl)-
Af'-diethylethylenediamine oxalate TS, shake, and allow to
stand for 5 minutes. Perform the test with this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution prepared with 1.0 mL of acetone in the same
manner as the blank: the absorbance at 525 nm is not more
than 0.10.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 30 mg each of Hydro-
chlorothiazide and Hydrochlorothiazide Reference Standard,
previously dried, and dissolve in 150 mL of the mobile phase,
add exactly 10 mL each of the internal standard solution,
then add the mobile phase to make 200 mL, and use these so-
lutions as the sample solution and the standard solution, re-
spectively. Perform the test with 20 /xL each of the sample so-
lution and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
hydrochlorothiazide to that of the internal standard.
Amount (mg) of C 7 H 8 C1N 3 4 S 2 = W s x (Q T /Q S )
W s : Amount (mg) of Hydrochlorothiazide Reference
Standard
Internal standard solution — A solution of 4-aminoacetophe-
none in acetonitrile (9 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
728
Hydrocortisone / Official Monographs
JP XV
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.1 mol/L sodium dihydrogen
phosphate TS, pH 3.0 and acetonitrile (9:1).
Flow rate: Adjust the flow rate so that the retention time of
hydrochlorothiazide is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, hydrochlorothiazide and the internal standard
are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of hydrochlorothiazide to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Hydrocortisone
t Hn=iJU3 1 7
C 21 H 30 O 5 : 362.46
1 1/U 7 ,2 1 -Trihydroxypregn-4-ene-3 ,20-dione [50-23-7]
Hydrocortisone, when dried, contains not less than
97.0% and not more than 102.0% of C21H30O5.
Description Hydrocortisone occurs as a white, crystalline
powder. It is odorless.
It is sparingly soluble in methanol, in ethanol (95) and in
1,4-dioxane, slightly soluble in chloroform, and very slightly
soluble in diethyl ether and in water.
Melting point: 212 - 220°C (with decomposition).
Identification (1) Add 2 mL of sulfuric acid to 2 mg of
Hydrocortisone: the solution shows a yellow-green fluores-
cence immediately, and the color of the solution changes
gradually from orange to dark red. Dilute carefully the solu-
tion with 10 mL of water: the color changes through yellow
to orange-yellow with green fluorescence, and a small amount
of a flocculent precipitate is formed.
(2) Dissolve 0.01 g of Hydrocortisone in 1 mL of
methanol, add 1 mL of Fehling's TS, and heat: a red
precipitate is formed.
(3) Determine the infrared absorption spectrum of
Hydrocortisone, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Hydrocortisone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve Hydrocortisone and
Hydrocortisone Reference Standard in ethanol (95), respec-
tively, then evaporate the ethanol to dryness, and repeat the
test on the residues.
Optical rotation <2.49> [a]™: +150
0.1 g, 1,4-dioxane, 10 mL, 100 mm).
156° (after drying,
Purity Related substances — Dissolve 20 mg of Hydrocorti-
sone in 10 mL of a mixture of chloroform and methanol
(9:1), and use this solution as the sample solution. Pipet 1 mL
of this solution, add a mixture of chloroform and methanol
(9:1) to make exactly 50 mL, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10 fiL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of chloroform
and ethanol (95) (17:3) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 20 mg each of Hydrocortisone and
Hydrocortisone Reference Standard, previously dried and ac-
curately weighed, in 20 mL each of a mixture of chloroform
and methanol (9:1), add 10 mL each of the internal standard
solution, then add a mixture of chloroform and methanol
(9:1) to make 50 mL, and use these solutions as the sample
solution and the standard solution. Perform the test with 5
/uL each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
hydrocortisone to that of the internal standard, respectively.
Amount (mg) of C 2 iH3 O 5
= W s x (Qt/Qs)
W s : Amount (mg) of Hydrocortisone Reference Standard
Internal standard solution — A solution of prednisone in a
mixture of chloroform and methanol (9:1) (9 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with silica gel for liquid
chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of chloroform, methanol and
acetic acid (100) (1000:20:1).
Flow rate: Adjust the flow rate so that the retention time of
hydrocortisone is about 15 minutes.
System suitability —
System performance: When the procedure is run with 5 /uL
of the standard solution under the above operating condi-
tions, the internal standard and hydrocortisone are eluted in
this order with the resolution between these peaks being not
less than 7.
JPXV
Official Monographs / Hydrocortisone Acetate 729
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of hydrocortisone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Hydrocortisone Acetate
t Hnzuu^V^B^iZxJU
C 23 H 32 6 : 404.50
11/3,17,21 -Trihydroxypregn-4-ene-3 ,20-dione 2 1 -acetate
[50-03-3]
Hydrocortisone Acetate, when dried, contains not
less than 97.0% and not more than 102.0% of
C23H 32 6 .
Description Hydrocortisone Acetate occurs as white crys-
tals or crystalline powder. It is odorless.
It is sparingly soluble in 1,4-dioxane, slightly soluble in
methanol, in ethanol (95) and in chloroform, very slightly
soluble in diethyl ether, and practically insoluble in water.
Melting point: about 220°C (with decomposition).
Identification (1) Add 2 mL of sulfuric acid to 2 mg of
Hydrocortisone Acetate: the solution shows a yellowish green
fluorescence immediately, and the color of the solution grad-
ually changes through orange-yellow to dark red. This solu-
tion shows a strong light green fluorescence under ultraviolet
light. Add carefully 10 mL of water to this solution: the color
changes from yellow to orange-yellow with a light green
fluorescence, and a yellow-brown, flocculent precipitate is
formed.
(2) Dissolve 0.01 g of Hydrocortisone Acetate in 1 mL of
methanol by warming, add 1 mL of Fehling's TS, and heat:
an orange to red precipitate is formed.
(3) To 0.05 g of Hydrocortisone Acetate add 2 mL of
potassium hydroxide-ethanol TS, and heat on a water bath
for 5 minutes. Cool, add 2 mL of diluted sulfuric acid (2 in
7), and boil gently for 1 minute: the odor of ethyl acetate is
perceptible.
(4) Determine the infrared absorption spectra of
Hydrocortisone Acetate and Hydrocortisone Acetate Refer-
ence Standard, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: both the sample and the Reference Standard exhibit
similar intensities of absorption at the same wave numbers. If
any difference appears, dissolve the sample and Reference
Standard in ethanol (95), respectively, evaporate to dryness,
and repeat the test on the residues.
Optical rotation <2.49> [a]^ : + 158 - + 165° (after drying,
50 mg, 1,4-dioxane, 10 mL, 100 mm).
Purity Related substances — Dissolve 40 mg of Hydrocorti-
sone Acetate in 25 mL of a mixture of chloroform and
methanol (9:1), and use this solution as the sample solution.
Pipet 2 mL of the sample solution, add a mixture of chlo-
roform and methanol (9:1) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 fiL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of
dichloromethane, diethyl ether, methanol and water
(160:30:8:1) to a distance of about 12 cm, and air-dry the
plate. Spray evenly alkaline blue tetrazolium TS on the plate:
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 20 mg each of Hydrocortisone
Acetate and Hydrocortisone Acetate Reference Standard,
previously dried and accurately weighed, in methanol, add
exactly 10 mL each of the internal standard solution, then
add methanol to make 100 mL, and use these solutions as the
sample solution and the standard solution. Perform the test
with 20 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Q s , of the peak area of hydrocortisone acetate to that of the
internal standard, respectively.
Amount (mg) of C 23 H 32 6 = W s x (Q T /Q S )
W s : Amount (mg) of Hydrocortisone Acetate Reference
Standard
Internal standard solution — A solution of benzyl para-
hydroxybenzoate in methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /urn in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (13:7).
Flow rate: Adjust the flow rate so that the retention time of
hydrocortisone acetate is about 8 minutes.
System suitability —
System performance: When the procedure is run with
20 fiL of the standard solution under the above operating
conditions, hydrocortisone acetate and the internal standard
are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of hydrocortisone acetate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
730
Hydrocortisone and Diphenhydramine Ointment / Official Monographs
JP XV
Hydrocortisone and
Diphenhydramine Ointment
Hydrocortisone Butyrate
Method of preparation
Hydrocortisone Acetate
Diphenhydramine
White Petrolatum
5g
5g
a sufficient quantity
To make 1000 g
Prepare as directed under Ointments, with the above in-
gredients.
Description Hydrocortisone and Diphenhydramine Oint-
ment is white to pale yellow in color.
Identification (1) To 1 g of Hydrocortisone and Diphen-
hydramine Ointment add 10 mL of ethanol (95), heat on a
water bath for 5 minutes with occasional shaking, cool, and
filter. Take 5 mL of the filtrate, distill off the ethanol, and to
the residue add 2 mL of sulfuric acid: the solution shows a
yellow-green fluorescence immediately and the color of the
solution gradually changes through yellow to yellow-brown.
Add carefully 10 mL of water to this solution: the color
changes to yellow with green fluorescence, and a light yellow,
flocculent precipitate is formed (hydrocortisone acetate).
(2) To 1 mL of the filtrate obtained in (1) add 5 mL of
potassium hydrogen phthalate buffer solution, pH 4.6, and 2
mL of bromophenol blue TS, and add further 5 mL of chlo-
roform. Shake well, and allow to stand: a yellow color de-
velops in the chloroform layer (diphenhydramine).
(3) To 1 g of Hydrocortisone and Diphenhydramine
Ointment add 5 mL of methanol, warm, and shake. After
cooling, separate the methanol layer, and use this layer as the
sample solution. Dissolve 0.01 g each of hydrocortisone
acetate and diphenhydramine in 10 mL each of methanol,
and use these solutions as standard solutions (1) and (2). Per-
form the test with the sample solution and standard solutions
(1) and (2) as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of these solutions on a plate of silica
gel with a complex fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and diethyl ether (4:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (broad
spectrum wavelength): two spots from the sample solution
show the same Ri value as the corresponding spots from
standard solutions (1) and (2).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
CH a
C 25 H 36 6 : 432.55
ll/?,17,21-Trihydroxypregn-4-ene-3,20-dione 17-butanoate
[13609-67-1]
Hydrocortisone Butyrate, when dried, contains not
less than 96.0% and not more than 104.0% of
C25H360 6 .
Description Hydrocortisone Butyrate occurs as a white
powder. It is odorless.
It is freely soluble in tetrahydrofuran, in chloroform and in
1,2-dichloroethane, soluble in methanol, sparingly soluble in
ethanol (99.5), slightly soluble in diethyl ether, and practical-
ly insoluble in water.
Melting point: about 200°C (with decomposition).
Identification (1) Add 2 mL of sulfuric acid to 2 mg of
Hydrocortisone Butyrate: the solution shows a yellowish
green fluorescence immediately, and the color of the solution
gradually changes through orange-yellow to dark red. This
solution shows a strong light green fluorescence under ultrav-
iolet light (main wavelength: 254 nm). Add carefully 10 mL
of water to this solution: the color changes from yellow to
orange-yellow with a light green fluorescence, and a yellow-
brown, flocculent precipitate is formed.
(2) Dissolve 0.01 g of Hydrocortisone Butyrate in 1 mL
of methanol by warming, add 1 mL of Fehling's TS, and
heat: an orange to red precipitate is formed.
(3) To 0.05 g of Hydrocortisone Butyrate add 2 mL of
potassium hydrox-ide-ethanol TS, and heat on a water bath
for 5 minutes. Cool, add 2 mL of diluted sulfuric acid (2 in
7), and boil gently for 1 minute: the odor of ethyl butyrate is
perceptible.
(4) Determine the infrared absorption spectrum of
Hydrocortisone Butyrate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a] 2 ^: +48 - +52° (after drying, 0.1
g, chloroform, 10 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Hydrocortisone Butyrate according to method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 25 mg of Hydrocorti-
sone Butyrate in 5 mL of tetrahydrofuran, and use this solu-
tion as the sample solution. Pipet 2 mL of this solution, and
add tetrahydrofuran to make exactly 50 mL. Pipet 5 mL of
JPXV
Official Monographs / Hydrocortisone Sodium Phosphate 731
this solution, add tetrahydrofuran to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /uL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 1,2-
dichloroethane, methanol and water (470:30:1) to a distance
of about 15 cm, and air-dry the plate. Spray evenly alkaline
blue tetrazolium TS on the plate: the spots other than the
principal spot from the sample solution are not more than
two in number, and not more intense than those from the
standard solution in color.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg of Hydrocortisone
Butyrate, previously dried, and dissolve in ethanol (99.5) to
make exactly 100 mL. Pipet 2 mL of this solution, and add
ethanol (99.5) to make exactly 50 mL. Determine the absor-
bance A of this solution at the wavelength of maximum ab-
sorption at about 241 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>.
Amount (mg) of C 25 H 36 6 = (,4/375) x 25,000
Containers and storage Containers — Tight containers.
Hydrocortisone Sodium Phosphate
,PO s Na 2
C 21 H 29 Na 2 8 P: 486.40
Disodium ll/?,17,21-trihydroxypregn-4-ene-3,20-dione
21-phosphate [6000-74-4]
Hydrocortisone Sodium Phosphate contains not less
than 96.0% and not more than 102.0% of
C 2 iH 2 9Na 2 8 P, calculated on the dried basis.
Description Hydrocortisone Sodium Phosphate occurs as a
white to light yellow powder. It is odorless.
It is freely soluble in water, sparingly soluble in methanol,
very slightly soluble in ethanol (95), and practically insoluble
in diethyl ether.
It is hygroscopic.
Identification (1) To 2 mg of Hydrocortisone Sodium
Phosphate add 2 mL of sulfuric acid: a yellowish green
fluorescence is exhibited initially, then gradually changes
through orange-yellow to dark red. Examine the solution un-
der ultraviolet light (main wavelength: 254 nm): an intense,
light green fluorescence is exhibited. To this solution add
carefully 10 mL of water: the color changes from yellow to
orange-yellow with a light green fluorescence and a yellow-
brown, fiocculent floating substance is formed.
(2) Determine the infrared absorption spectrum of
Hydrocortisone Sodium Phosphate as directed in the paste
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Hydrocortisone Sodium Phosphate Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, dissolve Hydrocortisone Sodium Phosphate and
Hydrocortisone Sodium Phosphate Reference Standard in
methanol, respectively, then evaporate the methanol to dry-
ness, and repeat the test on the residues.
(3) Moisten 1.0 g of Hydrocortisone Sodium Phosphate
with a small quantity of sulfuric acid, and incinerate by grad-
ual heating. After cooling, dissolve the residue in 10 mL of
dilute nitric acid, and heat in a water bath for 30 minutes. Af-
ter cooling, filter if necessary. This solution responds to the
Qualitative Tests <1.09> for sodium salt and for phosphate.
Optical rotation <2.49> [a]„: + 121 - + 129° (1 g, calculat-
ed on the dried basis, phosphate buffer solution, pH 7.0, 100
mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Hydrocortisone Sodium Phos-
phate in 100 mL of water: the pH of this solution is between
7.5 and 9.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Hydrocortisone Sodium Phosphate in 10 mL of water: the
solution is clear and colorless to pale yellow.
(2) Chloride <1.03> — Dissolve 0.30 g of Hydrocortisone
Sodium Phosphate in 20 mL of water, and add 6 mL of dilute
nitric acid and water to make 100 mL. To 5 mL of this solu-
tion add water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution with
0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.600%).
(3) Heavy metals <1.07> — Proceed with 0.5 g of
Hydrocortisone Sodium Phosphate according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 40 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Hydrocortisone Sodium Phosphate according to Method
3, and perform the test (not more than 2 ppm).
(5) Free phosphoric acid — Weigh accurately about 0.25 g
of Hydrocortisone Sodium Phosphate, dissolve in water to
make exactly 100 mL, and use this solution as the sample so-
lution. Pipet 5 mL each of the sample solution and Standard
Phosphoric Acid Solution into separate 25-mL volumetric
flasks, add 2.5 mL of hexaammonium heptamolybdate-sul-
furic acid TS and 1 mL of l-amino-2-naphthol-4-sulfonic
acid TS, shake, add water to make exactly 25 mL, and allow
to stand at 20 ± 1°C for 30 minutes. Perform the test with
these solutions as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using a solution prepared with 5 mL
of water in the same manner as the blank. Determine the ab-
sorbances, A T and A s , of each solution from the sample solu-
tion and Standard Phosphoric Acid Solution at 740 nm: the
amount of free phosphoric acid is not more than 1.0%.
Content (%) of free phosphoric acid (H 3 P0 4 )
= (i T M s ) x (1/W) x 257.8
W: Amount (mg) of Hydrocortisone Sodium Phosphate,
calculated on the dried basis.
(6) Free hydrocortisone — Dissolve 25 mg of Hydrocorti-
732
Hydrocortisone Sodium Succinate / Official Monographs
JP XV
sone Sodium Phosphate in the mobile phase to make exactly
20 mL, and use this solution as the sample solution.
Separately, weigh 25 mg of Hydrocortisone Reference Stan-
dard, previously dried at 105°C for 3 hours, and dissolve in
the mobile phase to make exactly 100 mL. Pipet 10 mL of
this solution, add the mobile phase to make exactly 200 mL,
and use this solution as the standard solution. Perform the
test with exactly 20 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine the
peak areas, A T and A s , of hydrocortisone from each solu-
tion: A T is not larger than A s .
Operating conditions-
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
System suitability —
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
hydrocortisone is not more than 1.0%.
Loss on drying <2.41> Not more than 5.0% (1 g, in vacuum,
80°C, 5 hours).
Assay Weigh accurately about 20 mg each of Hydrocorti-
sone Sodium Phosphate and Hydrocortisone Sodium Phos-
phate Reference Standard (previously determine the loss on
drying <2.41> in the same manner as Hydrocortisone Sodium
Phosphate), dissolve each in 50 mL of the mobile phase, add
exactly 10 mL of the internal standard solution, then add the
mobile phase to make 200 mL, and use these solutions as the
sample solution and the standard solution, respectively. Per-
form the test with 20 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of hydrocortisone
phosphate to that of the internal standard, respectively.
Amount (mg) of C 21 H 29 Na 2 8 P = W s x (g T /g s )
W s : Amount (mg) of Hydrocortisone Sodium Phosphate
Reference Standard, calculated on the dried basis
Internal standard solution — A solution of isopropyl para-
hydroxybenzoate in the mobile phase (3 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.05 mol/L sodium dihydro-
gen phosphate TS, pH 2.6 and methanol (1:1).
Flow rate: Adjust the flow rate so that the retention time of
hydrocortisone phosphate is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, hydrocortisone phosphate and isopropyl para-
hydroxybenzoate are eluted in this order with the resolution
between these peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of hydrocortisone phosphate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Hydrocortisone Sodium Succinate
t: h-p^Jif'/v^A^iixjJith U^A
X^
C0 2 Na
C 25 H 3 3Na0 8 : 484.51
Monosodium 1 1/6,1 7,21 -trihydroxypregn-4-ene-3,20-dione
21 -succinate [125-04-2]
Hydrocortisone Sodium Succinate, calculated on the
dried basis, contains not less than 97.0% and not more
than 103.0% of C 25 H 33 Na0 8 .
Description Hydrocortisone Sodium Succinate occurs as
white powder or masses. It is odorless.
It is freely soluble in water, in methanol and in ethanol
(95), and practically insoluble in diethyl ether.
It is hygroscopic.
It is gradually colored by light.
Identification (1) Dissolve 0.2 g of Hydrocortisone Sodi-
um Succinate in 20 mL of water, and add 0.5 mL of dilute
hydrochloric acid with stirring: a white precipitate is formed.
Collect the precipitate, wash it with two 10-mL portions of
water, and dry at 105°C for 3 hours. To 3 mg of this dried
matter add 2 mL of sulfuric acid: the solution shows a yel-
lowish green fluorescence immediately, and the color of the
solution gradually changes through orange-yellow to dark
red. This solution shows a strong light green fluorescence un-
der ultraviolet light. Add carefully 10 mL of water to this so-
lution: the color changes from yellow to orange-yellow with a
light green fluorescence, and a yellow-brown flocculent
precipitate is formed.
(2) Dissolve 0.01 g of the dried matter obtained in (1) in 1
mL of methanol, add 1 mL of Fehling's TS, and heat: an
orange to red precipitate is formed.
(3) To 0.1 g of the dried matter obtained in (1) add 2 mL
of sodium hydroxide TS, and allow to stand for 10 minutes.
Filter the solution to remove the precipitate formed, mix the
filtrate with 1 mL of dilute hydrochloric acid, filter if necessa-
ry, then adjust the solution to a pH of about 6 with diluted
ammonia TS (1 in 10), and add 2 to 3 drops of iron (III) chlo-
ride TS: a brown precipitate is formed.
(4) Determine the infrared absorption spectrum of the
dried matter obtained in (1) as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
JPXV
Official Monographs / Hydrocortisone Succinate 733
trum or the spectrum of previously dried Hydrocortisone
Succinate Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers. If any
difference appears between the spectra, dissolve Hydrocorti-
sone Sodium Succinate and Hydrocortisone Succinate Refer-
ence Standard in methanol, respectively, then evaporate the
methanol to dryness, and repeat the test on the residues.
(5) Hydrocortisone Sodium Succinate responds to the
Qualitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> [«]£>: + 135 - + 145° (0.1 g, calcu-
lated on the dried basis, ethanol (95), 10 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Hydrocortisone Sodium Succinate in 10 mL of water: the so-
lution is clear and colorless.
(2) Other steroids — Dissolve 25 mg of Hydrocortisone
Sodium Succinate in methanol to make exactly 10 mL, and
use this solution as the sample solution. Separately, dissolve
0.025 g of hydrocortisone in methanol to make exactly 10
mL. Pipet 1 mL of this solution, add methanol to make ex-
actly 20 mL, and use this solution as the standard solution
(1). Pipet 6 mL of the standard solution (1), add methanol to
make exactly 10 mL, and use this solution as the standard so-
lution (2). Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 3 fih each of
the sample solution and standard solutions (1) and (2) on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, ethanol (99.5) and formic acid (150:10:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spot from
the sample solution corresponding to the spot from the stan-
dard solution (1) is not more intense than the spot from the
standard solution (1). Any spot other than the principal spot
and the above spot obtained from the sample solution is not
more than one, and is not more intense than the spot from
the standard solution (2).
Loss on drying <2.41> Not more than 2.0% (0.5 g, 105°C,
3 hours).
Assay Weigh accurately about 10 mg of Hydrocortisone
Sodium Succinate, and dissolve in methanol to make exactly
100 mL. Pipet 5 mL of this solution, add methanol to make
exactly 50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.01 g of Hydrocortisone
Succinate Reference Standard, previously dried at 105°C for
3 hours, proceed in the same manner as directed for the sam-
ple solution, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and standard solution at 240 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>, respectively.
Amount (mg) of C25H 33 Na08
= W s x (Aj/A s ) x 1.0475
W s : Amount (mg) of Hydrocortisone Succinate Reference
Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Hydrocortisone Succinate
CO;H
C 25 H340 8 : 462.53
1 1/3, 1 7,2 1 -Trihydroxypregn-4-ene-3 ,20-dione
21-(hydrogen succinate) [2203-97-6]
Hydrocortisone Succinate, when dried, contains not
less than 97.0% and not more than 103. 0% of
C25H340 8 .
Description Hydrocortisone Succinate occurs as a white
crystalline powder.
It is very soluble in methanol, freely soluble in ethanol
(99.5), sparingly soluble in ethanol (95), and practically in-
soluble in water.
Identification (1) To 3 mg of Hydrocortisone Succinate
add 2 mL of sulfuric acid: the solution shows a yellowish
green fluorescence immediately, and the color of the solution
gradually changes through orange-yellow to dark red. This
solution shows a strong light green fluorescence under ultrav-
iolet light. Add carefully 10 mL of water to this solution: the
color changes from yellow to orange-yellow with a light green
fluorescence, and a yellow-brown flocculent precipitate is
formed.
(2) Determine the infrared absorption spectrum of
Hydrocortisone Succinate, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of previously dried
Hydrocortisone Succinate Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers. If any difference appears between the spectra, dis-
solve Hydrocortisone Succinate and Hydrocortisone Suc-
cinate Reference Standard in methanol, respectively, then
evaporate the methanol to dryness, and repeat the test on the
residues.
Optical rotation <2.49> [a]™: + 147 -
0.1 g, ethanol (99.5), 10 mL, 100 nm).
153° (after drying,
Purity Related substances — Dissolve 25 mg of Hydrocorti-
sone Succinate in exactly 10 mL of methanol, and use this so-
lution as the sample solution. Separately, dissolve 0.025 g of
hydrocortisone in exactly 10 mL of methanol. Pipet 1 mL of
this solution, dilute with methanol to exactly 50 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 3 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, ethanol (99.5) and formic acid (150:10:1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
734
Hydrocotarnine Hydrochloride Hydrate / Official Monographs
JP XV
der ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 2.0% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 50 mg each of Hydrocorti-
sone Succinate and Hydrocortisone Succinate Reference
Standard, previously dried, and dissolve in methanol to make
exactly 50 mL. Pipet 5 mL each of these solutions, add exact-
ly 5 mL each of the internal standard solution, then add
methanol to make 50 mL, and use these solutions as the sam-
ple solution and standard solution. Perform the test with 10
/nL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following operating conditions, and calculate the ratios,
Qj and Q s , of the peak area of hydrocortisone succinate to
that of the internal standard, respectively.
Amount (mg) of C25H 34 8
= W s x (Qt/Qs)
W s : Amount (mg) of Hydrocortisone Succinate Reference
Standard
Internal standard solution — A solution of butyl parahydroxy
benzoate in methanol (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
(10 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetic acid-sodium acetate
buffer solution, pH 4.0 and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
hydrocortisone succinate is about 5 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, hydrocortisone succinate and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of hydrocortisone succinate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Hydrocotarnine Hydrochloride
Hydrate
,CH a
C 12 H 15 N0 3 .HC1.H 2 0: 275.73
4-Methoxy-6-methyl-5 ,6 ,7 , 8-
tetrahydro[l,3]dioxolo[4,5-g]isoquinoline
monohydrochloride monohydrate [5985-55-7, anhydride]
Hydrocotarnine Hydrochloride Hydrate, when d-
ried, contains not less than 98.0% of hydrocotarnine-
hydrochloride (C 12 H 15 N0 3 HC1: 257.72).
Description Hydrocotarnine Hydrochloride Hydrate occurs
as white to pale yellow crystals or crystalline powder.
It is freely soluble in water, sparingly soluble in ethanol
(95) and in acetic acid (100), and slightly soluble in acetic an-
hydride.
Identification (1) Determine the absorption spectrum of a
solution of Hydrocotarnine Hydrochloride Hydrate (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Hydrocotarnine Hydrochloride Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Hydrocotarnine Hydrochloride Hy-
drate (1 in 50) responds to the Qualitative Tests <1.09> (2) for
chloride.
pH <2.54> Dissolve 1.0 g of Hydrocotarnine Hydrochloride
Hydrate in 20 mL of water: the pH of the solution is between
4.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Hydrocotarnine Hydrochloride Hydrate in 10 mL of water:
the solution is clear, and when perform the test with this solu-
tion as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using water as the blank, the absorbance at 400 nm is
not more than 0.17.
(2) Heavy metals <1.07> — Proceeds with 1.0 g of
Hydrocotarnine Hydrochloride Hydrate according to
Method 1, and perform the test. Prepare the control solution
with 2.0 mL of Standard Lead Solution (not more than 20
ppm).
(3) Related substances — Dissolve 0.10 g of
Hydrocotarnine Hydrochloride Hydrate in 10 mL of diluted
ethanol (99.5) (1 in 2), and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add diluted
ethanol (99.5) (1 in 2) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with these
JPXV
Official Monographs / Hydrophilic Ointment
735
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of acetone, toluene, ethanol (99.5) and ammonia water (28)
(20:20:3:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 7.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Hydrocotarnine
Hydrochloride Hydrate, previously dried. Dissolve in 50 mL
of a mixture of acetic anhydride and acetic acid (100) (7:3) by
warming. Cool, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation.
Each mL of 0.1 mol/L perchloric acid VS
= 25.77 mg of C 12 H 15 N0 3 HC1
Containers and storage Containers — Tight containers.
Hydrogenated Oil
wfca
Hydrogenated Oil is the fat obtained by hydrogena-
tion of fish oil or of other oils originating from animal
or vegetable.
Description Hydrogenated Oil occurs as a white mass or
powder and has a characteristic odor and a mild taste.
It is freely soluble in diethyl ether, very slightly soluble in
ethanol (95), and practically insoluble in water.
The oil obtained by hydrogenation of castor oil is slightly
soluble in diethyl ether, very slightly soluble in ethanol (95),
and practically insoluble in water.
Acid value <1.13> Not more than 2.0.
Purity (1) Moisture and coloration — Hydrogenated Oil
(5.0 g), melted by heating on a water bath, forms a clear liq-
uid, from which no water separates. In a 10-mm thick layer
of the liquid, it is colorless or slightly yellow.
(2) Alkalinity— To 2.0 g of Hydrogenated Oil add 10 mL
of water, melt by heating on a water bath, and shake
vigorously. After cooling, add 1 drop of phenolphthalein TS
to the separated water layer: no color develops.
(3) Chloride— To 1.5 g of Hydrogenated Oil add 30 mL
of ethanol (95), boil for 10 minutes under a reflux condenser,
and filter after cooling. To 20 mL of the filtrate add 5 drops
of a solution of silver nitrate in ethanol (95) (1 in 50): the tur-
bidity of the solution does not exceed that of the following
control solution.
Control solution: To 1.0 mL of 0.01 mol/L hydrochloric
acid VS add ethanol (95) to make 20 mL, then add 5 drops of
a solution of silver nitrate in ethanol (95) (1 in 50).
(4) Heavy metals — Heat 2.0 g of Hydrogenated Oil with
5 mL of dilute hydrochloric acid and 10 mL of water on a
water bath for 5 minutes with occasional shaking. After cool-
ing, filter, and make 5 mL of the filtrate weakly alkaline with
ammonia TS, then add 3 drops of sodium sulfide TS: the so-
lution remains unchanged.
(5) Nickel — Place 5.0 g of Hydrogenated Oil in a quartz
or porcelain crucible, heat slightly with caution at the begin-
ning, and, after carbonization, incinerate by strong heating
(500 ± 20°C). Cool, add 1 mL of hydrochloric acid,
evaporate on a water bath to dryness, dissolve the residue in 3
mL of dilute hydrochloric acid, and add 7 mL of water. Then
add 1 mL of bromine TS and 1 mL of a solution of citric acid
monohydrate (1 in 5), make alkaline with 5 mL of ammonia
TS, and cool in running water. To this solution add 1 mL of
dimethylglyoxime TS, add water to make 20 mL, and use this
solution as the test solution. Allow to stand for 5 minutes: the
solution has no more color than the following control solu-
tion.
Control solution: Evaporate 1 mL of hydrochloric acid on
a water bath to dryness, add 1 mL of Standard Nickel Solu-
tion and 3 mL of dilute hydrochloric acid, and add 6 mL of
water. Then proceed as directed in the test solution, add
water to make 20 mL, and allow to stand for 5 minutes.
Residue on ignition <2.44> Not more than 0.1% (5 g).
Containers and storage Containers — Well-closed contain-
ers.
Hydrophilic Ointment
&*»*
Method of preparation
White Petrolatum
250 g
Stearyl Alcohol
200 g
Propylene Glycol
120 g
Polyoxyethylene hydrogenated
castor oil 60
40 g
Glycerin Monostearate
10 g
Methyl Parahydroxybenzoate
lg
Propyl Parahydroxybenzoate
lg
Purified Water
a sufficient quantity
To make
1000 g
Melt White Petrolatum, Stearyl Alcohol, polyoxyethylene
hydrogenated castor oil 60 and Glycerin Monostearate by
heating on a water bath, stir, and keep temperature of the
mixture at about 75 °C. To Propylene Glycol add Methyl
Parahydroxybenzoate and Propyl Parahydroxybenzoate,
melt by warming if necessary, dissolve in Purified Water, and
warm to about 75°C. Add this solution to the above mixture,
stir to form emulsion, cool, and stir thoroughly until it con-
geals.
Description Hydrophilic Ointment is white in color. It has a
slight, characteristic odor.
Containers and storage Containers — Tight containers.
736 Hydroxocobalamin Acetate / Official Monographs
JP XV
Hydroxocobalamin Acetate
o
HjN
H ? N
H 2 \i
H H 3 C H3C , J
M O
T
o
NH2
• H 3 C-CO ? H
r^T <XX
"0--P — HO
o
ftj>
C 62 H 89 CoN 13 15 P.C 2 H 4 2 : 1406.41
Coa- [a-(5 ,6-Dimethyl- l/Z-benzoimidazol- 1 -yl)] -Cofi-
hydroxocobamide monoacetate
[13422-51-0, Hydroxocobalamin]
Hydroxocobalamin Acetate contains not less than
95.0% of C 62 H 89 CoN 1 30 1 5P.C 2 H 4 2 , calculated on the
dried basis.
Description Hydroxocobalamin Acetate occurs as dark red
crystals or powder. It is odorless.
It is freely soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Hydroxocobalamin Acetate in acetic acid-sodium
acetate buffer solution, pH 4.5 (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Mix 1 mg of Hydroxocobalamin Acetate with 0.05 g
of potassium hydrogen sulfate, and fuse by igniting. Cool,
break up the mass with a glass rod, add 3 mL of water, and
dissolve by boiling. Add 1 drop of phenolphthalein TS, and
add dropwise sodium hydroxide TS until the solution de-
velops a light red. Then add 0.5 g of sodium acetate trihy-
drate, 0.5 mL of dilute acetic acid and 0.5 mL of a solution
of disodium l-nitroso-2-naphthol-3,6-disulfonate (1 in 500):
a red to orange-red color develops immediately. Then add 0.5
mL of hydrochloric acid, and boil for 1 minute: the red color
does not disappear.
(3) Add 0.5 mL of ethanol (99.5) and 1 mL of sulfuric
acid to 0.02 g of Hydroxocobalamin Acetate, and heat the
mixture: the odor of ethyl acetate is perceptible.
Purity Cyanocobalamin and colored impurities — Dissolve
50 mg of Hydroxocobalamin Acetate in exactly 5 mL each of
acetic acid-sodium acetate buffer solution, pH 5.0, in two
tubes. To one tube add 0.15 mL of potassium thiocyanate
TS, allow to stand for 30 minutes, and use this solution as the
sample solution (1). To the other tube add 0.10 mL of potas-
sium cyanide TS, allow to stand for 30 minutes, and use this
solution as the sample solution (2). Separately, dissolve 3.0
mg of Cyanocobalamin Reference Standard in exactly 10 mL
of acetic acid-sodium acetate buffer solution, pH 5.0, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Apply 20 /uL each of the sample solution and standard
solution 25 mm in length along the starting line, 10 mm apart
from each other, on a plate of silica gel for thin-layer chro-
matography. Develop the plate for 18 hours with 2-butanol
saturated with water, while supporting the plate at an angle
of about 15° to a horizontal plane, and air-dry the plate: the
spot from the sample solution (1) corresponding to that from
the standard solution is not more intense than the spot from
the standard solution, and the spots other than the principal
spot from the sample solution (2) are not more intense than
the spot from the standard solution.
Loss on drying <2.41> Not more than 12% (50 mg, in vacu-
um at a pressure not exceeding 0.67 kP, phosphorus (V)
oxide, 100°C, 6 hours).
Assay Weigh accurately about 20 mg of Hydroxocobala-
min Acetate, and dissolve in acetic acid-sodium acetate buffer
solution, pH 5.0, to make exactly 50 mL. Pipet 2 mL of this
solution into a 50-mL volumetric flask, add 1 mL of a solu-
tion of potassium cyanide (1 in 1000), and allow to stand for
30 minutes at ordinary temperature. Add acetic acid-sodium
acetate buffer solution, pH 5.0, to make exactly 50 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 20 mg of Cyanocobalamin Reference Stan-
dard after determining the loss on drying in the same manner
as for Cyanocobalamin, and dissolve in water to make exact-
ly 50 mL. To 2 mL of this solution, exactly measured, add
acetic acid-sodium acetate buffer solution, pH 5.0, to make
exactly 50 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and standard solution at 361 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 62 H 89 CoNi30 15 P.C 2 H40 2
= W s x (A T /A S ) x 1.0377
W s : Amount (mg) of Cyanocobalamin Reference Stan-
dard, calculated on the dried basis
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Hydroxypropylcellulose
t: Kq+v"/p tfJHzJUP-7.
Hydroxypropylcellulose is a hydroxypropyl ether of
cellulose.
Hydroxypropylcellulose, when dried, contains not
less than 53.4% and not more than 77.5% of hydrox-
ypropoxy group (-OC 3 H 6 OH: 75.09).
Description Hydroxypropylcellulose occurs as a white to
JPXV
Official Monographs / Hydroxypropylcellulose 737
yellowish white powder.
It is practically insoluble in diethyl ether.
It forms a viscous liquid upon addition of water or ethanol
(95).
Identification (1) To 1 g of Hydroxypropylcellulose add
100 mL of water, heat in a water bath at 70°C for 5 minutes
with stirring, and cool while shaking. Allow to stand at room
temperature until it becomes more homogeneous and vis-
cous, and use this solution as the sample solution. To 2 mL of
the sample solution add 1 mL of anthrone TS gently: a blue
to green color develops at the zone of contact.
(2) Heat the sample solution obtained in (1): a white tur-
bidity or precipitate is produced, and the turbidity or
precipitate disappears when cooled.
(3) To 1 g of Hydroxypropylcellulose add 100 mL of
ethanol (95), and allow to stand after stirring: a homogene-
ous and viscous liquid is produced.
pH <2.54> Dissolve 1.0 g of Hydroxypropylcellulose in 50
mL of freshly boiled and cooled water: the pH of the solution
is between 5.0 and 7.5.
Purity (1) Clarity of solution — Use an outer glass cylin-
der, 250 mm in height, 25 mm in internal diameter, 2 mm in
thickness, with a high-quality glass plate 2 mm thick at the
bottom, and inner glass cylinder, 300 mm in height, 15 mm in
internal diameter, 2 mm in thickness, with a high-quality g-
lass plate 2 mm thick at the bottom. In the outer cylinder
place a solution prepared by adding 1.0 g of Hydroxypropyl-
cellulose to 100 mL of water, heat while stirring in a water
bath at 70°C, and then cool to room temperature. Place this
cylinder on a sheet of white paper on which 15 parallel,
black, 1-mm width lines are drawn at 1-mm intervals. Place
the inner cylinder, and move it up and down while viewing
downward through the bottom of the inner cylinder, and
measure the minimum height of the solution between the bot-
tom of the outer cylinder and the lower end of the inner cylin-
der at the time when the lines on the paper cannot be differen-
tiated. The average value obtained from three repeated
procedures is greater than that obtained from the following
control solution treated in the same manner.
Control solution: To 5.50 mL of 0.005 mol/L sulfuric acid
VS add 1 mL of dilute hydrochloric acid, 5 mL of ethanol
(95) and water to make 50 mL. To this solution add 2 mL of
barium chloride TS, mix, allow to stand for 10 minutes, and
shake well before use.
(2) Chloride <1.03>— Add 1.0 g of Hydroxypropylcellu-
lose to 30 mL of water, heat in a water bath with stirring for
30 minutes, and filter while being hot. Wash the residue with
three 15-mL portions of hot water, combine the washings
with the filtrate, and add water to make 100 mL after cooling.
To 10 mL of the sample solution add 6 mL of dilute nitric
acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.142%).
(3) Sulfate <1.I4>— To 40 mL of the sample solution ob-
tained in (2) add 1 mL of dilute hydrochloric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution with 0.40 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Hydrox-
ypropylcellulose according to Method 2 and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Hydroxypropylcellulose according to Method 3, and per-
form the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay (i) Apparatus — Reaction flask: A 5-mL screw-cap
pressure-tight glass bottle, having an inverted conical bottom
inside, 20 mm in outside diameter, 50 mm in height up to the
neck, 2 mL in capacity up to a height of about 30 mm,
equipped with a pressure-tight septum of heat-resisting resin
and also with an inside stopper or sealer of fluoroplastic.
Heater: A square aluminum block 60 to 80 mm thick, hav-
ing holes 20.6 mm in diameter and 32 mm in depth, capable
of maintaining the inside temperature within ± 1°C.
(ii) Procedure — Weigh accurately about 65 mg of
Hydroxypropylcellulose, previously dried, transfer to the
reaction flask, add 65 mg of adipic acid, 2.0 mL of the inter-
nal standard solution and 2.0 mL of hydroiodic acid, stopper
the flask tightly, and weigh accurately. Shake the flask for 30
seconds, heat at 150°C on the heater for 30 minutes with
repeated shaking at 5-minute intervals, and continue heating
for an additional 30 minutes. Allow the flask to cool, and
again weigh accurately. If the mass loss is less than 10 mg, use
the upper layer of the mixture as the sample solution.
Separately, take 65 mg of adipic acid, 2.0 mL of the internal
standard solution and 2.0 mL of hydroiodic acid in another
reaction flask, stopper tightly, and weigh accurately. Add 50
/xL of isopropyl iodide for assay, and again weigh accurately.
Shake the reaction flask for 30 seconds, and use the upper
layer of the content as the standard solution. Perform the test
as directed under Gas Chromatography <2.02> with 1 /uL each
of the sample solution and standard solution according to the
following conditions, and calculate the ratios, Q T and Q s , of
the peak area of isopropyl iodide to that of the internal stan-
dard.
Amount (%) of hydroxypropoxy group (C 3 H 7 2 )
= (W s /W T )x(Q T Q s )x 44.17
W s : Amount (mg) of isopropyl iodide in the standard solu-
tion.
W T : Amount (mg) of the sample
Internal standard solution — A solution of w-octane in o-xy-
lene (4 in 100).
Operating conditions —
Detector: A thermal conductivity detector or hydrogen
flame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 3 m in length, packed with siliceous earth for gas
chromatography, 180 to 250 ^m in particle diameter, coated
with methyl silicone polymer for gas chromatography at the
ratio of 20%.
Column temperature: A constant temperature of about
100°C.
Carrier gas: Helium (for thermal-conductivity detector);
helium or nitrogen (for hydrogen flame-ionization detector).
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 10 minutes.
Selection of column: Proceed with 1 /xh of the standard so-
738
Low Substituted Hydroxypropylcellulose / Official Monographs
JP XV
lution under the above operating conditions. Use a column
giving well-resolved peaks of isopropyl iodide and the inter-
nal standard in this order.
Containers and storage Containers — Well-closed contain-
ers.
Low Substituted
Hydroxypropylcellulose
filMt l*n*v-/Qtf;u-tz;ua-x
Low Substituted Hydroxypropylcellulose is a low
substituted hydroxypropyl ether of cellulose.
Low Substituted Hydroxypropylcellulose, when
dried, contains not less than 5.0% and not more than
16.0% of hydroxypropoxy group (-OC 3 H 6 OH: 75.09).
Description Low Substituted Hydroxypropylcellulose oc-
curs as a white to yellowish white powder or granules. It is
odorless or has a slight, characteristic odor. It is tasteless.
It is practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves in a solution of sodium hydroxide (1 in 10), and
produces a viscous solution.
It swells in water, in sodium carbonate TS and in 2 mol/L
hydrochloric acid TS.
Identification (1) To 0.02 g of Low Substituted Hydrox-
ypropylcellulose add 2 mL of water, shake, and produce a
turbid solution. Add 1 mL of anthrone TS gently: a blue to
blue-green color develops at the zone of contact.
(2) To 0.1 g of Low Substituted Hydroxypropylcellulose
add 10 mL of water, stir and produce a turbid solution. Add
1 g of sodium hydroxide, shake until it becomes homogene-
ous, and use this solution as the sample solution. To 0.1 mL
of the sample solution add 9 mL of diluted sulfuric acid (9 in
10), shake well, heat in a water bath for exactly 3 minutes,
immediately cool in an ice bath, add carefully 0.6 mL of nin-
hydrin TS, shake well, and allow to stand at 25°C: a red color
develops at first, and it changes to purple within 100 minutes.
(3) To 5 mL of the sample solution obtained in (2) add 10
mL of a mixture of acetone and methanol (4:1), and shake: a
white, flocculent precipitate is produced.
pH <2.54> To 1.0 g of Low Substituted Hydroxypropylcel-
lulose add 100 mL of freshly boiled and cooled water, and
shake: the pH of the solution is between 5.0 and 7.5.
Purity (1) Chloride <1.03>— To 0.5 g of Low Substituted
Hydroxypropylcellulose add 30 mL of hot water, stir well,
heat on a water bath for 10 minutes, and filter the super-
natant liquid by decantation while being hot. Wash the
residue thoroughly with 50 mL of hot water, combine the
washings with the filtrate, and add water to make 100 mL af-
ter cooling. To 5 mL of the sample solution add 6 mL of di-
lute nitric acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.25 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.335%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Low
Substituted Hydroxypropylcellulose according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Low Substituted Hydroxypropylcellulose, according to
Method 3, and perform the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 6.0% (1 g, 105°C,
1 hour).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay (i) Apparatus — Reaction flask: A 5-mL screw-cap
pressure-tight glass bottle, having an inverted conical bottom
inside, 20 mm in outside diameter, 50 mm in height up to the
neck, 2 mL in capacity up to a height of about 30 mm,
equipped with a pressure-tight septum of heat-resisting resin
and also with an inside stopper or sealer of fluoroplastic.
Heater: A square-shaped aluminum block 60 to 80 mm
thick, having holes 20.6 mm in diameter and 32 mm in depth,
capable of maintaining the inside temperature within ± 1°C.
(ii) Procedure — Weigh accurately about 65 mg of Low
Substituted Hydroxypropylcellulose, previously dried, trans-
fer to the reaction flask, add 65 mg of adipic acid, 2.0 mL of
the internal standard solution and 2.0 mL of hydroiodic acid,
stopper the flask tightly, and weigh accurately. Shake the
flask for 30 seconds, heat at 150°C on the heater for 30
minutes with repeated shaking at 5-minute intervals, and con-
tinue heating for an additional 30 minutes. Allow the flask to
cool, and again weigh accurately. If the mass loss is less than
10 mg, use the upper layer of the mixture as the sample solu-
tion. Separately, take 65 mg of adipic acid, 2.0 mL of the in-
ternal standard solution and 2.0 mL of hydroiodic acid in
another reaction flask, stopper tightly, and weigh accurately.
Add 15 fih of isopropyl iodide for assay, and again weigh ac-
curately. Shake the reaction flask for 30 seconds, and use the
upper layer of the content as the standard solution. Perform
the test as directed under Gas Chromatography <2.02> with 2
/xL each of the sample solution and standard solution accord-
ing to the following conditions, and calculate the ratios, Q T
and Q s , of the peak area of isopropyl iodide to that of the
internal standard.
Amount (%) of hydroxypropoxy group (C 3 H 7 2 )
= (W S /W T )X(Q T /Q s )x 44.17
W s : Amount (mg) of isopropyl iodide in the standard solu-
tion.
W T \ amount (mg) of the sample
Internal standard solution — A solution of M-octane in o-xy-
lene (1 in 50).
Operating conditions —
Detector: A thermal conductivity detector or hydrogen
flame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 3 m in length, packed with siliceous earth for gas
chromatography, 180 to 250 /um in particle diameter, coated
with methyl silicone polymer for gas chromatography at the
ratio of 20%.
Column temperature: A constant temperature of about
100°C.
Carrier gas: Helium (for thermal-conductivity detector);
helium or nitrogen (for hydrogen flame-ionization detector).
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 10 minutes.
Selection of column: Proceed with 2 /xh of the standard so-
lution under the above operating conditions. Use a column
JPXV
Official Monographs / Hydroxyzine Pamoate 739
giving well-resolved peaks of isopropyl iodide and the inter-
nal standard in this order.
Containers and storage Containers — Tight containers.
Hydroxyzine Hydrochloride
t:r-p+yy>i|iS
■2HCI
and enantiomer
C 21 H 27 C1N 2 2 .2HC1: 447.83
2-(2-{4-[(/?S)-(4-Chlorophenyl)phenylmethyl]piperazin-l-
ylj ethoxy)ethanol dihydrochloride [2192-20-3]
Hydroxyzine Hydrochloride, when dried, contains
not less than 98.5% of C 21 H 27 C1N 2 2 .2HC1.
Description Hydroxyzine Hydrochloride occurs as a white,
crystalline powder. It is odorless, and has a bitter taste.
It is very soluble in water, freely soluble in methanol, in
ethanol (95) and in acetic acid (100), very slightly soluble in
acetic anhydride, and practically insoluble in diethyl ether.
Melting point: about 200°C (with decomposition).
Identification (1) To 5 mL of a solution of Hydroxyzine
Hydrochloride (1 in 100) add 2 to 3 drops of ammonium
thiocyanate-cobaltous nitrate TS: a blue precipitate is
formed.
(2) Determine the absorption spectrum of a solution of
Hydroxyzine Hydrochloride in methanol (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) A solution of Hydroxyzine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Hydroxyzine Hydrochloride in
20 mL of water: the pH of this solution is between 1.3 and
2.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Hydroxyzine Hydrochloride in 10 mL of water: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Hydrox-
yzine Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Hydroxyzine
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
[iL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, ethanol (95) and ammo-
nia solution (28) (150:95:1) to a distance of about 10 cm, and
air-dry the plate. Allow the plate to stand in iodine vapor: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 3.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.1 g of Hydroxyzine
Hydrochloride, previously dried, dissolve in 60 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 22.39 mg of C 21 H 27 C1N 2 2 .2HC1
Containers and storage Containers — Tight containers.
Hydroxyzine Pamoate
COjH
CO f H
and enantiomer
C 21 H 27 C1N 2 2 .C 23 H 16 6 : 763.27
2-(2- {4- [(i?S)-(4-Chlorophenyl)phenylmethyl] piperazin- 1 -
yl} ethoxy)ethanol mono[4,4'-methylenebis(3-hydroxy-2-
naphthoate)] (1/1) [10246-75-0]
Hydroxyzine Pamoate contains not less than 98.0%
of C 2 iH 27 ClN 2 2 .C 23 H 16 6 , calculated on the anhy-
drous basis.
Description Hydroxyzine Pamoate occurs as a light yellow,
crystalline powder. It is odorless, and has a slightly bitter
taste.
It is freely soluble in 7V,iV-dimethylformamide, slightly
soluble in acetone, and practically insoluble in water, in
methanol, in ethanol (95) and in diethyl ether.
Identification (1) To 0.1 g of Hydroxyzine Pamoate add
25 mL of sodium hydroxide TS, and shake well. Extract with
20 mL of chloroform, and use the chloroform layer as the
sample solution. Use the water layer for test (4). To 5 mL of
the sample solution add 2 mL of ammonium thiocyanate-
cobaltous nitrate TS, shake well, and allow to stand: a blue
color is produced in the chloroform layer.
(2) Evaporate 2 mL of the sample solution obtained in (1)
on a water bath to dryness, and dissolve the residue in 0.1
mol/L hydrochloric acid TS to make 500 mL. Determine the
absorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Perform the test with Hydroxyzine Pamoate as direct-
740
Hymecromone / Official Monographs
JP XV
ed under Flame Coloration Test <1.04> (2): a green color ap-
pears.
(4) To 1 mL of the water layer obtained in (1), add 2 mL
of 1 mol/L hydrochloric acid TS: a yellow precipitate is
produced. Collect the precipitate, dissolve the precipitate in 5
mL of methanol, and add 1 drop of iron (III) chloride TS: a
green color is produced.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Hydroxyzine Pamoate in 10 mL of A^,A r -dimethylformamide:
the solution is clear, and shows a slightly greenish, light yel-
low-brown color.
(2) Chloride <1.03> — To 0.3 g of Hydroxyzine Pamoate
add 6 mL of dilute nitric acid and 10 mL of water, shake for
5 minutes, and filter. Wash the residue with two 10-mL por-
tions of water, combine the washings with the filtrate, and
add water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with
0.80 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.095%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Hydrox-
yzine Pamoate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Hydroxyzine Pamoate according to Method 3, and per-
form the test (not more than 1 ppm).
(5) Related substances — Dissolve 0.40 g of Hydroxyzine
Pamoate in 10 mL of a mixture of sodium hydroxide TS and
acetone (1:1), and use the solution as the sample solution.
Pipet 1 mL of this solution, add a mixture of sodium
hydroxide TS and acetone (1:1) to make exactly 20 mL. Pipet
5 mL of this solution, add a mixture of sodium hydroxide TS
and acetone (1 : 1) to make exactly 50 mL, and use the solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, ethanol (95)
and ammonia TS (150:95:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly hydrogen hexachloroplatinate
(IV)-potassium iodide TS on the plate: the spots other than
the spots from hydroxyzine and pamoic acid obtained from
the sample solution are not more intense than the spot from
the standard solution.
Water <2.48> Not more than 3.0% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately about 0.6 g of Hydroxyzine Pamo-
ate, add 25 mL of sodium hydroxide TS, shake well, and ex-
tract with six 25-mL portions of chloroform. Filter each ex-
tract through 5 g of anhydrous sodium sulfate on a pledget of
absorbent cotton. Combine the chloroform extracts, and
evaporate the combined chloroform extracts on a water bath
to about 30 mL. Add 30 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to blue-green
(indicator: 2 drops of crystal violet TS). Perform a blank de-
termination.
Each mL of 0.1 mol/L perchloric acid VS
= 38.16 mg of C 21 H 27 C1N 2 2 .C 23 H 16 6
Containers and storage Containers — Tight containers.
Hymecromone
by'7n I E>
Ci H 8 O 3 : 176.17
7-Hydroxy-4-methylchromen-2-one [90-33-5 ]
Hymecromone, when dried, contains not less than
98.0% of C 10 H 8 O 3 .
Description Hymecromone occurs as white crystals or crys-
talline powder. It is odorless and tasteless.
It is freely soluble in A^A^dimethylformamide, sparingly
soluble in ethanol (95), in ethanol (99.5) and in acetone,
slightly soluble in diethyl ether, and practically insoluble in
water.
Identification (1) Dissolve 2 mg of Hymecromone in 5 mL
of ammonia-ammonium chloride buffer solution, pH 11.0:
the solution shows an intense blue-purple fluorescence.
(2) Dissolve 0.025 g of Hymecromone in 5 mL of diluted
ethanol (95) (1 in 2), and add 1 drop of iron (III) chloride TS:
initially a blackish brown color develops, and when allowed
to stand the color changes to yellow-brown.
(3) Determine the absorption spectrum of a solution of
Himecromone in ethanol (99.5) (1 in 250,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of
Himecromone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 187 - 191 °C
Purity (1) Chloride <1.03>— Dissolve 0.8 g of Hymecro-
mone in 40 mL of a mixture of acetone and water (2:1), and
add 6 mL of dilute nitric acid and a mixture of acetone and
water (2:1) to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS add 6
mL of dilute nitric acid and a mixture of acetone and water
(2:1) to make 50 mL (not more than 0.011%).
(2) Sulfate <1.14> — Dissolve 0.8 g of Hymecromone in 40
mL of a mixture of acetone and water (2:1), and add 1 mL of
dilute hydrochloric acid and a mixture of acetone and water
(2:1) to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.40 mL of 0.005 mol/L sulfuric acid VS add 1 mL of dilute
hydrochloric acid and a mixture of acetone and water (2:1) to
make 50 mL (not more than 0.024%).
(3) Heavy metals <I.07>— Proceed with 2.0 g of
Hymecromone according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
JPXV
Official Monographs / Hypromellose 741
Solution (not more than 10 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Hymecromone according to Method 3, and perform the
test (not more than 2 ppm).
(5) Related substances — Dissolve 80 mg of Hymecro-
mone in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
ethanol (95) to make exactly 50 mL. Pipet 1 mL of this solu-
tion, add ethanol (95) to make exactly 20 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chloroform
and ethanol (95) (10:1) to a distance of about 10 cm, and air-
dry the plate. Allow the plate to stand in iodine vapor for 5
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Hymecromone,
previously dried, dissolve in 90 mL of 7V,A^-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L tetramethylam-
monium hydroxide VS (potentiometric titration). Separately,
perform a blank determination with a solution prepared by
adding 14 mL of water to 90 mL of A^A^-dimethylfor-
mamide, and make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 17.62 mg of C, H 8 O 3
Containers and storage Containers — Tight containers.
Hypromellose
Hydroxypropylmethylcellulose
Cellulose, 2-hydroxypropyl methyl ether
[9004-65-3]
This monograph is harmonized with the European Phar-
macopoeia and the U.S. Pharmacopeia. The parts of the text
that are not harmonized are marked with symbol (* ♦).
Hypromellose is a methyl and hydroxypropyl mixed
ether of cellulose.
There are four substitution types of Hypromellose,
1828, 2208, 2910 and 2910. They contain methoxy
(-OCH 3 : 31.03) and hydroxypropoxy (-OC 3 H 6 OH:
75.09) groups conforming to the limits for the types of
Hypromellose shown in the table below, calculated on
the dried basis.
The viscosity is shown in millipascal second (mPa-s)
on the label, together with the substitution type.
Methoxy Group
Hydroxypropoxy
Substitution
(%)
Group (%)
Type
Min.
Max.
Min.
Max.
1828
16.5
20.0
23.0
32.0
2208
19.0
24.0
4.0
12.0
2906
27.0
30.0
4.0
7.5
2910
28.0
30.0
7.0
12.0
♦Description Hypromellose occurs as a white to yellowish
white, powder or granules.
It is practically insoluble in ethanol (99.5).
It swells with water and becomes a clear or slightly turbid,
viscous solution.*
Identification (1) Disperse evenly 1.0 g of Hypromellose
over the surface of 100 mL of water in a beaker, while gently
tapping the top of the container, if necessary, and allow the
beaker to stand: it aggregates on the surface of water.
(2) Add 1.0 g of Hypromellose to 100 mL of hot water,
and stir: it becomes a suspension. Cool the suspension to
10°C, and stir: the resulting liquid is a clear or a slightly
cloudy, viscous fluid.
(3) To 0.1 mL of the viscous fluid obtained in (2) add 9
mL of diluted sulfuric acid (9 in 10), stir, heat in a water bath
for exactly 3 minutes, and immediately cool in ice water. Add
carefully 0.6 mL of ninhydrin TS, stir, and allow to stand at
25°C: the solution shows a light red color first, then changes
to purple color within 100 minutes.
(4) Pour and spread out 2 to 3 mL of the viscous fluid ob-
tained in (2) onto a glass plate, and allow the water to
evaporate: a transparent film results.
(5) Pipet 50 mL of water, add exactly 50 mL of the vis-
cous fluid obtained in (2), and warm to rise the temperature
at a rate of 2 to 5°C per minute while stirring: the tempera-
ture, when a white turbidity of the solution starts to increase,
is not less than 50°C.
Viscosity <2.53> Method I: Apply to Hypromellose having a
labeled viscosity of less than 600 mPa-s. Put exactly an
amount of Hypromellose, equivalent to 4.000 g on the dried
basis, in a tared, wide-mouth bottle, add hot water to make
200.0 g, stopper the bottle, stir by mechanical means at 350-
to 450-revolutions per minute for 10 to 20 minutes to get a
homogeneous dispersion. If necessary, take off the sample at-
tached on the walls of the bottle, put them in the dispersed
solution, and dissolve by continuing the stirring in a water
bath not exceeding 10°C for 20 to 40 minutes while stirring.
Add cooled water, if necessary, to make 200.0 g, and use this
solution as the sample solution. Centrifuge the solution if
necessary to expel any entrapped air bubbles. Perform the
test with the sample solution at 20±0.1°C as directed in
Method I under Viscosity Determination: not less than 80%
and not more than 120% of the labeled viscosity.
Method II: Apply to Hypromellose having a labeled vis-
cosity of not less than 600 mPa-s. Put exactly an amount of
Hypromellose, equivalent to 10.00 g on the dried basis, in a
tared, wide-mouth bottle, add hot water to make 500.0 g,
stopper the bottle, and prepare the sample solution in the
same manner as directed in Method I. Perform the test with
the sample solution at 20 ± 0. 1 °C as directed in Method II (2)
742 Hypromellose / Official Monographs
JP XV
under Viscosity Determination, using a single cylinder-type
rotational viscometer, according to the following operating
conditions: not less than 75% and not more than 140% of the
labeled viscosity.
Operating conditions —
Apparatus: Brookfield type viscometer LV model
Rotor No., rotation frequency, and conversion factor: Ac-
cording to the following table, depending on the labeled vis-
cosity.
Labeled viscosity
(mPa-s)
n . Rotation ~
R No° r ^0^ "™
/mm
Not less than 600 and less than 1400
3
60
20
// 1400 // 3500
3
12
100
" 3500 n 9500
4
60
100
'/ 9500 u 99500
4
6
1000
a 99500
4
3
2000
Procedure of apparatus: Read value after 2 minutes of ro-
tation, and stop the rotation for 2 minutes. Repeat this proce-
dure more two times, and average three observed values.
pH <2.54> Allow the sample solution obtained in the Viscosi-
ty to stand at 20±2°C for 5 minutes: the pH of the solution
thus obtained is between 5.0 and 8.0.
Purity Heavy metals — Put 1.0 g of Hypromellose in a
100-mL Kjeldahl flask, add a sufficient amount of a mixture
of nitric acid and sulfuric acid (5:4) to wet the sample, and
heat gently. Repeat this procedure until to use totally 18 mL
of the mixture of nitric acid and sulfuric acid. Then boil gent-
ly until the solution changes to black. After cooling, add 2
mL of nitric acid, and heat until the solution changes to
black. Repeat this procedure until the solution no longer
changes to black, and heat strongly until dense white fumes
are evolved. After cooling, add 5 mL of water, boil gently un-
til dense white fumes are evolved, then heat until the volume
of the solution becomes to 2 to 3 mL. After cooling, if the so-
lution reveals yellow color by addition of 5 mL of water, add
1 mL of hydrogen peroxide (30), and heat until the volume of
the solution becomes to 2 to 3 mL. After cooling, dilute the
solution with 2 to 3 mL of water, transfer to a Nessler tube,
add water to make 25 mL, and use this solution as the test so-
lution. Separately, put 2.0 mL of Standard Lead Solution in
a 100-mL kjeldahl flask, add 18 mL of the mixture of nitric
acid and sulfuric acid (5:4) and an amount of nitric acid equal
to that used for preparation of the test solution, and heat un-
til white fumes are evolved. After cooling, add 10 mL of
water. In the case where hydrogen peroxide (30) is added for
the preparation of the test solution, add the same amount of
hydrogen peroxide (30), then proceed in the same manner for
preparation of the test solution, and use so obtained solution
as the control solution. Adjust the test solution and the con-
trol solution to pH 3.0 to 4.0 with ammonia solution (28),
and add water to make 40 mL, respectively. To these solu-
tions add 1.2 mL of thioacetamide-alkaline glycerin TS, 2
mL of acetate buffer solution, pH 3.5 and water to make 50
mL, separately. After allowing to stand for 5 minutes, ob-
serve vertically both tubes on a white background: the color
obtained with the test solution is not more intense than that
with the control solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C, 1
hour).
Residue on ignition <2.44> Not more than 1.5% (1 g).
Assay (i) Apparatus — Reaction bottle: A 5-mL pressure-
tight glass vial, having 20 mm in outside diameter and 50 mm
in height, the neck 20 mm in outside diameter and 13 mm in
inside diameter, equipped with a septum of butyl-rubber
processed the surface with fluoroplastics, which can be fixed
tightly to vial with aluminum cap, or equivalent.
Heater: A square-shaped aluminum block, having holes 20
mm in diameter and 32 mm in depth, adopted to the reaction
bottle. Capable of stirring the content of the reaction bottle
by means of magnetic stirrer or of reciprocal shaker about
100 times per minute.
(ii) Procedure — Weigh accurately about 65 mg of
Hypromellose, transfer to the reaction bottle, add 0.06 to
0. 10 g of adipic acid, 2.0 mL of the internal standard solution
and 2.0 mL of hydroiodic acid, stopper the bottle immedi-
ately, and weigh accurately. Stir or shake for 60 minutes
while heating so that the temperature of the bottle content is
130±2°C. In the case when the stirrer or shaker is not availa-
ble, heat for 30 minutes with repeated shaking at 5-minute in-
tervals by hand, and continue heating for an additional 30
minutes. Allow the bottle to cool, and again weigh accurate-
ly. If the mass loss is less than 0.50% or there is no evidence
of a leak, use the upper layer of the mixture as the sample so-
lution. Separately, put 0.06 to 0.10 g of adipic acid, 2.0 mL
of the internal standard solution and 2.0 mL of hydroiodic
acid in a reaction bottle, stopper the bottle immediately, and
weigh accurately. Add 45 /xL of iodomethane for assay and
15 to 22 /xL of isopropyl iodide for assay through the septum
using micro-syringe with weighing accurately every time, stir
thoroughly, and use the upper layer of the mixture as the
standard solution. Perform the test with 1 to 2 /xL each of the
sample solution and standard solution as directed under Gas
Chromatography <2.02> according to the following condi-
tions, and calculate the ratios, Q Ta and Q Tb , of the peak area
of iodomethane and isopropyl iodide to that of the internal
standard obtained from the sample solution, and Q Sa and Q
sb , of the peak area of iodomethane and isopropyl iodide to
that of the internal standard from the standard solution.
Content (%) of methoxy group (-CH 3 0)
= (Sla/Qsa) x (W Sa /W) x 21.86
Content (%) of hydroxypropoxy group (-C 3 H 7 2 )
= (GTb/Ssb) x (W sb /W) x 44.17
W Slt : Amount (mg) of iodomethane for assay.
W sb : Amount (mg) of isopropyl iodide for assay.
W: Amount (mg) of sample, calculated on the dried basis
Internal standard solution — A solution of w-octane in o-xy-
lene (3 in 100).
Operating conditions —
Detector: A thermal conductivity detector or hydrogen
flame-ionization detector.
Column: A glass column 3-4 mm in inside diameter and
1.8 - 3 m in length, packed with siliceous earth for gas chro-
matography, 125 to 150 /xm in diameter, coated with methyl
silicone polymer at the ratio of 10 - 20%.
Column temperature: A constant temperature of about
100°C.
Carrier gas: Helium for thermal conductivity detector, or
Helium or Nitrogen for hydrogen, flame-ionization detector.
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 10 minutes.
JPXV
Official Monographs / Hypromellose Phthalate 743
System suitability —
System performance: When the procedure is run with 1-2
/uL of the standard solution under the above operating condi-
tions, iodomethane, isopropyl iodide and the internal stan-
dard are eluted in this order, with complete separation of
these peaks.
♦Containers and storage Containers — Well-closed contain-
ers. ♦
Hypromellose Phthalate
Hydroxypropyl methylcellulose benzene-l,2-dicarboxylate
[9050-31-1 ]
Hypromellose Phthalate is a monophthalic acid ester
of hypromellose.
It contains methoxy group (-OCH 3 : 31.03), hydrox-
ypropoxy group (-OC 3 H 6 OH: 75.09), and carboxyben-
zoyl group (-COC 6 H 4 COOH: 149.12).
There are two substitution types, 200731 and
220824, and each type contains indicated amount of
carboxybenzoyl group in the accompanying table, cal-
culated on the anhydrous basis.
Substitution
Type
200731
220824
Carboxybenzoyl group (%)
Min. Max.
27.0
21.0
35.0
27.0
Its substitution type and its kinematic viscosity are
shown in square mm per second (mm 2 /s) on the label.
Description Hypromellose Phthalate occurs as white pow-
der or granules. It is odorless and tasteless.
It is practically insoluble in acetonitrile, in ethanol (99.5),
and in hexane.
It becomes a viscous liquid when a mixture of methanol
and dichloromethane (1:1) or a mixture of ethanol (99.5) and
acetone (1:1) is added.
It dissolves in sodium hydroxide TS.
Identification Determine the infrared absorption spectrum
of Hypromellose Phthalate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or spectrum of Hypromellose Phthalate Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Viscosity <2.53> To 10 g of Hypromellose Phthalate, previ-
ously dried at 105°C for 1 hour, add 90 g of a mixture of
methanol and dichloromethane in equal mass ratio, and stir
to dissolve. Determine the viscosity at 20 ± 0. 1 °C as directed
in Method I under Viscosity Determination: the viscosity is
not less than 80% and not more than 120% of the labeled
unit.
Purity (1) Chloride <1.03>— Dissolve 1.0 g of Hypromel-
lose Phthalate in 40 mL of 0.2 mol/L sodium hydroxide VS,
add 1 drop of phenolphthalein TS, and add dilute nitric acid
dropwise with vigorous stirring until the red color is dis-
charged. Further add 20 mL of dilute nitric acid with stirring.
Heat on a water bath with stirring until the gelatinous
precipitate formed turns to granular particles. After cooling,
centrifuge, and take off the supernatant liquid. Wash the
precipitate with three 20-mL portions of water by centrifug-
ing each time, combine the supernatant liquid and the wash-
ings, add water to make 200 mL, and filter. Perform the test
with 50 mL of the filtrate. Control solution: To 0.50 mL of
0.01 mol/L hydrochloric acid VS add 10 mL of 0.2 mol/L
sodium hydroxide VS, 7 mL of dilute nitric acid and water to
make 50 mL (not more than 0.07%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Hypromellose Phthalate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Phthalic acid — Weigh accurately about 0.2 g of
Hypromellose Phthalate, add about 50 mL of acetonitrile to
dissolve partially with the aid of ultrasonicator, add 10 mL of
water, and dissolve further with the ultrasonicator. After
cooling, add acetonitrile to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 12.5 mg of phthalic acid, dissolve in about 125
mL of acetonitrile by stirring, add 25 mL of water and
acetonitrile to make exactly 250 mL, and use this solution as
the standard solution. Perform the test with 10 /uL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas of phthalic acid, A T
and A s , of both solutions: amount of phthalic acid (C 8 H 6 4 :
166.13) is not more than 1.0%.
Amount (%) of phthalic acid
= (C/W)x(A 1 /A s )xl0
C: concentration of phthalic acid in the standard solution
(jUg/mL)
W: amount (mg) of the sample, calculated on the anhy-
drous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (3 to 10 //m in
particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of 0.1 mol/L cyanoacetic acid
and acetonitrile (17:3).
Flow rate: About 2.0 mL per minute.
System repeatability: Repeat the test six times with the
standard solution under the above operating conditions: the
relative standard deviation of the peak area of phthalic acid is
not more than 1.0%.
Water <2.48> Not more than 5.0% (1 g, direct titration, us-
ing a mixture of ethanol (99.5) and dichloromethane (3:2) in-
stead of methanol for Karl Fischer method).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 1 g of Hypromellose Phtha-
late, dissolve in 50 mL of a mixture of ethanol (95), acetone
744
Ibuprofen / Official Monographs
JP XV
and water (2:2:1), and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS (indicator: 2 drops of phenolphthalein TS).
Perform a blank determination, and make any necessary cor-
rection.
Amount (%) of carboxybenzoyl group (C 8 H 5 3 )
= {(0.01 x 149.1 XVJ/W] - {(2x 149.1 XP)/166.1}
P: amount (%) of phthalic acid obtained in the Purity (3)
V: amount (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed
W: amount (g) of the sample, calculated on the anhydrous
basis
Containers and storage Containers — Tight containers.
Ibuprofen
and enantiomer
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of hexane, ethyl acetate and
acetic acid (100) (15:5:1) to a distance of about 10 cm, and
air-dry the plate. Examine the plate under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, phosphorus (V) oxide, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Ibuprofen, previ-
ously dried, dissolve in 50 mL of ethanol (95), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 20.63 mg of Ci 3 H lg 2
Containers and storage Containers — Well-closed contain-
ers.
C 13 H 18 2 : 206.28
(2/?5')-2-[4-(2-Methylpropyl)phenyl]propanoic acid
[15687-27-1]
Ibuprofen, when dried, contains not less than 98.5%
of C 13 H 18 2 .
Description Ibuprofen occurs as a white crystalline powder.
It is freely soluble in ethanol (95) and in acetone, and prac-
tically insoluble in water.
It dissolves in dilute sodium hydroxide TS.
Identification (1) Determine the absorption spectrum of a
solution of Ibuprofen in dilute sodium hydroxide TS (3 in
20,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Ibuprofen, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 75 - 77°C
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Ibuprofen according to Method 2, and perform the test. Pre-
pare the control solution with 3 .0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Ibuprofen according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.50 g of Ibuprofen in
exactly 5 mL of acetone , and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add acetone to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
Ichthammol
-1 ??=E-JU
Ichthammol, calculated on the dried basis, contains
not less than 2.5% of ammonia (NH 3 : 17.030), not
more than 8.0% of ammonium sulfate [(NH 4 ) 2 S0 4 :
132.14], and not less than 10.0%> of total sulfur (as S:
32.07).
Description Ichthammol is a red-brown to blackish brown,
viscous fluid. It has a characteristic odor.
It is miscible with water, and is partially soluble in ethanol
(95) and in diethyl ether.
Identification (1) To 4 mL of a solution of Ichthammol (3
in 10) add 8 mL of hydrochloric acid: a yellow-brown to
blackish brown, oily or resinous mass is produced. Cool the
mass with ice to solidify, and discard the water layer. Wash
the residue with diethyl ether: a part of the mass dissolves but
it does not dissolve completely even when it is washed until
almost no color develops in the washing. Perform the follow-
ing tests with this residue.
(i) To 0.1 g of the residue add 1 mL of a mixture of
diethyl ether and ethanol (95) (1:1): it dissolves.
(ii) To 0.1 g of the residue add 2 mL of water: it dis-
solves. To 1 mL of this solution add 0.4 mL of hydrochloric
acid: a yellow-brown to blackish brown oily or resinous sub-
stance is produced.
(iii) To 1 mL of the solution obtained in (ii) add 0.3 g of
sodium chloride: a yellow-brown or blackish brown oily or
resinous substance is produced.
(2) Boil 2 mL of a solution of Ichthammol (1 in 10) with 2
mL of sodium hydroxide TS: the gas evolved changes
moistened red litmus paper to blue.
Loss on drying <2.41> Not more than 50% (0.5 g, 105 °C, 6
hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
JPXV
Official Monographs / Idarubicin Hydrochloride
745
Assay (1) Ammonia — Weigh accurately about 5 g of
Ichthammol, transfer to a Kjeldahl flask, and add 60 mL of
water, 1 mL of 1-octanol and 4.5 mL of a solution of sodium
hydroxide (2 in 5). Connect the flask to a distilling tube with
a spray trap and a condenser, and immerse the lower outlet of
the condenser in the receiver containing exactly 30 mL of 0.25
mol/L sulfuric acid VS. Distil slowly, collect about 50 mL of
the distillate, and titrate <2.50> the excess sulfuric acid with
0.5 mol/L sodium hydroxide VS (indicator: 3 drops of
methyl red TS). Perform a blank determination, and make
any necessary correction.
Each mL of 0.25 mol/L sulfuric acid VS
= 8.515 mg of NH 3
(2) Ammonium sulfate — Weigh accurately about 1 g of
Ichthammol, add 25 mL of ethanol (95), stir thoroughly, and
filter. Wash with a mixture of diethyl ether and ethanol (95)
(1:1) until the washings are clear and colorless. Dry the filter
paper and the residue in air, dissolve the residue in 200 mL of
hot water acidified slightly with hydrochloric acid, and filter.
Boil the filtrate, add 30 mL of barium chloride TS slowly,
heat for 30 minutes on a water bath, and filter. Wash the
precipitate with water, dry, and ignite to constant mass.
Weigh the residue as barium sulfate (BaS0 4 : 233.39).
Amount (mg) of ammonium sulfate [(NH 4 ) 2 S0 4 ]
= amount (mg) of barium sulfate (BaS0 4 ) x 0.5662
(3) Total sulfur — Weigh accurately about 0.6 g of
Ichthammol, transfer to a 200-mL Kjeldahl flask, and add 30
mL of water and 5 g of potassium chlorate, then add slowly
30 mL of nitric acid, and evaporate the mixture to about 5
mL. Transfer the residue to a 300-mL beaker with the aid of
25 mL of hydrochloric acid, and evaporate again to 5 mL.
Add 100 mL of water, boil, filter, and wash with water. Heat
the combined filtrate and washings to boil, add gradually 30
mL of barium chloride TS, heat the mixture on a water bath
for 30 minutes, and filter. Wash the precipitate with water,
dry, and ignite to constant mass. Weigh the residue as barium
sulfate (BaS0 4 ).
Amount (mg) of total sulfur (S)
= amount (mg) of barium sulfate (BaS0 4 ) x 0.13739
Containers and storage Containers — Tight containers.
Idarubicin Hydrochloride
<f*OUbf->>£&£
OH -KCI
1 ,2,3,4-tetrahydrotetracene-6, 1 1-dione monohydrochloride
[57852-57-0]
Idarubicin Hydrochloride contains not less than 960
fig (potency) and not more than 1030 fig (potency) per
mg, calculated on the anhydrous basis. The potency of
Idarubicin Hydrochloride is expressed as mass (poten-
cy) of idarubicin hydrochloride (C26H27NO9.HCI).
Description Idarubicin Hydrochloride occurs as a yellow-
red powder.
It is sparingly soluble in methanol, slightly soluble in water
and in ethanol (95), and practically insoluble in acetonitrile
and in diethyl ether.
Identification (1) Determine the absorption spectra of a
solution of Idarubicin Hydrochloride in methanol (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Idarubicin
Hydrochloride Reference Standard prepared in the same
manner as the sample solution: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectra of Idarubi-
cin Hydrochloride and Idarubicin Hydrochloride Reference
Standard as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare these
spectra: both spectra exhibit similar intensities of absorption
at the same wave numbers.
(3) Dissolve 2 mg of Idarubicin Hydrochloride in 3 mL of
water, and add 1 mL of dilute nitric acid and 3 drops of silver
nitrate TS: a white turbidity is produced.
Absorbance <2.24> E\ 0/ ° m (482 nm): 204 - 210 (20 mg calcu-
lated on the anhydrous basis, methanol, 1000 mL).
Optical rotation <2.49> [a]^ :
lated on the anhydrous basis,
+ 191 - +197° (20 mg calcu-
methanol, 20 mL, 100 mm).
C 26 H 27 N0 9 .HC1: 533.95
(25,45)-2-Acetyl-4-(3-amino-2,3,6-trideoxy-a-L-/yxo-
hexopyranosyloxy)-2,5,12-trihydroxy-
pH <2.54> The pH of a solution of Idarubicin Hydrochlo-
ride (1 in 200) is between 5.0 and 6.5.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals — Being specified separately.
(3) Related substances — Being specified separately.
(4) Residual solvent — Being specified separately.
Water <2.48> Not more than 5.0% (0.1 g, coulometric titra-
tion).
Residue on ignition Being specified separately.
Bacterial endotoxins <4.01> Less than 8.9EU/mg (poten-
cy).
Assay Weigh accurately an amount of Idarubicin
Hydrochloride and Idarubicin Hydrochloride Reference
Standard, equivalent to about 10 mg (potency), dissolve each
in the mobile phase containing no sodium lauryl sulfate to
make exactly 50 mL, and use these solutions as the sample so-
lution and the standard solution, respectively. Perform the
test with exactly 20 fiL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
peak areas, A T and A s , of idarubicin of these solutions.
Amount [fig (potency)] of C26H27NO9.HCI
746 Idarubicin Hydrochloride for Injection / Official Monographs
JP XV
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Idarubicin Hydrochloride
Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (4//m in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 10.2 g of potassium dihydrogen-
phosphate in a suitable amount of water, add 1 mL of phos-
phoric acid and water to make 750 mL, and add 250 mL of
tetrahydrofuran. To 500 mL of this solution add 0.72 g of so-
dium lauryl sulfate and 0.5 mL of 7V,./V-dimethyl-«-octyla-
mine, and adjust to pH 4 with 2 mol/L sodium hydroxide
TS.
Flow rate: Adjust the flow rate so that the retention time of
idarubicin is about 15 minutes.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, the number of theoretical steps of the peak of idarubi-
cin is not less than 3000 steps.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of idarubicin is not more than 2.0%.
Containers and storage Containers — Tight containers.
Idarubicin Hydrochloride for
Injection
Idarubicin Hydrochloride for Injection is a prepara-
tion for injection, which is dissolved before use.
It contains not less than 90.0% and not more than
110.0% of the labeled amount of idarubicin
hydrochloride (C 26 H 27 N0 9 .HC1: 533.95).
Method of preparation Prepare as directed under Injec-
tions, with Idarubicin Hydrochloride.
Description Idarubicin Hydrochloride for Injection occurs
as yellow-red masses.
Identification (1) Dissolve an amount of Idarubicin
Hydrochloride for Injection, equivalent to 2 mg (potency) of
Idarubicin Hydrochloride according to the labeled amount,
in 5 mL of sodium hydroxide TS: the solution shows a blue-
purple color.
(2) Dissolve an amount of Idarubicin Hydrochloride for
Injection, equivalent to 1 mg (potency) of Idarubicin
Hydrochloride according to the labeled amount, in 1 mL of
water, and add methanol to make 100 mL. Determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 250 nm and 254 nm, between 285 nm and 289 nm,
between 480 nm and 484 nm, and between 510 nm and 520
nm.
pH <2.54> The pH of a solution prepared by dissolving an
amount of Idarubicin Hydrochloride for Injection, equiva-
lent to 5 mg (potency) of Idarubicin Hydrochloride according
to the labeled amount, in 5 mL of water is between 5.0 and
7.0.
Purity Clarity and color of solution — Dissolve an amount
of Idarubicin Hydrochloride for Injection, equivalent to 5
mg (potency) of Idarubicin Hydrochloride according to the
labeled amount, in 5 mL of water: the solution is clear and
yellow-red.
Water <2.48> Weigh accurately the mass of one Idarubicin
Hydrochloride for Injection, add 5 mL of methanol for Karl
Fischer method using a syringe, dissolve with thorough shak-
ing, and perform the test with 4 mL of this solution as direct-
ed in the Volumetric titration (direct titration). Use 4 mL of
methanol for Karl Fischer method as the blank. Determine
the mass of the content from the difference between the mass
of one Idarubicin Hydrochloride for Injection obtained
above and the mass of its bottle and rubber stopper, which
are weighed accurately after washing with water then with
ethanol (95), drying at 105°C for 1 hour and allowing to cool
to room temperature in a desiccator (not more than 4.0%).
Bacterial endotoxins <4.01> Less than 8.9EU/mg (poten-
cy).
Uniformity of dosage units <6.02> Perform the test according
to the following method: it meets the requirement of the Con-
tent uniformity test.
To one Idarubicin Hydrochloride for Injection add the
mobile phase prepared without addition of sodium lauryl sul-
fate to make exactly FmL so that each mL contains 0.2 mg
(potency) of idarubicin hydrochloride (C26H27NO9.HCI), and
use this solution as the sample solution. Separately, weigh ac-
curately an amount of Idarubicin Hydrochloride Reference
Standard, equivalent to about 10 mg (potency), dissolve in
the mobile phase without containing sodium lauryl sulfate to
make exactly 50 mL, and use this solution as the standard so-
lution. Proceed as directed in the Assay under Idarubicin
Hydrochloride.
Amount [mg (potency)] of idarubicin hydrochloride
(C 26 H 27 N0 9 .HC1) = W s x (A T /A S ) x (K/50)
W s : Amount [mg (potency)] of Idarubicin Hydrochloride
Reference Standard
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Idarubicin Hydrochloride for Injection. Weigh ac-
curately an amount of the content, equivalent to about 5 mg
(potency) according to the labeled amount, dissolve in the
mobile phase prepared without addition of sodium lauryl sul-
fate to make exactly 25 mL, and use this solution as the sam-
ple solution. Separately, weigh accurately an amount of
JP XV
Official Monographs / Idoxuridine Ophthalmic Solution
747
Idarubicin Hydrochloride Reference Standard, equivalent to
about 10 mg (potency), dissolve in the mobile phase without
containing sodium lauryl sulfate to make exactly 50 mL, and
use this solution as the standard solution. Proceed as directed
in the Assay under Idarubicin Hydrochloride.
Amount [mg (potency)] of idarubicin hydrochloride
(C 26 H 27 N0 9 .HC1)
= W s x (A T /A S ) x (1/2)
W s : Amount [mg (potency)] of Idarubicin Hydrochloride
Reference Standard
Containers and storage Containers — Hermetic containers.
Idoxuridine
C 9 H„IN 2 5 : 354.10
5-Iodo-2'-deoxyuridine [54-42-2]
Idoxuridine, when dried,
98.0% of C 9 H n IN 2 5 .
contains not less than
Description Idoxuridine occurs as colorless, crystals or a
white, crystalline powder. It is odorless.
It is freely soluble in dimethylamide, slightly soluble in
water, very slightly soluble in ethanol (95), and practically in-
soluble in diethyl ether.
It dissolves in sodium hydroxide TS.
Melting point: about 176°C (with decomposition).
Identification (1) Dissolve 0.01 g of Idoxuridine in 5 mL
of water by warming, add 5 mL of diphenylamine-acetic acid
TS, and heat for 5 minutes: a blue color develops.
(2) Heat 0.1 g of Idoxuridine: a purple gas evolves.
(3) Dissolve 2 mg of Idoxuridine in 50 mL of 0.01 mol/L
sodium hydroxide. Determine the absorption spectrum of the
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Idoxuridine Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
Optical rotation <2.49> [a]™: +28- +31° (after drying,
0.2 g, sodium hydroxide TS, 20 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Idoxuridine in 5 mL of a solution of sodium hydroxide (1
in 200): the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Idoxuri-
dine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Related substances — Dissolve 0.10 g of Idoxuridine in
exactly 10 mL of a mixture of dilute ethanol and ammonia
solution (28) (99:1), and use this solution as the sample solu-
tion. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 50,mL of the
sample solution on a plate of silica gel with fluorescent indi-
cator for thin-layer chromatography. Develop the plate with
a mixture of ethyl acetate and diluted 2-propanol (2 in 3) (4:1)
to a distance of about 10 cm, and air-dry the plate. Then de-
velop two-dimensionally at right angles to the first, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): any spot other than the principal spot
does not appear.
(4) Iodine and iodide — Dissolve 0.10 g of Idoxuridine in
20 mL of water and 5 mL of sodium hydroxide TS, and add
immediately 5 mL of dilute sulfuric acid under ice-cooling.
Allow to stand for 10 minutes with occasional shaking, and
filter. Transfer the filtrate into a Nessler tube, add 10 mL of
chloroform and 3 drops of a solution of potassium iodate (1
in 100), shake for 30 seconds, and allow to stand: the chlo-
roform layer has no more color than the following control so-
lution.
Control solution: Weigh accurately 0.111 g of potassium
iodide, and dissolve in water to make 1000 mL. To exactly 1
mL of this solution add 19 mL of water, 5 mL of sodium
hydroxide TS and 5 mL of dilute sulfuric acid, mix, and
filter. Transfer the filtrate to a Nessler tube, and proceed in
the same manner.
Loss on drying <2.41> Not more than 0.5% (2 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.7 g of Idoxuridine, previ-
ously dried, dissolve in 80 mL of A^Af-dimethylformamide,
and titrate <2.50> with 0.1 mol/L tetramethylammonium
hydroxide VS until the color of the solution changes from
yellow through yellow-green to blue (indicator: 5 drops of
thymol blue-dimethylformamide TS). Perform a blank deter-
mination, and make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 35.41 mg of C 9 H n IN 2 5
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Idoxuridine Ophthalmic Solution
A h'^z^U v>,6Bg>j£
Idoxuridine Ophthalmic Solution contains not less
than 90% and not more than 110% of the labeled
amount of idoxuridine (C9HHIN2O5: 354.10).
Method of preparation Prepare as directed under
Ophthalmic Solutions, with Idoxuridine.
Description Idoxuridine Ophthalmic Solution is a clear,
colorless liquid.
Identification (1) To a volume of Idoxuridine Opthalmic
Solution, equivalent to 5 mg of Idoxuridine according to the
748
Ifenprodil Tartrate / Official Monographs
JP XV
labeled amount, add 5 mL of diphenylamine-acetic acid TS,
and heat for 20 minutes: a light blue color develops.
(2) Place a volume of Idoxuridine Ophthalmic Solution,
equivalent to 5 mg of Idoxuridine according to the labeled
amount, in a porcelain crucible, add 0. 1 g of anhydrous sodi-
um carbonate, heat slowly, evaporate to dryness and ignite
until the residue is incinerated. Dissolve the residue in 5 mL
of water, acidify with hydrochloric acid, and add 2 to 3 drops
of sodium nitrite TS: a yellow-brown color develops. Then
add 2 to 3 drops of starch TS: a deep blue color develops.
(3) To a volume of Idoxuridine Ophthalmic Solution,
equivalent to 2 mg of Idoxuridine according to the labeled
amount, add 0.01 mol/L sodium hydroxide TS to make 50
mL. Determine the absorption spectrum of this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 277 nm and 281 nm.
pH <2.54> 4.5 - 7.0
Purity 5-Iodouracil and 2'-deoxyuridine — To a volume of
Idoxuridine Ophthalmic Solution, equivalent to 4.0 mg of
Idoxuridine according to the labeled amount, add water to
make exactly 5 mL, and use this solution as the sample solu-
tion. Separately, dissolve 12.0 mg of 5-iodouracil for liquid
chromatography and 4.0 mg of 2'-deoxyuridine for liquid
chromatography in water to make exactly 200 mL. Measure
exactly 5 mL of this solution, add water to make exactly
25 mL, and use this solution as the standard solution.
Perform the test with exactly 10 iiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas of 5-iodouracil and 2'-deox-
yuridine: the peak areas of 5-iodouracil and 2'-deoxyuridine
of the sample solution are not more than the peak areas of 5-
iodouracil and 2 '-deoxy uridine of the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 iim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and methanol (24:1).
Flow rate: Adjust the flow rate so that the retention time of
2'-deoxyuridine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10 iiL
of the standard solution under the above operating condi-
tions, 2'-deoxyuridine and 5-iodouracil are eluted in this ord-
er with the resolution between these peaks being not less than
2.0.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
2'-deoxyuridine is not more than 1.0%.
Assay Measure exactly a volume of Idoxuridine
Ophthalmic Solution, equivalent to 3 mg of idoxuridine
(C9H11IN2O5) according to the labeled amount, add exactly 2
mL of the internal standard solution, then add water to make
10 mL, and use this solution as the sample solution.
Separately weigh accurately about 10 mg of Idoxuridine
Reference Standard, previously dried at 60°C for 3 hours,
dissolve in water to make exactly 10 mL. Measure exactly
3 mL of this solution, add exactly 2 mL of the internal stan-
dard solution, then add water to make 10 mL, and use this
solution as the standard solution. Perform the test with 10 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of idoxuridine to that of the internal standard, re-
spectively.
Amount (mg) of idoxuridine (C9H U IN 2 5 )
= W s x (Qj/Qs) x (3/10)
W s : Amount (mg) of Idoxuridine Reference Standard
Internal standard solution — A solution of sulfathiazole in the
mobile phase (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and methanol (87:13).
Flow rate: Adjust the flow rate so that the retention time of
idoxuridine is about 9 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, idoxuridine and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of idoxuridine to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, in a cold place, and avoid freez-
ing.
Ifenprodil Tartrate
H OH
HOjC
COzK
H OH
(C 21 H 27 N0 2 ) 2 .C 4 H 6 6 : 800.98
(li?5',25'i?)-4-[2-(4-Benzylpiperidin-l-yl)-l-
hydroxypropyljphenol hemi-(2/?,3/?)-tartrate [23210-58-4]
Ifenprodil Tartrate contains not less than 98.5% of
(C2iH27N02)2-C 4 H 6 6 , calculated on the anhydrous ba-
sis.
Description Ifenprodil Tartrate occurs as a white crystalline
JPXV
Official Monographs / Imipenem Hydrate 749
powder. It is odorless.
It is freely soluble in acetic acid (100), soluble in ethanol
(95), slightly soluble in water and in methanol, and practical-
ly insoluble in diethyl ether.
Optical rotation [a]™ ■ + 11 - + 15° (1 g, calculated on the
anhydrous basis, ethanol (95), 20 mL, 100 mm).
Melting point: about 148°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ifenprodil Tartrate in methanol (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Ifen-
prodil Tartrate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Dissolve 0.4 g of Ifenprodil Tartrate in 40 mL of
water by warming. After cooling, add 0.5 mL of ammonia
TS to this solution, extract with two 40-mL portions of chlo-
roform, and collect the water layer. Evaporate 30 mL of the
water layer on a water bath to dryness, and after cooling, dis-
solve the residue in 6 mL of water: the solution responds to
the Qualitative Tests <1.09> for tartrate.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Ifenprodil Tartrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.30 g of Ifenprodil
Tartrate in 10 mL of diluted ethanol (95) (3 in 4), and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add diluted ethanol (95) (3 in 4) to make exactly 200
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 10 /uL each of the sample solu-
tion and standard solution on a plate of silica gel for thinlay-
er chromatography. Develop the plate with a mixture of ethyl
acetate, hexane, 1-butanol and ammonia solution (28)
(140:40:20:1) to a distance of about 10 cm, and air-dry the
plate. Spray hydrogen hexachloroplatinate (IV)-potassium
iodide TS evenly on the plate: the spots other than the prin-
cipal spot from the sample solution are not more intense than
the spot from the standard solution.
Water <2.48> Not more than 4.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Ifenprodil Tartrate,
dissolve in 50 mL of acetic acid (100), and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 40.05 mg of (C 2I H 27 N0 2 ) 2 .C 4 H 6 6
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Imipenem Hydrate
■i $ ^^atK^p^
COjH
>HjO
H H
C 12 H 17 N 3 4 S.H 2 0: 317.36
(5i?,6S)-3-[2-(Formimidoylamino)ethylsulfanyl]-6-[(l/?)-
l-hydroxyethyl]-7-oxo-l-azabicyclo [3.2.0] hept-2-ene-2-
carboxylic acid monohydrate [74431-23-5]
Imipenem Hydrate contains not less than980,ug
(potency) and not more than 1010 fig (potency) per mg,
calculated on the anhydrous basis. The potency of Im-
ipenem Hydrate is expressed as mass (potency) of im-
ipenem (C 12 H 17 N 3 4 S: 299.35).
Description Imipenem Hydrate occurs as white to light yel-
low crystalline powder.
It is sparingly soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Imipenem Hydrate in 0.1 mol/L 3-(iV-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0 (1 in
50,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Imipenem Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(2) Determine the infrared absorption spectrum of Im-
ipenem Hydrate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Imipenem Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [ a ] 2 ^: + 89 - +94° (50 mg calculat-
ed on the anhydrous basis, 0.1 mol/L 3-(/V-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0, 10
mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Imipenem Hydrate in 200 mL of water is between 4.5 and
7.0.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Imipenem Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Put 2.0 g of Imipenem Hydrate in a
crucible, add 5 mL of nitric acid and 1 mL of sulfuric acid,
and heat carefully until white fumes evolve. After cooling,
add 2 mL of nitric acid, heat, and repeat this procedure once
more. Then add 2 mL of hydrogen peroxide (30), heat, and
repeat this procedure several times until the color of the solu-
tion changes to colorless to pale yellow. After cooling, heat
again until white fumes evolve. After cooling, add water to
make 5 mL, and perform the test with this solution as the test
750
Imipenem and Cilastatin Sodium for Injection / Official Monographs
JP XV
solution (not more than 1 ppm).
(3) Related substances — Dissolve 50 mg of Imipenem
Hydrate in 50 mL of 0.1 mol/L 3-(./V-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add 0.1 mol/L 3-(./V-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0 to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 (iL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the peak area of thienamycin, having the rela-
tive retention time of about 0.8 with respect to imipenem, ob-
tained from the sample solution is not more than 1.4 times
the peak area of imipenem from the standard solution, the
area of the peak other than imipenem and thienamycin from
the sample solution is not more than 1/3 times the peak area
of imipenem from the standard solution, and the total area of
the peaks other than imipenem and thienamycin from the
sample solution is not more than the peak area of imipenem
from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of imipenem.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, add 0.1 mol/L 3-(-/V-morpholino)-
propanesulfonic acid buffer solution, pH 7.0 to make exactly
50 mL. Confirm that the peak area of imipenem from 10 iiL
of this solution is equivalent to 7 to 13% of that from the
standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
imipenem is not more than 2.0%.
Water <2.48> Not less than 5.0% and not more than 8.0%
(20 mg, coulometric titration, water evaporation tempera-
ture: 140°C).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately an amount of Imipenem Hydrate
and Imipenem Reference Standard, equivalent to about 50
mg (potency), dissolve each in 0.1 mol/L 3-(iV-morpholino)-
propanesulfonic acid buffer solution, pH 7.0 to make exactly
50 mL, and use these solutions as the sample solution and the
standard solution. Perform the test with exactly 10,mL each
of the sample solution and standard solution, within 30
minutes after preparation of these solutions, as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and A s , of
imipenem of these solutions.
Amount [/ug (potency)] of imipenem (C^Hn^C^S)
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Imipenem Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /um in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.1 mol/L 3-(N-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0 and
acetonitrile (100:1).
Flow rate: Adjust the flow rate so that the retention time of
imipenem is about 6 minutes.
System suitability —
System performance: Dissolve 50 mg of Imipenem and 75
mg of resorcinol in 50 mL of 0.1 mol/L 3-(N-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0. When
the procedure is run with 10 [iL of this solution under the
above operating conditions, imipenem and resorcinol are
eluted in this order with the resolution between these peaks
being not less than 4.
System repeatability: When the test is repeated 5 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
imipenem is not more than 0.80%.
Containers and storage Containers — Hermetic containers.
Imipenem and Cilastatin Sodium for
Injection
Imipenem and Cilastatin Sodium for Injection is a
preparation for injection which is dissolved or sus-
pended before use.
It contains not less than 93.0% and not more than
115.0% of the labeled amount of imipenem
(C 12 H 17 N304S: 299.35) and an amount of cilastatin so-
dium (C 16 H 2 5N2Na0 5 S: 380.43), equivalent to not less
than 93.0% and not more than 115.0% of the labeled
amount of cilastatin (Ci 6 H 2 6N20 5 S: 358.45).
Method of preparation Prepare as directed under Injec-
tions, with Imipenem Hydrate and Cilastatin Sodium.
Description Imipenem and Cilastatin Sodium for Injection
occurs as a white to light yellowish white powder.
Identification (1) To 1 mL of a solution of Imipenem and
Cilastatin Sodium for Injection (1 in 100) add 1 mL of nin-
hydrin TS, heat in a water bath for 5 minutes: a purple color
appears (cilastatin).
(2) To 2 mL of a solution of Imipenem and Cilastatin So-
dium for Injection (1 in 1000) add 0.1 mol/L 3-(/V-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0 to
make 50 mL, and determine the absorption spectrum of the
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 296 nm and 300
nm (imipenem).
pH <2.54> The pH of a solution prepared by dissolving an
JP XV
Official Monographs / Imipramine Hydrochloride 751
amount of Imipenem and Cilastatin Sodium for Injection, e-
quivalent to 0.5 g (potency) of Imipenem Hydrate according
to the labeled amount, in 100 mL of isotonic sodium chloride
solution is between 6.5 and 8.0. The pH of the Injection in-
tended for intramuscular use is between 6.0 and 7.5.
Purity Clarity and color of solution — Dissolve an amount
of Imipenem and Cilastatin Sodium for Injection, equivalent
to 0.5 g (potency) of Imipenem Hydrate according to the la-
beled amount, in 100 mL of isotonic sodium chloride solu-
tion: the solution is clear and colorless or pale yellow.
Loss on drying <2.41> Not more than 3.0% (1 g, in vacuum,
60°C, 3 hours).
Bacterial endotoxins <4.0I>
cy).
Less than 0.25 EU/mg (poten-
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test. Calculate the acceptance value by
using the average of the limits specified in the potency defini-
tion for T.
Dissolve the total amount of the content of one Imipenem
and Cilastatin Sodium for Injection in isotonic sodium chlo-
ride solution to make 100 mL. Measure exactly KmL of this
solution, equivalent to about 25 mg (potency) of Imipenem
Hydrate according to the labeled amount, add 0.1 mol/L 3-
(/V-morpholino)propanesulfonic acid buffer solution, pH7.0
to make exactly 50 mL, and use this solution as the sample
solution. Proceed hereafter as directed in the Assay.
Amount [mg (potency)] of imipenem (C 12 H 17 N 3 04S)
= W s x (y4 TI A4 SI ) x (100/ V)
W s : Amount [mg (potency)] of Imipenem Reference Stan-
dard
Amount (mg) of cilastatin (C I6 H 26 N 2 5 S)
= W s x (A TC /A SC ) x (100/ V) x 0.955
W s : Amount (mg) of cilastatin ammonium for assay, cal-
culated on the anhydrous basis and corrected on the
amount of the residual solvent
Foreign insoluble matter <6.06> Perform the test according to
the Method 2: the Injection which is dissolved before use
meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: the Injection which is dissolved before
use meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Imipenem and Cilastatin Sodium for Injections.
Weigh accurately an amount of the content, equivalent to
one Imipenem and Cilastatin Sodium for Injection, dissolve
in isotonic sodium chloride solution to make exactly 100 mL.
Measure exactly an amount of this solution, equivalent to
about 25 mg (potency) of imipenem, add 0.1 mol/L 3-(N-
morpholino)propanesulfonic acid buffer solution, pH 7.0 to
make exactly 50 mL, and use this as the sample solution.
Separately, weigh accurately an amount of Imipenem Refer-
ence Standard, equivalent to about 25 mg (potency), and
weigh accurately about 25 mg of cilastatin ammonium for as-
say, separately determined the water content, dissolve in 10
mL of isotonic sodium chloride solution, add 0.1 mol/L 3-
(/V-morpholino)propanesulfonic acid buffer solution, pH 7.0
to make exactly 50 mL, and use this solution as the standard
solution. Perform the test with exactly 10 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas of imipenem, A T1 and
^4 SI , and that of cilastatin, A TC and A sc .
Amount [mg (potency)] of imipenem (C 12 H 17 N 3 4 S)
= W s x (A TI /A SI )
W s : Amount [mg (potency)]of Imipenem Reference Stan-
dard
Amount (mg) of cilastatin (C 16 H 2 6N 2 05S)
= W s x (A TC /A SC ) x 0.955
W s : Amount (mg) of cilastatin ammonium for assay, cal-
culated on the anhydrous basis and corrected on the
amount of ethanol
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 250 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with octylsilanized silica
gel for liquid chromatography (10,wm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 0.836 g of 3-(A r -mor-
pholino)propanesulfonic acid, 1.0 g of sodium 1-hexane sul-
fonate and 50 mg of disodium dihydrogen ethylenediamine
tetraacetate dihydrate in 800 mL of water, adjust to pH 7.0
with 0.1 mol/L sodium hydrate TS, and add water to make
1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
imipenem is about 3 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, imipenem and cilastatin are eluted in this order with
the resolution between these peaks being not less than 2.0,
and the symmetry factors of the peak of imipenem and
cilastatin are not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviations of the peak area
of imipenem and cilastatin are not more than 2.0%, respec-
tively.
Containers and storage Containers — Hermetic containers.
Imipramine Hydrochloride
4 $^7$>
CH, ;
C 19 H 24 N 2 .HC1: 316.87
3-(10,ll-Dihydro-5//-dibenzo[6,/]azepin-5-yl)-
752
Imipramine Hydrochloride Tablets / Official Monographs
JP XV
7V,./V-dimethylpropylamine monohydrochloride [113-52-0]
Imipramine Hydrochloride, when dried, contains
not less than 98.5% of C 19 H 24 N 2 .HC1.
Description Imipramine Hydrochloride occurs as a white to
pale yellowish white, crystalline powder. It is odorless.
It is freely soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
The pH of the aqueous solution (1 in 10) is between 4.2 and
5.2.
It is gradually colored by light.
Identification (1) Dissolve 5 mg of Imipramine
Hydrochloride in 2 mL of nitric acid: a deep blue color de-
velops.
(2) Dissolve 5 mg of Imipramine Hydrochloride in 250
mL of 0.01 mol/L hydrochloric acid TS. Determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Imipramine Hydrochloride Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Dissolve 0.05 g of Imipramine Hydrochloride in 5 mL
of water, add 1 mL of ammonia TS, allow to stand for 5
minutes, filter, and acidify the filtrate with dilute nitric acid:
it responds to the Qualitative Tests <1.09> (2) for chloride.
Melting point <2.60> 170 - 174°C (with decomposition).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Imipramine Hydrochloride in 10 mL of water: the solution is
clear, and has no more color than the following control solu-
tion.
Control solution: Take exactly 1.0 mL of Cobaltous Chlo-
ride Colorimetric Stock Solution, 2.4 mL of Ferric Chloride
Colorimetric Solution, 0.4 mL of Cupric Sulfate Colorimet-
ric Stock Solution and 6.2 mL of diluted hydrochloric acid (1
in 40), and mix them. Pipet 0.5 mL of this solution, and add
exactly 9.5 mL of water.
(2) Iminodibenzyl — Dissolve 50 mg of Imipramine
Hydrochloride in 10 mL of a mixture of hydrochloric acid
and ethanol (95) (1:1) in a 25-mL brown volumetric flask.
Cool the flask in ice water, add 5 mL of an ethanol (95) solu-
tion of furfural (1 in 250) and 5 mL of hydrochloric acid, and
allow to stand at 25 °C for 3 hours. Add a mixture of
hydrochloric acid and ethanol (95) (1:1) to make 25 mL, and
determine the absorbance of this solution at 565 nm as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it is
not more than 0.16.
(3) Related substances — Dissolve 0.20 g of Imipramine
Hydrochloride in 10 mL of ethanol (95), and use this solution
as the sample solution. Pipet 1 mL of this solution, and add
ethanol (95) to make exactly 50 mL. Pipet 5 mL of this solu-
tion, add ethanol (95) to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, acetic acid (100), hydrochloric acid and water
(11:7:1:1) to a distance of about 12 cm, and air-dry the plate.
Spray evenly potassium dichromate-sulfuric acid TS on the
plate: the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Imipramine
Hydrochloride, previously dried, and dissolve in 20 mL of
water. Add 5 mL of sodium hydroxide TS, and extract with
three 20-mL portions of chloroform. Filter each extract
through a pledget of absorbent cotton on which a small quan-
tity of anhydrous sodium sulfate is placed. Combine the chlo-
roform extracts, and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the yellow solution changes to red-purple (indi-
cator: 10 drops of metanil yellow TS). Perform a blank deter-
mination.
Each mL of 0.1 mol/L perchloric acid VS
= 31.69 mg of C 19 H 24 N 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Imipramine Hydrochloride Tablets
Imipramine Hydrochloride Tablets contain not less
than 93% and not more than 107% of the labeled
amount of imipramine hydrochloride (Ci 9 H 24 N 2 .HCl:
316.87).
Method of preparation Prepare as directed under Tablets,
with Imipramine Hydrochloride.
Identification (1) Weigh a quantity of powdered Imipra-
mine Hydrochloride Tablets, equivalent to 0.25 g of Imipra-
mine Hydrochloride according to the labeled amount, add 25
mL of chloroform, shake thoroughly, and filter. Evaporate
the filtrate on a water bath, and proceed with the residue as
directed in the Identification (1) under Imipramine
Hydrochloride.
(2) Dissolve an amount of the residue obtained in (1),
equivalent to 5 mg of Imipramine Hydrochloride, in 250 mL
of 0.01 mol/L hydrochloric acid TS, and determine the ab-
sorption spectrum as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 249
nm and 253 nm, and a shoulder between 270 nm and 280 nm.
(3) Dry the residue obtained in (1) at 105°C for 2 hours:
the residue melts between 170°C and 174°C (with decomposi-
tion).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Imipramine Hydrochlo-
ride Tablet at 75 revolutions per minute according to the Pad-
dle method, using 900 mL of 2nd fluid for dissolution test as
the test solution. Take 20 mL or more of the dissolved solu-
tion after 60 minutes from the start of the dissolution test,
and filter through a membrane filter with pore size of not
more than 0.8 ^m. Discard the first 10 mL of the filtrate,
pipet the subsequent FmL, add 2nd fluid for dissolution test
JPXV
Official Monographs / Indenolol Hydrochloride 753
to make exactly V'vaL so that each mL of the filtrate contains
about 10 fig of imipramine hydrochloride (C I9 H 24 N 2 .HC1) ac-
cording to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 0.025 g
of Imipramine Hydrochloride Reference Standard, previous-
ly dried at 105 °C for 2 hours, dissolve in 2nd fluid for disso-
lution test to make exactly 100 mL. Pipet 4 mL of this solu-
tion, add 2nd fluid for dissolution test to make exactly 100
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and the
standard solution at 250 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>. The dissolution rate of Im-
ipramine Hydrochloride Tablets in 60 minutes should be not
less than 75%.
Dissolution rate (%) with respect to the labeled amount
of imipramine hydrochloride (Ci9H 24 N 2 .HCl)
= W s x (Aj/As) x (V'/V) x (1/Q x 36
W s : Amount (mg) of Imipramine Hydrochloride Refer-
ence Standard.
C: Labeled amount (mg) of imipramine hydrochloride
(C 19 H 24 N 2 .HC1) in 1 tablet.
Assay Take 20 Imipramine Hydrochloride Tablets, add ex-
actly 200 mL of 0.01 mol/L hydrochloric acid TS, and shake
well until the tablets are completely disintegrated. After cen-
trifuging the solution, pipet a volume of the supernatant liq-
uid, equivalent to about 25 mg of imipramine hydrochloride
(C 19 H 24 N 2 .HC1) according to the labeled amount, add 0.01
mol/L hydrochloric acid TS to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Imipramine Hydrochloride for As-
say, previously dried at 105°C for 2 hours, dissolve in 0.01
mol/L hydrochloric acid TS to make exactly 100 mL, and use
this solution as the standard solution. Pipet 3 mL each of
these solutions into separators which contain 15 mL of potas-
sium hydrogen phthalate buffer solution, pH 5.6, 8 mL of
bromocresol green-sodium hydroxide TS and 30 mL of chlo-
roform, and shake. Filter the chloroform layer through a
pledget of absorbent cotton into a 100-mL volumetric flask.
Repeat the extraction with two 30-mL portions of chlo-
roform, combine the chloroform layers in the 100-mL volu-
metric flask, and add chloroform to make exactly 100 mL.
Perform the test with these solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using a solu-
tion obtained by proceeding with 3 mL of 0.01 mol/L
hydrochloric acid TS in the same manner as the blank. Deter-
mine the absorbances, A T and A s , of these solutions at 416
nm.
Amount (mg) of imipramine hydrochloride (Ci 9 H 24 N 2 .HCl)
= W s x (A T /A S )
W s : Amount (mg) of Imipramine Hydrochloride for Assay
Containers and storage Containers — Tight containers.
Indenolol Hydrochloride
'Ot/p-jimi
■HCI
and enantiomers
C 15 H 21 N0 2 .HC1: 283.79
(2RS)- 1 -(3#-Inden-4-yloxy)-
3-(l-methylethyl)aminopropan-2-ol monohydrochloride
(2RS)- 1 -(3//-Inden-7-yloxy)-
3-(l-methylethyl)aminopropan-2-ol monohydrochloride
[68906-88-7]
Indenolol Hydrochloride is a mixture of (2RS)-l-
(3//-Inden-4-yloxy)-3-(l-methylethyl)aminopropan-2-
ol monohydrochloride and (2^?S)-l-(3//-Inden-7-
yloxy)-3-(l-methylethyl)aminopropan-2-ol mono-
hydrochloride.
When dried, it contains not less than 98.5% of
C 15 H 21 N0 2 .HC1.
Description Indenolol Hydrochloride occurs as white to
pale yellow crystals or crystalline powder.
It is freely soluble in water and in acetic acid (100), soluble
in ethanol (95) and in chloroform, slightly soluble in acetic
anhydride, very slightly soluble in ethyl acetate, and practi-
cally insoluble in diethyl ether.
The pH of a solution of Indenolol Hydrochloride (1 in 10)
is between 3.5 and 5.5.
It is colored by light.
Identification (1) Dissolve 0.1 g of Indenolol Hydrochlo-
ride in 1 to 2 drops of dilute hydrochloric acid and 5 mL of
water, and add 1 mL of Reinecke salt TS: a red-purple
precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Indenolol Hydrochloride (1 in 50,000) as directed under
Ultravioret-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum 1 : both spectra ex-
hibit similar intensities of absorption at the same
wavelengths. Separately, determine the absorption spectrum
of a solution of Indenolol Hydrochloride (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 2:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the infrared absorption spectrum of In-
denolol Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Indenolol Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
Absorbance <2.24> £}* (250 nm): 330 - 340 (after drying,
10 mg, water, 1000 mL).
Melting point <2.60> 140 - 143°C
754 Indigocarmine / Official Monographs
JP XV
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Indenolol Hydrochloride in 10 mL of water: the solution is
clear and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of In-
denolol Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Indenolol Hydrochloride according to Method 1, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Indenolol
Hydrochloride in 10 mL of chloroform, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add chloroform to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /uL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 1,2-
dichloroethane, ethanol (99.5) and ammonia solution (28)
(70:15:2) to a distance of about 12 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Isomer ratio Dissolve 5 mg of Indenolol Hydrochloride in
1.0 mL of a mixture of ethyl acetate and dehydrated
trifluoroacetic acid (9:1), and use this solution as the sample
solution. Perform the test with 2 /xh of the sample solution as
directed under Gas Chromatography <2.02> according to the
following conditions. Determine the areas of two adjacent
peaks, A^ and A b , having the retention times of about 16
minutes, where A R is the peak area of shorter retention time
and A b is the peak area of longer retention time: the ratio
^4a/G4a + A b ) is between 0.6 and 0.7.
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column about 2 mm in inside diameter
and about 2 m in length, packed with siliceous earth for gas
chromatography (150 to 180 /um in particle diameter) coated
with 65% phenyl-methyl silicon polymer for gas chro-
matography at the ratio of 2%.
Column temperature: A constant temperature between
150°C and 170°C.
Carrier gas: Helium.
Flow rate: Adjust the flow rate so that the retention time of
the peak showing earlier elution of the two peaks of indenolol
hydrochloride is about 16 minutes.
Selection of column: Proceed with 2 fiL of the sample solu-
tion under the above operating conditions, and calculate the
resolution. Use a column with the resolution between the two
peaks being not less than 1.1.
Assay Weigh accurately about 0.5 g of Indenolol
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (4:1), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to green (indi-
cator: 3 drops of crystal violet TS). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.38 mg of C 15 H 21 N0 2 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Indigocarmine
Nad 3 S
SO3N3
C 16 H 8 N 2 Na 2 8 S 2 : 466.35
Disodium 3,3'-dioxo-[zl 2 2 -biindoline]-5,5'-disulfonate
[860-22-0]
Indigocarmine, when dried, contains not less than
95.0% of C 16 H 8 N 2 Na 2 8 S 2 .
Description Indigocarmine occurs as blue to dark blue pow-
der or granules. It is odorless.
It is sparingly soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It is hygroscopic.
When compressed, it has a coppery luster.
Identification (1) A solution of Indigocarmine (1 in 100) is
dark blue in color. Perform the following tests with this solu-
tion as the sample solution: the dark blue color of each solu-
tion disappears.
( i ) Add 1 mL of nitric acid to 2 mL of the sample solu-
tion;
( ii ) Add 1 mL of bromine TS to 2 mL of the sample solu-
tion;
(iii) Add 1 mL of chlorine TS to 2 mL of the sample solu-
tion;
(iv) Add 2 mL of sodium hydroxide TS and 0.2 g of zinc
powder to 2 mL of the sample solution, and warm.
(2) Dissolve 0.1 g of Indigocarmine in 100 mL of a solu-
tion of ammonium acetate (1 in 650). To 1 mL of the solution
add a solution of ammonium acetate (1 in 650) to make 100
mL. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Ignite 1 g of Indigocarmine to carbonize. After cool-
ing, add 20 mL of water to the residue, shake, and filter the
mixture: the filtrate responds to the Qualitative Tests <1.09>
for sodium salt and for sulfate.
pH <2.54> Dissolve 0.10 g of Indigocarmine in 20 mL of
water: the pH of the solution is between 5.0 and 6.0.
Purity (1) Water-insoluble substances — To 1.00 g of In-
digocarmine add 200 mL of water, shake, and filter through a
tared glass filter (G4). Wash the residue with water until the
blue color of the filtrate becomes practically colorless, and
JPXV
Official Monographs / Indometacin 755
dry the residue at 105°C for 4 hours: the mass of the residue
does not exceed 5.0 mg.
(2) Arsenic <1.11> — Place 0.8 g of Indigocarmine in a
Kjeldahl flask, add 5 mL of sulfuric acid and 5 mL of nitric
acid, and ignite gently. Repeat the addition of 2 to 3 mL of
nitric acid occasionally, and continue to heat until a colorless
to light yellow solution is obtained. After cooling, add 15 mL
of a saturated ammonium oxalate solution, heat the solution
until dense white fumes are evolved, and concentrate to 2 to 3
mL. After cooling, dilute with water to 10 mL, and perform
the test with 5 mL of this solution as the test solution (not
more than 5 ppm).
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not less than 28.0% and not
more than 38.0% (after drying, 1 g).
Assay Weigh accurately about 0.5 g of Indigocarmine,
previously dried, add 15 g of sodium hydrogen tartrate
monohydrate, and dissolve in 200 mL of water, boil with
bubbling of a stream of carbon dioxide, and titrate <2.50>,
while being hot, with 0.1 mol/L titanium (III) chloride VS
until the color of the solution changes from blue through yel-
low to orange.
Each mL of 0.1 mol/L titanium (III) chloride VS
= 23.32 mg of C 16 H 8 N 2 Na 2 8 S 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Indigocarmine Injection
<<>>s=f2jJUS >>±ttfc
Indigocarmine Injection is an aqueous solution for
injection. It contains not less than 95% and not more
than 105% of the labeled amount of indigocarmine
(C 16 H 8 N 2 Na 2 8 S 2 : 466.35).
Method of preparation Prepare as directed under Injection,
with Indigocarmine.
Description Indigocarmine Injection is a dark blue liquid.
pH: 3.0-5.0
Identification (1) To a volume of Indigocarmine Injec-
tion, equivalent to 0.02 g of Indigocarmine according to the
labeled amount, add 1 mL of nitric acid: the dark blue color
of the liquid disappears, and a yellow-brown color develops.
(2) To a volume of Indigocarmine Injection, equivalent
to 0.02 g of Indigocarmine according to the labeled amount,
add 1 mL of bromine TS: the dark blue color disappears, and
a yellow-brown color develops.
(3) To a volume of Indigocarmine Injection, equivalent
to 0.02 g of Indigocarmine according to the labeled amount,
add 1 mL of chlorine TS: the dark blue color disappears, and
a yellow-brown color develops.
(4) To a volume of Indigocarmine Injection, equivalent
to 0.01 g of Indigocarmine according to the labeled amount,
add ammonium acetate solution (1 in 650) to make 1000 mL,
and determine the absorbance of the solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 610 nm and 614 nm.
Assay Measure exactly a volume of Indigocarmine Injec-
tion, equivalent to about 0.2 g of indigocarmine
(C 16 H 8 N 2 Na 2 8 S 2 ), add 6 g of sodium hydrogen tartrate
monohydrate, and dissolve in water to make 200 mL. Then
boil under a carbon dioxide stream, and proceed as directed
in the Assay under Indigocarmine.
Each mL of 0.1 mol/L titanium (III) chloride VS
= 23.32 mg of C 16 H 8 N 2 Na 2 8 S 2
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Indium ( m In) Chloride Injection
±M{b^>v^A( 111 In)a|«
Indium ( in In) Chloride Injection is an aqueous solu-
tion for injection
It contains indium-Ill ( m In) in the form of indium
chloride.
It conforms to the requirements of Indium ( ni In)
Chloride Injection in the Minimum Requirements for
Radiopharmaceuticals .
Test for Extractable Volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Indium ( nl In) Chloride Injection is a clear,
colorless liquid.
Indometacin
-f > K * 9 :
COpH
C 19 H, 6 C1N0 4 : 357.79
[l-(4-Chlorobenzoyl)-5-methoxy-2 -methyl- li/-indol-3-
yl]acetic acid [53-86-1]
Indometacin, when dried, contains not less than
98.0% of C 19 H 16 C1N0 4 .
Description Indometacin occurs as a white to light yellow-
ish white, very fine crystalline powder.
It is sparingly soluble in methanol, in ethanol (95) and in
diethyl ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
It is colored by light.
Melting point: 155 - 162°C
Identification (1) Dissolve 2 mg of Indometacin in 100 mL
of methanol. Determine the absorption spectrum of the solu-
tion as directed under Ultraviolet-visible Spectrophotometry
756 Indometacin Capsules / Official Monographs
JP XV
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Indometacin Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of In-
dometacin, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of dried Indometacin Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, recrystallize the sample and the Reference Stan-
dard with diethyl ether, filter and dry the crystals, and per-
form the test with the crystals.
(3) Perform the test with Indometacin as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Purity (1) Acidity — To 1.0 g of Indometacin add 50 mL
of water, shake for 5 minutes, and filter. To the filtrate add
0.20 mL of 0.1 mol/L sodium hydroxide VS and 1 drop of
phenolphthalein TS: a red color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of In-
dometacin according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Indometacin according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Indometacin
in 10 mL of methanol, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add methanol
to make exactly 50 mL. Pipet 5 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 25
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of de-
hydrated diethyl ether and acetic acid (100) (100:3) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Indometacin, previ-
ously dried, dissolve in 60 mL of methanol, add 30 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (indicator: 3 drops of phenolphthalein TS). Perform a
blank determination.
Each mL of 0.1 mol/L sodium hydroxide VS
= 35.78 mg of C 19 H 16 C1N0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Indometacin Capsules
-1 >H;<?->>;rj^zJU
Indometacin Capsules contain not less than 90% and
not more than 110% of the labeled amount of in-
dometacin (C 19 H 16 C1N0 4 : 357.79).
Method of preparation Prepare as directed under Capsules,
with Indometacin.
Identification Powder the contents of Indometacin Cap-
sules. To a quantity of the powder, equivalent to 0.1 g of In-
dometacin according to the labeled amount, add 20 mL of
chloroform, shake well, and centrifuge. Filter the super-
natant liquid, and evaporate the filtrate to dryness. After
cooling, dissolve the residue in 20 mL of methanol. To 10 mL
of this solution add methanol to make 50 mL, then to 2 mL
of this solution add methanol to make 100 mL, and use this
solution as the sample solution. Determine the absorption
spectrum of the sample solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 317 nm and 321 nm.
Purity Related substances — Powder the content of In-
dometacin Capsules. To a quantity of the powder, equivalent
to 0.10 g of Indometacin according to the labeled amount,
add exactly 10 mL of methanol, shake well, filter, and use the
filtrate as the sample solution. Dissolve 25 mg of Indometa-
cin Reference Standard in methanol to make exactly 50 mL.
Pipet 1 mL of the solution, add methanol to make exactly 10
mL, and use this solution as the standard solution. Proceed
as directed in the Purity (4) under Indometacin.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Take 1 capsule of Indometacin Capsules, and perform the
test using 900 mL of a mixture of water and phosphate buffer
solution, pH 7.2, (4:1) as the test solution at 100 revolutions
per minute as directed in the Basket method. Take 20 mL or
more of the dissolved solution at 20 minutes after starting the
test, and filter through a membrane filter (less than 0.8 //m in
pore size). Discard the first 10 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Separately, weigh
accurately about 30 mg of Indometacin Reference Standard,
previously dried at 105°C for 4 hours, dissolve in a mixture
of water and phosphate buffer solution, pH 7.2, (4:1) to
make exactly 1000 mL, and use this as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and the standard solution at 320 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>. The dissolu-
tion rate of Indometacin Capsules in 20 minutes should be
not less than 75%.
Dissolution rate (%) with respect to the labeled amount
of indometacin (Ci 9 H 16 ClN0 4 )
= W s x (A T /A S ) x (90/C)
W s : Amount (mg) of Indometacin Reference Standard.
C: Labeled amount (mg) of indometacin (C 19 H 16 C1N0 4 ) in
1 capsule.
Assay Weigh accurately the contents of not less than 20 In-
JPXV
Official Monographs / Indometacin Suppositories 757
dometacin Capsules. Powder the combined contents, and
weigh accurately a portion of the powder, equivalent to
about 50 mg of indometacin (C 19 H 16 C1N0 4 ). Dissolve in 40
mL of methanol, and add methanol to make exactly 50 mL.
Filter this solution, discarding the first 10-mL portion of the
filtrate. Pipet the subsequent 5 mL of the filtrate, add exactly
3 mL of the internal standard solution, add the mobile phase
to make 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 50 mg of Indometacin
Reference Standard, previously dried at 105 C C for 4 hours,
and dissolve in methanol to make exactly 50 mL. Pipet 5 mL
of the solution, add exactly 3 mL of the internal standard so-
lution, add the mobile phase to make 100 mL, and use this
solution as the standard solution. Perform the test with 20 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Qj and Q s , of the
peak area of indometacin to that of the internal standard,
respectively.
Amount (mg) of indometacin (C 19 H 16 C1N0 4 )
= Ws x (Qt/Qs)
W s : Amount (mg) of Indometacin Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 iim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted phos-
phoric acid (1 in 1000) (7:3).
Flow rate: Adjust the flow rate so that the retention time of
indometacin is about 8 minutes.
System suitability —
System performance: Dissolve 50 mg of 4-chlorobenzoic
acid, 30 mg of butyl parahydroxybenzoate and 50 mg of in-
dometacin in 50 mL of methanol. To 5 mL of this solution
add the mobile phase to make 100 mL. When the procedure is
run with 20 /xh of this solution under the above operating
conditions, 4-chlorobenzoic acid, butyl parahydroxybenzo-
ate and indometacin are eluted in this order, with the resolu-
tion between the peaks of 4-chlorobenzoic acid and butyl
parahydroxybenzoate being not less than 2.0, and between
the peaks of butyl parahydroxybenzoate and indometacin
being not less than 5.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of indometacin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Indometacin Suppositories
-1 > Ky^->>^IJ
Indometacin Suppositories contain not less than
90% and not more than 1 10% of the labeled amount of
indometacin (C 19 H 16 C1N0 4 : 357.79).
Method of preparation Prepare as directed under Supposi-
tories, with Indometacin.
Identification Dissolve a quantity of Indometacin Supposi-
tories, equivalent to 0.05 g of Indometacin according to the
labeled amount, in 20 mL of methanol by warming, add
methanol to make 50 mL, and filter if necessary. To 2 mL of
this solution add methanol to make 100 mL, and use this so-
lution as the sample solution. Determine the absorption spec-
trum of the sample solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 317 nm and 321 nm.
Assay Weigh accurately not less than 20 Indometacin Sup-
positories, cut into small pieces carefully, and mix well.
Weigh accurately a portion of the mass, equivalent to about
50 mg of indometacin (Ci 9 H 16 ClN0 4 ), add 40 mL of tetra-
hydrofuran, warm at 40°C, dissolve by shaking, cool, and
add tetrahydrofuran to make exactly 50 mL. Filter the solu-
tion, discard the first 10 mL of the filtrate, pipet the subse-
quent 5 mL of the filtrate, add exactly 3 mL of the internal
standard solution, and add the mobile phase to make
100 mL. Allow the solution to stand for 30 minutes, filter
through a membrane filter (0.5 Lira pore size), discard the first
10 mL of the filtrate, and use the subsequent filtrate as the
sample solution. Separately, weigh accurately about 50 mg of
Indometacin Reference Standard, previously dried at 105°C
for 4 hours, and dissolve in tetrahydrofuran to make exactly
50 mL. Pipet 5 mL of the solution, proceed in the same man-
ner as the sample solution, and use as the standard solution.
Perform the test with 20 liL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of indometacin to that
of the internal standard, respectively.
Amount (mg) of indometacin (Ci 9 H 16 ClN0 4 )
= W s x (Q T /g s )
W s : Amount (mg) of Indometacin Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 iim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted phos-
phoric acid (1 in 1000) (7:3).
Flow rate: Adjust the flow rate so that the retention time of
758
Influenza HA Vaccine / Official Monographs
JP XV
indometacin is about 8 minutes.
System suitability —
System performance: Dissolve 50 mg of 4-chlorobenzoic
acid, 30 mg of butyl parahydroxybenzoate and 50 mg of in-
dometacin in 50 mL of methanol. To 5 mL of this solution
add the mobile phase to make 100 mL. When the procedure is
run with 20 [iL of this solution under the above operating
conditions, 4-chlorobenzoic acid, butyl parahydroxybenzo-
ate and indometacin are eluted in this order with the resolu-
tion between the peaks of 4-chlorobenzoic acid and butyl
parahydroxybenzoate being not less than 2.0 and between the
peaks of parahydroxybenzoate and indometacin being not
less than 5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of indometacin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant, and in a cold place.
Influenza HA Vaccine
•1 >7JH>-lf HA rP<7^>
Influenza HA Vaccine is a liquid for injection con-
taining hemagglutinin of influenza virus.
It conforms to the requirements of Influenza HA
Vaccine in the Minimum Requirements for Biological
Products.
Description Influenza HA Vaccine is a clear liquid or a
slightly whitish turbid liquid.
Insulin
Insulin is obtained from the pancreas of healthy bo-
vine or porcine, that has blood sugar-decreasing activi-
ty.
Its potency, calculated on the dried basis, is not less
than 26 Insulin Units in each mg.
It is labeled to indicate the animal species from
which it is derived.
Description Insulin occurs as a white, crystalline powder. It
is odorless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in diluted hydrochloric acid (1 in 360) and in di-
lute sodium hydroxide TS.
It is hygroscopic.
Identification Dissolve 0.01 g of Insulin in 10 mL of 0.1
mol/L hydrochloric acid TS, and use this solution as the sam-
ple solution. Adjust the pH of the sample solution to between
5.1 and 5.3 with a solution of sodium hydroxide (1 in 100): a
precipitate is produced. Adjust the solution to a pH between
2.5 and 3.5 with dilute hydrochloric acid: the precipitate dis-
solves.
Purity Clarity and color of solution — Dissolve 0.10 g of In-
sulin in 10 mL of diluted hydrochloric acid (1 in 360): the so-
lution is clear and colorless to light yellow.
Zinc content Weigh accurately about 10 mg of Insulin, dis-
solve in 5 mL of 0.1 mol/L hydrochloric acid TS and water to
make exactly 50 mL. If necessary, dilute the solution with
water so as to contain 0.4 to 1.0 /ug of zinc (Zn: 65.41) per
mL, and use as the sample solution. Add water to an ac-
curately measured volume of Standard Zinc Solution for
atomic absorption spectrophotometry to make a solution
containing 0.3 to 1.2 /ug of zinc (Zn: 65.41) per mL, and use
as the standard solution. Perform the test with the sample
solution and standard solution as directed under Atomic
Absorption Spectrophotometry <2.23> according to the fol-
lowing conditions, and determine the amount of zinc in the
sample solution using the calibration curve obtained from the
absorbance of the standard solution: the amount of zinc is
not less than 0.27% and not more than 1.08%, calculated on
the dried basis.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
Loss on drying <2.41> Not more than 10.0% (0.2 g, 105 °C,
16 hours).
Residue on ignition <2.44> Weigh accurately 0.02 to 0.04 g
of Insulin in a tared platinum dish, add 2 drops of nitric acid,
and heat the dish at first very gently and then strongly to in-
cinerate. Place the dish in a muffle furnace, heat at 600°C for
15 minutes, cool in a desiccator (silica gel), and weigh: the
mass of the residue is not more than 2.5%.
Nitrogen content Weigh accurately about 20 mg of Insulin,
dissolve in 10 mL of 0.1 mol/L hydrochloric acid TS, and
perform the test as directed under Nitrogen Determination
<1.08>: not less than 14.5% and not more than 16.5% of
nitrogen (N: 14.01) is found, calculated on the dried basis.
Assay
(i) Animals: Select healthy rabbits each weighing not less
than 1.8 kg. Keep the rabbits in the laboratory not less than 1
week before use in the assay by feeding them with an ap-
propriate uniform diet and water.
(ii) Diluent for insulin: Dissolve 1.0 to 2.5 g of phenol or
ffj-cresol in 500 mL of 0.01 mol/L hydrochloric acid VS, and
add 14 to 18 g of glycerin and 0.01 mol/L hydrochloric acid
VS to make 1000 mL.
(iii) Standard stock solution: Weigh accurately about 20
mg of Insulin Reference Standard, and dissolve it in the dil-
uent for insulin to make a standard stock solution containing
exactly 20.0 Units in each mL. Preserve this solution between
1°C and 15°C, and use within 6 months.
(iv) Standard solution: Dilute two portions of the stan-
dard stock solution to make two standard solutions with the
diluent for insulin, one to contain exactly 2.0 Units in each
mL which is designated as the high-dose standard solution
S H , and the other to contain exactly 1.0 Unit in each mL
which is designated as the low-dose standard solution S L .
(v) Sample solution: Weigh accurately about 20 mg of In-
sulin according to the labeled Units, dissolve with the diluent
JP XV
Official Monographs / Insulin 759
for insulin to make two different sample solutions, one to
contain exactly 2.0 Units in each mL which is designated as
the high-dose sample solution, T H , and the other to contain
exactly 1.0 Unit in each mL which is designated as the low-
dose sample solution, T L .
(vi) Dose for injection: Select the dose for injection on
the basis of trial or experience. Inject a fixed identical
volume, usually 0.3 to 0.5 mL, of the standard solutions and
the sample solutions throughout the whole run.
(vii) Procedure: Divide the animals into 4 equal groups of
not less than 6 animals each, with least difference in body
mass. Withhold all food, except water, for not less than 14
hours before the injections, and withhold water during the
assay until the final blood sample is taken. Handle the
animals with care in order to avoid undue excitement.
Inject into each of the animals subcutaneously the dose of
the standard solutions and the sample solutions indicated in
the following design.
First group S H Third group T H
Second group S L Fourth group T L
The second injection should be made on the day after the
first injection or within 1 week, using the dose of the standard
solutions and the sample solutions indicated in the following
design.
First group T L Third group S L
Second group T H Fourth group S H
At 1 hour and 2.5 hours after the time of injection, obtain
a sufficient blood sample to perform the test from a marginal
ear vein of each animal, and determine the blood sugar con-
tent of the blood samples according to (viii).
(viii) Blood sugar determination: Place 5.0 mL of a solu-
tion of zinc sulfate heptahydrate (9 in 2000) in a test tube 18
mm in outside diameter and 165 mm in length, add 1.0 mL of
a solution of sodium hydroxide (1 in 250), and add gently
0.10 mL of the blood sample to the mixture in the test tube
using a blood sugar pipet. Suck up the supernatant liquid into
the pipet, wash out the remaining blood in the inner wall of
the pipet, and repeat this procedure. Shake thoroughly the
contents in the test tube, and heat the test tube in a water bath
for 3 minutes. Filter the mixture through a funnel 30 to 40
mm in diameter in which a pledget of absorbent cotton,
previously washed with two 3-mL portions of warm water,
has been placed, receive the filtrate into a test tube 30 mm in
inside diameter and 90 mm in length, wash the test tube and
the funnel with two 3-mL portions of water, and combine the
washings with the filtrate. Add 2.0 mL of alkaline potassium
hexacyanoferrate (III) TS, heat in a water bath for 15
minutes, cool immediately, add 3.0 mL of potassium iodide-
zinc sulfate TS and 2.0 mL of diluted acetic acid (100) (3 in
100), and titrate <2.50> the liberated iodine with 0.005 mol/L
sodium thiosulfate VS (indicator: 2 to 4 drops of starch-sodi-
um chloride TS). Perform a blank determination. From the
consumed volume (mL) of 0.005 mol/L sodium thiosulfate
VS, obtain the blood sugar content (%) according to the fol-
lowing table.
Conversion Table for Blood suger content (%)
mL*
0.0
0.385
0.1
0.355
0.2
0.331
0.3
0.310
0.4
0.290
0.5
0.270
0.6
0.251
0.7
0.232
o.s
0.213
0.9
0.195
1.0
0.177
1.1
0.159
1.2
0.141
1.3
0.124
1.4
0.108
1.5
0.088
1.6
0.070
1.7
0.052
1.8
0.034
1.9
0.017
1
0.382
0.352
0.329
0.308
0.288
0.268
0.249
0.230
0.211
0.193
0.175
0.157
0.139
0.122
0.104
0.086
0.068
0.050
0.032
0.015
2
3
0.379
0.376
0.350
0.348
0.327
0.325
0.306
0.304
0.286
0.284
0.266
0.264
0.247
0.245
0.228
0.226
0.209
0.208
0.191
0.190
0.173
0.172
0.155
0.154
0.138
0.136
0.120
0.119
0.102
0.101
0.084
0.083
0.066
0.065
0.048
0.047
0.031
0.029
0.014
0.012
0.373
0.345
0.323
0.302
0.282
0.262
0.243
0.224
0.206
0.188
0.170
0.152
0.134
0.117
0.099
0.081
0.063
0.045
0.027
0.010
0.370
0.343
0.321
0.300
0.280
0.260
0.241
0.222
0.204
0.186
0.168
0.150
0.132
0.115
0.097
0.079
0.061
0.043
0.025
0.008
0.367
0.341
0.318
0.298
0.278
0.259
0.240
0.221
0.202
0.184
0.166
0.148
0.131
0.113
0.095
0.077
0.059
0.041
0.024
0.007
0.364
0.338
0.316
0.296
0.276
0.257
0.238
0.219
0.200
0.182
0.164
0.146
0.129
0.111
0.093
0.075
0.057
0.039
0.022
0.005
0.361
0.336
0.314
0.294
0.274
0.255
0.236
0.217
0.199
0.181
0.163
0.145
0.127
0.110
0.092
0.074
0.056
0.038
0.020
0.003
0.358
0.333
0.312
0.292
0.272
0.253
0.234
0.215
0.197
0.179
0.161
0.143
0.125
0.108
0.090
0.072
0.054
0.036
0.019
0.002
■"Indicates the volume of 0.005 mol/L sodium thiosulfate VS
required in titration. For example, if the amount was 1.28
mL, the blood sugar content would be 0.127% from the
above table.
(ix) Calculation: Sum up the two blood sugar values of
each animal after each injection. Subtract the blood sugar
value effected by the first injection from that effected by the
second injection of each animal in the first group and the
third group. The differences are symbolized as y x and y it re-
spectively. Subtract the blood sugar value effected by the sec-
ond injection from that effected by the first injection of each
animal in the second group and the fourth group. The differ-
ences are symbolized as y2 and y4, respectively. Sum up not
less than 6 values of individual differences in the blood sugar
values y u y 2 , y 3 , and y 4 to obtain Y u Y 2 , Y it and Y 4 , respec-
tively.
Units in each mg of Insulin
= antilog Mx (Units in each mL of S H ) X (b/a)
M= 0.301 x(F a /r b )
Y !l =-Y 1 +Y 2 +Y 3 -Y 4
Y b =Y l + Y 2 +Yi+Y 4
a: Amount (mg) of the sample.
b: Total volume (mL) of the high-dose sample solution
prepared by dissolving the sample with the diluent
for insulin.
Compute L (P = 0.95) by using the following equation: L
should be not more than 0.1212. If L exceeds 0.1212, repeat
the assay by increasing the number of animals or improving
the assay conditions in a better way until L becomes not more
than 0.1212.
L = 2V(C- 1KCM 2 + 0.09062)
c=r b v(y b 2 -4/s a i a )
/: Number of the animals of each group.
s 2 ={Zy 2 -(Y/j)}/n
I>y 2 : The sum of squares of y u y 2 , y 3 , and y 4 in each
group.
760
Insulin Human (Genetical Recombination) / Official Monographs
JP XV
Y=Y l 2 +Y 2 2 +Yi 1 +Y 4 2
n = 4(/-l)
t 2 : Value shown in the following table against n for which s 2
is calculated.
n
t 2 = F l
n
fi=F x
n
fi=F x
1
161.45
13
AMI
25
4.242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
GO
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Contianers and storage Containers — Tight containers.
Storage — Not exceeding 8°C.
Insulin Human
(Genetical Recombination)
Gly- 1 le-Val-Glu-Gln-Cys-Cys-Thr-Se r-l le-Cys- Ser- La u-Tyr- Gin- Leu-G lu-
Asn-Tyr-Cys-Asn
I
Phe-Val- Asn-GI n -His- Leu • Cys-G ly-Ser-H is-Leu- Val -G I u -Ala-Leu-Tyi-Leu-
Va I- Cys- Gly-G lu- Arg-Gly-Phe- Ph a-Tyr-Th r-Pro-Lys-Thr
C 257 H3 83 N 65 77 S 6 : 5807.57
[11061-68-0]
Insulin Human (Genetical Recombination) is a hu-
man insulin prepared by genetical recombinant tech-
nology.
It has an activity to reduce the blood sugar concen-
tration. It contains not less than 27.5 Insulin Units per
mg, calculated on the dried basis.
Description Insulin Human (Genetical Recombination) oc-
curs as a white powder.
It is practically insoluble in water and in ethanol (95).
It dissolves in 0.01 mol/L hydrochloric acid TS and in
sodium hydroxide TS with decomposition.
It is hygroscopic.
Identification Weigh accurately a suitable amount of Insu-
lin Human (Genetical Recombination), and dissolve in 0.01
mol/L hydrochloric acid TS to make a solution so that each
mL contains 2.0 mg. Transfer 500 iiL of this solution into a
clean test tube, add 2.0 mL of HEPES buffer solution, pH
7.5 and 400 //L of V8-protease TS, react at 25 °C for 6 hours,
then add 2.9 mL of ammonium sulfate buffer solution to stop
the reaction, and use this solution as the sample solution.
Separately, proceed with Human Insulin Reference Standard
in the same manner as above, and use this solution as the
standard solution. Perform the test with exactly 50 //L each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and compare the chromatograms obtained
from these solutions: the peak appears just after the peak of
the solvent and the succeeding three peaks with apparently
higher peak height show the same retention time and similar
peak height each other on both chromatograms.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 214 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (3 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Solution A — A mixture of water, ammoni-
um sulfate buffer solution and acetonitrile (7:2:1). Solution
B — A mixture of water, acetonitrile and ammonium sulfate
buffer solution (2:2:1).
Change the mixing ratio of the solutions A and B linearly
from 9:1 to 3:7 in 60 minutes after sample injection, further
change to 0:10 linearly in 5 minutes, and then flow the solu-
tion B only for 5 minutes.
Flow rate: 1.0 mL per minute.
System suitability —
System performance: When the procedure is run with 50
fiL of the standard solution under the above operating condi-
tions, the symmetry factor of the two larger peaks which ap-
pear next to the first peak just after the solvent peak are not
more than 1.5, and the resolution between these peaks is not
less than 3.4.
Purity (1) Related substances — Perform this procedure
rapidly. Dissolve 7.5 mg of Insulin Human (Genetical
Recombination) in 2 mL of 0.01 mol/L hydrochloric acid
TS, and use this solution as the sample solution. Perform the
test with 20 iiL of the sample solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak area of human insulin, A u
the peak area of the desamide substance at the relative reten-
tion time of 1.3 to the human insulin, A D , and the total area
of the peaks other than the solvent peak, A T : the amounts of
the desamide substance and related substances other than the
desamide substance are each not more than 2.0%. Previous-
ly, perform the test with 0.01 mol/L hydrochloric acid TS in
the same manner to confirm the solvent peak.
Amount (%) of the desamide substance = (A D /A T ) x 100
Amount (%) of related substances other than the
desamide substance = [{A T - (A { + A D )}/A T ] x 100
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 214 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
JPXV
Official Monographs / Insulin Human (Genetical Recombination)
761
Mobile phase: Solution A — A mixture of phosphoric acid-
sodium sulfate buffer solution, pH 2.3 and acetonitrile for
liquid chromatography (41:9). Solution B — A mixture of
phosphoric acid-sodium sulfate buffer solution, pH 2.3 and
acetonitrile for liquid chromatography (1:1).
Flow a mixture of the solution A and the solution B (78:22)
for 36 minutes before and after the sample injection, then
change the mixing ratio to 33:67 linearly in 25 minutes, and
maintain this ratio for 6 minutes. Then flow the first mixture
(78:22) for the next 15 minutes. Adjust the mixing ratio of the
first mixture so that the retention time of human insulin is
about 25 minutes.
Flow rate: 1.0 mL per minute.
Time span of measurement: For about 75 minutes after the
sample is injected.
System suitability —
Test for required detection: Confirm that the peak height
of the desamide substance obtained from 20 liL of human in-
sulin desamide substance-containing TS is between 30% and
70% of the full scale.
System performance: When the procedure is run with 20
/uL of human insulin desamide substance-containing TS un-
der the above operating conditions, human insulin and hu-
man insulin desamide substance are eluted in this order with
the resolution between these peaks being not less than 2.0,
and the symmetry factor of the peak of human insulin is not
more than 1.8.
(2) High-molecular proteins — Dissolve 4 mg of Insulin
Human (Genetical Recombination) in 1 mL of 0.01 mol/L
hydrochloric acid TS. Perform the test with 100 iuL of this so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate each peak
area: the total of areas of the peaks having smaller retention
time than human insulin is not more than 1 .0% of the total
area of all peaks.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 276 nm).
Column: A stainless steel column 7.5 mm in inside di-
ameter and 30 cm in length, packed with hydrophilic silica gel
for liquid chromatography.
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of a solution of L-arginine (1 in
1000), acetonitrile and acetic acid (100) (13:4:3).
Flow rate: Adjust the flow rate so that the retention time of
human insulin is about 20 minutes.
Time span of measurement: Until the peak of human insu-
lin monomer has appeared.
System suitability —
Test for required detection: Confirm that the peak height
of the dimer obtained from 100 //L of human insulin dimer
containing TS is between 10% and 50% of the full scale.
System performance: When the procedure is run with 100
[iL of human insulin dimer containing TS under the above
operating conditions, polymer, dimer and monomer are elut-
ed in this order, and the ratio, HJH 2 , of the peak height of
the dimer Hi to the height of the bottom between the peaks of
the dimer and the monomer H 2 is not less than 2.0.
(3) Product related impurities — Within the limits speci-
fied in each application dossier.
(4) Process related impurities — Within the limits specified
in each application dossier.
Zinc content Weigh accurately about 50 mg of Insulin Hu-
man (Genetical Recombination), and dissolve in 0.01 mol/L
hydrochloric acid TS to make exactly 25 mL. If necessary, di-
lute with 0.01 mol/L hydrochloric acid TS to make a solution
so that each mL contains between 0.4 n% and 1.6 [ig of zinc
(Zn: 65.41), and use this solution as the sample solution.
Separately, take exactly a suitable amount of Standard Zinc
Solution for Atomic Absorption Spectrophotometry, dilute
with 0.01 mol/L hydrochloric acid TS to make solutions con-
taining 0.40 ^g, 0.80 //g, 1.20 ii% and 1.60 //g of zinc (Zn:
65.41) in each mL, respectively, and use these solutions as the
standard solutions. Perform the test with the sample solution
and standard solutions as directed under Atomic Absorption
Spectrophotometry <2.23>, and determine the amount of zinc
(Zn: 65.41) in the sample solution by using a calibration
curve obtained from the absorbances of the standard solu-
tions: not more than 1.0%, calculated on the dried basis.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Zinc hollow cathode lamp
Wavelength: 213.9 nm
Loss on drying <2.41> Not more than 10.0% (0.2 g, 105°C,
24 hours).
Bacterial endotoxins <4.01> Less than lOEU/mg.
Assay Perform this procedure quickly. Weigh accurately
about 7.5 mg of Insulin Human (Genetical Recombination),
dissolve in 0.01 mol/L hydrochloric acid TS to make exactly
5 mL, and use this solution as the sample solution. Separate-
ly, weigh accurately a suitable amount of Human Insulin
Reference Standard, dissolve exactly in 0.01 mol/L
hydrochloric acid TS to make a solution so that each mL con-
tains about 40 Insulin Units, and use this solution as the stan-
dard solution. Perform the test with exactly 20 liL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas of human insulin, A Ti
and A SI , and the peak areas of the desamide substance at the
relative retention time of 1.3 to the human insulin, A TD and
A SD , respectively, of these solutions.
Amount (Insulin Unit/mg) of human insulin
(C257H383N 65 077S 6 )
= {(W s x F)/D] x {(A Tl + A TD )/(A sl + ^sd)1
x (5/W-j)
F: Label unit (Insulin Unit/mg) of Human Insulin Refer-
ence Standard.
D: Volume (mL) of 0.01 mol/L hydrochloric acid TS used
to dissolve the reference standard.
W T : Amount (mg) of the sample calculated on the dried
basis.
Ws' Amount (mg) of Human Insulin Reference Standard.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 214 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of phosphoric acid-sodium sul-
762 Insulin Injection / Official Monographs
JP XV
fate buffer solution, pH 2.3 and acetonitrile for liquid chro-
matography (3:1). Adjust the mixing ratio of the component
of the mobile phase so that the retention time of human insu-
lin is between 10 minutes and 17 minutes.
Flow rate: 1.0 mL per minute.
System suitability —
System performance: When the procedure is run with 20
/uL of human insulin desamide substance-containing TS un-
der the above operating conditions, human insulin and hu-
man insulin desamide substance are eluted in this order with
the resolution between these peaks being not less than 2.0,
and the symmetry factor of the peak of human insulin is not
more than 1.8.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of human insulin is not more than 1.6%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and at -20°C or below.
Insulin Injection
Insulin Injection is an aqueous solution for injec-
tion.
It contains not less than 95% and not more than 105
% of the labeled Insulin Units.
Method of preparation Suspend Insulin in Water for Injec-
tion, dissolve by adding Hydrochloric Acid, and prepare as
directed under Injections. It contains 0.10 to 0.25 g of Phenol
or Cresol and 1.4 to 1.8 g of Concentrated Glycerin for each
100 mL of Insulin Injection. It should not contain sodium
chloride.
Description Insulin Injection is a clear, colorless or pale yel-
low liquid.
Identification Adjust Insulin Injection to pH between 5.1
and 5.3 with a solution of sodium hydroxide (1 in 100): a
precipitate is produced. Adjust the solution to a pH between
2.5 and 3.5 with dilute hydrochloric acid: the precipitate dis-
solves.
pH <2.54> 2.5-3.5
Residue on ignition <2.44> Measure exactly a volume of In-
sulin Injection, equivalent to 500 to 1000 Units according to
the labeled Units, in a tared platinum dish, and evaporate
slowly by heating on a water bath to dryness. Add 2 drops of
nitric acid to the residue, and heat at first very gently, then
strongly to incinerate. Place in a muffle furnace, and heat at
600°C for 15 minutes, cool in a desiccator (silica gel), and
weigh: the mass of the residue is not more than 1 mg for each
labeled 1000 Units.
Extractable volume <6.05> It meets the requirement.
Nitrogen content Perform the test as directed under Nitro-
gen Determination <1.08>: not less than 0.50 mg and not
more than 0.64 mg of nitrogen (N: 14.01) is found for each
labeled 100 Units.
Assay Proceed with Insulin Injection as directed in the As-
say under Insulin with alterations in (v) Sample solution and
(ix) Calculation as follows.
(v) Sample solution: According to the labeled Units, di-
lute two portions of Insulin Injection to make two sample so-
lutions with the diluent for insulin, one to contain exactly 2.0
Units in each mL which is designated as the high-dose sample
solution T H , and the other to contain exactly 1.0 Unit in each
mL which is designated as the low-dose sample solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, replacing the equation as follows.
Units in each mL of Insulin Injection
= antilog M x (Units in each mL of S H ) X (b/a)
a: Volume (mL) of the sample.
b: Total volume (mL) of the high-dose sample solution pre-
pared by diluting the volume of the sample with diluent
for insulin.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Isophane Insulin Injection
(Aqueous Suspension)
Isophane Insulin Injection (Aqueous Suspension) is
an aqueous suspension for injection.
It contains not less than 90% and not more than 110
% of the labeled Insulin Units, and not less than 0.01
mg and not more than 0.04 mg of zinc (Zn: 65.41) for
each labeled 100 Units.
When Sodium Chloride is used in the preparation of
Isophane Insulin Injection (Aqueous Suspension), this
should be stated on the label.
Method of preparation Prepare as directed under Injec-
tions, with Insulin and Protamine Sulfate. To each 100 mL
of Isophane Insulin Injection (Aqueous Suspension) add
either 0.38 to 0.63 g of Dibasic Sodium Phosphate, 1.4 to 1.8
g of Concentrated Glycerin, 0.15 to 0.17 g of Cresol, and
0.06 to 0.07 g of Phenol, or 0.38 to 0.63 g of Dibasic Sodium
Phosphate, 0.42 to 0.45 g of Sodium Chloride, 0.7 to 0.9 g of
Concentrated Glycerin, and 0.18 to 0.22 g of Cresol.
Description Isophane Insulin Injection (Aqueous Suspen-
sion) is a white aqueous suspension. When allowed to stand,
it separates into a white precipitate and colorless supernatant
liquid, and the precipitate returns easily to the suspension
state on gentle shaking.
When examined microscopically, the precipitate mostly
consists of fine, oblong crystals of 5 to 30 /xm in major axis,
and does not contain amorphous substances or large ag-
gregates.
Identification Proceed as directed in the Identification un-
der Insulin Zinc Protamine Injection (Aqueous Suspension).
pH <2.54> 1.0-1 A
Purity (1) Protein — Perform the test as directed under
Nitrogen Determination <1.08>: not exceeding 0.85 mg of
JPXV
Official Monographs / Insulin Zinc Injection (Aqueous Suspension)
763
nitrogen (N: 14.01) is found for each labeled 100 Units.
(2) Isophane ratio — (i) Buffer solution A: Dissolve 2.0 g
of anhydrous disodium hydrogenphosphate, 16 g of glycerin,
1.6 g of m-cresol, and 0.65 g of phenol in water to make ex-
actly 200 mL.
(ii) Buffer solution B: Dissolve 2.0 g of anhydrous disodi-
um hydrogenphosphate, 4.35 g of sodium chloride, 8.0 g of
glycerin, and 2.0 g of m-cresol in water to make exactly 200
mL.
(iii) Insulin solution: Weigh accurately 1000 Units of In-
sulin Reference Standard, dissolve in 1.5 mL of diluted
hydrochloric acid (1 in 360), and add 5.0 mL of buffer solu-
tion A and water to make 20 mL. Adjust the pH to 7.2 with
dilute hydrochloric acid or sodium hydroxide TS. The solu-
tion is clear. Dilute with water to make exactly 25 mL. The
solution is clear, and the pH is between 7.1 and 7.4. When it
is stated on the label that Sodium Chloride is used in the
preparation, use 5.0 mL of buffer solution B instead of buffer
solution A in the above procedure.
(iv) Protamine solution: Weigh accurately 50 mg of Pro-
tamine Sulfate Reference Standard, and dissolve in 2 mL of
buffer solution A and water to make 8 mL. Adjust the pH to
7.2 with dilute hydrochloric acid or sodium hydroxide TS,
and dilute with water to exactly 10 mL. The solution is clear,
and the pH is between 7.1 and 7.4. When it is stated on the
label that Sodium Chloride is used in the preparation, use 2
mL of buffer solution B instead of buffer solution A in the
above procedure.
(v) Procedure: When Isophane Insulin Injection (Aque-
ous Suspension) contains 40 Units per ml, centrifuge a por-
tion of the suspension, measure exactly two 10-mL portions
of the supernatant liquid in two tubes A and B, respectively,
add exactly 1 mL of the insulin solution to tube A, and 1 mL
of the protamine solution to tube B, mix the contents of each
tube, allow to stand for 10 minutes, and determine the tur-
bidity of each mixture by using a photometer or a nephelome-
ter: the turbidity of the mixture in tube B is not greater than
that in tube A. When Isophane Insulin Injection (Aqueous
Suspension) contains 80 Units per ml, measure exactly 5 mL
of the supernatant liquid, and proceed in the same manner.
Extractable volume <6.05> It meets the requirement.
Assay (1) Insulin — To Isophane Insulin Injection (Aque-
ous Suspension) add diluted hydrochloric acid (1 in 1000) to
adjust pH to about 2.5, and proceed with the clear solution as
directed in the Assay under Insulin, with alterations in (v)
Sample solution and (ix) Calculation as follows.
(v) Sample solution: According to the labeled Units, di-
lute Insulin Injection (Aqueous Suspension) to make two
sample solutions with the diluent for insulin, one to contain
exactly 2.0 Units in each mL which is designated as the high-
dose sample solution T H , and the other to contain exactly 1.0
Unit in each mL which is designated as the low-dose sample
solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, using the following equation,
Units in each mL of Isophane Insulin Injection (Aqueous
Suspension)
= antilog M x (Units in each mL of S H ) x (b/a)
a: Volume (mL) of the sample,
instead of the following equation,
Units in each mg of Insulin
= antilog M x (Units in each mL of S H ) x (b/a)
a: Mass (mg) of the sample.
(2) Zinc — Pipet a volume of Isophane Insulin Injection
(Aqueous Suspension), equivalent to about 400 Units accord-
ing to the labeled Units, add 1 mL of 0.1 mol/L hydrochloric
acid TS and water to make exactly 100 mL, dilute, if necessa-
ry, with water to contain 0.6 to 1.0 fig of zinc (Zn: 65.41) per
mL, and use this solution as the sample solution. Separately,
pipet a volume of Standard Zinc Solution for Atomic Ab-
sorption Spectrophotometry, dilute with water to contain 0.4
to 1.2 fig of zinc (Zn: 65.41) per mL, and use this solution as
the standard solution. Perform the test with the sample solu-
tion and the standard solution according to Atomic Absorp-
tion Spectrophotometry <2.23> under the following condi-
tions, and determine the amount of zinc in the sample solu-
tion using the analytical curve obtained from the absorbance
of the standard solution.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow - cathode lamp
Wavelength: 213.9 nm
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Insulin Zinc Injection
(Aqueous Suspension)
Insulin Zinc Injection (Aqueous Suspension) is an
aqueous suspension for injection.
It contains not less than 90% and not more than 110
% of the labeled Insulin Units, and not less than 0.12
mg and not more than 0.30 mg of zinc (Zn: 65.41) for
each labeled 100 Units.
Method of preparation Prepare as directed under Injec-
tions, with Insulin and Zinc Chloride. It contains 0.15 to 0.17
g of Sodium Acetate Hydrate, 0.65 to 0.75 g of Sodium Chlo-
ride and 0.09 to 0.11 g of Methyl Parahydroxybenzoate for
each 100 mL of Insulin Zinc Injection (Aqueous Suspension).
Description Insulin Zinc Injection (Aqueous Suspension) is
a white suspension. When allowed to stand, it separates into
a white precipitate and a colorless supernatant liquid, and it
readily becomes a suspension again on gentle shaking.
When it is examined microscopically, the majority of the
particles in the suspension are crystals, the dimension of
which is 10 to 40 //m. The rest is amorphous and does not ex-
ceed 2 nm. in dimension.
Identification Adjust the pH of Amorphous Insulin Zinc
Injection (Aqueous Suspension) to between 2.5 and 3.5 with
dilute hydrochloric acid: the particles dissolve, and the solu-
tion is clear and colorless.
pH <2.54> 7.1-7.5
Purity Dissolved insulin — Perform the following test with a
764
Amorphous Insulin Zinc Injection (Aqueous Suspension) / Official Monographs
JP XV
clear liquid obtained by centrifuging Insulin Zinc Injection
(Aqueous Suspension): not more than 2.5% of the labeled
units is found.
Use the clear liquid of Insulin Zinc Injection (Aqueous
Suspension) as the sample solution. Prepare the standard so-
lution having a concentration of 2.5% of the labeled units of
Insulin Zinc Injection (Aqueous Suspension by proceeding as
directed in the Assay (iv) under Insulin. Divide the healthy
rabbits weighing not less than 1.8 kg, fasted for not less than
14 hours before injection, into 2 equal groups of not less than
3. Inject subcutaneously an amount of the standard solution
or the sample solution equivalent to 0.3 units per kg of body
mass to the animals of each group. Collect blood before and
1 hour and 2.5 hours after injection, then proceed as directed
in the Assay (viii) under Insulin, and calculate the ratio of the
average blood sugar level of 1 hour and 2.5 hours after to
that of before injection of each animal: the mean value for
the group injected the sample solution is not less than that for
the group injected the standard solution.
Extractable volume <6.05> It meets the requirement.
Nitrogen content Perform the test as directed under Nitro-
gen Determination <1.08>: the amount of nitrogen (N: 14.01)
is not less than 0.50 mg and not more than 0.64 mg for each
labeled 100 Unites.
Assay (1) Insulin — Proceed as directed in the Assay under
Insulin with the clear liquid obtained from Insulin Zinc Injec-
tion (Aqueous Suspension) by adjusting the pH to about 2.5
with diluted hydrochloric acid (1 in 1000), with alterations in
(v) Sample solution and (ix) Calculation as follows.
(v) Sample solution: According to the labeled Units, di-
lute two portions of Insulin Zinc Injection (Aqueous Suspen-
sion) to make two sample solutions with the diluent for insu-
lin, one to contain exactly 2.0 Units in each mL which is
designated as the high-dose sample solution T H , and the other
to contain exactly 1.0 Unit in each mL which is designated as
the low-dose sample solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, using the following equation,
Units in each mL of Insulin Zinc Injection (Aqueous Suspen-
sion)
= antilog M x (Units in each mL of S H ) x (b/a)
a: Volume (mL) of the sample,
instead of the following equation,
Units in each mg of Insulin
= antilog M x (Units in each mL of S H ) x (b/a)
a: Mass (mg) of the sample.
(2) Zinc — Measure exactly a volume of Insulin Zinc In-
jection (Aqueous Suspension), equivalent to about 200 Units
according to the labeled units, add 1 mL of 0.1 mol/L
hydrochloric acid TS and sufficient water to make exactly 200
mL, then dilute with water to contain 0.6 to 1.0 fig of zinc
(Zn: 65.41) per mL, and use this solution as the sample solu-
tion. Separately, pipet a volume of Standard Zinc Solution
for atomic absorption spectrophotometry, dilute with water
to contain 0.4 to 1.2 jug of zinc (Zn: 65.41) per mL, and use
this solution as the standard solution. Perform the test with
the sample solution and standard solution as directed under
Atomic Absorption Spectrophotometry <2.23> according to
the following conditions, and determine the amount of zinc
in the sample solution using the calibration curve obtained
from the absorbance of the standard solution.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
(3) Crystalline insulin — Measure exactly a volume of
Amorphous Insulin Zinc Injection (Aqueous Suspension),
equivalent to about 600 Units according to the labeled units,
centrifuge, discard the supernatant liquid, suspend the
residue in 5 mL of water, add 10 mL of sodium acetate-ace-
tone TS, shake for 3 minutes, and centrifuge. Discard the su-
pernatant liquid, and repeat the above treatment on the
residue. Wash down the residue into a Kjeldahl flask with 15
mL of sulfuric acid, and perform the test as directed under
the Nitrogen Determination <1.08>: the amount of nitrogen
(N: 14.01) is not less than 55% and not more than 70% of the
total nitrogen content. Calculate the total nitrogen content
for insulin Units of the sample taken from the values of nitro-
gen obtained in the Nitrogen content.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Amorphous Insulin Zinc Injection
(Aqueous Suspension)
Amorphous Insulin Zinc Injection (Aqueous Sus-
pension) is an aqueous suspension for injection.
It contains not less than 90% and not more than 110
% of the labeled Insulin Units, and not less than 0.12
mg and not more than 0.30 mg of zinc (Zn: 65.41) for
each labeled 100 Units.
Method of preparation Prepare as directed under Injec-
tions, with Insulin and Zinc Chloride. Each 100 mL of
Amorphous Insulin Zinc Injection (Aqueous Suspension)
contains 0.15 to 0.17 g of Sodium Acetate Hydrate, 0.65 to
0.75 g of Sodium Chloride, and 0.09 to 0.11 g of Methyl
Parahydroxybenzoate.
Description Amorphous Insulin Zinc Injection (Aqueous
Suspension) is a white suspension. When allowed to stand, it
separates into a white precipitate and a colorless supernatant
liquid, and it readily becomes a suspension again on gentle
shaking.
When examined microscopically, most of the particles in
the suspension are amorphous and have no uniform shape,
and most of the dimension does not exceed 2/(m.
Identification Adjust the pH of Amorphous Insulin Zinc
Injection (Aqueous Suspension) to between 2.5 and 3.5 with
dilute hydrochloric acid: the particles dissolve, and the solu-
tion is clear and colorless.
pH <2.54> 7.1-7.5
Purity Dissolved insulin — Perform the following test with a
clear liquid obtained by centrifuging Amorphous Insulin
JPXV
Official Monographs / Crystalline Insulin Zinc Injection (Aqueous Suspension)
765
Zinc Injection (Aqueous Suspension): not more than 2.5% of
the labeled units is found.
Use the clear liquid of Amorphous Insulin Zinc Injection
(Aqueous Suspension) as the sample solution. Prepare the
standard solution having a concentration of 2.5% of the la-
beled units of Amorphous Insulin Zinc Injection (Aqueous
Suspension) by proceeding as directed in the Assay (iv) under
Insulin. Divide healthy rabbits weighing more than 1.8 kg,
fasted for not less than 14 hours before injection, into 2 equal
groups of not less than 3. Inject subcutaneously an amount
of the standard solution or the sample solution equivalent to
0.3 units per kg of body mass to the animals of each group.
Collect blood before and 1 hour and 2.5 hours after injec-
tion, then proceed as directed in the Assay (viii) under Insu-
lin, and calculate the ratio of the average blood sugar level of
1 hour and 2.5 hours after to that of before injection of each
animal: the mean value for the group injected the sample so-
lution is not less than that for the group injected the standard
solution.
Extractable volume <6.05> It meets the requirement.
Nitrogen content Perform the test as directed under Nitro-
gen Determination <1.08>: the amount of nitrogen (N: 14.01)
is not less than 0.50 mg and not more than 0.64 mg for each
labeled 100 Units.
Assay (1) Insulin — Proceed as directed in the Assay under
Insulin with the clear liquid obtained from Amorphous Insu-
lin Zinc Injection (Aqueous Suspension) by adjusting the pH
to about 2.5 with diluted hydrochloric acid (1 in 1000), with
alteration in (v) Sample solution and (ix) Calculation as fol-
lows.
(v) Sample solution: According to the labeled Units, di-
lute two portions of Amorphous Insulin Zinc Injection
(Aqueous Suspension) to make two sample solutions with the
diluent for insulin, one to contain exactly 2.0 Units in each
mL which is designated as the high-dose sample solution T H ,
and the other to contain exactly 1 .0 Unit in each mL which is
designated as the low-dose sample solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, using the following equation,
Units in each mL of Amorphous Insulin Zinc Injection
(Aqueous Suspension)
= antilog M x (Units in each mL of S H ) x (b/a)
a: Volume (mL) of the sample,
instead of the following equation,
Units in each mg of Insulin
= antilog M x (Units in each mL of S H ) x (b/a)
a: Mass (mg) of the sample.
(2) Zinc — Measure exactly a volume of Amorphous Insu-
lin Zinc Injection (Aqueous Suspension), equivalent to about
200 Units according to the labeled units, add 1 mL of 0.1
mol/L hydrochloric acid TS and sufficient water to make ex-
actly 200 mL, then dilute with water to contain 0.6 to 1.0 /ug
of zinc (Zn: 65.41) per mL, and use this solution as the sam-
ple solution. Separately, pipet a volume of Standard Zinc So-
lution for atomic absorption spectrophotometry, dilute with
water to contain 0.4 to 1.2//g of zinc (Zn: 65.41) per mL, and
use this solution as the standard solution. Perform the test
with the sample solution and the standard solution as direct-
ed under Atomic Absorption Spectrophotometry <2.23> ac-
cording to the following conditions, and determine the
amount of zinc in the sample solution using the calibration
curve obtained from the absorbance of the standard solution.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
(3) Crystalline insulin — Measure exactly a volume of
Amorphous Insulin Zinc Injection (Aqueous Suspension),
equivalent to about 1000 Units according to the labeled units,
centrifuge, discard the supernatant liquid, suspend the
residue in 5 mL of water, add 10 mL of sodium acetate-ace-
tone TS, shake for 3 minutes, and centrifuge. Discard the su-
pernatant liquid, and repeat the above treatment on the
residue. Wash down the residue into a Kjeldahl flask with 15
mL of sulfuric acid, and perform the test as directed under
the Nitrogen Determination <1.08>: the amount of nitrogen
(N: 14.01) is not more than 10% of the total nitrogen con-
tent. Calculate the total nitrogen content for insulin Units of
the sample taken from the values of nitrogen obtained in the
Nitrogen content.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Crystalline Insulin Zinc Injection
(Aqueous Suspension)
Crystalline Insulin Zinc Injection (Aqueous Suspen-
sion) is an aqueous suspension for injection.
It contains not less than 90% and not more than 110
% of the labeled Insulin Units, and not less than 0.12
mg and not more than 0.30 mg of zinc (Zn: 65.41) for
each labeled 100 Units.
Method of preparation Prepare as directed under Injec-
tions, with Insulin and Zinc Chloride. Each 100 mL of Crys-
talline Insulin Zinc Injection (Aqueous Suspension) contains
0.15 to 0.17 g of Sodium Acetate Hydrate, 0.65 to 0.75 g of
Sodium Chloride, and 0.09 to 0.11 g of Methyl Parahydrox-
ybenzoate.
Description Crystalline Insulin Zinc Injection (Aqueous
Suspension) is a white suspension. When allowed to stand, it
separates into a white precipitate and a colorless supernatant
liquid, and it readily becomes a suspension again on gentle
shaking.
When it is examined microscopically, most part of the par-
ticles in the suspension are crystals, the dimension of which is
mostly 10 to 40 //m.
Identification Adjust the pH of Crystalline Insulin Zinc In-
jection (Aqueous Suspension) to between 2.5 and 3.5 with di-
lute hydrochloric acid: the particles dissolve, and the solution
is clear and colorless.
pH <2.54> 7.1-7.5
Purity Dissolved insulin — Perform the following test with a
766
Insulin Zinc Protamine Injection (Aqueous Suspension) / Official Monographs
JP XV
clear liquid obtained by centrifuging Crystalline Insulin Zinc
Injection (Aqueous Suspension): not more than 2.5% of the
labeled units is found.
Use the clear liquid of Crystalline Insulin Zinc Injection
(Aqueous Suspension) as the sample solution. Prepare the
standard solution having a concentration of 2.5% of the la-
beled units of Crystalline Insulin Zinc Injection (Aqueous
Suspension) by proceeding as directed in the Assay (iv) under
Insulin. Divide healthy rabbits weighing more than 1.8 kg,
fasted for not less than 14 hours before injection, into 2 equal
groups of not less than 3. Inject subcutaneously an amount
of the standard solution or the sample solution equivalent to
0.3 units per kg of body mass to the animals of each group.
Collect blood before and 1 hour and 2.5 hours after injec-
tion, then proceed as directed in the Assay (viii) under Insu-
lin, and calculate the ratio of the average blood sugar level of
1 hour and 2.5 hours after to that of before injection of each
animal: the mean value for the group injected the sample so-
lution is not less than that for the group injected the standard
solution.
Extractable volume <6.05> It meets the requirement.
Nitrogen content Perform the test as directed under Nitro-
gen Determination <1.0S>: not less than 0.50 mg and not
more than 0.64 mg of nitrogen (N: 14.01) is found for each
labeled 100 Units.
Assay (1) Insulin — Proceed as directed in the Assay under
Insulin with the clear liquid obtained from Crystalline Insulin
Zinc Injection (Aqueous Suspension) by adjusting the pH to
about 2.5 with diluted hydrochloric acid (1 in 1000), with al-
terations in (v) Sample solution and (ix) Calculation as fol-
lows.
(v) Sample solution: According to the labeled Units, di-
lute two portions of Crystalline Insulin Zinc Injection (Aque-
ous Suspension) to make two sample solutions with the dil-
uent for insulin, one to contain exactly 2.0 Units in each mL
which is designated as the high-dose sample solution T H , and
the other to contain exactly 1.0 Unit in each mL which is
designated as the low-dose sample solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, using the following equation,
Units in each mL of Crystalline Insulin Zinc Injection (Aque-
ous Suspension)
= antilog M x (Units in each mL of S H ) x (b/a)
a: Volume (mL) of the sample,
instead of the following equation,
Units in each mg of Insulin
= antilog M x (Units in each mL of S H ) x (b/a)
a: Mass (mg) of the sample.
(2) Zinc — Measure exactly a volume of Crystalline Insu-
lin Zinc Injection (Aqueous Suspension), equivalent to about
200 Units according to the labeled units, add 1 mL of 0.1
mol/L hydrochloric acid TS and sufficient water to make ex-
actly 200 mL, then dilute with water to contain 0.6 to 1.0 /ug
of zinc (Zn: 65.41) per mL, and use this solution as the sam-
ple solution. Separately, pipet a volume of Standard Zinc So-
lution for atomic absorption spectrophotometry, dilute with
water to contain 0.4 to 1.2//g of zinc (Zn: 65.41) per mL, and
use this solution as the standard solution. Perform the test
with the sample solution and the standard solution as direct-
ed under Atomic Absorption Spectrophotometry <2.23> ac-
cording to the following conditions, and determine the
amount of zinc in the sample solution using the calibration
curve obtained from the absorbance of the standard solution.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
(3) Crystalline insulin — Measure accurately a volume of
Crystalline Insulin Zinc Injection (Aqueous Suspension),
equivalent to about 400 Units according to the labeled Units,
centrifuge, discard the supernatant liquid, suspend the
residue in 5 mL of water, add 10 mL of sodium acetate-ace-
tone TS, shake for 3 minutes, and centrifuge. Discard the su-
pernatant liquid, and repeat the above treatment on the
residue. Wash down the residue into a Kjeldahl flask with 15
mL of sulfuric acid, and perform the test as directed under
the Nitrogen Determination <1.08>: the amount of nitrogen
(N: 14.01) is not less than 85% of the total nitrogen content.
Calculate the total nitrogen content for insulin Units of a
sample from the values of nitrogen obtained in the Nitrogen
content.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Insulin Zinc Protamine Injection
(Aqueous Suspension)
Insulin Zinc Protamine Injection (Aqueous Suspen-
sion) is an aqueous suspension for injection.
It contains not less than 90% and not more than 110
% of the labeled Insulin Units, and not less than 0.20
mg and not more than 0.30 mg of zinc (Zn: 65.41) for
each labeled 100 Units.
Method of preparation Prepare as directed under Injec-
tions, with Insulin, Protamine Sulfate and Zinc Chloride. It
contains 0.38 to 0.63 g of Dibasic Sodium Phosphate Hy-
drate, 1.4 to 1.8 g of Concentrated Glycerin, and 0.18 to 0.22
g of Cresol or 0.22 to 0.28 g of Phenol for each 100 mL of In-
sulin Zinc Protamine Injection (Aqueous Suspension).
Description Insulin Zinc Protamine Injection (Aqueous
Suspension) is a white suspension. When allowed to stand, it
separates into a white precipitate and a colorless, supernatant
liquid, and it readily becomes suspension again on gentle
shaking.
When it is examined microscopically, no large particles are
seen.
Identification Adjust the pH of Insulin Zinc Protamine In-
jection (Aqueous Suspension) to between 2.5 and 3.5 with di-
lute hydrochloric acid: the particles dissolve, and the solution
is clear and colorless.
pH <2.54> 7.0-7.4
Purity (1) Protein — Perform the test as directed under
JPXV
Official Monographs / Iodamide 767
Nitrogen Determination <1.08>: not exceeding 1.25 mg of
nitrogen (N: 14.01) is found for each labeled 100 Units.
(2) Dissolved insulin — Perform the following test with
the clear liquid obtained by centrifuging Insulin Zinc Prota-
mine Injection (Aqueous Suspension): not more than 2.5%
of the labeled Units is found.
Use a clear liquid of Insulin Zinc Protamine Injection
(Aqueous Suspension) as the sample solution, and prepare
the standard solution by proceeding as directed in the Assay
(iv) under Insulin to adjust its concentration to 2.5% of the
labeled units of Insulin Zinc Protamine Injection (Aqueous
Suspension). Divide the healthy rabbits weighing not less
than 1.8 kg, fasted for not less than 14 hours before injec-
tion, into 2 equal groups of not less than 3. Inject subcutane-
ously an amount of the standard solution or the sample solu-
tion equivalent to 0.3 units per kg of body mass. Collect
blood before and 1 hour and 2.5 hours after injection, pro-
ceed as directed in the Assay (viii) under Insulin, and calcu-
late the ratios of the average blood sugar content in each rab-
bit measured 1 hour and 2.5 hours after injection to the con-
tent before injection: the mean value for the group injected
with the sample solution is not less than the mean value for
the group injected with the standard solution.
Extractable volume <6.0J> It meets the requirement.
Assay (1) Insulin — Proceed as directed in the Assay under
Insulin with the clear liquid obtained by adjusting the pH to
2.5 with diluted hydrochloric acid (1 in 1000), with altera-
tions in (v) Sample solution and (ix) Calculation as follows.
(v) Sample solution: According to the labeled Units, di-
lute Insulin Zinc Protamine Injection (Aqueous Suspension)
to make two sample solutions with the diluent for insulin,
one to contain exactly 2.0 Units in each mL which is designat-
ed as the high-dose sample solution T H , and the other to con-
tain exactly 1.0 Unit in each mL which is designated as the
low-dose sample solution T L .
(ix) Calculation: Proceed as directed in the Assay (ix)
Calculation under Insulin, using the following equation,
Units in each mL of Insulin Zinc Protamine Injection (Aque-
ous Suspension)
= antilog M x (Units in each mL of S H ) x (b/a)
a: Volume (mL) of the sample,
instead of the following equation,
Units in each mg of Insulin
= antilog M x (Units in each mL of S H ) x (b/a)
a: Mass (mg) of the sample.
(2) Zinc — Measure accurately a volume of Insulin Zinc
Protamine Injection (Aqueous Suspension), equivalent to
about 200 Units according to the labeled units, add 1 mL of
0.1 mol/L hydrochloric acid TS and sufficient water to make
exactly 200 mL, dilute with water to contain 0.6 to 1.0 /xg of
zinc (Zn: 65.41) in 1 mL, and use this solution as the sample
solution. Separately, pipet a volume of Standard Zinc Solu-
tion for Atomic Absorption Spectrophotometry, dilute with
water to contain 0.4 to 1.2 /xg of zinc (Zn: 65.41) per ml, and
use this solution as the standard solution. Perform the test
with the sample solution and standard solution according to
the Atomic Absorption Spectrophotometry <2.23> under the
following conditions, and determine the amount of zinc in
the sample solution using the analytical curve obtained from
the absorbance of the standard solution.
Gas: Combustible gas — Acetylene gas
Supporting gas — Air
Lamp: Zinc hollow-cathode lamp
Wavelength: 213.9 nm
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 24 months after preparation.
Iodamide
a-y'S K
H 3 C
O
A
i
Ct>;H
I
CH 3
Y
o
C 12 H„I 3 N 2 04: 627.94
3-Acetylamino-5-acetylaminomethyl-2,4,6-triiodobenzoic
acid [440-58-4]
Iodamide, calculated on the dried basis, contains not
less than 98.5% of C 12 H 11 I 3 N 2 4 .
Description Iodamide occurs as a white, crystalline powder.
It is odorless.
It is slightly soluble in water and in ethanol (95), and prac-
tically insoluble in diethyl ether.
It dissolves in sodium hydroxide TS and in sodium car-
bonate TS.
It gradually changes in color by light.
Identification (1) To 0.01 g of Iodamide add 5 mL of
hydrochloric acid, and heat in a water bath for 5 minutes: the
solution responds to the Qualitative Tests <1.09> for primary
aromatic amines.
(2) Heat 0.1 g of Iodamide over a flame: a purple gas
evolves.
(3) Determine the infrared absorption spectrum of Io-
damide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, dissolve 1 g of Iodamide in 100 mL of water by
heating, and concentrate the solution to about 30 mL by gen-
tle boiling. After cooling, collect the formed crystals by filtra-
tion, dry, and repeat the test on the dried crystals.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Iodamide in 10 mL of diluted sodium hydroxide TS (1 in 5):
the solution is clear and colorless.
(2) Primary aromatic amines — Dissolve 0.20 g of Io-
damide in 5 mL of water and 1 mL of sodium hydroxide TS,
add 4 mL of a solution of sodium nitrite (1 in 100) and 10 mL
of 1 mol/L hydrochloric acid TS, shake well, and allow to
stand for 2 minutes. To this solution add 5 mL of ammonium
amidosulfate TS, shake thoroughly, allow to stand for 1
minute, add 0.4 mL of a solution of 1-naphthol in ethanol
768
lodinated ( 131 I) Human Serum Albumin Injection / Official Monographs
JP XV
(95) (1 in 10), 15 mL of sodium hydroxide TS and water to
make exactly 50 mL, and determine the absorbance at 485
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using a solution, prepared in the same manner, as the
blank: the absorbance of the solution is not more than 0.12.
(3) Soluble halide — Dissolve 2.5 g of Iodamide in 20 mL
of water and 2.5 mL of ammonia TS, then add 20 mL of di-
lute nitric acid and water to make 100 mL. Allow to stand for
15 minutes with occasional shaking, and filter. Discard the
first 10 mL of the filtrate, transfer 25 mL of the subsequent
filtrate to a Nessler tube, and add ethanol (95) to make 50
mL. Use this solution as the test solution, and proceed as
directed under Chloride Limit Test <1.03>. Prepare the con-
trol solution with 0.10 mL of 0.01 mol/L hydrochloric acid
VS and 6 mL of dilute nitric acid, and dilute with water to 25
mL, then with ethanol (95) to 50 mL.
(4) Iodine — Dissolve 0.20 g of Iodamide in 2 mL of sodi-
um hydroxide TS, add 2.5 mL of 0.5 mol/L sulfuric acid TS,
allow to stand for 10 minutes with occasional shaking, then
add 5 mL of chloroform, shake vigorously and allow to
stand: the chloroform layer remains colorless.
(5) Heavy metals <1.07> — Proceed with 2.0 g of Iodamide
according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(6) Arsenic </.//> — Prepare the test solution with 1.0 g
of Iodamide according to Method 3, and perform the test
(not more than 2 ppm).
Loss on drying <2.41> Not more than 3.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Iodamide in a
saponification flask, dissolve in 40 mL of sodium hydroxide
TS, add 1 g of zinc powder, connect the flask with a reflux
condenser, boil for 30 minutes, cool, and filter. Wash the
flask and filter paper with 50 mL of water, and combine the
washings with the filtrate. Add 5 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L silver nitrate VS until the color
of the precipitate changes from yellow to green (indicator: 1
mL of tetrabromophenolphthalein ethyl ester TS).
Each mL of 0.1 mol/L silver nitrate VS
= 20.93 mg of C 12 H 11 I 3 N 2 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
lodinated ( 131 I) Human Serum
Albumin Injection
a^kAjJiJt Tju^s > ( 131 d &*l*
lodinated ( 131 I) Human Serum Albumin Injection is
an aqueous solution for injection containing normal
human serum albumin iodinated by iodine-131 ( 131 I).
It conforms to the requirements of Iodinated ( 131 I)
Human Serum Albumin Injection in the Minimum Re-
quirements for Radiopharmaceuticals.
Test for Extractable Volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Iodinated ( 131 I) Human Serum Albumin Injec-
tion is a clear, colorless or light yellow liquid.
Iodine
bom
I: 126.90
Iodine contains not less than 99.5% of I.
Description Iodine occurs as grayish black plates or heavy,
granular crystals, having a metallic luster and a characteristic
odor.
It is freely soluble in diethyl ether, soluble in ethanol (95),
sparingly soluble in chloroform, and very slightly soluble in
water.
It dissolves in potassium iodide TS.
Iodine sublimes at room temperature.
Identification (1) A solution of Iodine in ethanol (95) (1 in
50) shows a red-brown color.
(2) A solution of Iodine in chloroform (1 in 1000) shows
a red-purple to purple color.
(3) Add 0.5 mL of starch TS to 10 mL of a saturated so-
lution of Iodine: a dark blue color is produced. When the
mixture is boiled, the color disappears, and it reappears on
cooling.
Purity (1) Non-volatile residue — Sublime 2.0 g of Iodine
on a water bath, and dry the residue at 105°C for 1 hour: the
mass of the residue is not more than 1.0 mg.
(2) Chloride or bromide — Mix 1 .0 g of finely powdered
Iodine with 20 mL of water, and filter the mixture. To 10 mL
of the filtrate add dropwise diluted sulfurous acid solution (1
in 5) until the yellow color disappears. Add 1 mL of ammonia
TS, followed by 1 mL of silver nitrate TS in small portions,
and add water to make 20 mL. Shake well, filter, and after
discarding the first 2 mL of the filtrate, take 10 mL of the
subsequent filtrate. To the filtrate add 2.0 mL of nitric acid
and water to make 20 mL: the solution so obtained has no
more turbidity than the following control solution.
Control solution: To 0.20 mL of 0.01 mol/L hydrochloric
acid VS add 5 mL of water, 2.5 mL of ammonia TS, 1 mL of
silver nitrate TS, 2.0 mL of nitric acid and water to make 20
mL.
Assay Place 1 g of potassium iodide and 1 mL of water in a
glass-stoppered flask, weigh accurately, add about 0.3 g of
Iodine to the flask, and weigh accurately again. Dissolve the
iodine by gentle shaking, add 20 mL of water and 1 mL of di-
lute hydrochloric acid, and titrate <2.50> with 0.1 mol/L so-
dium thiosulfate VS (indicator: 1 mL of starch TS).
Each mL of 0.1 mol/L sodium thiosulfate VS
= 12.69 mg of I
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Dilute Iodine Tincture
769
Iodine Tincture
Dilute Iodine Tincture
Iodine Tincture contains not less than 5.7 w/v% and
not more than 6.3 w/v% of iodine (I: 126.90), and not
less than 3.8w/v% and not more than 4.2 w/v% of
potassium iodide (KI: 166.00).
Dilute Iodine Tincture contains not less than 2.8
w/v% and not more than 3.2w/v% of iodine (I:
126.90), and not less than 1.9 w/v% and not more than
2.1 w/v% of potassium iodide (KI: 166.00).
Method of preparation
Iodine
Potassium Iodide
70 vol% Ethanol
60 g
40 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Tinctures, with the above in-
gredients. It may be prepared with an appropriate quantity of
Ethanol or Ethanol for Disinfectant and Purified Water in
place of 70 vol% Ethanol.
Description Iodine Tincture is a dark red-brown liquid, and
has a characteristic odor.
Specific gravity cP° a : about 0.97
Identification (1) To a mixture of 1 mL of starch TS and
9 mL of water add 1 drop of Iodine Tincture: a dark blue-
purple color develops.
(2) Evaporate 3 mL of Iodine Tincture to dryness on a
water bath, and heat gently over a free flame: a white residue
is formed which responds to the Qualitative Tests <1.09> for
potassium salt and iodide.
Alcohol number <7.07> Not less than 6.6 (Method 2). Per-
form the pretreatment (ii) in the Method 1.
Assay (1) Iodine — Pipet 5 mL of Iodine Tincture, add
0.5 g of potassium iodide, 20 mL of water and 1 mL of dilute
hydrochloric acid, and titrate <2.50> with 0.1 mol/L sodium
thiosulfate VS (indicator: 2 mL of starch TS).
Each mL of 0.1 mol/L sodium thiosulfate VS
= 12.69 mg of I
(2) Potassium iodide — Pipet 5 mL of Iodine Tincture
into an iodine flask, add 20 mL of water, 50 mL of
hydrochloric acid and 5 mL of chloroform. Cool to room
temperature, and titrate <2.50> with 0.05 mol/L potassium
iodate VS until the red-purple color disappears from the chlo-
roform layer, with agitating the mixture vigorously and con-
tinuously. After the chloroform layer has been decolorized,
allow the mixture to stand for 5 minutes. If the color reap-
pears, the mixture should be titrated <2.50> further with 0.05
mol/L potassium iodate VS. Calculate the amount (mg) of
potassium iodide from the number of mL (a) of 0.05 mol/L
potassium iodate VS used as above and the number of mL (b)
of 0.1 mol/L sodium thiosulfate VS used in the titration un-
der the Assay (1).
Amount (mg) of potassium iodide (KI)
= 16.600 x {a-(b/2)}
Containers and storage Containers — Tight containers.
Method of preparation
Iodine
Potassium Iodide
70 vol% Ethanol
30 g
20 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients. It may be prepared with an appropriate
quantity of Ethanol or Ethanol for Disinfection and Purified
Water in place of 70 vol% Ethanol. It may also be prepared
by adding 70 vol% Ethanol to 500 mL of Iodine Tincture to
make 1000 mL.
Description Dilute Iodine Tincture is a dark red-brown liq-
uid, and has a characteristic odor.
Specific gravity df : about 0.93
Identification (1) To a mixture of 1 mL of starch TS and 9
mL of water add 1 drop of Dilute Iodine Tincture: a dark
blue-purple color develops.
(2) Evaporate 3 mL of Diluted Iodine Tincture to dryness
on a water bath, and heat gently over a free flame: a white
residue is formed which responds to the Qualitative Tests
<1.09> for potassium salt and iodide.
Alcohol number <7.07> Not less than 6.7 (Method 2). Per-
form the pretreatment (ii) in the Method 1 .
Assay (1) Iodine — Pipet exactly 10 mL of Dilute Iodine
Tincture, add 0.5 g of potassium iodide, 20 mL of water and
1 mL of dilute hydrochloric acid, and titrate <2.50> with 0.1
mol/L sodium thiosulfate VS (indicator: 2 mL of starch TS).
Each mL of 0.1 mol/L sodium thiosulfate VS
= 12.69 mg of I
(2) Potassium iodide — Pipet exactly 10 mL of Dilute Io-
dine Tincture into an iodine flask, add 20 mL of water, 50
mL of hydrochloric acid and 5 mL of chloroform. Cool to
room temperature, and titrate <2.50> with 0.05 mol/L potas-
sium iodate VS until the red-purple color in the chloroform
layer disappears while agitating vigorously and continuously.
After the chloroform layer has been decolorized, allow the
mixture to stand for 5 minutes. If the color reappears, the
mixture should be titrated <2.50> further with 0.05 mol/L
potassium iodate VS. Calculate the amount (mg) of potassi-
um iodide from the volume (amL) of 0.05 mol/L potassium
iodate VS consumed as above and the volume (b mL) of 0.1
mol/L sodium thiosulfate VS consumed in the titration under
Assay (1).
Amount (mg) of potassium iodide (KI)
= 16.600x la-(b/2)}
Containers and storage Containers — Tight containers.
770
Compound Iodine Glycerin / Official Monographs
JP XV
Compound Iodine Glycerin
«*a-H-yiJtu>
Compound Iodine Glycerin contains not less than
1.1 w/v% and not more than 1.3 w/v% of iodine (I:
126.90), not less than 2.2 w/v% and not more than 2.6
w/v% of potassium iodide (KI: 166.00), not less than
2.7 w/v% and not more than 3.3 w/v% of total iodine
(as I), and not less than 0.43 w/v% and not more than
0.53 w/v% of phenol (C 6 H 6 Q: 94.11).
Method of preparation
Iodine
Potassium Iodide
Glycerin
Mentha Water
Liquefied Phenol
Purified Water
12 g
24 g
900 mL
45 mL
5mL
a sufficient quantity
To make 1000 mL
Dissolve Potassium Iodide and Iodine in about 25 mL of
Purified Water. After adding Glycerin, add Mentha Water,
Liquefied Phenol and sufficient Purified Water to make 1000
mL, mixing thoroughly. It may be prepared with an ap-
propriate quantity of Concentrated Glycerin and Purified
Water in place of Glycerin.
Description Compound Iodine Glycerin is a red-brown, vis-
cous liquid. It has a characteristic odor.
Specific gravity c^„: about 1.23
Identification (1) The colored solution obtained in the As-
say (1) acquires a red color. Determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
510 nm and 514 nm (iodine).
(2) The colored solution obtained in the Assay (2) ac-
quires a red color. Determine the absorption spectrum of this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 510 nm and 514
nm (potassium iodide).
(3) The colored solution obtained in the Assay (4) has a
yellow color. Determine the absorption spectrum of this solu-
tion as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 401 nm and 405 nm
(phenol).
(4) Take 1 mL of Compound Iodine Glycerin in a glass-
stoppered test tube, add 10 mL of ethanol (95), and mix.
Then add 2 mL of sodium hydroxide TS, add 1 mL of a solu-
tion of copper (II) chloride dihydrate in ethanol (95) (1 in 10),
and shake: a blue color develops (glycerin).
Assay (1) Iodine — Measure the specific gravity <2.56> of
Compound Iodine Glycerin according to Method 2. Weigh
exactly about 7 mL of it, add ethanol (95) to make exactly
200 mL, and use this solution as the sample solution. On the
other hand, weigh accurately about 80 mg of iodine for assay
and about 0.17 g of potassium iodide for assay, previously d-
ried at 105 °C for 4 hours, dissolve in ethanol (95) to make ex-
actly 200 mL, and use this solution as the standard solution.
Pipet 3 mL each of the sample solution and the standard so-
lution into 50-mL separators, to each add exactly 10 mL of a
mixture of chloroform and hexane (2:1) and 15 mL of water
successively, and shake immediately and vigorously. Separate
the chloroform-hexane layers [use the water layers in (2)],
and filter through a pledget of cotton. Determine the absor-
bances of the filtrates, A T and A s , at 512 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, using a
mixture of chloroform and hexane (2:1) as the blank.
Amount (mg) of iodine (I) = W s x (A T /A S )
W s : Amount (mg) of iodine for assay
(2) Potassium iodide — Separate the water layers of the
sample solution and the standard solution obtained in (1),
pipet 10 mL of each of the water layers, and to each add 1
mL of diluted dilute hydrochloric acid (1 in 2), 1 mL of sodi-
um nitrite TS and exactly 10 mL of a mixture of chloroform
and hexane (2:1). Shake immediately and vigorously,
separate the chloroform-hexane layers, and filter through a
pledget of cotton. Determine the absorbances, A T and A s , of
both solutions at 512 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using a mixture of chloroform
and hexane (2:1) as the blank.
Amount (mg) of potassium iodide (KI)= W s x (A T /A S )
W s : Amount (mg) of potassium iodide for assay
(3) Total iodine — Measure the specific gravity <2.56> of
Compound Iodine Glycerin according to Method 2. Weigh
exactly about 5 mL of it, and add water to make exactly 50
mL. Pipet 5 mL of this solution into a 50-mL flask, and add
0.5 g of zinc powder and 5 mL of acetic acid (100). Shake un-
til the color of iodine disappears, and heat under a reflux con-
denser on a water bath for 30 minutes. Wash the condenser
with 10 mL of hot water, and filter through a glass filter (G3).
Wash the flask with two 10-mL portions of warm water, and
combine the filtrate and the washings. After cooling, add
water to make exactly 50 mL, and use this solution as the
sample solution. On the other hand, dissolve about 0.2 g of
potassium iodide for assay, previously dried at 105 °C for 4
hours and accurately weighed, in water to make exactly 50
mL. Pipet 5 mL of this solution, add 5 mL of acetic acid
(100) and water to make exactly 50 mL, and use this solution
as the standard solution. Pipet 4 mL each of the sample solu-
tion and standard solution into 30-mL separators, and to
each add 5 mL of water, 1 mL of diluted dilute hydrochloric
acid (1 in 2), 1 mL of sodium nitrite TS and 10 mL of a mix-
ture of chloroform and hexane (2:1). Shake well immediate-
ly, and proceed as directed in (2).
Amount (mg) of total iodine (I) = W s x (A T /A S ) x 0.7644
W s : Amount (mg) of potassium iodide for assay
(4) Phenol — Measure the specific gravity <2.56> of Com-
pound Iodine Glycerin according to Method 2. Weigh exactly
about 2 mL of it, add 3 mL of 0.1 mol/L sodium thiosulfate
VS, and shake. Add 2 mL of dilute hydrochloric acid, and
shake with two 10-mL portions of chloroform. Separate the
chloroform layer, and shake with two 10-mL portions of 0.5
mol/L sodium hydroxide TS. Separate the water layer, add
water to make exactly 500 mL, and use this solution as the
sample solution. Dissolve about 0.5 g of phenol for assay, ac-
curately weighed, in ethanol (95) to make exactly 100 mL,
JPXV
Official Monographs / Dental Iodine Glycerin 771
pipet 2 mL of this solution, proceed in the same manner as
the sample solution, and use so obtaind solution as the stan-
dard solution. Pipet 3 mL each of the sample solution and
standard solution, to each add 2 mL of dilute hydrochloric
acid, and place in a water bath at 30°C. Allow to stand for 10
minutes, and add exactly 2 mL of a solution of sodium nitrite
(1 in 100), shake, and allow to stand at 30°C for 60 minutes.
Add dilute potassium hydroxide-ethanol TS to make exactly
25 mL, and determine the absorbances of these solutions, A T
and A s , at 403 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using the solution prepared in the
same manner with 3 mL of water instead of the sample solu-
tion as the blank.
Amount (mg) of phenol (C 6 H 6 0)
= W s x(Aj/A s )x(l/50)
fV s : Amount (mg) of phenol for assay
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dental Iodine Glycerin
Sf4ffl a - K ■ f 'J it 'J >
Dental Iodine Glycerin contains not less than 9.0
w/v% and not more than 11.0 w/v% of iodine (I:
126.90), not less than 7.2 w/v% and not more than 8.8
w/v% of potassium iodide (KI: 166.00), and not less
than 0.9 w/v% and not more than 1.1 w/v% of zinc
sulfate hydrate (ZnS0 4 .7H 2 0: 287.58).
Method of preparation
Iodine
Potassium Iodide
Zinc Sulfate Hydrate
Glycerin
Purified Water
10 g
1
35 mL
a sufficient quantity
To make 100 mL
Dissolve and mix the above ingredients.
Description Dental Iodine Glycerin is a dark red-brown liq-
uid, having the odor of iodine.
Identification (1) The colored solution obtained in the As-
say (1) acquires a red color. Determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
510 nm and 514 nm (iodine).
(2) The colored solution obtained in the Assay (2) ac-
quires a red color. Determine the absorption spectrum of this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 510 nm and 514
nm (potassium iodide).
(3) Put 1 mL of Dental Iodine Glycerin in a glass-stop-
pered, test tube, add 10 mL of ethanol (95), and mix. Then
add 2 mL of sodium hydroxide TS, add 1 mL of a solution of
copper (II) chloride dihydrate in ethanol (95) (1 in 10), and
shake: a blue color develops (glycerin).
(4) The colored solution obtained in the Assay (3) ac-
quires a red-purple to purple color. Determine the absorption
spectrum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
618 nm and 622 nm (zinc sulfate hydrate).
Assay (1) Iodine — Pipet 5 mL of Dental Iodine Glycerin,
and add diluted ethanol (3 in 10) to make exactly 50 mL.
Pipet 5 mL of this solution, add water to make exactly 200
mL, and use this solution as the sample solution. On the
other hand, weigh accurately about 0.5 g of iodine for assay
and about 0.4 g of potassium iodide for assay, previously
dried at 105 °C for 4 hours, and dissolve in diluted ethanol (3
in 10) to make exactly 50 mL. Pipet 5 mL of this solution,
add water to make exactly 200 mL, and use this solution as
the standard solution. Pipet 10 mL each of the sample solu-
tion and standard solution, to each add exactly 20 mL of a
mixture of chloroform and hexane (2:1), shake immediately,
and separate the chloroform-hexane layer [use the water layer
in (2)]. Filter through a pledget of cotton. Determine the ab-
sorbances, A T and A s , of the filtrates obtained from the sam-
ple solution and standard solution, respectively, at 512 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a mixture of chloroform and hexane (2:1) as the blank.
Amount (mg) of iodine (I) = W s x (A T /A S )
W s : Amount (mg) of iodine for assay
(2) Potassium iodide — Separate the water layers of the
sample solution and standard solution obtained in (1), pipet 7
mL each of the water layers, and to each add exactly 1 mL of
diluted hydrochloric acid (1 in 2), 1 mL of sodium nitrite TS
and 10 mL of a mixture of chloroform and hexane (2:1), and
shake immediately. Separate the chloroform-hexane layer,
and filter through a pledget of cotton. Determine the absor-
bances, A-y and A s , of the filtrates obtained from the sample
solution and standard solution, respectively, at 512 nm as
directed under Ultraviolet-visible Spectrophotometry, using a
mixture of chloroform and hexane (2:1) as the blank.
Amount (mg) of potassium iodide (KI)= W s X (A T /A S )
W s : Amount (mg) of potassium iodide for assay
(3) Zinc sulfate Hydrate — Pipet 5 mL of Dental Iodine
Glycerin, and add diluted ethanol (3 in 10) to make exactly 50
mL. Pipet 5 mL of this solution, add water to make exactly
100 mL, and use this solution as the sample solution. On the
other hand, pipet 10 mL of Standard Zinc Stock Solution,
add diluted ethanol (3 in 200) to make exactly 1000 mL, and
use this solution as the standard solution. Pipet 10 mL each
of the sample solution and standard solution, to each add 10
mL of a mixture of chloroform and hexane (2:1), shake, and
allow to stand. Pipet 3 mL each of the water layers, and to
each add 2 mL of boric acid-potassium chloride-sodium
hydroxide buffer solution, pH 10.0, 2 mL of zincon TS and
water to make exactly 25 mL. Determine the absorbances, A T
and A s , obtained from the sample solution and standard so-
lution, respectively, at 620 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using the solution pre-
pared in the same manner with 3 mL of water as the blank.
Amount (mg) of zinc sulfate Hydrate (ZnS0 4 .7H 2 0)
= W s x(A T /A s )x 4.397
W s : Amount (mg) of zinc in 10 mL of Standard Zinc Stock
Solution
Containers and storage Containers — Tight containers.
772 Iodine, Salicylic Acid and Phenol Spirit / Official Monographs
JP XV
Storage — Light-resistant.
Iodine, Salicylic Acid and Phenol
Spirit
a-|«--tf-U5 L JU»-7x/-JUlt
Iodine, Salicylic Acid and Phenol Spirit contains not
less than 1.08 w/v% and not more than 1.32 w/v% of
iodine (I: 126.90), not less than 0.72 w/v% and not
more than 0.88 w/v% of potassium iodide (KI:
166.00), not less than 4.5 w/v% and not more than 5.5
w/v% of salicylic acid (C 7 H 6 3 : 138.12), not less than
1.8 w/v% and not more than 2.2w/v% of phenol
(C 6 H 6 0: 94.11), and not less than 7.2w/v% and not
more than 8.8 w/v% of benzoic acid (C 7 H 6 2 : 122.12).
Method of preparation
Iodine Tincture
200 mL
Salicylic Acid
50 g
Phenol
20 g
Benzoid Acid
80 g
Ethanol for Disinfection
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients. It may be prepared with an appropiate
quantity of Ethanol and Purified Water in place of Ethanol
for Disinfection.
Description Iodine, Salicylic Acid and Phenol Spirit is a
dark red-brown liquid, having the odor of phenol.
Identification (1) To a mixture of 1 mL of starch TS and 9
mL of water add 1 drop of Iodine, Salicylic Acid and Phenol
Spirit: a dark blue-purple color develops (iodine).
(2) To 1 mL of Iodine, Salicylic Acid and Phenol Spirit
add 5 mL of ethanol (95) and water to make 50 mL. To 1 mL
of this solution add hydrochloric acid-potassium chloride
buffer solution, pH 2.0, to make 50 mL, and to 15 mL of this
solution add 5 mL of a solution of iron (III) nitrate enneahy-
drate (1 in 200): a red-purple color is produced (salicylic
acid).
(3) Shake 1 mL of Iodine, Salicylic Acid and Phenol
Spirit with 1 mL of sodium thiosulfate TS, add 20 mL of
water and 5 mL of dilute hydrochloric acid, and extract with
25 mL of diethyl ether. Wash the diethyl ether extract with
two 25-mL portions of sodium hydrogen carbonate TS, and
extract with 10 mL of dilute sodium hydroxide TS. Shake 1
mL of the extract with 1 mL of sodium nitrite TS and 1 mL
of dilute hydrochloric acid, and add 3 mL of sodium
hydroxide TS: a yellow color is developed (phenol).
(4) Shake 1 mL of Iodine, Salicylic Acid and Phenol
Spirit with 1 mL of sodium thiosulfate TS, add 20 mL of
water and 5 mL of dilute hydrochloric acid, extract with 10
mL of diethyl ether, and use the diethyl ether extract as the
sample solution. Dissolve 25 mg of salicylic acid, 0.01 g of
phenol and 0.04 g of benzoic acid in 5 mL each of diethyl
ether, respectively, and use these solutions as the standard so-
lutions (1), (2) and (3). Perform the test with the sample solu-
tion and standard solutions (1), (2) and (3) as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL of each solu-
tion on a plate of silica gel with fluorescent indicator for thin-
layer chromatography. Develop the plate with a mixture of
chloroform, acetone and acetic acid (100) (45:5:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the 3 spots from
the sample solution show the same Rf value as the cor-
responding spots of the standard solutions (1), (2) and (3).
Spray evenly iron (III) chloride TS on the plate: the spot from
standard solution (1) and the corresponding spot from the
sample solution acquires a purple color.
Assay (1) Iodine — Pipet 4 mL of Iodine, Salicylic Acid
and Phenol Spirit, add ethanol (95) to make exactly 50 ml,
and use this solution as the sample solution. On the other
hand, weigh accurately about 1.2 g of iodine for assay and
about 0.8 g of potassium iodide for assay, previously dried at
105 °C for 4 hours, and dissolve in ethanol (95) to make ex-
actly 100 ml. Pipet 4 ml of this solution, add ethanol (95) to
make exactly 50 ml, and use this solution as the standard so-
lution. Pipet 3 ml each of the sample solution and standard
solution, to each add exactly 25 ml of a mixture of chlo-
roform and hexane (2:1), and shake. Further add exactly 10
ml of water, shake and separate the chloroform-hexane lay-
ers [use the water layers in (2)] . Filter through a pledget of ab-
sorbent cotton, and determine the absorbances of the filtrates
from the sample solution and standard solution, respectively,
A T and A s , at 512 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using a mixture of chloroform
and hexane (2:1) as the blank.
Amount (mg) of iodine (I) = W s x (A T /A S ) x (1/25)
W s : Amount (mg) of iodine for assay
(2) Potassium iodide — Separate the water layers of the
sample solution and standard solution obtained in the Assay
(1), pipet 8 ml each of the water layers, and add 1 ml of dilut-
ed dilute hydrochloric acid (1 in 2) and 1 ml of sodium nitrite
TS. Immediately after shaking, add exactly 10 ml of a mix-
ture of chloroform and hexane (2:1), shake, and proceed in
the same manner as for the Assay (1).
Amount (mg) of potassium iodide (KI)
= H A s x(.4 T A4 s )x(l/25)
W s : Amount (mg) of potassium iodide for assay
(3) Salicylic acid, phenol and benzoic acid — Pipet 2 mL
of Iodine, Salicylic Acid and Phenol Spirit, add 20 mL of
diluted methanol (1 in 2) and 0.1 mol/L soium thiosulfate VS
until the color of iodine disappears, add exactly 20 mL of the
internal standard solution, then add diluted methanol (1 in 2)
to make 200 mL, and use this solution as the sample solution.
Weigh accurately about 0.2 g of salicylic acid for assay,
previously dried in a desiccator (silica gel) for 3 hours, about
80 mg of phenol for assay, and 0.32 g of benzoic acid, previ-
ously dried in a desiccator (silica gel) for 3 hours, dissolve in
diluted methanol (1 in 2) to make exactly 50 mL. Pipet 25 mL
of this solution, add exactly 20 mL of the internal standard
solution and diluted methanol (1 in 2) to make 200 mL, and
use this solution as the standard solution. Perform the test
with 3 fiL of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the operating conditions in the Assay under Compound Sali-
cylic Acid Spirit. Calculate the ratios, Q Ta , Q Tb and g Tc , of
JP XV
Official Monographs / Iopamidol 773
the peak areas of salicylic acid, phenol and benzoic acid to
those of the internal standard of the sample solution, and the
ratios, Qsa. Qsb and Q Sc , of the peak areas of salicylic acid,
phenol and benzoic acid to those of the internal standard of
the standard solution.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= ^SaX(Q Ta /Gsa)X(l/2)
Amount (mg) of phenol (C 6 H 6 0)
= W sb x(Q Tb /Q sb )x(l/2)
Amount (mg) of benzoic acid (C 7 H 6 2 )
= ^scX(e T c/Qsc)x(l/2)
W S z- Amount (mg) of salicylic acid for assay
W sb : Amount (mg) of phenol for assay
W Sc : Amount (mg) of benzoic acid
Internal standard solution — A solution of theophylline in
methanol (1 in 1000).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Iodoform
h**JUA
i i
X,
CHI 3 : 393.73
Triiodomethane [75-47-8]
Iodoform, when dried, contains not less than 99.0%
of CHI 3 .
Description Iodoform occurs as lustrous, yellow crystals or
crystalline powder. It has a characteristic odor.
It is freely soluble in diethyl ether, sparingly soluble in
ethanol (95), and practically insoluble in water.
It is slightly volatile at ordinary temperature.
Melting point: about 120°C (with decomposition).
Identification
evolved.
Heat 0.1 g of Iodoform: a purple gas is
Purity (1) Water-soluble colored substances and acidity
or alkalinity — Shake well 2.0 g of Iodoform, previously pow-
dered, with 5 mL of water for 1 minute, allow to stand, and
filter the supernatant liquid: the filtrate is colorless and neu-
tral.
(2) Chloride <1.03>— Shake well 3.0 g of Iodoform,
previously powdered, with 75 mL of water for 1 minute, al-
low to stand, and filter the supernatant liquid. To 25 mL of
the filtrate add 6 mL of dilute nitric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.011%).
(3) Sulfate <1.14>— To 25 mL of the filtrate obtained in
(2) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 0.35 mL of 0.005
mol/L sulfuric acid VS (not more than 0.017%).
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Iodoform, previous-
ly dried, in a 500-mL glass-stoppered flask, and dissolve it in
20 mL of ethanol (95). Add exactly 30 mL of 0. 1 mol/L silver
nitrate VS and 10 mL of nitric acid, stopper the flask, shake
well, and allow to stand in a dark place over 16 hours. Add
150 mL of water, and titrate <2.50> the excess silver nitrate
with 0.1 mol/L ammonium thiocyanate VS (indicator: 5 mL
of ammonium iron (III) sulfate TS). Perform a blank deter-
mination.
Each mL of 0.1 mol/L silver nitrate VS
= 13.12 mg of CHI3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Iopamidol
OH
HO H
H 3 C
TXX,"
O^^NH
C 17 H 22 I 3 N 3 8 : 777.09
N, N ' -Bis [2-hydroxy- 1 -(hydroxymethyl)ethyl] -5- [(2S)-2-
hydroxypropanoylamino]-2,4,6-triiodoisophthalamide
[62883-00-5]
Iopamidol, when dried, contains not less than 99.0%
of C 17 H 22 I 3 N 3 8 .
Description Iopamidol occurs as a white crystalline pow-
der.
It is very soluble in water, sparingly soluble in methanol,
and very slightly soluble in ethanol (99.5).
Identification (1) To 0.05 g of Iopamidol add 5 mL of
hydrochloric acid, heat for 10 minutes in a water bath: the
test solution responds to the Qualitative Tests <1.09> for pri-
mary aromatic amines.
(2) Heat 0.1 g of Iopamidol over a flame: a purple gas is
evolved.
(3) Determine the infrared absorption spectrum of
Iopamidol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [ a ]f 36 : -4.6 - -5.2° (after drying,
4 g, water, warm, after cooling, 10 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Iopamidol in 10 mL of water: the solution is clear and color-
less.
(2) Primary aromatic amines — Dissolve 0.60 g of
774 Iotalamic Acid / Official Monographs
JP XV
Iopamidol in 8 mL of water, add 1 mL of a solution of sodi-
um nitrite (1 in 50) and 12 mL of 2 mol/L hydrochloric acid
TS, shake, and allow to stand for 2 minutes. Add 1 mL of a
solution of ammonium amidosulfate (1 in 10), shake well,
allow to stand for 1 minute, and add 1 mL of
naphthylethylenediamine TS and water to make exactly 50
mL. Determine the absorbance of this solution at 495 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>
using a solution, prepared in the same manner, as the blank:
the absorbance is not more than 0.12 (not more than
0.020%).
(3) Iodine — Dissolve 2.0 g of Iopamidol in 25 mL of
water, add 5 mL of 1 mol/L sulfuric acid and 5 mL of tol-
uene, shake well, and allow to stand: the toluene layer is
colorless.
(4) Free iodine ion — Weigh accurately about 5.0 g of
Iopamidol, dissolve in 70 mL of water, and adjust the pH to
about 4.5 with dilute acetic acid. To this solution add 2 mL of
0.1 mol/L sodium chloride TS, and titrate <2.50> with 0.001
mol/L silver nitrate VS (potentiometric titration).
Each mL of 0.001 mol/L silver nitrate VS
= 0.1269 mg of I
Content of iodine ion in Iopamidol is not more than
0.001%.
(5) Heavy metals <1.07> — Moisten 1.0 g of Iopamidol
with a small quantity of sulfuric acid, heat gradually to
almost incinerate by a possibly lower temperature. After
cooling, moisten again with a small quantity of sulfuric acid,
heat gradually until white fumes no longer are evolved, and
incinerate by ignition between 450 to 550°C. Proceed as
directed in Method 2, and perform the test. Prepare the con-
trol solution with 1.0 mL of Standard Lead Solution (not
more than 10 ppm).
(6) Related substances — Dissolve 0.10 g of Iopamidol in
water to make exactly 10 mL, and use this solution as the
sample solution. Separately, dissolve 10 mg of N,N' -bis[2-
hydroxy - 1 - (hydroxymethyl)ethyl] - 5 - hydroxyacetylamino -
2,4,6-triiodoisophthalamide in water to make exactly 100
mL. Pipet 5 mL of this solution, add water to make exactly
50 mL, and use this solution as the standard solution. Per-
form the test with exactly 20 iiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area of the both solutions by the au-
tomatic integration method: each area of the peaks other
than the peak of iopamidol from the sample solution is not
larger than the peak area of the standard solution, and the
total of these areas is not larger than 2.5 times of the peak
area of the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Use water as the mobile phase A, and a mix-
ture of water and methanol (3:1) as the mobile phase B.
Change the mixed ratios of the mobile phase A and the mo-
bile phase B stepwise as follows:
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0- 6
92
8
6-18
92^65
8^35
18-30
65^ 8
35^92
30-34
8
92
Flow rate: Adjust the flow rate to 1.5 mL per minute.
Time span of measurement: About 4.3 times as long as the
retention time of iopamidol.
System suitability —
System performance: Dissolve 1 mL of the sample solution
and 10 mg of 7V,./V'bis[2-hydroxy-l-(hydroxymethyl)ethyl]-5-
hydroxyacetylamino-2,4,6-triiodoisophthalamide in water to
make 100 mL. When the procedure is run with 20 ^L of this
solution under the above operating conditions, N,N' -bis[2-
hydroxy-l-(hydroxymethyl)ethyl]-5-hydroxyacetylamino-
2,4,6-triiodoisophthalamide and iopamidol are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak
areas of A^,A r '-bis[2-hydroxy-l-(hydroxymethyl)ethyl]-
5-hydroxyacetylamino-2,4,6-triiodoisophthalamide is not
more than 1.0%.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Iopamidol, previ-
ously dried, transfer to a saponification flask, dissolve in 40
mL of sodium hydroxide TS, add 1 g of zinc powder, boil for
30 minutes under a reflux confenser, cool, and filter. Wash
the flask and the filter paper with 50 mL of water, and com-
bine the washing with the filtrate. Add 5 mL of acetic acid
(100) to this solution, and titrate <2.50> with 0.1 mol/L silver
nitrate VS (potentiometric titration).
Each mL of 0.1 mol/L sliver nitrate VS
= 25.90 mg of C 17 H 22 I 3 N 3 8
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Iotalamic Acid
i
CnH 9 I 3 N 2 4 : 613.91
3-Acetylamino-2,4,6-triiodo-
5-(methylaminocarbonyl)benzoic acid [2276-90-6]
JPXV
Official Monographs / Iotroxic Acid 775
Iotalamic Acid, when dried, contains not less than
99.0% of C„H 9 I 3 N 2 04.
Description Iotalamic Acid occurs as a white powder. It is
odorless.
It is sparingly soluble in ethanol (95), very slightly soluble
in water, and practically insoluble in diethyl ether.
It dissolves in sodium hydroxide TS.
It gradually colored by light.
Identification (1) Heat 0.1 g of Iotalamic Acid over a
flame: a purple gas is evolved.
(2) Determine the infrared spectrum of Iotalamic Acid,
previously dried, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
Iotalamic Acid in 10 mL of sodium hydroxide TS: the solu-
tion is clear and colorless.
(2) Primary aromatic amines — To 0.50 g of Iotalamic
Acid add 15 mL of water, and dissolve it in 1 mL of sodium
hydroxide TS while ice-cooling. Add 4 mL of a solution of
sodium nitrite (1 in 100) to the solution, immediately add 12
mL of 1 mol/L hydrochloric acid TS, and shake gently. Then
allow the mixture to stand for exactly 2 minutes, add 8 mL of
ammonium amidosulfate TS, and shake occasionally for 5
minutes. Add 3 drops of a solution of 1-naphthol in ethanol
(95) (1 in 10), allow to stand for 1 minute, add 3.5 mL of am-
monia-ammonium chloride buffer solution, pH 10.7, mix,
and immediately add water to make 50 mL. Determine within
20 minutes the absorbance of this solution at 485 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution, prepared in the same manner, as the blank:
the absorbance is not more than 0.25.
(3) Soluble halides — Dissolve 0.5 g of Iotalamic Acid in
20 mL of diluted ammonia TS (1 in 40), add 6 mL of dilute
nitric acid, shake, allow to stand for 5 minutes, and filter.
Transfer the filtrate to a Nessler tube, wash the residue with
20 mL of water, combine the filtrate and the washings, and
add water to make 50 mL. Proceed as directed for the Chlo-
ride Limit Test <1.03> using this solution as the test solution.
Prepare the control solution as follows: to 0.10 mL of 0.01
mol/L hydrochloric acid VS and add 20 mL of diluted am-
monia TS (1 in 40), 6 mL of dilute nitric acid and water to
make 50 mL.
(4) Iodine — Dissolve 0.20 g of Iotalamic Acid in 2.0 mL
of sodium hydroxide TS, add 2.5 mL of 0.5 mol/L sulfuric
acid TS, and allow to stand for 10 minutes with occasional
shaking. Add 5 mL of chloroform, shake well, and allow to
stand: the chloroform layer remains colorless.
(5) Heavy metals <1.07> — Proceed with 1 .0 g of Iotalamic
Acid according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(6) Arsenic </.//> — Prepare the test solution with 0.6 g
of Iotalamic Acid according to Method 3, and perform the
test (not more than 3.3 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Iotalamic Acid,
previously dried, place it in a saponification flask, dissolve in
40 mL of sodium hydroxide TS, add 1 g of zinc powder, and
heat for 30 minutes under a reflux condenser. Cool, filter,
wash the flask and the filter paper with 50 mL of water, and
combine the washings and the filtrate. Add 5 mL of acetic
acid (100) to this solution, and titrate <2.50> with 0.1 mol/L
silver nitrate VS, until the color of the precipitate changes
from yellow to green (indicator: 1 mL of
tetrabromophenolphthalein ethyl ester TS).
Each mL of 0.1 mol/L silver nitrate VS
= 20.46 mg of CnH 9 I 3 N 2 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Iotroxic Acid
-ITl-r-n^Xi?
XC
Or*
C 22 H I8 I 6 N 2 9 : 1215.81
3,3'-(3,6,9-Trioxaundecanedioyl)diiminobis-(2,4,6-
triiodobenzoic acid) [51022-74-3]
Iotroxic Acid contains not less than 98.5% of
C 2 2H lg I 6 N 2 09, calculated on the anhydrous basis.
Description Iotroxic Acid occurs as a white crystalline pow-
der.
It is soluble in methanol, slightly soluble in ethanol (95),
and practically insoluble in water and in diethyl ether.
It is gradually colored by light.
Identification (1) Heat 0.1 g of Iotroxic Acid over a flame:
a purple gas evolves.
(2) Dissolve a suitable amount of Iotroxic Acid in a suita-
ble amount of methanol, evaporate the methanol under
reduced pressure, and determine the infrared absorption
spectrum of the residue so obtained as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Iotroxic Acid in 10 mL of diluted sodium hydroxide TS (1 in
5): the solution is clear and colorless.
(2) Primary aromatic amines — Dissolve 0.20 g of Iotrox-
ic Acid in 5 mL of water and 1 mL of sodium hydroxide TS,
add 4 mL of a solution of sodium nitrite (1 in 100) and 10 mL
of 1 mol/L hydrochloric acid TS, mix, and allow to stand for
2 minutes. Add 5 mL of ammonium amidosulfate TS, shake
well, allow to stand for 1 minute, then add 0.4 mL of a solu-
tion of a-naphthol in ethanol (95) (1 in 10), 15 mL of sodium
hydroxide TS and water to make exactly 50 mL. Read the ab-
sorbance of this solution at 485 nm as directed under Ultrav-
776 Ipratropium Bromide Hydrate / Official Monographs
JP XV
iolet-visible Spectrophotometry <2.24>, using a blank solu-
tion obtained in the same manner as above: the absorbance is
not more than 0.22.
(3) Iodine — Dissolve 0.20 g of Iotroxic Acid in 2.0 mL of
sodium hydrogen carbonate TS, add 5 mL of toluene, mix
well, and allow to stand: the toluene layer is colorless.
(4) Free iodine ion — Weigh accurately about 5.0 g of
Iotroxic Acid, dissolve in 12 mL of a solution of meglumine
(3 in 20), add water to make 70 mL, and adjust the pH to
about 4.5 with acetic acid (100). To this solution add 2 mL of
0.1 mol/L sodium chloride TS, and titrate <2.50> with 0.001
mol/L silver nitrate VS (potentiometric titration).
Each mL of 0.001 mol/L silver nitrate
= 0.1269 mg of I
Content of iodine ion in Iotroxic Acid, calculated on the
anhydrous basis, is not more than 0.004%.
(5) Heavy metals <1.07> — Heat strongly 1.0 g of Iotroxic
Acid as directed under Residue on Ignition Test, then pro-
ceed according to Method 2, and perform the test. Prepare
the control solution with 1.0 mL of Standard Lead Solution
(not more than 10 ppm).
(6) Related substances — Dissolve 0.15 g of Iotroxic Acid
in 10 mL of methanol, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add methanol to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of toluene, acetone and for-
mic acid (6:4:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Water <2.48> 1.0 - 2.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Iotroxic Acid, dis-
solve in 40 mL of sodium hydroxide TS in a saponification
flask, add 1 g of zinc powder, and boil for 30 minutes under a
reflux condenser. After cooling, filter, wash the flask and the
filter paper with 50 mL of water, and combine the washings
to the filtrate. To this solution add 5 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L silver nitrate VS (potentio-
metric titration).
Each mL of 0.1 mol/L silver nitrate VS
= 20.26 mg of C 22 H 18 I 6 N 2 9
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ipratropium Bromide Hydrate
^f^vr-ntf^A^b^KW^
r
HsC-N*-<;
CH 3 \ .,0
Br -HjO
C 2 oH 30 BrN0 3 .H 2 0: 430.38
(li?,3r,5S)-3-[(2i?5)-3-Hydroxy-2-phenylpropanoyloxy]-
8-methyl-8-(l-methylethyl)-8-azoniabicyclo[3.2.1]octane
bromide monohydrate [66985-17-9]
Ipratropium Bromide Hydrate, when dried, contains
not less than 99.0% of ipratropium bromide (C 2 oH 30
BrN0 3 : 412.36).
Description Ipratropium Bromide Hydrate occurs as a
white, crystalline powder.
It is freely soluble in water, soluble in ethanol (99.5),
slightly soluble in acetonitrile and in acetic acid (100), and
practically insoluble in diethyl ether.
The pH of a solution of Ipratropium Bromide Hydrate (1
in 20) is between 5.0 and 7.5.
Melting point: about 223 °C (with decomposition, after
drying).
Identification (1) To 5 mg of Ipratropium Bromide Hy-
drate add 0.5 mL of fuming nitric acid, and evaporate on a
water bath to dryness. After cooling, dissolve the residue in 5
mL of acetone, and add 2 drops of potassium hydroxide-
ethanol TS: a purple color develops.
(2) Determine the absorption spectrum of a solution of
Ipratropium Bromide Hydrate in 0.01 mol/L hydrochloric
acid TS (3 in 2000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Ipratropium Bromide Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) The solution of Ipratropium Bromide Hydrate (1 in
100) responds to the Qualitative Tests <1.09> for bromide.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ipratropium Bromide Hydrate in 20 mL of water: the solu-
tion is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 1.0 g of
Ipratropium Bromide Hydrate. Prepare the control solution
with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.024%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Ipratro-
pium Bromide Hydrate according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Ipratropium Bromide Hydrate according to Method 3,
JPXV
Official Monographs / Isepamicin Sulfate 777
and perform the test. Use a solution of magnesium nitrate
hexahydrate in ethanol (95) (1 in 10) (not more than 1 ppm).
(5) Isopropylatropine bromide — Dissolve 25 mg of
Ipratropium Bromide Hydrate in the mobile phase to make
exactly 100 mL, and use this solution as the sample solution.
Perform the test with 25 fiL of the sample solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine the peak area, A^, of ipratropi-
um and the peak area, A b , having a relative retention time to
ipratropium about 1.3 by the automatic integration method:
Ab/(Az + A b ) is not more than 0.01, and no peak other than
the peak of ipratropium and the peak having a relative reten-
tion time to ipratropium about 1.3 appears within about 14
minutes of the retention time after the solvent peak.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 10 to 15 cm in length, packed with octylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: Room temperature.
Mobile phase: A mixture of diluted phosphoric acid (1 in
200), acetonitrile and methanesulfonic acid (1000:120:1).
Flow rate: Adjust the flow rate so that the retention time of
ipratropium is about 7 minutes.
Selection of column: Heat a solution of Ipratropium
Bromide in 1 mol/L hydrochloric acid TS (1 in 100) at 100°C
for 1 hour, and cool. To 2.5 mL of this solution add the mo-
bile phase to make 100 mL. Proceed with 25 fiL of this solu-
tion under the above operating conditions, and calculate the
resolution. Use a column showing a resolution not less than 3
between the peak of ipratropium and the peak having a rela-
tive retention time to ipratropium about 0.6.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of ipratropium obtained from 25 /uL of
the sample solution composes 50 to 80% of the full scale.
(6) Apo-compounds — Dissolve 0.14 g of Ipratropium
Bromide in 0.01 mol/L hydrochloric acid TS to make 100
mL. Perform the test with this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and determine
the absorbances, Ay and A 2 , at 246 nm and 263 nm, respec-
tively: AJA 2 is not more than 0.91.
Loss on drying <2.4I> 3.9 - 4.4% (1 g, 105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Ipratropium
Bromide Hydrate, previously dried, dissolve in 40 mL of a-
cetic acid (100), add 40 mL of 1,4-dioxane and 2.5 mL of bis-
muth nitrate TS, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 41.24 mg of C 2 oH 30 BrN03
Containers and storage Containers — Tight containers.
Isepamicin Sulfate
• jr HjSOj
C 22 H 43 N 5 12 .a:H 2 S04
6-Amino-6-deoxy-a-D-glucopyranosyl-(l -> 4)-
[3-deoxy-4-C-methyl-3-methylamino-/?-L-arabinopyranosyl-
(l->6)]-2-deoxy-l-7V-[(2S)-3-amino-2-hydroxypropanoyl]-
D-streptamine sulfate [67814-76-0]
Isepamicin Sulfate is the sulfate of a derivative of
gentamycin B, an aminoglycoside substance, having
antibacterial activity produced by the growth of
Micromonospora purpurea.
It contains not less than 680 /ug (potency) and not
more than 780 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Isepamicin Sulfate is
expressed as mass (potency) of isepamicin
(C 22 H 43 N 5 12 : 569.60).
Description Isepamicin Sulfate occurs as a white to pale yel-
lowish white powder.
It is very soluble in water, and practically insoluble in
methanol and in ethanol (95).
It is hygroscopic.
Identification (1) Dissolve 0.02 g of Isepamicun Sulfate in
1 mL of water, add 3 mL of anthrone TS , shake, and allow to
stand: a blue-purple color develops.
(2) Dissolve 10 mg each of Isepamicin Sulfate and
Isepamicin Sulfate Reference Standard in 5 mL of water, and
use these solutions as the sample solution and the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer chromatography <2.03>. Spot 5 fiL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ammonia water (28), ethanol (99.5), 1-buthanol and
chloroform (5:5:4:2) to a distance of about 15 cm, and air-
dry the plate. Spray evenly 0.2% ninhydrin-water saturated
1-butanol TS on the plate, and heat at about 100°C for about
10 minutes: the principal spots from the sample solution and
the standard solution exhibit a red-brown color and show the
same Ri value.
(3) Dissolve 0.01 g of Isepamicin Sulfate in 1 mL of
water, and add 1 drop of barium chloride TS: a white
precipitate is produced.
Optical rotation <2.49> [a]™: + 100 - + 120° (0.25 g calcu-
lated on the anhydrous bases, water, 25 mL, 100 mm).
778
Isoflurane / Official Monographs
JP XV
pH <2.54> Dissolve 0.5 g of Isepamicin Sulfate in 5 mL of
water: the pH of the solution is between 5.5 and 7.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Isepamicin Sulfate in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Isepami-
cin Sulfate according to Method 4, and perform the test. Pre-
pare the control solution with 1.0 mL Standard Lead Solu-
tion (not more than 10 ppm).
(3) Related substances — Perform the test with 5 /xL of the
sample solution obtained in the Assay as directed under As-
say. Determine each peak area of the sample solution by the
automatic integration method, and calculate the amounts of
their peaks by the area percentage method: the amount of
HAPA-gentamine-B equivalent to about 0.4 of the relative
retention time to isepamicin is not more than 5.0%, and gen-
tamicin B equivalent to about 1 .3 of that is not more than 3.0
%. Correct the peak area of gentamicin B by multiplying the
sensitivity coefficient, 1.11.
Operating conditions —
Apparatus, detector, column, column temperature, reac-
tion coil, mobile phase, reagent, reaction temperature, flow
rate of the mobile phase, and flow rate of the reagent: Pro-
ceed as directed in the operating conditions in the Assay.
Time span of measurement: About 2 times as long as the
retention time of isepamicin.
System suitability —
Test for required detection: Pipet 1 mL of the standard so-
lution, add water to make exactly 10 mL, and use this solu-
tion as the solution for the test for required detection. Pipet 1
mL of the solution, and add water to make exactly 10 mL.
Confirm that the peak area of isepamicin obtained from 5 /uL
of this solution is equivalent to 7 to 13% of that obtained
from 5 /xL of the solution for the test for required detection.
System performance and system repeatability: Proceed as
directed in the system suitability in the Assay.
Water <2.48> Not more than 12.0% (0.2 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (2:1) in-
stead of methanol for water determination).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Weigh accurately an amount of Isepamicin Sulfate
and Isepamicin Sulfate Reference Standard, equivalent to
about 20 mg (potency), dissolve each in water to make exactly
100 mL, and use these solutions as the sample solution and
the standard solution, respectively. Perform the test with ex-
actly 5 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the peak areas, A T
and A s , of isepamicin of the solutions.
Amount L"g (potency)] of isepamicin (C 2 2H43N50 12 )
= W s x (A T /A S ) x 1000
W s : Amount [mg (potency)] of Isepamicin Sulfate Refer-
ence Standard
Operating conditions —
Apparatus: Consist of two pumps for the mobile phase and
the reagent transport, inject port, column, reaction coil, de-
tector and recorder. Use a reaction coil with thermostat.
Detector: Fluorometry (excitation wavelength: 360 nm, de-
tection wavelength: 440 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Reaction coil: A column 0.25 /xm in inside diameter and 5
m in length.
Mobile phase: Dissolve 28.41 g of anhydrous sodium sul-
fate and 5.23 g of sodium 1-pentane sulfonate in 900 mL of
water, add 1 mL of acetic acid (100), and add water to make
exactly 1000 mL.
Reagent: To 500 mL of boric acid-potassium chloride-so-
dium hydroxide buffer solution, pH 10.0, add 5 mL of a solu-
tion of o-phthalaldehyde in ethanol (95) (2 in 25), 1 mL of 2-
mercaptoethanol and 2 mL of a solution of lauromacrogol (1
in 4).
Reaction temperature: A constant temperature of about
45°C.
Flow rate of the mobile phase: About 0.6 mL per minute.
Flow rate of the reagent: About 0.5 mL per minute.
System suitability —
System performance: Dissolve 2 mg of Gentamicin B in 10
mL of the standard solution. When the procedure is run with
5 /xL of this solution under the above operating conditions,
isepamicin and gentamicin B are eluted in this order with the
resolution between these peaks being not less than 1.0.
System repeatability: When the test is repeated 5 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
isepamicin is not more than 3.0%.
Containers and storage Containers — Tight containers.
Isoflurane
■iV7Ji7:
h\\ F
F F
and enantiomer
C 3 H 2 C1F 5 0: 184.49
(2i?S)-2-Chloro-2-(difluoromethoxy)-l ,1,1 -trifluoroethane
[26675-46-7]
Isoflurane contains not less than 99.0% and not
more than 101.0% of C 3 H 2 C1F 5 0, calculated on the
anhydrous basis.
Description Isoflurane occurs as a clear, colorless fluid liq-
uid.
It is miscible with ethanol (99.5), with methanol and with
o-xylene.
It is slightly soluble in water.
It is volatile, and has no inflammability.
It shows no optical rotation.
Refractive index « D : about 1.30
Boiling point: about 47 - 50°C
Identification (1) The test solution obtained by the Oxyg-
en Flask Combustion Method <1.06> with 50 /xL of Isoflu-
rane, using 40 mL of water as the absorbing liquid, responds
JPXV
Official Monographs / Isoflurane 779
to the Qualitative Tests <1.09> for chloride and fluoride.
(2) Determine the infrared absorption spectrum of Isoflu-
rane as directed in the liquid film method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum or the spectrum of Isoflurane Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Specific gravity <2.56> d^: 1.500 - 1.520
Purity (1) Acidity or alkalinity — To 10 mL of Isoflurane
add 5 mL of freshly boiled and cooled water, and shake for 1
minute: the water layer is neutral.
(2) Soluble chloride — To 60 g of Isoflurane add 40 mL of
water, shake thoroughly, and separate the water layer. To 20
mL of the layer add 6 mL of dilute nitric acid and water to
make 50 mL. Perform the test with this solution as directed
under Chloride Limit Test <1.03>, Prepare the control solu-
tion with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not
more than 3 ppm).
(3) Soluble fluoride — To 6 g of Isoflurane add 12 mL of
diluted 0.01 mol/L sodium hydroxide TS (1 in 20), and shake
for 10 minutes. Transfer 4.0 mL of the water layer into a
Nessler tube, add 30 mL of a mixture of alizarin complexone
TS, acetic acid-potassium acetate buffer solution, pH 4.3 and
cerium (III) nitrate TS (1:1:1), add water to make 50 mL, al-
low to stand for 60 minutes, and use this solution as the sam-
ple solution. Separately, to 0.4 mL of the fluorine standard
solution and 4.0 mL of diluted 0.01 mol/L sodium hydroxide
TS (1 in 20) in a Nessler tube add 30 mL of the mixture of
alizarin complexone TS, acetic acid-potassium acetate buffer
solution, pH 4.3 and cerium (III) nitrate TS (1:1:1), then pro-
ceed in the same manner as for the preparation of the sample
solution, and use the solution so obtained as the standard so-
lution. Determine the absorbances of the sample solution and
standard solution at 600 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using a solution, obtained
by proceeding in the same manner as above with 4.0 mL of
diluted 0.01 mol/L sodium hydroxide TS (1 in 20), as the
blank: the absorbance of the sample solution is not more
than that of the standard solution (not more than 2 ppm).
Fluorine standard solution: Dissolve exactly 2.21 g of sodi-
um fluoride in water to make exactly 1000 mL. Pipet 10 mL
of this solution, and add water to make exactly 1000 mL.
Each mL of this solution contains 0.01 mg of fluorine (F).
(4) Peroxide — To 10 mL of Isoflurane add 1 mL of a
freshly prepared solution of potassium iodide (1 in 10), shake
vigorously, and allow to stand in a dark place for 1 hour: the
water layer is not yellow.
(5) Related substances — Use Isoflurane as the sample so-
lution. To exactly 1 mL of the sample solution add o-xylene
to make exactly 100 mL. Pipet 1 mL of this solution, add o-
xylene to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 5 fiL each of
the sample solution and standard solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions, and determine each peak area by the automatic in-
tegration method: the area of the peak other than isoflurane
is not more than the peak area of isoflurane from the stan-
dard solution, and the total area of the peaks other than
isoflurane is not more than 3 times the peak area of isoflurane
from the standard solution.
Operating conditions —
Detector, column, column temperature, carrier gas, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 5 times as long as the
retention time of isoflurane after injection of the sample solu-
tion.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add o-xylene to make exactly 2mL. Confirm
that the peak area of isoflurane obtained with 5 /iL of this so-
lution is equivalent to 35 to 65% of that with 5 /iL of the stan-
dard solution.
System performance and system repeatability: Proceed as
directed in the system suitability in the Assay.
(6) Residue on evaporation Pipet 65 mL of Isoflurane,
evaporate on a water bath, and dry the residue at 105 °C for 1
hour: not more than 1.0 mg.
Water <2.48> Not more than 0.1% (2 g, Courometric titra-
tion).
Assay To exactly 5 mL each of Isoflurane and Isoflurane
Reference Standard (separately determined water content
<2.48> in the same manner as Isoflurane), add exactly 3 mL of
ethyl acetate as the internal standard, then add o-xylene to
make exactly 50 mL each. To 5 mL each of these solutions
add o-xylene to make 50 mL, and use these solutions as the
sample solution and the standard solution, respectively. Per-
form the test with 2 /uL each of the sample solution and stan-
dard solution as directed under Gas Chromatography <2.02>
according to the following conditions, and determine the ra-
tios, Qj and Q s , of the peak area of isoflurane to that of the
internal standard.
Amount (mg) of isoflurane (C 3 H 2 C1F 5 0) in 5 mL of
Isoflurane
= V s x (Qj/Qs) x 1000 x 1 .506
V s : Amount (mL) of Isoflurane Reference Standard, cal-
culated on the anhydrous basis
1.506: Specific gravity (dlo) of isoflurane
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A stainless steel column 3 mm in inside diameter
and 3.5 m in length, packed with siliceous earth for gas chro-
matography (125- 149 //m in particle diameter), coated in
10% with nonylphenoxypoly(ethyleneoxy)ethanol for gas
chromatography and in 15% with polyalkylene glycol for gas
chromatography.
Column temperature: A constant temperature of about
80°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
isoflurane is about 7 minutes.
System suitability —
System performance: When the procedure is run with 2 fiL
of the standard solution under the above operating condi-
tions, isoflurane and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3
System repeatability: When the test is repeated 6 times with
2 /xL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
isoflurane is not more than 1.0%.
Containers and storage Containers — Tight containers.
780 L-Isoleucine / Official Monographs
Storage — At a temperature not exceeding 30°C.
L-Isoleucine
L--1 VP-f ->>
H CH 3
H NHj
C 6 H 13 N0 2 : 131.17
(2S,3S)-2-Ammo-3-methylpentanoic acid
[73-32-5]
L-Isoleucine,
98.5% of C.H
, when
13 N0 2 .
dried, contains not less than
Description L-Isoleucine occurs as white crystals or crystal-
line powder. It is odorless or has a faint characteristic odor,
and has a slightly bitter taste.
It is freely soluble in formic acid, sparingly soluble in
water, and practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Isoleucine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]o- +39.5 -+41.5° (after
drying, 1 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of L-Isoleucine in 100 mL of
water: the pH of this solution is between 5.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
L-Isoleucine in 10 mL of 1 mol/L hydrochloric acid TS: the
solution is clear and colorless.
(2) Chloride <1.03>— Perform the test with 0.5 g of L-
Isoleucine. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of l-
Isoleucine. Prepare the control solution with 0.35 mL of
0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium — Perform the test with 0.25 g of L-
Isoleucine. Prepare the control solution with 5.0 mL of Stan-
dard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Dissolve 1.0 g of L-Isoleucine
in 40 mL of water and 2 mL of dilute acetic acid by warming,
cool, and add water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 2.0 mL of Standard Lead Solution add 2 mL of
dilute acetic acid and water to make 50 mL (not more than 20
ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of L-Isoleucine according to Method 2, and perform the test
(not more than 2 ppm).
(7) Related substances — Dissolve 0.10 g of L-Isoleucine
in 25 mL of water, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add water to
make exactly 50 mL. Pipet 5 mL of this solution, add water
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
JP XV
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-butanol, water and acetic acid (100) (3:1:1) to a
distance of about 10 cm, and dry the plate at 80°C for 30
minutes. Spray evenly the plate with a solution of ninhydrin
in acetone (1 in 50), and heat at 80°C for 5 minutes: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 0.30% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0. 13 g of L-Isoleucine, previ-
ously dried, and dissolve in 3 mL of formic acid, add 50 mL
of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 13.12 mg of C 6 H 13 N0 2
Containers and storage Containers — Tight containers.
Isoniazid
-fV-7yH
C 6 H 7 N 3 0: 137.14
Pyridine-4-carbohydrazide
,NH 2
[54-85-3]
Isoniazid, when dried, contains not less than 98.5%
of C 6 H 7 N 3 0.
Description Isoniazid occurs as colorless crystals or a white,
crystalline powder. It is odorless.
It is freely soluble in water, sparingly soluble in ethanol
(95), slightly soluble in acetic anhydride, and very slightly
soluble in diethyl ether.
Identification (1) Dissolve about 20 mg of Isoniazid in
water to make 200 mL. To 5 mL of the solution add 1 mL of
0.1 mol/L hydrochloric acid TS and water to make 50 mL.
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Isoniazid, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> Dissolve 1.0 g of Isoniazid in 10 mL of freshly
boiled and cooled water: the pH of this solution is between
6.5 and 7.5.
JPXV
Official Monographs / Isoniazid Tablets
781
Melting point <2.60> 170 - 173 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
isoniazid in 20 mL of water: the solution is clear and color-
less.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Isoniazid
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Isoniazid according to Method 3, and perform the test. In
this case, add 10 mL of a solution of magnesium nitrate hexa-
hydrate in ethanol (95) (1 in 50), then add 1.5 mL of hydro-
gen peroxide (30), and ignite the ethanol to burn (not more
than 5 ppm).
(4) Hydrazine — Dissolve 0.10 g of isoniazid in 5 mL of
water, add 0.1 mL of a solution of salicylaldehyde in ethanol
(95) (1 in 20), shake immediately, and allow to stand for 5
minutes: no turbidity is produced.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of isoniazid, previously
dried, dissolve in 50 mL of acetic acid (100) and 10 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the color of the solution changes from yellow to
green (indicator: 0.5 mL of p-naphtholbenzein TS). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 13.71 mg of C 6 H 7 N 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Isoniazid Injection
Isoniazid Injection is an aqueous solution for injec-
tion.
It contains not less than 95% and not more than 105
% of the labeled amount of isoniazid (C 6 H 7 N 3 0:
137.14).
Method of preparation Prepare as directed under Injec-
tions, with Isoniazid.
Description Isoniazid Injection occurs as a clear, colorless
liquid.
pH: 6.5-7.5.
Identification To a volume of Isoniazid Injection, equiva-
lent to 20 mg of Isoniazid according to the labeled amount,
and add water to make 200 mL. To 5 mL of the solution add
1 mL of 0.1 mol/L hydrochloric acid TS and water to make
50 mL. Determine the absorption spectrum of this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 264 nm and 268 nm.
Extractable volume <6.05> It meets the requirement.
Assay To an exactly measured volume of Isoniazid Injec-
tion, equivalent to about 50 mg of isoniazid (C 6 H 7 N 3 0), add
water to make exactly 100 mL. Pipet 5 mL of the solution,
add exactly 5 mL of the internal standard and the mobile
phase to make 50 mL, and use this solution as the sample so-
lution. Separately, weigh accurately about 50 mg of isoniazid
for assay, previously dried at 105 °C for 2 hours, and dissolve
in water to make exactly 100 mL. Pipet 5 mL of this solution,
add exactly 5 mL of the internal standard and the mobile
phase to make 50 mL, and use this solution as the standard
solution. Perform the test with 5 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of
isoniazid to that of the internal standard.
Amount (mg) of isoniazid (C 6 H 7 N 3 0) = W s x (Q T /Q S )
W s : Amount (mg) of isoniazid for assay
Internal standard solution — A solution of propyl para-
hydroxybenzoate (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 6.80 g of potassium dihydrogen
phosphate in water to make 1000 mL. Separately, to 5.76 g of
phosphoric acid add water to make 1000 mL. Mix these solu-
tions to make a solution having pH 2.5. To 500 mL of this so-
lution add 500 mL of methanol, and add 2.86 g of sodium
tridecanesulfonate to dissolve.
Flow rate: Adjust the flow rate so that the retention time of
isoniazid is about 5 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, isoniazid and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of isoniazid to that of the internal standard is not
more than 1.3%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Isoniazid Tablets
Isoniazid Tablets contain not less than 95% and not
more than 105% of the labeled amount of isoniazid
(C 6 H 7 N 3 0: 137.14).
Method of preparation Prepare as directed under Tablets,
782
/-Isoprenaline Hydrochloride / Official Monographs
JP XV
with Isoniazid.
Identification Take a quantity of powdered Isoniazid
Tablets, equivalent to 0.02 g of Isoniazid according to the la-
beled amount, add 200 mL of water, shake well, and filter.
To 5 mL of the filtrate add 1 mL of 0.1 mol/L hydrochloric
acid TS and water to make 50 mL, and determine the absorp-
tion spectrum of this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 264 nm and 268 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Isoniazid Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of water as the test solution. Take 20 mL or more
of the dissolved solution 20 minutes after starting the test,
and filter through a membrane filter with pore size of not
more than 0.45 ^m. Discard the first 10 mL of the filtrate,
pipet 5 mL of the subsequent, add water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 0.1 g of isoniazid for assay, previous-
ly dried at 105°C for 2 hours, dissolve in water to make exact-
ly 100 mL, then pipet 5 mL of this solution, add water to
make exactly 50 mL, and then pipet 5 mL of this solution,
add water to make exactly 50 mL, and use this solution as the
standard solution. Determine the absorbances, A T and^4 s , of
the sample solution and standard solution at 267 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Isoniazid Tablets in 20 minutes is not
less than 75%.
Dissolution rate (%) with respect to the labeled
amount of isoniazid (C 6 H 7 N 3 0)
= W s x (A T /A S ) x (90/ Q
W s : Amount (mg) of isoniazid for assay.
C: Labeled amount (mg) of isoniazid (C 6 H 7 N 3 0) in 1
tablet.
Assay Weigh accurately and powder not less than 20 Isonia-
zid Tablets. Weigh accurately a quantity of the powder,
equivalent to about 0.1 g of isoniazid (C 6 H 7 N 3 0), add 150
mL of water, shake for 30 minutes, then add water to make
exactly 200 mL, and filter. Discard the first 10 mL of the
filtrate, pipet 5 mL of the subsequent filtrate, add the mobile
phase to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 50 mg of
isoniazid for assay, previously dried at 105°C for 2 hours,
dissolve in water to make exactly 100 mL. Pipet 5 mL of this
solution, add the mobile phase to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with exactly 10,mL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the peak
areas, A T and A s , of isoniazid of the sample solution and
standard solution.
Amount (mg) of isoniazid (C 6 H 7 N 3 0)
= W s x (A T /A S ) x 2
W s : Amount (mg) of isoniazid for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [im in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 6.80 g of potassium dihydrogen
phosphate in water to make 1000 mL. Separately, to 5.76 g of
phosphoric acid add water to make 1000 mL. Mix these solu-
tions to adjust the pH to 2.5. To 400 mL of this solution add
600 mL of methanol, and dissolve 2.86 g of sodium
tridecanesulfonate in this.
Flow rate: Adjust the flow rate so that the retention time of
isoniazid is about 5 minutes.
System suitability —
System performance: Dissolve 5 mg of Isoniazid and 5 mg
of isonicotinic acid in 100 mL of the mobile phase. When the
procedure is run with 10 fiL of this solution under the above
operating conditions, isonicotinic acid and isoniazid are elut-
ed in this order with the resolution between these peaks being
not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
isoniazid is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
/-Isoprenaline Hydrochloride
i-4 vyix-j-u >£K£
>HCI
247.72
4-{(l.R)-l-Hydroxy-
2-[(l-methylethyl)amino]ethyl}benzene-
1,2-diol monohydrochloride [51-30-9]
/-Isoprenaline Hydrochloride, when dried, contains
not less than 98.0% of C n H 17 N0 3 .HCl.
Description /-Isoprenaline Hydrochloride occurs as a white,
crystalline powder. It is odorless.
It is freely soluble in water, sparingly soluble in ethanol
(95), and practically insoluble in acetic acid (100), in acetic
anhydride, in diethyl ether and in chloroform.
It gradually changes in color by air and by light.
Identification (1) Dissolve 0.01 g of /-Isoprenaline
Hydrochloride in 5 mL of water, and add 1 drop of iron (III)
chloride TS: a deep green color develops, and changes
through yellow-green to brown on standing.
(2) Dissolve 1 mg each of /-Isoprenaline Hydrochloride in
1 mL of water in the test tubes A and B . Add 1 mL of potas-
sium hydrogen phthalate buffer solution, pH 3.5 to A, and
add 10 mL of phosphate buffer solution, pH 6.5 to B. To
each of the test tubes add 1 mL of iodine TS, allow to stand
for 5 minutes, and add 2 mL each of sodium thiosulfate TS: a
red color develops in the test tube A, and a deep red color de-
JPXV
Official Monographs / Isopropylantipyrine
783
velops in the test tube B.
(3) Dissolve 0.01 g of /-Isoprenaline Hydrochloride in 1
mL of water, and add 1 mL of phosphotungstic acid TS: a
light brown precipitate is produced.
(4) Determine the absorption spectrum of a solution of l-
Isoprenaline Hydrochloride in 0.1 mol/L hydrochloric acid
TS (1 in 20,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(5) A solution of /-Isoprenaline Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]™'-
0.25 g, water, 25 mL, 100 mm).
■36- -41° (after drying,
pH <2.54> Dissolve 0.01 g of /-Isoprenaline Hydrochloride
in 10 mL of water: the pH of the solution is between 4.5 and
5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
/-Isoprenaline Hydrochloride in 20 mL of 0.1 mol/L
hydrochloric acid TS: the solution is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.10 g of /-
Isoprenaline Hydrochloride. Prepare the control solution
with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.192%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of l-
Isoprenaline Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Isoproterenone — Dissolve 50 mg of /-Isoprenaline
Hydrochloride in 0.01 mol/L hydrochloric acid TS to make
exactly 25 mL, and determine the absorbance of the solution
at 310 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: not more than 0.040.
Loss on drying <2.41>
silica gel, 4 hours).
Not more than 0.5% (1 g, in vacuum,
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of /-Isoprenaline
Hydrochloride, previously dried, dissolve in 100 mL of a
mixture of acetic acid (100) and acetic anhydride (3:2) by
warming, cool, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation.
Each mL of 0.1 mol/L perchloric acid VS
= 24.77 mg of C U H I7 N0 3 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Isopropanol
Isopropyl Alcohol
OH
I
C 3 H s O: 60.10
Propan-2-ol [67-63-0]
Description Isopropanol is a clear, colorless liquid. It has a
characteristic odor.
It is miscible with water, with methanol, with ethanol (95),
and with diethyl ether.
It is flammable and volatile.
Identification (1) To 1 mL of Isopropanol add 2 mL of io-
dine TS and 2 mL of sodium hydroxide TS, and shake: a light
yellow precipitate is formed.
(2) To 5 mL of Isopropanol add 20 mL of potassium
dichromate and 5 mL of sulfuric acid with caution, and warm
gently on a water bath: the produced gas has the odor of ace-
tone, and the gas turns the filter paper, previously wetted
with a solution of salicylaldehyde in ethanol (95) (1 in 10) and
with a solution of sodium hydroxide (3 in 10), to red-brown.
Specific gravity <2.56> df : 0.785 - 0.788
Purity (1) Clarity of solution — To 2.0 mL of Isopropanol
add 8 mL of water, and shake: the solution is clear.
(2) Acidity — To 15.0 mL of Isopropanol add 50 mL of
freshly boiled and cooled water and 2 drops of
phenolphthalein TS, and add 0.40 mL of 0.01 mol/L sodium
hydroxide VS: a red color develops.
(3) Residue on evaporation — Evaporate 20.0 mL of
Isopropanol on a water bath to dryness, and dry at 105°C for
1 hour: the mass of the residue is not more than 1.0 mg.
Water <2.48> Not more than 0.75 w/v% (2 mL, direct titra-
tion).
Distilling range <2.57> 81 - 83°C, not less than 94 vol%.
Containers and storage Containers — Tight containers.
Storage — Remote from fire.
Isopropylantipyrine
Propyphenazone
HjC— ( O
CH,
C 14 H 18 N 2 0: 230.31
l,5-Dimethyl-4-(l-methylethyl)-2-phenyl-
l,2-dihydro-3//-pyrazol-3-one [479-92-5]
784
Isosorbide / Official Monographs
JP XV
Isopropylantipyrine, when dried, contains not less
than 98.0% of C 14 H 18 N 2 0.
Description Isopropylantipyrine occurs as white crystals or
crystalline powder. It is odorless, and has a slightly bitter
taste.
It is very soluble in acetic acid (100), freely soluble in
ethanol (95) and in acetone, soluble in diethyl ether, and
slightly soluble in water.
Identification (1) To 2 mL of a solution of Isopropylan-
tipyrine (1 in 500) add 1 drop of iron (III) chloride TS: a light
red color develops. Further add 3 drops of sulfuric acid to
this solution: the color changes to pale yellow.
(2) Add 5 mL of a solution of Isopropylantipyrine (1 in
500) to a mixture of 5 mL of potassium hexacyanoferrate
(III) TS and 1 to 2 drops of iron (III) chloride TS: a dark
green color gradually develops.
(3) To 2 mL of a solution of Isopropylantipyrine (1 in
500) add 2 to 3 drops of tannic acid TS: a white precipitate is
produced.
Melting point <2.60> 103 - 105°C
Purity (1) Chloride < 1.03 > —Dissolve 1.0 g of
Isopropylantipyrine in 30 mL of dilute ethanol, and add 6
mL of dilute nitric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 0.40 mL of 0.01 mol/L
hydrochloric acid VS add 6 mL of dilute nitric acid, 30 mL of
dilute ethanol and water to make 50 mL (not more than
0.014%).
(2) Sulfate <1.14> — Dissolve 1.0 g of Isopropylantipyrine
in 30 mL of dilute ethanol, and add 1 mL of dilute
hydrochloric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control so-
lution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS
add 1 mL of dilute hydrochloric acid and 30 mL of dilute
ethanol, and dilute with water to make 50 mL (not more than
0.019%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Isopropylan-
tipyrine in 25 mL of acetone, add 2 mL of dilute acetic acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 2.0 mL of Standard Lead Solution add 2 mL of di-
lute acetic acid, 25 mL of acetone, and dilute with water to
make 50 mL (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Isopropylantipyrine according to Method 3, and perform
the test (not more than 2 ppm).
(5) Antipyrine — Dissolve 1.0 g of Isopropylantipyrine in
10 mL of dilute ethanol, and add 1 mL of sodium nitrite TS
and 1 mL of dilute sulfuric acid: no green color develops.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Isopropylantipy-
rine, previously dried, dissolve in 60 mL of a mixture of acet-
ic acid (100) and acetic anhydride (2:1), and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 23.03 mg of C 14 H 18 N 2
Containers and storage Containers — Tight containers.
Isosorbide
4 yyjufi*
C 6 H I0 O 4 : 146.14
l,4:3,6-Dianhydro-D-glucitol [652-67-5]
Isosorbide contains not less than 98.5% of C 6 H 10 O 4 ,
calculated on the anhydrous basis.
Description Isosorbide occurs as white crystals or masses. It
is odorless, or has a faint, characteristic odor, and has a bit-
ter taste.
It is very soluble in water and in methanol, freely soluble in
ethanol (95), and slightly soluble in diethyl ether.
It is hygroscopic.
Identification (1) To 0.1 g of Isosorbide add 6 mL of
diluted sulfuric acid (1 in 2), and dissolve by heating in a
water bath. After cooling, shake well with 1 mL of a solution
of potassium permanganate (1 in 30), and heat in a water
bath until the color of potassium permanganate disappears.
To this solution add 10 mL of 2,4-dinitrophenylhydrazine
TS, and heat in a water bath: an orange precipitate is formed.
(2) To 2 g of Isosorbide add 30 mL of pyridine and 4 mL
of benzoyl chloride, boil under a reflux condenser for 50
minutes, cool, and pour gradually the solution into 100 mL
of cold water. Filter the formed precipitate by suction
through a glass filter (G3), wash with water, recrystallize
twice from ethanol (95), and dry in a desiccator (in vacuum,
silica gel) for 4 hours: it melts <2.60> between 102°C and 103°
C.
(3) Determine the infrared absorption spectrum of
Isosorbide as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [ a ]™: +45.0- +46.0° (5 g, calcu-
lated on the anhydrous basis, water, 50 mL, 100 mm).
Purity (1) Clarity and color of solution — Take 25 g of
Isosorbide in a Nessler tube, and dissolve in 50 mL of water:
the solution is clear, and has no more color than the follow-
ing control solution.
Control solution: To a mixture of 1.0 mL of Cobaltous
Chloride Stock CS, 3.0 mL of Ferric Chloride Stock CS and
2.0 mL of Cupric Sulfate Stock CS add water to make 10.0
mL. To 3.0 mL of this solution add water to make 50 mL.
(2) Sulfate <1.14>— Perform the test with 2.0 g of Isosor-
bide. Prepare the control solution with 1.0 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.024%).
(3) Heavy metals <1.07> — Proceed with 5.0 g of Isosor-
bide according to Method 1, and perform the test. Prepare
the control solution with 2.5 mL of Standard Lead Solution
JPXV
Official Monographs / Isosorbide Dinitrate
785
(not more than 5 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Isosorbide according to Method 1, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Isosorbide in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 2 mL of the sample solution, add methanol to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 fXL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethanol (95) and cyclohexane (1 : 1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly a mixture of
ethanol (95) and sulfuric acid (9:1) on the plate, and heat at
150°C for 30 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Water <2.48> Not more than 1.5% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 10 g of Isosorbide, calculat-
ed on the anhydrous basis, and dissolve in water to make ex-
actly 100 mL. Determine the optical rotation <2.49>, a D , of
this solution at 20 ± 1°C in a 100-mm cell.
Amount (g) of C 6 H 10 O 4 = a D x 2.1978
Containers and storage Containers — Tight containers.
Isosorbide Dinitrate
NO s
C 6 H 8 N 2 8 : 236.14
l,4:3,6-Dianhydro-D-glucitol dinitrate
[87-33-2]
Isosorbide Dinitrate contains not less than 95.0% of
C 6 H 8 N 2 O g , calculated on the anhydrous basis.
Description Isosorbide Dinitrate occurs as white crystals or
crystalline powder. It is odorless or has a faint odor like that
of nitric acid.
It is very soluble in AfTV-dimethylformamide and in ace-
tone, freely soluble in chloroform and in toluene, soluble in
methanol, in ethanol (95) and in diethyl ether, and practically
insoluble in water.
It explodes if heated quickly or subjected to percussion.
Identification (1) Dissolve 0.01 g of Isosorbide Dinitrate
in 1 mL of water, and dissolve by adding 2 mL of sulfuric
acid cautiously. After cooling, superimpose 3 mL of iron (II)
sulfate TS, and allow to stand for 5 to 10 minutes: a brown
ring is produced at the zone of contact.
(2) Dissolve 0.1 g of Isosorbide Dinitrate in 6 mL of
diluted sulfuric acid (1 in 2) by heating in a water bath. After
cooling, add 1 mL of a solution of potassium permanganate
(1 in 30), stir well, and heat in a water bath until the color of
potassium permanganate disappears. Add 10 mL of 2,4-
dinitro-phenylhydrazine TS, and heat in a water bath: an
orange precipitate is produced.
Optical rotation <2.49> [a]™: + 134 - + 139° (1 g, calculat-
ed on the anhydrous basis, ethanol (95), 100 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Isosorbide Dinitrate in 10 mL of acetone: the solution is clear
and colorless.
(2) Sulfate <1.14> — Dissolve 1.5 g of Isosorbide Dinitrate
in 15 mL of A^A'-dimethylformamide, add 60 mL of water,
cool, and filter. Wash the filter paper with three 20-mL por-
tions of water, combine the washings with the filtrate, and
add water to make 150 mL. To 40 mL of this solution add 1
mL of dilute hydrochloric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.40 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.048%).
(3) Nitrate — Dissolve 0.05 g of Isosorbide Dinitrate in 30
mL of toluene, and extract with three 20-mL portions of
water. Combine the aqueous layers, and wash with two
20-mL portions of toluene. To the aqueous layer add water to
make 100 mL, and use this solution as the sample solution.
Pipet 5.0 mL of Standard Nitric Acid Solution and 25 mL of
the sample solution in each Nessler tube, and add water to
make 50 mL, respectively. To each of them add 0.06 g of
Griss-Romijin's nitric acid reagent, stir well, allow to stand
for 30 minutes, and observe from the side of the Nessler tube:
the sample solution has no more color than the standard solu-
tion.
(4) Heavy metals <1.07> — Dissolve 1.0 g of Isosorbide
Dinitrate in 30 mL of acetone, and add 2 mL of dilute acetic
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 2.0 mL of Standard Lead Solution add 30 mL of ace-
tone, 2 mL of dilute acetic acid and water to make 50 mL
(not more than 20 ppm).
Water <2.48> Not more than 1.5% (0.3 g, direct titration).
Assay Weigh accurately about 0.1 g of Isosorbide Dinitrate
in a Kjeldahl flask as described under the Nitrogen Determi-
nation <1.08>, dissolve in 10 mL of methanol, add 3 g of
Devarda's alloy and 50 mL of water, and connect the flask
with the distillation apparatus as described under the Nitro-
gen Determination <1.08>. Measure exactly 25 mL of 0.05
mol/L sulfuric acid VS in an absorption flask, add 5 drops of
bromocresol green-methyl red TS, and immerse the lower end
of the condenser tube in it. Add 15 mL of a solution of sodi-
um hydroxide (1 in 2) through the funnel, cautiously rinse the
funnel with 20 mL of water, immediately close the clamp at-
tached to the rubber tubing, then begin the distillation with
steam gradually, and continue the distillation until the distil-
late measures 100 mL. Remove the absorption flask, rinse the
end of the condenser tube with a small quantity of water, and
titrate <2.50> the distillate and the rinsings with 0.1 mol/L so-
dium hydroxide VS until the color of the solution changes
from red through light red-purple to light blue-green. Per-
form a blank determination.
Each mL of 0.05 mol/L sulfuric acid VS
786
Isosorbide Dinitrate Tablets / Official Monographs
JP XV
= 11.81 mg of C 6 H 8 N 2 8
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Isosorbide Dinitrate Tablets
Isosorbide Dinitrate Tablets contain not less than
93% and not more than 107% of the labeled amount of
isosorbide dinitrate (C 6 H 8 N 2 8 : 236.14).
Method of preparation Prepare as directed under Tablets,
with Isosorbide Dinitrate.
Identification Weigh a quantity of powdered Isosorbide
Dinitrate Tablets, equivalent to 0.1 g of Isosorbide Dinitrate
according to the labeled amount, add 50 mL of diethyl ether,
shake well, and filter. Measure 5 mL of the filtrate, evaporate
to dryness cautiously, add 1 mL of water to the residue, and
dissolve by adding 2 mL of sulfuric acid cautiously. After
cooling, superimpose 3 mL of iron (II) sulfate TS, and allow
to stand for 5 to 10 minutes: a brown ring is produced at the
zone of contact.
Purity Free nitrate ion — Weigh accurately a quantity of
powdered Isosorbide Dinitrate Tablets, equivalent to 0.05 g
of Isosorbide Dinitrate according to the labeled amount,
transfer to a separator, add 30 mL of toluene, shake
thoroughly, extract with three 20-mL portions of water, and
proceed as directed in Purity (3) under Isosorbide Dinitrate.
Disintegration <6.09> It meets the requirement.
For sublingual tablets, the time limit of the test is 2
minutes, and omit the use of the disk.
Assay Weigh accurately and powder not less than 20 Isosor-
bide Dinitrate Tablets. Weigh accurately a portion of the
powder, equivalent to about 5 mg of isosorbide dinitrate
(C 6 H 8 N 2 8 ), add exactly 50 mL of acetic acid (100), shake for
15 minutes, filter, and use this filtrate as the sample solution.
Separately, weigh accurately about 90 mg of potassium ni-
trate, previously dried at 105°C for 4 hours, dissolve in 5 mL
of water, and add acetic acid (100) to make exactly 100 mL.
Measure exactly 10 mL of this solution, add acetic acid (100)
to make exactly 100 mL, and use this solution as the standard
solution. Measure exactly 2 mL each of the sample solution
and standard solution, add exactly 2.5 mL of salicylic acid
TS to each, shake well, allow to stand for 15 minutes, and
add 10 mL of water. Make them alkaline with about 12 mL
of a solution of sodium hydroxide (2 in 5) while cooling in an
ice bath, and add water to make exactly 50 mL. Perform the
test with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using a solution, prepared with 2
mL of glacial acetic in the same manner, as the blank. Deter-
mine the absorbances, A T and A s , of the subsequent solu-
tions of the sample solution and standard solution at 412 nm,
respectively.
Amount (mg) of isosorbide dinitrate (C 6 H 8 N 2 8 )
= W s x (A T /A S ) x (1/20) x 1.1678
W s : Amount (mg) of potassium nitrate
Containers and storage Containers — Tight containers.
Japanese Encephalitis Vaccine
B*flH5fe r 7^5 1 >
Japanese Encephalitis Vaccine is a liquid for injec-
tion containing inactivated Japanese encephalitis virus.
It conforms to the requirements of Japanese En-
cephalitis Vaccine in the Minimum Requirements for
Biological Products.
Description Japanese Encephalitis Vaccine is a clear or a
slightly whitish turbid and colorless liquid.
Freeze-dried Japanese Encephalitis
Vaccine
Freeze-dried Japanese Encephalitis Vaccine is a
preparation for injection which is dissolved before use.
It contains inactivated Japanese encephalitis virus.
It conforms to the requirements of Freeze-dried
Japanese Encephalitis Vaccine in the Minimum Re-
quirements for Biological Products.
Description Freeze-dried Japanese Encephalitis Vaccine is a
clear or a slightly whitish turbid and colorless liquid on
addition of solvent.
Josamycin
H OH
CH, o
H Mill/' J
HT "" * H
CHa
CH 3
(3R,4R,5S,6R,SR,9R,l0E,12E,l5R)-3-Acetoxy-
5-[2,6-dideoxy-4-0-(3-methylbutanoyl)-3-C-methyl-
a-L-n'£o-hexopyranosyl-(l->4)-3,6-dideoxy-3-
dimethylamino-/?-D-glucopyranosyloxy]-6-formylmethyl-9-
hydroxy-4-methoxy-8-methylhexadeca- 10,1 2-dien-
15-olide [16846-24-5]
Josamycin is a macrolide substance having antibac-
terial activity produced by the growth of Streptomyces
narboensis var. josamyceticus.
It contains not less than 900 fig (potency) and not
more than 1 100 fig (potency) per mg, calculated on the
JPXV
Official Monographs / Josamycin 787
dried basis. The potency of Josamycin is expressed as
mass (potency) of josamycin (C 4 2H 6 9N0 15 ).
Description Josamycin occurs as a white to yellowish white
powder.
It is very soluble in methanol and in ethanol (99.5), and
very slightly soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Josamycin in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Josamycin Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Dissolve 5 mg each of Josamycin and Josamycin
Reference Standard in 1 mL of methanol, add diluted
methanol (1 in 2) to make 100 mL, and use these solutions as
the sample solution and the standard solution, respectively.
Perform the test with 10 [iL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions: the retention
time of the main peak obtained from the sample solution is
the same as that of the peak of josamycin from the standard
solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (2).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Josamycin according to Method 2, and perform the test. Pre-
pare the control solution with 3 .0 mL of Standard Lead Solu-
tion (not more than 30 ppm).
(2) Related substances — Dissolve 50 mg of Josamycin in
5 mL of methanol, add diluted methanol (1 in 2) to make 50
mL, and use this solution as the sample solution. Perform the
test with 10 [iL of the sample solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions. Determine each peak area by the automatic integra-
tion method, and calculate the amounts of josamycin and the
related substances by the area percentage method: the
amounts of the peaks other than josamycin are not more than
6%, and the total of these peaks is not more than 20%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 231 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 5 cm in length, packed with octadecylsilanized sil-
ica gel for liquid chromatography (3 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C
Mobile phase: Dissolve 119 g of sodium perchlorate mono-
hydrate in water to make 1000 mL, and adjust the pH to 2.5
with 1 mol/L hydrochloric acid TS. To 600 mL of this solu-
tion add 400 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
josamycin is about 10 minutes.
Time span of measurement: About 4 times as long as the
retention time of josamycin beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 3 mL of
the sample solution, add diluted methanol (1 in 2) to make
exactly 50 mL, and use this solution as the solution for sys-
tem suitability test. Measure exactly 2 mL of the solution for
system suitability test, and add diluted methanol (1 in 2) to
make exactly 20 mL. Confirm that the peak area of josamy-
cin obtained from 10 /xL of this solution is equivalent to 8 to
12% of that from 10 /xL of the solution for system suitability
test.
System performance: Dissolve about 0.05 g of Josamycin
in 50 mL of 0.1 mol/L potassium dihydrogen phosphate TS,
pH 2.0, and allow to stand at 40°C for 3 hours. Adjust the
pH of this solution to 6.8 to 7.2 with 2 mol/L sodium
hydroxide TS, and add 50 mL of methanol. This solution
contains both josamycin and josamycin SI. When the proce-
dure is run with 10 [iL of this solution under the above oper-
ating conditions, the resolution between the peaks of josamy-
cin SI, which relative retention time to josamycin is about
0.9, and josamycin is not less thanl.5.
System repeatability: When the test is repeated 6 times with
10 [iL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak areas of josamycin is not more than 1.5%.
Loss on drying <2.41> Not more than 1.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium ii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.9 to
8.1 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Josamycin Reference Standard, equivalent to about 30 mg
(potency), dissolve in 5 mL of methanol, add water to make
exactly 100 mL, and use this solution as the standard stock
solution. Keep the standard stock solution at 5°C or below,
and use within 7 days. Take exactly a suitable amount of the
standard stock solution before use, add water to make solu-
tions so that each mL contains 30 fig (potency) and 7.5 fxg
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Josamycin, equivalent to about 30 mg (potency), dissolve in 5
mL of methanol, and add water to make exactly 100 mL.
Take exactly a suitable amount of this solution, add water to
make solutions so that each mL contains 30 /xg (potency) and
7.5 /xg (potency), and use these solutions as the high concen-
tration sample solution and low concentration sample solu-
tion, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
788
Josamycin Propionate / Official Monographs
JP XV
Josamycin Propionate
nJ^
CH 3
CH 3 O VJ-™ H / »-}•
Hi *H
O" "CH 3
CH S
C 45 H 73 N0 16 : 884.06
(3/?,4i?,5S,6i?,8i?,9i?,10£ , ,12£ , ,15/?)-3-Acetoxy-5-
[2,6-dideoxy-4-0-(3-methylbutanoyl)-3-C-methyl-
a-L-n'&o-hexopyranosyl-(l-»4)-3,6-dideoxy-3-
dimethylamino-/?-D-glucopyranosyloxy]-6-formylmethyl-4-
methoxy-8-methyl-9-propanoyloxyhexadeca- 10,12-
dien-15-olide [16846-24-5, Josamycin]
Josamycin Propionate is a derivative of josamycin.
It contains not less than 843 fig (potency) and not
more than 1000,ug (potency) per mg, calculated on the
dried basis. The potency of Josamycin Propionate is
expressed as mass (potency) of josamycin (C 4 2H 6 9N0 15 :
827.99).
Description Josamycin Propionate occurs as a white to
light yellowish white crystalline powder.
It is very soluble in acetonitrile, freely soluble in methanol
and in ethanol (99.5), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Josamycin Propionate in methanol (1 in 100,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Josamycin Propionate
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Dissolve 5 mg each of Josamycin Propionate and
Josamycin Propionate Reference Standard in 50 mL of dilut-
ed acetonitrile (1 in 2), and use these solutions as the sample
solution and the standard solution, respectively. Perform the
test with 10 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions: the retention time of the
peak of josamycin propionate obtained from the sample so-
lution is the same with that of the peak of josamycin
propionate from the standard solution.
Operating conditions—
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Purity (2).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Josamycin Propionate according to Method 2, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 30ppm).
(2) Related substances — Dissolve 50 mg of Josamycin
Propionate in the mobile phase to make 50 mL, and use this
solution as the sample solution. Perform the test with 10 /uL
of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Determine each peak area by the automatic integration
method, and calculate the amounts of each peak other than
josamycin propionate by the area percentage method: the
amount of any peak other than josamycin is not more than 6
%, and the total of these peaks is not more than 22%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 234 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 10 mL of triethylamine add water to
make 1000 mL, and adjust the pH to 4.3 with acetic acid
(100). To 500 mL of this solution add 500 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
josamycin propionate is about 24 minutes.
Time span of measurement: About 3.5 times as long as the
retention time of josamycin propionate beginning after the
solvent peak.
System suitability —
Test for required detectability: Measure exactly 3 mL of
the sample solution, add the mobile phase to make exactly 50
mL, and use this solution as the solution for system suitabil-
ity test. Measure exactly 2mL of the solution for system
suitability test, and add the mobile phase to make exactly 20
mL. Confirm that the peak area of josamycin propionate ob-
tained from 10 [iL of this solution is equivalent to 8 to 12%
of that from 10 /uL of the solution for system suitability test.
System performance: Dissolve 5 mg of josamycin pro-
pionate and 2 mg of josamycin in 50 mL of the mobile phase.
When the procedure is run with 10 /iL of this solution under
the above operating conditions, josamycin and josamycin
propionate are eluted in this order with the resolution be-
tween these peaks being not less than 25.
System repeatability: When the test is repeated 6 times with
10,mL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of josamycin propionate is not more than
1.5%.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium ii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.9 to
8.1 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Josamycin Propionate Reference Standard, equivalent to
about 20 mg (potency), dissolve in 10 mL of methanol, add
JPXV
Official Monographs / Kainic Acid Hydrate
789
1/15 mol/L phosphate buffer solution, pH 5.6 to make ex-
actly 50 mL, and use this solution as the standard stock solu-
tion. Keep the standard stock solution at 5°C or below, and
use within 3 days. Take exactly a suitable amount of the stan-
dard stock solution before use, add 1/15 mol/L phosphate
buffer solution, pH 5.6 to make solutions so that each mL
contains 80 /xg (potency) and 20 /ug (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Josamycin Propionate, equivalent to about 20 mg (potency),
dissolve in 10 mL of methanol, add 1/15 mol/L phosphate
buffer solution, pH 5.6 to make exactly 50 mL. Take exactly
a suitable amount of this solution, add 1/15 mol/L phos-
phate buffer solution, pH 5.6 to make solutions so that each
mL contains 80 /xg (potency) and 20 /xg (potency), and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Kainic Acid Hydrate
H;C
H 2
C 10 H 15 NO 4 .H 2 O: 231.25
(25,3S,4S)-3-(Carboxymethyl)-
4-( 1 -methylethenyl)pyrrolidine-2-carboxylic acid
monohydrate [487-79-6, anhydride]
Kainic Acid Hydrate, when dried, contains not less
than 99.0% of kainic acid (C 10 H 15 NO 4 : 213.23).
Description Kainic Acid Hydrate occurs as white crystals or
crystalline powder. It is odorless, and has an acid taste.
It is sparingly soluble in water and in warm water, very
slightly soluble in acetic acid (100) and in ethanol (95), and
practically insoluble in diethyl ether.
It dissolves in dilute hydrochloric acid and in sodium
hydroxide TS.
The pH of its solution (1 in 100) is between 2.8 and 3.5.
Melting point: about 252°C (with decomposition).
Identification (1) To 5 mL of a solution of Kainic Acid
Hydrate (1 in 5000) add 1 mL of ninhydrin TS, and warm in a
water bath at a temperature between 60°C and 70°C for 5
minutes: a yellow color is produced.
(2) Dissolve 0.05 g of Kainic Acid Hydrate in 5 mL of a-
cetic acid (100), and add 0.5 mL of bromine TS: the color of
bromine disappears immediately.
Optical rotation <2.49> [a]^ : - 13
mL, 200 mm).
■17° (0.5 g, water, 50
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Kainic Acid Hydrate in 10 mL of water: the solution is
clear and colorless.
(2) Chloride <1.03>— Take 0.5 g of Kainic Acid Hydrate
in a platinum crucible, dissolve in 5 mL of sodium carbonate
TS, and evaporate on a water bath to dryness. Heat the cruci-
ble slowly at first, and then ignite until the sample is almost
incinerated. After cooling, add 12 mL of dilute nitric acid to
the residue, dissolve by warming, and filter. Wash the residue
with 15 mL of water, combine the washings and the filtrate,
and add water to make 50 mL. Perform the test using this so-
lution as the test solution.
Control solution: Add 5 mL of sodium carbonate TS to
0.30 mL of 0.01 mol/L hydrochloric acid VS, and proceed as
directed above (not more than 0.021%).
(3) Sulfate <1.14>— Dissolve 0.5 g of Kainic Acid Hydrate
in 40 mL of water by warming. Cool, add 1 mL of dilute
hydrochloric acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.30 mL of 0.005 mol/L sulfuric acid VS
(not more than 0.028%).
(4) Ammonium — Take 0.25 g of Kainic Acid Hydrate,
and perform the test. Prepare the control solution with 5.0
mL of Standard Ammonium Solution (not more than 0.02
%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of Kainic
Acid Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(6) Arsenic <1.11> — Dissolve 1.0 g of Kainic Acid Hy-
drate in 5 mL of dilute hydrochloric acid, and perform the
test with this solution as the test solution (not more than 2
ppm).
(7) Amino acid and other imino acid — Dissolve 0.10 g of
Kainic Acid Hydrate in 10 mL of water, and use this solution
as the sample solution. Pipet 2 mL of this solution, and add
water to make exactly 100 mL. Pipet 1 mL of this solution,
add water to make exactly 20 mL, and use this solution as the
standard solution. Perform the test as directed under Thin-
layer Chromatography <2.03> with these solutions. Spot 10
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with the supernatant liquid of a mixture of water, 1-
butanol and acetic acid (100) (5:4:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly a solution of nin-
hydrin in acetone (1 in 50) on the plate, and dry the plate at
80°C for 5 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> 6.5 - 8.5% (1 g, 105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.4 g of Kainic Acid Hy-
drate, previously dried, and dissolve in 50 mL of warm water,
cool and titrate <2.50> with 0.1 mol/L sodium hydroxide VS
(indicator: 10 drops of bromothymol blue TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 21.32 mg of C 10 H 15 NO 4
Containers and storage Containers — Tight containers.
790
Kainic Acid and Santonin Powder / Official Monographs
JP XV
Kainic Acid and Santonin Powder
a 4 =>»■-«-> r-=>tt
Kainic Acid and Santonin Powder contains not less
than 9.0% and not more than 11.0% of santonin
(Ci 5 H 18 3 : 246.30), and not less than 1.80% and not
more than 2.20% of kainic acid hydrate (C 10 H 15 NO 4 .H
2 0: 231.25).
Method of preparation
Santonin
Kainic Acid Hydrate
Starch, Lactose Hydrate or
their mixture
100 g
20 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Kainic Acid and Santonin Powder occurs as a
white powder.
Identification (1) Shake 1 g of Kainic Acid and Santonin
Powder with 10 mL of chloroform, and filter [use the residue
for the test (2)]. Distil off the chloroform of the filtrate, and
dissolve the residue in 2 mL of potassium hydroxide-ethanol
TS: a red color is produced (santonin).
(2) Shake the residue obtained in (1) with 20 mL of warm
water, filter, and to 1 mL of the filtrate add 10 mL of water
and 1 mL of ninhydrin-L-ascorbic acid TS. Warm in a water
bath between 60°C and 70°C for 5 minutes: a yellow color is
produced (kainic acid).
Assay (1) Santonin — Weigh accurately about 0.25 g of
Kainic Acid and Santonin Powder, add 20 mL of ethanol
(95), shake thoroughly for 5 minutes, and filter. Wash the
residue with three 10-mL portions of ethanol (95), and filter.
Combine the filtrate and the washings, and add ethanol (95)
to make exactly 50 mL. Pipet 2 mL of this solution, add
ethanol (95) to make exactly 100 mL, and use this solution as
the sample solution. Weigh accurately about 25 mg of santo-
nin for assay, proceed in the same manner as the sample solu-
tion, and use the obtained solution as the standard solution.
Determine the absorbances, A T and A s , of these solutions at
240 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>.
Amount (mg) of santonin (C 15 H 18 3 )
= W s x(A T /A s )
W s : Amount (mg) of santonin for assay
(2) Kainic acid — Weigh accurately about 1 .25 g of Kainic
Acid and Santonin Powder, add 20 mL of diluted pyridine (1
in 10), shake thoroughly for 5 minutes, and filter. Wash the
residue with three 10-mL portions of diluted pyridine (1 in
10), and filter. Combine the filtrate and the washings, and
add diluted pyridine (1 in 10) to make exactly 50 mL. Pipet 2
mL of this solution, add diluted pyridine (1 in 10) to make ex-
actly 25 mL, and use this solution as the sample solution.
Dissolve about 25 mg of kainic acid for assay, previously
dried at 105°C for 4 hours and accurately weighed, in diluted
pyridine (1 in 10) to make exactly 50 mL. Pipet 2 mL of this
solution, add diluted pyridine (1 in 10) to make exactly 25
mL, and use this solution as the standard solution. Pipet 2
mL each of the sample solution and standard solution, add 2
mL of ninhydrin-L-ascorbic acid TS, and heat on a water
bath for 30 minutes. After cooling immediately, shake
vigorously for 2 minutes, add water to make exactly 20 mL,
and allow to stand for 15 minutes. Determine the absor-
bances, A T and A s , of these solutions at 425 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using the
solution prepared in the same manner with 2 mL of diluted
pyridine (1 in 10) instead of the sample solution as the blank.
Amount (mg) of kainic acid hydrate (QoH^NCvHjO)
= W s x(A T /A s )x 1.0845
W s : Amount (mg) of kainic acid for assay
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Kallidinogenase
[9001-01-8]
Kallidinogenase is an enzyme obtained from healthy
porcine pancreas, and has kinin-releasing activity
based on cleavage of kininogen.
It contains not less than 25 Kallidinogenase Units per
mg. Usually, it is diluted with Lactose Hydrate or the
like.
Kallidinogenase contains not less than 90% and not
more than 110% of the labeled Units.
Description Kallidinogenase occurs as a white to light
brown powder. It is odorless or has a faint, characteristic
odor.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
The pH of a solution of Kallidinogenase (1 in 300) is be-
tween 5.5 and 7.5.
Identification (1) Weigh accurately an appropriate
amount of Kallidinogenase according to the labeled Units,
and dissolve in 0.05 mol/L phosphate buffer solution, pH 7.0
to prepare a solution containing 10 Kallidinogenase Units per
mL. Pipet 5 mL of this solution, and add exactly 1 mL of
trypsin inhibitor TS and 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 10 mL. Pipet 4 mL each of this
solution into two separate test tubes, add exactly 1 mL each
of aprotinin TS and 0.05 mol/L phosphate buffer solution,
pH 7.0 separately to each test tube, allow them to stand at
room temperature for 20 minutes, and use these solutions as
the sample solutions 1 and 2. Separately, pipet 1 mL of tryp-
sin inhibitor TS, and add 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 10 mL. Pipet 4 mL each of this
solution into two separate test tubes, add exactly 1 mL each
of aprotinin TS and 0.05 mol/L phosphate buffer solution,
pH 7.0 separately to each tube, allow them to stand at room
temperature for 20 minutes, and use these solutions as the
JPXV
Official Monographs / Kallidinogenase 791
sample solutions 3 and 4. Then, pipet 2.5 mL of substrate TS
for kallidinogenase assay (1), previously warmed at 30.0 ±
0.5°C for 5 minutes, place in a 10-mm cell, add exactly 0.5
mL of the sample solution 1 warmed at 30.0 ± 0.5°C for 5
minutes, and start simultaneously a chronograph. Perform
the test at 30.0 ± 0.5°C as directed under Ultraviolet-visible
Spectrophotometry <2.24> using water as the blank, and de-
termine the absorbances at 405 nm, A t _ 2 and A,_ 6 , of this so-
lution, after having allowed it to stand for exactly 2 and 6
minutes. Perform the same test with the sample solutions 2, 3
and 4, and determine the absorbances, A 2 _ 2 , A 2 . s , A 3 _ 2 , A 3 _ 6 ,
A 4 _ 2 and A 4 _ 6 , of these solutions. Calculate /by using the fol-
lowing equation: the value of / does not exceed 0.2.
/= (^i-6
A i- 2 ) ~ (^3-6 ~ Aj_ 2 )
(2) Pipet 2.9 mL of substrate TS for kallidinogenase as-
say (2), previously warmed at 30.0 ± 0.5°C for 5 minutes,
place in a 10-mm cell, add exactly 0.1 mL of the sample solu-
tion obtained in the Assay, and start simultaneously a
chronograph. Perform the test at 30.0 ± 0.5°C as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and de-
termine the change of the absorbance at 253 nm for 4 to 6
minutes. Separately, pipet 1 mL of trypsin inhibitor TS, and
add 0.05 mol/L phosphate buffer solution, pH 7.0 to make
exactly 10 mL. Add exactly 0.1 mL of this solution to exactly
2.9 mL of substrate TS for kallidinogenase assay (2), previ-
ously warmed at 30.0 ± 0.5°C for 5 minutes, and use this so-
lution as the blank. If the rate of change in the absorbance
remains constant, determine the change of absorbance per 1
minute, A, and calculate R by using the following equation:
the value of R is between 0.12 and 0.16.
R = (,4/0.0383) x [\/(a - b)}
a: Amount (mg) of Kallidinogenase in 1 mL of the sample
solution.
b: Amount (Unit) of kallidinogenase in 1 mg of Kal-
lidinogenase obtained in the Assay.
Specific activity Perform the test with Kallidinogenase as
directed under Nitrogen Determination <1.08> to determine
the nitrogen content, convert lmg of nitrogen (N: 14.01) into
6.25 mg of protein, and calculate the specific activity using
the amount (Units) of Kallidinogenase obtained in the Assay:
it is not less than 100 Kallidinogenase Units per lmg of pro-
tein.
Purity (1) Fat — To 1 .0 g of Kallidinogenase add 20 mL of
diethyl ether, extract with occasional shaking for 30 minutes,
and filter. Wash the residue with 10 mL of diethyl ether, com-
bine the washing with the filtrate, evaporate the diethyl ether,
and dry the residue at 105°C for 2 hours: the mass of the
residue is not more than 1 mg.
(2) Kininase —
(i) Bradykinin solution: Weigh an appropriate amount of
bradykinin, and dissolve in gelatin-phosphate buffer solu-
tion, pH 7.4 to prepare a solution containing 0.200 /xg of
bradykinin per mL.
(ii) Kallidinogenase solution: Weigh accurately a suitable
amount of Kallidinogenase according to the labeled unit, dis-
solve in gelatin-phosphate buffer solution, pH 7.4 to make a
solution containing 1 unit of kallidinogenase per mL.
(iii) Sample solution: Pipet 0.5 mL of bradykinin solu-
tion, warm at 30 ± 0.5°C for 5 minutes, then add exactly 0.5
mL of kallidinogenase solution previously warmed at 30 ±
0.5°C for 5 minutes, and mix immediately. After allow this
solution to stand at 30 ± 0.5 °C for exactly 150 seconds, add
exactly 0.2 mL of a solution of trichloroacetic acid (1 in 5),
and shake. Boil for 3 minutes, then cool in ice immediately,
centrifuge, and allow to stand at a room temperature for 15
minutes. Pipet 0.5 mL of the supernatant liquid, add exactly
0.5 mL of gelatin-tris buffer solution, pH 8.0, and mix. Pipet
0. 1 mL of this solution, add exactly 0.9 mL of trichloroacetic
acid-gelatin-tris buffer solution, and mix. Pipet 0.2 mL of
this solution, add exactly 0.6 mL of trichloroacetic acid-
gelatin-tris buffer solution, shake, and use this solution as the
sample solution.
(iv) Control solution: Proceed with 0.5 mL of gelatin-
phosphate buffer solution, pH 7.4 as described in (iii), and
use the solution so obtained as the control solution.
(v) Procedure: Add 0.1 mL of anti-bradykinin antibody
TS to anti-rabbit antibody-coated wells of a 96-well
microplate, shake, and allow to stand at a constant tempera-
ture of about 25 C C for 1 hour. Remove the anti-bradykinin
antibody TS, add 0.3 mL of phosphate buffer solution for
microplate washing to the wells, then remove. Repeat this
procedure 3 times, take off the washings thoroughly, then
add 100 //L each of the sample solution and control solution,
and 50 [iL of gelatin-phosphate buffer solution, pH 7.0,
shake, and allow to stand at a constant temperature of about
25 °C for 1 hour. Then add 50 ^L of peroxidase-labeled
bradykinin TS, shake, and allow to stand in a cold place for a
night.
Take off the solution, add 0.3 mL of phosphate buffer so-
lution for microplate washing, and remove. Repeat this
procedure more 4 times, take off the washings thoroughly,
add 100 //L of substrate solution for peroxidase determina-
tion, and allow to stand at a constant temperature of about
25 °C for exactly 30 minutes while protecting from light.
Then add 100 /xL of diluted sulfuric acid (23 in 500), shake,
and determine the absorbance at 490 - 492 nm.
Separately, dissolve a suitable amount of bradykinin in
gelatin-phosphate buffer solution, pH 7.0 to make solutions
containing exactly 100 ng, 25 ng, 6.25 ng, 1.56 ng, 0.39 ng
and 0.098 ng of bradykinin per mL, and use these solutions
as the standard solution (1), the standard solution (2), the
standard solution (3), the standard solution (4), the standard
solution (5) and the standard solution (6), respectively. Use 1
mL of gelatin-phosphate buffer solution, pH 7.0 as the stan-
dard solution (7). To each of the well add 50 /xh each of the
standard solutions and 100 [iL of trichloroacetic acid-gelatin-
tris buffer solution, and proceed in the same manner as for
the sample solution and for the control solution.
Prepare the standard curve from the amounts of bradyki-
nin in the standard solutions and their absorbances, and de-
termine the amount of bradykinin, B T (pg) and B s (pg), of
the sample solution and the control solution.
The absorbance is usually determined by using a spec-
trophotometer for microplate. Since the wells are used as the
cell for absorbance determination, take care for dirt and
scratch of the well. Light pass length of the well is changeable
by the amount of the liquid, exact addition of the liquid is
necessary.
(vi) Judgment: The value R calculated by the following
equation is not less than 0.8.
R = (B T /B S )
792 Kallidinogenase / Official Monographs
JP XV
(3) Trypsin-like substances — Pipet 4 mL of the sample
stock solution prepared for the Assay, add exactly 1 mL of
trypsin inhibitor TS and 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make exactly 10 mL, and use this solution as
the sample solution. Pipet 2.5 mL of substrate TS for kal-
lidinogenase assay (1), previously warmed at 30 ± 0.5°C for
5 minutes, place in a 10-mm cell, add exactly 0.5 mL of the
sample solution, warmed at 30 ± 0.5°C for 5 minutes, and
start simultaneously a chronograph. Perform the test at 30 ±
0.5°C as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24> using water as the blank, and determine the absor-
bances at 405 nm, A 2 and A 6 , of this solution after having al-
lowed it to stand for exactly 2 and 6 minutes. Separately,
pipet 4 mL of the sample stock solution prepared for the As-
say, add 0.05 mol/L phosphate buffer solution, pH 7.0 to
make exactly 10 mL, and use this solution as the control solu-
tion. Perform the same test with the control solution, and de-
termine the absorbances, A 2 and A^. Calculate Tby using the
following equation: the value of T does not exceed 0.05.
T = {(A' 6 - Ai) - (A 6 - A 2 )/(A' 6 - AQ]
(4) Protease — Weigh accurately an appropriate amount
of Kallidinogenase according to the labeled Units, dissolve in
0.05 mol/L phosphate buffer solution, pH 7.0 to prepare a
solution containing 1 Kallidinogenase Unit per mL, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, place in a test tube, and allow to stand at 35 ±
0.5°C for 5 minutes. Then, pipet 5 mL of substrate TS for
kallidinogenase assay (3), previously warmed to 35 ± 0.5 °C,
add quickly to the sample solution in the test tube, and allow
to stand at 35 ± 0.5 C C for exactly 20 minutes. Then add ex-
actly 5 mL of trichloroacetic acid TS, shake well, allow to
stand at room temperature for 1 hour, and filter through a
membrane filter (5 jum in pore size). Discard the first 3 mL of
the filtrate, and determine the absorbance, A, of the subse-
quent filtrate at 280 nm within 2 hours as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using water as
the blank. Separately, pipet 1 mL of the sample solution, add
exactly 5 mL of trichloroacetic acid TS, shake well, and add
exactly 5 mL of the substrate TS for kallidinogenase assay
(3). Proceed in the same manner as described for the sample
solution, and determine the absorbance, A , of this solution.
Calculate the value of (A-A ): it is not more than 0.2.
Loss on drying <2.41> Not more than 2.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 3% (0.5g, 650 -
750°C).
Kinin-releasing activity
(i) Kallidinogenase solution: Weigh accurately a suitable
amount of Kallidinogenase, according to the labeled unit,
dissolve in 0.02 mol/L phosphate buffer solution, pH 8.0 to
make a solution containing 0.1 unit of kallidinogenase per
mL. Perform this procedure by using glassware.
(ii) Sample solution: Pipet 0.5 mL of kininogen TS,
warm at 30 ± 0.5 °C for 5 minutes, then add exactly 0.5 mL
of kallidinogenase solution previously warmed at 30 ± 0.5 °C
for 5 minutes, and mix immediately. After allow this solution
to stand at 30 ± 0.5°C for exactly 2 minutes, add exactly 0.2
mL of a solution of trichloroacetic acid (1 in 5), and shake.
Boil for 3 minutes, then cool in ice immediately, centrifuge,
and allow to stand at a room temperature for 15 minutes.
Pipet 0.5 mL of the supernatant liquid, add exactly 0.5 mL of
gelatin-tris buffer solution, pH 8.0, and shake. Pipet 0.1 mL
of this solution, add exactly 1.9 mL of trichloroacetic acid-
gelatin-tris buffer solution, shake, and use this solution as the
sample solution.
(iii) Procedure: Perform the test with the sample solution
as directed in the Purity (2), and determine the amount, B
(pg), of kinin per well. The kinin-releasing activity per 1 unit
of Kallidinogenase calculated by the following equation is
not less than 500 ng bradykinin equivalent/min/unit.
Kinin-releasing activity (ng bradykinin equivalent /min/
unit) per 1 unit of Kallidinogenase = B x 4.8
Assay Weigh accurately an appropriate amount of Kal-
lidinogenase according to the labeled Units, dissolve in 0.05
mol/L phosphate buffer solution, pH 7.0 to prepare a solu-
tion containing about 10 Kallidinogenase Units per mL, and
use this solution as the sample stock solution. Pipet 4 mL of
the sample stock solution, add exactly 1 mL of trypsin inhibi-
tor TS and 0.05 mol/L phosphate buffer solution, pH 7.0 to
make exactly 10 mL, and use this solution as the sample
solution. Pipet 2.5 mL of substrate TS for kallidinogenase
assay (1), previously warmed at 30±0.5°C for 5 minutes,
place in a 1-cm cell, add exactly 0.5 mL of the sample solu-
tion, warmed at 30±0.5°C for 5 minutes, and start simul-
taneously a chronograph. Perform the test at 30±0.5°C as
directed under the Ultraviolet-visible Spectrophotometry
<2.24> using water as the blank, and determine the absor-
bances at 405 nm, A T2 and A T6 , of this solution after allowing
to stand for exactly 2 and 6 minutes. Separately, dissolve Kal-
lidinogenase Reference Standard in 0.05 mol/L phosphate
buffer solution, pH 7.0 to make a solutin so that each mL
contains exactly 10 Units, and use this solution as the stan-
dard stock solution. Pipet 4 mL of the stock solution, add ex-
actly 1 mL of trypsin inhibitor TS and 0.05 mol/L phosphate
buffer solution, pH 7.0 to make exactly 10 mL, and use this
solution as the standard solution. Take exactly 0.5 mL of the
standard solution, perform the test in the same manner as de-
scribed for the sample solution, and determine the absor-
bances, A S2 and A S6 , of the solution after allowing to stand
for exactly 2 and 6 minutes. Separately, take exactly 1 mL of
the trypsin inhibitor TS, and add 0.05 mol/L phosphate
buffer solution, pH 7.0 to make exactly 10 mL. Pipet 0.5 mL
of this solution, perform the test in the same manner as de-
scribed for the sample solution, and determine the absor-
bances, A 02 and A 06 , of the solution after allowing to stand
for exactly 2 and 6 minutes.
Units per 1 mg of Kallidinogenase
= (A T6 -A T2 ) - (A Q6 -A 02 ) x Ws x j
(As 6 -A S2 )-(A 06 -A 02 ) a b
W s : Amount (Units) of Kallidinogenase Reference Standard
a: Volume (mL) of the standard stock solution
b: Amount (mg) of Kallidinogenase in 1 mL of the sample
stock solution
Cantainers and storage Containers — Tight containers.
JPXV
Official Monographs / Kanamycin Monosulfate
793
Kanamycin Monosulfate
t>-)-7^ >>—
• H 2 SOj
C 18 H3 6 N 4 Oii.H 2 S04: 582.58
3-Amino-3-deoxy-a-D-glucopyranosyl-(l— *6)-
[6-amino-6-deoxy-a-D-glucopyranosyl-(l— ►4)]-2-deoxy-
D-streptamine monosulfate [25389-94-0]
Kanamycin Monosulfate is the sulfate of an
aminoglycoside substance having antibacterial activity
produced by the growth of Streptomyces kanamyceti-
cus.
It contains not less than 750 fig (potency) and not
more than 832 fig (potency) per mg, calculated on the
dried basis. The potency of Kanamycin Monosulfate
is expressed as mass (potency) of kanamycin
(C 18 H 3 6N 4 0ii: 484.50).
Description Kanamycin Monosulfate occurs as a white
crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 50 mg of Kanamycin Monosul-
fate in 3 mL of water, and add 6 mL of anthrone TS: a blue-
purple color develops.
(2) Dissolve 20 mg each of Kanamycin Monosulfate and
Kanamycin Monosulfate Reference Standard in 1 mL of
water, and use these solutions as the sample solution and the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 ftL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with the supernatant layer of a mixture of chloroform, am-
monia solution (28) and methanol (2:1:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly a solution of
0.2% ninhydrin-water saturated 1-butanol TS on the plate,
and heat at 100°C for 10 minutes: the principal spots ob-
tained from the sample solution and the standard solution
show a purple-brown color and the same i?f value.
(3) To a solution of Kanamycin Monosulfate (1 in 5) add
1 drop of barium chloride TS: a white precipitate is formed.
Optical rotation <2.49> [«]": +112- +123° (0.2 g calcu-
lated on the dried basis, water, 20 mL, 100 mm).
Sulfuric acid Weigh accurately about 0.25 g of Kanamycin
Monosulfate, dissolve in 100 mL of water, adjust the pH to
11.0 with ammonia solution (28), add exactly 10 mL of
0.1 mol/L barium chloride VS, and titrate <2.50> with 0.1
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
until the color of the solution, blue-purple, disappears (indi-
cator: 0.5 mg of phthalein purple). At a near of the end-point
add 50 mL of ethanol (99.5). Perform a blank determination
in the same manner. The amount of sulfuric acid (S0 4 ) is not
less than 15.0% and not more than 17.0%, calculated on the
dried basis.
Each mL of 0.1 mol/L barium chloride VS
= 9.606 mg of S0 4
Purity (1) Heavy metals <1.07>— Proceed with 2.0 g of
Kanamycin Monosulfate according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Kanamycin Monosulfate according to Method 4, and per-
form the test (not more than 1 ppm).
(3) Related substances — Dissolve 0.30 g of Kanamycin
Monosulfate in water to make exactly 10 mL, and use this so-
lution as the sample solution. Separately, dissolve 45 mg of
Kanamycin Monosulfate Reference Standard in water to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 1 fiL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a so-
lution of potassium dihydrogen phosphate (3 in 40) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly a
solution of ninhydrin in 1-butanol (1 in 100) on the plate, and
heat at 110°C for 10 minutes: the spot other than the prin-
cipal spot obtained from the sample solution is not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 4.0% (5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Weigh accurately an amount of
Kanamycin Monosulfate Reference Standard, previously
dried, equivalent to about 20 mg (potency), dissolve in dilut-
ed phosphate buffer solution, pH 6.0 (1 in 2) to make exactly
50 mL, and use this solution as the standard stock solution.
Keep the standard stock solution between 5 and 15°C and use
within 30 days. Take exactly a suitable amount of the stan-
dard stock solution before use, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 20 fig (potency) and 5 fig (potency), and use these so-
lutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Kanamycin Monosulfate, equivalent to about 20 mg (poten-
cy), and dissolve in water to make exactly 50 mL. Take ex-
actly a suitable amount of this solution, add 0.1 mol/L phos-
phate buffer solution, pH 8.0 to make solutions so that each
794 Kanamycin Sulfate / Official Monographs
JP XV
mL contains 20 fig (potency) and 5 fig (potency), and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Well-closed contain-
ers.
Kanamycin Sulfate
xH 2 S0 4
NH;
C 18 H 36 N40„.xH 2 S04
3-Amino-3-deoxy-a-D-glucopyranosyl-(l-
[6-amino-6-deoxy-a-D-glucopyranosyl-(l-
D-streptamine sulfate [133-92-6]
•6)-
>4)]-2-deoxy-
Kanamycin Sulfate is the sulfate of an aminoglyco-
side substance having antibacterial activity produced
by the growth of Streptomyces kanamyceticus.
It contains not less than 690 fig (potency) and not
more than 740 fig (potency) per mg, calculated on the
dried basis. The potency of Kanamycin Sulfate is ex-
pressed as mass (potency) of kanamycin (Ci8H 36 N 4 0n:
484.50).
Description Kanamycin Sulfate occurs as a white to yellow-
ish white powder.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 20 mg each of Kanamycin Sul-
fate and Kanamycin Monosulfate Reference Standard in 1
mL of water, and use these solutions as the sample solution
and the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 fiL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of chloroform, ammonia solu-
tion (28) and methanol (2:1:1) to a distance of about 10 cm,
and air-dry the plate. Spray evenly 0.2% ninhydrin-water
saturated 1-butanol TS on the plate, and heat at 100°C for 10
minutes: the principal spots obtained from the sample solu-
tion and the standard solution show a purple-brown color
and the same Rf value.
(2) A solution of Kanamycin Sulfate (1 in 10) responds to
the Qualitative Test <1.09> (1) for sulfate.
Optical rotation <2.49> [ a ]g>: + 103 - + 115° (0.5 g calcu-
lated on the dried basis, water, 50 mL, 100 mm).
of Kanamycin Sulfate in 20 mL of water is between 6.0 and
7.5.
Purity (1) Clarity and color of solution — Dissolve 1.5 g of
Kanamycin Sulfate in 5 mL of water: the solution is clear and
colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Kanamy-
cin Sulfate according to Method 4, and perform the test. Pre-
pare the control solution with 3.0 mL of Standard Lead Solu-
tion (not more than 30 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Kanamycin Sulfate according to Method 3, and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve 0.30 g of Kanamycin
Sulfate in water to make exactly 10 mL, and use this solution
as the sample solution. Separately, dissolve 9.0 mg of
Kanamycin Monosulfate Reference Standard in water to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 1 fiL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a so-
lution of potassium dihydrogen phosphate (3 in 40) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly a
solution of ninhydrin in 1-butanol (1 in 100) on the plate, and
heat at 110°C for 10 minutes: the spot other than the prin-
cipal spot obtained from the sample solution is not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 5.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er having pH 7.8 to 8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Kanamycin Monosulfate Reference Standard, previously
dried, equivalent to about 20 mg (potency), dissolve in dilut-
ed phosphate buffer solution, pH 6.0 (1 in 2) to make exactly
50 mL, and use this solution as the standard stock solution.
Keep the standard stock solution at 5 to 15°C and use within
30 days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solu-
tion, pH 8.0 to make solutions so that each mL contains 20
fig (potency) and 5 fig (potency), and use these solutions as
the high concentration standard solution and low concentra-
tion standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Kanamycin Sulfate, equivalent to about 20 mg (potency),
and dissolve in water to make exactly 50 mL. Take exactly a
suitable amount of this solution, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 20 fig (potency) and 5 fig (potency), and use these so-
lutions as the high concentration sample solution and low
concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
JPXV
Official Monographs / Ketamine Hydrochloride
795
Kaolin
Kaolin is a native, hydrous aluminum silicate.
Description Kaolin occurs as white or nearly white, frag-
mentary masses or powder. It has a slightly clay-like odor.
It is practically insoluble in water, in ethanol (99.5) and in
diethyl ether.
It is insoluble in dilute hydrochloric acid and in sodium
hydroxide TS.
When moistened with water, it darkens and becomes plas-
tic.
Identification (1) Heat 1 g of Kaolin with 10 mL of water
and 5 mL of sulfuric acid in a porcelain dish, and evaporate
the mixture nearly to dryness. Cool, add 20 mL of water, boil
for 2 to 3 minutes, and filter: the color of the residue is gray.
(2) The filtrate obtained in (1) responds to the Qualitative
Tests <].09> (1), (2) and (4) for aluminum salt.
Purity (1) Acid or alkali — Add 25 mL of water to 1 .0 g of
Kaolin, agitate thoroughly, and filter: the pH <2.54> of the
filtrate is between 4.0 and 7.5.
(2) Acid-soluble substances — Add 20 mL of dilute
hydrochloric acid to 1.0 g of Kaolin, agitate for 15 minutes,
and filter. Evaporate 10 mL of the filtrate to dryness, and
heat strongly between 450°C and 550°C to constant mass: the
mass of the ignited residue is not more than 0.010 g.
(3) Carbonate — Stir 1.0 g of Kaolin with 5 mL of water,
then add 10 mL of diluted sulfuric acid (1 in 2): no efferves-
cence occurs.
(4) Heavy metals <1.07> — Boil 1.5 g of Kaolin gently with
50 mL of water and 5 mL of hydrochloric acid for 20 minutes
with frequent agitation, cool, centrifuge, and separate the su-
pernatant liquid. Wash the precipitate twice with 10 mL of
water, centrifuge each time, and combine the supernatant liq-
uid and the washings. Add dropwise ammonia solution (28)
to this solution until a slight precipitate occurs, then add di-
lute hydrochloric acid dropwise while agitating strongly to
complete solution. Add 0.45 g of hydroxylammonium chlo-
ride, and heat. Cool, add 0.45 g of sodium acetate trihydrate
and 6 mL of dilute acetic acid, filter if necessary, and wash
with 10 mL of water. Combine the filtrate and the washings,
and add water to make 150 mL. Perform the test using 50 mL
of this solution as the test solution. To 2.5 mL of Standard
Lead Solution add 0.15 g of hydroxylammonium chloride,
0.15 g of sodium acetate trihydrate, 2 mL of acetic acid (31)
and water to make 50 mL, and use this solution as the control
solution (not more than 50 ppm).
(5) Iron <1.10> — Add 10 mL of dilute hydrochloric acid
to 40 mg of Kaolin, and heat for 10 minutes with shaking in a
water bath. After cooling, add 0.5 g of L-tartaric acid, dis-
solve with shaking, prepare the test solution with this solu-
tion according to Method 2, and perform the test according
to Method B. Prepare the control solution with 2.0 mL of
Standard Iron Solution (not more than 500 ppm).
(6) Arsenic <1.11> — Add 5 mL of water and 1 mL of sul-
furic acid to 1.0 g of Kaolin, and heat on a sand bath until
white fumes begin to evolve. Cool, and add water to make 5
mL. Perform the test with this solution as the test solution
(not more than 2 ppm).
(7) Foreign matter — Place 5 g of Kaolin in a beaker, add
100 mL of water, stir, and decant to leave sand. Repeat this
procedure several times with 100-mL portions of water: no
sandy residue remains.
Loss on ignition <2.43> Not more than 15.0% (1 g, 600°C,
5 hours).
Plasticity Add 7.5 mL of water to 5.0 g of Kaolin, and
agitate thoroughly: the resultant mass has no remarkable
fluidity.
Containers and storage Containers — Well-closed contain-
ers.
Ketamine Hydrochloride
*r9 5 >i&m^
I
and enantiomer
C 13 H 16 ClNO.HCl: 274.19
(2i?S)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone
monohydrochloride [1867-66-9]
Ketamine Hydrochloride, when dried, contains not
less than 99.0% of C 13 H 16 ClNO.HCl.
Description Ketamine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is very soluble in formic acid, freely soluble in water and
in methanol, sparingly soluble in ethanol (95) and in acetic
acid (100), and practically insoluble in acetic anhydride and
in diethyl ether.
A solution of Ketamine Hydrochloride (1 in 10) shows no
optical rotation.
Melting point: about 258°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ketamine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 3000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Keta-
mine Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Ketamine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> (2) for chloride.
Absorbance <2.24> E\ 0/ ° m (269 nm): 22.0-24.5 (after
drying, 0.03 g, 0.1 mol/L hydrochloric acid TS, 100 mL).
pH <2.54> Dissolve 1.0 g of Ketamine Hydrochloride in 10
mL of freshly boiled and cooled water: the pH of the solution
is between 3.5 and 4.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
796 Ketoprofen / Official Monographs
JP XV
Ketamine Hydrochloride in 5 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Ketamine
Hydrochloride according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ketamine Hydrochloride, according to Method 1 , and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.5 g of Ketamine
Hydrochloride in 10 mL of methanol and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of cyclohexane and isopropylamine
(49:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly Dragendorff's TS for spraying on the plate, dry
the plate, and then spray evenly hydrogen peroxide TS: the
spots other than the principal spot from the sample solution
is not more intense than the spot from the standard solution.
Loss on drying <2.41> Not more then 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Ketamine
Hydrochloride, previously dried, dissolve in 1 mL of formic
acid, add 70 mL of a mixture of acetic anhydride and acetic
acid (100) (6:1), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 27.42 mg of C 13 H, 6 C1N0.HC1
Containers and storage Containers — Tight containers.
Ketoprofen
H CH 3
and enantiomer
C 16 H 14 3 : 254.28
(2/?5')-2-(3-Benzoylphenyl)propanoic acid [22071-15-4]
Ketoprofen, when dried, contains not less than
99.0% and not more than 100.5% of C 16 H 14 3 .
Description Ketoprofen occurs as a white, crystalline pow-
der.
It is very soluble in methanol, freely soluble in ethanol (95)
and in acetone, and practically insoluble in water.
A solution of Ketoprofen in ethanol (99.5) (1 in 100) shows
no optical rotation.
It is colored to pale yellow by light.
Identification (1) Determine the absorption spectrum of a
solution of Ketoprofen in methanol (1 in 200,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Ketoprofen, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 94 - 97°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ketoprofen in 10 mL of aceton: the solution is clear, and has
no more color than the following control solution.
Control solution: To a mixure of 0.6 mL of Cobalt (II)
Chloride Colorimetric Stock Solution and 2.4 mL of Iron
(III) Chloride Colorimetric Stock Solution add diluted
hydrochloric acid (1 in 10) to make 10 mL. To 5.0 mL of this
solution add diluted hydrochloric acid (1 in 10) to make 100
mL.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Ketoprofen according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Conduct this procedure with a
minimum of exposure to light, using light-resistant vessels.
Dissolve 20 mg of Ketoprofen in 20 mL of the mobile phase,
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, and add the mobile phase to make ex-
actly 50 mL. Pipet 1 mL of this solution, add the mobile
phase to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with exactly 20 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the areas of the peaks, having the rela-
tive retention time of about 1.5 and about 0.3 with respect to
ketoprofen, are not larger than 4.5 times and not larger than
2 times the peak area of ketoprofen from the standard solu-
tion, respectively, the area of the peak other than ketoprofen
and the peaks mentioned above is not larger than the peak
area of ketoprofen from the standard solution, and the total
area of these peaks is not larger than 2 times the peak area of
ketoprofen from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 233 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 68.0 g of potassium dihydrogen
phosphate in water to make 1000 mL, and adjust the pH to
3.5 with phosphoric acid. To 20 mL of this solution add 430
mL of acetonitrile and 550 mL of water.
Flow rate: Adjust the flow rate so that the retention time of
JP XV
Official Monographs / Ketotifen Fumarate
797
ketoprofen is about 7 minutes.
Time span of measurement: About 7 times as long as the
retention time of ketoprofen.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make exactly 10 mL.
Confirm that the peak area of ketoprofen obtained with 20
fiL of this solution is equivalent to 9 to 11% of that with 20
fiL of the standard solution.
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of ketoprofen are not less than 8000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ketoprofen is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, 60 °C, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Ketoprofen, previ-
ously dried, dissolve in 25 mL of ethanol (95), add 25 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS ( potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 25.43 mg of C 16 Hi 4 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ketotifen Fumarate
^=^,C0 2 H
H0 2 C
Ci 9 H 19 NOS.C4H 4 04: 425.50
4-( 1 -Methylpiperidin-4-ylidene)-4//-
benzo[4,5]cyclohepta[l,2-d]thiophen-10(9//)-one
monofumarate [34580-14-8]
Ketotifen Fumarate, when dried, contains not less
than 99.0% and not more than 101.0% of ketotifen
fumarate (QsH^NOS^H^).
Description Ketotifen Fumarate occurs as a white to light
yellowish white crystalline powder.
It is sparingly soluble in methanol and in acetic acid (100),
and slightly soluble in water, in ethanol (99.5) and in acetic
anhydride.
Melting point: about 190°C (with decomposition).
Identification (1) Prepare the test solution with 0.03 g of
Ketotifen Fumarate as directed under Oxygen Flask Combus-
tion Method using 20 mL of water as the absorbing liquid:
the test solution responds to the Qualitative Tests <1.09> for
sulfate.
(2) Determine the absorption spectrum of a solution of
Ketotifen Fumarate in methanol (1 in 50,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Ketotifen Fumarate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Purity (1) Chloride <1.03>— Dissolve 0.6 g of Ketotifen
Fumarate in 2.5 mL of sodium carbonate TS in a crucible,
heat on a water bath to dryness, and ignite at about 500°C.
Dissolve the residue in 15 mL of water, filter if necessary,
neutralize with diluted nitric acid (3 in 10), and add 6 mL of
dilute nitric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control so-
lution as follows: To 0.25 mL of 0.01 mol/L hydrochloric
acid VS add 2.5 mL of sodium carbonate TS, the used
amount of diluted nitric acid (3 in 10) for the neutralization,
6 mL of dilute nitric acid and water to make 50 mL (not more
than 0.015%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Ketotifen
Fumarate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Ketotifen
Fumarate in 10 mL of a mixture of methanol and ammonia
TS (99:1), and use this solution as the sample solution. Pipet
1 mL of the sample solution, and add a mixture of methanol
and ammonia TS (99:1) to make exactly 25 mL. Pipet 1 mL
of this solution, add a mixture of methanol and ammonia TS
(99:1) to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of acetonitrile, water and ammonia solu-
tion (28) (90:10:1) to a distance of about 15 cm, and air-dry
the plate. Spray evenly Dragendorff's TS for spraying and
then hydrogen peroxide TS on the plate: the number of the
spot other than the principal spot obtained from the sample
solution is not more than four, and they are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Ketotifen
Fumarate, previously dried, dissolve in 80 mL of a mixture of
acetic anhydride and acetic acid (100) (7:3), and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
798 Kitasamycin / Official Monographs
= 42.55 mg of Ci 9 H 19 NOS.C 4 H 4 04
Containers and storage Containers — Tight containers.
Kitasamycin
Leucomycin
H OH
Leucomycin
Aj:
R' =
— «L
o Ch3
R s =
— c.
||
Leucomycin
A*:
R' =
II
— c.
CH 3
tf =
— C
11
— c
Leucomycin
As:
R 1 -
-H
R 2 =
Leucomycin
A 6 :
R' =
It
— C
CH 3
R z =
11
— c
Leucomycin
A T :
R' =
-H
R J =
Jl
— c
II
— c.
II
— c.
Leucomycin
As:
R> =
II
— c,
CH 3
R J =
Leucomycin
A»:
R 1 =
-H
R? =
Leucomycin
A, 3 :
R 1 =
-H
R 2 =
II
— 0,
CH 3
CH 3
CH 3
,CH 3
„CH 3
(Leucomycins Ai, A 5 , A 7 , A 9 and A 13 )
(3i?,4i?,5S,6i?,8i?,9i?,10£',12£',15i?)-5-[4-O-Acyl-
2,6-dideoxy-3-C-methyl-a-L-n'£>o-hexopyranosyl-(l->4)-
3,6-dideoxy-3-dimethylamino-/?-D-glucopyranosyloxy]-6-
formylmethyl-3,9-dihydroxy-4-methoxy-8-methylhexadeca-
10,12-dien-15-olide
Leucomycin A!
Leucomycin A 5
Leucomycin A 7
Leucomycin A 9
Leucomycin A 13
acyl = 3-methylbutanoyl
acyl = butanoyl
acyl = propanoyl
acyl = acetyl
acyl = hexanoyl
(Leucomycins A 3 , A 4 , A 6 and A 8 )
(3RAR,5S,6R,8R,9R,lOE,l2E,l5R)-3-Acetoxy-5-
[4-0-acyl-2,6-dideoxy-3-C-methyl-a-L-n7jo-
hexopyranosyl-(1^4)-3,6-dideoxy-3-dimethylamino-jS-D-
glucopyranosyloxy]-6-formylmethyl-9-hydroxy-4-methoxy-
8-methylhexadeca-10,12-dien-15-olide
Leucomycin A 3 :
Leucomycin A 4 :
Leucomycin A 6 :
Leucomycin A 8 :
acyl = 3-methylbutanoyl
acyl = butanoyl
acyl = propanoyl
acyl = acetyl
[1392-21-8, Kitasamycin]
JP XV
Kitasamycin is a mixture of macrolide substances
having antibacterial activity produced by the growth of
Streptomyces kitasatoensis.
It contains not less than 1450 fig (potency) and not
more than 1700 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Kitasamycin is ex-
pressed as mass (potency) of kitasamycin corre-
sponding to the mass of leucomycin A 5 (C39H 65 N0 14 :
771.93). One mg (potency) of kitasamycin is equivalent
to 0.530 mg of leucomycin A 5 (C3 9 H 65 NOi4).
Description Kitasamycin occurs as a white to light yellow-
white powder.
It is very soluble in acetonitrile, in methanol and in ethanol
(95), and practically insoluble in water.
Identification Determine the absorption spectrum of a solu-
tion of Kitasamycin in methanol (1 in 40,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
Content ratio of the active principle Dissolve 0.02 g of
Kitasamycin in diluted acetonitrile (1 in 2) to make 20 mL,
and use this solution as the sample solution. Perform the test
with 5 fiL of the sample solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and measure each peak area by the automatic integra-
tion method. Calculate the amounts of leucomycin A 5 , leu-
comycin A 4 and leucomycin A t by the area percentage
method: the amounts of leucomycin A 5 , leucomycin A 4 and
leucomycin A] are 40 to 70%, 5 to 25% and 3 to 12%, respec-
tively. Relative retention times of leucomycin A 4 and leu-
comycin A! to that of leucomycin A 5 are 1.2 and 1.5, respec-
tively.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 ftm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To a volume of a solution of ammonium
acetate (77 in 500) add diluted phosphoric acid (1 in 150) to
adjust to pH 5.5. To 370 mL of this solution add 580 mL of
methanol and 50 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
leucomycin A 5 is about 8 minutes.
Time span of measurement: About 3 times as long as the
retention time of leucomycin A 5 .
System suitability —
System performance: Dissolve about 20 mg each of Leu-
comycin A 5 Reference Standard and Josamycin Reference
Standard in 20 mL of diluted acetonitrile (1 in 2). When the
procedure is run with 5 fXL of this solution under the above
operating conditions, leucomycin A 5 and josamycin are elut-
ed in this order with the resolution between these peaks being
not less than 5.
System repeatability: When the test is repeated 6 times with
5 fiL of the sample solution under the above operating condi-
tions, the relative standard deviation of the peak area of leu-
JP XV
Official Monographs / Kitasamycin Acetate 799
comycin A 5 is not more than 1.0%.
Water <2.48> Not more than 3.0% (0.1 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Weigh accurately an amount of
Leucomycin A 5 Reference Standard equivalent to about
30 mg (potency), dissolve in 10 mL of methanol, add water to
make exactly 100 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at 5°C or be-
low and use within 3 days. Take exactly a suitable amount of
the standard stock solution before use, add phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
30 fig (potency) and 7.5 fig (potency), and use these solutions
as the high concentration standard solution and low concen-
tration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Kitasamycin equivalent to about 0.03 g (potency), dissolve in
10 mL of methanol, and add water to make exactly 100 mL.
Take exactly a suitable amount of the solution, add phos-
phate buffer solution, pH 8.0 to make solutions so that each
mL contains 30 fig (potency) and 7 .5 fig (potency), and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Kitasamycin Acetate
Leucomycin Acetate
CH 3
h J — o
CH a
O
T
R =
Leucomycin Ai Acetate
Leucomycin A3 Acetate
Leucomycin A4 Acetate
Leucomycin As Acetate
Leucomycin A6 Acetate- R =
Leucomycin A? Acetate ' R =
R =
R.
R =
(3R,4R,5S,6R,SR,9R,l0E,l2E,15R)-3,9-
Diacetoxy-5-[4-0-acyl-2,6-dideoxy-3-C-methyl-a-
L-n7j>o-hexopyranosyl-(l->4)-2-0-acetyl-3,6-dideoxy-
3-dimethylamino-/?-D-glucopyranosyloxy]-6-formylmethyl-
4-methoxy-8-methylhexadeca- 10,1 2-dien- 1 5-olide
Leucomycin Aj and A 3 Acetates: acyl = 3-methylbutanoyl
Leucomycin A 4 and A 5 Acetates: acyl = butanoyl
Leucomycin A 6 and A 7 Acetates: acyl = propanoyl
[1 78234-32-7, Kitasamycin Acetate]
Kitasamycin Acetate is a derivative of kitasamycin.
It contains not less than 680 fig (potency) and not
more than 790 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Kitasamycin Acetate
is expressed as mass (potency) of kitasamycin corre-
sponding to the mass of leucomycin A 5 (C39H 65 N0 14 :
771.93). One mg (potency) of kitasamycin is equivalent
to 0.530 mg of leucomycin A 5 (C3 9 H 65 NOi4).
Description Kitasamycin Acetate occurs as a white to light
yellow-white powder.
It is very soluble in methanol and in ethanol (95), and
practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Kitasamycin Acetate in methanol (1 in 40,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
800
Kitasamycin Tartrate / Official Monographs
JP XV
(2) Determine the infrared absorption spectrum of
Kitasamycin Acetate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Water <2.48> Not more
titration, direct titration).
than 5.0% (0.1 g, volumetric
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer.
(iii) Standard solution - Weigh accurately an amount of
Leucomycin A 5 Reference Standard equivalent to about
30 mg (potency), dissolve in 10 mL of methanol, add water to
make exactly 100 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at 5°C or
below and use within 3 days. Take exactly a suitable amount
of the standard stock solution before use, add 0.1 mol/L
phosphate buffer solution, pH 8.0 to make solutions so that
each mL contains 30,Mg (potency) and 7.5 /ug (potency), and
use these solutions as the high concentration standard solu-
tion and low concentration standard solution, respectively.
(iv) Sample solution — Weigh accurately an amount of
Kitasamycin Acetate equivalent to about 30 mg (potency),
dissolve in 25 mL of methanol, add water to make exactly 50
mL, shake well, and allow to stand at 37 ± 2°C for 24 hours.
Take exactly a suitable amount of the solution, add 0.1 mol/
L phosphate buffer solution, pH 8.0 to make solutions so that
each mL contains 30 /ug (potency) and 7.5 /ug (potency), and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Kitasamycin Tartrate
Leucomycin Tartrate
Leucomycin «a: R 1 = — c^ n £ = — c J^
Leucomycin A*. R 1 ± - -c v
Leucomycin ^'- R^-H
R z = — <L
O
R 2 = — (L
Leucomycin As; R' - ■
Leucomycin A r ; R 1 = -M
Leucomycin Ag: R' = —
Leucomycin Ag: R 1 =-H
Leucomycin A 13 : r 1 = -h
::;
R'= — C ,CKs
CHj
I
-c
C.Hj
CK
(Leucomycin A b A 5 , A 7 , A 9 and A 13 Tartrates)
(3R,4R,5S,6R,8R,9R,l0E,12E,15R)-5-[4-O-Acyl-
2,6-dideoxy-3-C-methyl-a-L-n£>o-hexopyranosyl-(l— >4)-
3,6-dideoxy-3-dimethylamino-/?-D-glucopyranosyloxy]-6-
formylmethyl-3,9-dihydroxy-4-methoxy-8-methylhexadeca-
10,1 2-dien- 1 5-olide mono-(2i? , 3R )-tartrate
Leucomycin Aj Tartrate: acyl = 3-methylbutanoyl
Leucomycin A 5 Tartrate: acyl = butanoyl
Leucomycin A 7 Tartrate: acyl = propanoyl
Leucomycin A 9 Tartrate: acyl = acetyl
Leucomycin A 13 Tartrate: acyl = hexanoyl
(Leucomycin A 3 , A 4 , A 6 and A 8 Tartrates)
(3RAR,5S,6R,8R,9R,l0E,12E,15R)-3-Acetoxy-5-[4-O-
acyl-2,6-dideoxy-3-C-methyl-a-L-n'£>o-
hexopyranosyl-(l— ►4)-3,6-dideoxy-3-dimethylamino-y6-D-
glucopyranosyloxy]-6-formylmethyl-9-hydroxy-
4-methoxy-8-methylhexadeca- 10,1 2-dien- 1 5-olide
mono-(2/?,3i?)-tartrate
acyl = 3-methylbutanoyl
acyl = butanoyl
acyl = propanoyl
acyl = acetyl
Leucomycin A 3 Tartrate:
Leucomycin A 4 Tartrate:
Leucomycin A 6 Tartrate:
Leucomycin A 8 Tartrate:
[37280-56-1, Kitasamycin Tartrate]
Kitasamycin Tartrate is the tartrate of kitasamycin.
It contains not less than 1300 fig (potency) per mg,
JPXV
Official Monographs / Lactic Acid
801
calculated on the anhydrous basis. The potency of
Kitasamycin Tartrate is expressed as mass (potency) of
kitasamycin based on the amount of leucomycin A 5
(C3 9 H 6 5N0 14 : 771.93). One mg (potency) of Kitasamy-
cin Tartrate is equivalent to 0.530 mg of leucomycin A 5
(C 39 H 65 N0 14 ).
Description Kitasamycin Tartrate occurs as a white to light
yellowish white powder.
It is very soluble in water, in methanol and in ethanol
(99.5).
Identification (1) Determine the absorption spectrum of a
solution of Kitasamycin Tartrate in methanol (1 in 40,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Kitasamycin Tartrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) Dissolve 1 g of Kitasamycin Tartrate in 20 mL of
water, add 3 mL of sodium hydroxide TS, add 20 mL of n-
butyl acetate, shake well, and discard the w-butyl acetate lay-
er. To the aqueous layer add 20 mL of w-butyl acetate, and
shake well. The aqueous layer so obtained responds to the
Qualitative Tests <1.09> (1) for tartrate.
pH <2.54> Dissolve 3.0 g of Kitasamycin Tartrate in 100 mL
of water: the pH of the solution is between 3.0 and 5.0.
Content ratio of the active principle Dissolve 20 mg of
Kitasamycin Tartrate in diluted acetonitrile (1 in 2) to make
20 mL, and use this solution as the sample solution. Perform
the test with 5 /uL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, determine the peak areas by the automatic in-
tegration method, and calculate the amounts of leucomycin
A 5 , leucomycin A 4 and leucomycin A t by the area percentage
method: the amount of leucomycin A 5 is 40 - 70%, leucomy-
cin A 4 is 5 - 25%, and leucomycin Ai is 3 - 12%. The relative
retention times of leucomycin A 4 and leucomycin A[ with
respect to leucomycin A 5 are 1.2 and 1.5, respectively.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To a suitable amount of a solution of am-
monium acetate (77 in 500) add diluted phosphoric acid (1 in
150) to adjust the pH to 5.5. To 370 mL of this solution add
580 mL of methanol and 50 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
leucomycin A 5 is about 8 minutes.
Time span of measurement: About 3 times as long as the
retention time of leucomycin A 5 .
System suitability —
System performance: Dissolve about 20 mg of Leucomycin
A 5 Reference Standard and about 20 mg of Josamycin Refer-
ence Standard in 20 mL of diluted acetonitrile (1 in 2). When
the procedure is run with 5 /uL of this solution under the
above operating conditions, leucomycin A 5 and josamycin
are eluted in this order with the resolution between these
peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
5 fiL of the sample solution under the above operating condi-
tions, the relative standard deviation of the peak area of leu-
comycin A 5 is not more than 1.0%.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Kitasamycin Tartrate in 10 mL of water: the solution is clear
and colorless or light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Kitasamycin Tartrate according to Method 2, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 30 ppm).
Water <2.48> Not more than 3.0% (0.1 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er.
(iii) Standard solutions — Weigh accurately an amount of
Leucomycin A 5 Reference Standard, equivalent to about 30
mg (potency), dissolve in 10 mL of methanol, add water to
make exactly 100 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at not ex-
ceeding 5°C, and use within 3 days. Take exactly a suitable
amount of the standard stock solution before use, add phos-
phate buffer solution, pH 8.0 to make solutions so that each
mL contains 30 /ug (potency) and 7.5/ug (potency), and use
these solutions as the high concentration standard solution
and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Kitasamycin Tartrate, equivalent to about 30 mg (potency),
and dissolve in water to make exactly 100 mL. Take exactly a
suitable amount of this solution, add phosphate buffer solu-
tion, pH 8.0 to make solutions so that each mL contains 30
fig (potency) and 7.5 fig (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Lactic Acid
H OH
H 3 C CO z H and enantiomer
C 3 H 6 3 : 90.08
(2i?5')-2-Hydroxypropanoic acid [50-27-5]
Lactic Acid is a mixture of lactic acid and lactic an-
hydride.
It contains not less than 85.0% and not more than
92.0% of C 3 H 6 3 .
802
Anhydrous Lactose / Official Monographs
JP XV
Description Lactic Acid occurs as a clear, colorless or light
yellow, viscous liquid. It is odorless or has a faint, unpleasant
odor.
It is miscible with water, with ethanol (95) and with diethyl
ether.
It is hygroscopic.
Specific gravity d^j: about 1.20
Identification A solution of Lactic Acid (1 in 50) changes
blue litmus paper to red and responds to the Qualitative Tests
<1.09> for lactate.
Purity (1) Chloride <1.03>— Perform the test with 1.0 g of
Lactic Acid. Prepare the control solution with 1 .0 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.036%).
(2) Sulfate <1.14>— Perform the test with 2.0 g of Lactic
Acid. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.010%).
(3) Heavy metals <1.07>— To 2.0 g of Lactic Acid add 10
mL of water and 1 drop of phenolphthalein TS, and add am-
monia TS dropwise until a pale red color appears. Add 2 mL
of dilute acetic acid and water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution from 2.0 mL of Standard Lead Solution and
2 mL of dilute acetic acid, and dilute with water to 50 mL
(not more than 10 ppm).
(4) Iron <1.10> — Prepare the test solution with 4.0 g of
Lactic Acid according to Method 1, and perform the test ac-
cording to Method A. Prepare the control solution with 2.0
mL of Standard Iron Solution (not more than 5 ppm).
(5) Sugars — To 1 .0 g of Lactic Acid add 10 mL of water,
and neutralize with sodium hydroxide TS. Boil the mixture
with 10 mL of Fehling's TS for 5 minutes: no red precipitate
is produced.
(6) Citric, oxalic, phosphoric and L-tartaric acid — To 1.0
g of Lactic Acid add 1.0 mL of water, followed by 40 mL of
calcium hydroxide TS. Boil the mixture for 2 minutes: no
change occurs.
(7) Glycerin or mannitol — Shake 10 mL of Lactic Acid
with 12 mL of diethyl ether: no turbidity is produced.
(8) Volatile fatty acids — Warm Lactic Acid: it does not
produce any acetic acid-like or butyric acid-like odor.
(9) Cyanide — Transfer 1 .0 g of Lactic Acid to a Nessler
tube, add 10 mL of water and 1 drop of phenolphthalein TS,
add dropwise a solution of sodium hydroxide (1 in 10) by
shaking until a pale red color develops, add 1.5 mL of a solu-
tion of sodium hydroxide (1 in 10) and water to make 20 mL,
and heat in a water bath for 10 minutes. Cool, add dropwise
dilute acetic acid until a red color of the solution disappears,
add 1 drop of dilute acetic acid, add 10 mL of phosphate
buffer solution, pH 6.8, and 0.25 mL of sodium toluensul-
fonchloramide TS, stopper immediately, mix gently, and
allow to stand for 5 minutes. To the solution add 15 mL of
pyridine-pyrazolone TS and water to make 50 mL, and allow
to stand at 25°C for 30 minutes: the solution has no more
color than the following control solution.
Control solution: Pipet 1.0 mL of Standard Cyanide
Solution, and add water to make exactly 20 mL. Transfer 1.0
mL of this solution to a Nessler tube, add 10 mL of water and
1 drop of phenolphthalein TS, and then proceed as described
above.
(10) Readily carbonizable substances — Superimpose
slowly 5 mL of Lactic Acid, previously kept at 15°C, upon 5
mL of sulfuric acid for readily carbonizable substances,
previously kept at 15°C, and allow to stand at 15°C for 15
minutes: no dark color develops at the zone of contact.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 3 g of Lactic Acid, transfer
in a conical flask, add accurately measured 40 mL of 1 mol/L
sodium hydroxide VS, invert a watch glass over the flask, and
heat on a water bath for 10 minutes. Titrate <2.50> the excess
sodium hydroxide with 0.5 mol/L sulfuric acid VS immedi-
ately (indicator: 2 drops of phenolphthalein TS). Perform a
blank determination.
Each mL of 1 mol/L sodium hydroxide VS
= 90.08 mg of C 3 H 6 3
Containers and storage Containers — Tight containers.
Anhydrous Lactose
«7k!L*f
HO
T ' \ Fr
H0 N OH OH
HO Jr-\
ff-Lactose:R'=H. R^OH
^-Lactose: R'=OH, R £ =H
C 12 H 22 0„: 342.30
/?-D-Galactopyranosyl-(l ->4)-/?-D-glucopyranose OS-lactose)
j5-D-Galactopyranosyl-(l ->4)-a-D-glucopyranose (a-lactose)
[63-42-3, Anhydrous Lactose]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Anhydrous Lactose is ^-lactose or a mixture of /?-
lactose and a-lactose.
The relative quantities of a-lactose and ^-lactose in
Anhydrous Lactose is indicated as the isomer ratio.
♦Description Anhydrous Lactose occurs as white crystals
or powder.
It is freely soluble in water, and practically insoluble in-
ethanol (99. 5). ♦
Identification Determine the infrared absorption spectrum
of Anhydrous Lactose, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with *the
Reference Spectrum or* the spectrum of Anhydrous Lactose
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: +54.4- +55.9°. Weigh ac-
curately about 10 g of Anhydrous Lactose, calculated on the
anhydrous basis, dissolve in 80 mL of water warmed to 50°C,
JPXV
Official Monographs / Lactose Hydrate
803
and add 0.2 mL of ammonia TS after cooling. After standing
for 30 minutes, add water to make exactly 100 mL, and deter-
mine the optical rotation of this solution in a 100-mm cell.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Anhydrous Lactose in 10 mL of hot water: the solution is
clear, and colorless or nearly colorless. Determine the absor-
bance at 400 nm of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
control solution: not more than 0.04.
(2) Acidity or alkalinity — Dissolve 6 g of Anhydrous
Lactose in 25 mL of freshly boiled and cooled water by heat-
ing, and after cooling, add 0.3 mL of phenolphthalein TS:
the solution is colorless. To this solution add 0.40 mL of 0.1
mol/L sodium hydroxide VS: a red color develops.
*(3) Heavy metals <1.07> — Proceed with 4.0 g of Anhy-
drous Lactose according to Method 2, and perform the test.
Prepare the control solution with 2 mL of Standard Lead So-
lution (not more than 5 ppm).»
(4) Proteins and light absorbing substances — Dissolve 1 .0
g of Anhydrous Lactose in water to make 100 mL, and use
this solution as the sample solution. Determine the absor-
bances as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using water as the control solution: not more
than 0.25 at between 210 nm and 220 nm, and not more than
0.07 at between 270 nm and 300 nm.
Loss on drying <2.41> Not more than 0.5% (1 g, 80 C C,
2 hours).
Water <2.48> Not more than 1 .0% (1 g, direct titration. Use
a mixture of methanol for Karl Fischer method and for-
mamide for Karl Fischer method (2:1) instead of methanol
for Karl Fischer method).
Residue on ignition <2.44> Not more than 0.1% (1 g).
♦Microbial limit <4.05> The total viable aerobic microbial
count is not more than 100 per g, and the total count of fungi
and yeast is not more than 50 per g, and Salmonella and Es-
cherichia coli should not be observed. »
Isomer ratio Place 1 mg of Anhydrous Lactose in an 5-mL
screw capped reaction vial for gas chromatography, add 0.45
mL of dimethylsulfoxide, stopper, and shake well. Add 1.8
mL of a mixture of pyridine and trimethylsilylimidazole
(72:28), mix, and allow to stand for 20 minutes, and use this
solution as the sample solution. Perform the test with 2 /xL of
the sample solution as directed under Gas Chromatography
<2.02> according to the following conditions, and determine
peak areas of a-lactose and /?-actose, A^ and A b , and calcu-
late the content (%) of /Mactose in Anhydrous Lactose by the
following equation.
Content (%) of /Mactose = [A b /(A a + A b )] x 100
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Sample injection port: about 275°C
Column: A column 4 mm in inside diameter and 0.9 m in
length, packed with siliceous earth for gas chromatography
coated at the ratio of 3% with 25% phenyl-25%
cyanopropyl-methylsilicone polymer for gas chro-
matography.
Column temperature: A constant temperature of about
215°C.
Carrier gas: Helium
Flow rate: A constant flow rate of about 40 mL per minute.
System suitability —
System performance: Prepare a solution with 1 mg of a
mixture of a-lactose and /Mactose (1:1) in the same manner
as for preparing the sample solution, and proceed with 2 /uL
of this solution under the above operating conditions, and
determine the retention times of the peaks of a-lactose and
/Mactose: a ratio of the retention time of a-lactose to that of
/Mactose is about 0.7 with the resolution between these peaks
being not less than 3.0.
♦Containers and storage Containers — Well-closed contain-
ers. ♦
Lactose Hydrate
Lactose
?L*f7k;frJ^
■H s O
C 12 H 22 0„.H 2 0: 360.31
/?-D-Galactopyranosyl-( 1 -> 4)-a-D-glucopyranose
monohydrate
[64044-51-5, Mixture of a- and /Mactose monohydrate]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Lactose Hydrate is the monohydrate of /?-D-galac-
topyranosyl-(l->4)-a-D-glucopyranose.
♦It is a disaccharide obtained from milk, consist of
one unit of glucose and one unit of galactose. ♦
♦The label states the effect where it is the granulated
powder. ♦
♦Description Lactose Hydrate occurs as white, crystals,
powder or granulated powder.
It is freely soluble in water, and practically insoluble in-
ethanol (99. 5). »
Identification Determine the infrared absorption spectrum
of Lactose Hydrate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with ♦the
Reference Spectrum or* the spectrum of Lactose Hydrate
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [aft : +54.4- +55.9°. Weigh ac-
curately about 10 g of Lactose Hydrate, calculated on the an-
hydrous basis, dissolve in 80 mL of water warmed to 50°C,
and add 0.2 mL of ammonia TS after cooling. After standing
for 30 minutes, add water to make exactly 100 mL, and deter-
804
Lactulose / Official Monographs
JP XV
mine the optical rotation of this solution in a 100-mm cell.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Lactose Hydrate in 10 mL of hot water: the solution is clear,
and colorless or nearly colorless. Determine the absorbance
at 400 nm of this solution as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, using water as the control so-
lution: not more than 0.04.
(2) Acidity or alkalinity — Dissolve 6 g of Lactose Hy-
drate in 25 mL of freshly boiled and cooled water by heating,
and after cooling, add 0.3 mL of phenolphthalein TS: the so-
lution is colorless. To this solution add 0.4 mL of 0.1 mol/L
sodium hydroxide VS: a red color develops.
*(3) Heavy metals <1.07> — Dissolve 4.0 g of Lactose Hy-
drate in 20 mL of warm water, add 1 mL of 0.1 mol/L
hydrochloric acid TS and water to make 50 mL. Proceed with
this solution according to Method 1, and perform the test.
Prepare the control solution with 1 mL of 0.1 mol/L
hydrochloric acid TS and 2.0 mL of Standard Lead Solution
(not more than 5 ppm).»
(4) Proteins and light absorbing substances — Dissolve 1 .0
g of Lactose Hydrate in water to make 100 mL, and use this
solution as the sample solution. Determine the absorbances
as directed under Ultraviolet-visible Spectrophotometry <2.24
>, using water as the control solution: not more than 0.25 at
between 210 nm and 220 nm, and not more than 0.07 at be-
tween 270 nm and 300 nm.
*Loss on drying <2.41> Not more than 0.5%. For the
granulated powder, not more than 1.0% (1 g, 80°C, 2
hours). ♦
Water <2.48> 4.5 - 5.5%. *For the granulated powder, 4.0
- 5.5%» (1 g, direct titration. Use a mixture of methanol for
Karl Fischer method and formamide for Karl Fischer method
(2:1) instead of methanol for Karl Fischer method).
Residue on ignition <2.44> Not more than 0.1% (1 g).
♦Microbial limit <4.05> The total viable aerobic microbial
count is not more than 100 per g, and the total count of fungi
and yeast is not more than 50 per g. Salmonella and Es-
cherichia coli should not be observed. »
♦Containers and storage Containers — Well-closed contain-
ers.*
Lactulose
V77P-7.
C 12 H 22 O u : 342.30
/?-D-Galactopyranosyl-(1^4)-D-fructose [4618-18-2]
Lactulose is a solution of lactulose prepared by
isomerizing lactose under the existing of alkaline and
purified by ion-exchange resin.
It contains not less than 50.0% and not more than
56.0% of C 12 H 22 O n .
Description Lactulose occurs as a clear, colorless or light
yellow, viscous liquid. It is odorless, and has a sweet taste.
It is miscible with water and with formamide.
Identification (1) To 0.7 g of Lactulose add 10 mL of
water, 10 mL of a solution of hexaammonium heptamolyb-
date tetrahydrate (1 in 25) and 0.2 mL of acetic acid (100),
and heat in a water bath for 5 to 10 minutes: a blue color de-
velops.
(2) Mix 0.3 g of Lactulose and 30 mL of water, add 16
mL of 1 mol/L iodine TS, then immediately add 2.5 mL of 8
mol/L sodium hydroxide TS, allow to stand for 7 minutes,
and add 2.5 mL of diluted sulfuric acid (3 in 20). To this solu-
tion add a saturated solution of sodium sulfite heptahydrate
until the solution turns light yellow, then add 3 drops of
methyl orange TS, neutralize with a solution of sodium
hydroxide (4 in 25), and add water to make 100 mL. To 10
mL of this solution add 5 mL of Fehling's TS, and boil for 5
minutes: a red precipitate is produced.
pH <2.54> To 2.0 g of Lactulose add water to make 15 mL:
the pH of the solution is between 3.5 and 5.5.
Specific gravity <2.56> df : 1.320 - 1.360
Purity (1) Heavy metals <1.07> — Proceed with 5.0 g of
Lactulose according to Method 4, and perform the test. Pre-
pare the control solution with 2.5 mL of Standard Lead Solu-
tion (not more than 5 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Lactulose according to Method 1, and perform the test
(not more than 2 ppm).
(3) Glactose and lactose — Determine the heights of the
peaks corresponding to D-galactose and lactose respectively,
on the chromatogram obtained in Assay from the sample so-
lution and the standard solution, and calculate the ratios of
the peak heights of D-galactose and lactose to that of the in-
ternal standard from the sample solution, g Ta and Q Tb , and
then from the standard solution, Qsa and Q sb : it contains D-
galactose of not more than 11%, and lactose of not more
than 6%.
Amount (mg) of D-galactose (C 6 H 12 6 )
= w s x (e Ta /e Sa )
W s : Amount (mg) of D-galactose
Amount (mg) of lactose (Ci 2 H 22 O n .H 2 0)
= Ws x (GTb/Ssb)
W s : Amount (mg) of lactose Hydrate
Loss on drying <2.41> Not more than 35% (0.5 g, in vacu-
um, 80°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Lactulose, add exactly
10 mL of the internal standard solution and water to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 0.5 g of Lactulose Reference Stan-
dard, accurately about 80 mg of D-galactose and accurately
about 40 mg of lactose monohydrate, add exactly 10 mL of
the internal standard solution and water to make 50 mL, and
JPXV
Official Monographs / Lanatoside C
805
use this solution as the standard solution. Perform the test
with 20 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Q s , of the peak height of lactulose to that of the internal stan-
dard, respectively.
Amount (mg) of C I2 H 2 20n
= W s x (Q T /Q S )
W s : Amount (mg) of Lactulose Reference Standard
Internal standard solution — A solution of D-mannitol (1 in
20).
Operating conditions —
Detector: A differential refractometer.
Column: A stainless steel column 8 mm in inside diameter
and 50 cm in length, packed with gel type strong acid ion-ex-
change resin for liquid chromatography (degree of crosslink-
age: 6%) (11 //m in particle diameter).
Column temperature: A constant temperature of about
75°C.
Mobile phase: Water.
Flow rate: Adjust the flow rate so that the retention time of
lactulose is about 18 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, lactulose and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak heights of lactulose, galactose and lactose to the height
of the internal standard are not more than 2.0%, respective-
ly.
Containers and storage Containers — Tight containers.
Lanatoside C
=>i- Y -> K c
H OH
C 49 H 76 2 „: 985.12
3/?-[/?-D-Glucopyranosyl-(l->4)-3-0-acetyl-2,6-dideoxy-
/?-D-n7j>o-hexopyranosyl-(l->4)-2,6-dideoxy-/?-D-n'do-
hexopyranosyl-( 1 -* 4)-2,6-dideoxy-0-D-ribo-
hexopyranosyloxy]-12/?, 14-dihydroxy-5/?,14/?-card-
20(22)-enolide [17575-22-3]
Lanatoside C, when dried, contains not less than
90.0% and not more than 102.0% of C 49 H 76 O 20 .
Description Lanatoside C occurs as colorless or white crys-
tals or a white, crystalline powder. It is odorless.
It is soluble in methanol, slightly soluble in ethanol (95),
and practically insoluble in water and in diethyl ether.
It is hygroscopic.
Identification Place 1 mg of Lanatoside C to a small test
tube having an internal diameter of about 10 mm, dissolve in
1 mL of a solution of iron (III) chloride hexahydrate in acetic
acid (100) (1 in 10,000), and underlay gently with 1 mL of sul-
furic acid: at the zone of contact of the two liquids, a brown
ring is produced, and the color of the upper layer near the
contact zone gradually changes to blue through purple. Final-
ly the color of the entire acetic acid layer changes to blue-
green through deep blue.
Purity Related substances — Dissolve 10 mg of Lanatoside
C in exactly 5 mL of methanol, and use this solution as the
sample solution. Separately, dissolve 1.0 mg of Lanatoside C
Reference Standard in exactly 5 mL of methanol, and use this
solution as the standard solution. Perform the test as directed
under Thin-layer Chromatography <2.03> with these solu-
tions. Spot 20 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of
dichloromethane, methanol and water (84:15:1) to a distance
of about 13 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat the plate at 110°C for 10
minutes: any spots other than the principal spot from the
sample solution are neither larger nor darker than the spot
from the standard solution.
Optical rotation <2.49> [a]o- +32- +35° (after drying,
0.5 g, methanol, 25 mL, 100 mm).
Loss on drying <2.41> Not more than 7.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Weigh accurately about 50 mg each of Lanatoside C
and Lanatoside C Reference Standard, previously dried, and
dissolve in methanol to make exactly 25 mL. Pipet 5 mL each
of these solutions, add methanol to make exactly 100 mL,
and use these solutions as the sample solution and the stan-
dard solution, respectively. Pipet 5 mL each of the sample so-
lution and standard solution into 25-mL light-resistant, volu-
metric flasks, and add 5 mL of 2,4,6-trinitrophenol TS and
0.5 mL of a solution of sodium hydroxide (1 in 10), shake
well, and add methanol to make 25 mL. Allow these solu-
tions to stand between 18°C and 22°C for 25 minutes, and
determine the absorbances, A T and A s , of the solutions at 485
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using a solution prepared with 5 mL of methanol in
the same manner as the blank solution.
Amount (mg) of C 49 H 76 O20
= W s x (A T /A S )
806
Lanatoside C Tablets / Official Monographs
JP XV
W s : Amount (mg) of Lanatoside C Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Lanatoside C Tablets
7-J- h •> P c ft
Lanatoside C Tablets contain not less than 90% and
not more than 110% of the labeled amount of lanato-
side C (C 49 H 76 O 20 : 985.12).
Method of preparation Prepare as directed under Tablets,
with Lanatoside C.
Identification (1) Shake a quantity of powdered Lanato-
side C Tablets, equivalent to 1 mg of Lanatoside C according
to the labeled amount, with 3 mL of diethyl ether, and filter.
Wash the residue with two 3-mL portions of diethyl ether,
and air-dry. To the remaining residue add 10 mL of a mixture
of chloroform and methanol (9:1), shake, and filter. Wash
the residue with two 5-mL portions of a mixture of chlo-
roform and methanol (9:1), combine the filtrate and wash-
ings, and evaporate on a water bath to a smaller volume.
Transfer the solution to a small test tube having an internal
diameter of about 10 mm, further evaporate on a water bath
to dryness, and proceed as directed in the Identification under
Lanatoside C.
(2) Perform the test with the sample solution and the
standard solution obtained in the Assay as directed under
Thin-layer Chromatography <2.03>. Spot 25 /xL each of these
solutions on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of
dichloromethane, methanol and water (84:15:1) to a distance
of about 13 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat the plate at 110°C for 10
minutes: the spots obtained from the sample solution and the
standard solution show a black color, and have the same i?f
values.
Uniformity of dosage unit <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Warm 1 tablet of Lanatoside C Tablets with 5 mL of water
until the tablet is disintegrated, add 30 mL of ethanol (95),
disperse finely the particles with the aid of ultrasonic radia-
tion, add ethanol (95) to make exactly KmL of a solution
containing about 5 [ig of lanatoside C (C 4 9H 76 02o) in each
mL, and filter. Discard the first 10 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Separately,
weigh accurately about 25 mg of Lanatoside C Reference
Standard, previously dried in vacuum over phosphorus (V)
oxide at 60°C for 4 hours, and dissolve in ethanol (95) to
make exactly 100 mL. Pipet 2 mL of this solution, add 10 mL
of water, add ethanol (95) to make exactly 100 mL, and use
this solution as the standard solution. Pipet 2 mL each of the
sample solution, the standard solution and diluted ethanol
(95) (17 in 20) into three brown glass-stoppered test tubes T, S
and B, previously containing exactly 10 mL of 0.012 w/v%
L-ascorbic acid-hydrochloric acid TS, add exactly 1 mL each
of dilute hydrogen peroxide TS immediately, shake vigorous-
ly, and allow to stand at a constant temperature between 25°
C and 30 °C for 40 minutes. Determine the fluorescence inten-
sities, F T , F s and F B , of the subsequent solutions from the
sample solution and the standard solution and the diluted
ethanol (95) (17 in 20) at 355 nm of the excitation wavelength
and at 490 nm of the fluorescence wavelength as directed un-
der Fluorometry <2.22>, respectively.
Amount (mg) of lanatoside C (C 49 H 76 2 o)
= W s x {(F T - Fb)/(F s - F B )} x (F/5000)
W 5 : Amount (mg) of Lanatoside C Reference Standard
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Take 1 tablet of Lanatoside C Tablets, and perform the
test with 500 mL of diluted hydrochloric acid (3 in 500)
deaired by a suitable method as the test solution at 100 revo-
lutions per minute as directed in the Paddle method. Take 20
mL of the dissolved solution at 60 minutes after starting the
test, and filter through a membrane filter (not more than 0.8
/urn). Discard the first 10 mL of the filtrate, and use the subse-
quent filtrate as the sample solution. Separately, dry Lanato-
side C Reference Standard in vacuum over phosphorus (V)
oxide at 60°C for 4 hours, weigh accurately a portion of it, e-
quivalent to 100 times an amount of the labeled amount of
lanatoside C (C 49 H 76 02o), dissolve in ethanol (95) to make ex-
actly 100 mL. Pipet 1 mL of this solution, add the test solu-
tion to make exactly 500 mL, warm at 37±0.5°C for 60
minutes, and use this solution as the standard solution. Pipet
3 mL each of the sample solution, the standard solution and
the test solution, and transfer to glass-stoppered brown test
tubes T, S and B, respectively. To these solutions add exactly
10 mL each of 0.012 w/v% L-ascorbic acid-hydrochloric acid
TS, and shake. Immediately add exactly 0.2 mL each of dilut-
ed hydrogen peroxide TS (1 in 100), shake well, and allow to
stand at a constant temperature between 30°C and 37°C for
45 minutes. Determine immediately the fluorescence intensi-
ties, F T , F s and F B , of the sample solution and the standard
solution at 355 nm of the excitation wavelength and at 490
nm of the fluorescence wavelength as directed under Fluoro-
metry <2.22>.
Dissolution rate of each of six Lanatoside C Tablets after
60 minutes should be not less than 65%.
Requirement of retest is not applied to Lanatoside C
Tablets.
Dissolution rate (%) to labeled amount of
lanatoside C (C 49 H 76 2 o)
= W s x {(Ft - F B )/(F S - F B )} x (1/Q
W s : Amount (mg) of Lanatoside C Reference Standard.
C: Labeled amount (mg) of lanatoside C (C 49 H 76 O 20 ) in
each tablet.
Assay Weigh accurately and powder not less than 20
Lanatoside C Tablets. Weigh accurately a portion of the
powder, equivalent to about 5 mg of lanatoside C
(C 45 H 76 2 o), into a 100-mL light-resistant volumetric flask,
add 50 mL of ethanol (95), and shake for 15 minutes. Then
dilute with ethanol (95) to make exactly 100 mL. Filter this
solution, discard the first 20 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Separately, weigh
accurately about 5 mg of Lanatoside C Reference Standard,
previously dried in vacuum over phosphorus (V) oxide at
60°C for 4 hours, dissolve in ethanol (95) to make exactly 100
JP XV
mL, and use this solution as the standard solution. Pipet 5
mL each of the sample solution and the standard solution
into light-resistant, glass-stoppered test tubes, add 3 mL each
of alkaline 2,4,6-trinitrophenol TS, shake well and allow
these solutions to stand between 22°C and 28 °C for 25
minutes. Determine the absorbances, A T and A s , of the sub-
sequent sample solution and the subsequent standard solu-
tion at 490 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using a solution, prepared by the same
manner with 5 mL of ethanol (95), as the blank.
Amount (mg) of lanatoside C (C49H 76 2 o)
= W s x (A T /A S )
W s : Amount (mg) of Lanatoside C Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Hydrous Lanolin
Hydrous Lanolin is Purified Lanolin to which water
is added. It contains not less than 70% and not more
than 75% of Purified Lanolin (as determined by the
test for Residue on evaporation).
Description Hydrous Lanolin is a yellowish white, oin-
tment-like substance, and has a slight, characteristic odor,
which is not rancid.
It is soluble in diethyl ether and in cyclohexane, with the
separation of water.
When melted by heating on a water bath, it separates into a
clear oily layer and a clear water layer.
Melting point: about 39°C
Identification Dissolve 1 g of Hydrous Lanolin in 50 mL of
cyclohexane, and remove the separated water. Superimpose
carefully 1 mL of the cyclohexane solution on 2 mL of sulfur-
ic acid: a red-brown color develops at the zone of contact,
and sulfuric acid layer shows a green fluorescence.
Acid value <1.13> Not more than 1.0.
Iodine value 18-36 Heat a suitable amount of Hydrous
Lanolin on a water bath to remove its almost moisture, then
weigh accurately about 0.8 g of the treated Hydrous Lanolin
in a glass-stoppered 500-mL flask, and add 10 mL of cyclo-
hexane to dissolve, and add exactly 25 mL of Hanus's TS,
and mix well. If a clear solution is not obtained, add more cy-
clohexane to make clear, and allow the mixture to stand for 1
hour between 20°C and 30°C in a light-resistant, well-closed
container while occasional shaking. Add 20 mL of a solution
of potassium iodide (1 in 10) and 100 mL of water, shake,
and titrate <2.50> the liberated iodine with 0.1 mol/L sodium
thiosulfate VS (indicator: 1 mL of starch TS). Perform a
blank determination in the same manner.
Iodine value= [(a-b) x \.269]/W
W: amount (g) of sample.
a: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
sumed in the blank determination.
b: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
Official Monographs / Hydrous Lanolin 807
sumed in the titration.
Purity (1) Acidity or alkalinity — To 5 g of Hydrous Lano-
lin add 25 mL of water, boil for 10 minutes, and cool. Add
water to restore the previous mass, and separate the aqueous
layer: the aqueous layer is neutral.
(2) Chloride <1.03>— To 2.0 g of Hydrous Lanolin add 40
mL of water, boil for 10 minutes, and cool. Add water to re-
store the previous mass, and filter. To 20 mL of the filtrate
add 6 mL of dilute nitric acid and water to make 50 mL. Use
this solution as the test solution, and perform the test. Pre-
pare the control solution with 1.0 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(3) Ammonia — To 10 mL of the aqueous layer obtained
in (1) add 1 mL of sodium hydroxide TS, and boil: the gas
evolved does not turn moistened red litmus paper to blue.
(4) Water-soluble organic substances — To 5 mL of the
aqueous layer obtained in (1) add 0.25 mL of 0.002 mol/L
potassium permanganate VS, and allow to stand for 5
minutes: the red color of the solution does not disappear.
(5) Petrolatum — Dissolve 1 .0 g of the dried residue ob-
tained in the Residue on evaporation in 10 mL of a mixture
of tetrahydrofuran and isooctane (1:1), and use this solution
as the sample solution. Add dissolve 20 mg of vaseline in 10
mL of a mixture of tetrahydrofuran and isooctane (1:1), and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 25 /xL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with isooctane to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly
diluted sulfuric acid (1 in 2) on the plate, heat the plate at
80°C for 5 minutes, cool, and examine under ultraviolet light
(main wavelength: 365 nm): no fluorescent spot is observed in
the same level with the spot of standard solution. For this test
use a thin-layer plate previously developed with isooctane to
the upper end, dried in air, and heated at 110°C for 60
minutes.
Residue on evaporation Weigh accurately about 12.5 g of
Hydrous Lanolin, dissolve in 50 mL of diethyl ether, place it
in a separator, transfer the separated aqueous layer to
another separator, add 10 mL of diethyl ether, shake, and
combine the diethyl ether layer and diethyl ether in the first
separator. Shake the diethyl ether layer with 3 g of anhydrous
sodium sulfate, and filter through dry filter paper. Wash the
separator and the filter paper with two 20-mL portions of
diethyl ether, combine the washings with the filtrate,
evaporate on a water bath until the odor of diethyl ether is no
longer perceptible, and dry in a desiccator (in vacuum, silica
gel) for 24 hours: the content is not less than 70% and not
more than 75%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Not exceeding 30°C.
808
Purified Lanolin / Official Monographs
JP XV
Purified Lanolin
Adeps Lanae Rurificatus
Purified Lanolin is the purified product of the fat-
like substance obtained from the wool of Ovis aries
Linne (Bovidae).
Description Purified Lanolin is a light yellow to yellowish
brown, viscous, ointment-like substance, and has a faint,
characteristic but not rancid odor.
It is very soluble in diethyl ether and in cyclohexane, freely
soluble in tetrahydrofuran and in toluene, and very slightly
soluble in ethanol (95). It is practically insoluble in water, but
miscible without separation with about twice its mass of
water, retaining ointment-like viscosity.
Melting point: 37-43°C
Identification Superimpose carefully 1 mL of a solution of
Purified Lanolin in cyclohexane (1 in 50) on 2 mL of sulfuric
acid: a red-brown color develops at the zone of contact, and
the sulfuric acid layer shows a green fluorescence.
Acid value <1.13> Not more than 1.0.
Iodine value 18-36 Weigh accurately about 0.8 g of
Purified Lanolin in a glass-stoppered 500-mL flask, add 20
mL of cyclohexane to dissolve, and add exactly 25 mL of
Hanus' TS, and mix well. If a clear solution is not obtained,
add more cyclohexane to make clear, and allow the mixture
to stand for 1 hour between 20°C and 30°C in light-resistant,
well-closed containers, with occasional shaking. Add 20 mL
of a solution of potassium iodide (1 in 10) and 100 mL of
water, shake, and titrate the liberated iodine with 0.1 mol/L
sodium thiosulfate VS (indicator: 1 mL of starch TS). Per-
form a blank determination.
Iodine value = [(a - b) x 1 .269]/ W
W: amount (g) of sample.
a: Volume (mL) of 0.1 mol/L sodium thiosulfate VS used
in the blank determination.
b: Volume (mL) of 0.1 mol/L sodium thiosulfate VS used
in the titration of the sample.
Purity (1) Acid or alkali — To 5 g of Purified Lanolin add
25 mL of water, boil for 10 minutes, and cool. Add water to
restore the previous mass, and separate the aqueous layer: the
aqueous layer is neutral.
(2) Chloride <1.03>— To 2.0 g of Purified Lanolin add 40
mL of water, boil for 10 minutes, and cool. Add water to re-
store the previous mass, and filter. To 20 mL of the filtrate
add 6 mL of dilute nitric acid and water to make 50 mL. Use
this solution as the test solution, and perform the test. Pre-
pare the control solution with 1.0 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(3) Ammonia — To 10 mL of the aqueous layer obtained
in (1) add 1 mL of sodium hydroxide TS, and boil: the gas
evolved does not turn moistened red litmus paper to blue.
(4) Water-soluble organic substances — To 5 mL of the
aqueous layer obtained in (1) add 0.25 mL of 0.002 mol/L
potassium permanganate VS, and allow to stand for 5
minutes: the red color of the solution does not disappear.
(5) Petrolatum — Dissolve 1.0 g of Purified Lanolin in 10
mL of a mixture of tetrahydrofuran and isooctane (1:1), and
use this solution as the sample solution. And dissolve 20 mg
of vaseline in 10 mL of a mixture of tetrahydrofuran and
isooctane (1:1), and use this solution as the standard solu-
tion. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 25 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel for thin-layer chromatography. Develop the plate with
isooctane to a distance of about 10 cm, and air-dry the plate.
Spray evenly diluted sulfuric acid (1 in 2) on the plate, heat
the plate at 80°C for 5 minutes, cool, and examine under
ultraviolet light (main wavelength: 365 nm): no fluorescent
spot is observable same level of the spot of standard solution.
Use a thin-layer plate previously developed with isooctane to
the upper end, dried in air, and heated at 110°C for 60
minutes.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C,
2 hours).
Total ash <J.07> Not more than 0.1%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Not exceeding 30°C.
Lard
Adeps Suillus
Lard is the fat obtained from Sus scrofa Linne var.
domesticus Gray (Suidae).
Description Lard occurs as a white, soft, unctuous mass,
and has a faint, characteristic odor and a bland taste.
It is freely soluble in diethyl ether and in petroleum ether,
very slightly soluble in ethanol (95), and practically insoluble
in water.
Melting point: 36 - 42°C
Congealing point of the fatty acids: 36 - 42°C
Acid value <1.13> Not more than 2.0.
Saponification value <1.13> 195 - 203
Iodine value <7.75> 46 - 70
Purity (1) Moisture and coloration — Melt 5 g of Lard by
heating on a water bath: it forms a clear liquid, from which
no water separates. Observe the liquid in a layer 10 mm thick:
the liquid is colorless to slightly yellow.
(2) Alkalinity— To 2.0 g of Lard add 10 mL of water,
melt by warming on a water bath, and shake vigorously. Af-
ter cooling, add 1 drop of phenolphthalein TS to the separat-
ed water layer: the layer is colorless.
(3) Chloride <1.03>— To 1.5 g of Lard add 30 mL of
ethanol (95), boil for 10 minutes under a reflux condenser,
and filter after cooling. To 20 mL of the filtrate add 5 drops
of a solution of silver nitrate in ethanol (95) (1 in 50): the
opalescence of the mixture does not exceed that of the follow-
JPXV
Official Monographs / Latamoxef Sodium
809
ing control solution.
Control solution: To 1.0 mL of 0.01 mol/L hydrochloric
acid VS add ethanol (95) to make 20 mL, and add 5 drops of
a solution of silver nitrate in ethanol (95) (1 in 50).
(4) Beef tallow — Dissolve 5 g of Lard in 20 mL of diethyl
ether, stopper lightly with absorbent cotton, and allow to
stand at 20°C for 18 hours. Collect the separated crystals,
moisten them with ethanol (95), and examine under a micro-
scope of 200 magnifications: the crystals are in the form of
rhomboidal plates grouped irregularly, and do not contain
prisms or needles grouped in fan-shaped clusters.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Not exceeding 30°C.
Latamoxef Sodium
=7 9tt-\z-7i- h U^7A
C0 2 Na
CQ>Na 'V- N""^
'! I I j
CH,
C 20 H 18 N 6 Na 2 O 9 S: 564.44
Disodium (6/?,7R)-7-[2-carboxylato-
2-(4-hydroxyphenyl)acetamino]-7-methoxy-3-(l-methyl-
l//-tetrazol-5-ylsulfanylmethyl)-8-oxo-5-oxa-
l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate [64953-12-4]
Latamoxef Sodium contains not less than 830 fig
(potency) and not more than 940 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Latamoxef Sodium is expressed as mass (potency) of
latamoxef (C 2 oH 20 N 6 9 S: 520.47).
Description Latamoxef Sodium occurs as white to light yel-
lowish white, powder or masses.
It is very soluble in water, freely soluble in methanol, and
slightly soluble in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Latamoxef Sodium (3 in 100,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Latamoxef Sodium as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Determine the spectrum of a solution of Latamoxef
Sodium in heavy water for nuclear magnetic resonance spec-
troscopy (1 in 10) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits single signals, A and B, at around <53.5 ppm and at
around <54.0 ppm. The ratio of the integrated intensity of
these signals, A:B, is about 1:1.
(4) Latamoxef Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [«]£?: - 32 - - 40° (0.5 g calculated
on the anhydrous basis, phosphate buffer solutiuon, pH 7.0,
50 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Latamoxef Sodium in 10 mL of water is between 5.0 and
7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Latamoxef Sodium in 10 mL of water: the solution is clear
and pale yellow.
(2) Heavy metals <1.07> — Carbonize 1.0 g of Latamoxef
Sodium by heating gently, previously powdered if it is mass-
es. After cooling, add 10 mL of a solution of magnesium ni-
trate hexahydrate in ethanol (1 in 10), and burn the ethanol.
After cooling, add 1 mL of sulfuric acid. Proceed according
to Method 4, and perform the test. Prepare the control solu-
tion with 2.0 mL of Standard Lead Solution (not more than
20 ppm).
(3) Arsenic </.//> — Prepare the test solution by dissolv-
ing 1.0 g of Latamoxef Sodium in 20 mL of water, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve an amount of
Latamoxef Sodium, equivalent to about 25 mg (potency), in
water to make exactly 50 mL, and use this solution as the
sample solution. Pipet 2 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 5 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine each peak area by the automatic
integration method: the peak area of l-methyl-l//-tetrazole-
5-thiol, having the relative retention time of about 0.5 with
respect to the first eluted peak of the two peaks of latamoxef,
obtained from the sample solution is not more than the peak
area of latamoxef from the standard solution, and the peak
area of decarboxylatamoxef, having the relative retention
time of about 1.7 with respect to the first peak of the two
peaks of latamoxef, is not more than 2 times that of latamox-
ef from the standard solution. For this calculation, use the
peak area for l-methyl-l//-tetrazole-5-thiol after multiplying
by its response factor, 0.52.
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
latamoxef is not more than 2.0%.
Water <2.48> Not more than 5.0% (0.5 g, volumetric titra-
tion, back titration).
Isomer ratio Dissolve 25 mg of Latamoxef Sodium in water
to make 50 mL, and use this solution as the sample solution.
Perform the test with 5 fiL of the sample solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the areas, A a and A b , of the
810
Lauromacrogol / Official Monographs
JP XV
two peaks in order of elution, which appear close to each
other at the retention time of about 10 minutes: AJA h is be-
tween 0.8 and 1.4.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 7.7 g of ammonium acetate in
water to make 1000 mL. To 950 mL of this solution add
50 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
the first eluted peak of latamoxef is about 8 minutes.
System suitability —
System performance: When the procedure is run with 5 /uL
of the sample solution under the above operating conditions,
the resolution between the two peaks of latamoxef is not less
than 3.
System repeatability: When the test is repeated 3 times with
5 /uL of the sample solution under the above operating condi-
tions, the relative standard deviation of the area of the first
eluted peak of latamoxef is not more than 2.0%.
Assay Weigh accurately an amount of Latamoxef Sodium
and Latamoxef Ammonium Reference Standard, equivalent
to about 25 mg (potency) each, dissolve in exactly 5 mL of
the internal standard solution, add water to make 50 mL, and
use these solutions as the sample solution and the standard
solution. Perform the test with 5 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, g T and Q s , of the peak area of
latamoxef to that of the internal standard.
Amount [/ug (potency)] of latamoxef (C2oH 2 oN 6 9 S)
= W s x (<2 T /<2s) x 1000
W s : Amount [mg (potency)] of Latamoxef Ammonium
Reference Standard
Internal standard solution — A solution of m-cresol (3 in 200).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.94 g of potassium dihydrogen
phosphate, 3.22 g of disodium hydrogen phosphate dodeca-
hydrate and 1.60 g of tetra M-butylammonium bromide in
water to make exactly 1000 mL. To 750 mL of this solution
add 250 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
latamoxef is about 7 minutes.
System suitability —
System performance: When the procedure is run with 5 /uL
of the standard solution under the above operating condi-
tions, latamoxef and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
5 ,uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of latamoxef to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Not exceeding 5°C.
Lauromacrogol
Polyoxyethylene Lauryl Alcohol Ether
Lauromacrogol is a polyoxyethylene ether prepared
by the polymerization of ethylene oxide with laury al-
cohol.
Description Lauromacrogol is a colorless or light yellow,
clear liquid or a white, petrolatum-like or waxy solid. It has a
characteristic odor, and a somewhat bitter and slightly irrita-
tive taste.
It is very soluble in ethanol (95), in diethyl ether and in car-
bon tetrachloride.
It is freely soluble or dispersed as fine oily drops in water.
Identification (1) Shake well 0.5 g of Lauromacrogol with
10 mL of water and 5 mL of ammonium thiocyanate-cobalt
nitrate TS, then shake with 5 mL of chloroform, and allow to
stand: the chloroform layer becomes blue in color.
(2) Dissolve 0.35 g of Lauromacrogol in 10 mL of carbon
tetrachloride, and perform the test as directed in the Solution
method under Infrared Spectrophotometry <2.25> using a
0.1-mm fixed cell: it exhibits absorption at the wave numbers
of about 1347 cm -1 , 1246 cm -1 and 1110 cm -1 .
Purity (1) Acidity — Transfer 10.0 g of Lauromacrogol
into a flask, and add 50 mL of neutralized ethanol. Heat on a
water bath nearly to boil, shaking once or twice while heat-
ing. Cool, and add 5.3 mL of 0.1 mol/L sodium hydroxide
VS and 5 drops of phenolphthalein TS: a red color develops.
(2) Unsaturated compound — Shake 0.5 g of
Lauromacrogol with 10 mL of water, and add 5 drops of bro-
mine TS: the color of the solution does not disappear.
Residue on ignition <2.44> Not more than 0.2% (1 g).
Containers and storage Containers — Tight containers.
Lenampicillin Hydrochloride
>HCI
C 21 H 23 N 3 7 S.HC1: 497.95
5-Methyl-2-oxo[l ,3]dioxol-4-ylmethyl (2S,5R,6R )-i
[(2R )-2-amino-2-phenylacetylamino]-3 ,3-dimethyl-7-
JPXV
Official Monographs / Lenampicillin Hydrochloride 811
oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate
monohydrochloride [80734-02-7]
Lenampicillin Hydrochloride is the hydrochloride of
ampicillin methyloxodioxolenylmethyl ester.
It contains not less than 653 fig (potency) and not
more than 709 fig (potency) per mg, calculated on the
anhydrous basis and corrected by the amount of the
residual solvents. The potency of Lenampicillin
Hydrochloride is expressed as mass (potency) of am-
picillin (C 16 H 19 N 3 4 S: 349.40).
Description Lenampicillin Hydrochloride occurs as a white
to light yellowish white powder.
It is very soluble in water, in methanol and in ethanol (95),
and freely soluble in iV,./V-dimethylformamide.
Identification (1) Determine the infrared absorption spec-
trum of Lenampicillin Hydrochloride as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Lenampicillin
Hydrochloride Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(2) To 1 mL of a solution of Lenampicillin Hydrochlo-
ride (1 in 100) add 0.5 mL of dilute nitric acid and 1 drop of
silver nitrate TS: a white precipitate is formed.
Optical rotation <2.49> [ a ]g>: + 174 - + 194° (0.2 g calculated
on the anhydrous de-residual solventization basis, ethanol
(95), 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Lenampicillin Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Lenampicillin Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(3) Free ampicillin — Weigh accurately about 0.1 g of
Lenampicillin Hydrochloridein, dissolve in exactly 10 mL of
the internal standard solution, and use this solution as the
sample solution. Separately, weigh accurately an amount of
Ampicillin Reference Standard, equivalent to about 25 mg
(potency), and dissolve in water to make exactly 100 mL.
Pipet 2 mL of this solution, add exactly 10 mL of the internal
standard solution, and use this solution as the standard solu-
tion. The sample solution should be used to the following test
immediately after the solution is prepared. Perform the test
with 10 fiL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Q T and
Q s , of the peak height of ampicillin to that of the internal
standard: the amount of ampicillin is not more than 1.0%.
Amount (%) of ampicillin (C 16 H 19 N 3 04S)
= (W S /W T ) x (Q T /Q S ) x 2
W s : Amount [mg (potency)] of Ampicillin Reference Stan-
dard
W T : Amount (mg) of the sample
Internal standard solution — A solution of anhydrous caffeine
in the mobile phase (1 in 50,000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.22 g of potassium dihydrogen
phosphate in water to make 900 mL, and add 100 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ampicillin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, ampicillin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of ampicillin to that of the internal standard is
not more than 5%.
(4) Penicilloic acid — Weigh accurately about 0.1 g of
Lenampicillin Hydrochloride, dissolve in water to make ex-
actly 100 mL, and use this solution as the sample solution.
Pipet 10 mL of the sample solution, add 10 mL of potassium
hydrogen phthalate buffer solution, pH 4.6 and exactly 10
mL of 0.005 mol/L iodine VS, allow to stand for exactly 15
minutes while protecting from exposure to light, and titrate
<2.50> with 0.01 mol/L sodium thiosulfate VS (indicator: 1
mL of starch TS). Perform a blank determination, and make
any necessary correction: the amount of penicilloic acid
(C 16 H 21 N 3 5 S: 367.42) is not more than 3.0%.
Each mL of 0.01 mol/L sodium thiosulfate VS
= 0.45 mg of C 16 H 21 N 3 5 S
(5) Residual solvent <2.46> — Weigh accurately about 0.25
g of Lenampicillin Hydrochloride, dissolve in exactly 1 mL
of the internal standard solution, add A^TV-dimethylfor-
mamide to make 5 mL, and use this solution as the sample so-
lution. Separately, weigh accurately about 80 mg of 2-
propanol and about 0.12 g of ethyl acetate, and add N,N-
dimethylformamide to make exactly 100 mL. Pipet 1 mL and
3 mL of this solution, add exactly 1 mL each of the internal
standard solution, add 7V,7V-dimethylformamide to make 5
mL, and use these solutions as the standard solution (1) and
the standard solution (2), respectively. Perform the test with
4 fiL each of the sample solution, standard solution (1) and
standard solution (2) as directed under Gas Chromatography
<2.02> according to the following conditions, and determine
the ratios, Q Ta and Q Tb , of the peak height of 2-propanol and
ethyl acetate to that of the internal standard of the sample so-
lution, the ratios, Q S ai and Qsm. of the peak height of 2-
propanol and ethyl acetate to that of the internal standard of
the standard solution (1) and the ratios, gsa2 and Q S bi, of the
peak height of 2-propanol and ethyl acetate to that of the in-
ternal standard of the standard solution (2). Calculate the
amounts of 2-propanol and ethyl acetate by the following
equations: not more than 0.7% and not more than 1.7%,
respectively.
Amount (%) of 2-propanol
= (W S JW T ) X {(2g Ta - 3Q Sal + gsa2)/(Gsa2 ~ 2 S al)}
812
L-Leucine / Official Monographs
JP XV
Amount (%) of ethyl acetate
= {W^/Wj) x {(2g Tb - 3Q sbl + esb 2 )/(Gsb2 - Gsm)}
W^'- Amount (g) of 2-propanol
W sb : Amount (g) of ethyl acetate
W T : Amount (g) of the sample
Internal standard solution — A solution of cyclohexane in
AT.Af-dimethylformamide (1 in 1000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 3 m
in length, packed with siliceous earth for gas chromato-
graphy (180-250/^m in particle diameter) coated with
tetrakishydroxypropylethylenediamine for gas chromato-
graphy at the ratio of 10 to 15%.
Column temperature: A constant temperature of about
80°C.
Injection port temperature: A constant temperature of
about 160°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 1 minute.
System suitability —
System performance: When the procedure is run with 4//L
of the standard solution (2) under the above operating condi-
tions, the internal standard, ethyl acetate and 2-propanol are
eluted in this order, and the resolution between the peaks of
the internal standard and ethyl acetate is not less than 2.0.
System repeatability: When the test is repeated 3 times with
4 fiL of the standard solution (2) under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of ethyl acetate to that of the internal standard is
not more than 5.0%.
Water <2.48> Not more than 1.5% (1 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately an amount of Lenampicillin
Hydrochloride and Lenampicillin Hydrochloride Reference
Standard, equivalent to about 0.1 g (potency), dissolve each
in the internal standard solution to make exactly 10 mL, and
use these solutions as the sample solution and the standard
solution. Perform the test with 5 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, Q T and Q s , of the peak area of
lenampicillin to that of the internal standard.
Amount [fig (potency)] of ampicillin (C 16 H 19 N 3 04S)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Lenampicillin Hydrochlo-
ride Reference Standard
Internal standard solution — A solution of ethyl aminobenzo-
ate in the mobile phase (1 in 4000).
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 9.53 g of potassium dihydrogen
phosphate in water to make exactly 700 mL, and add acetoni-
trile to make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
lenampicillin is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, lenampicillin and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of lenampicillin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
L-Leucine
l- n -i -> >
C 6 H 13 N0 2 : 131.17
(2<S)-2-Amino-4-methylpentanoic acid
[61-90-5]
L-Leucine, when dried, contains not less than 98.5%
of C 6 H 13 N0 2 .
Description L-Leucine occurs as white crystals or crystalline
powder. It is odorless or has a faint characteristic odor, and
has a slightly bitter taste.
It is freely soluble in formic acid, sparingly soluble in
water, and practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Leucine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [ a ]g>: + 14.5 - + 16.0° (after
drying, 1 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of L-Leucine in 100 mL of water:
the pH of this solution is between 5.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
L-Leucine in 10 mL of 1 mol/L hydrochloric acid TS: the so-
lution is clear and colorless.
(2) Chloride <1.03>— Dissolve 0.5 g of L-Leucine in 40
mL of water and 6 mL of dilute nitric acid, and add water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Dissolve 0.6 g of L-Leucine in 40 mL
of water and 1 mL of dilute hydrochloric acid, and add water
to make 50 mL. Perform the test using this solution as the
JPXV
Official Monographs / Levallorphan Tartrate
813
test solution. Prepare the control solution with 0.35 mL of
0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Leucine. Prepare the control solution with 5.0 mL of Stan-
dard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of L-Leu-
cine according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of L-Leucine according to Method 2, and perform the test
(not more than 2 ppm).
(7) Related substances — Dissolve 0.10 g of L-Leucine by
warming, after cooling, add water to make 25 mL, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, and add water to make exactly 50 mL. Pipet 5 mL
of this solution, add water to make exactly 20 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 ,uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (3:1:1) to a distance of about 10
cm, and dry the plate at 80°C for 30 minutes. Spray evenly a
solution of ninhydrin in acetone (1 in 50) on the plate, and
heat at 80°C for 5 minutes: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.13 g of L-Leucine, previ-
ously dried, and dissolve in 3 mL of formic acid, add 50 mL
of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 13.12 mg of C 6 H I3 N0 2
Containers and storage Containers — Well-closed contain-
ers.
Levallorphan Tartrate
UM'njL.7 7>>S5gt±£
H0 2 C
C0 2 H
C 19 H 25 NO.C 4 H 6 6 : 433.49
17-Allylmorphinan-3-ol monotartrate [71-82-9]
Levallorphan Tartrate, when dried, contains not less
than 98.5% of C 19 H 25 NO.C 4 H 6 6 .
yellow, crystalline powder. It is odorless.
It is soluble in water and in acetic acid (100), sparingly
soluble in ethanol (95), and practically insoluble in diethyl
ether.
Identification (1) Determine the absorption spectrum of a
solution of Levallorphan Tartrate in 0.01 mol/L hydrochlor-
ic acid TS (1 in 10,000) as directed under Ultraviolet-visible
Spectrophotometry, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Leval-
lorphan Tartrate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Levallorphan Tartrate (1 in 30)
responds to the Qualitative Tests <1.09> (1) and (2) for tar-
trate.
Optical rotation <2.49> [a]™: -37.0
drying, 0.2 g, water, 10 mL, 100 mm).
■39.2° (after
pH <2.54> Dissolve 0.2 g of Levallorphan Tartrate in 20 mL
of water: the pH of this solution is between 3.3 and 3.8.
Melting point <2.60> 174 - 178°C
Purity (1) Clarity and color of solution — Dissolve 0.2 g of
Levallorphan Tartrate in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Levallor-
phan Tartrate according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Levallorphan
Tartrate in 10 mL of water, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of methanol and ammonia TS (200:3) to a distance
of about 10 cm, and air-dry the plate. Spray evenly Dragen-
dorff's TS for spraying on the plate: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 80°C, 4 hours).
Residue on ignition <2.44> Not more than 0.10% (1 g).
Assay Weigh accurately about 0.5 g of Levallorphan Tar-
trate, previously dried, dissolve in 30 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (indica-
tor: 2 drops of crystal violet TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 43.35 mg of C 19 H 25 NO.C 4 H 6 6
Containers and storage Containers — Well-closed contain-
ers.
Description Levallorphan Tartrate occurs as a white to pale
814
Levallorphan Tartrate Injection / Official Monographs
JP XV
Levallorphan Tartrate Injection
Levallorphan Tartrate Injection is an aqueous solu-
tion for injection.
It contains not less than 93% and not more than 107
% of the labeled amount of levallorphan tartrate (Ci 9
H 25 NO.C 4 H 6 6 : 433.49).
Method of preparation Prepare as directed under Injection,
with Levallorphan Tartrate.
Description Levallorphan Tartrate Injection is a clear,
colorless liquid.
pH: 3.0-4.5
Identification Take an exact volume of Levallorphan Tar-
trate Injection, equivalent to 3 mg of Levallorphan Tartrate
according to the labeled amount, add 5 mL of water and 2
drops of dilute hydrochloric acid, and wash with five 15-mL
portions of diethyl ether by a vigorous shaking. Take the
water layer, evaporate the diethyl ether remained by warming
on a water bath, and after cooling, add 0.01 mol/L
hydrochloric acid TS to make 50 mL. Determine the absorp-
tion spectrum of this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 277 nm and 281 nm.
Extractable volume <6.05> It meets the requirement.
Assay Take wxactly a volume of Levallorphan Tartrate In-
jection, equivalent to about 2 mg of levallorphan tartrate
(C 19 H 2 5NO.C 4 H 6 6 ), add exactly 10 mL of the internal stan-
dard solution, and use this solution as the sample solution.
Separately, weigh accurately about 0.1 g of levallorphan tar-
trate for assay, previously dried at 80°C for 4 hours on phos-
phorus (V) oxide under reduced pressure, and dissolve in
water to make exactly 100 mL. Pipet 2 mL of this solution,
add exactly 10 mL of the internal standard solution, and use
this solution as the standard solution. Perform the test with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following operating conditions, and calculate the ratios,
Q T and Q s , of the peak area of levallorphan to that of the in-
ternal standard:
Amount (mg) of C 19 H 2 5NO.C 4 H 6 6
= W s x (Q T /Q S ) x (1/50)
W s : Amount (mg) of levallorphan tartrate for assay
Internal standard solution — Dissolve 0.04 g of isobutyl para-
hydroxybenzoate in 10 mL of ethanol (95), add water to
make 100 mL, and to 10 mL of this solution add water to
make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 300 mL of this solu-
tion add 200 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
levallorphan is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the internal standard and levallorphan are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of levallorphan to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Levodopa
UtK' H/\
CO;H
H NHj
C 9 H,,N0 4 : 197.19
3-Hydroxy-L-tyrosine [59-92-7]
Levodopa, when dried, contains not less than 98.5%
of C 9 H„N0 4 .
Description Levodopa occurs as white or slightly grayish
white crystals or crystalline powder. It is odorless.
It is freely soluble in formic acid, slightly soluble in water,
and practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
The pH of a saturated solution of Levodopa is between 5.0
and 6.5.
Melting point: about 275°C (with decomposition).
Identification (1) To 5 mL of a solution of Levodopa (1 in
1000) add 1 mL of ninhydrin TS, and heat for 3 minutes in a
water bath: a purple color develops.
(2) To 2 mL of a solution of Levodopa (1 in 5000) add 10
mL of 4-aminoantipyrine TS, and shake: a red color de-
velops.
(3) Dissolve 3 mg of Levodopa in 0.001 mol/L
hydrochloric acid TS to make 100 mL. Determine the absorp-
tion spectrum of the solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
Absorbance <2.24> E\ 0/ ° m (280 nm): 136 - 146 (after drying,
30 mg, 0.001 mol/L hydrochloric acid TS, 1000 mL).
Optical rotation <2.49> [ a \^: -11.5- -13.0° (after
drying, 2.5 g, 1 mol/L hydrochloric acid TS, 50 mL, 100
nm).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
JPXV
Official Monographs / Levomepromazine Maleate
815
Levodopa in 20 mL of 1 mol/L hydrochloric acid TS: the so-
lution is clear and colorless.
(2) Chloride <1.03> — Dissolve 0.5 g of Levodopa in 6 mL
of dilute nitric acid, and add water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 0.3 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Dissolve 0.40 g of Levodopa in 1 mL
of dilute hydrochloric acid and 30 mL of water, and add
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 0.25 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.030%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Levodo-
pa according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(5) Arsenic <1.11> — Dissolve 1.0 g of Levodopa in 5 mL
of dilute hydrochloric acid, and perform the test with this so-
lution as the test solution (not more than 2 ppm).
(6) Related sulstances — Dissolve 0.10 g of Levodopa in
10 mL of sodium disulfite TS, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add sodi-
um disulfite TS to make exactly 25 mL. Pipet 1 mL of this so-
lution, add sodium disulfite TS to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /XL each of the sample solution
and standard solution on a plate of cellulose for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, water, acetic acid (100) and methanol (10:5:5:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly a
solution of ninhydrin in acetone (1 in 50) on the plate and
heat at 90°C for 10 minutes: the spots other than the prin-
cipal spot from the sample solution are not more intense than
the spot from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Levodopa, previ-
ously dried, dissolve in 3 mL of formic acid, add 80 mL of a-
cetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the color of the solution changes from purple
through blue-green to green (indicator: 3 drops of crystal vio-
let TS). Perform a blank determination, and make any neces-
sary correction.
Each mL of 0.1 mol/L perchloric acid
= 19.72 mg of C 9 H u N0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Levomepromazine Maleate
UtKV ~fn -7 v>v V4 >mik
H CH 3 CH 3
CH :J
ft
CH 3
(
COjH-
CO;H
C 19 H 2 4N 2 OS.G,H 4 04: 444.54
(2i?)-3-(2-Methoxy-10//-phenothiazin-10-yl)-
N,N, 2-trimethylpropylamine monomaleate
[7 J 04-38-3]
Levomepromazine Maleate, when dried, contains
not less than 98.0% of C19H24N2OS.C4H4O4.
Description Levomepromazine Maleate occurs as white
crystals or crystalline powder. It is odorless, and has a slight-
ly bitter taste.
It is freely soluble in acetic acid (100), soluble in chlo-
roform, sparingly soluble in methanol, slightly soluble in
ethanol (95) and in acetone, very slightly soluble in water,
and practically insoluble in diethyl ether.
Melting point: 184 - 190°C (with decomposition).
Identification (1) Dissolve 5 mg of Levomepromazine
Maleate in 5 mL of sulfuric acid: a red-purple color develops,
which slowly becomes deep red-purple. To this solution add 1
drop of potassium dichromate TS: a brownish yellow-red
color is produced.
(2) To 0.2 g of Levomepromazine Maleate add 5 mL of
sodium hydroxide TS and 20 mL of diethyl ether, and shake
well. Separate the diethyl ether layer, wash twice with 10-mL
portions of water, add 0.5 g of anhydrous sodium sulfate,
filter, evaporate the diethyl ether on a water bath, and dry the
residue at 105 °C for 2 hours: the residue melts <2.60> be-
tween 124°C and 128°C.
(3) To 0.5 g of Levomepromazine Maleate add 5 mL of
water and 2 mL of ammonia solution (28), extract with three
5-mL portions of chloroform, separate and evaporate the
water layer to dryness. To the residue add 2 to 3 drops of di-
lute sulfuric acid and 5 mL of water, and extract with four
25-mL portions of diethyl ether. Combine all the diethyl
ether extracts, evaporate the diethyl ether in a water bath at a
temperature of about 35 °C with the aid of a current of air:
the residue melts <2.60> between 128°C and 136°C.
Optical rotation <2.49> [ a ]%: -13.5- -16.5° (after
drying, 0.5 g, chloroform, 20 mL, 200 mm).
Purity (1) Clarity and color of solution — To 0.5 g of
Levomepromazine Maleate add 10 mL of methanol, and dis-
solve by warming: the solution is clear, and colorless or pale
yellow.
(2) Chloride <1.03> — Dissolve 0.5 g of Levomepromazine
Maleate in 40 mL of methanol, and add 6 mL of dilute nitric
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution with
0.40 mL of 0.01 mol/L hydrochloric acid VS, 40 mL of
methanol, 6 mL of dilute nitric acid and water to make 50
mL (not more than 0.028%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of
816
Levothyroxine Sodium Hydrate / Official Monographs
JP XV
Levomepromazine Maleate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Levomepromazine
Maleate, previously dried, and dissolve in a mixture of 40 mL
of acetic acid (100) and 20 mL of acetone for nonaqueous
titration. Titrate <2.50> with 0.1 mol/L perchloric acid VS
until the color of the solution changes from red-purple
through blue-purple to blue (indicator: 5 drops of
bromocresol green-methylrosaniline chloride TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 44.45 mg of C^H^OS.QH^
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Levothyroxine Sodium Hydrate
i
COjNa
H NH 2
• lH s O
C 15 H 10 I 4 NNaO4.xH 2 O
Monosodium 0-(4-hydroxy-3 ,5-diiodophenyl)-3 ,5-diiodo-
L-tyrosinate hydrate [25416-65-3]
Levothyroxine Sodium Hydrate contains not less
than 97.0% of levothyroxine sodium (C 15 H 10 l4NNaO 4 :
798.85), calculated on the dried basis.
Description Levothyroxine Sodium Hydrate occurs as a
pale yellowish white to light yellow-brown powder. It is odor-
less.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water and in diethyl ether.
It dissolves in sodium hydroxide TS.
It is gradually colored by light.
Identification (1) Heat 0.1 g of Levothyroxine Sodium
Hydrate over a flame: a purple gas evolves.
(2) To 0.5 mg of Levothyroxine Sodium Hydrate add 8
mL of a mixture of water, ethanol (95), hydrochloric acid
and sodium hydroxide TS (6:5:2:2), warm in a water bath for
2 minutes, cool, and add 0.1 mL of sodium nitrite TS. Allow
to stand in a dark place for 20 minutes, and add 1.5 mL of
ammonia solution (28): a yellowish red color is produced.
(3) Determine the absorption spectrum of a solution of
Levothyroxine Sodium Hydrate in dilute sodium hydroxide
TS (1 in 10,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(4) Moisten Levothyroxine Sodium Hydrate with sulfuric
acid, and ignite: the residue responds to the Qualitative Tests
<1.09> (1) and (2) for sodium salt.
Optical rotation <2.49> [a]™: -5- -6° (0.3 g, calculated
on the dried basis, a mixture of ethanol (95) and sodium
hydroxide TS (2:1), 10 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.3 g of
Levothyroxine Sodium Hydrate in 10 mL of a mixture of
ethanol (95) and sodium hydroxide TS (2:1) by warming: the
solution is clear and pale yellow to pale yellow-brown in
color.
(2) Soluble halides — Dissolve 0.01 g of Levothyroxine
Sodium Hydrate in 10 mL of water and 1 drop of dilute nitric
acid, shake for 5 minutes, and filter. To the filtrate add water
to make 10 mL, then add 3 drops of silver nitrate TS, and
mix: the solution has no more opalescence than the following
control solution.
Control solution: To 0.20 mL of 0.01 mol/L hydrochloric
acid VS add 10 mL of water and 1 drop of dilute nitric acid,
and proceed as directed above.
(3) Related substances — Dissolve 20 mg of
Levothyroxine Sodium Hydrate in 2 mL of a mixture of
ethanol (95) and ammonia solution (28) (14:1), and use this
solution as the sample solution. Pipet 1 mL of this solution,
add a mixture of ethanol (95) and ammonia solution (28)
(14:1) to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of Z-butanol, ?-amyl alcohol, water, ammonia
solution (28) and 2-butanone (59:32:17:15:7) to a distance of
about 12 cm, and air-dry the plate. Spray evenly a solution of
0.3 g of ninhydrin in 100 mL of a mixture of 1-butanol and a-
cetic acid (100) (97:3) on the plate, and heat at 100°C for 3
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> 1 - 11% (0.5 g, in vacuum, phospho-
rus (V) oxide, 60°C, 4 hours).
Assay Weigh accurately about 25 mg of Levothyroxine So-
dium Hydrate, and proceed as directed under Oxygen Flask
Combustion Method <1.06>, using a mixture of 10 mL of so-
dium hydroxide solution (1 in 100) and 1 mL of a freshly pre-
pared sodium bisulfate solution (1 in 100) as the absorbing
liquid, and prepare the test solution. Apply a small amount
of water to the upper part of apparatus A, pull out C careful-
ly, and wash C, B and the inner wall of A with 40 mL of
water. To the test solution add 1 mL of bromine-acetic acid
TS, insert the stopper C, and shake vigorously for 1 minute.
Remove the stopper, rinse the stopper, the sample holder and
the inner wall of the flask with 40 mL of water, and add 0.5
mL of formic acid. Stopper the flask with C, and shake
vigorously for 1 minute again. Remove the stopper, and rinse
the stopper, the sample holder and the inner wall of the flask
with 40 mL of water. Bubble the solution with enough nitro-
gen gas in the flask to remove the oxygen and excess bromine,
add 0.5 g of potassium iodide to the solution, and dissolve.
Add immediately 3 mL of dilute sulfuric acid, mix, and allow
to stand for 2 minutes. Titrate <2.50> the solution with 0.02
mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS).
Perform a blank determination, and make any necessary cor-
rection.
JPXV
Official Monographs / Levothyroxine Sodium Tablets
817
Each mL of 0.02 mol/L sodium thiosulfate VS
= 0.6657 mg of C 15 H 10 I 4 NNaO4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Levothyroxine Sodium Tablets
Levothyroxine Sodium Tablets contain not less than
90% and not more than 1 10% of the labeled amount of
levothyroxine sodium (C 15 H 10 l4NNaO 4 : 798.85).
Method of preparation Prepare as directed under Tablets,
with Levothyroxine Sodium Hydrate.
Identification (1) Weigh a quantity of powdered
Levothyroxine Sodium Tablets, equivalent to 0.5 mg of
Levothyroxine Sodium Hydrate according to the labeled
amount, add 8 mL of a mixture of water, ethanol (95),
hydrochloric acid and sodium hydroxide TS (6:5:2:2), warm
in a water bath for 2 minutes, cool, and filter. To the filtrate
add 0. 1 mL of sodium nitrite TS, and allow to stand in a dark
place for 20 minutes. Add 1.5 mL of ammonia solution (28):
a yellowish red color develops.
(2) To a quantity of powdered Levothyroxine Sodium
Tablets, equivalent to 1 mg of Levothyroxine Sodium Hy-
drate according to the labeled amount, add 10 mL of ethanol
(95), shake, filter, and use the filtrate as the sample solution.
Dissolve 0.01 g of levothyroxine sodium for thin-layer chro-
matography in 100 mL of ethanol (95), and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of Z-butanol, t-amyl alcohol,
water, ammonia solution (28) and 2-butanone
(59:32:17:15:7) to a distance of about 12 cm, and air-dry the
plate. Spray a solution of 0.3 g of ninhydrin in 100 mL of a
mixture of 1-butanol and acetic acid (100) (97:3) on the plate,
and heat at 100°C for 3 minutes: the spots obtained from the
sample solution and the standard solution show a red-purple
color, and has the same Rf value.
Purity Soluble halides — Weigh a quantity of powdered
Levothyroxine Sodium Tablets, equivalent to 2.5 mg of
Levothyroxine Sodium Hydrate according to the labeled
amount, add 25 mL of water, warm to 40 C C, shake for 5
minutes, add 3 drops of dilute nitric acid, and filter. To the
filtrate add 3 drops of silver nitrate TS, and mix: the solution
has no more opalescence than the following control solution.
Control solution: To 0.25 mL of 0.01 mol/L hydrochloric
acid VS add 25 mL of water and 3 drops of dilute nitric acid,
and proceed as directed above.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Place 1 tablet of Levothyroxine Sodium Tablets in a glass-
stoppered centrifuge tube, add exactly 10 mL of 0.01 mol/L
sodium hydroxide TS, warm at 50°C for 15 minutes, and
shake vigorously for 20 minutes. Centrifuge this solution,
pipet 5 mL of the supernatant liquid, add 1 mL of the inter-
nal standard solution, and use this solution as the sample so-
lution. Perform the test with 20 /xL of the sample solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratio of the peak
area of levothyroxine to that of the internal standard. Calcu-
late the mean value from the ratios of each peak area of 10
samples: the deviation (%) of the mean value and the ratio of
each peak area should be not more than 15%. When the devi-
ation (%) is more than 15%, and 1 sample shows not more
than 25%, perform another test with 20 samples. Calculate
the deviation (%) of the mean value of the 30 samples used in
the 2 tests and the ratio of each peak area: there should be not
more than 1 sample with the deviation more than 15% but
not more than 25%, and no sample should deviate by more
than 25%.
Internal standard solution — A solution of ethinylestradiol in
a mixture of acetonitrile and diluted phosphoric acid (1 in 10)
(9:1) (3 in 40,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: a constant wavelength between 220 nm and 230
nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 10 to 25 cm in length, packed with octadecyl-
silanized silica gel.
Column temperature: A constant temperature at about
25°C.
Mobile phase: A mixture of methanol, water and phos-
phoric acid (1340:660:1).
Flow rate: Adjust the flow rate so that the retention time of
levothyroxine is about 9 minutes.
Selection of column: To 5 mL of a solution of
levothyroxine sodium in 0.01 mol/L sodium hydroxide TS (1
in 200,000) add 1 mL of the internal standard solution. Pro-
ceed with 20 iuL of this solution under the above operating
conditions, and calculate the resolution. Use a column giving
elution of levothyroxine and the internal standard in this ord-
er with the resolution between these peaks being not less than
2.0.
Assay Weigh accurately and powder not less than 20
Levothyroxine Sodium Tablets. Weigh accurately a portion
of the powder, equivalent to about 3 mg of levothyroxine so-
dium (C 15 Hiol4NNa0 4 ), into a crucible, and add potassium
carbonate amounting to twice the mass of the powder. In the
case that the weighed powder is less than 4 g, add 8 g of
potassium carbonate to the crucible. Mix well, and gently tap
the crucible on the bench to compact the mixture. Overlay
with 10 g of potassium carbonate, and compact again by tap-
ping. Heat the crucible strongly at a temperature between
675 °C and 700°C for 25 minutes. Cool, add 30 mL of water,
heat gently to boiling, and filter into a flask. To the residue
add 30 mL of water, boil, and filter into the same flask. Rinse
the crucible and the char on the funnel with hot water until
the filtrate measures 300 mL. Add slowly 7 mL of freshly pre-
pared bromine TS and diluted phosphoric acid (1 in 2) in the
ratio of 3.5 mL to 1 g of the added potassium carbonate, and
boil until starch-potassium iodide paper is no longer colored
blue by the evolved gas. Wash the inside of the flask with
water, and continue boiling for 5 minutes. During the boiling
add water from time to time to maintain a volume of not less
than 250 mL. Cool, add 5 mL of a solution of phenol (1 in
818
Lidocaine / Official Monographs
JP XV
20), again rinse the inside of the flask with water, and allow
to stand for 5 minutes. Add 2 mL of diluted phosphoric acid
(1 in 2) and 5 mL of potassium iodide TS, and titrate <2.50>
immediately the liberated iodine with 0.01 mol/L sodium
thiosulfate VS (indicator: 3 mL of starch TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.01 mol/L sodium thiosulfate VS
= 0.3329 mg of C 15 H 10 I 4 NNaO4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Lidocaine
'J K -h <f >
CH 3
rVVr
,
CHj
CH,
C 14 H 22 N 2 0: 234.34
2-Diethylamino-/V-(2,6-dimethylphenyl)acetamide
[137-58-6]
Lidocaine, when dried, contains not less than 99.0%
of C 14 H 22 N 2 0.
Description Lidocaine occurs as white to pale yellow crys-
tals or crystalline powder.
It is very soluble in methanol and in ethanol (95), soluble in
acetic acid (100) and in diethyl ether, and practically insolu-
ble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Dissolve 0.04 g of Lidocaine in 10 mL of
1 mol/L hydrochloric acid TS, and add water to make 100
mL. Determine the absorption spectrum of the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Lidocaine as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Melting point <2.60> 66 - 69°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Lidocaine in 2 mL of dilute hydrochloric acid, and add water
to make 10 mL: the solution is clear and colorless to light yel-
low.
(2) Chloride <1.03> — Dissolve 0.6 g of Lidocaine in 6 mL
of dilute nitric acid, add water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution with 0.70 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.041%).
(3) Sulfate <1.14> — Dissolve 0.5 g of Lidocaine in 5 mL
of dilute hydrochloric acid, add water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 1 .0 mL of 0.005 mol/L sulfuric
acid VS, 5 mL of dilute hydrochloric acid and water to make
50 mL (not more than 0.096%).
(4) Heavy metals <1.07> — Carbonize 2.0 g of Lidocaine
by gentle ignition. After cooling, add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10), and
fire the ethanol to burn. After cooling, add 1 mL of sulfuric
acid, proceed according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Related substances — Dissolve 0.10 g of Lidocaine in 2
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add methanol to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, 2-butanone, water
and formic acid (5:3:1:1) to a distance of about 10 cm, air-d-
ry the plate, and dry more at 80°C for 30 minutes. After cool-
ing, examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41>
silica gel, 24 hours).
Not more than 0.5% (1 g, in vacuum,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 0.5 g of Lidocaine, previously dried
and accurately weighed, in 20 mL of acetic acid (100), and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (indicator: 1
drop of crystal violet TS) until the color of the solution
changes from purple to blue-green through blue. Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 23.43 mg of C 14 H 22 N 2
Containers and storage Containers — Tight containers.
Lidocaine Injection
Lidocaine Hydrochloride Injection
Lidocaine Injection is an aqueous injection.
It contains not less than 95% and not more than 105
% of the labeled amount of lidocaine hydrochloride
(C 14 H 22 N 2 O.HCl: 270.80).
Method of preparation Prepare as directed under Injec-
tions, with Lidocaine and an equivalent amount of
Hydrochloric Acid.
No preservative is added in the case of intravenous injec-
tions.
Description Lidocaine Injection is a colorless, clear liquid.
pH: 5.0-7.0
Identification To a volume of Lidocaine Injection, equiva-
lent to 0.02 g of Lidocaine Hydrochloride (C 14 H 22 N 2 O.HCl)
according to the labeled amount, add 1 mL of sodium
hydroxide TS, and extract with 20 mL of hexane. To 10 mL
JPXV
Official Monographs / Limaprost Alfadex
819
of the hexane extract add 20 mL of 1 mol/L hydrochloric
acid TS, and shake vigorously. Determine the absorption
spectrum of the water layer as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>: it exhibits a maximum be-
tween 261 nm and 265 nm.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Perform the test with Lidocaine Injection
stored in a container in a volume exceeding 10 mL and in-
tended to intravenous injection: it meets the requirements of
the Pyrogen Test.
Assay To an exactly measured volume of Lidocaine Injec-
tion, equivalent to about 0.1 g of lidocaine hydrochloride
(C14H22N2O.HCI), add exactly 10 mL of the internal standard
solution and 0.001 mol/L hydrochloric acid TS to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 85 mg of lidocaine for assay, previ-
ously dried in a desiccator (in vacuum, silica gel) for 24
hours, dissolve in 0.5 mL of 1 mol/L hydrochloric acid TS
and a suitable volume of 0.001 mol/L hydrochloric acid TS,
and add exactly 10 mL of the internal standard solution, then
add 0.001 mol/L hydrochloric acid TS to make 50 mL, and
use this solution as the standard solution. Perform the test
with 5 [iL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Qs, of the peak area of lidocaine to that of the internal stan-
dard.
Amount (mg) of lidocaine hydrochloride
(C 14 H 22 N 2 O.HCl)
= W s x (Qj/Q s ) x 1.1556
W s : Amount (mg) of lidocaine for assay
Internal standard solution — A solution of benzophenone in
methanol (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (10 /an in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.88 g of sodium lauryl sulfate in
1000 mL of a mixture of 0.02 mol/L phosphate buffer solu-
tion, pH 3.0 and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
lidocaine is about 6 minutes.
System suitability —
System performance: When proceed with 5 /xL of the stan-
dard solution under the above operating conditions,
lidocaine and the internal standard are eluted in this order
with the resolution between these peaks being not less than 6.
System repeatability: When the test is repeated 6 times with
5 nL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of lidocaine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Limaprost Alfadex
°, H
OH
hJ OH
C22H36U5 ■ XC36H60O30
(2£)-7-{(l/?,2i?,3/?)-3-Hydroxy-2-[(l£,3S,5S)-3-
hydroxy-5-methylnon- 1 -en-1 -yl]-
5-oxocyclopentyl} hept-2-enoic acid — a-cyclodextrin
[100459-01-6 (limaprost: alfadex = 1:1; clathrate compound)]
Limaprost Alfadex is a a-cyclodextrin clathrate com-
pound of limaprost.
It contains not less than 2.8% and not more than 3.2
% of limaprost (C22H 36 5 : 380.52), calculated on the
anhydrous basis.
Description Limaprost Alfadex occurs as a white powder.
It is freely soluble in water, slightly soluble in methanol,
very slightly soluble in ethanol (99.5), and practically insolu-
ble in ethyl acetate.
It is hygroscopic.
Identification (1) Dissolve 20 mg of Limaprost Alfadex in
5 mL of water, add 5 mL of ethyl acetate, shake, centrifuge,
and use the upper layer as the sample solution (1). Separately,
to 20 mg of Limaprost Alfadex add 5 mL of ethyl acetate,
shake, centrifuge, and use the supernatant liquid as the sam-
ple solution (2). Evaporate the solvent of the sample solu-
tions (1) and (2) under reduced pressure, add 2 mL of sulfuric
acid to each of the residue, and shake them for 5 minutes: the
solution obtained from the sample solution (1) develops an
orange-yellow color while the solution from the sample solu-
tion (2) does not develop any color.
(2) Dissolve 20 mg of Limaprost Alfadex in 5 mL of
water, add 5 mL of ethyl acetate, shake, centrifuge, and
evaporate the solvent of the upper layer under reduced pres-
sure. Dissolve the residue in 2 mL of ethanol (95), 5 mL of
1,3-dinitrobenzene TS, add 5 mL of a solution of potassium
hydroxide in ethanol (95) (17 in 100) while ice-cooling, and
allow to stand in a dark place while ice-cooling for 20
minutes: a purple color develops.
(3) To 50 mg of Limaprost Alfadex add 1 mL of iodine
TS, dissolve by heating in a water bath, and allow to stand: a
dark blue precipitate is formed.
(4) Determine the absorption spectrum of a solution of
Limaprost Alfadex in dilute ethanol (3 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it does
not exhibit a maximum between 200 nm and 400 nm. To 10
mL of this solution add 1 mL of potassium hydroxide-
ethanol TS, and allow to stand for 15 minutes. Determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
820
Lincomycin Hydrochloride Hydrate / Official Monographs
JP XV
Optical rotation <2.49> [«]£: + 125 - 135° (0.1 g, calculat-
ed on the anhydrous basis, dilute ethanol, 20 mL, 100 mm)
Purity Related substances — Perform the test immediately
after preparation of the sample solution. Dissolve 0.10 g of
Limaprost Alfadex in 2 mL of water, add 1 mL of ethanol
(95), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add dilute ethanol to make exactly
100 mL, and use this solution as the standard solution (1).
Pipet 3 mL of the standard solution (1), add dilute ethanol to
make exactly 10 mL, and use this solution as the standard so-
lution (2). Perform the test with exactly 3 /uL each of the sam-
ple solution and standard solutions (1) and (2) as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing operating conditions, and determine each peak area by
the automatic integration method: the area of the peak of 17-
epi-isomer, having the relative retention time of about 1.1
with respect to limaprost, and the area of the peak of 11-
deoxy substance, having the relative retention time of about
2.1 with respect to limaprost, are not larger than the peak
area of limaprost from the standard solution (2), and the area
of the peak other than the principal peak and other than the
peaks mentioned above is not larger than 1/3 times the peak
area of limaprost from the standard solution (2). The total
area of the peaks other than limaprost from the samples solu-
tion is not larger than the peak area of limaprost from the
standard solution (1).
Operating conditions —
Detector, column, column temperature, mobile phase and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of limaprost beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution (1) add dilute ethanol to make exactly 10 mL.
Confirm that the peak area of limaprost obtained from 3 juL
of this solution is equivalent to 8 to 12% of that of limaprost
obtained from 3 juL of the standard solution (1).
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
3 /uL of the standard solution (1) under the above conditions,
the relative standard deviation of the peak area of limaprost
is not more than 2.0%.
Water <2.48> Not more than 6.0% (0.2 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately about 0.1 g of Limaprost Afladex,
dissolve in 5 mL of water, add exactly 5 mL of the internal
standard solution, and use this solution as the sample solu-
tion. Separately, weigh accurately about 3 mg of Limaprost
Reference Standard, dissolve in 5 mL of water, add exactly 5
mL of the internal standard solution, and use this solution as
the standard solution. Perform the test with 3 /xL each of the
sample solution and the standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the ratios, Q T and Q s , of the peak
area of limaprost to that of the internal standard.
Amount (mg) of limaprost (C 22 H 36 5 ) = W s x (Q T /Q S )
W s : Amount (mg) of Limaprost Reference Standard
Internal standard solution— A solution of propyl para-
hydroxybenzoate in ethanol (95) (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.02 mol/L potassium di-
hydrogen phosphate TS, acetonitrile for liquid chro-
matography and 2-propanol for liquid chromatography
(9:5:2)
Flow rate: Adjust the flow rate so that the retention time of
limaprost is about 12 minutes.
System suitability —
System performance: When the procedure is run with 3 /uL
of the standard solution under the above operating condi-
tions, the internal standard and limaprost are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
3 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of limaprost to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at a temperature not exceeding
-10°C.
Lincomycin Hydrochloride Hydrate
u>3v 4 •>>£»£*»*
HjC
,CH 3
,H OH
N-lX
CH :!
H
,OH H j
• HCI ■ HjO
CH 3
HO
C 18 H 34 N 2 6 S.HC1.H 2 0: 461.01
Methyl 6 , 8-dideoxy-6- [(2 S,4R )- 1 -methyl-4-
propylpyrrolidine-2-carboxamido] - 1 -thio-D-erythro-
a-D-galacto-octopyranoside monohydrochloride
monohydrate [7179-49-9]
Lincomycin Hydrochloride Hydrate is the
hydrochloride of a substance having antibacterial ac-
tivity produced by the growth of Streptomyces lincol-
nensis var. lincolnensis.
It contains not less than 825 Lig (potency) per mg,
calculated on the anhydrous basis. The potency of Lin-
comycin Hydrochloride Hydrate is expressed as mass
(potency) of lincomycin (C 18 H3 4 N 2 6 S: 406.54).
Description Lincomycin Hydrochloride Hydrate occurs as
white, crystals or crystalline powder.
It is freely soluble in water and in methanol, sparingly solu-
ble in ethanol (95), and very slightly soluble in acetonitrile.
JPXV
Official Monographs / Liothyronine Sodium
821
Identification (1) Determine the infrared absorption spec-
trum of Lincomycin Hydrochloride Hydrate as directed in
the paste method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum or
the spectrum of Lincomycin Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) A solution of Lincomycin Hydrochloride Hydrate (1
in 100) responds to the Qualitative Tests <1.09> (2) for chlo-
ride.
Optical rotation <2.49> [ a ]o- + 135 - + 150° (0.5 g, water,
25 mL, 100 mm).
pH <2.54> Dissolve 0.10 g of Lincomycin Hydrochloride
Hydrate in 1 mL of water: 3.0 - 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Lincomycin Hydrochloride Hydrate in 10 mL of water: the
solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Lin-
comycin Hydrochloride Hydrate according to Method 4, and
perform the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 5 ppm).
(3) Lincomycin B — Perform the test with 20 /xL of the
sample solution obtained in the Assay as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas of lincomycin and lin-
comycin B, having the relative retention time of about 0.5
with respect to lincomycin, by the automatic integration
method: the peak area of lincomycin B is not more than 5.0%
of the sum of the peak areas of lincomycin and lincomycin B.
Operating conditions-
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, and add the mobile phase to make ex-
actly 20 mL. Confirm that the peak area of lincomycin ob-
tained from 20,mL of this solution is equivalent to 3.5 to
6.5% of that obtained from 20 [iL of the sample solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Water <2.48> 3.0 - 6.0% (0.5 g, volumetric titration, direct
titration).
Assay Weigh accurately an amount of Lincomycin
Hydrochloride Hydrate and Lincomycin Hydrochloride
Reference Standard, equivalent to about 10 mg (potency),
dissolve each in the mobile phase to make exactly 10 mL, and
use these solutions as the sample solution and the standard
solution. Perform the test with exactly 20 iiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of lincomy-
cin.
Amount [fig (potency)] of lincomycin (C 18 H34N 2 6 S)
= W s x (Aj/As) x 1000
W s : Amount [mg (potency)] of Lincomycin Hydrochloride
Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octylsilanized silica gel for
liquid chromatography (5 ftm in particle diameter).
Column temperature: A constant temperature of about
46°C.
Mobile phase: To 13.5 mL phosphoric acid add water to
make 1000 mL, and adjust the pH to 6.0 with ammonia TS.
To 780 mL of this solution add 150 mL of acetonitrile and
150 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
lincomycin is about 9 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the theoretical plates and the symmetrical factor of the
peak of lincomycin are not less than 4000 and not more than
1.3, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
lincomycin is not more than 2.0%.
Containers and storage Containers — Tight containers.
Liothyronine Sodium
COjNa
H NH ?
CisHnLNNaCV 672.96
Monosodium 0-(4-hydroxy-3-iodophenyl)-3 ,5-diiodo-
L-tyrosinate [55-06-1]
Liothyronine Sodium contains not less than 95.0%
of C 15 H n l3NNa04, calculated on the dried basis.
Description Liothyronine Sodium occurs as a white to light
brown powder. It is odorless.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water and in diethyl ether.
It dissolves in sodium hydroxide TS and in ammonia TS.
Identification (1) To 5 mL of a solution of Liothyronine
Sodium in ethanol (95) (1 in 1000) add 1 mL of ninhydrin TS,
and warm in a water bath for 5 minutes: a purple color de-
velops.
(2) Heat 0.02 g of Liothyronine Sodium with a few drops
of sulfuric acid over a flame: a purple gas is evolved.
(3) Determine the absorption spectrum of a solution of
Liothyronine Sodium in ethanol (95) (1 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(4) Ignite 0.02 g of Liothyronine Sodium until thorough-
ly charred. After cooling, add 5 mL of water to the residue,
shake, and filter: the filtrate responds to the Qualitative Tests
<1.09> (1) for sodium salt.
822
Liothyronine Sodium Tablets / Official Monographs
JP XV
Optical rotation <2.49> [«]£: + 18 - +22° (0.2 g, calculat-
ed on the dried basis, a mixture of ethanol (95) and 1 mol/L
hydrochloric acid TS (4:1), 10 mL, 100 mm).
Purity (1) Soluble halide — To 10 mg of Liothyronine So-
dium add 10 mL of water and 1 drop of dilute nitric acid,
shake for 5 minutes, and filter. Add water to the filtrate to
make 10 mL, and mix with 3 drops of silver nitrate TS: the
solution shows no more turbidity than the following control
solution.
Control solution: To 0.35 mL of 0.01 mol/L hydrochloric
acid VS add 1 drop of dilute nitric acid and water to make 10
mL, and add 3 drops of silver nitrate TS.
(2) Iodine and iodide — Dissolve 0.10 g of Liothyronine
Sodium in 10 mL of dilute sodium hydroxide TS and 15 mL
of water, add 5 mL of dilute sulfuric acid, and allow to stand
for 10 minutes with occasional shaking. Filter the mixture
into a Nessler tube, add 10 mL of chloroform and 3 drops of
a solution of potassium iodate (1 in 100) to the filtrate, mix
for 30 seconds, and allow to stand: the chloroform layer has
no more color than the following control solution.
Control solution: Weigh exactly 0.111 g of potassium
iodide, and dissolve in water to make 1000 mL. Pipet 1 mL of
this solution, add 10 mL of dilute hydroxide TS, 14 mL of
water and 5 mL of dilute sulfuric acid, and mix. Filter the
mixture into a Nessler tube, and perform the test with the
filtrate in the same manner as for the sample.
(3) Related substances — Dissolve 0.15 g of Liothyronine
Sodium in 5 mL of diluted ammonia TS (1 in 3), and use this
solution as the sample solution. Pipet 1 mL of this solution,
add diluted ammonia TS (1 in 3) to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 1 /uL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of t-
butanol, f-amyl alcohol, water, ammonia solution (28) and 2-
butanone (59:32:17:15:7) to a distance of about 12 cm, and
air-dry the plate. Spray evenly a solution of 0.3 g of nin-
hydrin in 100 mL of a mixture of 1-butanol and acetic acid
(100) (97:3) on the plate, and dry the plate at 100°C for 3
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 4.0% (0.2 g, 105 °C,
2 hours).
Assay Weigh accurately about 25 mg of Liothyronine Sodi-
um, and proceed as directed under Oxygen Flask Combus-
tion Method <1.06>, using a mixture of 10 mL of a solution of
sodium hydroxide (1 in 100) and 1 mL of a freshly prepared
solution of sodium bisulfate (1 in 100) as the absorbing liq-
uid, and prepare the test solution. Apply a small amount of
water to the upper part of apparatus A, pull out C carefully,
and wash C, B and the inner wall of A with 40 mL of water.
To the test solution add 1 mL of bromine-acetic acid TS, in-
sert the stopper C, and shake vigorously for 1 minute. Re-
move the stopper, rinse the stopper, the sample holder and
the inner wall of the flask with 40 mL of water, and add 0.5
mL of formic acid. Stopper the flask with C, and shake
vigorously for 1 minute again. Remove the stopper, and rinse
the stopper, the sample holder and the inner wall of the flask
with 40 mL of water again. Bubble the solution with enough
nitrogen gas in the flask to remove the oxygen and excess bro-
mine, add 0.5 g of potassium iodide to the solution, and dis-
solve. Add immediately 3 mL of dilute sulfuric acid, mix,
and allow to stand for 2 minutes. Titrate <2.50> the solution
with 0.02 mol/L sodium thiosulfate VS (indicator: 3 mL of
starch TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.02 mol/L sodium thiosulfate VS
= 0.7477 mg of C 15 H u I 3 NNa04
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Liothyronine Sodium Tablets
Liothyronine Sodium Tablets contain not less than
90% and not more than 1 10% of the labeled amount of
liothyronine sodium (CnHnLNNaCV 672.96).
Method of preparation Prepare as directed under Tablets,
with Liothyronine Sodium.
Identification (1) To a glass-stoppered centrifuge tube add
a portion of finely powdered Liothyronine Sodium Tablets,
equivalent to 0.1 mg of Liothyronine Sodium according to
the labeled amount, add 30 mL of dilute sodium hydroxide
TS, shake vigorously, and centrifuge. Transfer the super-
natant liquid to a separator, add 10 mL of dilute hydrochlor-
ic acid, and extract with two 20-mL portions of ethyl acetate.
Filter each extract successively through absorbent cotton
previously overlaid with 8 g of anhydrous sodium sulfate.
Evaporate the filtrate on a water bath to dryness with the aid
of a current of nitrogen. Dissolve the residue in 0.5 mL of
methanol, and use this solution as the sample solution.
Separately, dissolve 10 mg of liothyronine sodium for thin-
layer chromatography in methanol to make 50 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 20 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of Z-butanol, t-
amyl alcohol, water, ammonia solution (28) and 2-butanone
(59:32:17:15:7) to a distance of about 12 cm, and air-dry the
plate. Spray evenly a solution of 0.3 g of ninhydrin in 100 mL
of a mixture of 1-butanol and acetic acid (100) (97:3) on the
plate, and dry the plate at 100°C for 3 minutes: the spots ob-
tained from the sample solution and the standard solution
show a red-purple color, and has the same Ri value.
(2) The colored solution obtained in the Assay is blue in
color.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Place 1 tablet of Liothyronine Sodium Tablets in a glass-
stoppered centrifuge tube, add exactly 10 mL of 0.01 mol/L
sodium hydroxide TS, warm at 50°C for 15 minutes, and
shake vigorously for 20 minutes. Centrifuge for 5 minutes,
and filter the supernatant liquid, if necessary. Pipet a definite
volume of this solution, and add a volume of 0.01 mol/L so-
JP XV
Official Monographs / Lisinopril Hydrate
823
dium hydroxide VS to prepare a definite volume of a solution
containing about 0.5 /ug of liothyronine sodium (C 15 H U I 3
NNa0 4 ) per mL. Pipet 5 mL of this solution, add exactly 1
mL of the internal standard solution, and use this solution as
the sample solution. Perform the test with 200 iuL of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and calculate the ratio
of the peak area of the liothyronine to that of the internal
standard. Calculate the mean value of the ratios of each peak
area of 10 samples: the deviation (%) of each ratio of the
peak area from the mean value should be not more than 15
%. When the deviation (%) is more than 15%, and 1 sample
shows not more than 25%, perform another test with 20 sam-
ples. Calculate the deviation (%) of each ratio of the peak
area from the mean value of the 30 samples used in the two
tests: there should be not more than 1 sample with the devia-
tion more than 15% but not more than 25%, and no sample
should deviate by more than 25%.
Internal standard solution — A solution of propylparahy-
droxybenzoate in a mixture of methanol and diluted phos-
phoric acid (1 in 10) (9:1) (1 in 250,000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsylanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Diluted methanol (57 in 100).
Flow rate: Adjust the flow rate so that the retention time of
liothyronine is about 9 minutes.
System suitability —
System performance: To 5 mL of a solution of liothyro-
nine sodium in 0.01 mol/L sodium hydroxide TS (1 in
2,000,000) add 1 mL of the internal standard solution, and
use this solution as the solution for system suitability test.
When the procedure is run with 200 /th of this solution under
the above operating conditions, the internal standard and
liothyronine are eluted in this order with the resolution be-
tween these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
200 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the ratios of the peak area of liothyronine to that of the in-
ternal standard is not more than 1.0%.
Assay Weigh accurately not less than 20 Liothyronine Sodi-
um Tablets, and finely powder. Place an accurately weighed
portion of the powder, equivalent to about 50 /xg of liothyro-
nine sodium (CuHn^NNaO^, in an agate mortar, add 1 g of
powdered potassium carbonate, and mix well. Transfer the
mixture cautiously to a porcelain crucible, and compact the
contents by gently tapping the crucible on a table. Add an ad-
ditional 1.5 g of powdered potassium carbonate to the same
agate mortar, mix well with any content adhering to the mor-
tar, cautiously overlay the mixture on the top of the same
porcelain crucible, and compact the charge again in the same
manner. Ignite the combined mixture in the crucible between
675°C and 700°C for 30 minutes. Cool, add a few mL of
water to the crucible, heat gently to boiling, and filter the
contents of the crucible through a glass filter (G4) into a
20-mL volumetric flask. Wash the residue with water, and
combine the washings with the filtrate. Cool, add water to
make 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 75 mg of potassium
iodide for assay, previously dried at 105°C for 4 hours, and
dissolve in water to make exactly 200 mL. Measure exactly 5
mL of the solution, and add a solution of potassium car-
bonate (1 in 8) to make exactly 100 mL. To 2 mL of this solu-
tion, exactly measured, add a solution of potassium car-
bonate (1 in 8) to make exactly 20 mL, and use the solution as
the standard solution. Pipet 5 mL each of the sample solution
and the standard solution into glass-stoppered test tubes, add
3.0 mL of diluted sulfuric acid (4 in 25) and 2.0 mL of potas-
sium permanganate TS, and heat on a water bath for 15
minutes. Cool, add 1.0 mL of diluted sodium nitrite TS (1 in
10), swirl to mix, and add 1 .0 mL of a solution of ammonium
amidosulfate (1 in 10). Allow to stand at room temperature
for 10 minutes with occasional shaking. Then add 1.0 mL of
potato starch TS and 1.0 mL of a freshly prepared, diluted
potassium iodide TS (1 in 40), swirl to mix, and transfer each
solution to a 20-mL volumetric flask. Rinse the test tube with
water, collect the washings in the volumetric flask, add water
to make 20 mL, and allow to stand for 10 minutes. Perform
the test with these solutions as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, using a solution prepared with
5 mL of potassium carbonate (1 in 8) in the same manner as
the sample solution as the blank. Determine the absorbances,
A T and^4 s , of the subsequent solutions of the sample solution
and the standard solution at the wavelength of maximum ab-
sorption at about 600 nm, respectively.
Amount (mg) of liothyronine sodium (C I5 H n l3NNa0 4 )
= W s x (At/As) x (1/2000) x 1.3513
W s : Amount (mg) of potassium iodide for assay
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Lisinopril Hydrate
EHjO
C 2 iH 31 N305-2H 2 0: 441.52
(2S)-l-{(25)-6-Amino-2-[(15)-l-carboxy-
3-phenylpropylamino] hexanoyl} pyrrolidine-2-carboxylic
acid dihydrate
[83915-83-7]
Lisinopril Hydrate contains not less than 98.5% and
not more than 101.0% of lisinopril (C2iH 31 N 3 05:
405.49), calculated on the anhydrous basis.
Description Lisinopril Hydrate occurs as a white crystalline
powder, having a slight characteristic odor.
It is soluble in water, sparingly soluble in methanol, and
824
Lisinopril Tablets / Official Monographs
JP XV
practically insoluble in ethanol (99.5).
Melting point: about 160°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Lisinopril Hydrate in methanol (1 in 1000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Lisinopril Hydrate as directed in the paste method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]": -43.0- -47.0° (0.25 g cal-
culated on the anhydrous basis, 0.25 ml/L zinc acetate buffer
solution, pH 6.4, 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Lisinopril Hydrate according to Method 4, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve about 0.10 g of
Lisinopril Hydrate in 50 mL of water, and use this solution as
the sample solution. Pipet 3 mL of the sample solution, add
water to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with exactly 15 fiL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak, having the
relative retention time of about 1.2 with respect to lisinopril,
is not larger than 1/5 times the peak area of lisinopril from
the standard solution, the area of the peak other than
lisinopril and the peak mentioned above is not larger than
2/15 times the peak area of lisinopril from the standard solu-
tion, and the total area of the peaks other than lisinopril is
not larger than the peak area of lisinopril from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase A: Diluted 0.05 mol/L sodium dihydrogen
phosphate TS (1 in 2).
Mobile phase B: A mixture of diluted 0.05 mol/L sodium
dihydrogen phosphate TS (1 in 2) and acetonitrile for liquid
chromatography (3:2).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-10
10-25
90->50
50
10^50
50
Flow rate: About 1.5 mL per minute.
Time span of measurement: About 2.5 times as long as the
retention time of lisinopril beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 2.5 mL of
the standard solution, and add water to make exactly 50 mL.
Confirm that the peak area of lisinopril obtained with 15 /ah
of this solution is equivalent to3.5to6.5%of that with 1 5 /uL
of the standard solution.
System performance: To 10 mg of Lisinopril Hydrate and
2 mL of a solution of anhydrous caffeine (1 in 1000) add
water to make 200 mL. When the procedure is run with 15 fiL
of this solution under the above operating conditions,
lisinopril and caffeine are eluted in this order with the resolu-
tion between these peaks being not less than 6.
System repeatability: When the test is repeated 6 times with
15 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
lisinopril is not more than 2.0%.
Water <2.48> Not less than 8.0% and not more than 9.5%
(0.3 g, volumetric titration, back titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.66 g of Lisinopril Hydrate,
dissolve in 80 mL of water, and titrate <2.50> with 0.1 mol/L
sodium hydroxide VS (potentiometric titration). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.55 mg of C 21 H 31 N 3 5
Containers and storage Containers — Well-closed containers.
Lisinopril Tablets
Lisinopril Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of
lisinopril (C 21 H 31 N 3 05: 405.49).
Method of preparation Prepare as directed under Tablets,
with Lisinopril Hydrate.
Identification To an amount of powdered Lisinopril
Tablets, equivalent to 10 mg of lisinopril, add 10 mL of
methanol, shake for 20 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 10 mg of Lisinopril
in 10 mL of methanol, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 30 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of acetonitrile, acetic acid (100), water and ethyl
acetate (2:2:1:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly ninhydrin TS on the plate, and heat at 120
°C: the principal spot with the sample solution and the spot
with the standard solution show a red-purple color and their
Rf values are the same.
Purity Related substances Powder not less than 20
Lisinopril Tablets. Take a portion of the powder, equivalent
JPXV
Official Monographs / Lisinopril Tablets
825
to about 25 mg of lisinopril (C 21 H 31 N 3 5 ), add exactly 25 mL
of water, shake for 20 minutes, filter, and use the filtrate as
the sample solution. Pipet 3 mL of the sample solution, add
water to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with exactly 15 /uL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak, having the
relative retention time of about 2.0 with respect to lisinopril,
is not more than 2/3 times the peak area of lisinopril from the
standard solution.
Operating conditions —
Proceed as directed in the Purity (2) under Lisinopril Hy-
drate.
System suitability —
Test for required detectability: To exactly 2.5 mL of the
standard solution add water to make exactly 50 mL. Confirm
that the peak area of lisinopril obtained with 15 fiL of this so-
lution is equivalent to 3.5 to 6.5% of that with 15 /uL of the
standard solution.
System performance: Proceed as directed in the Purity (2)
under Lisinopril Hydrate.
System repeatability: When the test is repeated 6 times with
15 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
lisinopril is not more than 2.0%.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Lisinopril Tablets add exactly 5 mL each of
the internal standard solution per every 1 mg of lisinopril ac-
cording to the labeled amount, shake for 20 minutes, cen-
trifuge, and use the supernatant liquid as the sample solution.
Hereafter, proceed as directed in the Assay.
Amount (mg) of lisinopril (C 21 H 31 N 3 5 )
= W s x (Q T /Q S ) x (C/10)
W s : Amount (mg) of lisinopril for assay, calculated on the
anhydrous basis
C: Labeled amount (mg) of lisinopril (C 21 H 31 N 3 5 ) in 1
tablet
Internal standard solution — A solution of anhydrous caffeine
(1 in 20,000)
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Lisinopril Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of water as the dissolution medium. Withdraw 20
mL or more of the dissolution medium 60 minutes after start-
ing the test for a 5-mg tablet, 90 minutes after starting the test
for a 10-mg tablet and a 20-mg tablet, and filter through a
membrane filter with pore size of not more than 0. 5 /xm. Dis-
card the first 10 mL of the filtrate, pipet the subsequent V
mL, add water to make exactly V mL so that each mL con-
tains about 5.6 /xg of lisinopril (C 2 iH 3 iN 3 5 ) according to the
labeled amount, and use this solution as the sample solution.
Separately, weigh accurately about 15 mg of lisinopril for as-
say, separately determined the water content <2.48> in the
same manner as Lisinopril Hydrate, and dissolve in water to
make exactly 100 mL. Pipet 2 mL of this solution, add water
to make exactly 50 mL, and use this solution as the standard
solution. Perform the test with exactly 50 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of lisinopril.
The dissolution rate for a 5-mg tablet in 60 minutes is not less
than 80%, for a 10-mg tablet in 90 minutes is not less than
80%, and for a 20-mg tablet in 90 minutes is not less than
75%.
Dissolution rate (%) with respect to the labeled amount of
lisinopril (C 2 iH 31 N 3 5 )
= W s x (A T /A S ) x (V'/V) x (I/O x 36
W s : Amount (mg) of lisinopril for assay, calculated on the
anhydrous basis
C: Labeled amount (mg) of lisinopril (C 2 iH 31 N 3 5 ) in 1
tablet
Operating conditions —
Detector, column temperature, and mobile phase: Proceed
as directed in the operating conditions in the Assay.
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Flow rate: Adjust the flow rate so that the retention time of
lisinopril is about 7 minutes.
System suitability —
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of lisinopril are not less than 1000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
50 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
lisinopril is not more than 2.0%.
Assay Weigh accurately the mass of not less than 20
Lisinopril Tablets, and powder. Weigh accurately a portion
of the powder, equivalent to about 5 mg of lisinopril
(C 21 H 31 N 3 5 ), add exactly 25 mL of the internal standard so-
lution, shake for 20 minutes, centrifuge, and use the super-
natant liquid as the sample solution. Separately, weigh ac-
curately about 10 mg of lisinopril for assay, separately deter-
mined the water content <2.48> in the same manner as
Lisinopril Hydrate, add exactly 50 mL of the internal stan-
dard solution to dissolve, and use this solution as the stan-
dard solution. Perform the test with 10 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Q T and Q s , of the peak area
of lisinopril to that of the internal standard.
Amount (mg) of lisinopril (C 2I H 31 N 3 5 )
= W s x (Q T /Q S ) x (1/2)
W s : Amount (mg) of lisinopril for assay, calculated on the
anhydrous basis
Internal standard solution — A solution of anhydrous caffeine
(1 in 20,000)
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm).
826
Lithium Carbonate / Official Monographs
JP XV
Column: A stainless steel column 4.0 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase: A mixture of diluted 0.05 mol/L sodium di-
hydrogen phosphate TS (1 in 2) and acetonitrile for liquid
chromatography (19:1).
Flow rate: Adjust the flow rate so that the retention time of
lisinopril is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, lisinopril and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 7.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of lisinopril to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Lithium Carbonate
Li 2 C0 3 : 73.89
Lithium Carbonate, when dried, contains not less
than 99.5% of Li 2 C0 3 .
Description Lithium Carbonate occurs as a white, crystal-
line powder. It is odorless.
It is sparingly soluble in water, slightly soluble in hot
water, and practically insoluble in ethanol (95) and in diethyl
ether.
It dissolves in dilute acetic acid.
The pH of a solution of Lithium Carbonate (1 in 100) is be-
tween 10.9 and 11.5.
Identification (1) Perform the test as directed under
Flame Coloration Test <1.04> (1) with Lithium Carbonate: a
persistent red color appears.
(2) Dissolve 0.2 g of Lithium Carbonate in 3 mL of dilute
hydrochloric acid, and add 4 mL of sodium hydroxide TS
and 2 mL of disodium hydrogenphosphate TS: a white
precipitate is produced. To the precipitate add 2 mL of
hydrochloric acid: it dissolves.
(3) A solution of Lithium Carbonate (1 in 100) responds
to the Qualitative Tests <1.09> for carbonate.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Lithium Carbonate in 10 mL of water by warming: the so-
lution is clear and colorless.
(2) Acetic acid-insoluble substances — Take 1 .0 g of Lithi-
um Carbonate, dissolve in 40 mL of dilute acetic acid, filter
the insoluble substances using filter paper for quantitative
analysis, wash with five 10-mL portions of water, and ignite
the insoluble substances together with the filter paper to in-
cinerate: the mass of the residue is not more than 1.5 mg.
(3) Chloride <1.03>— To 0.40 g of Lithium Carbonate
add 10 mL of water and 7 mL of dilute nitric acid, and dis-
solve by heating to boil. After cooling, add 6 mL of dilute
nitric acid, and dilute with water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.25 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.022%).
(4) Sulfate <1.14>— To 0.40 g of Lithium Carbonate add
10 mL of water and 4 mL of dilute hydrochloric acid, and
dissolve by heating to boil. After cooling, add 1 mL of dilute
hydrochloric acid, and dilute with water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution with 0.40 mL of 0.005 mol/L sulfuric
acid VS (not more than 0.048%).
(5) Heavy metals <1.07> — To 4.0 g of Lithium Carbonate
add 5 mL of water, gradually add 10 mL of hydrochloric acid
while mixing, and dissolve. Evaporate the solution on a water
bath to dryness. To the residue add 10 mL of water, and dis-
solve. Place the solution in a Nessler tube, add 1 drop of
phenolphthalein TS, add ammonia TS until the solution
shows a slight red color, then add 2 mL of dilute acetic acid,
and dilute with water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows: Evaporate 10 mL of hydrochloric acid on a water
bath to dryness. To the residue add 10 mL of water, and dis-
solve. Place the solution in a Nessler tube, add 1 drop of
phenolphthalein TS, add ammonia TS until the solution
shows a pale red color, then add 2.0 mL of Standard Lead
Solution and 2 mL of dilute acetic acid, and dilute with water
to make 50 mL (not more than 5 ppm).
(6) Iron — Prepare the test solution with 1.0 g of Lithium
Carbonate according to Method 2 using 11 mL of dilute
hydrochloric acid, and perform the test according to Method
B. Prepare the control solution with 1 .0 mL of Standard Iron
Solution (not more than 10 ppm).
(7) Aluminum — To 10 mL of solution A obtained in (6)
add 10 mL of water and 5 mL of acetic acid-sodium acetate
buffer solution, pH 4.5, and shake. Add 1 mL of a solution
of L-ascorbic acid (1 in 100), 2 mL of aluminon TS and water
to make 50 mL, shake well, and allow to stand for 10
minutes: the solution has no more color than the following
control solution.
Control solution: Dissolve 0.1758 g of aluminum potassi-
um sulfate 12-water in water to make 1000 mL. To 1.0 mL of
this solution add 10 mL of solution B obtained in (6) and
water to make 20 mL, add 5 mL of acetic acid-sodium acetate
buffer solution, pH 4.5, and proceed in the same manner.
(8) Barium — To 20 mL of solution A obtained in (6) add
6 mL of water, 0.5 mL of dilute hydrochloric acid, 3 mL of
ethanol (95) and 2 mL of potassium sulfate TS, and allow to
stand for 1 hour: the solution has no more turbidity than the
following control solution.
Control solution: Dissolve 17.8 mg of barium chloride di-
hydrate in water to make 1000 mL. To 6 mL of this solution
add 20 mL of solution B, 0.5 mL of dilute hydrochloric acid
and 3 mL of ethanol (95), and proceed in the same manner.
(9) Calcium — Weigh accurately about 5 g of Lithium
Carbonate, add 50 mL of water and 15 mL of hydrochloric
acid, and dissolve. Remove carbon dioxide from the solution
by boiling, add 5 mL of ammonium oxalate TS, then make
alkaline with ammonia TS, and allow to stand for 4 hours.
Filter the produced precipitate through a glass filter (G4),
JP XV
Official Monographs / Lorazepam
827
wash with warm water until the turbidity of the washing is
not produced with calcium chloride TS within 1 minute.
Transfer the precipitate and the glass filter into a beaker, add
water until the glass filter is covered with water, then add 3
mL of sulfuric acid, heat between 70°C and 80°C, and titrate
with 0.02 mol/L potassium permanganate VS until a pale red
color persists for 30 seconds: the amount of calcium (Ca:
40.08) is not more than 0.05%.
Each mL of 0.02 mol/L potassium permanganate VS
= 2.004 mg of Ca
(10) Magnesium — To 3.0 mL of solution A obtained in
(6) add 0.2 mL of a solution of titan yellow (1 in 1000) and
water to make 20 mL, then add 5 mL of sodium hydroxide (3
in 20), and allow to stand for 10 minutes: the solution has no
more color than the following control solution.
Control solution: Dissolve 49.5 mg of magnesium sulfate
heptahydrate, previously dried at 105 °C for 2 hours and
heated at 450°C for 3 hours, in water to make 1000 mL. To
this solution add 3 mL of solution B obtained in (6), 0.2 mL
of a solution of titanium yellow (1 in 1000) and water to make
20 mL, and proceed in the same manner.
(11) Potassium — Dissolve 1.0 g of Lithium Carbonate in
water to make 100 mL, and use this solution as the sample so-
lution. To 5 mL of the sample solution add 1.0 mL of dilute
acetic acid, shake, add 5 mL of a solution of sodium
tetraphenylborate (1 in 30), shake immediately, and allow to
stand for 10 minutes: the solution has no more turbidity than
the following control solution.
Control solution: Dissolve 9.5 mg of potassium chloride in
water to make 1000 mL. To 5 mL of this solution add 1.0 mL
of dilute acetic acid, shake, and proceed in the same manner.
(12) Sodium — Weigh accurately about 0.8 g of Lithium
Carbonate, dissolve in water to make exactly 100 mL, and
use this solution as the sample stock solution. Measure exact-
ly 25 mL of the sample stock solution, add water to make ex-
actly 100 mL, and use this solution as the sample solution (1).
Separately, weigh accurately 25.4 mg of sodium chloride, dis-
solve in water to make exactly 1000 mL, and use this solution
as the standard solution. Measure exactly 25 mL of the sam-
ple stock solution, add exactly 20 mL of the standard solu-
tion, then add water to make exactly 100 mL, and use this
solution as the sample solution (2). Determine emission inten-
sities of sodium using a flame photometer with the sample so-
lution (1) and the sample solution (2) under the following
conditions. Adjust the wavelength dial to 589 nm, atomize
the sample solution (2) into the flame, then adjust the sen-
sitivity so that the emission intensity Z. s shows 100 adjust-
ment, and determine emission intensity L T of the sample so-
lution (1). Then, make the other conditions identical, change
the wavelength dial to 580 nm, determine emission intensity
L B of the sample solution (1): the amount of sodium, calcu-
lated from the following equation, is not more than 0.05%.
Amount (%) of sodium (Na)
= {(L T - L B )/(L S - L T )} x (W'/W) x 100
W: Amount (mg) of the sample in 25 mL of the sample
stock solution
W : Amount (mg) of sodium in 20 mL of the standard so-
lution
(13) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Lithium Carbonate, add 2 mL of water and 3 mL of
hydrochloric acid, and perform the test (not more than 2
ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C,
3 hours).
Assay Weigh accurately about 1 g of Lithium Carbonate,
previously dried, add exactly 100 mL of water and 50 mL of
0.5 mol/L sulfuric acid VS, remove carbon dioxide by boil-
ing gently, cool, and titrate <2.50> the excess sulfuric acid
with 1 mol/L sodium hydroxide VS until the color of the so-
lution changes from red to yellow (indicator: 3 drops of
methyl red TS). Perform a blank determination.
Each mL of 0.5 mol/L sulfuric acid VS
= 36.95 mg of Li 2 C0 3
Containers and storage Containers — Well-closed contain-
ers.
Lorazepam
and enanttomer
C 15 H 10 Cl 2 N 2 O 2 : 321.16
(3i?S)-7-Chloro-5-(2-chlorophenyl)-3-hydroxy-
l,3-dihydro-2/f-l,4-benzodiazepin-2-one [846-49-1]
Lorazepam, when dried, contains not less than
98.5% of C 15 H 10 Cl 2 N 2 O 2 .
Description Lorazepam occurs as a white, crystalline pow-
der. It is odorless.
It is sparingly soluble in ethanol (95) and in acetone, slight-
ly soluble in diethyl ether, and practically insoluble in water.
It is gradually colored by light.
Identification (1) To 0.02 g of Lorazepam add 15 mL of
dilute hydrochloric acid, boil for 5 minutes, and cool: the so-
lution responds to the Qualitative Tests <1.09> for primary
aromatic amines.
(2) Determine the absorption spectrum of a solution of
Lorazepam in ethanol (95) (1 in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Lorazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) Perform the test with Lorazepam as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Absorbance <2.24> E
i%
(229 nm): 1080-1126 (after
drying, 1 mg, ethanol (95), 200 mL).
828
Loxoprofen Sodium Hydrate / Official Monographs
JP XV
Purity (1) Chloride <I.03> — To 1.0 g of Lorazepam add
50 mL of water, allow to stand for 1 hour with occasional
shaking, and filter. To 25 mL of the filtrate add 6 mL of di-
lute nitric acid and water to make 50 mL. Perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not
more than 0.014%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Lorazep-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Lorazepam according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Lorazepam in
20 mL of ethanol (95), and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add ethanol (95) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform, 1,4-dioxane
and acetic acid (100) (91:5:4) to a distance of about 15 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
105°C, 3 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.4 g of Lorazepam, previ-
ously dried, dissolve in 50 mL of acetone, and titrate <2.50>
with 0.1 mol/L tetrabutylammonium hydroxide VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L tetrabutylammonium
hydroxide VS
= 32. 12 mg of C 15 H I0 Cl 2 N 2 O 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Loxoprofen Sodium Hydrate
CH;
C 15 H 17 Na0 3 .2H 2 0: 304.31
Monosodium 2-{4-[(2-
oxocyclopentyl)methyl]phenylj propanoate dihydrate
[80382-23-6]
Loxoprofen Sodium Hydrate contains not less than
98.5% of loxoprofen sodium (C 15 H 17 Na0 3 : 268.28),
calculated on the anhydrous basis.
Description Loxoprofen Sodium Hydrate occurs as white
to yellowish white crystals or crystalline powder.
It is very soluble in water and in methanol, freely soluble in
ethanol (95), and practically insoluble in diethyl ether.
A solution of Loxoprofen Sodium Hydrate (1 in 20) does
not show optical rotation.
The pH of a solution of Loxoprofen Sodium Hydrate in
freshly boiled and cooled water (1 in 20) is between 6.5 and
8.5.
Identification (1) Determine the absorption spectrum of a
solution of Loxoprofen Sodium Hydrate (1 in 55,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Lox-
oprofen Sodium Hydrate as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Loxoprofen Sodium Hydrate (1 in 10)
responds to the Qualitative Tests <1.09> for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Loxoprofen Sodium Hydrate in 10 mL of water: the solution
is clear and colorless or pale yellow. The color is not darker
than that of diluted Matching Fluid for Color A (1 in 2).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Lox-
oprofen Sodium Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 1.0 g of Loxoprofen
Sodium Hydrate in 10 mL of methanol, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add methanol to make exactly 200 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 1,2-
dichloroethane and acetic acid (100) (9:1) to a distance of
about 15 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Water <2.48> 11.0 - 13.0% (0.2 g, direct titration).
Assay Weigh accurately about 60 mg of Loxoprofen Sodi-
um Hydrate, and dissolve in diluted methanol (3 in 5) to
make exactly 100 mL. Pipet 5 mL of this solution, add exact-
ly 10 mL of the internal standard solution, add diluted
methanol (3 in 5) to make 100 mL, and use this solution as
the sample solution. Separately, weigh accurately about 50
mg of Loxoprofen Reference Standard, previously dried in a
desiccator (in vacuum, 60°C) for 3 hours, and dissolve in
diluted methanol (3 in 5) to make exactly 100 mL. Pipet 5 mL
of this solution, proceed in the same manner as directed for
the preparation of the sample solution, and use so obtained
solution as the standard solution. Perform the test with 10 /uL
each of the sample solution and standard solution as directed
JPXV
Official Monographs / L-Lysine Hydrochloride
829
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak area of loxoprofen to that of the internal standard.
Amount (mg) of loxoprofen sodium (C I5 H 17 Na0 3 )
= W s x (<2 T /<2s) x 1.089
W s : Amount (mg) of Loxoprofen Reference Standard
Internal Standard Solution — A solution of ethyl benzoate in
diluted methanol (3 in 5) (7 in 50,000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 222 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of methanol, water, acetic acid
(100) and triethylamine (600:400:1:1).
Flow rate: Adjust the flow rate so that the retention time of
loxoprofen is about 7 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, loxoprofen and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 5 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of loxoprofen to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
L-Lysine Hydrochloride
Lysine Hydrochloride
• hc:
C 6 H 14 N 2 2 .HC1: 182.65
(25')-2,6-Diaminohexanoic acid monohydrochloride
[657-27-2]
L-Lysine Hydrochloride, when dried,
less than 98.5% of C 6 H 14 N 2 2 .HC1.
contains not
Description L-Lysine Hydrochloride occurs as a white pow-
der. It is odorless, and has a slight, characteristic taste.
It is freely soluble in water and in formic acid, and practi-
cally insoluble in ethanol (95).
Identification (1) Determine the infrared absorption spec-
trum of L-Lysine Hydrochloride, previously dried, as direct-
ed in the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers. If any differ-
ence appears between the spectra, dissolve L-Lysine
Hydrochloride in water, evaporate the water to dryness at
60°C, and repeat the test with the residue.
(2) A solution of L-Lysine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [ a ]$: + 19.0 - +21.5° (after
drying, 2 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of L-Lysine Hydrochloride in 10
mL of water: the pH of this solution is between 5.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
L-Lysine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.6 g of L-Ly-
sine Hydrochloride. Prepare the control solution with 0.35
mL of 0.005 mol/L sulfuric acid VS (not more than 0.028%).
(3) Ammonium <1.02> — Perform the test with 0.25 g of
L-Lysine Hydrochloride. Prepare the control solution with
5.0 mL of Standard Ammonium Solution (not more than
0.02%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of L-Lysine
Hydrochloride according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of L-Lysine Hydrochloride according to Method 1, and per-
form the test (not more than 2 ppm).
(6) Related substances — Dissolve 0.10 g of L-Lysine
Hydrochloride in 25 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 50 mL, pipet 5 mL of this solution,
add water to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of 1-propanol and ammonia water (28) (67:33)
to a distance of about 10 cm, and dry the plate at 100°C for
30 minutes. Spray evenly the plate with a solution of nin-
hydrin in acetone (1 in 50) and heat at 80°C for 5 minutes: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of L-Lysine
Hydrochloride, previously dried, dissolve in 2 mL of formic
acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS, and
heat on a water bath for 30 minutes. After cooling, add 45
mL of acetic acid (100), and titrate the excess perchloric acid
with 0.1 mol/L sodium acetate VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 9.132 mg of C 6 H 14 N 2 2 HC1
Containers and storage Containers — Tight containers.
830
Lysozyme Hydrochloride / Official Monographs
JP XV
Lysozyme Hydrochloride
V V>-A±£it±£
Lys -Val - Phe -Gty - Arg-Cys - Gl u - Loir Ala ■ Ala Afa Met Lys ■ Arg - His- Gly - Leu - Asp -Asn -Tyr-
Arg Gly Tyf Ser-Leu Gly ■Asn-TrpVal-Cys-Ala-Ara-LyB-Phe-GlL*-Sef-AsnPti&-Asn-"Thr-
I
Gin - Ala -Thr - Asn -Arg- Asn-Thr - Asp -Gly - Ser -Thr -Asp -Tyr- Gly - 1 la -Leu- Gin- tie -Asn - Set 1 -
Arg-TYp-TYp-Cys-Asn-Asp-GlyArg-Tnr-Pro GlySerArg Asn Leu Cys-Asn ItePro Cys
J
Ser-Ala-Leu-Leu - Ser -5er- Asp - He -Th r-Ala - Se r- Val- Asn - Cys - Ala- Lys - Lys - lie -Val Ser
Asp -Gly - Asn -Gly -Met - Asn ■ Ala -Trp -Val -Ala-Trp - Arg- Asn ■ Arg- Cys - Lys - Gly -Thr - Asp-Val -
Gln-Ala-Trp-lla-Arg-Gfy-Cys-Arg-LGU ■ tHCI
C616H963Ni 93 Oi 8 2Sio.XHCl
[12650-88-3, egg white lysozyme]
Lysozyme Hydrochloride is a hydrochloride of a
basic polypeptide obtained from albumen of hen's egg,
and has an activity to hydrolyze mucopolysaccharides.
It contains not less than 0.9 mg (potency) of lyso-
zyme per mg, calculated on the dried basis.
Description Lysozyme Hydrochloride occurs as white, crys-
tals, or crystalline or amorphous powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
It is hygroscopic.
The pH of a solution of Lysozyme Hydrochloride (3 in
200) is between 3.0 and 5.0.
Identification (1) To 5 mL of a solution of Lysozyme
Hydrochloride in acetate buffer solution, pH 5.4 (1 in 500)
add 1 mL of ninhydrin TS, and heat for 10 minutes: a blue-
purple color develops.
(2) Determine the absorption spectrum of a solution of
Lysozyme Hydrochloride in acetate buffer solution, pH 5.4 (1
in 10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
Purity (1) Clarity of solution — To 5 mL of a solution of
Lysozyme Hydrochloride (3 in 200) add, if necessary, dilute
hydrochloric acid to adjust the pH to 3: the solution is clear.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Lyso-
zyme Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 8.0% (0.1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 2.0% (0.5 g).
Nitrogen Perform the test as directed under Nitrogen De-
termination <1.0S>: the amount of nitrogen (N: 14.01) is be-
tween 16.8% and 18.6%, calculated on the dried basis.
Assay Weigh accurately an amount of Lysozyme
Hydrochloride, equivalent to about 25 mg (potency), dissolve
in phosphate buffer solution, pH 6.2 to make exactly 100 mL.
Pipet 2 mL of this solution, add phosphate buffer solution,
pH 6.2 to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately an amount of
Lysozyme Reference Standard (separately determine its loss
on drying <2.41> in the same manner as Lysozyme
Hydrochloride), equivalent to about 25 mg (potency), and
dissolve in phosphate buffer solution, pH 6.2 to make exactly
100 mL. Pipet 1 mL and 2 mL of this solution, add phos-
phate buffer solution, pH 6.2 to them to make exactly 50 mL,
and use these solutions as the standard solution (1) and the
solution (2), respectively. Keep the sample solution and the
standard solutions in an ice-bath. Pipet 4 mL of substrate so-
lution for lysozyme hydrochloride, previously warmed in a
water bath of 35°C for about 5 minutes, add exactly 100 //L
of the sample solution, previously warmed in a water bath of
35°C for about 3 minutes, and allow to stand at 35°C for ex-
actly 10 minutes, then add exactly 0.5 mL of 1 mol/L
hydrochloric acid TS, and immediately shake. Determine the
absorbance under Ultraviolet-visible Spectrophotometry
<2.24>, A T , of this solution at 640 nm, using water as the
blank. Determine the absorbances, ^4 S i and A S2 , of the solu-
tions obtained with the standard solution (1) and the stan-
dard solution (2) in the same manner as the sample solution.
Amount [mg (potency)] of lysozyme per mg,
calculated on the dried basis
= (W S /2W T ) x {(A Si - Aj)/(A sl - A S2 ) + 1)
W s : Amount (mg) of Lysozyme Reference Standard, cal-
culated on the dried basis.
W T : Amount (mg) of the sample, calculated on the dried
basis.
Containers and storage Containers — Tight containers.
Macrogol 400
Polyethylene Glycol 400
v-7n=f-jU400
Macrogol 400 is a polymer of ethylene oxide and
water, represented by the formula HOCH 2
(CH 2 OCH 2 )„CH 2 OH, in which the value of n ranges
from 7 to 9.
Description Macrogol 400 occurs as a clear, colorless and
viscous liquid. It has no odor or a slight, characteristic odor.
It is miscible with water, with methanol, with ethanol (95)
and with pyridine.
It is soluble in diethyl ether.
It is slightly hygroscopic.
Congealing point: 4 - 8°C
Specific gravity d? : 1.110-1.140
Identification Dissolve 0.05 g of Macrogol 400 in 5 mL of
dilute hydrochloric acid, add 1 mL of barium chloride TS,
shake, and filter, if necessary. To the filtrate add 1 mL of a
solution of phosphomolybdic acid M-hydrate (1 in 10): a yel-
low-green precipitate is formed.
pH <2.54> Dissolve 1.0 g of Macrogol 400 in 20 mL of
water: the pH of this solution is between 4.0 and 7.0.
Purity (1) Acidity — Dissolve 5.0 g of Macrogol 400 in 20
mL of neutralized ethanol, and add 2 drops of
phenolphthalein TS and 0.20 mL of 0.1 mol/L sodium
hydroxide VS: the solution is red in color.
JPXV
Official Monographs / Macrogol 1500 831
(2) Ethylene glycol and diethylene glycol — Dissolve 4.0 g
of Macrogol 400 in water to make exactly 10 mL, and use this
solution as the sample solution. Weigh accurately about 50
mg each of ethylene glycol and diethylene glycol, dissolve in
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 2 fiL each of
the sample solution and standard solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions. Determine the peak heights, H Ta and // Sa , of ethylene
glycol of each solution, and the peak heights, H Tb and H sb , of
diethylene glycol, and calculate the amount of ethylene glycol
and diethylene glycol: the sum of the contents of ethylene
glycol and diethylene glycol is not more than 0.25%.
Amount (mg) of ethylene glycol
= ^ Sa x(// Ta /// Sa )x(l/l0)
Amount (mg) of diethylene glycol
= W sb x(H Tb /H sb )x(l/W)
W Sa : Amount (mg) of ethylene glycol for gas chro-
matography
W sb : Amount (mg) of diethylene glycol for gas chro-
matography
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A colum about 3 mm in inside diameter and
about 1.5 m in length, packed with siliceous earth for gas
chromatography, 150 to 180 /um in particle diameter, coated
with D-sorbitol at the ratio of 12%.
Column temperature: A constant temperature of about 165
°C.
Carrier gas: Nitrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
diethylene glycol is about 8 minutes.
Selection of column: Proceed with 2 fiL of the standard so-
lution under the above operating conditions, and calculate
the resolution. Use a column clearly dividing peaks of ethy-
lene glycol and diethylene glycol in this order.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of diethylene glycol obtained from 2//L
of the standard solution composes about 80% of the full
scale.
Average molecular mass Add 42 g of phthalic anhydride to
300 mL of freshly distilled pyridine, exactly measured, in a
1-L light-resistant glass-stoppered bottle. Shake the bottle
vigorously to dissolved the solid, and allow to stand for 16
hours or more. Pipet 25 mL of this solution into an about
200-mL glass-stoppered pressure bottle. Add about 1.5 g of
Macrogol 400, accurately weighed, stopper the bottle, wrap it
securely with strong cloth, and immerse in a water bath, hav-
ing a temperature of 98 ± 2°C, to the level so that the mix-
ture in the bottle soaks completely in water. Maintain the
temperature of the bath at 98 ± 2°C for 30 minutes. Remove
the bottle from the bath, and allow to cool in air to room
temperature. Add exactly 50 mL of 0.5 mol/L sodium
hydroxide VS and 5 drops of a solution of phenolphthalein in
pyridine (1 in 100). Titrate <2.50> with 0.5 mol/L sodium
hydroxide VS until a light red color remains for not less than
15 seconds. Perform a blank determination.
Average molecular mass
= (Wx4000)/(a-b)
W: Amount (g) of sample.
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS used
in the blank determination.
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS used
in the test of the sample.
Average molecular mass is between 380 and 420.
Water <2.48> Not more than 1.0% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Containers and storage Containers — Tight containers.
Macrogol 1500
Polyethylene Glycol 1500
v?a=f— ;n5oo
Macrogol 1500 is a mixture containing equal
amounts of lower and higher polymers of ethylene
oxide and water, represented by the formula HOCH 2
(CH 2 OCH 2 )„CH 2 OH, in which the value of n is 5 or 6
for the lower polymers and from 28 to 36 for the
higher.
Description Macrogol 1500 occurs as a white, smooth
petrolatum-like solid. It is odorless or has a faint, charac-
teristic odor.
It is very soluble in water, in pyridine and in diphenyl
ether, freely soluble in methanol, sparingly soluble in ethanol
(95), very slightly soluble in ethanol (99.5), and practically in-
soluble in diethyl ether.
Congealing point: 37 - 41 °C
Identification Dissolve 0.05 g of Macrogol 1500 in 5 mL of
dilute hydrochloric acid, add 1 mL of barium chloride TS,
shake, and filter, if necessary. To the filtrate add 1 mL of a
solution of phosphomolybdic acid M-hydrate (1 in 10): a yel-
low-green precipitate is formed.
pH <2.54> Dissolve 1.0 g of Macrogol 1500 in 20 mL of
water: the pH of the solution is between 4.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 5.0 g of
Macrogol 1500 in 50 mL of water: the solution is clear and
colorless.
(2) Acidity— Dissolve 5.0 g of Macrogol 1500 in 20 mL of
neutralized ethanol, and add 2 drops of phenolphthalein TS
and 0.20 mL of 0.1 mol/L sodium hydroxide VS: the solu-
tion is red in color.
(3) Ethylene glycol and diethylene glycol — Place 50.0 g
of Macrogol 1500 in a distilling flask, add 75 mL of diphenyl
ether, warm to dissolve if necessary, distil slowly under a
reduced pressure of 0.13 to 0.27 kPa and take 25 mL of the
distillate in a 100-mL container with 1-mL graduation. To the
distillate add exactly 20 mL of water, shake vigorously, cool
in ice water, congeal the diphenyl ether, and filtrate into a
25-mL volumetric flask. Wash the residue with 5.0 mL of ice-
cold water, combine the washings with the filtrate, warm to
room temperature, and add water to make 25 mL. Transfer
this solution to a glass-stoppered flask, shake with 25.0 mL
of freshly distilled acetonitrile, and use this solution as the
sample solution. Separately, to 62.5 mg of diethylene glycol
832 Macrogol 4000 / Official Monographs
JP XV
add a mixture of water and freshly distilled acetonitrile (1:1)
to make exactly 25 mL, and use this solution as the standard
solution. Take exactly 10 mL each of the sample solution and
the standard solution, and add to each exactly 15 mL of ceri-
um (IV) diammonium nitrate TS. Perform the test with this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> within 2 to 5 minutes: the absorbance of the so-
lution obtained from the sample solution at the wavelength
of maximum absorption at about 450 nm is not larger than
the absorbance of the solution obtained from the standard
solution.
Water <2.48> Not more than 1.0% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Containers and storage Containers — Tight containers.
Macrogol 4000
Polyethylene Glycol 4000
T-7P3-JU4000
Macrogol 4000 is a polymer of ethylene oxide and
water, represented by the formula HOCH 2
(CH 2 OCH 2 )„CH 2 OH, in which the value of n ranges
from 59 to 84.
Description Macrogol 4000 is a white, paraffin-like solid,
occurring as flakes or powder. It is odorless or has a faint,
characteristic odor.
It is very soluble in water, freely soluble in methanol and in
pyridine, and practically insoluble in ethanol (99.5) and in
diethyl ether.
Congealing point: 53 - 57°C
Identification Dissolve 0.05 g of Macrogol 4000 in 5 mL of
dilute hydrochloric acid, add 1 mL of barium chloride TS,
shake, and filter, if necessary. To the filtrate add 1 mL of a
solution of phosphomolybdic acid w-hydrate (1 in 10): a yel-
low-green precipitate is formed.
pH <2.54> Dissolve 1.0 g of Macrogol 4000 in 20 mL of
water: the pH of this solution is between 4.0 and 7.5.
Purity (1) Clarity and color of solution — A solution of
5.0 g of Macrogol 4000 in 50 mL of water is clear and color-
less.
(2) Acidity— Dissolve 5.0 g of Macrogol 4000 in 20 mL of
neutralized ethanol by warming, cool, and add 0.20 mL of
0.1 mol/L sodium hydroxide VS and 1 drop of
phenolphthelein TS: the color of the solution is red.
Average molecular mass Weigh accurately about 12.5 g of
Macrogol 4000, transfer to an about 200-mL glass-stoppered
pressure bottle, add about 25 mL of pyridine, dissolve by
warming, and allow to cool. Separately, pipet 300 mL of
freshly distilled pyridine into a 1000-mL light-resistant, glass-
stoppered bottle, add 42 g of phthalic anhydride, dissolve
with vigorous shaking, and allow to stand for 16 hours or
more. Pipet 25 mL of this solution, transfer to the former
pressure bottle, stopper the bottle tightly, wrap it securely
with strong cloth, and immerse in a water bath, previously
heated at 98 ± 2°C, to the level so that the mixture in the
bottle soaks completely in water. Maintain the temperature
of the bath at 98 ± 2°C for 30 minutes. Remove the bottle
from the bath, and allow to cool in air to room temperature.
Add exactly 50 mL of 0.5 mol/L sodium hydroxide VS and 5
drops of a solution of phenolphthalein in pyridine (1 in 100).
Titrate <2.50> with 0.5 mol/L sodium hydroxide VS until a
light red color remains for not less than 15 seconds. Perform
a blank determination.
Average molecular mass
= (Wx4000)/(a-b)
W: Amount (g) of sample.
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the blank determination.
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the test of the sample.
Average molecular mass is between 2600 and 3800.
Water <2.48> Not more than 1.0% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Containers and storage Containers — Well-closed contain-
ers.
Macrogol 6000
Polyethylene Glycol 6000
v7n=f-jU6000
Macrogol 6000 is a polymer of ethylene oxide
and water, represented by the formula
HOCH 2 (CH 2 OCH 2 )„CH 2 OH, in which the value of n
ranges from 165 to 210.
Description Macrogol 6000 is a white, paraffin-like solid,
occurring as flakes or powder. It is ordorless or has a faint,
characteristic odor.
It is very soluble in water, freely soluble in pyridine, and
practically insoluble in methanol, in ethanol (95), in ethanol
(99.5) and in diethyl ether.
Congealing point: 56 - 61 °C
Identification Dissolve 0.05 g of Macrogol 6000 in 5 mL of
dilute hydrochloric acid, add 1 mL of barium chloride TS,
shake, and filter, if necessary. To the filtrate add 1 mL of a
solution of phosphomolybdic acid M-hydrate (1 in 10): a yel-
low-green precipitate is formed.
pH <2.54> Dissolve 1.0 g of Macrogol 6000 in 20 mL of
water: the pH of this solution is between 4.5 and 7.5.
Purity (1) Clarity and color of solution — Dissolve 5.0 g of
Macrogol 6000 in 50 mL of water: the solution is clear and
colorless.
(2) Acidity— Dissolve 5 .0 g of Macrogol 6000 in 20 mL of
neutralized ethanol by warming, cool, and add 0.20 mL of
0.1 mol/L sodium hydroxide VS and 1 drop of
phenolphthalein TS: the color of the solution is red.
Average molecular mass Weigh accurately about 12.5 g of
Macrogol 6000, transfer to an about 200-mL glass-stoppered
pressure bottle, add about 25 mL of pyridine, dissolve by
warming, and allow to cool. Separately, pipet 300 mL of
JP XV
Official Monographs / Macrogol Ointment
833
freshly distilled pyridine into a 1000-mL light-resistant, glass-
stoppered bottle, add 42 g of phthalic anhydride, dissolve
with vigorous shaking, and allow to stand for 16 hours or
more. Pipet 25 mL of this solution, transfer to the former
pressure bottle, stopper the bottle tightly, wrap it securely
with strong cloth, and immerse in a water bath, previously
heated at 98 ± 2°C, to the level so that the mixture in the
bottle soaks completely in water. Maintain the temperature
of the bath at 98 ± 2°C for 30 minutes. Remove the bottle
from the bath, and allow to cool in air to room temperature.
Add exactly 50 mL of 0.5 mol/L sodium hydroxide VS and 5
drops of a solution of phenolphthalein in pyridine (1 in 100).
Titrate <2.50> with 0.5 mol/L sodium hydroxide VS until a
light red color remains for not less than 15 seconds. Perform
a blank determination in the same manner.
Average molecular mass
= (Wx4000)/(a-b)
W: Amount (g) of sample.
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the blank determination.
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
sumed in the test of the sample.
Average molecular mass is between 7300 and 9300.
Water <2.48> Not more than 1.0% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Containers and storage Containers — Well-closed contain-
ers.
Macrogol 20000
Polyethylene Glycol 20000
y"?qa-JU20000
of neutralized ethanol by warming, cool, and add 0.20 mL of
0.1 mol/L sodium hydroxide VS and 1 drop of
phenolphthalein TS: the color of the solution is red.
Average molecular mass Weigh accurately about 15 g of
Macrogol 20000, transfer to an about 200-mL glass-stop-
pered pressure bottle, add about 25 mL of pyridine, dissolve
by warming, and allow to cool. Separately, pipet 300 mL of
freshly distilled pyridine into a 1000-mL light-resistant glass-
stoppered bottle, add 42 g of phthalic anhydride, dissolve
with vigorous shaking, and allow to stand for 16 hours or
more. Pipet 25 mL of this solution, transfer to the former
pressure bottle, stopper the bottle tightly, wrap it securely
with strong cloth, and immerse in a water bath, having a tem-
perature of 98 ± 2°C, to the same depth as the mixture in the
bottle. Maintain the temperature of the bath at 98 ± 2°C for
60 minutes. Remove the bottle from the bath, and allow to
cool in air to room temperature. Add exactly 50 mL of 0.5
mol/L sodium hydroxide VS and 5 drops of a solution of
phenolphthalein in pyridine (1 in 100). Titrate <2.50> with 0.5
mol/L sodium hydroxide VS until a light red color remains
for not less than 15 seconds. Perform a blank determination.
Average molecular mass
= (Wx4000)/(a-b)
W: Amount (g) of sample.
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS used
in the blank determination.
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS used
in the test of the sample.
Average molecular mass is between 15000 and 25000.
Water <2.48> Not more than 1.0% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Containers and storage Containers — Well-closed contain-
ers.
Macrogol 20000 is a polymer of ethylene oxide
and water, represented by the formula
HOCH 2 (CH 2 OCH 2 )„CH 2 OH, in which the value of n
lies between 340 and 570.
Description Macrogol 20000 occurs as white, paraffin-like
flakes or powder. It is ordorless or has a faint, characteristic
odor.
It is freely soluble in water and in pyridine, and practically
insoluble in methanol, in ethanol (95), in anhydrous diethyl
ether, in petroleum benzine and in macrogol 400.
Congealing point: 56 - 64°C
Identification Dissolve 0.05 g of Macrogol 20000 in 5 mL of
dilute hydrochloric acid, add 1 mL of barium chloride TS,
shake, and filter, if necessary. To the filtrate add 1 mL of a
solution of phosphomolybdic acid w-hydrate (1 in 10): a yel-
low-green precipitate is formed.
pH <2.54> Dissolve 1.0 g of Macrogol 20000 in 20 mL of
water: the pH of this solution is between 4.5 and 7.5.
Purity (1) Clarity and color of solution — Dissolve 5.0 g of
Macrogol 20000 in 50 mL of water: the solution is clear and
colorless.
(2) Acidity— Dissolve 5.0 g of Macrogol 20000 in 20 mL
Macrogol Ointment
Polyethylene Glycol Ointment
Method of preparation
Macrogol 4000
Macrogol 400
500 |
500 i
To make 1000 g
Melt Macrogol 4000 and Macrogol 400 by warming on a
water bath at 65 °C, and mix well until it congeals. Less than
100 g of Macrogol 4000 or Macrogol 400 may be replaced by
an equal amount of Macrogol 400 or Macrogol 4000 to pre-
pare 1000 g of a proper soft ointment.
Description Macrogol Ointment is white in color. It has a
faint, characteristic odor.
Identification Dissolve 0.05 g of Macrogol Ointment in 5
mL of dilute hydrochloric acid, add 1 mL of barium chloride
TS, shake, filter if necessary, and add 1 mL of a solution of
phosphomolybdic acid w-hydrate (1 in 10) to the filtrate: a
yellow-green precipitate is formed.
834
Magnesium Carbonate / Official Monographs
JP XV
Containers and storage Containers — Tight containers.
Magnesium Carbonate
Magnesium Carbonate is a basic hydrated magnesi-
um carbonate or a normal hydrated magnesium car-
bonate.
Magnesium Carbonate contains not less than 40.0%
and not more then 44.0% of magnesium oxide (MgO:
40.30).
"Heavy magnesium carbonate" may be used as
commonly used name for Magnesium Carbonate
which shows the height of the precipitate below the
12.0-mL graduation line in the Precipitation test.
Description Magnesium Carbonate occurs as white, friable
masses or powder. It is odorless.
It is practically insoluble in water, in ethanol (95), in
diethyl ether and in 1-propanol.
It dissolves in dilute hydrochloric acid with effervescence.
Its saturated solution is alkaline.
Identification (1) Dissolve 1 g of Magnesium Carbonate
in 10 mL of dilute hydrochloric acid, boil, then cool, neutral-
ize with sodium hydroxide TS, and filter, if necessary: the so-
lution responds to the Qualitative Tests <1.09> for magnesium
salt.
(2) Magnesium Carbonate responds to the Qualitative
Tests <1.09> (1) for carbonate.
Purity (1) Soluble salts — To 2.0 g of Magnesium Car-
bonate add 40 mL of 1-propanol and 40 mL of water, heat to
boil with constant stirring, cool, and filter. Wash the residue
with water, combine the washings with the filtrate, and add
water to make exactly 100 mL. Evaporate 50 mL of the solu-
tion on a water bath to dryness, and dry at 105°C for 1 hour:
the mass of the residue does not exceed 10.0 mg.
(2) Heavy metals <1.07> — Moisten 1.0 g of Magnesium
Carbonate with 4 mL of water, dissolve by addition of 10 mL
of dilute hydrochloric acid, and evaporate on a water bath to
dryness. Dissolve the residue in 35 mL of water, 2 mL of di-
lute acetic acid, 1 drop of ammonia TS, filter, if necessary,
wash the filter paper with water, combine the washings with
the filtrate, and add water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: evaporate 10 mL of dilute
hydrochloric acid on a water bath to dryness, add 2 mL of di-
lute acetic acid and 3.0 mL of Standard Lead Solution, and
dilute with water to make 50 mL (not more than 30 ppm).
(3) Iron — Prepare the test solution with 0.10 g of Mag-
nesium Carbonate according to Method 1, and perform the
test according to Method A. Prepare the control solution
with 2.0 mL of Standard Iron Solution (not more than 200
ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Magnesium Carbonate, previously moistened with 1.5 mL
of water, add 3.5 mL of dilute hydrochloric acid, and per-
form the test (not more than 5 ppm).
(5) Calcium oxide — Weigh accurately about 0.6 g of
Magnesium Carbonate, and dissolve in 35 mL of water and 6
mL of dilute hydrochloric acid. Add 250 mL of water and 5
mL of a solution of L-tartaric acid (1 in 5), then add 10 mL of
a solution of 2,2',2"-nitrilotrisethanol (3 in 10) and 10 mL of
8 mol/L potassium hydroxide TS, allow to stand for 5
minutes, and titrate <2.50> with 0.01 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS until the color of
the solution changes form red-purple to blue (indicator: 0.1 g
of NN indicator). Perform a blank determination, and make
any necessary correction.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 0.5608 mg of CaO
The content of calcium oxide (CaO: 56.08) is not more
than 0.6%.
(6) Acid-insoluble substances — Mix 5.0 g of Magnesium
Carbonate and 75 mL of water, add 10 mL of hydrochloric
acid dropwise while stirring, boil for 5 minutes, and cool.
Collect the insoluble residue using filter paper for quantita-
tive analysis, wash well with water until the last washing
shows no turbidity with silver nitrate TS, and ignite the
residue together with the filter paper: the mass of the residue
is not more than 2.5 mg.
Precipitation test Transfer 1.0 g of Magnesium Carbonate,
previously sifted through a No. 100 (150 /am) sieve to a glass-
stoppered measuring cylinder with a 50-mL graduation line at
150 mm from the bottom, and add water to make 50 mL.
Shake vigorously for exactly 1 minute, allow to stand for 15
minutes, and measure the height of the precipitate (in gradua-
tion in ml).
Assay Weigh accurately about 0.4 g of Magnesium Car-
bonate, dissolve in 10 mL of water and 3.5 mL of dilute
hydrochloric acid, and add water to make exactly 100 mL.
Pipet 25 mL of the solution, add 50 mL of water and 5 mL of
ammonia-ammonium chloride buffer solution, pH 10.7, and
titrate <2.50> with 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS (indicator: 0.04 g of
eriochrome black T-sodium chloride indicator). Perform a
blank determination, and make any necessary correction.
From the volume of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS consumed deduct the
volume of 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS corresponding to the content of calcium
oxide (CaO) obtained in the Purity (5).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.015 mg of MgO
Each mg of calcium oxide (CaO)
= 0.36 mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
Containers and storage Containers — Well-closed contain-
JPXV
Official Monographs / Magnesium Oxide
835
Magnesium Oxide
MgO: 40.30
Magnesium Oxide, when ignited, contains not less
than 96.0% of MgO.
When 5 g of Magnesium Oxide has a volume not
more than 30 mL, it may be labeled heavy magnesium
oxide.
Description Magnesium Oxide occurs as a white powder or
granules. It is odorless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in dilute hydrochloric acid.
It absorbs moisture and carbon dioxide in air.
Identification A solution of Magnesium Oxide in dilute
hydrochloric acid (1 in 50) responds to the Qualitative Tests
<1.09> for magnesium salt.
Purity (1) Alkali and soluble salts — Transfer 2.0 g of
Magnesium Oxide to a beaker, add 100 mL of water, cover
the beaker with a watch-glass, heat on a water bath for 5
minutes, and filter immediately. After cooling, to 50 mL of
the filtrate add 2 drops of methyl red TS and 2.0 mL of 0.05
mol/L sulfuric acid VS: a red color develops. Evaporate 25
mL of the remaining filtrate to dryness, and dry the residue at
105 °C for 1 hour: the mass of the residue is not more than 10
mg.
(2) Carbonate — Boil 0.10 g of Magnesium Oxide with 5
mL of water, cool, and add 5 mL of acetic acid (31): almost
no effervescence occurs.
(3) Heavy metals <1.07> — Dissolve 1.0 g of Magnesium
Oxide in 20 mL of dilute hydrochloric acid, and evaporate on
a water bath to dryness. Dissolve the residue in 35 mL of
water, add 1 drop of phenolphthalein TS, neutralize with am-
monia TS, add 2 mL of dilute acetic acid, and filter, if neces-
sary. Wash the filter paper with water, add water to the com-
bined washing and the filtrate to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 20 mL of dilute hydrochloric
acid add 1 drop of phenolphthalein TS, neutralize with am-
monia TS, and add 2 mL of dilute acetic acid, 4.0 mL of
Standard Lead Solution and water to make 50 mL (not more
than 40 ppm).
(4) Iron <1.10> — Prepare the test solution with 40 mg of
Magnesium Oxide according to Method 1, and perform the
test according to Method A. Prepare the control solution
with 2.0 mL of Standard Iron Solution (not more than 500
ppm).
(5) Calcium oxide — Weigh accurately about 0.25 g of
Magnesium Oxide, previously ignited, dissolve in 6 mL of di-
lute hydrochloric acid by heating. Cool, add 300 mL of water
and 3 mL of a solution of L-tartaric acid (1 in 5), then add 10
mL of a solution of 2,2',2"-nitrilotrisethanol (3 in 10) and 10
mL of 8 mol/L potassium hydroxide TS, allow to stand for 5
minutes, and titrate <2.50> with 0.01 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS until the color of
The figures are in mm.
A: Distilling flask of about 300-mL capacity.
B: Steam generator of about 1000- ml- capacity, contain-
ing a few boiling tips to prevent bumping
C: Condenser
D: Receiver: 200-mL volumetric flask
E: Steam-introducing tube having an internal diameter
of about 8 mm
K G: Rubber tube with a clamp
H: Thermometer
the solution changes from red-purple to blue (indicator: 0.1 g
of NN indicator). Perform a blank determination, and make
any necessary correction.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 0.5608 mg of CaO
The mass of calcium oxide (CaO: 56.08) is not more than
1.5%.
(6) Arsenic <1.11> — Dissolve 0.20 g of Magnesium Oxide
in 5 mL of dilute hydrochloric acid, and perform the test with
this solution as the test solution (not more than 10 ppm).
(7) Acid-insoluble substances — Mix 2.0 g of Magnesium
Oxide with 75 mL of water, add 12 mL of hydrochloric acid
dropwise, while shaking, and boil for 5 minutes. Collect the
insoluble residue using filter paper for quantitative analysis,
wash well with water until the last washing shows no turbidity
with silver nitrate TS, and ignite the residue together with the
filter paper: the mass of the ignited residue does not more
than 2.0 mg.
(8) Fluoride — (i) Apparatus: Use a hard glass apparatus
as illustrated in the figure. Ground-glass joints may be used.
(ii) Procedure: Transfer 5.0 g of Natural Aluminum Sili-
cate to the distilling flask A with the aid of 20 mL of water,
add about 1 g of glass wool and 50 mL of diluted purified sul-
furic acid (1 in 2), and connect A to the distillation appara-
tus, previously washed with steam streamed through the
steam introducing tube E. Connect the condenser C with the
receiver D containing 10 mL of 0.01 mol/L sodium
hydroxide VS and 10 mL of water so that the lower end of C
is immersed in the solution. Heat A gradually until the tem-
perature of the solution in A reaches 130°C, then open the
836
Magnesium Silicate / Official Monographs
JP XV
rubber tube F, close the rubber tube G, boil water in the
steam generator B vigorously, and introduce the generated
steam into F. Simultaneously, heat A, and maintain the tem-
perature of the solution in A between 135°C and 145°C. Ad-
just the distilling rate to about 10 mL per minute. Collect
about 170 mL of the distillate, then stop the distillation, wash
C with a small quantity of water, combine the washings with
the distillate, add water to make exactly 200 mL, and use this
solution as the test solution. Perform the test with the test so-
lution as directed in the procedure of determination for fluo-
ride under the Oxygen Flask Combustion Method. No cor-
rective solution is used in this procedure.
Amount (mg) of fluoride (F: 19.00) in the test solution
= amount (mg) of fluoride in 5 mL of
the standard solution
x (A T /A S ) x (200/ V)
The content of fluoride (F) is not more than 0.08%.
Loss on ignition <2.43> Not more than 10% (0.25 g, 900°C,
constant mass).
Assay Ignite Magnesium Oxide to constant mass at 900°C,
weigh accurately about 0.2 g of the residue, dissolve in 10 mL
of water and 4.0 mL of dilute hydrochloric acid, and add
water to make exactly 100 mL. Pipet 25 mL of this solution,
add 50 mL of water and 5 mL of ammonia-ammonium chlo-
ride buffer solution, pH 10.7, and titrate <2.50> with 0.05 mol
/L disodium dihydrogen ethylenediamine tetraacetate VS (in-
dicator: 0.04 g of eriochrome black T-sodium chloride indi-
cator). Perform a blank determination, and make any neces-
sary correction.
From the volume of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS consumed, deduct the
volume of 0.05 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS corresponding to the content of calcium
oxide (CaO) obtained in the Purity (5).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.015 mg of MgO
Each mg of calcium oxide (CaO)
= 0.36 mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
Containers and storage Containers — Tight containers.
Magnesium Silicate
Magnesium Silicate contains not less than 45.0% of
silicon dioxide (Si0 2 : 60.08) and not less than 20.0% of
magnesium oxide (MgO: 40.30), and the ratio of per-
centage (%) of magnesium oxide to silicon dioxide is
not less than 2.2 and not more than 2.5.
Description Magnesium Silicate occurs as a white, fine pow-
der. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Identification (1) Mix 0.5 g of Magnesium Silicate with 10
mL of dilute hydrochloric acid, filter, and neutralize the
filtrate with ammonia TS: the solution responds to the
Qualitative Tests <1.09> for magnesium salt.
(2) Prepare a bead by fusing ammonium sodium
hydrogenphosphate tetrahydrate on a platinum loop. Place
the bead in contact with Magnesium Silicate, and fuse again:
an infusible matter appears in the bead, which changes to an
opaque bead with a web-like structure upon cooling.
Purity (1) Soluble salts— Add 150 mL of water to 10.0 g
of Magnesium Silicate, heat on a water bath for 60 minutes
with occasional shaking, then cool, dilute with water to 150
mL, and centrifuge. Dilute 75 mL of the resultant transpar-
ent liquid with water to 100 mL, and use this solution as the
sample solution. Evaporate 25 mL of the sample solution on
a water bath to dryness, and ignite the residue at 700°C for 2
hours: the mass of the ignited residue is not more than 0.02 g.
(2) Alkalinity — To 20 mL of the sample solution ob-
tained in (1) add 2 drops of phenolphthalein TS and 1 .0 mL
of 0.1 mol/L hydrochloric acid VS: no color develops.
(3) Chloride <1.03> — Take 10 mL of the sample solution
obtained in (1), add 6 mL of dilute nitric acid, dilute with
water to 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution with 0.75 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.053%).
(4) Sulfate <1.14> — To the residue obtained in (1) add
about 3 mL of dilute hydrochloric acid, and heat on a water
bath for 10 minutes. Add 30 mL of water, filter, wash the
residue on the filter with water, combine the washings with
the filtrate, and dilute to 50 mL with water. To 4 mL of the
solution add 1 mL of dilute hydrochloric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 1.0 mL of 0.005
mol/L sulfuric acid VS (not more than 0.480%).
(5) Heavy metals <1.07> — To 1.0 g of Magnesium Silicate
add 20 mL of water and 3 mL of hydrochloric acid, and boil
for 2 minutes. Filter, and wash the residue on the filter with
two 5-mL portions of water. Evaporate the combined filtrate
and washings on a water bath to dryness, add 2 mL of dilute
acetic acid to the residue, warm until solution is complete,
filter, if necessary, add water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution with 3.0 mL of Standard Lead Solution, 2
mL of dilute acetic acid and water to make 50 mL (not more
than 30 ppm).
(6) Arsenic </.//> — To 0.4 g of Magnesium Silicate add 5
mL of dilute hydrochloric acid, heat gently to boiling while
shaking well, cool rapidly, and centrifuge. Mix the residue
with 5 mL of dilute hydrochloric acid with shaking, cen-
trifuge, then add 10 mL of water to the residue, and repeat
the extraction in the same manner. Concentrate the combined
extracts on a water bath to 5 mL. Use this solution as the test
solution, and perform the test (not more than 5 ppm).
Loss on ignition <2.43> Not more than 34% (0.5 g, 850°C,
3 hours).
Acid-consuming capacity <6.04> Place about 0.2 g of Mag-
nesium Silicate, accurately weighed, in a glass-stoppered
flask, add exactly 30 mL of 0.1 mol/L hydrochloric acid VS
and 20 mL of water, shake at 37 ± 2°C for 1 hour, and cool.
Pipet 25 mL of the supernatant liquid, and titrate <2.50> the
excess hydrochloric acid, while stirring well, with 0.1 mol/L
sodium hydroxide VS until the pH becomes 3.5.
JPXV
Official Monographs / Magnesium Stearate
837
1 g of Magnesium Silicate, calculated on the anhydrous ba-
sis by making allowance for the observed loss on ignition de-
termined as directed in the preceding Loss on ignition, con-
sumes not less than 140 mL and not more than 160 mL of 0.1
mol/L hydrochloric acid VS.
Assay (1) Silicon dioxide — Weigh accurately about 0.7 g
of Magnesium Silicate, add 10 mL of 0.5 mol/L sulfuric acid
TS, evaporate on a water bath to dryness, add 25 mL of
water to the residue, and heat on a water bath for 15 minutes
with occasional stirring. Filter the supernatant liquid through
filter paper for assay, add 25 mL of hot water to the residue,
stir, and decant the supernatant liquid on the filter paper to
filter. Wash the residue in the same manner with two 25-mL
portions of hot water, transfer the residue onto the filter
paper, and wash with hot water until the last washing does
not respond to the Qualitative Tests <1.09> (1) for sulfate.
Place the residue and the filter paper in a platinum crucible,
incinerate with strong heating, and ignite between 775 °C and
825 °C for 30 minutes, then cool, and weigh the residue as a
(g). Moisten the residue with water, and add 6 mL of
hydrofluoric acid and 3 drops of sulfuric acid. Evaporate to
dryness, ignite for 5 minutes, cool, and weigh the residue as b
(g).
Content (%) of silicon dioxide (Si0 2 )
= {(a - b)/W} x 100
W: Mass (g) of the sample
(2) Magnesium oxide — Weigh accurately about 0.3 g of
Magnesium Silicate, transfer to a 50-mL conical flask, add 10
mL of 0.5 mol/L sulfuric acid VS, and heat on a water bath
for 15 minutes. Cool, transfer to a 100-mL volumetric flask,
wash the conical flask with water, add the washings to the
volumetric flask, dilute with water to 100 mL, and filter.
Pipet 50 mL of the filtrate, shake with 50 mL of water and 5
mL of diluted 2,2',2"-nitrilotrisethanol (1 in 2), add 2.0 mL
of ammonia TS and 10 mL of ammonia-ammonium chloride
buffer solution, pH 10.7, and titrate <2.50> with 0.05 mol/L
disodium dihydrogen ethylenediamine tetraacetate VS (indi-
cator: 0.04 g of eriochrome black T-sodium chloride indica-
tor).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.015 mg of MgO
(3) Ratio of percentage (%) of magnesium oxide (MgO)
to silicon dioxide (Si0 2 ) — Calculate the quotient from the
percentages obtained in (1) and (2).
Containers and storage Containers — Well-closed contain-
ers.
Magnesium Stearate
Magnesium Stearate consists chiefly magnesium salts
of stearic acid (C 18 H 36 2 : 284.48) and palmitic acid
(C 16 H 32 2 : 256.42).
It contains, when dried, not less than 4.0% and not
more than 5.0% of magnesium (Mg: 24.31).
Description Magnesium Stearate occurs as a white, light,
bulky powder.
It is smooth to the touch and sticky to the skin. It has no
odor or a faint, characteristic odor.
It is practically insoluble in water and in ethanol (95).
Identification (1) Mix 5.0 g of Magnesium Stearate with
50 mL of peroxide-free diethyl ether, 20 mL of dilute nitric
acid, and 20 mL of water in a round-bottom flask, and heat
to dissolve completely under a reflux condenser. After cool-
ing, transfer the contents of the flask to a separator, shake,
allow the layers to separate, and transfer the aqueous layer to
a flask. Extract the diethyl ether layer with two 4-mL portions
of water, and combine these extracts to the main aqueous ex-
tract. After washing the combined aqueous extract with 15
mL of peroxide-free diethyl ether, transfer to a 50-mL volu-
metric flask, add water to make exactly 50 mL, mix, and use
this solution as the sample solution: the sample solution
responds to the Qualitative Tests <1.09> for magnesium.
(2) The retention times of the peaks corresponding to
stearic acid and palmitic acid in the chromatogram of the
sample solution correspond to those of methyl stearate and
methyl palmitate in the chromatogram of the system suitabil-
ity solution, as obtained in the Purity (5).
Purity (1) Acidity or alkalinity — Heat 1.0 g of Magnesi-
um Stearate in 20 mL of freshly boiled and cooled water on a
water bath for 1 minute while shaking, and filter after cool-
ing. To 10 mL of the filtrate add 0.05 mL of bromothymol
blue TS, and add exactly 0.05 mL of 0.1 mol/L hydrochloric
acid VS or 0.1 mol/L sodium hydroxide VS: the color of the
solution changes.
(2) Chloride <1.03>— Perform the test with 10.0 mL of
the sample solution obtained in Identification (1). Prepare the
control solution with 1.40 mL of 0.02 mol/L hydrochloric
acid VS (not more than 0.10%).
(3) Sulfate <1.14>— Perform the test with 10.0 mL of the
sample solution obtained in Identification (1). Prepare the
control solution with 10.2 mL of 0.01 mol/L sulfuric acid VS
(not more than 1.0%).
(4) Heavy metals <1.07> — Heat 1.0 g of Magnesium
Stearate weakly first, then incinerate at about 500 ± 25 °C.
After cooling, add 2 mL of hydrochloric acid, evaporate on a
water bath to dryness, add 20 mL of water and 2 mL of dilute
acetic acid to the residue, and heat for 2 minutes. After cool-
ing, filter this solution through a filter paper, wash the filter
paper with 15 mL of water, and combine the washing with
the filtrate. To the filtrate add water to make 50 mL, and per-
form the test with this solution as the test solution. Prepare
the control solution as follows: evaporate 2 mL of
hydrochloric acid on a water bath to dryness, add 2 mL of di-
lute acetic acid, 2.0 mL of Standard Lead Solution and water
to make 50 mL (not more than 20 ppm).
(5) Relative content of stearic acid and palmitic
acid — Transfer exactly 0.10 g of Magnesium Stearate to a
small conical flask fitted with a reflux condenser. Add 5.0 mL
of boron trifluoride-methanol TS, mix, and reflux for about
10 minutes to dissolve the solids. Add 4.0 mL of heptane
through the condenser, and reflux for about 10 minutes. Af-
ter cooling, add 20 mL of saturated sodium chloride solu-
tion, shake, and allow the layers to separate. Transfer the
heptane layer through about 0.1 g of anhydrous sodium sul-
fate, previously washed with heptane, to another flask.
838
Magnesium Sulfate Hydrate / Official Monographs
JP XV
Transfer 1 .0 mL of this solution to a 10-mL volumetric flask,
dilute with heptane to volume, mix, and use this solution as
the sample solution. Perform the test with 1 juL of the sample
solution as directed under Gas chromatography <2.02> ac-
cording to the following conditions, and determine the area,
A, of the methyl stearate peak and the total of the areas, B,
of all of fatty acid ester peaks. Calculate the percentage of
stearic acid in the fatty acid fraction of Magnesium Stearate
by the following formula.
Content (%) of stearic acid = (A/B) x 100
Similarly, calculate the percentage of palmitic acid in Mag-
nesium Stearate. The methyl stearate peak, and the total of
the methyl stearate and methyl palmitate peaks are not less
than 40% and not less than 90% of the total area of all fatty
acid ester peaks, respectively, in the chromatogram.
Operating conditions —
Detector: A hydrogen fiame-ionization detector main-
tained at a constant temperature of about 260°C.
Sample injection port: A splitless injection system main-
tained at a constant temperature of about 220°C.
Column: A fused silica capillary column 0.32 mm in inside
diameter and 30 m in length, the inside coated with a 0.5-,um
layer of polyethylene glycol 15000-diepoxide for gas chro-
matography.
Column temperature: Maintain at 70°C for 2 minutes after
injection, then program to increase the temperature at the
rate of 5°C per minute to 240°C and to maintain this temper-
ature for 5 minutes.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
methyl stearate is about 32 minutes.
Split ratio: Splitless
Time span of measurement: About 1.5 time as long as the
retention time of methyl stearate beginning after the solvent
peak.
System suitability —
Test for required detection: Place exactly 50 mg each of
stearic acid for gas chromatography and palmitic acid for gas
chromatography, each previously dried in a desiccator (silica
gel) for 4 hours, in a small conical flask fitted with a reflux
condenser. Add 5.0 mL of boron trifluoride-methanol TS,
mix, and proceed in the same manner as directed for the
preparation of the sample solution, and use the solution so
obtained as the solution for system suitability test. To exactly
1 mL of the solution add heptane to make exactly 10 mL.
Confirm that the peak area of methyl stearate obtained from
1 /uL of this solution is equivalent to 5 to 15% of that from
the solution for system suitability test.
System performance: When the procedure is run with 1 /xL
of the solution for system suitability test under the above
operating conditions, methyl palmitate and methyl stearate
are eluted in this order, with the relative retention time of
methyl palmitate to methyl stearate being about 0.86, and
with the resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
the solution for system suitability test under the above oper-
ating conditions, the relative standard deviation of the peak
areas of methyl palmitate and methyl stearate are not more
than 6.0%, respectively, and the relative standard deviation
of the ratios of the peak area of methyl palmitate to methyl
stearate is not more than 1.0%.
Loss on drying <2.41> Not more than 6.0% (2 g, 105°C,
constant mass).
Microbial limit <4.05> The total viable aerobic microbial
count is not more than 1000 per g, and the total count of
fungi and yeasts is not more than 500 per g. Salmonella and
Escherichia coli should not be observed.
Assay Transfer about 0.5 g of previously dried Magnesium
Stearate, accurately weighed, to a 250-mL flask, add 50 mL
of a mixture of 1-butanol and ethanol (99.5) (1:1), 5 mL of
ammonia solution (28), 3 mL of ammonium chloride buffer
solution, pH 10, 30.0 mL of 0.1 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS, and 1 to 2 drops of
eriochrome black T TS, and mix. Heat at 45°C to 50°C to
make the solution clear, and after cooling, titrate <2.50> the
excess disodium dihydrogen ethylenediamine tetraacetate
with 0.1 mol/L zinc sulfate VS until the solution changes
from blue to purple in color. Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.431 mg of Mg
Containers and storage Containers — Tight containers.
Magnesium Sulfate Hydrate
MgS0 4 .7H 2 0: 246.47
Magnesium Sulfate Hydrate, when ignited, contains
not less than 99.0% of magnesium sulfate (MgS0 4 :
120.37).
Description Magnesium Sulfate Hydrate occurs as colorless
or white crystals. It has a cooling, saline, bitter taste.
It is very soluble in water, and practically insoluble in
ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification A solution of Magnesium Sulfate Hydrate (1
in 40) responds to the Qualitative Tests <1.09> for magnesium
salt and for sulfate.
pH <2.54> Dissolve 1.0 g of Magnesium Sulfate Hydrate in
20 mL of water: the pH of this solution is between 5.0 and
8.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Magnesium Sulfate Hydrate in 20 mL of water: the solution
is clear and colorless.
(2) Chloride <1.03>— Perform the test with 1 .0 g of Mag-
nesium Sulfate Hydrate. Prepare the control solution with
0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.014%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Magnesi-
um Sulfate Hydrate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than lOppm).
(4) Zinc — Dissolve 2.0 g of Magnesium Sulfate Hydrate
in 20 mL of water, and add 1 mL of acetic acid and 5 drops
of potassium hexacyanoferrate (II) TS: no turbidity is
produced.
JPXV
Official Monographs / Magnesium Sulfate Mixture
839
(5) Calcium — Dissolve 1.0 g of Magnesium Sulfate Hy-
drate in 5.0 mL of dilute hydrochloric acid, add water to
make 100 mL, and use this solution as the sample solution.
Separately, dissolve 1.0 g of Magnesium Sulfate Hydrate in
2.0 mL of standard calcium solution and 5.0 mL of dilute
hydrochloric acid, add water to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with the sample solution and standard solution as directed
under Atomic Absorption Spectrophotometry <2.23> accord-
ing to the following conditions, and determine the absor-
bances, A T and A s , of both solutions: A T is not bigger than
A S -A T (not more than 0.02%).
Gas: Combustible gas — Acetylene or hydrogen
Supporting gas — Air
Lamp: Calcium hollow-cathod lamp
Wavelength: 422.7 nm
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Magnesium Sulfate Hydrate according to Method 1, and
perform the test (not more than 2 ppm).
Loss on ignition <2.43> 45.0-52.0% (1 g, after drying at
105 °C for 2 hours, ignite at 450°C for 3 hours).
Assay Weigh accurately about 0.6 g of Magnesium Sulfate
Hydrate, previously ignited at 450°C for 3 hours after drying
at 105°C for 2 hours, and dissolve in 2 mL of dilute
hydrochloric acid and water to make exactly 100 mL. Pipet
25 mL of this solution, add 50 mL of water and 5 mL of am-
monia-ammonium chloride buffer solution, pH 10.7, and ti-
trate <2.50> with 0.05mol/L disodium dihydrogen
ethylenediamine tetraacetate VS (indicator: 0.04 g of
eriochrome black T-sodium chloride indicator). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 6.018 mg of MgS0 4
Containers and storage Containers — Well-closed contain-
ers.
Magnesium Sulfate Injection
fM^y^y^AMS
Magnesium Sulfate Injection is an aqueous solution
for injection.
It contains not less than 95% and not more than 105
% of the labeled amount of magnesium sulfate hydrate
(MgS0 4 .7H 2 0: 246.47).
Method of preparation Prepare as directed under Injec-
tions, with Magnesium Sulfate Hydrate.
Description
less liquid.
Magnesium Sulfate Injection is a clear, color-
Identification Measure a volume of Magnesium Sulfate In-
jection, equivalent to 0.5 g of Magnesium Sulfate Hydrate
according to the labeled amount, and add water to make 20
mL: the solution responds to the Qualitative Tests <1.09> for
magnesium salt and for sulfate.
pH <2.54> 5.5-7.0 When the labeled concentration ex-
ceeds 5%, prepare a solution of 5% with water, and perform
the test.
Bacterial endotoxins <4.01> Less than 0.09 EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Magnesium Sulfate In-
jection, equivalent to about 0.3 g of magnesium sulfate hy-
drate (MgS0 4 .7H 2 0), and add water to make 75 mL. Then
add 5 mL of ammonia-ammonium chloride buffer solution,
pH 10.7, and proceed as directed in the Assay under Mag-
nesium Sulfate Hydrate.
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 12.32 mg of MgS0 4 .7H 2
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Magnesium Sulfate Mixture
Magnesium Sulfate Mixture contains not less than
13.5 w/v% and not more than 16.5 w/v% of magnesi-
um sulfate hydrate (MgS0 4 .7H 2 0: 246.47).
Method of preparation
Magnesium Sulfate Hydrate
Bitter Tincture
Dilute Hydrochloric Acid
Purified Water
150 g
20 mL
5mL
a sufficient quantity
To make 1000 mL
Prepare before use, with the above ingredients.
Description Magnesium Sulfate Mixture is a light yellowish
clear liquid. It has a bitter and acid taste.
Identification (1) Magnesium Sulfate Mixture responds to
the Qualitative Tests <1.09> for magnesium salt.
(2) Magnesium Sulfate Mixture responds to the Qualita-
tive Tests <1.09> (2) for chloride.
Assay Pipet 10 mL of Magnesium Sulfate Mixture, and add
water to make exactly 100 mL. Pipet 10 mL of this solution,
add 50 mL of water and 5 mL of pH 10.7 ammonia-ammoni-
um chloride buffer solution, and titrate <2.50> with 0.05
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
(indicator: 0.04 g of eriochrome black T-sodium chloride in-
dicator).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 12.32 mg of MgS0 4 .7H 2
Containers and storage Containers — Tight containers.
840
Maltose Hydrate / Official Monographs
JP XV
Maltose Hydrate
>H s O
H OH
H OH
C 12 H 22 0„.H 2 0: 360.31
a-D-Glucopyranosyl-(l— >-4)-/?-D-glucopyranose
monohydrate [6363-53-7]
Maltose Hydrate, when dried, contains not less than
98.0% of C 12 H 22 0„.H 2 0.
Description Maltose Hydrate occurs as white crystals or
crystalline powder.
It has a sweet taste.
It is freely soluble in water, very slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
Identification (1) Dissolve 0.5 g of Maltose Hydrate in 5
mL of water, add 5 mL of ammonia TS, and heat for 5
minutes on a water bath: an orange color develops.
(2) Add 2 to 3 drops of a solution of Maltose Hydrate (1
in 50) to 5 mL of boiling Fehling TS: a red precipitate is
formed.
Optical rotation <2.49> [a]™: +126- +131° Weigh ac-
curately about 10 g of Maltose Hydrate, previously dried,
dissolve in 0.2 mL of ammonia TS and water to make exactly
100 mL, and determine the optical rotation of this solution in
a 100-mm cell.
pH <2.54> The pH of a solution of Maltose Hydrate (1 in
10) is between 4.5 and 6.5.
Purity (1) Clarity and color of solution — Put 10 g of Mal-
tose Hydrate in 30 mL of water in a Nessler tube, warm at 60
°C in a water bath to dissolve, and after cooling, add water to
make 50 mL: the solution is clear, and has no more color
than the following control solution.
Control solution: Add water to a mixture of 1.0 mL of
Cobaltous Chloride Stock CS, 3.0 mL of Ferric Chloride
Stock CS and 2.0 mL of Cupric Sulfate Stock CS to make
10.0 mL. To 1.0 mL of this solution add water to make 50
mL.
(2) Chloride <1.03>— Perform the test with 2.0 g of Mal-
tose Hydrate. Prepare the control solution with 1.0 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.018%).
(3) Sulfate <1.14>— Perform the test with 2.0 g of Mal-
tose Hydrate. Prepare the control solution with 1.0 mL of
0.005 mol/L sulfuric acid VS (not more than 0.024%).
(4) Heavy metals <1.07> — Proceed with 5.0 g of Maltose
Hydrate according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 4 ppm).
(5) Arsenic </.//> — Dissolve 1.5 g of Maltose Hydrate in
5 mL of water, add 5 mL of dilute sulfuric acid and 1 mL of
bromine TS, heat on a water bath for 5 minutes, then heat to
concentrate to 5 mL, and use this solution as the test solution
after cooling. Perform the test (not more than 1.3 ppm).
(6) Dextrin, soluble starch and sulfite — Dissolve 1.0 g of
Maltose Hydrate in 10 mL of water, and add 1 drop of iodine
TS: a yellow color appears, and the color changes to a blue by
adding 1 drop of starch TS.
(7) Nitrogen — Weigh accurately about 2 g of Maltose
Hydrate, and perform the test as directed under Nitrogen De-
termination <1.08> using 10 mL of sulfuric acid for the
decomposition and 45 mL of a solution of sodium hydroxide
(2 in 5) for the addition: the amount of nitrogen (N: 14.01) is
not more than 0.01%.
(8) Related substances — Dissolve 0.5 g of Maltose Hy-
drate in 10 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following operating
conditions. Determine the peak areas from both solutions by
the automatic integration method: the total area of the peaks
which appear before the peak of maltose from the sample so-
lution is not larger than 1.5 times of the peak area of maltose
from the standard solution, and the total area of the peaks
which appear after the peak of maltose from the sample solu-
tion is not larger than 1/2 time of the peak area of maltose
from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed as directed in the
operating conditions in the Assay.
Detection sensitivity: Adjust the sensitivity so that the peak
height of maltose obtained from 20 /xL of the standard solu-
tion is about 30 mm.
Time span of measurement: About 2 times as long as the
retention time of maltose.
Loss on drying <2.41> Not more than 0.5% (1 g, 80°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g each of Maltose Hy-
drate and Maltose Reference Standard, previously dried, dis-
solve in exactly 10 mL each of the internal standard solution,
and use these solutions as the sample solution and the stan-
dard solution, respectively. Perform the test with 20 jXL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing operating conditions, and determine the ratios, Q T and
Q s , of the peak area of maltose to that of the internal stan-
dard.
= W s x(Q T /Q s )
W s : Amount (mg) of Maltose Reference Standard
Internal standard solution — A solution of ethylene glycol (1
in 50).
Operating conditions —
Detector: A differential refractometer.
Column: A stainless steel column about 8 mm in inside di-
ameter and about 55 cm in length, packed with gel-type
strong acid cation-exchange resin for liquid chromatography
(degree of cross-linking: 8 %) (10 /xm in particle diameter).
Column temperature: A constant temperature of about
JPXV
Official Monographs / D-Mannitol
841
50°C.
Mobile phase: Water
Flow rate: Adjust the flow rate so that the retention time of
maltose is about 18 minutes.
Selection of column: Dissolve 0.25 g of maltose, 0.25 g of
glucose and 0.4 g of ethylene glycol in water to make 100 mL.
Proceed with 20 /xh of this solution under the above operat-
ing conditions, and calculate the resolution. Use a column
giving elution of maltose, glucose and ethylene glycol in this
order with the resolution of between the peaks of maltose and
glucose being not less than 4.
Containers and storage Containers — Tight containers.
Freeze-dried Mamushi Antivenom,
Equine
Freeze-dried Mamushi Antivenom, Equine, is a
preparation for injection which is dissolved before use.
It contains Agkistrodon Halys antivenom in im-
munoglobulin of horse origin.
It conforms to the requirements of Freeze-dried
Mamushi Antivenom, Equine, in the Minimum Re-
quirements for Biological Products.
Description Freeze-dried Mamushi Antivenom, Equine,
becomes a colorless or light yellow-brown, clear liquid, or a
slightly white-turbid liquid on addition of solvent.
D-Mannitol
d-t>- Y—)l
HO H H OH
HO H H OH
C 6 H 14 6 : 182.17
D-Mannitol [69-65-8]
D-Mannitol, when dried, contains not less than
98.0% of C 6 H 14 6 .
Description D-Mannitol occurs as white crystals or powder.
It is odorless, and has a sweet taste with a cold sensation.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It dissolves in sodium hydroxide TS.
Identification (1) To 5 drops of a saturated solution of D-
Mannitol add 1 mL of iron (III) chloride TS and 5 drops of a
solution of sodium hydroxide (1 in 5): a yellow precipitate is
produced. Shake this solution vigorously: a clear solution is
produced. On addition of a solution of sodium hydroxide (1
in 5), no precipitate is produced.
(2) Determine the infrared absorption spectrum of D-
Mannitol as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers. If
any difference appears between the spectra, dissolve 1 g of D-
Mannitol in 3 mL of warm water, then allow to stand at 5°C
for 24 hours or until crystals appear, and filter. Wash the
crystals so obtained with a few amount of cold water, dry at
105 °C for 4 hours, and perform the test with the crystals.
Optical rotation <2.49> [ a ] D °: + 137 - + 145° Weigh ac-
curately about 1 .0 g of D-Mannitol, previously dried, dissolve
in 80 mL of a solution of hexaammonium heptamolybdate
tetrahydrate (1 in 20), and add diluted sulfuric acid (1 in 35)
to make exactly 100 mL. Measure the optical rotation of this
solution in a 100-mm cell.
Melting point <2.60> 166 - 169°C
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
D-Mannitol in 10 mL of water by warming: the solution is
clear and colorless.
(2) Acidity — Dissolve 5.0 g of D-Mannitol in 50 mL of
freshly boiled and cooled water, and add 1 drop of
phenolphthalein TS and 0.50 mL of 0.01 mol/L sodium
hydroxide VS: a red color develops.
(3) Chloride <1.03>— Perform the test with 2.0 g of d-
Mannitol. Prepare the control solution with 0.40 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.007%).
(4) Sulfate <1.14>— Perform the test with 2.0 g of D-Man-
nitol. Prepare the control solution with 0.40 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.010%).
(5) Heavy metals <1.07> — Proceed with 5.0 g of D-Man-
nitol according to Method 1, and perform the test. Prepare
the control solution with 2.5 mL of Standard Lead Solution
(not more than 5 ppm).
(6) Nickel — Dissolve 0.5 g of D-Mannitol in 5 mL of
water, add 3 drops of dimethylglyoxime TS and 3 drops of
ammonia TS, and allow to stand for 5 minutes: no red color
develops.
(7) Arsenic <1.11> — Prepare the test solution with 1.5 g
of D-Mannitol according to Method 1, and perform the test
(not more than 1.3 ppm).
(8) Sugars — To 5.0 g of D-Mannitol add 15 mL of water
and 4.0 mL of dilute hydrochloric acid, and heat under a
reflux condenser in a water bath for 3 hours. After cooling,
neutralize with sodium hydroxide TS (indicator: 2 drops of
methyl orange TS), and add water to make 50 mL. Pipet 10
mL of this solution into a flask, boil gently with 10 mL of
water and 40 mL of Fehling's TS for 3 minutes, and allow to
stand to precipitate copper (I) oxide. Filter the supernatant
liquid through a glass filter (G4), wash the precipitate with
hot water until the last washing no longer shows an alkaline
reaction, and filter the washings through the glass filter
described above. Dissolve the precipitate in 20 mL of iron
(III) sulfate TS in the flask, filter through the glass filter
described above, and wash the filter with water. Combine the
washings and the filtrate, heat to 80°C, and titrate <2.50> with
0.02 mol/L potassium permanganate: the consumed volume
is not more than 1.0 mL.
Loss on drying <2.4I> Not more than 0.30% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of D-Mannitol, previ-
ously dried, and dissolve in water to make exactly 100 mL.
Pipet 10 mL of the solution into an iodine flask, add exactly
842 D-Mannitol Injection / Official Monographs
JP XV
50 mL of potassium periodate TS, and heat for 15 minutes in
a water bath. After cooling, add 2.5 g of potassium iodide,
stopper tightly, and shake well. Allow to stand for 5 minutes
in a dark place, and titrate <2.50> with 0.1 mol/L sodium
thiosulfate VS (indicator: 1 mL of starch TS). Perform a
blank determination.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.822 mg of C 6 H 14 6
Containers and storage Containers — Tight containers.
D-Mannitol Injection
D-Mannite Injection
d-t>- h-ju&fctfft
D-Mannitol Injection is an aqueous solution for in-
jection.
It contains not less than 95% and not more than
105% of the labeled amount of D-mannitol
(C 6 H 14 6 : 182.17).
Method of preparation Prepare as directed under Injec-
tions, with D-Mannitol. No preservative is added.
Description D-Mannitol Injection is a clear, colorless liq-
uid. It has a sweet taste.
It may precipitate crystals.
Identification Concentrate D-Mannitol Injection on a water
bath to make a saturated solution. Proceed with 5 drops of
this solution as directed in the Identification (1) under D-
Mannitol.
pH <2.54> 4.5 - 7.0
Bacterial endotoxins <4.01> Less than 0.50 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of D-Mannitol Injection,
equivalent to about 5 g of D-Mannitol (C 6 Hi 4 6 ), and add
water to make exactly 250 mL. To exactly 10 mL of this solu-
tion add water to make exactly 100 mL. Measure exactly 10
mL of this solution into an iodine flask, and proceed as
directed in the Assay under D-Mannitol.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.822 mg of C 6 H 14 6
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Maprotiline Hydrochloride
C 20 H 23 N.HC1: 313.86
3-(9,10-Dihydro-9,10-ethanoanthracen-9-yl)-
TV-methylpropylamine monohydrochloride [10347-81-6]
Maprotiline Hydrochloride, when dried, contains
not less than 99.0% of C 20 H 23 N.HC1.
Description Maprotiline Hydrochloride occurs as a white
crystalline powder.
It is soluble in methanol and in acetic acid (100), sparingly
soluble in ethanol (99.5), and slightly soluble in water.
Melting point: about 244°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Maprotiline Hydrochloride in methanol (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Maprotiline Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, recrystallize the sample with
ethanol (99.5), filter, dry the crystals so obtained, and per-
form the test with the crystals.
(3) To 5 mL of a solution of Maprotiline Hydrochloride
(1 in 200) add 2 mL of ammonia TS, heat on a water bath for
5 minutes, cool, and filter. Acidify the filtrate with dilute
nitric acid: the solution responds to the Qualitative Tests
<1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Maprotiline Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.10 g of Maprotiline
Hydrochloride in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 [iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop with a mixture of 2-butanol, dilut-
ed ammonia solution (28) (1 in 3) and ethyl acetate (14:5:4) to
a distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): the number
of the spot other than the principal spot from the sample so-
lution is not more than 2 and they are not more intense than
the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Maprotiline
Hydrochloride, previously dried, dissolve in 180 mL of acetic
acid (100), add 8 mL of a solution of bismuth nitrate penta-
hydrate in acetic acid (100) (1 in 50), and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
JPXV
Official Monographs / Meclofenoxate Hydrochloride
843
= 31.39 mg of C 20 H 23 N.HC1
Containers and storage Containers — Well-closed contain-
ers.
Freeze-dried Live Attenuated
Measles Vaccine
Freeze-dried Live Attenuated Measles Vaccine is a
preparation for injection which is dissolved before use.
It contains live attenuated measles virus.
It conforms to the requirements of Freeze-dried Live
Attenuated Measles Vaccine in the Minimum Require-
ments for Biological Products.
Description Freeze-dried Live Attenuated Measles Vaccine
becomes a colorless, yellowish or reddish clear liquid on addi-
tion of solvent.
Meclofenoxate Hydrochloride
XX
°~.-K
"CHs
•HO
C 12 H 16 C1N0 3 .HC1: 294.17
2-(Dimethylamino)ethyl (4-chlorophenoxy)acetate
monohydrochloride [3685-84-5]
Meclofenoxate Hydrochloride contains not less than
98.0% of C 12 H 16 C1N0 3 .HC1, calculated on the anhy-
drous basis.
Description Meclofenoxate Hydrochloride occurs as white
crystals or crystalline powder. It has a faint, characteristic
odor and a bitter taste.
It is freely soluble in water and in ethanol (95), sparingly
soluble in acetic anhydride, and practically insoluble in
diethyl ether.
The pH of a solution of Meclofenoxate Hydrochloride (1
in 20) is between 3.5 and 4.5.
Identification (1) To 0.01 g of Meclofenoxate Hydrochlo-
ride add 2 mL of ethanol (95), dissolve by warming if neces-
sary, cool, add 2 drops of a saturated solution of hydrox-
ylammonium chloride in ethanol (95) and 2 drops of a satu-
rated solution of potassium hydroxide in ethanol (95), and
heat in a water bath for 2 minutes. After cooling, render the
solution slightly acidic with dilute hydrochloric acid, and add
3 drops of iron (III) chloride TS: a red-purple to dark purple
color develops.
(2) Dissolve 0.05 g of Meclofenoxate Hydrochloride in 5
mL of water, and add 2 drops of Reinecke salt TS: a light red
precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Meclofenoxate Hydrochloride (1 in 10,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) A solution of Meclofenoxate Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 139 - 143°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Meclofenoxate Hydrochloride in 10 mL of water: the solu-
tion is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 1.0 g of
Meclofenoxate Hydrochloride. Prepare the control solution
with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than
0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Meclofenoxate Hydrochloride according to Method 1, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Meclofenoxate Hydrochloride according to method 3, and
perform the test (not more than 2 ppm).
(5) Organic acids — To 2.0 g of Meclofenoxate
Hydrochloride add 50 mL of diethyl ether, shake for 10
minutes, filter through a glass filter (G3), wash the residue
with two 5-mL portions of diethyl ether, and combine the
washings with the filtrate. To this solution add 50 mL of neu-
tralized ethanol and 5 drops of phenolphthalein TS, and neu-
tralize with 0.1 mol/L sodium hydroxide VS: the volume of
0.1 mol/L sodium hydroxide VS consumed is not more than
0.54 mL.
Water <2.48> Not more than 0.50% (1 g, dirct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Meclofenoxate
Hydrochloride, dissolve in 70 mL of acetic anhydride, and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS until the color
of the solution changes from blue-green through yellow-
green to pale greenish yellow [indicator: 3 drops of a solution
of malachite green oxalate in acetic acid (100) (1 in 100)]. Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 29.42 mg of C 12 H 16 C1N0 3 .HC1
Containers and storage Containers — Tight containers.
844
Mecobalamin / Official Monographs
JP XV
Mecobalamin
/ziM'vS >
J-
«CH
? "h
-O-P O HO
II
M
HO'
C 63 H 91 CoN 13 0,4P: 1344.38
Coa- [a-(5 ,6-Dimethyl- l//-benzoimidazol- 1 -yl)]-Cofi-
methylcobamide [13422-55-4]
Mecobalamin contains not less than 98.0% of
C 6 3H 91 CoN 1 30 14 P, calculated on the anhydrous basis.
Description Mecobalamin occurs as dark red crystals or
crystalline powder.
It is sparingly soluble in water, slightly soluble in ethanol
(99.5), and practically insoluble in acetonitrile.
It is affected by light.
Identification (1) Conduct this procedure without ex-
posure to light, using light-resistant vessels. Determine the
absorption spectrum of a solution of Mecobalamin in
hydrochloric acid-potassium chloride buffer solution, pH 2.0
(1 in 20,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum 1 or the spectrum of a solution of
Mecobalamin Reference Standard prepared in the same man-
ner as the sample solution: both spectra exhibit similar inten-
sities of absorption at the same wavelengths. Separately, de-
termine the absorption spectrum of a solution of Mecobala-
min in phosphate buffer solution, pH 7.0 (1 in 20,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 2 or
the spectrum of a solution of Mecobalamin Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Mix 1 mg of Mecobalamin with 0.05 g of potassium
bisulfate, and fuse by igniting. Cool, break up the mass with
a glass rod, add 3 mL of water, and dissolve by boiling. Add
1 drop of phenolphthalein TS, then add dropwise sodium
hydroxide TS until a light red color just develops. Add 0.5 g
of sodium acetate, 0.5 mL of dilute acetic acid and 0.5 mL of
a solution of disodium l-nitroso-2-naphthol-3,6-disulfonate
(1 in 500): a red to orange-red color is immediately produced.
Then add 0.5 mL of hydrochloric acid, and boil for 1 minute:
the red color does not disappear.
Purity (1) Clarity and color of solution — Dissolve 20 mg
of Mecobalamin in 10 mL of water: the solution is clear and
red color.
(2) Related substances — Perform the test with 10 /xL of
the sample solution obtained in the Assay as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine the peak area of mecobalamin and
others of the sample solution by the automatic integration
method: each area of the peaks other than mecobalamin is
not larger than 0.5% of the peak area of mecobalamin, and
the total area of the peaks other than mecobalamin is not
larger than 2.0%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2.5 times as long as the
retention time of mecobalamin.
System suitability —
Test for required detection: To exactly 1 mL of the sample
solution add the mobile phase to make exactly 100 mL, and
use this solution as the test solution for system suitability.
Pipet 1 mL of the test solution for system suitability, add the
mobile phase to make exactly 10 mL. Confirm that the peak
area of mecobalamin obtained from 10 fiL of this solution is
equivalent to 7 to 13% of that of mecobalamin obtained
from 10 //L of the test solution for system suitability.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 /uL of the test solution for system suitability under the
above operating conditions, the relative standard deviation
of the peak areas of mecobalamin is not more than 3.0%.
Water <2.48> Not more than 12% (0.1 g, direct titration).
Assay Conduct this procedure without exposure to light,
using light-resistant vessels. Weigh accurately about 50 mg of
Mecobalamin and Mecobalamin Reference Standard
(separately, determine the water <2.48> in the same manner as
mecobalamin), dissolve each in the mobile phase to make ex-
actly 50 mL, and use these solutions as the sample solution
and the standard solution, respectively. Perform the test with
exactly 10 /uL of each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of mecobalamin in each solution.
Amount (mg) of C^H^CoNoOhP
= W s x(A T /A s )
PF s :Amount (mg) of Mecobalamin Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 266 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
JPXV
Official Monographs / Medicinal Carbon
845
Mobile phase: To 200 mL of acetonitrile add 800 mL of
0.02 mol/L phosphate buffer solution, pH 3.5, then add
3.76 g of sodium 1-hexane sulfonate to dissolve.
Flow rate: Adjust the flow rate so that the retention time of
mecobalamin is about 12 minutes.
System suitability —
System performance: Dissolve 5 mg each of cyanocobala-
min and hydroxocobalamin acetate in the mobile phase to
make 100 mL. When the procedure is run with 10 iiL of this
solution under the above operating conditions, cyanocobala-
min and hydroxocobalamin are eluted in this order with the
resolution between these peaks being not less than 3. And
when the procedure is run with 10 fiL of the standard solution
under the above operating conditions, the number of theoret-
ical plates of the peak of mecobalamin is not less than 6000.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of mecobalamin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Medazepam
yy-t£n°A
C 16 H 15 C1N 2 : 270.76
7-Chloro-l-methyl-5-phenyl-2,3-dihydro-l//-l,4-
benzodiazepine [2898-12-6]
Medazepam, when dried, contains not less than
98.5% of C 16 H 15 C1N 2 .
Description Medazepam occurs as white to light yellow
crystals or crystalline powder. It is odorless.
It is freely soluble in methanol, in ethanol (95), in acetic
acid (100) and in diethyl ether, and practically insoluble in
water.
It gradually changes in color by light.
Identification (1) Dissolve 0.01 g of Medazepam in 3 mL
of citric acid-acetic acid TS: a deep orange color develops.
Heat in a water bath for 3 minutes: the color changes to dark
red.
(2) Determine the absorption spectrum of a solution of
Medazepam in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Perform the test with Medazepam as directed under
Flame Coloration Test <1.04> (2): a green color is produced.
Melting point <2.60> 101 - 104°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Medazepam in 10 mL of methanol: the solution is clear and
light yellow to yellow in color.
(2) Chloride <1.03>— Dissolve 1.5 g of Medazepam in 50
mL of diethyl ether, add 46 mL of water and 4 mL of sodium
carbonate TS, shake, and collect the water layer. Wash the
water layer with two 20-mL portions of diethyl ether, and
filter. To 20 mL of the filtrate add dilute nitric acid to neutral-
ize, add 6 mL of dilute nitric acid and water to make 50 mL,
and perform the test using this solution as the test solution.
Prepare the control solution with 0.30 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.018%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Medazepam according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Medazepam according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.25 g of Medazepam
in 10 mL of methanol, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add methanol
to make exactly 20 mL. Pipet 2 mL of this solution, add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
iiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of cyclo-
hexane, acetone and ammonia solution (28) (60:40: 1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Medazepam, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 27.08 mg of C I6 H 15 C1N 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Medicinal Carbon
Description Medicinal Carbon occurs as a black, odorless
and tasteless powder.
Identification Place 0.5 g of Medicinal Carbon in a test
tube, and heat by direct application of flame with the aid of a
current of air: it burns without any flame. Pass the evolved
gas through calcium hydroxide TS: a white turbidity is
produced.
Purity (1) Acidity or alkalinity — Boil 3.0 g of Medicinal
846
Medicinal Soap / Official Monographs
JP XV
Carbon with 60 mL of water for 5 minutes, allow to cool, di-
lute to 60 mL with water, and filter: the filtrate is colorless
and neutral.
(2) Chloride <1.03>— Take 4.0 mL of the filtrate obtained
in (1) in a Nessler tube, add 6 mL of dilute nitric acid and
sufficient water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 0.80 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.142%).
(3) Sulfate <1.14>— Take 5 mL of the filtrate obtained in
(1) in a Nessler tube, add 1 mL of dilute hydrochloric acid
and sufficient water to make 50 mL, and perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more
than 0.192%).
(4) Sulfide — Boil 0.5 g of Medicinal Carbon with a mix-
ture of 15 mL of dilute hydrochloric acid and 10 mL of
water: lead (II) acetate paper does not become brown when
held in the evolved gas within 5 minutes.
(5) Cyanogen compounds — Place a mixture of 5 g of
Medicinal Carbon, 2 g of L-tartaric acid and 50 mL of water
in a distilling flask connected to a condenser provided with a
tightly fitting adapter, the end of which dips below the sur-
face of a mixture of 2 mL of sodium hydroxide TS and 10 mL
of water, contained in a small flask surrounded by ice. Heat
the mixture in the distilling flask to boiling, and distil to 25
mL. Dilute the distillate with water to 50 mL. To 25 mL of
the diluted distillate add 1 mL of a solution of iron (II) sul-
fate heptahydrate (1 in 20), heat the mixture almost to boil-
ing, cool, and filter. To the filtrate add 1 mL of hydrochloric
acid and 0.5 mL of dilute iron (III) chloride TS: no blue color
is produced.
(6) Acid soluble substances — To about 1 g of Medicinal
Carbon, accurately weighed, add 20 mL of water and 5 mL
of hydrochloric acid, boil for 5 minutes, filter, wash the
residue with 10 mL of hot water, and add the washings to the
filtrate. Add 5 drops of sulfuric acid to the filtrate, evaporate
to dryness, and ignite the residue strongly: the mass of the
residue is not more than 3.0%.
(7) Heavy metals <1.07> — Proceed with 0.5 g of Medici-
nal Carbon according to Method 2, and perform the test.
Prepare the control solution with 2.5 mL of Standard Lead
Solution (not more than 50 ppm).
(8) Zinc — Ignite 0.5 g of Medicinal Carbon to ash, add 5
mL of dilute nitric acid to the residue, boil gently for 5
minutes, filter, wash with 10 mL of water, and combine the
washings and the filtrate. Add 3 mL of ammonia TS to the
solution, filter again, wash with water, combine the washings
and the filtrate, add another washing to make 25 mL, add 1
drop of sodium sulfide TS, and allow to stand for 3 minutes:
the liquid produces no turbidity.
(9) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Medicinal Carbon according to Method 3, and perform
the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 15.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 4.0% (1 g).
Adsorptive power (1) Add 1.0 g of Medicinal Carbon,
previously dried, to 100 mL of water containing 120 mg of
quinine sulfate, shake the mixture vigorously for 5 minutes,
filter immediately, and reject the first 20 mL of the filtrate.
Add 5 drops of iodine TS to 10 mL of the subsequent filtrate:
no turbidity is produced.
(2) Dissolve 250 mg of methylene blue trihydrate, exactly
weighed, in water to make exactly 250 mL. Measure two
50-mL portions of this solution into each of two glass-stop-
pered flasks. To one flask add exactly 250 mg of Medicinal
Carbon, previously dried, and shake vigorously for 5
minutes. Filter the contents of each flask, rejecting the first 20
mL of each filtrate. Pipet 25-mL portions of the remaining
filtrate into two 250-mL volumetric flasks. To each volumet-
ric flask add 50 mL of a solution of sodium acetate trihydrate
(1 in 10), then add exactly 35 mL of 0.05 mol/L iodine VS
with swirling. Allow them to stand for 50 minutes, shaking
vigorously from time to time. Dilute each mixture to exactly
250 mL with water, allow to stand for 10 minutes, and filter
each solution at a temperature not exceeding 20 °C, rejecting
the first 30 mL of each filtrate. Titrate <2.50> the excess iodine
in a 100-mL aliquot of each filtrate with 0.1 mol/L sodium
thiosulfate VS. The difference between the two titrations is
not less than 1.2 mL.
Containers and storage Containers — Well-closed contain-
ers.
Medicinal Soap
Medicinal Soap is sodium salts of fatty acids.
Description Medicinal Soap occurs as white to light yellow
powder or granules. It has a characteristic odor free from
rancidity.
Medicinal Soap is sparingly soluble in water, and slightly
soluble in ethanol (95).
A solution of Medicinal Soap (1 in 100) is alkaline.
Fatty acid Dissolve 25 g of Medicinal Soap in 300 mL of
hot water, add 60 mL of dilute sulfuric acid slowly, and
warm in a water bath for 20 minutes. After cooling, filter off
the precipitate, and wash with warm water until the washing
no longer shows acidity to methyl orange TS. Transfer the
precipitate to a small beaker, and heat on a water bath to
complete separation of water and transparent fatty acids.
Filter the fatty acid into a small beaker while warm, dry at
100°C for 20 minutes, and perform the test with this material
as directed under Fats and Fatty Oils <1.13>. The congealing
point of the fatty acid is between 18°C and 28 °C. The acid
value is 185 - 205. The iodine value is 82 - 92.
Purity (1) Acidity or alkalinity — Dissolve 5.0 g of Medici-
nal Soap in 85 mL of neutralized ethanol by warming on a
water bath, filter while hot through absorbent cotton, and
wash the filter and the residue with three 5-mL portions of
hot neutralized ethanol. Combine the filtrate and the wash-
ings, add hot neutralized ethanol to make exactly 100 mL,
and perform the following tests quickly using this as the sam-
ple solution at 70°C.
(i) Add 3 drops of phenolphthalein TS and 0.20 mL of
0. 1 mol/L sodium hydroxide VS to 40 mL of the sample solu-
tion: a red color develops.
(ii) Add 3 drops of phenolphthalein TS and 0.20 mL of
0.05 mol/L sulfuric acid VS to 40 mL of the sample solution:
JPXV
Official Monographs / Mefenamic Acid
847
no red color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Medici-
nal Soap according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Ethanol-insoluble substances — Weigh accurately
about 2 g of Medicinal Soap, dissolve by warming in 100 mL
of neutralized ethanol, filter the solution through a glass filter
(G4), wash the residue with hot neutralized ethanol, and dry
at 105°C for 4 hours: the mass of the residue is not more than
1.0%.
(4) Water-insoluble substances — Wash thoroughly the
dried substances obtained in (3) with 200 mL of water, and
dry at 105°C for 4 hours: the mass of the residue is not more
than 0.15%.
(5) Alkali carbonates — To the washings obtained in (4)
add 3 drops of methyl orange TS and 2 mL of 0.05 mol/L
sulfuric acid VS: a red color develops.
Loss on drying Not more than 5.0% in the case of the pow-
der, and not more than 10.0% in the case of the granules.
Weigh accurately about 0.5 g of Medicinal Soap in a tared
beaker, add 10 g of sea sand (No. 1), previously dried at
105 °C for 1 hour, and again weigh the beaker. Add 10 mL of
ethanol (95), evaporate on a water bath to dryness with
thorough stirring, and dry at 105°C for 3 hours.
Containers and storage Containers — Well-closed contain-
ers.
Mefenamic Acid
^7itAl
COjH
C 15 H 15 N0 2 : 241.29
2-(2,3-Dimethylphenylamino)benzoic acid
[61-68-7]
Mefenamic Acid, when dried, contains not less than
99.0% of C 15 H 15 N0 2 .
Description Mefenamic Acid occurs as a white to light yel-
low powder. It is odorless and tasteless at first, but leaves a
slightly bitter aftertaste.
It is sparingly soluble in diethyl ether, slightly soluble in
methanol, in ethanol (95) and in chloroform, and practically
insoluble in water.
It dissolves in sodium hydroxide TS.
Melting point: about 225°C (with decomposition).
Identification (1) Dissolve 0.01 g of Mefenamic Acid in 1
mL of methanol by warming, cool, add 1 mL of a solution of
/^-nitrobenzene diazonium fluoroborate (1 in 1000) and 1 mL
of sodium hydroxide TS, and mix thoroughly: an orange-red
color is produced.
(2) Dissolve 0.01 g of Mefenamic Acid in 2 mL of sulfur-
ic acid, and heat: the solution shows a yellow color and a
green fluorescence.
(3) Dissolve 7 mg of Mefenamic Acid in a solution of
hydrochloric acid in methanol (1 in 1000) to make 500 mL.
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
Purity (1) Chloride <1.03>— To 1.0 g of Mefenamic Acid
add 20 mL of sodium hydroxide TS, and dissolve by warm-
ing. Cool, add 2 mL of acetic acid (100) and water to make
100 mL, and mix well. Remove the produced precipitate by
filtration, discard the first 10 mL of the filtrate, and to subse-
quent 25 mL of the filtrate add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.50 mL of 0.01 mol/L hydrochloric acid VS add 5 mL of so-
dium hydroxide TS, 0.5 mL of acetic acid (100), 6 mL of
nitric acid and water to make 50 mL (not more than 0.071%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Mefenamic Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Mefenamic Acid according to Method 3, and perform the
test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Mefenamic
Acid, in 5 mL of a mixture of chloroform and methanol
(3:1), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add a mixture of chloroform and
methanol (3:1) to make exactly 200 mL, pipet 10 mL of this
solution, add a mixture of chloroform and methanol (3:1) to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 25 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of 2-methyl-l-propanol and
ammonia solution (28) (3:1) to a distance of about 10 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Mefenamic Acid,
previously dried, and dissolve in 100 mL of ethanol (95),
previously neutralized to phenol red TS with 0.1 mol/L sodi-
um hydroxide VS, by warming gently. Cool, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS until the color of
the solution changes from yellow through yellow-red to red-
purple (indicator: 2 to 3 drops of phenol red TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 24.13 mg of C 15 H 15 N0 2
Containers and storage Containers — Well-closed contain-
ers.
848
Mefloquine Hydrochloride / Official Monographs
JP XV
Mefloquine Hydrochloride
/7U*>i%m^
HCI
h and enantiomer
HUL
C 17 H 16 F 6 N 2 O.HCl: 414.77
(l.RS)-[2,8-Bis(trifluoromethyl)quinolin-4-yl][(2S'.R)-
piperidin-2-yl]methanol monohydrochloride
[51773-92-3]
Mefloquine Hydrochloride, when dried, contains not
less than 99.0% and not more than 101.0% of
C 17 H 16 F 6 N 2 O.HCl.
Description Mefloquine Hydrochloride occurs as white
crystals or a white crystalline powder.
It is freely soluble in methanol, soluble in ethanol (99.5),
and slightly soluble in water.
It dissolves in sulfuric acid.
A solution of Mefloquine Hydrochloride in methanol (1 in
20) shows no optical rotation.
Melting point: about 260°C (with decomposition).
Identification (1) Dissolve 2 mg of Mefloquine
Hydrochloride in 1 mL of sulfuric acid: the solution shows a
blue fluorescence under ultraviolet light (main wavelength:
365 nm).
(2) Determine the absorption spectrum of a solution of
Mefloquine Hydrochloride in methanol (1 in 25,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the infrared absorption spectrum of Meflo-
quine Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) To 5 mL of a solution of Mefloquine Hydrochloride
(1 in 1000) add 1 mL of dilute nitric acid and 1 mL of silver
nitrate TS: a white precipitate is formed, and the separated
precipitate dissolves on the addition of an excess amount of
ammonia TS.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Mefloquine Hydrochloride according to Method 2 using a
quartz crucible, and perform the test. Prepare the control so-
lution with 2.0 mL of Standard Lead Solution (not more than
20 ppm).
(2) Arsenic <1.11> — To 1.0 g of Mefloquine Hydrochlo-
ride add 10 mL of a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 10), burn the ethanol, gradually
heat, and incinerate by ignition at 800°C. If a carbonized
residue still retains, moisten the residue with a little amount
of nitric acid, and ignite again to incinerate. After cooling, to
the residue add 3 mL of hydrochloric acid, warm on a water
bath to dissolve, and perform the test using this solution as
the test solution (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Mefloquine
Hydrochloride in 50 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, and add the mobile phase to make exactly 50 mL. Pipet
2 mL of this solution, add the mobile phase to make exactly
20 mL, and use this solution as the standard solution. Per-
form the test with exactly 10,mL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the area of the peak other than mefloquine and the
peak eluted first from the sample solution is not larger than
the peak area of mefloquine from the standard solution, and
the total area of the peaks other than the peak of mefloquine
and the peak eluted first from the sample solution is not larg-
er than 2.5 times the peak area of mefloquine from the stan-
dard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 282 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with aminopropyl-
silanized silica gel for liquid chromatography (10 /xm in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of acetonitrile and diluted phos-
phoric acid (1 in 14) (24:1).
Flow rate: Adjust the flow rate so that the retention time of
mefloquine is about 10 minutes.
Time span of measurement: About 3 times as long as the
retention time of mefloquine.
System suitability —
Test for required detectability: To exactly 10 mL of the
standard solution add the mobile phase to make exactly 20
mL. Confirm that the peak area of mefloquine obtained with
10,aL of this solution is equivalent to 40 to 60% of that ob-
tained with 10 /xL of the standard solution.
System performance: Dissolve 10 mg of mefloquine
hydrochloride and 5 mg of diprophylline in 50 mL of the mo-
bile phase. To 2 mL of this solution add the mobile phase to
make 20 mL. When the procedure is run with 10 ^L of this
solution under the above operating conditions, diprophylline
and mefloquine are eluted in this order with the resolution be-
tween these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
mefloquine is not more than 2.0%.
(4) Residual solvent — The test is specified separately.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.5 g of Mefloquine
Hydrochloride, previously dried, dissolve in 100 mL of a
mixture of acetic anhydride and acetic acid (100) (7:3), and ti-
JPXV
Official Monographs / Mefruside Tablets
849
trate <2.50> with 0.1 mol/L perchloric acid VS (potentiomet-
ric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 41.48 mg of C 17 Hi 6 F 6 N 2 O.HCl
Containers and storage Containers — Well-closed contain-
ers.
Mefruside
/7Jk> K
(p"
o o
CH : ,
CH a O
and enantiomer
C 13 H 19 C1N 2 5 S 2 : 382.88
4-Chloro-A r -methyWV-[(2i?S')-2-methyltetrahydrofuran-2-
ylmethyl]-3-sulfamoylbenzenesulfonamide [7195-27-9]
Mefruside, when dried, contains not less than 98.5%
of C 13 H 19 C1N 2 5 S 2 .
Description Mefruside occurs as a white crystalline powder.
It is very soluble in dimethylformamide, freely soluble in
acetone, soluble in methanol, sparingly soluble in ethanol
(95), and practically insoluble in water.
A solution of Mefruside in dimethylformamide (1 in 10)
has no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Mefruside in methanol (1 in 40,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Mefruside, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Perform the test with Mefruside as directed under
Flame Coloration Test (2): a green color appears.
Melting point <2.60> 149 - 152°C
Purity (1) Heavy metals <1.07> — Dissolve 1.0 g of Mefru-
side in 30 mL of acetone, and add 2 mL of dilute acetic acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: to 2.0 mL of Standard Lead Solution add 30 mL of ace-
tone, 2 mL of dilute acetic acid and water to make 50 mL
(not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Mefruside according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.20 g of Mefruside in
10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add acetone to make
exactly 200 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and acetone
(5:2) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41>
2 hours).
Not more than 0.5% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Mefruside, previ-
ously dried, dissolve in 80 mL of A^TV-dimethylformamide,
and titrate <2.50> with 0.1 mol/L tetramethylammonium
hydroxide VS (potentiometric titration). Separately, perform
a blank determination with a solution prepared by adding 13
mL of water to 80 mL of A^Af-dimethylformamide, and
make any necessary correction.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 38.29 mg of C 13 H 19 C1N 2 5 S 2
Containers and storage Containers — Well-closed contain-
ers.
Mefruside Tablets
/7)i-> HIS
Mefruside Tablets contain not less than 95% and not
more than 105% of the labeled amount of mefruside
(C 13 H 19 C1N 2 5 S 2 : 382.88).
Method of preparation
with Mefruside.
Prepare as directed under Tablets,
Identification (1) Weigh a quantity of powdered Mefru-
side Tablets, equivalent to 0.3 g of Mefruside according to
the labeled amount, shake with 15 mL of heated methanol
for 20 minutes, and filter. Add 25 mL of water to the filtrate,
and allow to stand while ice-cooling for 30 minutes. Filter the
white precipitate formed, wash with water, and dry at 105°C
for 2 hours: the precipitate melts <2.60> between 149°C and
152°C.
(2) Weigh a quantity of powdered Mefruside Tablets,
equivalent to 0.01 g of Mefruside according to the labeled
amount, shake with 70 mL of methanol strongly for 15
minutes, add methanol to make 100 mL, and filter. Deter-
mine the absorption spectrum of the filtrate as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 274 nm and 278 nm, and between 283 nm and
287 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Mefruside Tablets at 50
revolutions per minute according to the Paddle method using
900 mL of water as the test solution. Take 20 mL or more of
850
Meglumine / Official Monographs
JP XV
the dissolved solution 45 minutes after starting the test, and
filter through a filter paper for quantitative analysis (5C).
Discard the first 5 mL of the filtrate, and use the subsequent
filtrate as the sample solution. Separately, weigh accurately
about 70 mg of mefruside for assay, previously dried at
105 °C for 2 hours, dissolve in methanol to make exactly 50
mL. Pipet 2 mL of this solution, add water to make exactly
100 mL, and use this solution as the standard solution. Deter-
mine the absorbances, A T and A s , of the sample solution and
the standard solution at 285 nm in a layer of 5 cm in length as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Mefruside Tablets in 45 minutes is not
less than 85%.
Dissolution rate (%) with respect to the labeled amount
of mefruside (C 13 H 19 C1N 2 5 S 2 )
= ^ s x(y4 T M s )x(l/Qx36
W s : Amount (mg) of mefruside for assay.
C: Labeled amount (mg) of mefruside (Ci3H 19 ClN 2 5 S2) in
1 tablet.
Assay Weigh accurately not less than 20 Mefruside Tablets,
and powder. Weigh accurately a portion of the powder,
equivalent to about 65 mg of mefruside (CoH^CUS^OsSJ,
shake with 70 mL of methanol for 15 minutes, then add
methanol to make exactly 100 mL, and filter. Discard the first
20 mL of the filtrate, take exactly 10 mL of the subsequent
filtrate, add methanol to make exactly 50 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 65 mg of mefruside for assay, previously dried at
105 °C for 2 hours, and dissolve in methanol to make exactly
100 mL. Pipet 10 mL of this solution, add methanol to make
exactly 50 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and the standard solution at 285 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of mefruside (C 13 H 19 C1N 2 5 S 2 )
= W s x(Aj/A s )
W s : Amount (mg) of mefruside for assay
Containers and storage Containers — Tight containers.
Meglumine
HO H H OH
HO H H OH
CH :i
C 7 H 17 N0 5 : 195.21
1-Deoxy-l-methylamino-D-glucitol [6284-40-8]
Meglumine, when dried,
99.0% of C 7 H 17 NO s .
contains not less than
Description Meglumine occurs as a white, crystalline pow-
der. It is odorless, and has a slightly bitter taste.
It is freely soluble in water, and slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
The pH of a solution of Meglumine (1 in 10) is between
11.0 and 12.0.
Identification (1) To 1 mL of a solution of Meglumine (1
in 10) add 1 mL of potassium l,2-naphthoquinone-4-sul-
fonate TS: a deep red color develops.
(2) To 2 mL of a solution of Meglumine (1 in 10) add 1
drop of methyl red TS, and add 0.5 mL of dilute sodium
hydroxide TS and 0.5 g of boric acid after neutralizing with
0.5 mol/L sulfuric acid TS: a deep red color develops.
(3) Dissolve 0.5 g of Meglumine in 1 mL of diluted
hydrochloric acid (1 in 3), and add 10 mL of ethanol (99.5): a
white precipitate is produced. Then, rubbing the inside wall
of the container with a glass rod, cool with ice and produce
more precipitate. Filter the precipitate by suction through a
glass filter (G3), wash the precipitate with a small volume of
ethanol (99.5), and dry at 105°C for 1 hour: the residue thus
obtained melts <2.60> between 149°C and 152°C.
Optical rotation <2.49> [a]™: - 16.0 - - 17.0° (after
drying, 1 g, water, 10 mL, 100 mm).
Melting point <2.60> 128-131°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Meglumine in 10 mL of water: the solution is clear and color-
less.
(2) Chloride <1.03>— Dissolve 1.0 g of Meglumine in 30
mL of water, and add 10 mL of dilute nitric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.25 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.009%).
(3) Sulfate <1. 14>— Dissolve 1 .0 g of Meglumine in 30 mL
of water, and add 5 mL of dilute hydrochloric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution with 0.40 mL of
0.005 mol/L sulfuric acid VS (not more than 0.019%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Meglu-
mine according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Meglumine according to Method 3, and perform the test
(not more than 1 ppm).
(6) Reducing substances — To 5 mL of a solution of
Meglumine (1 in 20) add 5 mL of Fehling's TS, and boil for 2
minutes: no red-brown precipitate is produced.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Meglumine, previ-
ously dried, dissolve in 25 mL of water, and titrate <2.50>
with 0.1 mol/L hydrochloric acid VS (indicator: 2 drops of
methyl red TS).
Each mL of 0.1 mol/L hydrochloric acid VS
= 19.52 mg of C 7 H 17 N0 5
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Meglumine Amidotrizoate Injection
851
Meglumine Amidotrizoate
Injection
Meglumine Amidotrizoate Injection is an aqueous
solution for injection.
It contains not less than 46.9 w/v% and not more
than 51.8 w/v% of amidotrizoic acid (CnH^^C),!:
613.91).
Method of preparation
Amidotrizoic Acid (anhydrous) 493.2 g
Meglumine 156.8 g
Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Meglumine Amidotrizoate Injection is a clear,
colorless to pale yellow, slightly viscous liquid.
It gradually changes in color by light.
Identification (1) To 2 mL of Meglumine Amidotrizoate
Injection add 25 mL of water, and add 2.5 mL of dilute
hydrochloric acid with stirring: a white precipitate is
produced. Filter the precipitate by suction through a glass
filter (G4), wash with two 10-mL portions of water, and dry
at 105°C for 1 hour. Proceed with the precipitate so obtained
as directed in the Identification (2) under Amidotrizoic Acid.
(2) To 1 mL of Meglumine Amidotrizoate Injection add
1 mL of potassium l,2-naphthoquinone-4-sulfonate TS and
0.2 mL of sodium hydroxide TS: a deep red color develops.
Optical rotation <2.49> c^ : -3.63 - -4.20° (100 mm).
pH <2.54> 6.0 - 7.7
Purity (1) Primary aromatic amines — Mix 0.40 mL of
Meglumine Amidotrizoate Injection with 6 mL of water, add
4 mL of a solution of sodium nitrite (1 in 100) and 10 mL of 1
mol/L hydrochloric acid TS, and shake. Proceed as directed
in the Purity (2) under Amidotrizoic Acid: the absorbance is
not more than 0.19.
(2) Iodine and iodide — To 0.50 mL of Meglumine
Amidotrizoate Injection add water to make 20 mL, shake
with 5 mL of dilute nitric acid, filter by suction through a
glass filter (G4). Add 5 mL of chloroform to the filtrate, and
shake vigorously: no color develops in the chloroform layer.
Then add 1 mL of hydrogen peroxide (30), and shake
vigorously: the chloroform layer has no more color than the
following control solution.
Control solution: Dissolve 0.10 g of potassium iodide in
water to make 100 mL. Add 20 mL of water to 0.10 mL of
this solution, add 5 mL of dilute nitric acid, 5 mL of chlo-
roform and 1 mL of hydrogen peroxide (30), and shake
vigorously.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Prepare a solution with isotonic sodium
chloride solution so as to contain 0.40 mL of Meglumine
Amidotrizoate Injection per 1 mL, and perform the test: it
meets the requirements.
Assay To an exactly measured 1 mL of Meglumine
Amidotrizoate Injection add water to make exactly 200 mL,
pipet 2 mL of this solution, add exactly 10 mL of the internal
standard solution and water to make 100 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 0.25 g of amidotrizoic acid for assay (previously deter-
mine the loss on drying <2.41> in the same manner as
Amidotrizoic Acid), dissolve in a solution of meglumine (3 in
1000) to make exactly 100 mL. Pipet 2 mL of this solution,
add exactly 10 mL of the internal standard solution and
water to make 100 mL, and use this solution as the standard
solution. Perform the test with 5 [iL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
amidotrizoic acid to that of the internal standard.
Amount (mg) of amidotrizoic acid (CnH^NjC^)
= W S X(Q 1 /Q S )X2
W s : Amount (mg) of amidotrizoic acid for assay, calculat-
ed on the dried basis
Internal standard solution — Dissolve 0.06 g of acetrizoic acid
in a solution of meglumine (3 in 1000) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.7 g of tetrabutylammonium
phosphate and 7.0 g of dipotassium hydrogenphosphate in
750 mL of water, adjust the pH to 7.0 with diluted phosphor-
ic acid (1 in 10), add water to make 800 mL, then add 210 mL
of acetonitrile, and mix.
Flow rate: Adjust the flow rate so that the retention time of
amidotrizoic acid is about 5 minutes.
System suitability —
System performance: When the procedure is run with 5 /iL
of the standard solution under the above operating condi-
tions, amidotrizoic acid and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 6.
System repeatability: When the test is repeated 6 times with
5 /XL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of amidotrizoic acid to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
852
Meglumine Iotalamate Injection / Official Monographs
JP XV
Meglumine Iotalamate Injection
Meglumine Iotalamate Injection is an aqueous solu-
tion for injection.
It contains not less than 95% and not more than
105% of the labeled amount of iotalamic acid (C n H 9 I 3
N 2 4 : 613.91).
Method of preparation
(1)
Iotalamic Acid
Meglumine
Water for Injection
227.59 g
72.41 g
a sufficient quantity
(2)
Iotalamic Acid
Meglumine
Water for Injection
To make 1000 mL
455 g
145 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients (1) or (2).
Description Meglumine Iotalamate Injection is a clear,
colorless to pale yellow, slightly viscous liquid.
It gradually changes in color by light.
Identification (1) To 1 mL of Meglumine Iotalamate In-
jection add 1 mL of potassium naphthoquinone sulfonate TS
and 0.2 mL of sodium hydroxide TS: a deep red color de-
velops.
(2) To a volume of Meglumine Iotalamate Injection,
equivalent to 1 g of Iotalamic Acid according to the labeled
amount, add 25 mL of water, and add 2.5 mL of dilute
hydrochloric acid while shaking: a white precipitate is
produced. Filter the precipitate by suction through a glass
filter (G4), wash the precipitate with two 10-mL portions of
water, and dry at 105 °C for 4 hours. Proceed with the
precipitate so obtained as directed in the Identification (2) un-
der Iotalamic Acid.
Optical rotation <2.49>
Method of preparation (1) a 2 ^'-
mm).
Method of preparation (2) a 2 ^:
mm).
pH <2.54> 6.5 - 7.7
-1.67
-3.35
-1.93° (100
-3.86° (100
Purity (1) Primary aromatic amines — To a volume of
Meglumine Iotalamate Injection, equivalent to 0.20 g of
Iotalamic Acid according to the labeled amount, add 15 mL
of water, shake, add 4 mL of a solution of sodium nitrite (1
in 100) under ice-cooling, and proceed as directed in the Puri-
ty (2) under Iotalamic Acid: the absorbance is not more than
0.17.
(2) Iodine and iodide — Take a volume of Meglumine
Iotalamate Injection, equivalent to 1.5 g of Iotalamic Acid
according to the labeled amount, and proceed as directed in
the Purity (2) under Sodium Iotalamate Injection.
Bacterial endotoxins <4.01> Less than 0.90 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay To an exactly measured volume of Meglumine
Iotalamate Injection, equivalent to about 4 g of iotalamic
acid (CnH^NjC^), add water to make exactly 200 mL. Pipet
2 mL of this solution, add water to make exactly 200 mL. To
exactly 5 mL of this solution add exactly 5 mL of the internal
standard solution, add the mobile phase to make 100 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 0.4 g of iotalamic acid for assay,
previously dried at 105°C for 4 hours, dissolve in 100 mL of
water and 1 mL of sodium hydroxide TS, and add water to
make exactly 200 mL. Pipet 5 mL of this solution, add water
to make exactly 50 mL. To exactly 5 mL of this solution add
exactly 5 mL of the internal standard solution, add the mo-
bile phase to make 100 mL, and use this solution as the stan-
dard solution. Perform the test with 10 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, g T and Q s , of the peak area of
iotalamic acid to that of the internal standard.
Amount (mg) of iotalamic acid (C11H9I3N2O4)
= W s x(Q J /Q s )
W s : Amount (mg) of iotalamic acid for assay
Internal standard solution — A solution of L-tryptophan in
the mobile phase (3 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: Dissolve 3.9 g of phosphoric acid and 2.8
mL of triethylamine in water to make 2000 mL. To this solu-
tion add 100 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
iotalamic acid is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, iotalamic acid and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of iotalamic acid to that of the internal standard is
not more than 1.0%.
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
•Hermetic containers,
JPXV
Official Monographs / Meglumine Sodium Amidotrizoate Injection
853
Meglumine Sodium Amidotrizoate
Injection
Meglumine Sodium Amidotrizoate Injection is an
aqueous solution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of amidotrizoic acid
(CiiH 9 I 3 N 2 4 : 613.91).
Method of preparation
(1)
Amidotrizoic Acid (anhydrous)
Sodium Hydroxide
Meglumine
Water for Injection
522.77 g
25.16g
43.43 g
a sufficient quantity
(2)
Amidotrizoic Acid (anhydrous)
Sodium Hydroxide
Meglumine
Water for Injection
To make 1000 mL
471.78 g
5.03 g
125.46 g
a sufficient quantity
(3)
Amidotrizoic Acid (anhydrous)
Sodium Hydroxide
Meglumine
Water for Injection
To make 1000 mL
597.30 g
6.29 g
159.24 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients (1), (2) or (3).
Description Meglumine Sodium Amidotrizoate Injection is
a clear, colorless to pale yellow, slightly viscous liquid.
It gradually changes in color by light.
Identification (1) To a volume of Meglumine Sodium
Amidotrizoate Injection, equivalent to 1 g of Amidotrizoic
Acid according to the labeled amount, add 25 mL of water,
and add 2.5 mL of dilute hydrochloric acid with stirring: a
white precipitate is produced. Filter the precipitate by suction
through a glass filter (G4), wash with two 10-mL portions of
water, and dry at 105°C for 1 hour. Proceed with the
precipitate so obtained as directed in the Identification (2) un-
der Amidotrizoic Acid.
(2) To 1 mL of Meglumine Sodium Amidotrizoate Injec-
tion add 1 mL of potassium l,2-naphthoquinone-4-sulfonate
TS and 0.2 mL of sodium hydroxide TS: a deep red color de-
velops.
(3) Meglumine Sodium Amidotrizoate Injection responds
to the Qualitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49>
Method of preparation (1) o£°: -1.01
Method of preparation (2) o£°: -2.91
Method of preparation (3) o^ : - 3.69
pH <2.54> 6.0 - 7.7
-1.17° (100 mm).
-3.36° (100 mm).
-4.27° (100 mm).
Purity (1) Primary aromatic amines — To a volume of
Meglumine Sodium Amidotrizoate Injection, equivalent to
0.20 g of Amidotrizoic Acid according to the labeled
amount, add 6 mL of water, mix, add 4 mL of a solution of
sodium nitrite (1 in 100) and 10 mL of 1 mol/L hydrochloric
acid TS, and shake. Proceed as directed in the Purity (2) un-
der Amidotrizoic Acid: the absorbance is not more than 0.19.
(2) Iodine and iodide — To a volume of Meglumine Sodi-
um Amidotrizoate Injection, equivalent to 0.25 g of
Amidotrizoic Acid according to the labeled amount, add
water to make 20 mL, add 5 mL of dilute nitric acid, shake
well, and filter by suction through a glass filter (G4). Add 5
mL of chloroform to the filtrate, and shake vigorously: no
color develops in the chloroform layer. Then add 1 mL of
hydrogen peroxide (30), and shake vigorously: the chlo-
roform layer has no more color than the following control so-
lution.
Control solution: Dissolve 0.10 g of potassium iodide in
water to make 100 mL. Add 20 mL of water to 0.10 mL of
this solution, add 5 mL of dilute nitric acid, 5 mL of chlo-
roform and 1 mL of hydrogen peroxide (30), and shake
vigorously.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Dilute Maglumine Sodium Amidotrizoate
Injection with isotonic sodium chloride solution so as to con-
tain 0.20 g of amidotrizoic acid (CnHgL^O,,) per mL ac-
cording to the labelled amount, and perform the test: it meets
the requirements.
Assay To an exactly measured volume of Meglumine Sodi-
um Amidotrizoate Injection, equivalent to about 0.5 g of
amidotrizoic acid (CnHgL^C^), add water to make exactly
200 mL. Pipet 2 mL of this solution, add exactly 10 mL of
the internal standard solution and water to make 100 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 0.25 g of amidotrizoic acid for assay
(previously determine the loss on drying <2.41> in the same
manner as Amidotrizoic Acid), dissolve in a solution of
meglumine (3 in 1000) to make exactly 100 mL, then pipet 2
mL of this solution, add exactly 10 mL of the internal stan-
dard solution and water to make 100 mL, and use this solu-
tion as the standard solution. Perform the test with 5 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the ratios, Q T and Q s , of the
peak area of amidotrizoic acid to that of the internal stan-
dard.
Amount (mg) of amidotrizoic acid (CnHgL^C^)
= W s x(Q 1 /Q s )
W s : Amount (mg) of amidotrizoic acid for assay, calculat-
ed on the dried basis
Internal standard solution — Dissolve 0.06 g of acetrizoic acid
in a solution of meglumine (3 in 1000) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
854
Meglumine Sodium Iodamide Injection / Official Monographs
JP XV
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.7 g of tetrabutylammonium
phosphate and 7.0 g of dipotassium hydrogenphosphate in
750 mL of water, adjust the pH to 7.0 with diluted phosphor-
ic acid (1 in 10), add water to make 800 mL, then add 210 mL
of acetonitrile, and mix.
Flow rate: Adjust the flow rate so that the retention time of
amidotrizoic acid is about 5 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, amidotrizoic acid and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 6.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of amidotrizoic acid to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Meglumine Sodium Iodamide
Injection
Meglumine Sodium Iodamide Injection is an aque-
ous solution for injection.
It contains not less than 59.7 w/v% and not more
than 65.9 w/v% of iodamide (CnHn^NjC^: 627.94).
Method of preparation
Iodamide
Sodium Hydroxide
Meglumine
Water for Injection
627.9 g
6.0 g
I65.9g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Meglumine Sodium Iodamide Injection is a
clear, colorless to pale yellow, slightly viscous liquid.
It gradually changes in color by light.
Identification (1) To 2 mL of Meglumine Sodium Io-
damide Injection add 25 mL of water, and add 3 mL of dilute
hydrochloric acid with thorough stirring: a white precipitate
is formed. Filter the precipitate by suction through a glass
filter (G3), and wash with two 10-mL portions of water.
Transfer the precipitate to a suitable flask, add 100 mL of
water, dissolve by heating, and gently boil until the volume
becomes about 30 mL. After cooling, collect the separated
crystals by filtration, dry at 105°C for 1 hour, and proceed as
directed in the Identification (1) and (2) under Iodamide.
(2) Determine the infrared absorption spectrum of the d-
ried crystals obtained in (1) as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
3390 cm" 1 , 1369 cm" 1 , 1296 cm" 1 , 1210 cm" 1 and
1194 cm" 1 .
(3) To 1 mL of Meglumine Sodium Iodamide Injection
add 1 mL of potassium l,2-naphthoquinone-4-sulfonate TS
and 0.2 mL of sodium hydroxide TS: a deep red color is
produced.
(4) Meglumine Sodium Iodamide Injection responds to
the Qualitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49> c^ : -3.84- -4.42° (100 mm).
pH <2.54> 6.5-7.5
Purity (1) Primary aromatic amines — Mix 0.30 mL of
Meglumine Sodium Iodamide Injection and 6 mL of water,
then add 4 mL of a solution of sodium nitrite (1 in 100) and
10 mL of 1 mol/L hydrochloric acid TS, shake well, and pro-
ceed as directed in the Purity (2) under Iodamide: the absor-
bance is not more than 0.22.
(2) Iodine and iodide — To 0.40 mL of Meglumine Sodi-
um Iodamide Injection add water to make 20 mL, then add 5
mL of dilute nitric acid, shake well, filter by suction through
a glass filter (G3). To the filtrate add 5 mL of chloroform,
and shake vigorously: no color develops in the chloroform
layer. Then add 1 mL of a strong hydrogen peroxide solu-
tion, and shake vigorously: the chloroform layer has no more
color than the control solution.
Control solution: Dissolve 0.10 g of potassium iodide in
water to make 100 mL. To a 0. 10-mL portion of this solution
add 20 mL of water, 5 mL of dilute nitric acid, 5 mL of chlo-
roform and 1 mL of strong hydrogen peroxide solution, and
shake vigorously.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Dilute Meglumine Sodium Iodamide Injec-
tion with isotonic sodium chloride solution so as to contain
0.30 mL of Meglumine Sodium Iodamide Injection per mL
according to the labeled amount, and perform the test: it
meets the requirements.
Assay To an exactly measured 8 mL of Meglumine Sodium
Iodamide Injection add sodium hydroxide TS to make exact-
ly 100 mL, and use this solution as the sample solution. Pipet
10 mL of the sample solution into a saponification flask, add
30 mL of sodium hydroxide TS and 1 g of zinc powder, and
proceed as directed in the Assay under Iodamide.
Each mL of 0.1 mol/L silver nitrate VS
= 20.93 mg of C 12 H„I 3 N 2 04
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
•Hermetic containers,
JPXV
Official Monographs / Menatetrenone
855
Melphalan
t)\,7r=7>
C 13 H 18 C1 2 N 2 2 : 305.20
4-Bis(2-chloroethyl)amino-L-phenylalanine [148-82-3 ]
Melphalan contains not less than 93.0%
C 13 H 18 Cl2N 2 02, calculated on the dried basis.
of
Description Melphalan occurs as a white, to light yellowish
white, crystalline powder.
It is slightly soluble in water, in methanol and in ethanol
(95), and practically insoluble in diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute sodium
hydroxide TS.
It is gradually colored by light.
Optical rotation [a]g>: about - 32° (0.5 g, calculated on the
dried basis, methanol, 100 mL, 100 mm).
Identification (1) To 0.02 g of Melphalan add 50 mL of
methanol, dissolve by warming, add 1 mL of a solution of 4-
(4-nitrobenzyl)pyridine in acetone (1 in 20), and evaporate on
a water bath to dryness. Dissolve the residue in 1 mL of
warmed methanol and add 2 drops of ammonia solution (28):
a purple color develops.
(2) Dissolve 0.1 g of Melphalan in 10 mL of dilute sodi-
um hydroxide TS, and heat on a water bath for 10 minutes.
After cooling, add dilute nitric acid to acidify, and filter: the
filtrate responds to the Qualitative Tests <1.09> for chloride.
(3) Determine the absorption spectrum of a solution of
Melphalan in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and conpare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
Purity (1) Ionisable chloride — Weigh accurately about 0.5
g of Melphalan, dissolve in 80 mL of diluted nitric acid (1 in
40), stir for 2 minutes, and titrate <2.50> with 0.1 mol/L sil-
ver nitrate VS (potentiometric titration): the consumed
volume is not more than 1 .0 mL to 0.50 g of Melphalan.
(2) Heavy metals <1. 07>— Proceed with 1.0 g of Melpha-
lan according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Melphalan according to Method 3, and perform the test
(not more than 2 ppm).
Loss on drying <2.41> Not more than 7.0% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 105 °C, 2 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.25 g of Melphalan, add 20
mL of a solution of potassium hydroxide (1 in 5), and heat
under a reflux condenser on a water bath for 2 hours. After
cooling, add 75 mL of water and 5 mL of nitric acid, cool,
and titrate <2.50> with 0.1 mol/L silver nitrate VS (potentio-
metric titration). Make any necessary correction by using the
results obtained in the Purity (1).
Each mL of 0.1 mol/L silver nitrate VS
= 15.26 mg of C 13 H 18 C1 2 N 2 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Menatetrenone
^txhU/:
C 3I H 40 O 2 : 444.65
2-Methyl-3-[(2£,6£,10£)-3,7,ll,15-tetramethylhexadeca-
2,6,10, 14-tetraen-l-yl]-l,4-naphthoquinone [863-61-6]
Menatetrenone contains not less than 98.0% of
C 31 H 40 O2, calculated on the dehydrated basis.
Description Menatetrenone occurs as yellow, crystals, crys-
talline powder, waxy mass or oily material.
It is very soluble in hexane, soluble in ethanol (99.5), spar-
ingly soluble in 2-propanol, slightly soluble in methanol, and
practically insoluble in water.
It decomposes and the color becomes more intense by
light.
Melting point: about 37 °C
Identification (1) Dissolve 0.1 g of Menatetrenone in 5
mL of ethanol (99.5) by warming, cool, and add 1 mL of a
solution of potassium hydroxide in ethanol (95) (1 in 10): a
blue color develops, and upon standing it changes from blue-
purple to red-brown through red-purple.
(2) Determine the infrared absorption spectrum of
Menatetrenone, after melting by warming if necessary, as
directed in the liquid film method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Menatetrenone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Menatetrenone according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Menadione — To 0.20 g of Menatetrenone add 5 mL
of diluted ethanol (99.5) (1 in 2), shake well, and filter. To 0.5
mL of the filtrate add 1 drop of a solution of 3-methyl-l-
phenyl-5-pyrazorone in ethanol (99.5) (1 in 20) and 1 drop of
ammonia water (28), and allow to stand for 2 hours: no blue-
purple color develops.
(3) cis Isomer — Dissolve 0.10 g of Menatetrenone in 10
mL of hexane, and use this solution as the sample solution.
Pipet 1 mL of this solution, add hexane to make exactly 50
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
856
eW-Menthol / Official Monographs
JP XV
Chromatography <2.03>. Spot 10 fiL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
chromatogram with a mixture of hexane and dibutyl ether
(17:3) to a distance of about 12 cm, and air-dry the plate. Ex-
amine under ultraviolet light (main wavelength: 254 nm): the
spot corresponding to relative i?f value 1.1 regarding to the
principal spot from the sample solution is not more intense
than the spot from the standard solution.
(4) Related substances — Conduct this procedure without
exposure to daylight, using a light-resistant vessel. Dissolve
0.10 g of Menatetrenone in 100 mL of ethanol (99.5), and use
this solution as the sample solution. Pipet 1 mL of this solu-
tion, add ethanol (99.5) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 [iL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine each peak area of
these solutions by the automatic integration method: the total
area of peaks other than the peak of menatetrenone from the
sample solution is not larger than the peak area of menatetre-
none from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 6 times as long as the
retention time of menatetrenone beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add ethanol (99.5) to make exactly 50 mL.
Confirm that the peak area of menatetrenone obtained from
20 /xL of this solution is equivalent to 7 to 13% of that of
menatetrenone obtained from 20 /xL of the standard solu-
tion.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of menatetrenone is not more than 1.0%.
Water <2.48> Not more than 0.5% (0.5 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Conduct this procedure without exposure to day-
light, using a light-resistant vessel. Weigh accurately about
0.1 g each of Menatetrenone and Menatetrenone Reference
Standard (separately, determine the water <2.4S> in the same
manner as Menatetrenone), dissolve each in 50 mL of 2-
propanol, and add ethanol (99.5) to make exactly 100 mL.
Pipet 10 mL of these solutions, and add ethanol (99.5) to
make exactly 100 mL. Pipet 2 mL each of these solutions,
add exactly 4 mL each of the internal standard solution, and
use these solutions as the sample solution and standard solu-
tion. Perform the test with 20 jxL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of
menatetrenone to that of the internal standard.
Amount (mg) of C 31 H 40 O 2
= W s x (Q T /Q S )
W s : Amount (mg) of Menatetrenone Reference Standard,
calculated on the dehydrated basis
Internal standard solution — A solution of phytonadione in 2-
propanol (1 in 20,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 270 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Methanol
Flow rate: Adjust the flow rate so that the retention time of
menatetrenone is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, menatetrenone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of menatetrenone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
^//-Menthol
dl-/> I — JU
H OH
and enantiomer
C 10 H 2 „O: 156.27
(1RS,2SR, 5R 5')-5-Methyl-2-(l-methylethyl)cyclohexanol
[89-78-1]
cW-Menthol contains not less than 98.0%
CioH 20 0.
of
Description rf/-Menthol occurs as colorless crystals. It has a
characteristic and refreshing odor and a burning taste, fol-
lowed by a cool taste.
It is very soluble in ethanol (95) and in diethyl ether, and
very slightly soluble in water.
It sublimes gradually at room temperature.
Identification (1) Triturate c?/-Menthol with an equal
amount of camphor, chloral hydrate or thymol: the mixture
liquefies.
(2) Shake 1 g of dl-Menthol with 20 mL of sulfuric acid:
the mixture becomes turbid with a yellow-red color. Allow to
stand for 3 hours: a clear, oily layer possesses no aroma of
menthol is separated.
JPXV
Official Monographs / /-Menthol 857
Congealing point <2.42> 27 - 28 °C
Optical rotation <2.49> [a]„°: -2.0 ■
(95), 25 mL, 100 mm).
+ 2.0° (2.5 g, ethanol
Purity (1) Non-volatile residue — Volatilize 2.0 g of dl-
Menthol on a water bath, and dry the residue at 105°C for 2
hours: the residue weighs not more than 1.0 mg.
(2) Thymol— Add 0.20 g of dl-Menthol to a cold mixture
of 2 mL of acetic acid (100), 6 drops of sulfuric acid and 2
drops of nitric acid: no green to blue-green color immediately
develops.
(3) Nitromethane or nitroethane — To 0.5 g of c?/-Men-
thol placed in a flask add 2 mL of a solution of sodium
hydroxide (1 in 2) and 1 mL of strong hydrogen peroxide,
connect a reflux condenser to the flask, and boil the mixture
gently for 10 minutes. After cooling, add water to make ex-
actly 20 mL, and filter. Take 1 mL of the filtrate in a Nessler
tube, add water to make 10 mL, neutralize with dilute
hydrochloric acid, then add 1 mL of dilute hydrochloric acid,
and cool. To the mixture add 1 mL of a solution of sulfanilic
acid (1 in 100), allow to stand for 2 minutes, and then add 1
mL of a solution of iV,7V-diethyl-/V'-l-naphthylethylenedia-
mine oxalate (1 in 1000) and water to make 25 mL: no red-
purple color immediately develops.
Assay Weigh accurately about 2 g of (//-Menthol, add ex-
actly 20 mL of a mixture of dehydrated pyridine and acetic
anhydride (8:1), connect a reflux condenser, and heat on a
water bath for 2 hours. Wash down the condenser with 20
mL of water, and titrate <2.50> with 1 mol/L sodium
hydroxide VS (indicator: 5 drops of phenolphthalein TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 1 mol/L sodium hydroxide VS
= 156.3 mg of C 10 H 20 O
Containers and storage Containers — Tight containers.
Storage — In a cold place.
/-Menthol
l-/> V-)l
H OH
C 10 H 20 O: 156.27
( 1 R ,2 S, 5R )-5-Methyl-2-(l -methylethyl)cyclohexanol
[2216-51-5]
/-Menthol contains not less than 98.0% of C 10 H 20 O.
Description /-Menthol occurs as colorless crystals. It has a
characteristic and refreshing odor and a burning taste, fol-
lowed by a cool taste.
/-Menthol is very soluble in ethanol (95) and in diethyl
ether, and very slightly soluble in water.
/-Menthol sublimes gradually at room temperature.
Identification (1) Triturate /-Menthol with an equal
amount of camphor, chloral hydrate or thymol: the mixture
liquefies.
(2) Shake 1 g of /-Menthol with 20 mL of sulfuric acid:
the mixture becomes turbid with a yellow-red color. Allow to
stand for 3 hours: a clear, oily layer which possesses no aro-
ma of menthol is separated.
Optical rotation <2.49> [a]™:
ethanol (95), 25 mL, 100 mm).
Melting point <2.60> 42 - 44°C
-45.0-
■51.0° (2.5 g,
Purity (1) Non-volatile residue — Volatilize 2.0 g of /-
Menthol on a water bath, and dry the residue at 105°C for 2
hours: the residue weighs not more than 1.0 mg.
(2) Thymol— Add 0.20 g of /-Menthol to a cold mixture
of 2 mL of acetic acid (100), 6 drops of sulfuric acid and 2
drops of nitric acid: no green to blue-green color immediately
develops.
(3) Nitromethane or nitroethane — To 0.5 g of /-Menthol
placed in a flask add 2 mL of sodium hydroxide solution (1 in
2) and 1 mL of strong hydrogen peroxide, connect a reflux
condenser to the flask, and boil the mixture gently for 10
minutes. After cooling, add water to make exactly 20 mL,
and filter. Take 1 mL of the filtrate in a Nessler tube, add
water to make 10 mL, neutralize with dilute hydrochloric
acid, add another 1 mL of dilute hydrochloric acid, and cool.
To the mixture add 1 mL of a solution of sulfanilic acid (1 in
100), allow to stand for 2 minutes, and then add 1 mL of a
solution of TV.A'-diethyl-./V '-1-naphthylethylenediamine oxa-
late (1 in 1000) and water to make 25 mL: no red-purple color
immediately develops.
Assay Weigh accurately about 2 g of /-Menthol, add exactly
20 mL of a mixture of dehydrated pyridine and acetic anhy-
dride (8:1), connect a reflux condenser, and heat on a water
bath for 2 hours. Wash the condenser with 20 mL of water,
and titrate <2.50> with 1 mol/L sodium hydroxide VS (indica-
tor: 5 drops of phenolphthalein TS). Perform a blank deter-
mination and make any necessary correction.
Each mL of 1 mol/L sodium hydroxide VS
= 156.3 mg of C 10 H 20 O
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Mepenzolate Bromide
M>'/7-hM*
N-CH a
and enantfomer
C 21 H 26 BrN0 3 : 420.34
(3 RS)-3- [(Hydroxy)(diphenyl)acetoxy] -1,1-
dimethylpiperidinium bromide [76-90-4]
Mepenzolate Bromide, when dried, contains not less
than 98.5% of mepenzolate bromide (C2iH 2 6BrN0 3 ).
Description Mepenzolate Bromide is white to pale yellow
crystals or crystalline powder. It is odorless, and has a bitter
858
Mepitiostane / Official Monographs
JP XV
taste.
It is very soluble in formic acid, freely soluble in methanol,
soluble in hot water, slightly soluble in water and in ethanol
(95), very slightly soluble in acetic anhydride, and practically
insoluble in diethyl ether.
Melting point: about 230°C (with decomposition).
Identification (1) To 0.03 g of Mepenzolate Bromide add
10 drops of sulfuric acid: a red color develops.
(2) Dissolve 0.01 g of Mepenzolate Bromide in 20 mL of
water and 5 mL of dilute hydrochloric acid, and to 5 mL of
this solution add 1 mL of Dragendorff's TS: an orange
precipitate is produced.
(3) Determine the absorption spectrum of a solution of
Mepenzolate Bromide in 0.01 mol/L hydrochloric acid TS (1
in 2000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(4) Dissolve 0.5 g of Mepenzolate Bromide in 50 mL of
water and 3 mL of nitric acid by heating. This solution
responds to the Qualitative Tests <1.09> for Bromide.
Purity (1) Heavy Metals <1. 07>— Proceed with 1.0 g of
Mepenzolate Bromide according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not less than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Mepenzolate Bromide according to Method 3, and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.40 g of Mepenzolate
Bromide in exactly measured 10 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 200 mL, and use this
solution as the standard solution (1). Separately, dissolve 40
mg of benzophenone in methanol to make exactly 100 mL.
Pipet 2 mL of this solution, add methanol to make exactly 10
mL, and use this solution as the standard solution (2). Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution, standard solutions (1) and (2) on a plate of silica gel
with fluorecent indicator for thin-layer chromatography. De-
velop the plate with a mixture of 1-butanol, methanol, water
and acetic acid (100) (3:3:2:1) to a distance of about 10 cm,
and air-dry the plate and then at 80 °C for 30 minutes. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than either the principal spot or the spot cor-
responding to benzophenone from the sample solution are
not more intense than the spot from standard solution (1),
and the spot corresponding to benzophenone from the sam-
ple solution is not more intense than the spot from standard
solution (2). Spray evenly Dragendorff's TS on the plate: the
spots other than the principal spot from the sample solution
are not more intense than the spot from standard solution
(1).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Mepenzolate
Bromide, previously dried, dissolve in 2 mL of formic acid,
add 60 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 42.03 mg of C 21 H 26 BrN0 3
Containers and storage Containers — Tight containers.
Mepitiostane
/ ifttx9>
O CHj
C 25 H 40 O 2 S: 404.65
2a,3a-Epithio- 1 7/3-( 1 -methoxycyclopentyloxy)-5a-
androstane [21362-69-6]
Mepitiostane contains not less than 96.0% and not
more than 102.0% of C25H40O2S, calculated on the an-
hydrous basis.
Description Mepitiostane occurs as white to pale yellow
crystals or crystalline powder.
It is freely soluble in triethylamine, in chloroform, in
diethyl ether and in cyclohexane, soluble in diethylene glycol
dimethyl ether and in petroleum ether, sparingly soluble in
acetone, slightly soluble in methanol and in ethanol (99.5),
and practically insoluble in water.
It is hydrolyzed in moist air.
Identification (1) Dissolve 1 mg of Mepitiostane in 1 mL
of methanol, and add 0.5 mL of palladium (II) chloride TS:
an orange precipitate is formed. To this suspension add 1 mL
of water and 2 mL of chloroform, shake well, and allow to
stand: an orange color develops in the chloroform layer.
(2) Dissolve 0.1 g of Mepitiostane in 2 mL of diethylene
glycol dimethyl ether, shake with 1 mL of 1 mol/L
hydrochloric acid TS, and filter. To the filtrate add 1.5 mL of
2,4-dinitrophenylhydrazine-diethylene glycol dimethyl ether
TS and 1.5 mL of diluted ethanol (95) (2 in 3): an orange-yel-
low precipitate is formed. Filter the precipitate, recrystallize
from ethanol (99.5), and dry in a desiccator (in vacuum,
phosphorus (V) oxide) for 4 hours: the crystals melt <2.60>
between 144°C and 149°C.
(3) Determine the infrared absorption spectrum of
Mepitiostane as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™:
roform, 10 mL, 100 mm).
+ 20- +23° (0.1 g, chlo-
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Mepitiostane in 4 mL of petroleum ether: the solution is
clear and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
JPXV
Official Monographs / Mepivacaine Hydrochloride
859
Mepitiostane according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — Dissolve 20 mg of Mepitiostane
in exactly 5 mL of a mixture of acetone and triethylamine
(1000:1), and use this solution as the sample solution.
Separately, dissolve 10 mg of Epitiostanol Reference Stan-
dard in a mixture of acetone and triethylamine (1000:1) to
make exactly 10 mL. Pipet 1 mL and 3 mL of this solution,
to each add a mixture of acetone and triethylamine (1000:1)
to make exactly 25 mL, and use these solutions as the stan-
dard solution (1) and the standard solution (2), respectively.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 fiL each of the sample
solution and standard solutions (1) and (2) on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of hexane and acetone (3:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly diluted sulfuric acid (1 in 5) on the plate, heat between
120°C and 130°C for 5 minutes, and examine under ultravio-
let light (main wavelength: 365 nm): the spots other than the
principal spot from the sample solution showing the same Rf
value as the standard solutions are not more intense than the
spot from the standard solution (2), and the remaining spots
other than the principal spot are not more intense than the
spot from the standard solution (1).
Water <2.4S> Not more than 0.7% (0.3 g, back titration).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.3 g of Mepitiostane, and
dissolve in cyclohexane to make exactly 10 mL. Pipet 2 mL of
this solution, add 10 mL of ethanol (99.5), mix with exactly 2
mL each of 0.01 mol/L hydrochloric acid TS and the internal
standard solution, add ethanol (99.5) to make 20 mL, allow
to stand at ordinary temperature for 30 minutes, and use this
solution as the sample solution. Separately, weigh accurately
about 45 mg of Epitiostanol Reference Standard, dissolve in
exactly 2 mL of the internal standard solution, add ethanol
(99.5) to make 20 mL, and use this solution as the standard
solution. Perform the test with 10 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chroma-
tography <2.01> according to the following conditions, and
calculate the ratios, Q T and Q s , of the peak area of
epitiostanol to that of the internal standard, respectively.
Amount (mg) of C 25 H 4 o0 2 S = W s x (Q T /Q S ) X 5 x 1 .3202
W s : Amount (mg) of Epitiostanol Reference Standard,
calculated on the anhydrous basis
Internal standard solution — A solution of w-octylbenzene in
ethanol (99.5) (1 in 300).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (20:3).
Flow rate: Adjust the flow rate so that the retention time of
epitiostanol is about 6 minutes.
System suitability —
System performance: When the procedure is run with
10 fiL of the standard solution under the above operating
conditions, epitiostanol and the internal standard are eluted
in this order with the resolution between these peaks being
not less than 4.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of epitiostanol to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, under Nitrogen atmosphere, and
in a cold place.
Mepivacaine Hydrochloride
>tfM" ;/]•<>
and enantiomer
C 15 H 22 N 2 0.HC1: 282.81
(2i?S)-A f -(2,6-Dimethylphenyl)-l-methylpiperidine-2-
carboxamide monohydrochloride [1722-62-9]
Mepivacaine Hydrochloride, when dried, contains
not less than 98.5% of C 15 H 22 N 2 O.HCl.
Description Mepivacaine Hydrochloride occurs as white
crystals or crystalline powder.
It is freely soluble in water and in methanol, soluble in
acetic acid (100), sparingly soluble in ethanol (99.5), and
practically insoluble in diethyl ether.
A solution of Mepivacaine Hydrochloride (1 in 10) shows
no optical rotation.
Melting point: about 256°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Mepivacaine Hydrochloride (1 in 2500) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Mepivacaine Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Mepivacaine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 0.2 g of Mepivacaine Hydrochloride in
10 mL of water: the pH of this solution is between 4.0 and
5.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Mepivacaine Hydrochloride in 10 mL of water: the solution
is clear and colorless.
860
Mepivacaine Hydrochloride Injection / Official Monographs
JP XV
(2) Sulfate <1.14>— Perform the test with 0.5 g of
Mepivacaine Hydrochloride. Prepare the control solution
with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.038%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of
Mepivacaine Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(4) Related substances — Dissolve 0.10 g of Mepivacaine
Hydrochloride in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, and
add methanol to make exactly 20 mL. Pipet 4 mL of this so-
lution, add methanol to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of diethyl
ether, methanol and ammonia solution (28) (100:5:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly bis-
muth nitrate-potassium iodide TS on the plate: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 1.0% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Mepivacaine
Hydrochloride, previously dried, dissolve in 10 mL of acetic
acid (100) and add 70 mL of acetic anhydride. Titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 28.28 mg of C 15 H 22 N 2 0.HC1
Containers and storage Containers — Tight containers.
Mepivacaine Hydrochloride
Injection
Mepivacaine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of mepivacaine
hydrochloride (C 15 H 22 N 2 0.HC1: 282.81).
Method of preparation Prepare as directed under Injec-
tions, with Mepivacaine Hydrochloride.
Description Mepivacaine Hydrochloride
clear, colorless liquid.
pH: 4.5-6.8
Injection is a
Identification To a volume of Mepivacaine Hydrochloride
Injection, equivalent to 0.02 g of Mepivacaine Hydrochloride
according to the labeled amount, add 1 mL of sodium
hydrochloride TS, and extract with 20 mL of hexane. To 8
mL of the hexane extract add 20 mL of 1 mol/L hydrochloric
acid TS, shake vigorously, and determine the absorption
spectrum of the water layer separated as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 261 nm and 265 nm, and between 270 nm and
273 nm.
Extractable volume <6.05> It meets the requirement.
Assay To an exactly measured volume of Mepivacaine
Hydrochloride Injection, equivalent to about 40 mg of
Mepivacaine Hydrochloride according to the labeled
amount, add exactly 4 mL of the internal standard solution
and 0.001 mol/L hydrochloric acid TS to make 20 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 40 mg of mepivacaine hydrochloride for as-
say, previously dried at 105°C for 3 hours, dissolve in 0.001
mol/L hydrochloric acid TS, add exactly 4 mL of the internal
standard solution and 0.001 mol/L hydrochloride TS to
make 20 mL, and use this solution as the standard solution.
Perform the test with 5 [iL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of mepivacaine to that
of the internal standard.
Amount (mg) of C 15 H 22 N 2 O.HCl
= W s x(Q T /Q s )
W s : Amount (mg) of mepivacaine hydrochloride for assay
Internal standard solution — A solution of benzophenone in
methanol (1 in 4000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (10 /xm in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.88 g of sodium lauryl sulfate in
1000 mL of a mixture of 0.02 mol/L phosphate buffer solu-
tion, pH 3.0 and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
mepivacaine is about 6 minutes.
Selection of column: Proceed with 5 fiL of the standard so-
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of mepivacaine
and benzophenone in this order with the resolution between
these peaks being not less than 6.
Containers and storage Containers — Hermetic containers.
JPXV
Official Monographs / Mercaptopurine Hydrate
861
Mequitazine
,N„
01
and enantiomer
C 20 H 22 N 2 S: 322.47
10-[(3flS>l-Azabicyclo[2.2.2]oct-3-ylmethyl]-10//-
phenothiazine [29216-28-2]
Mequitazine, when dried, contains not less than
98.5% of C 20 H 22 N 2 S.
Description Mequitazine occurs as white crystals or crystal-
line powder.
It is freely soluble in methanol and in acetic acid (100),
soluble in ethanol (95), and practically insoluble in water.
It is gradually colored by light.
A solution of Mequitazine in methanol (1 in 50) shows no
optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Mequitazine in ethanol (95) (1 in 250,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Me-
quitazine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 146 - 150°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Mequitazine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve 0.05 g
of Mequitazine in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 50 mL, then pipet 5 mL of this so-
lution, add methanol to make exactly 50 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop with a mixture of ethyl
acetate, methanol and diethylamine (7:2:2) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the number of the spot
other than the principal spot from the sample solution is not
more than 3 and they are not more intense than the spot from
the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Mequitazine, dis-
solve in 50 mL of acetic acid (100), titrate <2.50> with 0.1 mol
/L perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.25 mg of C 20 H 22 N 2 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Mercaptopurine Hydrate
i.u
■ HjO
C 5 H 4 N 4 S.H 2 0: 170.19
1 ,7-Dihydro-6//-purine-6-thione monohydrate
[6112-76-1]
Mercaptopurine Hydrate contains not less than
98.0% of mercaptopurine (C 5 H 4 N 4 S: 152.18), calculat-
ed on the anhydrous basis.
Description Mercaptopurine Hydrate occurs as light yellow
to yellow crystals or crystalline powder. It is odorless.
It is practically insoluble in water, in acetone and in diethyl
ether.
It dissolves in sodium hydroxide TS and in ammonia TS.
Identification (1) Dissolve 0.6 g of Mercaptopurine Hy-
drate in 6 mL of sodium hydroxide solution (3 in 100), and
add slowly 0.5 mL of iodomethane with vigorous stirring.
Stir well for 10 minutes, cool in an ice bath, and adjust the
pH with acetic acid (31) to about 5. Collect the separated
crystals by filtration, recrystallize from water, and dry at
120 C C for 30 minutes: the crystals melt <2.60> between 218°C
and 222 °C (with decomposition).
(2) Determine the absorption spectrum of a solution of
Mercaptopurine Hydrate in 0.1 mol/L hydrochloric acid TS
(1 in 200,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
Purity (1) Clarity of solution — Dissolve 0.20 g of Mercap-
topurine Hydrate in 10 mL of ammonia TS: the solution is
clear.
(2) Sulfate <1.14>— Dissolve 0.05 g of Mercaptopurine
Hydrate in 10 mL of dilute hydrochloric acid, add 5 drops of
barium chloride TS, and allow to stand for 5 minutes: no tur-
bidity is produced.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Mercap-
topurine Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
862
Mercurochrome / Official Monographs
JP XV
(4) Hypoxanthine — Dissolve 50 mg of Mercaptopurine
Hydrate in exactly 10 mL of a solution of ammonia solution
(28) in methanol (1 in 10), and use this solution as the sample
solution. Separately, dissolve 5.0 mg of hypoxanthine in a so-
lution of ammonia solution (28) in methanol (1 in 10) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 fXL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of methanol, chloroform, n-
butyl formate and ammonia solution (28) (8:6:4:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spot from
the sample solution is not observed at the same place as that
from the standard solution, or if a spot is observed at the
same place, it is not larger than that from the standard solu-
tion.
(5) Phosphorus — Take 0.20 g of Mercaptopurine Hy-
drate in a crucible, add 2 mL of diluted sulfuric acid (3 in 7),
then heat gently, slowly adding dropwise several 0.5-mL por-
tions of nitric acid, until the liquid becomes colorless. Con-
tinue to heat until most of the liquid has evaporated, cool,
and dissolve the residue in 10 mL of water. Transfer the solu-
tion to a 25-mL volumetric flask, wash the crucible with two
4-mL portions of water, combine the washings with the solu-
tion in the volumetric flask, and use this solution as the sam-
ple solution. Separately, dissolve 0.4396 g of potassium di-
hydrogenphosphate in water to make exactly 200 mL. To 2.0
mL of this solution add water to make exactly 100 mL.
Transfer 2.0 mL of this solution to a 25-mL volumetric flask,
add 16 mL of water, and use this solution as the standard so-
lution. To the sample solution and the standard solution add
1 mL of diluted sulfuric acid (3 in 7), 0.5 mL of nitric acid,
0.75 mL of hexaammonium heptamolybdate TS, 1 mL of 1-
amino-2-naphthol-4-sulfonic acid TS and water to make 25
mL, and allow to stand for 5 minutes. Perform the test with
these solutions as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using water as the blank: the absor-
bance of the subsequent solution of the sample solution at
750 nm is not larger than that of the subsequent solution of
the standard solution.
Water <2.48> 10.0 - 12.0% (0.2 g, back titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Mercaptopurine
Hydrate, dissolve in 90 mL of 7V,iV-dimethylformamide, and
titrate <2.50> with 0.1 mol/L tetramethylammonium
hydroxide VS (potentiometric titration). Perform a blank de-
termination with a mixture of 90 mL of AfN-dimethylfor-
mamide and 15 mL of water, and make any necessary correc-
tion.
Each mL of 0.1 mol/L tetramethylammonium
hydroxide VS
= 15.22 mg of C5H4N4S
Containers and storage Containers — Well-closed contain-
ers.
Mercurochrome
Merbromin
"7— +3. p^nA
Mercurochrome is a sodium salt of a mixture of
brominated and mercurized fluoresceins.
When dried, it contains not less than 18.0% and not
more than 22.4% of bromine (Br: 79.90), and not less
than 22.4% and not more than 26.7% of mercury (Hg:
200.59).
Description Mercurochrome occurs as blue-green to green-
ish red-brown scales or granules. It is odorless.
It is freely soluble in water, but sometimes leaves a small
amount of insoluble matter. It is practically insoluble in
ethanol (95) and in diethyl ether.
Identification (1) A solution of Mercurochrome (1 in
2000) shows a red color and a yellow-green fluorescence.
(2) To 5 mL of a solution of Mercurochrome (1 in 250)
add 3 drops of dilute sulfuric acid: a reddish orange
precipitate is produced.
(3) Heat 0.1 g of Mercurochrome with small crystals of
iodine in a test tube: red crystals are sublimed on the upper
part of the tube. If yellow crystals are produced, scratch with
a glass rod: the color of the crystals changes to red.
(4) Place 0.1 g of Mercurochrome in a porcelain crucible,
add 1 mL of a solution of sodium hydroxide (1 in 6),
evaporate to dryness with stirring, and ignite. Dissolve the
residue in 5 mL of water, acidify with hydrochloric acid, and
shake with 3 drops of chlorine TS and 2 mL of chloroform: a
yellowish brown color develops in the chloroform layer.
Purity (1) Dyestuff — Dissolve 0.40 g of Mercurochrome
in 20 mL of water, add 3 mL of dilute sulfuric acid, and
filter: the filtrate has no more color than Matching Fluid C.
(2) Soluble halides — Dissolve 5.0 g of Mercurochrome in
80 mL of water, add 10 mL of dilute nitric acid and water to
make 100 mL, shake, and filter. Transfer 40 mL of the filtrate
to a Nessler tube, add 6 mL of dilute nitric acid and water to
make 50 mL, then add 1 mL of silver nitrate TS, mix well,
and allow to stand for 5 minutes protected from direct sun-
light: no turbidity is produced, or even if produced, it is not
more than that of the following control solution.
Control solution: To 0.25 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 50
mL, then add 1 mL of silver nitrate TS, and proceed as
directed above.
(3) Soluble mercury salts — To 5 mL of the filtrate ob-
tained in (1) add 5 mL of water, and use this solution as the
sample solution. Dissolve 40 mg of mercury (II) chloride,
weighed accurately, in water to make 1000 mL, and add 3 mL
of dilute sulfuric acid to 20 mL of this solution. To 5 mL of
the solution add 5 mL of water, and use this as the control so-
lution. Add 1 drop each of sodium sulfide TS to these solu-
tions, and compare: the sample solution has no more color
than the control solution.
(4) Insoluble mercury compounds — Dissolve 2.5 g of
Mercurochrome in 50 mL of water, allow to stand for 24
JP XV
Official Monographs / Meropenem Hydrate
863
hours, centrifuge, and wash the precipitate with small por-
tions of water until the last washing becomes colorless.
Transfer the precipitate to a glass-stoppered flask, add exact-
ly 5 mL of 0.05 mol/L iodine VS, allow to stand for 1 hour
with frequent agitation, add 4.3 mL of 0.1 mol/L sodium
thiosulfate VS dropwise with shaking, and add 1 mL of
starch TS: a blue color develops.
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C,
5 hours).
Assay (1) Mercury — Weigh accurately about 0.6 g of
Mercurochrome, previously powdered and dried, transfer to
an iodine flask, dissolve in 50 mL of water, add 8 mL of acet-
ic acid (31), 20 mL of chloroform and exactly 30 mL of 0.05
mol/L iodine VS, stopper tightly, and allow to stand for 1
hour with frequent, vigorous shaking. Titrate <2.50> the ex-
cess iodine with 0.1 mol/L sodium thiosulfate VS with
vigorous shaking (indicator: 1 mL of starch TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L iodine VS = 10.03 mg of Hg
(2) Bromine — Weigh accurately about 0.5 g of Mer-
curochrome, previously powdered and dried, in a porcelain
crucible, add 2 g of potassium nitrate, 3 g of potassium car-
bonate and 3 g of anhydrous sodium carbonate, mix well,
cover the surface of the mixture with 3 g of a mixture of equal
amounts of potassium carbonate and anhydrous sodium car-
bonate, and ignite almost to fusion. Cool, dissolve the ignited
mixture in 80 mL of warm water, acidify with nitric acid, and
add exactly 25 mL of 0.1 mol/L silver nitrate VS. Shake well,
and titrate <2.50> the excess silver nitrate with 0.1 mol/L am-
monium thiocyanate VS (indicator: 2 mL of ammonium iron
(III) sulfate TS). Perform a blank determination and make
any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS = 7.990 mg of Br
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Mercurochrome Solution
Merbromin Solution
Mercurochrome Solution contains not less than 0.42
w/v% and not more than 0.56 w/v% of mercury (Hg:
200.59).
Method of preparation
Mercurochrome
Purified Water
20 g
a sufficient quantity
To make 1000 mL
Prepare by mixing the above ingredients.
Description Mercurochrome Solution is a dark red liquid.
Identification (1) To 1 mL of Mercurochrome Solution
add 40 mL of water: the resulting solution shows a red color
and a yellow-green fluorescence.
(2) Dilute 1 mL of Mercurochrome Solution with 4 mL
of water, and add 3 drops of dilute sulfuric acid: a red-orange
precipitate is produced.
(3) Evaporate 5 mL of Mercurochrome Solution to dry-
ness, and proceed with the residue as directed in the Identifi-
cation (3) under Mercurochrome.
(4) To 5 mL of Mercurochrome Solution add 1 mL of a
solution of sodium hydroxide (1 in 6), and proceed as direct-
ed in the Identification (4) under Mercurochrome.
Purity Dyestuff — To 20 mL of Mercurochrome Solution
add 3 mL of dilute sulfuric acid, and filter: the filtrate has no
more color than Matching Fluid C.
Assay Transfer exactly measured 30 mL of Mercurochrome
Solution to an iodine flask, dilute with 20 mL of water, add 8
mL of acetic acid (31) and 20 mL of chloroform, and proceed
as directed in the Assay (1) under Mercurochrome.
Each mL of 0.05 mol/L iodine VS= 10.03 mg of Hg
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Meropenem Hydrate
,CH 3
NH CH 3
HO
HaG H H * CH-,
n
■3H f O
C 17 H 25 N 3 5 S.3H 2 0: 437.51
(4i?,5S,6S)-3-[(3S,5S)-5-(Dimethylcarbamoyl)pyrrolidin-
3-ylsulf anyl]-6- [(IR)- 1 -hydroxyethyl] -4-methyl-7-oxo- 1 -
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
trihydrate [119478-56-7]
Meropenem Hydrate contains not less than 980 fig
(potency) and not more than 1010 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Meropenem Hydrate is expressed as mass (potency) of
meropenem (CnH^NjOjS: 383.46).
Description Meropenem Hydrate occurs as a white to light
yellow crystalline powder.
It is sparingly soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
Identification (1) Dissolve 0.01 g of Meropenem Hydrate
in 2 mL of water, add 3 mL of hydroxylammonium chloride-
ethanol TS, allow to stand for 5 minutes, add 1 mL of acidic
ammonium iron (III) sulfate TS, and shake: a red-brown
color develops.
(2) Determine the absorption spectra of solutions of
Meropenem Hydrate and Meropenem Reference Standard (3
in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectra: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectra of
Meropenem Hydrate and Meropenem Reference Standard as
directed in the potassium bromide disk method under In-
864
Mestranol / Official Monographs
JP XV
frared Spectrophotometry <2.25>, and compare the spectra:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Optical rotation <2.49> [alp : - 17 - -21° (0.22 g calculat-
ed as the anhydrous basis, water, 50 mL, 100 mm).
pH <2.54> Dissolve 0.2 g of Meropenem Hydrate in 20 mL
of water: the pH of the solution is between 4.0 and 6.0.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Meropenem Hydrate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Related substances — Being specified separately.
Water <2.4S> Not less than 11.4% and not more than
13.4% (0.35 g, volumetric titration, direct titration).
Residue on ignition <2.44> Being specified separately.
Bacterial endotoxins <4.01> Less than 0.12EU/mg (poten-
cy).
Assay Weigh accurately an amount of Meropenem Hydrate
and Meropenem Reference Standard, equivalent to about 50
mg (potency), add exactly 10 mL of the internal standard so-
lution to dissolve, add triethylamine-phosphate buffer solu-
tion, pH 5.0 to make 100 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 5 /xL of the sample solution and the standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of meropenem to that of the in-
ternal standard.
Amount [/xg (potency)] of meropenem (Q7H25N3O5S)
= W s x(Q J /Q s )x 1000
W s : Amount [mg (potency)] of Meropenem Reference
Standard
Internal standard solution — A solution of benzyl alcohol in
triethylamine-phosphate buffer solution, pH 5.0 (1 in 300).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 6.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of triethylamine-phosphate
buffer solution, pH 5.0 and methanol (5:1).
Flow rate: Adjust the flow rate so that the retention time of
meropenem is about 7 minutes.
System suitability —
System performance: When the procedure is run with 5 juL
of the standard solution under the above operating condi-
tions, meropenem and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 20.
System repeatability: When the test is repeated 5 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of meropenem to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Tight containers.
Mestranol
/x v^y -ii
=CH
C 21 H 26 2 : 310.43
3-Methoxy-19-nor-17a-pregna-l,3,5(10)-trien-20-yn-17-ol
[72-33-3]
Mestranol, when dried, contains not less than 97.0%
and not more than 102.0% of C 2 iH 2 60 2 .
Description Mestranol occurs as a white to pale yellowish
white, crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in 1,4-dioxane,
sparingly soluble in ethanol (99.5) and in diethyl ether, and
practically insoluble in water.
Identification (1) Dissolve 2 mg of Mestranol in 1 mL of a
mixture of sulfuric acid and ethanol (99.5) (2:1): a red-purple
color develops with a yellow-green fluorescence.
(2) Determine the absorption spectrum of a solution of
Mestranol in ethanol (99.5) (1 in 10,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Mestranol Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Mes-
tranol, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of previously dried Mestranol Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
Optical rotation <2.49> [a]™: +2- +8° (after drying, 0.2
g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 148 - 154 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Mestranol according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Mestranol according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Mestranol in
20 mL of chloroform, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add chloroform to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
JPXV
Official Monographs / Metenolone Acetate
865
gel for thin-layer chromatography. Develop the plate with a
mixture of chloroform and ethanol (99.5) (29:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly diluted
sulfuric acid (1 in 5) on the plate, and heat at 105 °C for 15
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, 105 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 10 mg each of Mestranol
and Mestranol Reference Standard, previously dried, dis-
solve in ethanol (99.5) to make exactly 100 mL, and use these
solutions as the sample solution and the standard solution,
respectively. Determine the absorbances, A T and A s , of the
sample solution and the standard solution at 279 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C2iH 2 60 2
= W s x(A T /A s )
W s : Amount (mg) of Mestranol Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Metenolone Acetate
O CH a
C 22 H 32 3 : 344.49
l-Methyl-3-oxo-5a-androst-l-en-17/?-yl acetate
[434-05-9]
Metenolone Acetate, when dried, contains not less
than 97.0% and not more than 103.0%, of C 22 H3 2 3 .
Description Metenolone Acetate occurs as a white to pale
yellowish white, crystalline powder. It is odorless.
It is freely soluble in acetone, in 1,4-dioxane and in chlo-
roform, soluble in ethanol (95) and in methanol, sparingly
soluble in diethyl ether and in sesame oil, slightly soluble in
hexane and in petroleum ether, and practically insoluble in
water.
Identification (1) Dissolve 1 mg of Metenolone Acetate in
5 mL of a mixture of ethanol (95) and sulfuric acid (1:1), and
heat for 30 minutes in a water bath: a red-brown color de-
velops.
(2) To 0.01 g of Metenolone Acetate add 0.5 mL of dilute
sodium hydroxide-ethanol TS, and heat for 1 minute on a
water bath. After cooling, add 0.5 mL of diluted sulfuric acid
(1 in 2), and boil gently for 1 minute: the odor of ethyl acetate
is perceptible.
(3) Dissolve 0.05 g of Metenolone Acetate in 3 mL of
methanol, add 0.3 mL of a solution of potassium carbonate
(1 in 6), and boil for 2 hours under a reflux condenser. After
cooling, add this solution gradually to 50 mL of cold water,
and stir for 15 minutes. Filter the precipitate so obtained by
suction through a glass filter (G4), wash with 10 mL of water,
and dry at 105°C for 1 hour: it melts <2.60> between 157°C
and 161 °C.
(4) Determine the infrared absorption spectrum of
Metenolone Acetate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a] 2 ^: +39 - +42° (after drying, 0.2
g, chloroform, 10 mL, 100 mm).
Melting point <2.60> 141 - 144°C
Purity (1) Clarity and color of solution — Dissolve 0.50 g
of Metenolone Acetate in 10 mL of 1,4-dioxane: the solution
is clear and colorless to pale yellow.
(2) Heavy metals <1.07>— Proceed with 2.0 g of Meteno-
lone Acetate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Dissolve 35 mg of Metenolone
Acetate in 20 mL of chloroform, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, dilute
with chloroform to exactly 250 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of cyclo-
hexane and ethyl acetate (1:1) to a distance of about 12 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 0.5% (0.5 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 10 mg of Metenolone
Acetate, previously dried, and dissolve in methanol to make
exactly 100 mL. Pipet 5 mL of this solution, and dilute with
methanol to exactly 50 mL. Determine the absorbance A of
this solution at the wavelength of maximum absorption at
about 242 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of C 22 H 32 3 =(.4/391) x 10,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
866
Metenolone Enanthate / Official Monographs
JP XV
Metenolone Enanthate
C27H42O3: 414.62
1 -Methyl-3-oxo-5a-androst- 1 -en- 1 7/?-yl heptanoate
[303-42-4]
Metenolone Enanthate, when dried, contains not
less than 97.0% and not more than 103.0% of
C27H42O3.
Description Metenolone Enanthate occurs as white crystals
or crystalline powder. It is odorless.
It is very soluble in ethanol (95), in acetone, in 1,4-dioxane
and in chloroform, freely soluble in methanol, in ethyl
acetate, in diethyl ether, in cyclohexane, in petroleum ether
and in toluene, soluble in sesame oil, and practically insolu-
ble in water.
Identification (1) Heat 1 mg of Metenolone Enanthate
with 5 mL of a mixture of ethanol (95) and sulfuric acid (1:1)
on a water bath for 30 minutes: a red-brown color develops.
(2) Dissolve 0.05 g of Metenolone Enanthate in 3 mL of
methanol, add 0.3 mL of a solution of potassium carbonate
(1 in 6), boil under a reflux condenser for 2 hours, cool, add
slowly this solution to 50 mL of cold water, and stir for 15
minutes. Filter the produced precipitate by suction through a
glass filter (G4), wash with water until the washings become
neutral, and dry at 105°C for 1 hour: it melts <2.60> between
156°C and 162°C.
+ 43° (after drying, 0.2
Optical rotation <2.49> [a]^ : +39
g, chloroform, 10 mL, 100 mm).
Melting point <2.60> 67 - 72°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Metenolone Enanthate in 10 mL of 1,4-dioxane: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Meteno-
lone Enanthate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Dissolve 20 mg of Metenolone
Enanthate in exactly 10 mL of chloroform, and use this solu-
tion as the sample solution. Perform the test with the sample
solution as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL of the sample solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of cyclohexane and ethyl
acetate (1:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): any spot other than the principal spot does not appear.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.1 g of Metenolone Enan-
thate, previously dried, and dissolve in methanol to make ex-
actly 100 mL. Pipet 10 mL of this solution, and dilute with
methanol to make exactly 100 mL. Pipet 10 mL of this solu-
tion, and dilute again with methanol to make exactly 100 mL.
Determine the absorbance A of this solution at the
wavelength of maximum absorption at about 242 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 27 H 42 03 =(.4/325) x 100,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Metenolone Enanthate Injection
Metenolone Enanthate Injection is an oily solution
for injection.
It contains not less than 90% and not more than
110% of the labeled amount of metenolone enanthate
(C 27 H 42 3 : 414.62).
Method of preparation Prepare as directed under Injec-
tions, with Metenolone Enanthate.
Description Metenolone Enanthate Injection is a clear, pale
yellow, oily liquid.
Identification (1) Measure a volume of Metenolone Enan-
thate Injection, equivalent to 0.1 g of Metenolone Enanthate
according to the labeled amount, add 20 mL of petroleum
ether, and extract with three 20-mL portions of diluted acetic
acid (100) (5 in 7). Combine the extracts, wash with 20 mL of
petroleum ether, add 300 mL of cold water while cooling in
an ice bath, and stir sufficiently. Filter the produced
precipitate by suction through a glass filter (G4), wash with
water until the last washing becomes neutral, and dry in a
desiccator (in vacuum, phosphorus (V) oxide) for 6 hours.
With this sample, proceed as directed in the Identification (1)
under Metenolone Enanthate.
(2) Measure a volume of Metenolone Enanthate Injec-
tion, equivalent to 0.01 g of Metenolone Enanthate accord-
ing to the labeled amount, dissolve in 10 mL of chloroform,
and use this solution as the sample solution. Separately dis-
solve 0.01 g of metenolone enanthate in 10 mL of chlo-
roform, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with toluene to a distance of about 15 cm, and air-
dry the plate. Again develop this plate with a mixture of cy-
clohexane and ethyl acetate (1:1) to a distance of about 15
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the principal spot from the sam-
ple solution and the spot from the standard solution show the
same Rf value.
Extractable volume <6.05> It meets the requirement.
JPXV
Official Monographs / Metformin Hydrochloride Tablets
867
Assay To an exactly measured volume of Metenolone
Enanthate Injection, equivalent to about 0.1 g of metenolone
enanthate (C27H42O3), add chloroform to make exactly 100
mL. Pipet 5 mL of this solution, add chloroform to make ex-
actly 50 mL, and use this solution as the sample solution.
Weigh accurately about 0. 1 g of metenolone enanthate for as-
say, previously dried in a desiccator (in vacuum, phosphorus
(V) oxide) for 4 hours, and prepare the standard solution in
the same manner as directed for the preparation of the sam-
ple solution. Pipet 3 mL each of the sample solution and
standard solution, and treat each solution as follows: add 10
mL of isoniazid TS, exactly measured, add methanol to make
exactly 20 mL, and allow to stand for 60 minutes. Determine
the absorbances, A T and A s , of the solutions from the sample
solution and standard solution, respectively, at 384 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution obtained by proceeding with 3 mL of chlo-
roform as the blank.
Amount (mg) of metenolone enanthate (C27H42O3)
= W s x(A T /A s )
W s : Amount (mg) of metenolone enanthate for assay
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Metformin Hydrochloride
* r**JU$>£&J£
NH NH
A X
,CH 3
HCI
CHa
QHnNj.HCl: 165.62
1 , 1-Dimethylbiguanide monohydrochloride
[1115-70-4]
Metformin Hydrochloride, when dried, contains not
less than 98.5% and not more than 101.0% of
C 4 H U N5.HC1.
Description Metformin Hydrochloride occurs as white crys-
tals or crystalline powder.
It is freely soluble in water, sparingly soluble in acetic acid
(100), and slightly soluble in ethanol (99.5).
Melting point: about 221 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Metformin Hydrochloride (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Met-
formin Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) A solution of Metformin Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Metformin Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 2.5 g of Metformin
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
water to make exactly 50 mL. Pipet 1 mL of this solution,
add water to make exactly 10 mL, and use this solution as the
standard solution (1). Pipet 5 mL of the standard solution
(1), add water to make exactly 10 mL, and use this solution as
the standard solution (2). Separately, to 0.10 g of 1-
cyanoguanidine add water to make exactly 50 mL. Pipet 1
mL of this solution, add water to make exactly 20 mL, and
use this solution as the standard solution (3). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solutions (1), (2) and (3) on a plate of cellulose
for thin-layer chromatography. Develop the plate with a mix-
ture of 4-methyl-2-pentanone, 2-methoxyethanol, water and
acetic acid (100) (30:20:5:3) to a distance of about 10 cm, air-
dry the plate, then dry at 105°C for 10 minutes. Spray evenly
sodium pentacyanonitrosylferrate (Ill)-potassium hexac-
yanoferrate (III) TS on the plate: the spot other than the prin-
cipal spot is not more intense than the spot with the standard
solution (1), the number of them showing more intense than
the spot with the standard solution (2) is not more than two,
and the spot appeared at the position corresponding to the
spot with the standard solution (3) is not more intense than
the spot with the standard solution (3).
Loss on drying <2.41>
hours).
Not more than 0.5% (1 g, 105 °C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of Metformin
Hydrochloride, previously dried, dissolve in 40 mL of acetic
acid (100), add 40 mL of acetic anhydride, and titrate <2.50>
with 0.05 mol/L perchloric acid VS (potentiometric titra-
tion). Perform a blank determination in the same manner,
and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 4.141 mg of QHnNj.HCl
Containers and storage Containers — Tight containers.
Metformin Hydrochloride Tablets
Metformin Hydrochloride Tablets contain not less
than 95.0% and not more than 105.0% of the labeled
amount of metformin hydrochloride (C 4 H n N 5 .HCl:
165.62).
Method of preparation Prepare as directed under Tablets,
with Metformin Hydrochloride.
Identification Shake an amount of powdered Metformin
Hydrochloride Tablets, equivalent to 250 mg of Metformin
Hydrochloride according to the labeled amount, with 25 mL
of 2-propanol, and filter. Evaporate the filtrate under
reduced pressure in a water bath at 40°C, and determine the
infrared absorption spectrum of the residue as directed in the
868
Methamphetamine Hydrochloride / Official Monographs
JP XV
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 3370 cm" 1 , 3160 cm" 1 , 1627 cm" 1 , 1569
cm" 1 and 1419 cm" 1 .
Uniformity of dosage unit <6.02> It meets the requirement
of the Mass variation test.
Dissolution Being specified separately.
Assay Weigh accurately the mass of not less than 20 Met-
formin Hydrochloride Tablets, and powder. Weigh accurate-
ly a portion of the powder, equivalent to about 0.15 g of met-
formin hydrochloride (C4H11N5.HCI), add 70 mL of a mix-
ture of water and acetonitrile (3:2), shake for 10 minutes, add
the mixture of water and acetonitrile (3:2) to make exactly
100 mL, and filter through a membrane filter with a pore size
of not more than 0.45 //m. Discard the first 10 mL of the
filtrate, pipet 3 mL of the subsequent filtrate, add exactly 3
mL of the internal standard solution and the mixture of water
and acetonitrile (3:2) to make exactly 50 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 0.15 g of metformin hydrochloride for assay, previous-
ly dried at 105 °C for 3 hours, and dissolve in the mixture of
water and acetonitrile (3:2) to make exactly 100 mL. Pipet 3
mL of this solution, add exactly 3 mL of the internal stan-
dard solution and the mixture of water and acetonitrile (3:2)
to make 50 mL, and use this solution as the standard solu-
tion. Perform the test with 5 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, g T and Q s , of the peak area of met-
formin to that of the internal standard.
Amount (mg) of metformin hydrochloride (C 4 H U N 5 .HC1)
= ^ s x(g T /e s )
W s : Amount (mg) of metformin hydrochloride for assay
Internal standard solution — Dissolve 0.3 g of isobutyl para-
hydroxybenzoate in 100 mL of the mixture of water and
acetonitrile (3:2).
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 0.8 g of sodium lauryl sulfate in
620 mL of diluted phosphoric acid (1 in 2500), and add 380
mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
metformin is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, metformin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of metformin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Methamphetamine Hydrochloride
CH a • HCI
C 10 H 15 N.HC1: 185.69
(2 <S)--/V-Methyl- 1 -phenylpropan-2-amine
monohydrochloride [57-57-0]
Methamphetamine Hydrochloride, when dried, con-
tains not less than 98.5% of C 10 H 15 N.HC1.
Description Methamphetamine Hydrochloride occurs as
colorless crystals or a white, crystalline powder. It is odor-
less.
It is freely soluble in water, in ethanol (95) and in chlo-
roform, and practically insoluble in diethyl ether.
The pH of a solution of Methamphetamine Hydrochloride
(1 in 10) is between 5.0 and 6.0.
Identification (1) To 5 mL of a solution of Methampheta-
mine Hydrochloride (1 in 100) add 0.5 mL of hydrogen hex-
achloroplatinate (IV) TS: an orange-yellow, crystalline
precipitate is produced.
(2) To 5 mL of a solution of Methamphetamine
Hydrochloride (1 in 100) add 0.5 mL of iodine TS: a brown
precipitate is produced.
(3) To 5 mL of a solution of Methamphetamine
Hydrochloride (1 in 100) add 0.5 mL of 2,4,6-trinitrophenol
TS: a yellow, crystalline precipitate is produced.
(4) A solution of Methamphetamine Hydrochloride (1 in
20) responds to the Qualitative Tests <1.09> for chloride.
Optical rotation <2.49> [a]™: + 16 - + 19° (after drying, 0.2
g, water, 10 mL, 100 mm).
Melting point <2.60> 171 - 175°C
Purity (1) Acidity or alkalinity — Dissolve 2.0 g of
Methamphetamine Hydrochloride in 40 mL of freshly boiled
and cooled water, add 2 drops of methyl red TS, and use this
solution as the sample solution.
(i) To 20 mL of the sample solution add 0.20 mL of 0.01
mol/L sulfuric acid VS: a red color develops.
(ii) To 20 mL of the sample solution add 0.20 mL of 0.02
mol/L sodium hydroxide VS: a yellow color develops.
(2) Sulfate <1.14> — Dissolve 0.05 g of Methamphetamine
Hydrochloride in 40 mL of water, add 1 mL of dilute
hydrochloric acid and 1 mL of barium chloride TS, and allow
to stand for 10 minutes: the solution remains unchanged.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Methamphetamine
Hydrochloride, previously dried, and dissolve in 50 mL of a
JPXV
Official Monographs / Methotrexate
869
mixture of acetic anhydride and acetic acid (100) (7:3). Ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (potentiomet-
ric titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.57 mg of C 10 H 15 N.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
L-Methionine
C 5 H„N0 2 S: 149.21
(2S)-2-Amino-4-(methylsulfanyi)butanoic acid
[63-68-3]
L-Methionine, when dried, contains not less than
98.5% of C 5 H„N0 2 S.
Description L-Methionine occurs as white crystals or crys-
talline powder. It has a characteristic odor.
It is freely soluble in formic acid, soluble in water, and very
slightly soluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Methionine, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Optical rotation <2.49> [a]o- +21.0 -+25.0° (after
drying, 0.5 g, 6 mol/L hydrochloric acid TS, 25 mL, 100
mm).
pH <2.54> Dissolve 0.5 g of L-Methionine in 20 mL of
water: the pH of this solution is between 5.2 and 6.2.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
L-Methionine in 20 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Dissolve 0.5 g of L-Methionine in 20
mL of water, and add 6 mL of dilute nitric acid and water to
make 40 mL. Perform the test using this solution as the test
solution.
Prepare the control solution with 0.30 mL of 0.01 mol/L
hydrochloric acid VS, 6 mL of dilute nitric acid and water to
make 40 mL. In this test, to the test solution and the control
solution add 10 mL each of silver nitrate TS (not more than
0.021%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of L-
Methionine. Prepare the control solution with 0.35 mL of
0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Methionine. Prepare the control solution with 5.0 mL of
Standard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Dissolve 1.0 g of L-Methionine
in 40 mL of water and 2 mL of dilute acetic acid, dissolve by
warming, cool, and add water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 2 mL of dilute acetic acid and water to make 50 mL (not
more than 20 ppm).
(6) Arsenic <1.11> — Transfer 1.0 g of L-Methionine to a
100-mL decomposition flask, add 5 mL of nitric acid and 2
mL of sulfuric acid, put a small funnel on the mouth of the
flask, and heat carefully until white fumes are evolved. After
cooling, add two 2-mL portions of nitric acid, heat, add
2-mL portions of hydrogen peroxide (30) several times, and
heat until the solution becomes colorless or pale yellow. After
cooling, add 2 mL of saturated ammonium oxalate monohy-
drate solution, and heat again until white fumes are evolved.
After cooling, add water to make 5 mL, and perform the test
with this solution as the test solution (not more than 2 ppm).
(7) Related substances — Dissolve 0.10 g of L-Methionine
in 10 mL of water, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add water to
make exactly 50 mL. Pipet 5 mL of this solution, add water
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. After air-drying, immedi-
ately develop the plate with a mixture of 1-butanol, water and
acetic acid (100) (3:1:1) to a distance of about 10 cm, and dry
the plate at 80°C for 30 minutes. Spray evenly a solution of
ninhydrin in acetone (1 in 50) on the plate, and heat at 80°C
for 5 minutes: the spots other than the principal spot from
the sample solution are not more intense than the spot from
the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.15 g of L-Methionine,
previously dried, and dissolve in 3 mL of formic acid, add 50
mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 14.92 mg of C 5 H„N0 2 S
Containers and storage Containers — Tight containers.
Methotrexate
9 H CO,H
CO;H
C 20 H 22 N 8 O 5 : 454.44
N- {4-[(2,4-Diaminopteridin-
6-ylmethyl)(methyl)amino]benzoylj -L-glutamic acid
[59-05-2]
Methotrexate is a mixture of 4-amino-10-methylfolic
870
Methoxsalen / Official Monographs
JP XV
acid and closely related compounds.
It contains not less than 94.0% and not more than
102.0% of C 2 oH 2 2N 8 5 , calculated on the anhydrous
basis.
Description Methotrexate occurs as a yellow-brown, crys-
talline powder.
It is slightly soluble in pyridine, and practically insoluble in
water, in acetonitrile, in ethanol (95) and in diethyl ether.
It dissolves in dilute sodium hydroxide TS and in dilute so-
dium carbonate TS.
It is gradually affected by light.
Identification (1) Dissolve 1 mg of Methotrexate in 100
mL of 0.1 mol/L hydrochloric acid TS. Determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Methotrexate Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Methotrexate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Methotrexate Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
Water <2.48> Take 5 mL of pyridine for water determina-
tion and 20 mL of methanol for Karl Fischer method in a
dried titration flask, and titrate with water determination TS
until the end point. Weigh accurately about 0.2 g of
Methotrexate, immediately place in the titration flask, and
add a known excess volume of Karl Fischer TS. Mix well for
30 minutes, and perform the test: the water content is not
more than 12.0%.
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 25 mg each of Methotrexate
and Methotrexate Reference Standard, dissolve in the mobile
phase to make exactly 250 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
exactly 10 /uL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and measure the peak areas, A T and A s , of meth-
otrexate in each solution.
Amount (mg) of C 20 H 22 N 8 O 5 = W s x (A T /A S )
W s : Amount (mg) of Methotrexate Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 302 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of disodium hydrogen phos-
phate-citric acid buffer solution, pH 6.0 and acetonitrile
(89:11).
Flow rate: Adjust the flow rate so that the retention time of
methotrexate is about 8 minutes.
System suitability —
System performance: Dissolve 10 mg each of Methotrexate
and folic acid in 100 mL of the mobile phase. When the
procedure is run with 10 /xL of this solution under the above
operating conditions, folic acid and methotrexate are eluted
in this order with the resolution between these peaks being
not less than 8.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
methotrexate is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Methoxsalen
/ r-++fU>
C 12 H 8 4 : 216.19
9-Methoxy-7//-furo[3,2-g]chromen-7-one
[298-81-7]
Methoxsalen contains not less than 98.0% and not
more than 102.0% of C 12 H 8 4 , calculated on the anhy-
drous basis.
Description Methoxsalen occurs as white to pale yellow
crystals or crystalline powder. It is odorless and tasteless.
It is freely soluble in chloroform, slightly soluble in
methanol, in ethanol (95) and in diethyl ether, and practically
insoluble in water.
Identification (1) To 0.01 g of Methoxsalen add 5 mL of
dilute nitric acid, and heat: a yellow color develops. Make
this solution alkaline with a solution of sodium hydroxide (2
in 5): the color changes to red-brown.
(2) To 0.01 g of Methoxsalen add 5 mL of sulfuric acid,
and shake: a yellow color develops.
(3) Determine the absorption spectrum of a solution of
Methoxsalen in ethanol (95) (I in 200,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Methoxsalen Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Melting point <2.60> 145 - 149 C C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Methoxsalen according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Methoxsalen according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Methoxsalen
in 10 mL of chloroform, and use this solution as the sample
JPXV
Official Monographs / Methylcellulose 871
solution. Pipet 2 mL of the sample solution, add chloroform
to make exactly 50 mL. Pipet 1 mL of this solution, add chlo-
roform to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of chlo-
roform, hexane and ethyl acetate (40:10:3) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Water <2.48> Not more than 0.5% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Methoxsalen
and Methoxsalen Reference Standard, and dissolve each in
ethanol (95) to make exactly 100 mL. Pipet 2 mL each of
these solutions, and dilute each with ethanol (95) to make ex-
actly 25 mL. Pipet 10 mL each of these solutions, and dilute
each again with ethanol (95) to make exactly 50 mL, and use
these solutions as the sample solution and the standard solu-
tion, respectively. Determine the absorbances, A T and^4 s , of
the sample solution and the standard solution at 300 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C I2 H 8 4
= W s x(A T /A s )
W s : Amount (mg) of Methoxsalen Reference Standard,
calculated on the anhydrous basis
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Methylbenactyzium Bromide
CH 3 Br
C 21 H 28 BrN0 3 : 422.36
A^,A^-Diethyl-2-[(hydroxyl)(diphenyl)acetoxy]-Af-
methylethylaminium bromide [3166-62-9]
Methylbenactyzium Bromide, when dried, contains
not less than 99.0% of C 21 H 28 BrN0 3 .
Description Methylbenactyzium Bromide occurs as white
crystals or crystalline powder. It is odorless, and has an ex-
tremely bitter taste.
It is freely soluble in water and in acetic acid (100), soluble
in ethanol (95), slightly soluble in acetic anhydride, and prac-
tically insoluble in diethyl ether.
The pH of a solution of Methylbenactyzium Bromide (1 in
50) is between 5.0 and 6.0.
Identification (1) Shake 0.5 mL of a solution of Methyl-
benactyzium Bromide (1 in 100) with 5 mL of phosphate
buffer solution, pH 7.0, 2 to 3 drops of bromothymol blue TS
and 5 mL of chloroform: a yellow color develops in the chlo-
roform layer.
(2) To about 1 g of Methylbenactyzium Bromide add 5
mL of water and 10 mL of sodium hydroxide TS, allow to
stand for 5 minutes, add 5 mL of dilute hydrochloric acid,
collect the precipitate, wash well with water, recrystallize
from a mixture of water and ethanol (95) (10:3), and dry at
105°C for 1 hour: the crystals melt <2.60> between 145°C and
150°C. Continue the heating up to about 200°C: a red color
develops.
(3) Add 2 mL of dilute nitric acid to 5 mL of a solution of
Methylbenactyzium Bromide (1 in 10): the solution responds
to the Qualitative Tests <1.09> (1) for bromide.
Melting point <2.60> 168 - 172°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Methylbenactyzium Bromide in 10 mL of water: the solution
is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.5 g of Methyl-
benactyzium Bromide. Prepare the control solution with 0.40
mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%).
(3) Heavy metals <I.07>— Proceed with 2.0 g of Methyl-
benactyzium Bromide according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Methylbenactyzium
Bromide, previously dried, and dissolve in 80 mL of a mix-
ture of acetic anhydride and acetic acid (100) (4:1). Titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 42.24 mg of C 2I H 28 BrN0 3
Containers and storage Containers — Tight containers.
Methylcellulose
^^JHzJUP-X
Cellulose, methyl ether
[9004-67-5]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Methylcellulose is a methyl ether of cellulose.
It contains not less than 26.0% and not more than
33.0% of methoxy group (-OCH 3 : 31.03), calculated
on the dried basis.
The viscosity of Methylcellulose is shown in millipas-
872 Methylcellulose / Official Monographs
JP XV
cal second (mPa-s).
♦Description Methylcellulose occurs as a white to yellowish
white, powder or granules.
It is practically insoluble in ethanol (99.5).
Methylcellulose swells, when water is added, and forms a
clear or slightly turbid, viscous liquid. »
Identification (1) Disperse evenly 1.0 g of Methylcellulose
over the surface of 100 mL of water in a beaker, while gently
tapping the top of the container, if necessary, and allow the
beaker to stand: it aggregates on the surface of water.
(2) Add 1.0 g of Methylcellulose to 100 mL of hot water,
and stir: it becomes a suspension. Cool the suspension to
5°C, and stir: the resulting liquid is a clear or a slightly
cloudy, viscous fluid.
(3) To 0.1 mL of the viscous fluid obtained in (2) add 9
mL of diluted sulfuric acid (9 in 10), stir, heat in a water bath
for exactly 3 minutes, and immediately cool in ice water. Add
carefully 0.6 mL of ninhydrin TS, stir, and allow to stand at
25 °C: the solution shows a light red color first, then changes
to purple color within 100 minutes.
(4) Pour and spread out 2 to 3 mL of the viscous fluid ob-
tained in (2) onto a glass plate, and allow the water to
evaporate: a transparent film results.
(5) Pipet 50 mL of water, add exactly 50 mL of the vis-
cous fluid obtained in (2), and warm to rise the temperature
at a rate of 2 to 5°C per minute while stirring: the tempera-
ture, when a white turbidity of the solution starts to increase,
is not less than 50°C.
Viscosity <2.53> Method I: Apply to Methylcellulose having
a labeled viscosity of less than 600mPa-s. Put exactly an
amount of Methylcellulose, equivalent to 4.000 g on the dried
basis, in a tared, wide-mouth bottle, add hot water to make
200.0 g, stopper the bottle, stir by mechanical means at 350-
to 450-revolutions per minute for 10 to 20 minutes to get a
homogeneous dispersion. If necessary, take off the sample at-
tached on the walls of the bottle, put them in the dispersed
solution, and dissolve by continuing the stirring in a water
bath not exceeding 5°C for 20 to 40 minutes. Add cooled
water, if necessary, to make 200.0 g, and use this solution as
the sample solution. Centrifuge the solution if necessary to
expel any entrapped air bubbles. Perform the test with the
sample solution at 20±0.1°C as directed in Method I under
Viscosity Determination: not less than 80% and not more
than 120% of the labeled viscosity.
Method II: Apply to Methylcellulose having a labeled vis-
cosity of not less than 600 mPa-s. Put exactly an amount of
Methylcellulose, equivalent to 10.00 g on the dried basis, in a
tared, wide-mouth bottle, add hot water to make 500.0 g,
stopper the bottle, and prepare the sample solution in the
same manner as directed in Method I. Perform the test with
the sample solution at 20±0.1°C as directed in Method II (2)
under Viscosity Determination, using a single cylinder-type
rotational viscometer, according to the following operating
conditions: not less than 75% and not more than 140% of the
labeled viscosity.
Operating conditions —
Apparatus: Brookfield type viscometer LV model
Rotor No., rotation frequency, and conversion factor: Ac-
cording to the following table, depending on the labeled vis-
cosity.
Labeled viscosity
(mPa-s)
D , Retation ~
R ° tor frequency Conversion
No ' /min factor
Not less than 600 i
and less than 1400
3
60
20
'/ 1400
" 3500
3
12
100
n 3500
// 9500
4
60
100
II 9500
"99,500
4
6
1000
ii 99,500
4
3
2000
Procedure of apparatus: Read value after 2 minutes of ro-
tation, and stop the rotation for 2 minutes. Repeat this proce-
dure more two times, and average three observed values.
pH <2.54> Allow the sample solution obtained in the Vis-
cosity to stand at 20±2°C for 5 minutes: the pH of the solu-
tion thus obtained is between 5.0 and 8.0.
Purity Heavy metals — Put 1.0 g of Methylcellulose in a
100-mL Kjeldahl flask, add a sufficient amount of a mixture
of nitric acid and sulfuric acid (5:4) to wet the sample, and
heat gently. Repeat this procedure until to use totally 18 mL
of the mixture of nitric acid and sulfuric acid. Then boil gent-
ly until the solution changes to black. After cooling, add 2
mL of nitric acid, and heat until the solution changes to
black. Repeat this procedure until the solution no longer
changes to black, and heat strongly until dense white fumes
are evolved. After cooling, add 5 mL of water, boil gently un-
til dense white fumes are evolved, then heat until the volume
of the solution becomes to 2 to 3 mL. After cooling, if the so-
lution reveals yellow color by addition of 5 mL of water, add
1 mL of hydrogen peroxide (30), and heat until the volume of
the solution becomes to 2 to 3 mL. After cooling, dilute the
solution with 2 to 3 mL of water, transfer to a Nessler tube,
add water to make 25 mL, and use this solution as the test so-
lution. Separately, put 2.0 mL of Standard Lead Solution in
a 100-mL kjeldahl flask, add 18 mL of the mixture of nitric
acid and sulfuric acid (5:4) and an amount of nitric acid equal
to that used for preparation of the test solution, and heat un-
til white fumes are evolved. After cooling, add 10 mL of
water. In the case where hydrogen peroxide (30) is added for
the preparation of the test solution, add the same amount of
hydrogen peroxide (30), then proceed in the same manner for
preparation of the test solution, and use so obtained solution
as the control solution. Adjust the test solution and the con-
trol solution to pH 3.0 to 4.0 with ammonia solution (28),
and add water to make 40 mL, respectively. To these solu-
tions add 1.2 mL of thioacetamide-alkaline glycerin TS, 2
mL of acetate buffer solution, pH 3.5 and water to make 50
mL, separately. After allowing to stand for 5 minutes, ob-
serve vertically both tubes on a white background: the color
obtained with the test solution is not more intense than that
with the control solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 5.0% (1 g, 105°C, 1
hour).
Residue on ignition <2.44> Not more than 1.5% (1 g).
Assay (i) Apparatus — Reaction bottle: A 5-mL pressure-
tight glass vial, having 20 mm in outside diameter and 50 mm
in height, the neck 20 mm in outside diameter and 13 mm in
inside diameter, equipped with a septum of butyl-rubber
processed the surface with fluoroplastics, which can be fixed
tightly to vial with aluminum cap, or equivalent.
Heater: A square-shaped aluminum block, having holes 20
JPXV
Official Monographs / Methyldopa Hydrate
873
mm in diameter and 32 mm in depth, adopted to the reaction
bottle. Capable of stirring the content of the reaction bottle
by means of magnetic stirrer or of reciprocal shaker about
100 times per minute.
(ii) Procedure — Weigh accurately about 65 mg of
Methylcellulose, transfer to the reaction bottle, add 0.06 to
0.10 g of adipic acid, 2.0 mL of the internal standard solution
and 2.0 mL of hydroiodic acid, stopper the bottle immedi-
ately, and weigh accurately. Stir or shake for 60 minutes
while heating so that the temperature of the bottle content is
130 ± 2°C. In the case when the stirrer or shaker is not availa-
ble, heat for 30 minutes with repeated shaking at 5-minute in-
tervals by hand, and continue heating for an additional 30
minutes. Allow the bottle to cool, and again weigh accurate-
ly. If the mass loss is less than 0.50% or there is no evidence
of a leak, use the upper layer of the mixture as the sample so-
lution. Separately, put 0.06 to 0.10 g of adipic acid in a reac-
tion bottle, 2.0 mL of the internal standard solution and 2.0
mL of hydroiodic acid, stopper the bottle immediately, and
weigh accurately. Add 45 /xL of iodomethane for assay
through the septum using micro-syringe, weigh accurately,
stir thoroughly, and use the upper layer of the mixture as the
standard solution. Perform the test with 1 to 2 /xL each of the
sample solution and standard solution as directed under Gas
Chromatography <2.02> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
iodomethane to that of the internal standard.
Content (%) of methoxy group (-CH 3 0)
= (.Qt/Qs)x(Ws/W)x 21.86
W s : Amount (mg) of iodomethane in the standard solution
W: Amount (mg) of sample, calculated on the dried basis
Internal standard solution — A solution of «-octane in o-xy-
lene (3 in 100).
Operating conditions —
Detector: A thermal conductivity detector or hydrogen
flame-ionization detector.
Column: A glass column 3-4 mm in inside diameter and
1.8 - 3 m in length, packed with siliceous earth for gas chro-
matography, 125 to 150 /xm in diameter, coated with methyl
silicone polymer at the ratio of 10 - 20%.
Column temperature: A constant temperature of about
100°C.
Carrier gas: Helium for thermal conductivity detector, or
Helium or Nitrogen for hydrogen, flame-ionization detector.
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 10 minutes.
System suitability —
System performance: When the procedure is run with 1-2
[iL of the standard solution under the above operating condi-
tions, iodomethane, isopropyl iodide and the internal stan-
dard are eluted in this order, with complete separation of
these peaks.
♦Containers and storage Containers — Well-closed contain-
ers.*
Methyldopa Hydrate
^JL-K/^jcfotl
CO = H
H 3 C NH 2
HjO
Q0Hj3NO4.il/2H2O: 238.24
(2<S)-2-Amino-3-(3,4-dihydroxyphenyl)-2-methylpropanoic
acid sesquihydrate [413 72-08-1}
Methyldopa Hydrate contains not less than 98.0%
of methyldopa (C 10 H 13 NO 4 : 211.21), calculated on the
anhydrous basis.
Description Methyldopa Hydrate occurs as a white to pale
grayish white, crystalline powder.
It is slightly soluble in water, in methanol and in acetic acid
(100), very slightly soluble in ethanol (95), and practically in-
soluble in diethyl ether.
It dissolves in dilute hydrochloric acid.
Identification (1) To 0.01 g of Methyldopa Hydrate add 3
drops of ninhydrin TS, and heat in a water bath for 3
minutes: a purple color develops.
(2) Determine the absorption spectrum of a solution of
Methyldopa Hydrate in 0.1 mol/L hydrochloric acid TS (1 in
25,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.44>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Methyldopa Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Methyldopa Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Methyldopa Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [ a ] 2 °: -25 - -28° (calculated on
the anhydrous basis, 1 g, aluminum (III) chloride TS, 20 mL,
100 mm).
Purity (1) Acidity — Shake 1 .0 g of Methyldopa Hydrate
with 100 mL of freshly boiled and cooled water, and add 0.20
mL of 0.1 mol/L sodium hydroxide VS and 2 drops of
methyl red TS: a yellow color develops.
(2) Chloride <1.03>— Perform the test with 0.5 g of
Methyldopa Hydrate. Prepare the control solution with 0.40
mL of 0.01 mol/L hydrochloric acid VS (not more than
0.028%).
(3) Heavy metals <1.07>— Proceed with 2.0 g of Methyl-
dopa Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Methyldopa Hydrate in 5 mL of dilute hydrochloric acid,
and perform the test (not more than 2 ppm).
(5) 3-O-Methylmethyldopa— Dissolve 0.10 g of Methyl-
874
Methyldopa Tablets / Official Monographs
JP XV
dopa Hydrate in methanol to make exactly 10 mL, and use
this solution as the sample solution. Separately, dissolve 5 mg
of 3-O-methylmethyldopa for thin-layer chromatography in
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
20 /xL each of the sample solution and standard solution on a
plate of cellulose for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, water and acetic acid (100)
(13:5:3) to a distance of about 10 cm, and air-dry the plate.
Spray evenly 4-nitroaniline-sodium nitrite TS on the plate,
and air-dry the plate, then spray evenly a solution of sodium
carbonate decahydrate (1 in 4) on the plate: the spot from the
sample solution corresponding to that from the standard so-
lution is not more intense than the spot from the standard so-
lution.
Water <2.48> 10.0 - 13.0% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Methyldopa Hy-
drate, dissolve in 80 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to blue-green
(indicator: 2 to 3 drops of crystal violet TS). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 21.12 mg of C 10 H 13 NO 4
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Methyldopa Tablets
yfjiFKi
Methyldopa Tablets contain not less than 90% and
not more than 110% of the labeled amount of methyl-
dopa (Ci H 13 NO 4 : 211.21).
Method of preparation Prepare as directed under Tablets,
with Methyldopa Hydrate.
Identification (1) To a quantity of powdered Methyldopa
Tablets, equivalent to 0.1 g of Methyldopa Hydrate accord-
ing to the labeled amount, add 10 mL of water, and heat in a
water bath for 5 minutes with occasional shaking. After cool-
ing, centrifuge for 5 minutes at 2000 rotations per minute,
apply 1 drop of the supernatant solution to a filter paper, and
dry with warm air. Place 1 drop of ninhydrin TS over the
spot, and heat for 5 minutes at 100°C: a purple color de-
velops.
(2) To 0.5 mL of the supernatant liquid obtained in the
Identification (1) add 2 mL of 0.05 mol/L sulfuric acid TS, 2
mL of iron (II) tartrate TS and 4 drops of ammonia TS, and
shake well: a deep purple color develops.
(3) To 0.7 mL of the supernatant liquid obtained in the
Identification (1) add 0.1 mol/L hydrochloric acid TS to
make 20 mL. To 10 mL of this solution add 0.1 mol/L
hydrochloric acid TS to make 100 mL, and determine the ab-
sorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits a maxi-
mum between 277 nm and 283 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Methyldopa Tablets at 50
revolutions per minute according to the Paddle method using
900 mL of water as the test solution. Take 30 mL or more of
the dissolved solution 60 minutes after start of the test, and
filter through a membrane filter with a pore size not exceeding
0.8 /xm. Discard the first 10 mL of the filtrate, pipet the subse-
quent KmL, add water to make exactly V mL so that each
mL contains about 25 /xg of methyldopa (C I0 H 13 NO 4 ) accord-
ing to the labeled amount, and use this solution as the sample
solution. Separately, weigh accurately about 56 mg of
methyldopa for assay (its loss on drying <2.41> is determined,
separately, at 125°C for 2 hours), and dissolve in water to
make exactly 200 mL. Pipet 10 mL of this solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and ^4 S , of
the sample solution and the standard solution at 280 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Methyldopa Tablets in 60 minutes is
not less than 75%.
Dissolution rate (%) with respect to
the labeled amount of methyldopa (Ci H 13 NO 4 )
= W s x (A T /A S ) x(V'/V)x (1/ C) x 45
W s : Amount (mg) of methyldopa for assay, calculated on
the dried basis
C: Labeled amount (mg) of methyldopa (C 10 H 13 NO 4 ) in 1
tablet
Assay Weigh accurately and powder not less than 20
Methyldopa Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 0.1 g of methyldopa (Ci Hi 3 NO 4 ),
add 50 mL of 0.05 mol/L sulfuric acid TS, shake thoroughly
for 15 minutes, add 0.05 mol/L sulfuric acid TS to make ex-
actly 100 mL, and filter through a dry filter paper. Discard
the first 20 mL of the filtrate, and use the subsequent filtrate
as the sample solution. Separately, weigh accurately about
0.11 g of Methyldopa Reference Standard (previously dry at
125 °C for 2 hours, and determine the loss on drying <2.41>),
dissolve in 0.05 mol/L sulfuric acid TS to make exactly 100
mL, and use this solution as the standard solution. Pipet 5
mL each of the sample solution and the standard solution,
add exactly 5 mL of iron (II) tartrate TS, and add ammonia-
ammonium acetate buffer solution, pH 8.5, to make exactly
100 mL. Perform the test with these solutions as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, using a so-
lution prepared with 5 mL of 0.05 mol/L sulfuric acid TS in
the same manner, as the blank. Determine the absorbances,
A-y and^4 s , of the subsequent solutions of the sample solution
and the standard solution at 520 nm, respectively.
Amount (mg) of methyldopa (C 10 H I3 NO 4 )
= W s x(Aj/A s )
W s : amount (mg) of Methyldopa Reference Standard, cal-
culated on the dried basis
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / ^/-Methylephedrine Hydrochloride
875
e?/-Methylephedrine Hydrochloride
WfJH7i h* U >ikWk
H OH CH 3
• HCI
H CH a
and enantiomer
C n H 17 NO.HCl: 215.72
( 1 RS,2SR )-2-Dimethylamino- 1 -phenylpropan-1 -ol
monohydrochloride [18760-80-0]
cW-Methylephedrine Hydrochloride, when dried,
contains not less than 99.0% and not more than
101.0% of C„H 17 NO.HCl.
Description (//-Methylephedrine Hydrochloride occurs as
colorless crystals or a white, crystalline powder.
It is freely soluble in water, sparingly soluble in ethanol
(99.5), slightly soluble in acetic acid (100), and practically
insoluble in acetic anhydride.
A solution of (//-Methylephedrine Hydrochloride (1 in 20)
shows no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of (//-Methylephedrine Hydrochloride (1 in 2000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of (//-
Methylephedrine Hydrochloride, previously dried, as direct-
ed in the potassium chloride disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
(3) A solution of (//-Methylephedrine Hydrochloride
(1 in 10) responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of (//-Methylephedrine Hydrochloride in 20 mL of water is
between 4.5 and 6.0.
Melting point <2.60> 207 - 21 1 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
(//-Methylephedrine Hydrochloride in 10 mL of water: the so-
lution is clear and colorless.
(2) Heavy metals <1.07>— Proceed with 1.0 g of (//-
Methylephedrine Hydrochloride according to Method 4, and
perform the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 50 mg of (//-
Methylephedrine Hydrochloride in 20 mL of water, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add water to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with exactly
20 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total area of the peaks
other than the peak of methylephedrine is not more than the
peak area of methylephedrine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 257 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 13.6 g of potassium dihydrogen
phosphate and 3 g of sodium 1 -heptane sulfonate in 1000 mL
of water, and adjust the pH to 2.5 with phosphoric acid. To
900 mL of this solution add 200 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
methylephedrine is about 10 minutes.
Time span of measurement: About 2 times as long as the
retention time of methylephedrine beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add water to make exactly 20 mL. Confirm that
the peak area of methylephedrine obtained from 20 /uL of this
solution is equivalent to 7 to 13% of that of methylephedrine
obtained from 20 /xh of the standard solution.
System performance: Dissolve 50 mg of (//-Methylephe-
drine Hydrochloride and 0.4 mg of methyl parahydroxyben-
zoate in 50 mL of water. When the procedure is run with
20 fxh of this solution under the above operating conditions,
methylephedrine and methyl parahydroxybenzoate are eluted
in this order with the resolution between these peaks being
not less than 3.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
methylephedrine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of (//-Methylephedrine
Hydrochloride, previously dried, dissolve in 80 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 21.57 mg of C„H 17 NO.HCl
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
876
10% ^/-Methylephedrine Hydrochloride Powder / Official Monographs
JP XV
10% ^/-Methylephedrine
Hydrochloride Powder
(//-Methylephedrine Hydrochloride Powder
rf/-yfJH7i K U >&&&%. 10%
10% cf/-Methylephedrine Hydrochloride Powder
contains not less than 9.3% and not more than 10.7%
of cW-methylephedrine hydrochloride (CnH 17 NO.HCl:
215.72).
Method of preparation
(//-Methylephedrine Hydrochloride
Starch, Lactose Hydrate or
their mixture
100 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification Determine the absorption spectrum of a
solution of 10% (//-Methylephedrine Hydrochloride Powder
(1 in 200) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits maxima between 250 nm and
253 nm, between 255 nm and 259 nm, and between 261 nm
and 264 nm.
Assay Weigh accurately about 0.5 g of 10% (//-Methylephe-
drine Hydrochloride Powder, add exactly 4 mL of the inter-
nal standard solution and 25 mL of water, shake vigorously
for 20 minutes to dissolve, add water to make 50 mL, filter
through a membrane filter with pore size of 0.45 fim, if neces-
sary, discard the first 10 mL of the filtrate, and use the subse-
quent filtrate as the sample solution. Separately, weigh ac-
curately about 50 mg of (//-methylephedrine hydrochloride
for assay, previously dried at 105°C for 3 hours, add exactly
4 mL of the internal standard solution and water to make 50
mL, and use this solution as the standard solution. Perform
the test with 20 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios
of the peak area, Q T and Q s , of methylephedrine to that of
the internal standard.
Amount (mg) of (//-methylephedrine hydrochloride
(C„H 17 NO.HCl)
= W s x(Q T /Q s )
W s : Amount (mg) of (//-methylephedrine hydrochloride
for assay
Internal standard solution — A solution of methyl para-
hydroxybenzoate in acetonitrile (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 257 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 13.6 g of potassium dihydrogen
phosphate and 3 g of sodium 1 -heptane sulfonate in 1000 mL
of water, and adjust the pH to 2.5 with phosphoric acid. To
900 mL of this solution add 200 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
methylephedrine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the standard solution under the above operating
conditions, methylephedrine and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of methylephedrine to that of the internal standard
is not more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Methylergometrine Maleate
(
COi>H
C 20 H 25 N3O 2 .C4H 4 O4: 455.50
(8S)-Aq(lS>l-(Hydroxymethyl)propyl]-6-methyl-9,10-
didehydrolergoline-8-carboxamide monomaleate
[7054-07-1]
Methylergometrine Maleate, when dried, contains
not less than 95.0% and not more than 105.0% of
C 20 H 25 N3O 2 .C4H 4 O4.
Description Methylergometrine Maleate occurs as a white
to pale yellow, crystalline powder. It is odorless.
It is slightly soluble in water, in methanol and in ethanol
(95), and practically insoluble in diethyl ether.
It gradually changes to yellow by light.
Melting point: about 190°C (with decomposition).
Identification (1) A solution of Methylergometrine Male-
ate (1 in 200) shows a blue fluorescence.
(2) The solution obtained in the Assay develops a deep
blue in color. Determine the absorption spectrum of the solu-
tion as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Methylergometrine
Maleate Reference Standard prepared in the same manner as
the sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(3) To 5 mL of a solution of Methylergometrine Maleate
JPXV
Official Monographs / Methylergometrine Maleate Tablets
877
(1 in 500) add 1 drop of potassium permanganate TS: the red
color of the test solution fades immediately.
Optical rotation <2.49> [a]™: +44- +50° (after drying, 0.1
g, water, 20 mL, 100 mm).
Purity Related substances — Conduct this procedure
without exposure to daylight, using light-resistant vessels.
Dissolve 8 mg of Methylergometrine Maleate in 2 mL of a
mixture of ethanol (95) and ammonia solution (28) (9:1), and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add a mixture of ethanol (95) and ammonia
solution (28) (9:1) to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test immediately
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography, and immediately
develop the plate with a mixture of chloroform, methanol
and water (75:25:3) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
365 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 2.0% (0.2 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Assay Weigh accurately about 10 mg each of Methyler-
gometrine Maleate and Methylergometrine Maleate Refer-
ence Standard, previously dried, add water to make exactly
250 mL, and use these solutions as the sample solution and
the standard solution. Pipet 2 mL each of the sample solution
and the standard solution separately into brown glassstop-
pered test tubes, add exactly 4 mL each of 4-
dimethylaminobenzaldehyde-iron (III) chloride TS while ice
cooling, warm for 10 minutes at 45°C, and allow to stand for
20 minutes at room temperature. Perform the test with these
solutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using a solution, prepared with 2.0 mL of water
in the same manner, as the blank. Determine the absor-
bances, A T and A s , of the subsequent solutions of the sample
solution and the standard solution at 545 nm, respectively.
Amount (mg) of C20H25N3O2.C4H4O4
= W s x(A T /A s )
W s : Amount (mg) of Methylergometrine Maleate Refer-
ence Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Methylergometrine Maleate Tablets
Methylergometrine Maleate Tablets contain not less
than 90.0% and not more than 1 10.0%, of the labeled
amount of methylergometrine maleate (C20H25N3O2.C4
H 4 4 : 455.50).
Method of preparation Prepare as directed under Tablets,
with Methylergometrine maleate.
Identification (1) The sample solution obtained in the As-
say shows a blue fluorescence.
(2) The colored solution obtained in the Assay shows a
deep blue color. Determine the absorption spectrum of the
colored solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 543 nm
and 547 nm and between 620 nm and 630 nm.
Uniformity of dosage unit <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
Transfer 1 tablet of Methylergometrine Maleate Tablets to
a brown glass-stoppered centrifuge tube, add 10 mL of water,
shake for 10 minutes vigorously, and disintegrate the tablet.
Add 3 g of sodium chloride and 2 mL of ammonia solution
(28), add exactly 25 mL of chloroform, and after vigorous
shaking for 10 minutes, centrifuge for 5 minutes. Discard the
water layer, take the chloroform extracts, add chloroform to
make exactly KmL of a solution containing about 5 fig of
methylergometrine maleate (C20H25N3O2.C4H4O4) per mL,
and use this solution as the sample solution. Separately,
weigh accurately about 1.25 mg of Methylergometrine Male-
ate Reference Standard, previously dried in a desiccator ( in
vacuum, phosphorus (V) oxide) for 4 hours, and dissolve in
water to make exactly 100 mL. Pipet 10 mL of this solution
into a brown glass-stoppered centrifuge tube, and add 3 g of
sodium chloride and 2 mL of ammonia solution (28). Add ex-
actly 25 mL of chloroform, shake vigorously for 10 minutes,
and centrifuge for 5 minutes. Discard the water layer, and use
the chloroform layer as the standard solution. Pipet 20 mL
each of the sample solution and the standard solution
separately into brown glass-stoppered centrifuge tubes, add
immediately exactly 10 mL of dilute 4-dimethylaminobenzal-
dehyde-iron (III) chloride TS, and shake for 5 minutes
vigorously. Centrifuge these solutions for 5 minutes, take the
water layers, and allow them to stand for 1 hour. Perform the
test with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using dilute 4-dimethylaminoben-
zaldehyde-iron (III) chloride TS as the blank. Determine the
absorbances, A T and A s , of the subsequent solutions of the
sample solution and standard solution at 545 nm, respec-
tively.
Amount (mg) of methylergometrine maleate
(C 20 H 25 N 3 O 2 .C 4 H 4 O 4 )
= W s x(A T / A s ) x(V/250)
W s : Amount (mg) of Methylergometrine Maleate Refer-
ence Standard
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Methylergometrine Male-
ate Tablets at 100 revolutions per minute according to the
Paddle method using 900 mL of water as the test solution.
Take 20 mL or more of the dissolved solution 30 minutes af-
ter starting the test, and filter through a membrane filter with
a pore size of not more than 0.8 /xm. Discard the first 10 mL
of the filtrate, to exactly FmL of the subsequent filtrate add
water to make exactly V mL so that each mL contains about
0.13 nz of methylergometrine maleate (C20H25N3O2.C4H4O4)
according to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 25 mg of
Methylergometrine Maleate Reference Standard, previously
dried in a desiccator for 4 hours (in vacuum, phosphorus (V)
oxide), and dissolve in water to make exactly 100 mL. Pipet 5
878
Methyl Parahydroxybenzoate / Official Monographs
JP XV
mL of this solution, add water to make exactly 100 mL, then
pipet 1 mL of this solution, add water to make exactly 100
mL, and use this solution as the standard solution. Determine
immediately the intensities of the fluorescence, F T and F s , of
the sample solution and the standard solution at 338 nm as
the excitation wavelength and at 427 nm as the fluorescence
wavelength as directed under Fluorometry <2.22>.
The dissolution rate of Methylergometrine Maleate Tablets
in 30 minutes should be not less than 70%.
Dissolution rate (%) with respect to the labeled amount
of methylergometrine maleate (C20H25N3O2.C4H4O4)
= W s x (Fj/F s ) x(V'/V)x(l/Qx (9/20)
W s : Amount (mg) of Methylergometrine Maleate Refer-
ence Standard
C: Labeled amount (mg) of methylergometrine maleate
(C 20 H 25 N3O 2 .C4H4O 4 ) in 1 tablet
Assay Weigh accurately and powder not less than 20
Methylergometrine Maleate Tablets. Weigh accurately a por-
tion of the powder, equivalent to about 0.3 mg of methyler-
gometrine maleate (C20H25N3O2.C4H4O4), transfer to a
brown separator, add 15 mL of sodium hydrogen carbonate
solution (1 in 20), and extract with four 20-mL portions of
chloroform. Filter each portion of the chloroform extracts
through a pledget of absorbent cotton, previously moistened
with chloroform, into another dried, brown separator, com-
bine all the extracts, and use this extract as the sample solu-
tion. Separately, weigh accurately about 10 mg of Methyler-
gometrine Maleate Reference Standard, previously dried in a
desiccator (silica gel) for 4 hours, dissolve in water, and add
water to make exactly 100 mL. Pipet 3 mL of this solution,
and transfer to a brown separator, proceed in the same man-
ner as the preparation of the sample solution, and use this ex-
tract as the standard solution. To each total volume of the
sample solution and the standard solution add exactly 25 mL
each of dilute p-dimethylaminobenzadehyde-ferric chloride
TS, and after vigorous shaking for 5 minutes, allow to stand
for 30 minutes. Draw off the water layer, centrifuge, and al-
low to stand for 1 hour. Perform the test with these solutions
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using dilute 4-dimethylaminobenzaldehyde-ferric
chloride TS as the blank. Determine the absorbances, A T and
^4 S > of the subsequent solutions of the sample solution and
standard solution at 545 nm, respectively.
Amount (mg) of methylergometrine maleate
(C20H25N3O2.C4H4O4)
= W s x (A T /A S ) x (3/100)
W s : Amount (mg) of Methylergometrine Maleate Refer-
ence Standard
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Methyl Parahydroxybenzoate
rt = 7-**ri' , &&W&**)l
HO"
C 8 H 8 3 : 152.15
Methyl 4-hydroxybenzoate
I-
[98-76-3]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Methyl Parahydroxybenzoate, when dried, contains
not less than 98.0% and not more than 102. 0% of
C8H 8 3 .
♦Description Methyl Parahydroxybenzoate, occurs as
colorless crystals or a white, crystalline powder.
It is freely soluble in ethanol (95) and in acetone, and
slightly soluble in water. »
Identification (1) The melting point <2.60> of Methyl
Parahydroxybenzoate is between 125°C and 128°C.
*(2) Determine the infrared absorption spectrum of
Methyl Parahydroxybenzoate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.*
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Methyl Parahydroxybenzoate in 10 mL of ethanol (95): the
solution is clear and not more intensely colored than the fol-
lowing control solution.
Control solution: To 5.0 mL of cobalt (II) chloride colori-
metric stock solution, 12.0 mL of iron (III) chloride colori-
metric stock solution and 2.0 mL of cupper (II) sulfate colori-
metric stock solution add water to make 1000 mL.
(2) Acidity — Dissolve 0.20 g of Methyl Parahydroxyben-
zoate in 5 mL of ethanol (95), add 5 mL of freshly boiled and
cooled water and 0.1 mL of bromocresol green-sodium
hydroxide-ethanol TS, then add 0. 1 mL of 0. 1 mol/L sodium
hydroxide VS: the solution shows a blue color.
♦(3) Heavy metals <1.07>— Dissolve 1.0 g of Methyl
Parahydroxybenzoate in 25 mL of acetone, add 2 mL of di-
lute acetic acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 25 mL of acetone, 2 mL of dilute acetic acid, and water
to make 50 mL (not more than 20 ppm).»
(4) Related substances — Dissolve 0.10 g of Methyl Para-
hydroxybenzoate in 10 mL of acetone, and use this solution
as the sample solution. Pipet 0.5 mL of the sample solution,
add acetone to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
/iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
JPXV
Official Monographs / Methylprednisolone
879
methanol, water and acetic acid (100) (70:30:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot other than the
principal spot is not more intense than the spot obtained with
the standard solution.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 .0 g of Methyl Parahydrox-
ybenzoate, add exactly 20 mL of 1 mol/L sodium hydroxide
VS, heat at about 70°C for 1 hour, and immediately cool in
ice. Titrate <2.50> the excess sodium hydroxide with 0.5
mol/L sulfuric acid VS up to the second equivalent point
(potentiometric titration). Perform a blank determination.
Each mL of 1 mol/L sodium hydroxide VS
= 152.1 mg of C 8 H 8 3
♦Containers and storage Containers — Well-closed contain-
ers.*
Compound Methyl Salicylate Spirit
Methylprednisolone
Wi j &m-)vm.n-)vn
Method of preparation
Methyl Salicylate
Capsicum Tincture
d- or tf/-Camphor
Ethanol
40 mL
100 mL
50 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients.
Description Compound Methyl Salicylate Spirit is a reddish
yellow liquid, having a characteristic odor and a burning
taste.
Identification (1) Shake 1 mL of Compound Methyl
Salicylate Spirit with 5 mL of dilute ethanol, and add 1 drop
of iron (III) chloride TS: a purple color is produced (methyl
salicylate).
(2) Shake thoroughly 0.5 mL of Compound Methyl
Salicylate Spirit with 10 mL of chloroform, and use this solu-
tion as the sample solution. Dissolve 0.04 g of methyl salicy-
late in 10 mL of chloroform, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 5 [iL each
of the sample solution and standard solution on the plate of
silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of hexane and
chloroform (4:1) to a distance of about 10 cm, air-dry the
plate, and examine under ultraviolet light (main wavelength:
254 nm): the spots from the sample solution and the standard
solution show the same Rf value. Spray evenly iron (III) chlo-
ride TS upon the plate: the spot from the standard solution
and the corresponding spot from the sample solution reveal a
purple color.
Containers and storage Containers — Tight containers.
H CH S
C 22 H 30 O 5 : 374.47
1 1/8,17, 21-Trihydroxy-6a-methylpregna-l,4-diene-
3,20-dione [83-43-2]
Methylprednisolone, when dried, contains not less
than 96.0% and not more than 104.0% of C 22 H3 O 5 .
Description Methylprednisolone occurs as a white, crystal-
line powder. It is odorless.
It is sparingly soluble in methanol and in 1,4-dioxane,
slightly soluble in ethanol (95) and in chloroform, and practi-
cally insoluble in water and in diethyl ether.
Melting point: 232 - 240°C (with decomposition).
Identification (1) Add 2 mL of sulfuric acid to 2 mg of
Methylprednisolone: a deep red color develops with no
fluorescence. Then add 10 mL of water to this solution: the
color fades, and a gray, flocculent precipitate is produced.
(2) Dissolve 0.01 g of Methylprednisolone in 1 mL of
methanol, add 1 mL of Fehling's TS, and heat: a red
precipitate is produced.
(3) Determine the absorption spectrum of a solution of
Methylprednisolone in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
Optical rotation <2.49> [a]™: +79- +86° (after drying, 0.1
g, 1,4-dioxane, 10 mL, 100 mm).
Purity Related substances — Dissolve 50 mg of Methylpred-
nisolone in 5 mL of a mixture of chloroform and methanol
(9:1), and use this solution as the sample solution. Pipet 1 mL
of this solution, add a mixture of chloroform and methanol
(9:1) to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of dichloromethane, diethyl ether,
methanol and water (385:75:40:6) to a distance of about 12
cm, and air-dry the plate. Then heat at 105°C for 10 minutes,
cool, and spray evenly alkaline blue tetrazolium TS on the
plate: the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.2 g).
Assay Weigh accurately about 10 mg of Methylpredniso-
880
Methylprednisolone Succinate / Official Monographs
JP XV
lone, previously dried, and dissolve in methanol to make ex-
actly 100 mL. To exactly 5 mL of this solution add methanol
to make exactly 50 mL, and determine the absorbance A at
the wavelength of maximum absorption at about 243 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C22H30O5
= (,4/400) x 1000
Containers and storage Containers — Tight containers.
Methylprednisolone Succinate
y^JL-TV Kzv'n>ay\^liXxJi.
COjH
C 26 H 34 8 : 474.54
11/6,17,21 -Trihydroxy-6a-methylpregna- 1 ,4-diene-
3,20-dione 21-(hydrogen succinate) [2921-57-5]
Methylprednisolone Succinate, when dried, contains
not less than 97.0% and not more than 103.0% of
C26H340 8 .
Description Methylprednisolone Succinate occurs as a
white, crystals or crystalline powder.
It is soluble in methanol, sparingly soluble in ethanol (95),
and practically insoluble in water.
Melting point: about 235 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Methylprednisolone Succinate in methanol
(1 in 50,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of
Methylprednisolone Succinate Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Methylprednisolone Succinate, previously dried, as directed
in the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of previously dried
Methylprednisolone Succinate Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers. In case when some differences are found be-
tween the spectra, repeat the test with residues obtained by
dissolving these substances in ethanol (95), evaporating to
dryness, and drying.
Optical rotation <2.49> [a]": +99- +103° (after drying,
0.2 g, ethanol (95), 20 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Methylprednisolone Succinate according to Method 4, and
perform the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Methylprednisolone Succinate according to Method 3, and
perform the test (not more than 1 ppm).
(3) Related substances — Dissolve 15 mg of Methylpred-
nisolone Succinate in 5 mL of methanol, add a mixture of
0.05 mol/L phosphate buffer solution, pH 3.5 and acetoni-
trile (1:1) to make 50 mL, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add the mixture
of 0.05 mol/L phosphate buffer solution, pH 3.5 and
acetonitrile (1:1) to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 5
IxL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of the peaks other
than the peak of methylprednisolone succinate is not more
than 1/2 of the peak area of methylprednisolone succinate
from the standard solution, and the total area of the peaks
other than the peak of methylprednisolone succinate is not
more than the peak area of methylprednisolone succinate
from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of methylprednisolone succinate.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add the mixture of 0.05 mol/L phosphate
buffer solution, pH 3.5 and acetonitrile (1:1) to make exactly
10 mL. Confirm that the peak area of methylprednisolone
succinate obtained from 5 fiL of this solution is equivalent to
7 to 13% of that obtained from 5 fiL of the standard solu-
tion.
System performance: Proceed as directed in the System
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
methylprednisolone succinate is not more than 1.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 15 mg each of Methylpred-
nisolone Succinate and Methylprednisolone Succinate
Reference Standard, previously dried, dissolve separately in 5
mL of methanol, and add the mixture of 0.05 mol/L phos-
phate buffer solution, pH 3.5 and acetonitrile (1:1) to make
exactly 50 mL. Pipet 5 mL each of these solutions, add
exactly 5 mL of the internal standard solution, and use these
solutions as the sample solution and the standard solution,
respectively. Perform the test with 5 fiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Qt and Q s , of the peak area
of methylprednisolone succinate to that of the internal
standard.
Amount (mg) of C 26 H 34 8 = W s x (Q T /Qs)
W s : Amount (mg) of Methylprednisolone Succinate
JP XV
Official Monographs / Methyl Salicylate
881
Reference Standard
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in a mixture of 0.05 mol/L phosphate buffer solu-
tion, pH 3.5 and acetonitrile (1:1) (3 in 20,000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 1000 mL of 0.05 mol/L potassium di-
hydrogen phosphate TS add a suitable amount of 0.05 mol/L
disodium hydrogen phosphate TS to make a solution having
pH 5.5. To 640 mL of this solution add 360 mL of acetoni-
trile.
Flow rate: Adjust the flow rate so that the retention time of
methylprednisolone succinate is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, methylprednisolone succinate and the internal stan-
dard are eluted in this order with the resolution between these
peaks being not less than 6.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of methylprednisolone succinate to that of the in-
ternal standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Methylrosanilinium Chloride
Crystal Violet
C 2 5H3oClN 3 : 407.98
Methylrosanilinium Chloride is hexamethyl-
pararosaniline chloride, and is usually admixed with
pentamethylpararosaniline chloride and tetramethyl-
pararosaniline chloride.
It contains not less than 96.0% of methylrosanilini-
um chloride [as hexamethylpararosaniline chloride
(C25H30CIN3)], calculated on the dried basis.
Description Methylrosanilinium Chloride occurs as green
fragments having a metallic luster or a dark green powder. It
is odorless or has a slight odor.
It is soluble in ethanol (95), sparingly soluble in water, and
practically insoluble in diethyl ether.
Identification (1) To 1 mL of sulfuric acid add 1 mg of
Methylrosanilinium Chloride: it dissolves, and shows an
orange to red-brown color. To this solution add water drop-
wise: the color of the solution changes from brown through
green to blue.
(2) Dissolve 0.02 g of Methylrosanilinium Chloride in 10
mL of water, add 5 drops of hydrochloric acid, and use this
solution as the sample solution. To 5 mL of the sample solu-
tion add tannic acid TS dropwise: an intense blue precipitate
is formed.
(3) To 5 mL of the sample solution obtained in (2) add
0.5 g of zinc powder, and shake: the solution is decolorized.
Place 1 drop of this solution on filter paper, and apply 1 drop
of ammonia TS adjacent to it: a blue color is produced at the
zone of contact of the both solutions.
Purity (1) Ethanol-insoluble substances — Weigh ac-
curately about 1 g of Methylrosanilinium Chloride, previous-
ly dried at 105°C for 4 hours, heat with 50 mL of ethanol (95)
under a reflux condenser for 15 minutes in a water bath, and
filter the mixture through a tared glass filter (G4). Wash the
residue on the filter with warm ethanol (95) until the last
washing does not show a purple color, and dry at 105°C for 2
hours: the mass of the residue is not more than 1.0%.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Methyl-
rosanilinium Chloride according to Method 2, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 30ppm).
(3) Zinc — To 0.10 g of Methylrosanilinium Chloride add
0.1 mL of sulfuric acid, and incinerate by ignition. After
cooling, boil with 5 mL of dilute hydrochloric acid, 0.5 mL
of dilute nitric acid and 4 mL of water, add 5 mL of ammo-
nia TS, boil again, and filter. To the filtrate add 2 to 3 drops
of sodium sulfide TS: no turbidity is produced.
(4) Arsenic </.//> — Prepare the test solution with 0.40 g
of Methylrosanilinium Chloride, according to Method 3, and
perform the test (not more than 5 ppm).
Loss on drying <2.41> Not more than 7.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 1.5% (0.5 g).
Assay Transfer about 0.4 g of Methylrosanilinium Chlo-
ride, accurately weighed, to a wide-mouthed, conical flask,
add 25 mL of water and 10 mL of hydrochloric acid, dis-
solve, and add exactly 50 mL of 0.1 mol/L titanium (III)
chloride VS while passing a stream of carbon dioxide through
the flask. Heat to boil, and boil gently for 15 minutes,
swirling the liquid frequently. Cool while passing a stream of
carbon dioxide through the flask, titrate <2.50> the excess
titanium (III) chloride with 0.05 mol/L ammonium iron (III)
sulfate VS until a faint, red color is produced (indicator: 5
mL of ammonium thiocyanate TS). Perform a blank deter-
mination.
Each mL of 0.1 mol/L titanium (III) chloride VS
= 20.40 mg of C 25 H 3 oClN 3
Containers and storage Containers — Tight containers.
Methyl Salicylate
v -CH a
OH
C 8 H r O,: 152.15
882
Methyltestosterone / Official Monographs
JP XV
Methyl 2-hydroxybenzoate [119-36-8]
Methyl Salicylate contains not less than 98.0% of
C 8 H 8 3 .
Description Methyl Salicylate is a colorless to pale yellow
liquid. It has a strong, characteristic odor.
It is miscible with ethanol (95) and with diethyl ether.
It is very slightly soluble in water.
Specific gravity df - 1.182-1.192
Boiling point: 219-224°C
Identification Shake 1 drop of Methyl Salicylate thoroughly
with 5 mL of water for 1 minute, and add 1 drop of iron (III)
chloride TS: a purple color develops.
Purity (1) Acidity— Shake 5.0 mL of Methyl Salicylate
thoroughly with 25 mL of freshly boiled and cooled water
and 1.0 mL of 0.1 mol/L sodium hydroxide VS for 1 minute,
add 2 drops of phenol red TS, and titrate <2.50> with 0.1 mol
/L hydrochloric acid VS until the red color disappears: not
more than 0.45 mL of 0.1 mol/L sodium hydroxide VS is
consumed.
(2) Heavy metals <1. 07>— Shake 10.0 mL of Methyl
Salicylate thoroughly with 10 mL of water, add 1 drop of
hydrochloric acid, and saturate with hydrogen sulfide by
passing it through the mixture: neither the oily layer nor the
aqueous layer shows a dark color.
Assay Weigh accurately about 2 g of Methyl Salicylate, add
an exactly measured 50 mL of 0.5 mol/L potassium hydrox-
ide-ethanol VS, and heat on a water bath for 2 hours under a
reflux condenser. Cool, and titrate <2.50> the excess potassi-
um hydroxide with 0.5 mol/L hydrochloric acid VS (indica-
tor: 3 drops of phenolphthalein TS). Perform a blank deter-
mination.
Each mL of 0.5 mol/L potassium hydroxide-ethanol VS
= 76.07 mg of C 8 H 8 3
Containers and storage Containers — Tight containers.
Methyltestosterone
C 2 „H 30 O 2 : 302.45
1 7/?-Hydroxy- 1 7«-methylandrost-4-en-3 -one [58-18-4]
Methyltestosterone, when dried, contains not less
than 98.0% and not more than 102.0% of C 2 oH3 2 .
Description Methyltestosterone occurs as white to pale
yellow, crystals or crystalline powder.
It is freely soluble in methanol and in ethanol (95), and
practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Methyltestosterone in ethanol (95) (1 in 100,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Methyltestosterone
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Methyltestosterone, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.55>, and compare the spectrum with the
Reference Spectrum or the spectrum of dried
Methyltestosterone Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: +79 - +85° (after drying, 0.1
g, ethanol (95), 10 mL, 100 mm).
Melting point <2.60> 163 - 168°C
Purity Related substances — Dissolve 40 mg of
Methyltestosterone in 2 mL of ethanol (95), and use this solu-
tion as the sample solution. Pipet 1 mL of this solution, add
ethanol (95) to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 iuL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and diethylamine (19:1) to a distance of about 15 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 10 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 20 mg each of
Methyltestosterone and Methyltestosterone Reference
Standard, previously dried in a desiccator (in vacuum, phos-
phorus (V) oxide) for 10 hours, dissolve each in methanol to
make exactly 200 mL. Pipet 5 mL each of these solutions,
add exactly 5 mL of the internal standard solution, add
methanol to make 50 mL, and use these solutions as the
sample solution and the standard solution. Perform the test
with 10 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Qj and
Q s , of the peak area of methyltestosterone to that of the in-
ternal standard.
Amount (mg) of C20H30O2
= W s x (Q T /Q S )
W s : Amount (mg) of Methyltestosterone Reference
Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in methanol (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 241 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
JPXV
Official Monographs / Methyltestosterone Tablets
883
35°C.
Mobile phase: A mixture of acetonitrile and water (11:9).
Flow rate: Adjust the flow rate so that the retention time of
methyltestosterone is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating
conditions, the internal standard and methyltestosterone are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of methyltestosterone to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Methyltestosterone Tablets
/fJl^xX l-XxP/S
Methyltestosterone Tablets contain not less than
90.0% and not more than 110.0% of the labeled
amount of methyltestosterone (C20H30O2: 302.45).
Method of preparation Prepare as directed under Tablets,
with Methyltestosterone.
Identification To a portion of powdered Methyltestosterone
Tablets, equivalent to 10 mg of Methyltestosterone according
to the labeled amount, add 50 mL of acetone, shake for 30
minutes, and filter. Evaporate the filtrate to dryness, dissolve
the residue in 10 mL of acetone, and use this solution as the
sample solution. Separately, dissolve 10 mg of
Methyltestosterone Reference Standard in 10 mL of acetone,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of
chloroform and ethanol (95) (9:1) to a distance of about
12 cm, and air-dry the plate. Spray evenly dilute sulfuric acid
on the plate, and heat at 110°C for 10 minutes: the spot from
the sample solution and the standard solution show the same
Rf value.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Methyltestosterone Tablets add 5 mL of
water to disintegrate, add 50 mL of methanol, and shake for
30 minutes. Add methanol to make exactly 100 mL, and cen-
trifuge. Measure exactly FmL of the supernatant liquid, add
methanol to make exactly V mL of a solution containing
about 10 n% of methyltestosterone (C 2 oH 30 2 ) per ml, and use
this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of Methyltestosterone Reference Stan-
dard, previously dried in a desiccator (in vacuum, phospho-
rus (V) oxide) for 10 hours, and dissolve in 5 mL of water and
50 mL of methanol, then add methanol to make exactly 100
mL. Pipet 5 mL of this solution, add methanol to make ex-
actly 50 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and the standard solution at the wavelength of maximum
absorption at about 241 nm, respectively, as directed under
Ultraviolet-visible Spectrophotometry <2.25>.
Amount (mg) of methyltestosterone (C20H30O2)
= W s x(A T /A s )x(V'/V)x(l/lO)
W s : Amount (mg) of Methyltestosterone Reference Stan-
dard
Assay Weigh accurately the mass of not less than 20
Methyltestosterone Tablets, and powder. Weigh accurately a
portion of the powder, equivalent to about 25 mg of
methyltestosterone (C20H30O2), add about 70 mL of
methanol, shake for 30 minutes, and add methanol to make
exactly 100 mL. Pipet 2 mL of this solution, add exactly 5
mL of the internal standard solution and methanol to make
50 mL, filter through a membrane filter (not exceeding 0.45
/xm in pore size), and use the filtrate as the sample solution.
Separately, weigh accurately about 20 mg of Methyltestoster-
one Reference Standard, previously dried in a desiccator (in
vacuum, phosphorus (V) oxide) for 10 hours, dissolve in
methanol to make exactly 200 mL. Pipet 5 mL of this solu-
tion, add exactly 5 mL of the internal standard solution, add
methanol to make 50 mL, and use this solution as the
standard solution. Perform the test with 10 /xh each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following
conditions, and determine the ratios, g T and Q s , of the peak
area of methyltestosterone to that of the internal standard.
Amount (mg) of methyltestosterone (C20H30O2)
= W s x (Q T /Q S ) x (5/4)
W s : Amount (mg) of Methyltestosterone Reference
Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in methanol (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 241 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of acetonitrile and water (11:9).
Flow rate: Adjust the flow rate so that the retention time of
methyltestosterone is about 10 minutes.
System suitability —
System performance: When the procedure is run with
10 [iL of the standard solution under the above operating
conditions, the internal standard and methyltestosterone are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of methyltestosterone to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
884
Meticrane / Official Monographs
JP XV
Meticrane
/^v>
3f
°w°
-coo
C 10 H 13 NO 4 S 2 : 275.34
6-Methylthiochromane-7-sulfonamide 1 , 1 -dioxide
[1084-65-7]
Meticrane, when dried, contains not less than 98.0%
of C 10 H 13 NO 4 S 2 .
Description Meticrane occurs as white, crystals or crystal-
line powder. It is odorless and has a slight bitter taste.
It is freely soluble in dimethylformamide, slightly soluble
in acetonitrile and in methanol, very slightly soluble in
ethanol (95), and practically insoluble in water.
Melting point: about 234°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Meticrane in methanol (3 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Meti-
crane, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Purity (1) Ammonium <1.02> — Perform the test with 0.10
g of Meticrane. Prepare the control solution with 3.0 mL of
Standard Ammonium Solution (not more than 0.03%).
(2) Heavy metals <1.07>— Proceed with 1.0 g of Meti-
crane according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Meticrane according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.05 g of Meticrane in
50 mL of acetonitrile. To 5 mL of this solution add the mo-
bile phase to make 25 mL, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add the mobile
phase to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly lO/iL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area of both solu-
tions by the automatic integration method: the total area of
the peaks other than meticrane from the sample solution is
not larger than the peak area of meticrane from the standard
solution.
Operating conditions 1 —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (17:3).
Flow rate: Adjust the flow rate so that the retention time of
meticrane is about 7 minutes.
Time span of measurement: About 4 times as long as the
retention time of meticrane beginning after the solvent peak.
System suitability 1 —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of meticrane obtained from 10 [xL
of this solution is equivalent to 7 to 13% of that of meticrane
obtained from 10 /xL of the standard solution.
System performance: Dissolve 0.01 g each of Meticrane
and caffeine in 100 mL of acetonitrile. To exactly 2 mL of
this solution add the mobile phase to make exactly 10 mL.
When the procedure is run with 10 /xL of this solution under
the above operating conditions 1, caffeine and meticrane are
eluted in this order with the resolution between these peaks
being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions 1 , the relative standard deviation of the peak area
of meticrane is not more than 2.0%.
Operating conditions 2 —
Detector, column, and column temperature: Proceed as
directed in the operating conditions 1.
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
meticrane is about 2 minutes.
Time span of measurement: About 10 times as long as the
retention time of meticrane beginning after the solvent peak.
System suitability 2 —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of meticrane obtained from 10 /uL
of this solution is equivalent to 7 to 13% of that of meticrane
obtained from 10 /xL of the standard solution.
System performance: Dissolve 0.02 g each of Meticrane
and methyl parahydroxybenzoate in 100 mL of acetonitrile.
To exactly 2 mL of this solution add the mobile phase to
make exactly 10 mL. When the procedure is run with 10 /xL
of this solution under the above operating conditions 2, meti-
crane and methyl parahydroxybenzoate are eluted in this ord-
er with the resolution between these peaks being not less than
4.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions 2, the relative standard deviation of the peak area
of meticrane is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Meticrane, previ-
ously dried, dissolve in 50 mL of dimethylformamide, add 5
mL of water, and titrate <2.50> with 0.1 mol/L potassium
hydroxide-ethanol VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
JPXV
Official Monographs / Metildigoxin
885
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 27.54 mg of C^H^NC^
Containers and storage Containers — Well-closed contain-
ers.
Metildigoxin
r_A
H3G CHa
C 4 2H 66 1 4. 1 /2C 3 H 6 0: 824.00
3/?-[2,6-Dideoxy-4-0-methyl-/?-D-n7jo-hexopyranosyl-
(l->4)-2,6-dideoxy-/?-D-n7j>o-hexopyranosyl-(l->4)-
2,6-dideoxy-/?-D-n7jo-hexopyranosyloxy]-12/?,14-
dihydroxy-5/?-card-20(22)-enolide — acetone (V\)
[30685-43-9, acetone-free]
Metildigoxin contains not less than 96.0% and not
more than 103.0% of C 42 H 66 14 .i/2C3H 6 0, calculated
on the anhydrous basis.
Description Metildigoxin occurs as a white to light yellow-
ish white, crystalline powder.
It is freely soluble in iV,Af-dimethylformamide, in pyridine
and in acetic acid (100), soluble in chloroform, sparingly
soluble in methanol, slightly soluble in ethanol (95) and in
acetone, very slightly soluble in water, and practically insolu-
ble in diethyl ether.
Identification (1) Dissolve 2 mg of Metildigoxin in 2 mL
of acetic acid (100), shake well with 1 drop of iron (III) chlo-
ride TS, and add gently 2 mL of sulfuric acid to divide into
two layers: a brown color develops at the interface, and a
deep blue color gradually develops in the acetic acid layer.
(2) Dissolve 2 mg of Metildigoxin in 2 mL of 1,3-
dinitrobenzene TS, add 2 mL of a solution of tetramethylam-
monium hydroxide in ethanol (95) (1 in 200), and shake: a
purple color gradually develops, and changes to blue-purple.
(3) Determine the absorption spectrum of a solution of
Metildigoxin in methanol (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Metildigoxin Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectrum of Metil-
digoxin as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Metildigoxin Reference Standard: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers. If any
difference appears between the spectra, dissolve Metildigoxin
and Metildigoxin Reference Standard in acetone, respective-
ly, then evaporate the acetone to dryness, and repeat the test
on the residues.
Optical rotation <2.49> [a]f 46A : +22.0- +25.5° (1 g, calcu-
lated on the anhydrous basis, pyridine, 10 mL, 100 mm).
Purity (1) Arsenic <1.11> — Prepare the test solution with
0.5 g of Metildigoxin according to Method 3, and perform
the test (not more than 4 ppm).
(2) Related substances — Dissolve 10 mg of Metildigoxin
in 10 mL of chloroform, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add chloroform
to make exactly 50 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 2-butanone and chloroform (3:1) to a distance of
about 15 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 110°C for 10 minutes: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Acetone Weigh accurately about 0.1 g of Metildigoxin, dis-
solve in exactly 2 mL of the internal standard solution, add
TV, A r -dimethylformamide to make 10 mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 0.4 g of acetone in a 50-mL volumetric flask containing
about 10 mL of 7V,./V-dimethylformamide, and add N,N-
dimethylformamide to make 50 mL. Pipet 5 mL of this solu-
tion, add exactly 20 mL of the internal standard solution,
then add A^TV-dimethylformamide to make 100 mL, and use
this solution as the standard solution. Perform the test with 1
/xL each of the sample solution and the standard solution as
directed under Gas Chromatography <2.02>, and calculate
the ratios, Qt and Q s , of the peak area of acetone to that of
the internal standard: the amount of acetone is between 2.0%
and 5.0%.
Amount (%) of acetone
= (W S /W T )X(Q T /Q S )
W s : Amount (g) of acetone
W T : amount (g) of the sample
Internal standard solution — A solution of ?-butanol in N,N-
dimethylformamide (1 in 2000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column about 2 mm in inside diameter
and 1 to 2 m in length, packed with porous ethylvinylben-
zene-divinylbenzene copolymer for gas chromatography (150
to 180 [im in particle diameter).
Column temperature: A constant temperature between
170°C and 230°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
acetone is about 2 minutes.
Selection of column: Proceed with 1 /xL of the standard so-
886
Metoclopramide / Official Monographs
JP XV
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of acetone and t-
butanol in this order with the resolution between these peaks
being not less than 2.0.
Water <2.48> Not more than 3.0% (0.3 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately 0.1 g each of Metildigoxin and
Metildigoxin Reference Standard, and dissolve each in
methanol to make exactly 50 mL. Pipet 5 mL each of the so-
lutions, add methanol to each to make exactly 100 mL, and
use these solutions as the sample solution and the standard
solution, respectively. Pipet 5 mL each of the sample solution
and the standard solution, add 15 mL of 2,4,6-
trinitrophenol-ethanol TS and 2 mL of sodium hydroxide TS
to each, shake well, add methanol to make exactly 25 mL,
and allow to stand at 20±0.5°C for 20 minutes. Perform the
test with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24> using a solution prepared by mix-
ing 15 mL of 2,4,6-trinitrophenol-ethanol TS and 2 mL of so-
dium hydroxide TS and adding methanol to make exactly 25
mL as the blank. Determine the maximum absorbances, A T
and A s , of the subsequent solutions obtained from the sam-
ple solution and the standard solution, respectively, by meas-
uring every 5 minutes, at 495 nm.
Amount (mg) of C 42 H 66 Oi 4 . 1 /2C3H 6
= W s x(A T /A s )
W s : Amount (mg) of Metildigoxin Reference Standard,
calculated on the anhydrous basis
Containers and storage Containers — Tight containers.
Metoclopramide
CHu
C 14 H 22 C1N 3 2 : 299.80
4-Amino-5-chloro-iV-[2-(diethylamino)ethyl]-2-
methoxybenzamide [364-62-5]
Metoclopramide, when dried, contains not less than
99.0% of C 14 H 22 C1N 3 2 .
Description Metoclopramide occurs as white crystals or a
crystalline powder, and is odorless.
It is freely soluble in acetic acid (100), soluble in methanol
and in chloroform, sparingly soluble in acetic anhydride, in
ethanol (95) and in acetone, very slightly soluble in diethyl
ether, and practically insoluble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Dissolve 0.01 g of Metoclopramide in 1
mL of dilute hydrochloric acid and 4 mL of water: the solu-
tion responds to the Qualitative Tests <1.09> for Primary
Aromatic Amines.
(2) Dissolve 0.01 g of Metoclopramide in 5 mL of dilute
hydrochloric acid and 20 mL of water, and to 5 mL of this
solution add 1 mL of Dragendorff's TS: a reddish orange
precipitate is produced.
(3) Dissolve 0. 1 g of Metoclopramide in 1 mL of 1 mol/L
hydrochloric acid TS, and dilute with water to make 100 mL.
To 1 mL of the solution add water to make 100 mL, deter-
mine the absorption spectrum of the solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
Melting point <2.60> 146 - 149 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Metoclopramide in 10 mL of 1 mol/L hydrochloric acid TS:
the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Metoclopramide as directed under Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Dissolve 1.0 g of Metoclopramide in
5 mL of 1 mol/L hydrochloric acid TS, and use this solution
as the sample solution. Perform the test (not more than 2
ppm).
(4) Related substances — Dissolve 0.10 g of
Metoclopramide in 10 mL of methanol, and use this solution
as the sample solution. Dilute 1 mL of this solution, exactly
measured, with methanol to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of 1-butanol and ammonia solution (28) (19:1) to a distance
of about 10 cm. Dry the plate, first in air and then at 80°C for
30 minutes. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 0.4 g of Metoclopramide, previously
dried and accurately weighed, in 50 mL of acetic acid (100),
add 5 mL of acetic anhydride, and warm for 5 minutes. Al-
low to cool, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (indicator: 2 drops of crystal violet TS). Perform the
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.98 mg of d 4 H 22 ClN 3 2
Containers and storage Containers — Well-closed contain-
Metoclopramide Tablets
> r-^PT^S Kffi
Metoclopramide Tablets contain not less than 95.0%
and not more than 105.0% of the labeled amount of
JPXV
Official Monographs / Metoprolol Tartrate
887
metoclopramide (C 14 H 22 C1N 3 2 : 299.80).
Method of preparation Prepare as directed under Tablets,
with Metoclopramide.
Identification (1) To a quantity of powdered
Metoclopramide Tablets, equivalent to 50 mg of
Metoclopramide according to the labeled amount, add 15 mL
of 0.5 mol/L hydrochloric acid TS, and heat in a water bath
at 70°C for 15 minutes while frequent shaking. After cooling,
centrifuge for 10 minutes, and to 5 mL of the supernatant liq-
uid add 1 mL of 4-dimethylaminobenzaldehyde-hydrochloric
acid TS: a yellow color develops.
(2) Determine the absorption spectrum of the sample
solution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits maxima between
270 nm and 274 nm, and between 306 nm and 310 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Metoclopramide Tablets add 10 mL of 0.1
mol/L hydrochloric acid TS, disperse the particles with the
aid of ultrasonic waves, then add 0.1 mol/L hydrochloric
acid TS to make exactly 25 mL, and centrifuge for 10
minutes. Pipet 4 mL of the supernatant liquid, add
0.1 mol/L hydrochloric acid TS to make exactly FmL of a
solution so that each mL contains about 12 fig of
metoclopramide (C14H22CIN3O2), and use this solution as the
sample solution. Separately, weigh accurately about 80 mg of
metoclopramide for assay, previously dried at 105°C for 3
hours, and dissolve in 0.1 mol/L hydrochloric acid TS to
make exactly 500 mL. Pipet 4 mL of this solution, add 0.1
mol/L hydrochloric acid TS to make exactly 50 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and the standard
solution at 308 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of metoclopramide (C14H22CIN3O2)
= W s x (A T /A S ) x (F/1000)
W s : Amount (mg) of metoclopramide for assay
Dissolution <6.10> Being specified separately.
Assay Weigh accurately not less than 20 Metoclopramide
Tablets, and powder. Weigh accurately a portion of the pow-
der, equivalent to about 75 mg of metoclopramide
(C14H22CIN3O2), add 300 mL of 0.1 mol/L hydrochloric acid
TS, shake for 1 hour, and add 0.1 mol/L hydrochloric acid
TS to make exactly 500 mL. Centrifuge for 10 minutes, pipet
4 mL of the supernatant liquid, add 0.1 mol/hydrochloric
acid TS to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 80 mg of
metoclopramide for assay, previously dried at 105°C for 3
hours, and dissolve in 0.1 mol/L hydrochloric acid TS to
make exactly 500 mL. Pipet 4 mL of this solution, add 0.1
mol/hydrochloric acid TS to make exactly 50 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and the standard
solution at 308 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of metoclopramide (Q4H22CIN3O2)
= W s x(A T /A s )
W s : Amount (mg) of metoclopramide for assay
Containers and storage Containers — Tight containers.
Metoprolol Tartrate
H OH
H
M^ ,CH a
Y
and enantiomer
H OH
HO^C
CO;H
(C 15 H2 5 N03)2-C 4 H 6 6 : 684.81
(2RS)- 1 - [4-(2-Methoxyethyl)phenoxy]-
3-[(l-methylethyl)amino]propan-2-ol
hemi-(2i?,3i?)-tartrate
[56392-17-7]
Metoprolol Tartrate, when dried, contains not less
than 99.0% and not more than 101.0% of
(C 15 H 25 N0 3 ) 2 .C 4 H 6 6 .
Description Metoprolol Tartrate occurs as a white crystal-
line powder.
It is very soluble in water, and freely soluble in methanol,
in ethanol (95) and in acetic acid (100).
Optical rotation [«]£>: + 7.0 - + 10.0° (after drying, 1 g,
water, 50 mL, 100 mm).
Identification (1) Determine the absorption spectrum of a
solution of Metoprolol Tartrate (1 in 10,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Metoprolol Tartrate, previously dried, as directed in the
paste method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers. If any difference appears between the spectra,
recrystallize the sample from a solution in acetone (23 in
1000), filter and dry the crystals, and perform the test with
the crystals.
(3) A solution of Metoprolol Tartrate (1 in 5) responds to
the Qualitative Tests <1.09> (1) for tartrate.
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Metoprolol Tartrate in 10 mL of water is between 6.0 and
7.0.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Metoprolol Tartrate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.10 g of Metoprolol
Tartrate in 5 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
methanol to make exactly 100 mL. Pipet 2 mL of this solu-
tion, add methanol to make exactly 10 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
888
Metoprolol Tartrate Tablets / Official Monographs
JP XV
on a plate of silica gel for thin-layer chromatography. After
saturating the plate with the atmosphere by allowing to stand
in a developing vessel, which contains the developing solvent
and a glass vessel containing ammonia water (28), develop
with the developing solvent, a mixture of ethyl acetate and
methanol (4:1), to a distance of about 12 cm, and air-dry the
plate. Allow to stand the plate in an iodine vapors until the
spot with the standard solution appears obviously: the spot
other than the principal spot and other than the spot on the
original point is not more than three spots, and they are not
more intense than the spot with the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Metoprolol Tar-
trate, previously dried, dissolve in 50 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination in the
same manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.24 mg of (C 15 H 25 N0 3 )2.C 4 H 6 6
Containers and storage Containers — Well-closed contain-
ers.
Metoprolol Tartrate Tablets
Metoprolol Tartrate Tablets contain not less than
93.0% and not more than 107.0% of the labeled
amount of metoprolol tartrate ((C 15 H25N03)2.C 4 H 6 6 :
684.81).
Method of preparation Prepare as directed under Tablets,
with Metoprolol Tartrate.
Identification To an amount of powdered Metoprolol Tar-
trate Tablets, equivalent to 10 mg of Metoprolol Tartrate ac-
cording to the labeled amount, add 100 mL of ethanol (95),
shake for 15 minutes, and filter. Determine the absorption
spectrum of the filtrate as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 274
nm and 278 nm and between 281 nm and 285 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
To 1 tablet of Metoprolol Tartrate Tablets add 1 mL of
water for every 10 mg of Metoprolol Tartrate, shake for 20
minutes, then add 75 mL of ethanol (95), shake for 15
minutes, add ethanol (95) to make exactly 100 mL, and cen-
trifuge. Pipet KmL of the supernatant liquid, add ethanol
(95) to make exactly V so that each mL contains about 0.1
mg of metoprolol tartrate ((Qsl-^NO^.QI-LCv), and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of metoprolol tartrate for assay, previ-
ously dried in vacuum at 60°C for 4 hours, dissolve in 5 mL
of water, and add ethanol (95) to make exactly 100 mL. Pipet
10 mL of this solution, add ethanol (95) to make exactly 50
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and the
standard solution at 276 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using ethanol (95) as the
blank.
Amount (mg) of metoprolol tartrate ((C 15 H25NC) 3 )2.C 4 ]-[ 6 6 )
= W s x (A T /A S ) x(V'/V)x (1/5)
W s : Amount (mg) of metoprolol tartrate for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Metoprolol Tartrate
Tablets at 50 revolutions per minute according to the Paddle
method using 900 mL of water as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 30
minutes after starting the test, and filter through a membrane
filter with a pore size not exceeding 0.5 //m. Discard the first
10 mL of the filtrate, pipet the subsequent KmL, add water
to make exactly V mL so that each mL contains about 22 /ug
of metoprolol tartrate ((C 15 H2 5 N0 3 )2.C 4 H 6 6 ) according to
the labeled amount, and use this solution as the sample solu-
tion. Separately, weigh accurately about 56 mg of metoprolol
tartrate for assay, previously dried in vacuum at 60°C for 4
hours, and dissolve in water to make exactly 200 mL. Pipet 8
mL of this solution, add water to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 50 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of metoprolol. The dissolution rate in 30
minutes is not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
metoprolol tartrate ((C 15 H 2 5N0 3 )2.C 4 H 6 6 )
= W s x (A T /A S ) x(V'/V)x (1/Q x 36
W s : Amount (mg) of metoprolol tartrate for assay
C: Labeled amount (mg) of metoprolol tartrate
((C 15 H 25 N0 3 )2.C 4 H 6 6 ) in 1 tablet
Operating conditions —
Proceed as directed in the Assay.
System suitability —
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of metoprolol are not less than 2000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
50 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
metoprolol is not more than 2.0%.
Assay Weigh accurately the mass of not less than 20
Metoprolol Tartrate Tablets, and powder. Weigh accurately
a portion of the powder, equivalent to about 0.12 g of
metoprolol tartrate ((C 15 H 25 N0 3 )2.C 4 H 6 6 ), add 60 mL of a
mixture of ethanol (99.5) and 1 mol/L hydrochloric acid TS
(100:1) and exactly 10 mL of the internal standard solution,
shake for 15 minutes, and add the mixture of ethanol (99.5)
and 1 mol/L hydrochloric acid TS (100:1) to make 100 mL.
Centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 0.12 g of metoprolol
tartrate for assay, previously dried in vacuum at 60°C for 4
hours, dissolve in 60 mL of the mixture of ethanol (99.5) and
JPXV
Official Monographs / Metronidazole Tablets
889
1 mol/L hydrochloric acid TS (100:1), add exactly 10 mL of
the internal standard solution, then add the mixture of
ethanol (99.5) and 1 mol/L hydrochloric acid TS (100:1) to
make 100 mL, and use this solution as the standard solution.
Perform the test with 10,mL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, g T and Q s , of the peak area of metoprolol to that
of the internal standard.
Amount (mg) of metoprolol tartrate ((C 15 H 2 5N03)2.C 4 H 6 6 )
= W s X(Q r /Q£
W s : Amount (mg) of metoprolol tartrate for assay
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in the mixture of ethanol (99.5) and 1 mol/L
hydrochloric acid TS (100:1) (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 14.0 g of sodium perchlorate
monohydrate in 1000 mL of water, and adjust to pH 3.2 with
diluted perchloric acid (17 in 2000). To 750 mL of this solu-
tion add 250 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
metoprolol is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
[iL of the standard solution under the above operating condi-
tions, metoprolol and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of metoprolol to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Metronidazole
/ hp-y7-JU
C 6 H 9 N 3 3 : 171.15
2-(2-Methyl-5-nitro-l//-imidazol-l-yl)ethanol [443-48-1]
Metronidazole, when dried, contains not less than
99.0% and not more than 101.0% of QH9N3O3.
Description Metronidazole occurs as white to pale yellow-
ish white crystals or crystalline powder.
It is freely soluble in acetic acid (100), sparingly soluble in
ethanol (99.5) and in acetone, and slightly soluble in water.
It dissolves in dilute hydrochloric acid.
It is colored to yellow-brown by light.
Identification (1) Determine the absorption spectrum of a
solution of Metronidazole in 0.1 mol/L hydrochloric acid TS
(1 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Metronidazole as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 159 - 163 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Metronidazole according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) 2-Methyl-5-nitroimidazol— Dissolve 0.10 g of
Metronidazole in acetone to make exactly 10 mL, and use this
solution as the sample solution. Separately, dissolve 20 mg of
2-methyl-5-nitroimidazole for thin-layer chromatography in
acetone to make exactly 20 mL, then pipet 5 mL of this solu-
tion, add acetone to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Immediately develop the plate with a mix-
ture of acetone, water and ethyl acetate (8:1:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot from the sam-
ple solution corresponding to the spot from the standard so-
lution is not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Metronidazole,
previously dried, and dissolve in 30 mL of acetic acid (100).
Titrate <2.50> with 0.1 mol/L perchloric acid VS (indicator:
0.5 mL of j9-naphtholbenzein TS) until the color of the solu-
tion changes from orange-yellow to green. Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 17.12 mg of C 6 H 9 N 3 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Metronidazole Tablets
Metronidazole Tablets contain not less than 93.0%
890
Metyrapone / Official Monographs
JP XV
and not more than 107.0% of the labeled amount of
metronidazole (C 6 H 9 N 3 3 : 171.15).
Method of preparation Prepare as directed under Tablets,
with Metronidazole.
Identification (1) Determine the absorption spectrum of
the sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 275 nm and 279 nm.
(2) Shake vigorously a quantity of powdered Metronida-
zole Tablets, equivalent to 0.20 g of Metronidazole according
to the labeled amount, with 20 mL of acetone for 10 minutes,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 0.10 g of metronidazole in 10 mL
of acetone, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 [iL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography, develop
the plate immediately with a mixture of acetone, water and
ethyl acetate (8:1:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the Rf value of the principal spots obtained from
the sample solution and the standard solution is the same.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Metronidazole Tablets add 25 mL of a mix-
ture of water and methanol (1:1), shake vigorously for 25
minutes, and add the mixture of water and methanol (1:1) to
make exactly 50 mL. Pipet 5 mL of this solution, and add a
mixture of water and methanol (4:1) to make exactly 100 mL.
Filter the solution through a membrane filter with pore size
of 0.45 //m, discard the first 3 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Hereinafter, pro-
ceed as directed in the Assay.
Amount (mg) of metronidazole (C6H9N3O3)
= W s x(A T /A s )x 10
W s : Amount (mg) of metronidazole for assay
Assay Weigh accurately the mass of not less than 20
Metronidazole Tablets, and powder. Weigh accurately a por-
tion of the powder, equivalent to about 0.25 g of metronida-
zole (QH9N3O3), add 25 mL of a mixture of water and
methanol (1:1), shake vigorously for 10 minutes, and add the
mixture of water and methanol (1:1) to make exactly 50 mL.
Pipet 5 mL of this solution, and add a mixture of water and
methanol (4:1) to make exactly 100 mL. Filter this solution
through a membrane filter with pore size of 0.45 //m, discard
the first 3 mL of the filtrate, and use the subsequent filtrate as
the sample solution. Separately, weigh accurately about 25
mg of metronidazole for assay, previously dried in vacuum
on silica gel for 24 hours, dissolve in the mixture of water and
methanol (4:1) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 10 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of metronidazole.
Amount (mg) of metronidazole (C 6 H 9 N 3 3 )
= W s x(A T /A s )x 10
W s : Amount (mg) of metronidazole for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 320 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and methanol (4:1).
Flow rate: Adjust the flow rate so that the retention time of
metronidazole is about 5 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of metronidazole are not less than 3000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
metronidazole is not more than 1.0%.
Containers and storage Containers — Tight containers.
Metyrapone
y^*°>
H3C CHa
2-Methyl-l,2-di(pyridin-3-yl)propan-l-one [54-36-4]
Metyrapone, when dried, contains not less than
98.0% of C 14 H 14 N 2 0.
Description Metyrapone occurs as a white to pale yellow,
crystalline powder. It has a characteristic odor and a bitter
taste.
It is very soluble in methanol, in ethanol (95), in acetic an-
hydride, in chloroform, in diethyl ether and in nitrobenzene,
and sparingly soluble in water.
It dissolves in 0.5 mol/L sulfuric acid TS.
Identification (1) Mix 5 mg of Metyrapone with 0.01 g of
l-chloro-2,4-dinitrobenzene, melt by gently heating for 5 to 6
seconds, cool, and add 4 mL of potassium hydroxide-ethanol
TS: a dark red color develops.
(2) Determine the absorption spectrum of a solution of
Metyrapone in 0.5 mol/L sulfuric acid TS (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
Melting point <2.60> 50 - 54°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
JPXV
Official Monographs / Mexiletine Hydrochloride
891
Metyrapone in 5 mL of methanol: the solution is clear and
colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Metyra-
pone according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than lOppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Metyrapone, according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.25 g of Metyrapone
in 5 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add methanol to
make exactly 50 mL. Pipet 5 mL of this solution, add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2//L
each of the sample solution and standard solution on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of chloroform
and methanol (15:1) to a distance of about 10 cm, and air-dry
the plate for about 15 minutes. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution is not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Metyrapone, previ-
ously dried, dissolve in 10 mL of nitrobenzene and 40 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 11.31 mg of C 14 H 14 N 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Mexiletine Hydrochloride
^+->Uf>M^
CH 3 H CH 3
• HCI
and enant iomer
C n H 17 NO.HCl: 215.72
( 1 RS)-2-(2 ,6-Dimethylphenoxy)- 1 -methylethylamine
monohydrochloride [5370-01-4]
Mexiletine Hydrochloride, when dried, contains not
less than 98.0% and not more than 102.0% of
C n H 17 NO.HCl.
Description Mexiletine Hydrochloride occurs as a white
powder.
It is freely soluble in water and in ethanol (95), slightly
soluble in acetonitrile, and practically insoluble in diethyl
ether.
A solution of Mexiletine Hydrochloride (1 in 20) shows no
optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Mexiletine Hydrochloride in 0.01 mol/L
hydrochloric acid TS (1 in 2000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
solution of Mexiletine Hydrochloride Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Mex-
iletine Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of dried Mexiletine
Hydrochloride Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers. If
any difference appears between the spectra, recrystallize the
sample from ethanol (95), filter, dry the crystals, and repeat
the test on the crystals.
(3) A solution of Mexiletine Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Mexiletine Hydrochloride in 10
mL of water: the pH of this solution is between 3.8 and 5.8.
Melting point <2.60> 200 - 204°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Mexiletine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy Metals <7. 07>— Proceed with 2.0 g of Mexile-
tine Hydrochloride according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 20 mg of Mexiletine
Hydrochloride in 20 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 250 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of both solutions by the automatic integration method:
each peak area of the peaks other than the peak of mexiletine
from the sample solution is not larger than the peak area of
mexiletine from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed as directed in the
operating conditions in the Assay.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of mexiletine obtained from 20 fiL of the
standard solution is between 5 mm and 10 mm.
Time span of measurement: About 3 times as long as the
retention time of mexiletine beginning after peaks of the sol-
vent.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 20 mg each of Mexiletine
Hydrochloride and Mexiletine Hydrochloride Reference
892
Miconazole / Official Monographs
JP XV
Standard, each previously dried, and dissolve each in the mo-
bile phase to make exactly 20 mL. Pipet 5 mL each of these
solutions, add exactly 5 mL of the internal standard solution,
then add the mobile phase to make 100 mL, and use these so-
lutions as the sample solution and the standard solution,
respectively. Perform the test with 20 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Qt and Q s , of the peak area of
mexiletine to that of the internal standard, respectively.
Amount (mg) of C u H 17 NO.HCl
= W s x «2t/<2s)
W s : Amount (mg) of Mexiletine Hydrochloride Reference
Standard
Internal standard solution — A solution of phenetylamine
hydrochloride in the mobile phase (3 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octylsilanized
silica gel for liquid chromatography (about 1 fim in particle
diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 2.5 g of sodium lauryl sulfate and
3 g of sodium dihydrogenphosphate dihydrate in 600 mL of
water, and add 420 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
mexiletine is about 6 minutes.
Selection of column: Proceed with 20 iiL of the standard
solution under the above conditions, and calculate the resolu-
tion. Use a column giving elution of the internal standard and
mexiletine in this order with the resolution between these
peaks being not less than 9.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Miconazole
and enantiomer
C 18 H 14 Cl4N 2 0: 416.13
l-[(2i?S)-2-(2,4-Dichlorobenzyloxy)-2-(2,4-
dichlorophenyl)ethyl]-l//-imidazole [22916-47-8]
acid (100), soluble in diethyl ether, and practically insoluble
in water.
A solution of Miconazole in methanol (1 in 20) shows no
optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Miconazole in methanol (1 in 2500) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Miconazole, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
turn: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 84 - 87°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Miconazole according to Method 2, and perform the test.
Prepare the control solution with 1.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Miconazole according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0. 10 g of Miconazole in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 20 mL. Pipet 1 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 50
iiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of hexane, chloroform, methanol and
ammonia solution (28) (60:30:10:1) to a distance of about 12
cm, and air-dry the plate. Allow the plate to stand in iodine
vapor for 20 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 60%, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Miconazole, previ-
ously dried, dissolve in 40 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 3 drops
of p-naphtholbenzein TS) until the color of the solution
changes from light yellow-brown to light yellow-green. Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 41.61 mg of C I8 H 14 C1 4 N 2
Containers and storage Containers — Tight containers.
Miconazole, when dried, contains not less than
98.5% of C 18 H 14 C1 4 N 2 0.
Description Miconazole occurs as a white to pale yellowish
white, crystalline powder.
It is freely soluble in methanol, in ethanol (95) and in acetic
JPXV
Official Monographs / Micronomicin Sulfate
893
Miconazole Nitrate
and enantiomer
C 18 H 14 C1 4 N 2 0.HN0 3 : 479.14
l-[(2i?S)-2-(2,4-Dichlorobenzyloxy)-2-(2,4-
dichlorophenyl)ethyl]-l//-imidazole mononitrate
[22832-87-7]
Miconazole Nitrate, when dried, contains not less
than 98.5% of C 18 H 14 Cl 4 N 2 O.HN03.
Description Miconazole Nitrate occurs as a white crystal-
line powder.
It is freely soluble in 7V,./V-dimethylformamide, sparingly
soluble in methanol, slightly soluble in ethanol (95), in ace-
tone and in acetic acid (100), and very slightly soluble in
water and in diethyl ether.
Melting point: about 180°C (with decomposition).
Identification (1) To 2 mL of a solution of Miconazole
Nitrate in methanol (1 in 100) add 2 mL of Reinecke salt TS:
a light red precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Miconazole Nitrate in methanol (1 in 2500) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Perform the test with a solution of Miconazole Ni-
trate in methanol (1 in 100) as directed under Flame Colora-
tion Test <1.04> (2): a green color appears.
(4) A solution of Miconazole Nitrate in methanol (1 in
100) responds to the Qualitative Tests <1.09> for nitrate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Miconazole Nitrate in 100 mL of methanol: the solution is
clear and colorless.
(2) Chloride <1.03>— Dissolve 0.10 g of Miconazole Ni-
trate in 6 mL of dilute nitric acid and 7V,7V-dimethylfor-
mamide to make 50 mL. Perform the test using this solution
as the test solution. Prepare the control solution as follows:
to 0.25 mL of 0.01 mol/L hydrochloric acid VS add 6 mL of
dilute nitric acid and 7V,7V-dimethylformamide to make 50
mL (not more than 0.09%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Micona-
zole Nitrate according to Method 2, and perform the test.
Prepare the control solution with 1.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Miconazole Nitrate according to Method 3, and perform
the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Miconazole
Nitrate in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 20 mL, pipet 1 mL of this solution,
add methanol to make exactly 20 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
50 iuL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of M-hexane, chloroform, methanol and
ammonia solution (28) (60:30:10:1) to a distance of about 12
cm, and air-dry the plate. Allow the plate in iodine vapor for
20 minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Miconazole Ni-
trate, previously dried, dissolve in 50 mL of acetic acid (100)
by warming, cool, and titrate <2.50> with 0.1 mol/L perchlor-
ic acid VS (potentiometric titration). Perform a blank deter-
mination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 47.91 mg of C 18 H 14 C1 4 N 2 0.HN0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Micronomicin Sulfate
?uj -7<{'»vmm
2-j-H 2 SO,
C 20 H 41 N 5 O 7 .2i/ 2 H 2 SO 4 : 708.77
2-Amino-2,3,4,6-tetradeoxy-6-methylamino-a-D-
erythro-hexopyranosyl-(l -> 4)- [3 -deoxy-4-C-methyl-
3-methylamino-/?-L-arabinopyranosyl-(l -> 6)]-2-deoxy-D-
streptamine hemipentasulfate
[52093-21-7, Micronomicin]
Micronomicin Sulfate is the sulfate of an
aminoglycoside substance having antibacterial activity
produced by the growth of Micromonospora
sagamiensis.
It contains not less than 590 ^g (potency) and not
more than 660 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Micronomicin Sulfate
is expressed as mass (potency) of micronomicin
(C 20 H 41 N 5 O 7 : 463.57).
Description Micronomicin Sulfate occurs as a white to light
894
Midecamycin / Official Monographs
JP XV
yellowish white powder.
It is very soluble in water, sparingly soluble in ethylene
glycol, and practically insoluble in methanol and in ethanol
(99.5).
It is hygroscopic.
Identification (1) Dissolve 50 mg each of Micronomicin
Sulfate and Micronomicin Sulfate Reference Standard in 10
mL of water, and use these solutions as the sample solution
and the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethanol (99.5), 1-buthanol and ammo-
nia solution (28) (10:8:7) to a distance of about 10 cm, and
air-dry the plate. Spray evenly a solution of ninhydrin in a
mixture of acetone and pyridine (25:1) (1 in 500), and heat at
100°C for 10 minutes: the spots obtained from the sample so-
lution and the standard solution are red-purple to red-brown
and their Rf values are the same.
(2) To 5 mL of a solution of Micronomicin Sulfate (1 in
100) add 1 mL of barium chloride TS: a white precipitate is
formed, and it does not dissolve by addition of dilute nitric
acid.
Optical rotation <2.49> [a]o- + 110 - + 130° (0.25 g calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Micronomicin Sulfate in 10 mL of water is between 3.5
and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .5 g of
Micronomicin Sulfate in 10 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Micronomicin Sulfate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.40 g of Micronomicin
Sulfate in 10 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /xL of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethanol (99.5), 1-buthanol and ammonia solution (28)
(10:8:7) to a distance of about 10 cm, and air-dry the plate.
Spray evenly a solution of ninhydrin in a mixture of acetone
and pyridine (25:1) (1 in 500), and heat at 100°C for 10
minutes: the spot other than the principal spot obtained from
the sample solution is not more intense than the spot from the
standard solution.
Water <2.48> Not more than 10.0% (0.2 g, volumetric titra-
tion, back titration). Use a mixture of methanol for water de-
termination and ethylene glycol for water determination (1:1)
instead of methanol for water determination.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er.
(iii) Standard solutions — Weigh accurately an amount of
Micronomicin Sulfate Reference Standard, equivalent to
about 20 mg (potency), dissolve in 0.1 mol/L phosphate
buffer solution for antibiotics, pH 8.0 to make exactly 20
mL, and use this solution as the standard stock solution.
Keep the standard stock solution at 5 - 15°C, and use within
30 days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solution
for antibiotics, pH 8.0 to make solutions so that each mL
contains 2 ng (potency) and 0.5 fig (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Micronomicin Sulfate, equivalent to about 20 mg (potency),
and dissolve in 0.1 mol/L phosphate buffer solution for an-
tibiotics, pH 8.0 to make exactly 20 mL. Take exactly a suita-
ble amount of this solution, add 0.1 mol/L phosphate buffer
solution for antibiotics, pH 8.0 to make solutions so that
each mL contains 2//g (potency) and 0.5 fig (potency), and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Midecamycin
Tt>~?4
H OH
H 3 C^A o J „
CH 3
C 4 iH 67 N0 15 : 813.97
(3R,4R,5S,6R,8R,9R,10E,12E,15R)-
5-[2,6-Dideoxy-3-C-methyl-4-0-propanoyl-a-L-n7j>o-
hexopyranosyl-(l- > 4)-3,6-dideoxy-3-dimethylamino-/6-D-
glucopyranosyloxy]-6-formylmethyl-9-hydroxy-4-methoxy-
8-methyl-3-propanoyloxyhexadeca-10,12-dien-15-olide
[35457-80-8]
Midecamycin is a macrolide substance having an-
tibacterial activity produced by the growth of Strep-
tomyces mycarofaciens.
It contains not less than 950 Lig (potency) and not
more than 1020 Lig (potency) per mg, calculated on the
dried basis. The potency of Midecamycin is expressed
as mass (potency) of midecamycin (C 41 H 67 N0 15 ).
Description Midecamycin occurs as a white crystalline pow-
der.
It is very soluble in methanol, freely soluble in ethanol
(95), and very slightly soluble in water.
Identification (1) Determine the absorption spectrum of a
JPXV
Official Monographs / Midecamycin Acetate
895
solution of Midecamycin in methanol (1 in 50,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Midecamycin Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Midecamycin as directed in the potassium bromide disk
method under the Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Midecamycin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 153 - 158°C
Purity Heavy metals <1.07> — Proceed with 1.0 g of
Midecamycin according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
Loss on drying <2.41> Not more than 2.0% (1.0 g, in vacu-
um not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Weigh accurately an amount of
Midecamycin Reference Standard, previously dried, equiva-
lent to about 20 mg (potency), dissolve in 10 mL of
methanol, add water to make exactly 50 mL, and use this so-
lution as the standard stock solution. Keep the standard stock
solution at 5°C or below and use within 7 days. Take exactly
a suitable amount of the standard stock solution before use,
add 0.1 mol/L phosphate buffer solution, pH 8.0 to make so-
lutions so that each mL contains 20 fig (potency) and 5 fig
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Midecamycin, previously dried, equivalent to about 20 mg
(potency), dissolve in 10 mL of methanol, and add water to
make exactly 50 mL. Take exactly a suitable amount of the
solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to
make solutions so that each mL contains 20 fig (potency) and
5 ug (potency), and use these solutions as the high concentra-
tion sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Midecamycin Acetate
fa"
o
C 45 H 71 N0 17 : 898.04
(3i?,4S,5S,6fl,8i?,9i?,10£',12£',15i?)-9-Acetoxy-5-
[3-0-acetyl-2,6-dideoxy-3-C-methyl-4-0-propanoyl-
a-L-ri£o-hexopyranosyl-(l->4)-3,6-dideoxy-3-
dimethylamino-/?-D-glucopyranosyloxy]-6-formylmethyl-
4-methoxy-8-methyl-3-propanoyloxyhexadeca-10,12-dien-
15-olide [55881-07-7]
Midecamycin Acetate is a derivative of midecamy-
cin.
It contains not less than 950 fig (potency) and not
more than 1010 fig (potency) per mg, calculated on the
dried basis. The potency of Midecamycin Acetate is ex-
pressed as mass of midecamycin acetate (C 4 5H 71 N0 1 7).
Description Midecamycin Acetate occurs as white, crystals
or crystalline powder.
It is sparingly soluble in methanol, slightly soluble in
ethanol (95), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Midecamycin Acetate in methanol (1 in 50,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Midecamycin Acetate Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Midecamycin Acetate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or spectrum of dried Midecamycin
Acetate Reference Standard: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Purity Heavy metals <1.07> — Proceed with 1.0 g of
Midecamycin Acetate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 2.0% (0.1 g, in vacu-
um not exceeding 0.67 kPa, 60°C, 3 hours).
896
Migrenin / Official Monographs
JP XV
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Micrococcus luteus ATCC 9341
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base lay-
er.
(iii) Standard solutions — Weigh accurately an amount of
Midecamycin Acetate Reference Standard, previously dried,
equivalent to about 25 mg (potency), and dissolve in
methanol to make exactly 50 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
5 - 15 °C and use within 7 days. Take exactly a suitable
amount of the standard stock solution before use, add 0.1
mol/L phosphate buffer solution, pH 4.5 to make solutions
so that each mL contains 20 /ug (potency) and 5 /ug (potency),
and use these solutions as the high concentration standard so-
lution and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Midecamycin Acetate, previously dried, equivalent to about
25 mg (potency), and dissolve in methanol to make exactly 50
mL. Take exactly a suitable amount of the solution, add 0.1
mol/L phosphate buffer solution, pH 4.5 to make solutions
so that each mL contains 20 /ug (potency) and 5 /ug (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Migrenin
Migrenin is composed of 90 parts of antipyrine, 9
parts of caffeine, and 1 part of citric acid in mass.
Migrenin, when dried, contains not less than 87.0%
and not more than 93.0% of antipyrine (C n H 12 N20:
188.23) and not less than 8.6% and not more than
9.5% of caffeine (C 8 H 10 N 4 O 2 : 194.19).
Description Migrenin occurs as a white powder or crystal-
line powder. It is odorless and has a bitter taste.
It is very soluble in water, freely soluble in ethanol (95) and
in chloroform, and slightly soluble in diethyl ether.
The pH of a solution of Migrenin (1 in 10) is between 3.0
and 4.0.
It is affected by moisture and light.
Identification (1) To 5 mL of a solution of Migrenin (1 in
100) add 2 drops of sodium nitrite TS and 1 mL of dilute sul-
furic acid: a deep green color develops.
(2) To 5 mL of a solution of Migrenin (1 in 50) add 1
drop of hydrochloric acid and 0.2 mL of formaldehyde solu-
tion, heat in a water bath for 30 minutes, add an excess of
ammonia TS, and filter. Acidify the filtrate with hydrochloric
acid, shake with 3 mL of chloroform, and separate the chlo-
roform layer. Evaporate the chloroform solution on a water
bath, add 10 drops of hydrogen peroxide TS and 1 drop of
hydrochloric acid to the residue, and evaporate on a water
bath to dryness: the residue shows a yellow-red color. Invert
the residue over a vessel containing 3 drops of ammonia TS: a
red-purple color develops, disappearing on the addition of 2
to 3 drops of sodium hydroxide TS.
(3) A solution of Migrenin (1 in 10) responds to the
Qualitative Tests <1.09> for citrate.
Melting point <2.60> 104 - 110°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Migrenin in 40 mL of water: the solution is clear and color-
less to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Migrenin
according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay (1) Antipyrine — Weigh accurately about 0.25 g of
Migrenin, previously dried in an iodine flask, dissolve in 25
mL of sodium acetate TS, add exactly 30 mL of 0.05 mol/L
iodine VS, and allow to stand for 20 minutes with occasional
shaking. Add 15 mL of chloroform to dissolve the precipitate
so obtained, and titrate <2.50> the excess iodine with 0.1 mol/
L sodium thiosulfate VS (indicator: 3 mL of starch TS). Per-
form a blank determination.
Each mL of 0.05 mol/L iodine VS
= 9.411 mg of C n H 12 N 2
(2) Caffeine — To about 1 g of Migrenin, previously dried
and accurately weighed, add exactly 5 mL of the internal
standard solution, dissolve in chloroform to make 10 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 90 mg of Caffeine Reference Stan-
dard, previously dried at 80°C for 4 hours, add exactly 5 mL
of the internal standard solution, dissolve in chloroform to
make 10 mL, and use this solution as the standard solution.
Perform the test with 1 /uL each of the sample solution and
standard solution as directed under Gas Chromatography
<2.02> according to the following conditions, and calculate
the ratios, g T and Q s , of the peak area of caffeine to that of
the internal standard.
Amount (mg) of caffeine (C 8 H 10 N 4 O 2 )= W s x (Q T /Q S )
W s : Amount (mg) of Caffeine Reference Standard
Internal standard solution — A solution of ethenzamide in
chloroform (1 in 50).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 2.6 mm in inside diameter and
210 cm in length, packed with siliceous earth for gas chro-
matography (180 to 250 /um in particle diameter) coated with
50% phenyl-methyl silicon polymer for gas chromatography
at the ratio of 15%.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethenzamide is about 4 minutes.
System suitability —
System performance: Dissolve 0.9 g of antipyrine and 0.09
JPXV
Official Monographs / Minocycline Hydrochloride
897
g of caffeine in 10 mL of chloroform. When the procedure is
run with 1 /uL of this solution under the above operating con-
ditions, caffeine and antipyrine are eluted in this order with
the resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
1 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of caffeine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Minocycline Hydrochloride
$ / tK ? U >±£Sgi£
NH S
N— CH 3
HjC
C 23 H 27 N 3 7 .HC1: 493.94
(45,4a5,5ai?, 12aS)-4,7-Bis(dimethylamino)-3, 10, 12,12a-
tetrahydroxy-l,ll-dioxo-l,4,4a,5,5a,6,ll,12a-
octahydrotetracene-2-carboxamide monohydrochloride
[13614-98-7]
Minocycline Hydrochloride is the hydrochloride of a
derivative of tetracycline.
It contains not less than 890 /ug (potency) and not
more than 950 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Minocycline
Hydrochloride is expressed as mass (potency) of
minocycline (C 23 H 27 N 3 7 : 457.48).
Description Minocycline Hydrochloride occurs as a yellow
crystalline powder.
It is freely soluble in A^TV-dimethylformamide, soluble in
methanol, sparingly soluble in water, and slightly soluble in
ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Minocycline Hydrochloride in a solution of
hydrochloric acid in methanol (19 in 20,000) (1 in 62,500) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Minocycline Hydrochloride
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Minocycline Hydrochloride as directed in the potassium chlo-
ride disk method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum or
the spectrum of Minocycline Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Minocycline Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Minocycline Hydrochloride in
100 mL of water: the pH of the solution is between 3.5 and
4.5.
Purity (1) A solution of Minocycline Hydrochloride (1 in
100) is clear, and when the test is performed within 1 hour af-
ter preparation of this solution, the absorbance of the solu-
tion at 560 nm, determined as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, is not more than 0.06.
(2) Heavy metals <1.07> — Proceed with 0.5 g of Minocy-
cline Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.5 mL of Standard
Lead Solution (not more than 50 ppm).
(3) Related substances — Dissolve 50 mg of Minocycline
Hydrochloride in 100 mL of the mobile phase, and use this
solution as the sample solution. Perform the test immediately
after the preparation of the sample solution with 20 fiL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and measure
each peak area by the automatic integration method. Calcu-
late the amount of each peak area by the area percentage
method: the amount of epiminocycline is not more than 1.2
%, the amount of each peak other than minocycline and
epiminocycline is not more than 1.0%, and the total area of
the peaks other than minocycline and epiminocycline is not
more than 2.0%.
Operating conditions —
Detector, column, column temperature, and mobile phase:
Proceed as directed in the operating conditions in the Assay.
Flow rate: Adjust the flow rate so that the retention time of
minocycline is about 12 minutes. The retention time of
epiminocycline is about 10 minutes under this condition.
Time span of measurement: About 2.5 times as long as the
retention time of minocycline beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the sample
solution add the mobile phase to make exactly 100 mL, and
use this solution as the solution for system suitability test.
Pipet 5 mL of the solution for system suitability test, and add
the mobile phase to make exactly 100 mL. Confirm that the
peak area of minocycline obtained from 20 fiL of this solu-
tion is equivalent to 3.5 to 6.5% of that of minocycline ob-
tained from 20,mL of the solution for system suitability test.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 fiL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of minocycline is not more than 2.0%.
Water <2.48> Not less than 4.3% and not more than 8.0%
(0.3 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately an amount of Minocycline
Hydrochloride and Minocycline Hydrochloride Reference
Standard, equivalent to about 50 mg (potency), dissolve each
in the mobile phase to make exactly 100 mL, and use these so-
lutions as the sample solution and the standard solution. Per-
form the test with exactly 20 fiL each of the sample solution
and the standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the peak areas, A T and A s , of minocycline of
these solutions.
898
Mitomycin C / Official Monographs
JP XV
Amount [fig (potency)] of minocycline (C23H 2 7N 3 7 )
= W s x(A T /A s )x 1000
W s : Amount [mg (potency)] of Minocycline Hydrochlo-
ride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Adjust the pH of a mixture of a solution of
ammonium oxalate monohydrate (7 in 250), A^iV-dimethyl-
formamide and 0.1 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate TS (11:5:4) to 6.2 with tetrabutylammoni-
um hydroxide TS.
Flow rate: Adjust the flow rate so that the retention time of
minocycline is about 12 minutes.
System suitability —
System performance: Dissolve 0.05 g (potency) of Minocy-
cline Hydrochloride Reference Standard in 25 mL of water.
Heat 5 mL of this solution on a water bath for 60 minutes,
then add water to make 25 mL. When the procedure is run
with 20 fiL of this solution under the above operating condi-
tions, epiminocycline and minocycline are eluted in this order
with the resolution between these peaks being not less than
2.0.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of peak areas of
minocycline is not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Mitomycin C
"7 -4 h "7 4 -> > C
C 15 H 18 N 4 05: 334.33
(laS,8S,8a.R,8bS)-6-Amino-4 ) 7-dioxo-8a-methoxy-5-
methyl-l,la,2,8,8a,8b-
hexahydroazirino[2',3':3,4]pyrrolo[l,2-a]indol-
8-ylmethyl carbamate [50-07-7]
Mitomycin C is a substance having antitumor activi-
ty produced by the growth of Streptomyces caespito-
sus.
It contains not less than 91 '0 fig (potency) and not
more than 1030 fig (potency) per mg, calculated on the
dried basis. The potency of Mitomycin C is expressed
as mass (potency) of mitomycin C (C 15 H 18 N 4 5 ).
Description Mitomycin C occurs as blue-purple, crystals or
crystalline powder.
It is freely soluble in Af.Af-dimethylacetamide, slightly solu-
ble in water and in methanol, and very slightly soluble in
ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Mitomycin C (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Mitomycin C Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Mitomycin C as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Mitomycin C Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity Related substances — Conduct this procedure rapidly
after the sample and the standard solutions are prepared.
Dissolve 50 mg of Mitomycin C in 10 mL of methanol, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add methanol to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 10 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: each area of the
peak other than mitomycin C obtained from the sample solu-
tion is not more than the peak area of mitomycin C from the
standard solution, and the total area of the peaks other than
mitomycin C from the sample solution is not more than 3
times the peak area of mitomycin C from the standard solu-
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ftm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase A: To 20 mL of 0.5 mol/L ammonium
acetate TS add water to make 1000 mL. To 800 mL of this so-
lution add 200 mL of methanol.
Mobile phase B: To 20 mL of 0.5 mol/L ammonium
acetate TS add water to make 1000 mL. To this solution add
1000 mL of methanol.
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following ta-
ble.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0- 10
10-30
30-45
100
100^0
0^100
100
Flow rate: About l.OmL/min
Time span of measurement: About 2 times as long as the
JPXV
Official Monographs / Morphine and Atropine Injection
899
retention time of mitomycin C beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 10 mL of the standard
solution, and add methanol to make exactly 100 mL. Con-
firm that the peak area of mitomycin C obtained from 10 iiL
of this solution is equivalent to 7 to 13% of that from 10,mL
of the standard solution.
System performance: Dissolve 25 mg of Mitomycin C and
40 mg of 3-ethoxy-4-hydroxybenzaldehyde in 50 mL of
methanol. When the procedure is run with 10 iiL of this solu-
tion under the above operating conditions, mitomycin C and
3-ethoxy-4-hydroxybenzaldehyde are eluted in this order with
the resolution between these peaks being not less than 15.
System repeatability: When the test is repeated 3 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
mitomycin C is not more than 3.0%.
Loss on drying <2.41> Not more than 1.0% (0.1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Assay Weigh accurately an amount of Mitomycin C and
Mitomycin C Reference Standard, equivalent to about 25 mg
(potency), dissolve each in TV.Af-dimethylacetamide to make
exactly 50 mL, and use these solutions as the sample solution
and the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of mitomycin C.
Amount [/ug (potency)] of C 15 H 18 N 4 5
= W s x(A T /A s )x 1000
W s : Amount [mg (potency)] of Mitomycin C Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 365 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with phenylated silica gel for liq-
uid chromatography (10 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 40 mL of 0.5 mol/L ammonium acetate
TS add 5 mL of diluted acetic acid (100) (1 in 20) and water to
make 1000 mL. To 600 mL of this solution add 200 mL of
methanol.
Flow rate: Adjust the flow rate so that the retention time of
mitomycin C is about 7 minutes.
System suitability —
System performance: Dissolve about 25 mg of Mitomycin
C Reference Standard and about 0.375 g of 3-ethoxy-4-
hydroxybenzaldehyde in 50 mL of A^Af-dimethylacetamide.
When the procedure is run with 10 iiL of this solution under
the above operating conditions, mitomycin C and 3-ethoxy-4-
hydroxybenzaldehyde are eluted in this order with the resolu-
tion between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
mitomycin C is not more than 1.0%.
Containers and storage Containers — Tight containers.
Morphine and Atropine Injection
=EJUt:*-7hntf>aj=t>£
Morphine and Atropine Injection is an aqueous so-
lution for injection.
It contains not less than 0.91 w/v% and not more
than 1.09w/v% of morphine hydrochloride
hydrate (C 17 H 19 N0 3 .HC1.3H 2 0: 375.84), and not less
than 0.027 w/v% and not more than 0.033 w/v% of
atropine sulfate hydrate [(Cn^NOj^.HjSCVHzO:
694.83].
Method of preparation
Morphine Hydrochloride Hydrate 10 g
Atropine Sulfate Hydrate 0.3 g
Water for Injection a significant quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Morphine and Atropine Injection is a clear,
colorless liquid.
It is gradually colored by light.
pH: 2.5-5.0
Identification To 2 mL of Morphine and Atropine Injection
add 2 mL of ammonia TS, and extract with 10 mL of diethyl
ether. Filter the extract with a filter paper, evaporate the
filtrate on a water bath to dryness, dissolve the residue in 1
mL of ethanol (99.5), and use this solution as the sample so-
lution. Separately, dissolve 0.1 g of morphine hydrochloride
in 10 mL of water, perform with 2 mL of this solution the
same procedure as used for preparation of the sample solu-
tion, and use the solution so obtained as the standard solu-
tion (1). Separately, dissolve 3 mg of atropine sulfate in 10
mL of water, perform with 2 mL of this solution the same
procedure as used for preparation of the sample solution,
and use the solution so obtained as the standard solution (2).
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solutions (1) and (2) on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of methanol and ammonia solution (28) (200:3) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
Dragendorff's TS on the plate: the two spots obtained from
the sample solution show the same color tone and the same
Rf value with either spot of orange color obtained from the
standard solution (1) or the standard solution (2) (morphine
and atropine).
Extractable volume <6.05> It meets the requirement.
Assay (1) Morphine hydrochloride hydrate — Pipet 2 mL
of Morphine and Atropine Injection, add exactly 10 mL of
the internal standard solution, then add water to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 25 mg of morphine hydrochloride for
assay, add exactly 10 mL of the internal standard solution to
dissolve, then add water to make 50 mL, and use this solution
as the standard solution. Perform the test with 20 iiL of the
900
Morphine Hydrochloride Hydrate / Official Monographs
JP XV
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, g T and Qs, of the peak area
of morphine to that of the internal standard.
Amount (mg) of morphine hydrochloride hydrate
(C 17 H 19 N0 3 .HC1.3H 2 0)
= W s x(Q T /Q s )x 1.1679
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrine hydro-
chloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH with sodium hydroxide TS to 3.0. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, morphine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 1.0%.
(2) Atropine sulfate hydrate — Pipet 2 mL of Morphine
and Atropine Injection, add exactly 2 mL of the internal
standard solution, and use this solution as the sample solu-
tion. Separately, weigh accurately about 15 mg of Atropine
Sulfate Reference Standard (separately determine its loss on
drying <2.41> in the same manner as directed under Atropine
Sulfate Hydrate), and dissolve in water to make exactly 50
mL. Pipet 2 mL of this solution, add exactly 2 mL of the in-
ternal standard solution, and use this solution as the standard
solution. Perform the test with 20 /iL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, Q T and Q s , of the peak areas of atro-
pine to that of the internal standard.
Amount (mg) of atropine sulfate hydrate
[(C 17 H 23 N0 3 ) 2 .H 2 S0 4 .H 2 0]
= W r s x(e r /Qg)x (1/25) x 1.027
W s : Amount (mg) of Atropine Sulfate Reference Stand-
ard, calculated on the dried basis
Internal standard solution — A solution of etilefrine hydro-
chloride (1 in 12,500).
Operating conditions —
Column, column temperature, and mobile phase: Proceed
as directed in the operating conditions in the Assay (1).
Detector: An ultraviolet absorption photometer (wave-
length: 225 nm).
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 7 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the sample solution under the above operating con-
ditions, morphine, the internal standard and atropine are
eluted in this order, and the resolution between morphine and
the internal standard is not less than 3.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of atropine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Morphine Hydrochloride Hydrate
=EJUt:*^fek5f-n!&i
• KCI • 3HsO
C 17 H 19 N0 3 .HC1.3H 2 0: 375.84
(5i?,6S)-4,5-Epoxy-17-methyl-7,8-didehydromorphinan-3,6-
diol monohydrochloride trihydrate [6055-06-7]
Morphine Hydrochloride Hydrate contains not less
than 98.0% and not more than 102.0% of morphine
hydrochloride (C 17 H 19 N0 3 .HC1: 321.80), calculated on
the anhydrous basis.
Description Morphine Hydrochloride Hydrate occurs as
white, crystals or crystalline powder.
It is freely soluble in formic acid, soluble in water, sparing-
ly soluble in methanol, and slightly soluble in ethanol (95).
It is colored by light.
Identification (1) Determine the absorption spectrum of a
solution of Morphine Hydrochloride Hydrate (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 1:
both spectra exhibit similar intensities of absorption at the
same wavelengths. Separately, determine the absorption
spectrum of a solution of Morphine Hydrochloride in dilute
sodium hydroxide TS (1 in 10,000) as directed under Ultrav-
iolet-visible Spectrophotometry, and compare the spectrum
with the Reference Spectrum 2: both spectra exhibit similar
intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Mor-
phine Hydrochloride Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
JPXV
Official Monographs / Morphine Hydrochloride Injection
901
the same wave numbers.
(3) A solution of Morphine Hydrochloride Hydrate (1 in
50) responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]™: - 111 - - 116° (0.5 g calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Morphine Hydrochloride Hydrate in 10 mL of water is
between 4.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Morphine Hydrochloride Hydrate in 10 mL of water: the
solution is clear and colorless.
(2) Sulfate < 1. 14>— Dissolve 0.20 g of Morphine
Hydrochloride Hydrate in 5 mL of water, and add 2 to 3
drops of barium chloride TS: no turbidity is produced.
(3) Meconic acid — Dissolve 0.20 g of Morphine
Hydrochloride Hydrate in 5 mL of water, and add 5 mL of
dilute hydrochloric acid and 2 drops of iron (III) chloride TS:
no red color develops.
(4) Related substances — Dissolve 0.1 g of Morphine
Hydrochloride Hydrate in 10 mL of diluted ethanol (95) (1 in
2), and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add diluted ethanol (95) (1 in 2) to make
exactly 200 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of ethanol (99.5), toluene,
acetone and ammonia solution (28) (14:14:7:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spots other than
the principal spot from the sample solution are not more in-
tense than the spot from the standard solution.
Water <2.48> 13 - 15% (0.1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.5 g of Morphine
Hydrochloride Hydrate, dissolve in 3.0 mL of formic acid,
add 100 mL of a mixture of acetic anhydride and acetic acid
(100) (7:3), mix, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.18 mg of C 17 H 19 N0 3 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Morphine Hydrochloride Injection
Morphine Hydrochloride Injection is an aqueous so-
lution for injection.
It contains not less than 93% and not more than
107% of the labeled amount of morphine hydrochlo-
ride hydrate (C 17 H 19 N0 3 .HC1.3H 2 0: 375.84).
Method of preparation Prepare as directed under Injec-
tions, with Morphine Hydrochloride Hydrate.
Description Morphine Hydrochloride Injection is a clear,
colorless liquid.
It is affected by light.
pH: 2.5-5.0
Identification Take a volume of Morphine Hydrochloride
Injection, equivalent to 0.04 g of Morphine Hydrochloride
Hydrate according to the labeled amount, add water to make
20 mL, and use this solution as the sample solution. To 5 mL
of the sample solution add water to make 100 mL, and deter-
mine the absorption spectrum as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 283 nm and 287 nm. And to 5 mL of the sample solu-
tion add dilute sodium hydroxide TS to make 100 mL, and
determine the absorption spectrum: it exhibits a maximum
between 296 nm and 300 nm.
Extractable volume <6.05> It meets the requirement.
Assay Take exactly a volume of Morphine Hydrochloride
Injection, equivalent to about 80 mg of morphine hydrochlo-
ride hydrate (C 17 H 19 N0 3 .HC1.3H 2 0), and add water to make
exactly 20 mL. Pipet 5 mL of this solution, add exactly 10
mL of the internal standard solution and water to make 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 25 mg of morphine hydrochloride for
assay, dissolve in exactly 10 mL of the internal standard solu-
tion, add water to make 50 mL, and use this solution as the
standard solution. Perform the test with 20 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, g T and g s , of the peak area
of morphine to that of the internal standard.
Amount (mg) of morphine hydrochloride
(C 17 H 19 N0 3 .HC1.3H 2 0)
= W S X(Q T /Q S )X4X 1.1679
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the standard solution under the above operating
conditions, morphine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 3.
902
Morphine Hydrochloride Tablets / Official Monographs
JP XV
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
-Hermetic containers,
Morphine Hydrochloride Tablets
=E;ut:*tt»tttt
Morphine Hydrochloride Tablets contain not less
than 93% and not more than 107% of the labeled
amount of morphine hydrochloride hydrate
(C 17 H 19 N0 3 .HC1.3H 2 0: 375.84).
Method of preparation Prepare as directed under Tablets,
with Morphine Hydrochloride Hydrate.
Identification Weigh a quantity of powdered Morphine
Hydrochloride Tablets equivalent to 0.01 g of Morphine
Hydrochloride Hydrate, add 100 mL of water, shake for 10
minutes, and filter. Determine the absorption spectrum of the
filtrate as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 283 nm and 287
nm. And weigh a quantity of powdered Morphine
Hydrochloride Tablets equivalent to 0.01 g of Morphine
Hydrochloride Hydrate, add 100 mL of dilute sodium
hydroxide TS, shake for 10 minutes, and filter. Determine the
absorption spectrum of the filtrate: it exhibits a maximum be-
tween 296 nm and 300 nm.
Assay Take not less than 20 Morphine Hydrochloride
Tablets, weigh accurately, and powder. Weigh accurately a
quantity of the powder, equivalent to about 20 mg of mor-
phine hydrochloride hydrate (C 17 H 19 N0 3 .HC1.3H 2 0), add
exactly 10 mL of the internal standard solution, extract the
mixture with ultrasonic waves for 10 minutes, and add water
to make 50 mL. Filter this solution, and use the filtrate as the
sample solution. Separately, weigh accurately about 25 mg of
morphine hydrochloride for assay, dissolve in exactly 10 mL
of the internal standard solution, add water to make 50 mL,
and use this solution as the standard solution. Perform the
test with 20 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of morphine to that of the inter-
nal standard.
Amount (mg) of morphine hydrochloride
(C 17 H 19 N0 3 .HC1.3H 2 0)
= W s x(Q T /Q s )x 1.1679
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [im in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with
20 /uL of the standard solution under the above operating
conditions, morphine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Freeze-dried Live Attenuated
Mumps Vaccine
tttt95S£&fc-& < fr-tf 9 f- >
Freeze-dried Live Attenuated Mumps Vaccine is a
dried preparation containing live attenuated mumps
virus.
It conforms to the requirements of Freeze-dried Live
Attenuated Mumps Vaccine in the Minimum Require-
ments of Biologic Products.
Description Freeze-dried Live Attenuated Mumps Vaccine
becomes a clear, colorless, yellowish or reddish liquid on
addition of solvent.
Mupirocin Calcium Hydrate
Ca !K -2H20
H OH H
C 52 H 86 Ca0 18 .2H 2 0: 1075.34
Monocalcium bis[9-((2£)-4- {(2S,3R,4R,5S)-5-
[(25,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4-
dihydroxy-3,4,5,6-tetrahydro-2//-pyran-2-yl}-3-methylbut-
2-enoyloxy)nonanoate] dihydrate [115074-43-6]
Mupirocin Calcium Hydrate is the calcium salt of a
substance having antibacterial activity produced by the
growth of Pseudomonas fluorescens.
JPXV
Official Monographs / Mupirocin Calcium Hydrate
903
It contains not less than 895 ,ug (potency) and not
more than 970 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Mupirocin Calcium
Hydrate is expressed as mass (potency) of mupirocin
(C 26 H 44 9 : 500.62).
Description Mupirocin Calcium Hydrate occurs as a white
powder and has a bitter taste.
It is freely soluble in methanol and slightly soluble in water
and in ethanol (95).
Identification (1) To 1 mL of a solution of Mupirocin
Calcium Hydrate in methanol (1 in 200) add 4 mL of hydrox-
ylamine perchlorate-ethanol TS and 1 mL of 7V,7V'-dicyclo-
hexylcarbodiimide-ethanol TS, shake well, and allow to
stand in lukewarm water for 20 minutes. After cooling, add 1
mL of iron (III) perchorate hexahydrate-ethanol TS to the so-
lution, and shake: a dark purple color develops.
(2) Determine the absorption spectrum of a solution of
Mupirocin Calcium Hydrate (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 219 nm and 224 nm.
(3) Determine the infrared absorption spectrum of
Mupirocin Calcium Hydrate as directed in the paste method
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 1708 cm -1 , 1648 cm -1 ,
1558 cm" 1 , 1231 cm" 1 , 1151cm" 1 and 894 cm" 1 .
(4) A solution of Mupirocin Calcium Hydrate (3 in 1000)
responds to the Qualitative Tests <1.09> (3) for calcium salt.
Optical rotation <2.49> [a]™: -16- -20° (1 g calculated
on the anhydrous basis, methanol, 20 mL, 100 mm).
Purity (1) Related substances — Dissolve 50 mg of
Mupirocin Calcium Hydrate in a mixture of 0.1 mol/L acetic
acid-sodium acetate buffer solution, pH 4.0, and a solution
of tetrahydrofuran (3 in 4) (1:1) to make 10 mL, and use this
solution as the sample solution (1). Pipet 2 mL of this solu-
tion, add a mixture of 0.1 mol/L acetic acid-sodium acetate
buffer solution, pH 4.0, and a solution of tetrahydrofuran (3
in 4) (1:1) to make exactly 100 mL, and use this solution as
the sample solution (2). Preserve these sample solutions at a
temperature between 4°C and 8°C. Perform the test with ex-
actly 20 /uL of the sample solution (1) and the sample solution
(2) as directed under Liquid Chromatography <2.01> accord-
ing to the following conditions, and determine the areas of
each peak of the sample solution (1) and the sample solution
(2) by the automatic integration method. Calculate the
amount of the related substances by the following formula:
the amount of principal related substance (appeared at about
0.7 of the relative retention time to mupirocin) is not more
than 4.0%, and the total amount of related substances (the
total area of the peaks other than of the solvent and mupiro-
cin) is not more than 6.0%.
Amount (%) of principal related substance
a. P x 100
= z+^ xl00x
100
A x 100
A + A m
Total amount (%) of related substances
a P x 100
= 2 x 100 x
A + A. _ A x 100
A + A m
A: Total peak areas other than of the solvent and mupiro-
cin from the sample solution (1)
A { : Peak area of the relative retention time of about 0.7 to
mupirocin from the sample solution (1)
A m : A value of 50 times of peak area of mupirocin from
the sample solution (2)
P: Potency per mg obtained from the assay.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of mupirocin beginning after the solvent peak.
System suitability —
Test for required detection: Pipet 1 mL of the sample solu-
tion (2), and add a mixture of 0.1 mol/L acetic acid-sodium
acetate buffer solution, pH 4.0, and a solution of tetra-
hydrofuran (3 in 4) (1:1) to make exactly 20 mL. Confirm
that the peak area of mupirocin obtained from 20 [iL of this
solution is equivalent to 4 to 6% of that obtained from 20 /uL
of the sample solution (2).
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 [iL of the sample solution (2) under the above operating
conditions, the relative standard deviation of the peak areas
of mupirocin is not more than 2.0%.
(2) Inorganic salt from manufacturing process — Being
specified separately.
Water <2.48> Not less than 3.0% and not more than 4.5%
(0.5 g, volumetric titration, direct titration).
Assay Weigh accurately an amount of Mupirocin Calcium
Hydrate and Mupirocin Lithium Reference Standard,
equivalent to about 20 mg (potency), dissolve in a mixture of
0.1 mol/L acetic acid-sodium acetate buffer solution, pH 4.0
and a solution of tetrahydrofuran (3 in 4) (1 : 1) to make exact-
ly 200 mL, and use these solutions as the sample solution and
the standard solution. Preserve these solutions at a tempera-
ture between 4°C and 8°C. Perform the test with exactly 20
fiL of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the peak areas, A T and A s ,
of mupirocin of each solution.
Amount [/ug (potency)] of mupirocin (C26H44O9)
= W s x(,4 T A4 s )xl000
W s : Amount [mg (potency)] of Mupirocin Lithium Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 7.71 g of ammonium acetate in 750
mL of water, adjust the pH to 5.7 with acetic acid (100), and
add water to make 1000 mL. To 300 mL of this solution add
100 mL of tetrahydrofuran.
Flow rate: Adjust the flow rate so that the retention time of
904
Nadolol / Official Monographs
JP XV
mupirocin is about 12.5 minutes.
System suitability —
System performance: Dissolve about 20 mg of Mupirocin
Lithium Reference Standard and about 5 mg of ethyl para-
hydroxybenzoate in a mixture of 0.1 mol/L acetic acid-sodi-
um acetate buffer solution, pH 4.0 and a solution of tetra-
hydrofuran (3 in 4) (1:1) to make 200 mL. When the proce-
dure is run with 20 /xL of this solution under the above oper-
ating conditions, mupirocin and ethyl parahydroxybenzoate
are eluted in this order with the resolution between these
peaks being not less than 12.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of mupirocin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Nadolol
tHP-Il
and enantiomer
C 17 H 27 N0 4 : 309.40
R 1 = OH, R 2 = H
(2i?S > 3Si?)-5-{(2S J R)-3-[(l,l-Dimethylethyl)amino]-
2-hydroxypropyloxy} -1,2,3, 4-tetrahydronaphthalene-
2,3-diol
R 1 = H, R 2 = OH
(2i?S,3Si?)-5-{(2jRS)-3-[(l,l-Dimethylethyl)amino]-
2-hydroxypropyloxy} -1,2,3, 4-tetrahydronaphthalene-
2,3-diol [42200-33-9]
Nadolol, when dried, contains not less than 98.0%
of C 17 H 27 N0 4 .
Description Nadolol occurs as a white to yellow-brownish
white crystalline powder.
It is freely soluble in methanol and in acetic acid (100),
soluble in ethanol (95), and slightly soluble in water and in
chloroform.
A solution of Nadolol in methanol (1 in 100) shows no op-
tical rotation.
Melting point: about 137°C
Identification (1) Determine the absorption spectrum of a
solution of Nadolol in methanol (1 in 5000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Nadolol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>: it exhibits absorption at the wave numbers of about
1585 cm" 1 , 1460 cm" 1 , 1092 cm" 1 , 935 cm" 1 and 770 cm" 1 .
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Nadolol according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 0.5 g of Nadolol in 10
mL of a mixture of methanol and chloroform (1:1), and use
this solution as the sample solution. Perform the test with the
sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 100 /uL each of the sample solution
and a mixture of methanol and chloroform (1:1) as a control
solution with 25 mm each of width at an interval of about 10
mm on the starting line of a plate 0.25 mm in thickness of sili-
ca gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of acetone,
chloroform and diluted ammonia TS (1 in 3) (8:1:1) to a dis-
tance of about 15 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm), and confirm the
positions of the principal spot and the spots other than the
principal spot from the sample solution. Scratch and collect
the silica gel of the positions of the plate corresponding to the
principal spot and the spots other than the principal spot. To
the silica gel collected from the principal spot add exactly 30
mL of ethanol (95), and to the silica gel from the spots other
than the principal spot add exactly 10 mL of ethanol (95). Af-
ter shaking them for 60 minutes, centrifuge, and determine
the absorbances of these supernatant liquids at 278 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Separately, proceed in the same manner with each position of
the silica gel from the control solution corresponding to the
principal spot and the spots other than the principal spot of
the sample solution, and perform a blank determination to
make correction. Amount of the related substances calculat-
ed by the following equation is not more than 2.0%.
Amount (%) of related substances = {A b /(A b + 3A R )} x 100
A a : Corrected absorbance of the principle spot.
A b : Corrected absorbance of the spots other than the prin-
ciple spot.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Isomer ratio Prepare a paste with 0.01 g of Nadolol as
directed in the paste method under Infrared Spectrophoto-
metry <2.25> so that its transmittance at an absorption band
at a wave number of about 1585 cm -1 is 25 to 30%, and de-
termine the infrared absorption spectrum between 1600 cm -1
and 1100 cm -1 . Determine the absorbances, A 116i and A n50 ,
from the transmittances, T U 65 and T l2 so, at wave numbers of
about 1265 cm -1 (racemic substance A) and 1250 cm" 1 (ra-
cemic substance B), respectively: the ratio A l26i /A l2 5o is be-
tween 0.72 and 1.08.
Assay Weigh accurately about 0.28 g of Nadolol, previous-
ly dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes from purple through blue to green-blue
(indicator: 3 drops of crystal violet TS). Perform a blank de-
termination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.94 mg of C 17 H 27 N0 4
Containers and storage Containers — Tight containers.
JPXV
Storage — Light-resistant.
Nalidixic Acid
Official Monographs / Nalidixic Acid
905
CHa
■N-s.
T ;
COjH
C 12 H 12 N 2 3 : 232.24
1 -Ethyl-7-methyl-4-oxo- 1 ,4-dihydro- 1 , 8-naphthyridine-3-
carboxylic acid [389-08-2]
Nalidixic Acid, when dried, contains not less than
99.0% and not more than 101.0% of C 12 H 12 N 2 3 .
Description Nalidixic Acid occurs as white to light yellow
crystals or crystalline powder.
It is sparingly soluble in iV,./V-dimethylformamide, very
slightly soluble in ethanol (99.5), and practically insoluble in
water.
It dissolves in sodium hydroxide TS.
Identification (1) Determine the absorption spectrum of a
solution of Nalidixic Acid in 0.01 mol/L sodium hydroxide
TS (1 in 200,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Nali-
dixic Acid, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 225 - 23 1 °C
Purity (1) Chloride <1.03>— To 2.0 g of Nalidixic Acid
add 50 mL of water, warm at 70°C for 5 minutes, cool
quickly, and filter. To 25 mL of the filtrate add 6 mL of dilute
nitric acid and water to make 50 mL, and perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion with 0.35 mL of 0.01 mol/L hydrochloric acid VS (not
more than 0.012%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Nalidixic
Acid according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Related substances — Dissolve 20 mg of Nalidixic Acid
in 20 mL of 0.01 mol/L sodium hydroxide TS. Pipet 5 mL of
this solution, add water to make exactly 10 mL, and use this
solution as the sample solution. Pipet 2 mL of the sample so-
lution, add water to make exactly 1000 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/nL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of the peak other
than nalidixic acid with the sample solution is not larger than
the peak area of nalidixic acid with the standard solution,
and the total area of the peaks other than the peak of nalidix-
ic acid with the sample solution is not larger than 2.5 times
the peak area of nalidixic acid with the standard solution.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 260 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 6.24 g of sodium dihydrogen phos-
phate dihydrate in 950 mL of water, adjust the pH to 2.8 with
phosphoric acid, and add water to make 1000 mL. To 300
mL of this solution add 200 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
nalidixic acid is about 19 minutes.
Time span of measurement: About 3 times as long as the
retention time of nalidixic acid beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 5 mL of the standard
solution, and add water to make exactly 10 mL. Confirm that
the peak area of nalidixic acid obtained with 10 /xh of this so-
lution is equivalent to 40 to 60% of that with 10 /xL of the
standard solution.
System performance: Dissolve 25 mg of methyl para-
hydroxybenzoate in 100 mL of a mixture of water and
methanol (1:1). To 1 mL of this solution add water to make
10 mL. To 5 mL of this solution add 5 mL of the standard so-
lution. When the procedure is run with 10 [iL of this solution
under the above operating conditions, methyl parahydrox-
ybenzoate and nalidixic acid are eluted in this order with the
resolution between these peaks being not less than 13.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nalidixic acid is not more than 2.0%.
Loss on drying <2.41> Not more than 0.20% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.3 g of Nalidixic Acid,
previously dried, dissolve in 50 mL of 7V,,/V-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L tetramethyl am-
monium hydroxide VS (potentiometric titration). Separately,
to 50 mL of 7V,7V-dimethylformamide add 13 mL of a mix-
ture of water and methanol (89: 1 1), perform a blank determi-
nation with the solution, and make any necessary correction.
Each mL of 0.1 mol/L tetramethyl ammonium hydroxide VS
= 23.22 mg of C 12 H 12 N 2 3
Containers and storage Containers — Tight containers.
906
Naloxone Hydrochloride / Official Monographs
JP XV
Naloxone Hydrochloride
Cl-a
C 19 H 21 N0 4 .HC1: 363.84
(5.R,14S)-17-Allyl-4,5-epoxy-3,14-dihydroxymorphinan-
6-one monohydrochloride [357-08-4]
Naloxone Hydrochloride contains not less than
98.5% of C 19 H 2 iN0 4 .HCl, calculated on the dried ba-
sis.
Description Naloxone Hydrochloride occurs as white to
yellowish white, crystals or crystalline powder.
It is freely soluble in water, soluble in methanol, slightly
soluble in ethanol (99.5) and in acetic acid (100), and very
slightly soluble in acetic anhydride.
It is hygroscopic.
It is gradually colored by light.
Identification (1) Determine the absorption spectrum of a
solution of Naloxone Hydrochloride (1 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Naloxone Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Naloxone Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [a]": - 170 - - 181° (0.25 g calcu-
lated on the dried basis, water, 10 mL, 100 mm).
pH <2.54> Dissolve 0.10 g of Naloxone Hydrochloride in 10
mL of freshly boiled and cooled water: the pH of the solution
is between 4.5 and 5.5.
Purity Related substances — Conduct this procedure as rap-
idly as possible without exposure to light, using light-resistant
containers. Dissolve 0.08 g of Naloxone Hydrochloride in 10
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add methanol to make ex-
actly 200 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop with a mixture of am-
monia-saturated 1-butanol TS and methanol (20:1) to a dis-
tance of about 12 cm, and air-dry the plate. Spray evenly iron
(III) chloride-potassium hexacyanoferrate (III) TS on the
plate: the number of the spot other than the principal spot
from the sample solution is not more than 1 and it is not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 2.0% [0.1 g, 105°C, 5
hours. Use a desiccator (phosphorus (V) oxide) for cooling].
Residue on ignition <2.44> Not more than 0.2% (0.1 g).
Assay Weigh accurately about 0.3 g of Naloxone
Hydrochloride, dissolve in 80 mL of acetic acid (100) by
warming. After cooling, add 80 mL of acetic anhydride, and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 36.38 mg of C 19 H 21 N0 4 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Naphazoline and Chlorpheniramine
Solution
t7r'/ l J >-<7a)l~7.
■ -? ^ >w
Naphazoline and Chlorpheniramine Solution con-
tains not less than 0.045 w/v% and not more than
0.055 w/v% of naphazoline nitrate (C 14 H 14 N 2 .HN0 3 :
273.29), and not less than 0.09 w/v% and not more
than 0.11 w/v% of chlorpheniramine maleate
(Ci 6 H 19 ClN 2 .C4H 4 4 : 390.86).
Method of preparation
Naphazoline Nitrate
0.5 g
Chlorpheniramine Maleate
lg
Chlorobutanol
2g
Glycerin
50 mL
Purified Water
a sufficient quantity
To make 1000 mL
Dissolve, and mix the above ingredients.
Description Naphazoline and Chlorpheniramine Solution is
a clear, colorless liquid.
Identification (1) To 20 mL of Naphazoline and Chlor-
pheniramine Solution add 2 mL of a solution of potassium
hydroxide (7 in 10) and 5 mL of pyridine, and heat at 100°C
for 5 minutes: a red color is produced (chlorobutanol).
(2) Place 10 mL of Naphazoline and Chlorpheniramine
Solution in a glass-stoppered test tube, add 10 mL of ethanol
(95), 2 mL of sodium hydroxide TS and 1 mL of a solution of
copper (II) chloride dihydrate in ethanol (95) (1 in 10), and
shake: a blue color is produced (glycerin).
(3) To 20 mL of Naphazoline and Chlorpheniramine So-
lution add 5 mL of sodium hydroxide TS, extract with 10 mL
of diethyl ether, and separate the diethyl ether layer. Take 5
mL of this solution, distil off the solvent, dissolve the residue
in 5 mL of methanol, and use this solution as the sample solu-
tion. Separately, dissolve 0.01 g each of naphazoline nitrate
and Chlorpheniramine Maleate Reference Standard in 10 mL
and 5 mL of methanol, respectively, and use these solutions
JPXV
Official Monographs / Naphazoline Hydrochloride
907
as standard solutions (1) and (2). Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 ,uL each of the sample solution and standard
solutions on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol, acetone and ammonia solution
(28) (73:15:10:2) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): two spots from the sample solution exhibit the same Rf
values as the spots from standard solutions (1) and (2). Spray
evenly Dragendorff's TS on the plate: the spots from stan-
dard solutions (1) and (2) and the corresponding spot from
the sample solutions reveal an orange color.
Assay Pipet 4 mL of Naphazoline and Chlorpheniramine
Solution, add exactly 4 mL of the internal standard solution,
then add water to make 10 mL, and use this solution as the
sample solution. Weigh accurately about 50 mg of naphazo-
line nitrate for assay, dried at 105°C for 2 hours, and about
0.1 g of Chlorpheniramine Maleate Reference Standard, d-
ried at 105°C for 3 hours, dissolve in water to make exactly
100 mL. Pipet 4 mL of this solution, add exactly 4 mL of the
internal standard solution, then add water to make 10 mL,
and use this solution as the standard solution. Perform the
test with 10 /xL each of the sample solution and standard so-
lutions as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Q T z and Q Tb , of the peak height of naphazoline nitrate and
chlorpheniramine maleate to that of the internal standard of
the sample solution, and the ratios, Qsa and g sb , of the peak
height of naphazoline nitrate and chlorpheniramine maleate
to that of the internal standard of the standard solution.
Amount (mg) of naphazoline nitrate (C I4 H 14 N 2 .HN03)
= ^SaX(e T a/Gsa)X(l/25)
Amount (mg) of chlorpheniramine maleate
(C 16 H 19 C1N 2 .C 4 H 4 4 )
= W sh X(Q Tb /Q sh )X(l/25)
W S!i : Amount (mg) of naphazoline nitrate for assay
W sb : Amount (mg) of Chlorpheniramine Maleate Refer-
ence Standard
Internal standard solution — A solution of ethenzamide in
methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column, about 4 mm in inside di-
ameter and 25 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of acetonitrile and a solution of
sodium laurylsulfate (1 in 500) in diluted phosphoric acid (1
in 1000) (1:1).
Flow rate: Adjust the flow rate so that the retention time of
chlorpheniramine maleate is about 10 minutes.
Selection of column: Proceed with 10 /xL of the standard
solution under the above operating conditions. Use a column
giving well-resolved peaks of the internal standard, naphazo-
line nitrate and chlorpheniramine maleate in this order.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Naphazoline Hydrochloride
1-7 7 7"U>±^ig±g
■ HCI
C l4 H l4 N 2 .HCl: 246.74
2-(Naphthalen-l-ylmethyl)-4,5-dihydro-l//-imidazole
monohydrochloride [550-99-2]
Naphazoline Hydrochloride, when dried, contains
not less than 98.5% of C l4 H l4 N 2 .HCl.
Description Naphazoline Hydrochloride occurs as a white,
crystalline powder. It is odorless, and has a bitter taste.
It is freely soluble in water, soluble in ethanol (95) and in
acetic acid (100), very slightly soluble in acetic anhydride,
and practically insoluble in diethyl ether.
Melting point: 255 - 260°C (with decomposition).
Identification (1) To 10 mL of a solution of Naphazoline
Hydrochloride (1 in 100) add 5 mL of bromine TS, and boil:
a deep purple color develops.
(2) To 30 mL of a solution of Naphazoline Hydrochlo-
ride (1 in 100) add 2 mL of sodium hydroxide TS, and extract
with two 25-mL portions of diethyl ether. Evaporate the
combined diethyl ether extracts to dryness with the aid of a
current of air. Dry the residue at 80°C for 1 hour: the residue
melts <2.60> between 117°C and 120°C.
(3) Dissolve 0.02 g of the residue obtained in (2) in 2 to 3
drops of dilute hydrochloric acid and 5 mL of water, and add
2 mL of Reinecke salt TS: a red-purple, crystalline precipitate
is formed.
(4) A solution of Naphazoline Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 0. 10 g of Naphazoline Hydrochloride in
10 mL of freshly boiled and cooled water: the pH of the solu-
tion is between 5.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Naphazoline Hydrochloride in 10 mL of water: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Naphazoline Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Naphazoline
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
908
Naphazoline Nitrate / Official Monographs
JP XV
= 24.67 mg of C 14 H 14 N 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
= 27.33 mg of C 14 H 14 N 2 .HN0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Naphazoline Nitrate
Naproxen
•HNO-j
C l4 H l4 N 2 .HN0 3 : 273.29
2-(Naphthalen- 1 -ylmethyl)-4, 5-dihydro- l//-imidazole
mononitrate [5144-52-5]
Naphazoline Nitrate, when dried, contains not less
than 98.5% of C 14 H 14 N 2 .HN03.
Description Naphazoline Nitrate occurs as a white, crystal-
line powder. It is odorless, and has a bitter taste.
It is freely soluble in acetic acid (100), soluble in ethanol
(95), sparingly soluble in water, slightly soluble in acetic an-
hydride, and practically insoluble in diethyl ether.
Identification (1) To 10 mL of a solution of Naphazoline
Nitrate (1 in 100) add 5 mL of bromine TS, and boil: a deep
purple color develops.
(2) To 20 mL of a solution of Naphazoline Nitrate (1 in
100) add 5 mL of sodium hydroxide TS, and extract with two
25-mL portions of diethyl ether. Combine the diethyl ether
extracts, evaporate to dryness with the aid of a current of air,
and dry the residue at 80°C for 1 hour: the residue so ob-
tained melts <2.60> between 117°C and 120°C.
(3) A solution of Naphazoline Nitrate (1 in 20) responds
to the Qualitative Tests <1.09> for nitrate.
pH <2.54> Dissolve 0.1 g of Naphazoline Nitrate in 10 mL
of freshly boiled and cooled water: the pH of the solution is
between 5.0 and 7.0.
Melting point <2.60> 167 - 170°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Naphazoline Nitrate in 50 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Naphazoline Nitrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41>
2 hours).
Not more than 0.5% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Naphazoline Ni-
trate, previously dried, dissolve in 10 mL of acetic acid (100)
and 40 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L perchloric acid VS (indicator: 3 drops of crystal violet
TS). Perform a blank determination, and make any necessary
correction.
Each mL of 0.1 mol/L perchloric acid VS
h s c.
C 14 H 14 3 : 230.26
(2S)-2-(6-Methoxynaphthalen-2-yl)propanoic acid
[22204-53-1]
Naproxen, when dried, contains not less than 98.5%
of C 14 H 14 3 .
Description Naproxen occurs as white crystals or crystalline
powder. It is odorless.
It is freely soluble in acetone, soluble in methanol, in
ethanol (99.5) and in chloroform, sparingly soluble in diethyl
ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 0.01 g of Naproxen in 5 mL of
methanol, add 5 mL of water, then add 2 mL of potassium
iodide TS and 5 mL of a solution of potassium iodate (1 in
100), and shake: a yellow to yellow-brown color develops. To
this solution add 5 mL of chloroform, and shake: a light red-
purple color develops in the chloroform layer.
(2) To 1 mL of a solution of Naproxen in ethanol (99.5)
(1 in 300) add 4 mL of hydroxylamine perchlorate-dehydrat-
ed ethanol TS and 1 mL of 7V,/V-dicyclohexylcarbodiimide-
dehydrated ethanol TS, shake well, and allow to stand in
lukewarm water for 20 minutes. After cooling, add 1 mL of
iron (III) perchlorate-dehydrated ethanol TS, and shake: a
red-purple color develops.
(3) Determine the absorption spectrum of a solution of
Naproxen in ethanol (99.5) (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of
Naproxen, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]": +63.0- +(
drying, 0.1 g, chloroform, 10 mL, 100 mm).
Melting point <2.60> 154 - 158 °C
1.5° (after
Purity (1) Clarity of solution — Dissolve 2.0 g of Naprox-
en in 20 mL of acetone: the solution is clear. Perform the test
with this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: the absorbance at 400 nm is not more
than 0.070.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Naprox-
JPXV
Official Monographs / Neostigmine Methylsulfate
909
en according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Naproxen according to Method 3, and perform the test
(not more than 1 ppm).
(4) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Naproxen in 10 mL of a mixture of chloroform and
ethanol (99.5) (1:1), and use this solution as the sample solu-
tion. Pipet 2 mL of the sample solution, and add a mixture of
chloroform and ethanol (99.5) (1:1) to make exactly 100 mL.
Pipet 5 mL of this solution, add a mixture of chloroform and
ethanol (99.5) (1:1) to make exactly 50 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /uL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
hexane, dichloromethane, tetrahydrofuran and acetic acid
(100) (50:30:17:3) to a distance of about 12 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot and the spot
of the starting point from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5g of Naproxen, previous-
ly dried, add 100 mL of diluted methanol (4 in 5), dissolve by
gentle warming if necessary, and titrate <2.50> with 0.1 mol/
L sodium hydroxide VS (indicator: 3 drops of phenolphtha-
lein TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 23.03 mg of C 14 H 14 3
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Neostigmine Methylsulfate
*-tx*v$. >/^)iffi.mi&
HaC-SO.,
Y " CH 3
C, 3 H 22 N 2 6 S: 334.39
3-(Dimethylcarbamoyloxy)-A r ,A r ,7V-
trimethylanilinium methyl sulfate [51-60-5]
Neostigmine Methylsulfate, when dried, contains
not less than 98.0% and not more than 102.0% of
C 13 H 22 N 2 6 S.
Description Neostigmine Methylsulfate occurs as a white,
crystalline powder.
It is very soluble in water, and freely soluble in acetonitrile
and in ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Neostigmine Methylsulfate (1 in 2000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Neostigmine Methylsulfate Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Ne-
ostigmine Methylsulfate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of dried Neostigmine
Methylsulfate Reference Standard: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
pH <2.54> Dissolve 1.0 g of Neostigmine Methylsulfate in
10 mL of freshly boiled and cooled water: the pH of the solu-
tion is between 3.0 and 5.0.
Melting point <2.60> 145 - 149 C C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Neostigmine Methylsulfate in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate — Dissolve 0.20 g of Neostigmine Methylsul-
fate in 10 mL of water, add 1 mL of dilute hydrochloric acid
and 1 mL of barium chloride TS: no turbidity is produced im-
mediately.
(3) Dimethylaminophenol — Dissolve 0.10 g of Neostig-
mine Methylsulfate in 5 mL of water, add 1 mL of sodium
hydroxide TS, and while cooling with ice, add 1 mL of dia-
zobenzenesulfonic acid TS: no color develops.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 25 mg each of Neostigmine
Methylsulfate and Neostigmine Methylsulfate Reference
Standard, previously dried, dissolve each in the mobile phase
to make exactly 50 mL, and use these solutions as the sample
solution and the standard solution, respectively. Perform the
test with exactly 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of neostigmine in each solution.
Amount (mg) of C 13 H22N 2 6 S
= W s x(A T /A s )
W s : Amount (mg) of Neostigmine Methylsulfate Refer-
ence Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 259 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 3.12 g of sodium dihydrogen-
910
Neostigmine Methylsulfate Injection / Official Monographs
JP XV
phosphate dihydrate in 1000 mL of water, adjust to pH 3.0
with phosphoric acid, and add 0.871 g of sodium 1-pen-
tanesulfonate to dissolve. To 890 mL of this solution add 110
mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
neostigmine is about 9 minutes.
System suitability —
System performance: Dissolve 25 mg of Neostigmine
Methylsulfate and 4 mg of dimethylaminophenol in 50 mL of
the mobile phase. When the procedure is run with 10 /uL of
this solution under the above operating conditions,
dimethylaminophenol and neostigmine are eluted in this ord-
er with the resolution between these peaks being not less than
6.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of neostigmine methylsulfate is not more than 1.0%.
Containers and storage Containers — Tight containers.
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Netilmicin Sulfate
*?-)\>^4*/>wmM
Hr.N
Neostigmine Methylsulfate
Injection
C 21 H 41 N 5 7 .2i/2H 2 S04: 720.78
3-Deoxy-4-C-methyl-3-methylamino-/?-L-
arabinopyranosyl-(l -> 6)-[2,6-diamino-
2,3,4,6-tetradeoxy-a-D-g/> , cero-hex-4-enopyranosyl-(l->4)]-
2-deoxy- 1 -/V-ethyl-D-streptamine hemipentasulf ate
[56391-57-2]
Neostigmine Methylsulfate Injection is an aqueous
solution for injection.
It contains not less than 93% and not more than
107% of the labeled amount of neostigmine methylsul-
fate (C 13 H 22 N 2 6 S: 334.39).
Method of preparation Prepare as directed under Injec-
tions, with Neostigmine Methylsulfate.
Description Neostigmine Methylsulfate Injection is a clear,
colorless liquid.
It is slowly affected by light.
pH: 5.0-6.5
Identification Take a volume of Neostigmine Methylsulfate
Injection equivalent to 5 mg of neostigmine methylsulfate ac-
cording to the labeled amount, add water to make 10 mL if
necessary, and determine the absorption spectrum of this so-
lution as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24>: it exhibits a maximum between 257 nm and 261
nm.
Extractable volume <6.05> It meets the requirement.
Bacterial endotoxins <4.01> Less than 5 EU/mg.
Assay Use Neostigmine Methylsulfate Injection as the sam-
ple solution. Separately, weigh accurately about 25 mg of Ne-
ostigmine Methylsulfate Reference Standard, previously d-
ried at 105°C for 3 hours, dissolve in the mobile phase to
make exactly 50 mL, and use this solution as the standard so-
lution. Proceed as directed in the Assay under Neostigmine
Methylsulfate.
Amount (mg) of neostigmine methylsulfate (CoP^^C^S)
= W s x(A T /A s )
W s : Amount (mg) of Neostigmine Methylsulfate Refer-
ence Standard
Netilmicin Sulfate is the sulfate of a derivative of
sisomicin.
It contains not less than 595 fig (potency) and not
more than 720 fig (potency) per mg, calculated on the
dried basis. The potency of Netilmicin Sulfate is ex-
pressed as mass (potency) of netilmicin (C2iH 41 N 5 07:
475.58).
Description Netilmicin Sulfate occurs as a white to light yel-
lowish white powder.
It is very soluble in water, and practically insoluble in
ethanol (95).
It is hygroscopic.
Identification (1) Dissolve 30 mg of Netilmicin Sulfate in
3 mL of water, and add 0.2 mL of bromine TS: the solution
is immediately decolorized.
(2) Dissolve 15 mg each of Netilmicin Sulfate and Netil-
micin Sulfate Reference Standard in 5 mL of water, and use
these solutions as the sample solution and the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer chromatography <2.03>. Spot 5 fiL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of methanol, chloroform, ammonia water (28) and ace-
tone (2:2:1:1) to a distance of about 15 cm, and air-dry the
plate. Spray evenly 0.2% ninhydrin-water saturated 1-
butabol TS on the plate, and heat at 100°C for 5 minutes: the
principal spots from the sample solution and the standard so-
lution exhibit a red-purple to red-brown color and show the
same i?f value.
(3) A solution of Netilmicin Sulfate (1 in 100) responds to
the Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [a]^°: +88 - +96° (0.1 g calculated
on the dried basis, water, 10 mL, 100 mm).
JP XV
Official Monographs / Nicardipine Hydrochloride 911
pH <2.54> Dissolve 0.5 g of Netilmicin Sulfate in 5 mL of
water: the pH of this solution is between 3.5 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Netilmicin Sulfate in 5 mL of water: the solution is clear and
colorless to light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Netilmi-
cin Sulfate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Related substances — Dissolve 50 mg of Netilmicin
Sulfate, calculated on the dried basis, in water to make 5 mL,
and use this solution as the sample solution. Pipet 0.5 mL, 1
mL, and 1.5 mL of the sample solution, add water to each to
make exactly 50 mL, and use these solutions as the standard
solution (1), the standard solution (2) and the standard solu-
tion (3), respectively. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solutions (1), (2)
and (3) on a plate of silica gel for thin-layer chromatography.
Develop with a mixture of methanol, chloroform, ammonia
water (28) and acetone (2:2:1:1) to a distance of about 10 cm,
and air -dry the plate. Spray evenly 0.2% ninhydrin-water
saturated 1-butabol TS on the plate, and heat at 100°C for 5
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution (3), and the total amount of the intensity of
the spots other than the principal spot from the sample solu-
tion is not more than 6%.
Loss on drying <2.41> Not more than 15.0% (0.15 g, in
vacuum not exceeding 0.67 kPa, 110°C, 3 hours). Sampling
should be carried out in a manner to avoid moisture absorp-
tion.
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC
6538 P
(ii) Culture medium — Use the medium ii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Netilmicin Sulfate Reference Standard equivalent to about 25
mg (potency), dissolve in 0.1 mol/L phosphate buffer solu-
tion, pH 8.0 to make exactly 25 mL, and use this solution as
the standard stock solution. Keep the standard stock solution
at 5°C or below and use within 7 days. Take exactly a suitable
amount of the standard stock solution before use, add 0.1
mol/L phosphate buffer solution, pH 8.0 to make solutions
so that each mL contains 4//g (potency) and 1 [ig (potency),
and use these solutions as the high concentration standard so-
lution and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Netilmicin Sulfate equivalent to about 25 mg (potency), dis-
solve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make
exactly 25 mL. Take exactly a suitable amount of the solu-
tion, add 0.1 mol/L phosphate buffer solution, pH 8.0 to
make solutions so that each mL contains 4 /ug (potency) and 1
H% (potency), and use these solutions as the high concentra-
tion sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, not exceeding 5°C, under nitro-
gen or argon atmosphere.
Nicardipine Hydrochloride
HaC'
and enantiomer
C 26 H 29 N 3 6 .HC1: 515.99
2-[Benzyl(methyi)amino]ethyl methyl (4RS)-
2,6-dimethyl-4-(3-nitrophenyl)-l,4-dihydropyridine-
3,5-dicarboxylate monohydrochloride [54527-84-3]
Nicardipine hydrochloride, when dried, contains not
less than 98.5% of C 26 H 29 N 3 6 .HC1.
Description Nicardipine Hydrochloride occurs as a pale
greenish yellow crystalline powder.
It is freely soluble in methanol and in acetic acid (100),
sparingly soluble in ethanol (99.5), and slightly soluble in
water, in acetonitrile and in acetic anhydride.
A solution of Nicardipine Hydrochloride in methanol (1 in
20) shows no optical rotation.
It is gradually affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Nicardipine Hydrochloride in ethanol (99.5) (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Nicardipine Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Dissolve 0.02 g of Nicardipine Hydrochloride in 10
mL of water and 3 mL of nitric acid: the solution responds to
the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 167 - 171 °C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Nicardipine Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Conduct this procedure without
exposure to daylight, using light-resistant vessels. Dissolve
0.10 g of Nicardipine Hydrochloride in 50 mL of the mobile
phase, and use this solution as the sample solution. Pipet 1
mL of the sample solution, add the mobile phase to make
exactly 50 mL, then take exactly 1 mL of this solution, add
the mobile phase to make exactly 10 mL, and use this solu-
912
Nicardipine Hydrochloride Injection / Official Monographs
JP XV
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area of both
solutions by the automatic integration method: the area of
each peak other than the peak of nicardipine from the sample
solution is not larger than the peak area of nicardipine from
the standard solution, and the total area of each peak other
than the peak of nicardipine from the sample solution is not
more than twice the peak area of nicardipine from the stan-
dard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 lim in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of a solution of perchloric acid
(43 in 50,000) and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
nicardipine is about 6 minutes.
Time span of measurement: About 4 times as long as the
retention time of nicardipine beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of nicardipine obtained from 10
[iL of this solution is equivalent to 8 to 12% of that of
nicardipine obtained from 10 /iL of the standard solution.
System performance: Dissolve 2 mg each of Nicardipine
Hydrochloride and nifedipine in 50 mL of the mobile phase.
When the procedure is run with 10 /iL of this solution under
the above operating conditions, nicardipine and nifedipine
are eluted in this order with the resolution between these
peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 /iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of nicardipine is not more than 3%.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately about
0.9 g of Nicardipine Hydrochloride, previously dried, dis-
solve in 100 mL of a mixture of acetic anhydride and acetic
acid (100) (7:3), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 51.60 mg of C 26 H 29 N 3 6 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Nicardipine Hydrochloride Injection
-ajuyt?>£K£a*t*
Nicardipine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 93% and not more than
107% of the labeled amount of nicardipine hydrochlo-
ride (C 26 H 29 N 3 6 .HC1: 515.99).
Method of preparation Prepare as directed under Injec-
tions, with Nicardipine Hydrochloride.
Description Nicardipine Hydrochloride Injection occurs as
a clear pale yellow liquid.
It is gradually changed by light.
Identification To a volume of Nicardipine Hydrochloride
Injection, equivalent to 1 mg of Nicardipine Hydrochloride
according to the labeled amount, add ethanol (99.5) to make
100 mL. Determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits maxima between 235 nm and 239 nm, and
between 351 nm and 355 nm.
pH <2.54> 3.0-4.5
Purity Related substances — Conduct the procedure with-
out exposure to day-light using light-resistant vessels. To a
volume of Nicardipine Hydrochloride Injection, equivalent
to 5 mg of Nicardipine Hydrochloride according to the la-
beled amount, add the mobile phase to make 10 mL, and use
this solution as the sample solution. To exactly 2 mL of the
sample solution add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 10 /iL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas of these solutions by the automatic integration
method: the areas of the peaks other than nicardipine from
the sample solution are not more than the peak area of
nicardipine from the standard solution, and the total of the
areas of the peaks other than nicardipine from the sample so-
lution is not more than 2 times of the peak area of nicardipine
from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of nicardipine beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of nicardipine obtained from 10
fiL of this solution is equivalent to 8 to 12% of that from the
standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 5 times with
10 /iL of the standard solution under the above operating
JPXV
Official Monographs / Nicergoline 913
conditions, the relative standard deviation of the peak areas
of nicardipine is not more than 1.0%.
Bacterial endotoxins <4.01> Less than 8.33 EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay Conduct the procedure without exposure to day-light
using light-resistant vessels. To an exact volume of Nicardi-
pine Hydrochloride Injection, equivalent to about 2 mg of
nicardipine hydrochloride (C26H29N3CVHCI), add exactly 5
mL of the internal standard solution and methanol to make
50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 50 mg of nicardipine
hydrochloride for assay, previously dried at 105°C for 2
hours, dissolve in methanol to make exactly 50 mL. Pipet 2
mL of this solution, add exactly 5 mL of the internal stan-
dard solution and methanol to make 50 mL, and use this so-
lution as the standard solution. Perform the test with 10 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, g T and Q s , of the
peak area of nicardipine to that of the internal standard.
Amount (mg) of nicardipine hydrochloride (C26H29N3O6.HCI)
= W s x(Q T /Q s )x(l/25)
W s : Amount (mg) of nicardipine hydrochloride for assay
Internal standard solution — A solution of di-M-butyl phtha-
late in methanol (1 in 625).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 11m in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.36 g of potassium dihydrogen
phosphate in water to make 1000 mL. To 320 mL of this so-
lution add 680 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
nicardipine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, nicardipine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 5 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of nicardipine is not more than 1.0%.
Nicergoline
--tzJUzfU >
Containers and storage Containers-
Colored containers may be used.
Storage — Light-resistant.
•Hermetic containers.
C 24 H 26 BrN 3 03: 484.39
l(SR , 1 OS)- 1 0-Methoxy- 1 ,6-dimethylergolin-8-yl]methyl
5-bromopyridine-3-carboxylate
[27848-84-6]
Nicergoline, when dried, contains not less than
98.5% and not more than 101.0% of C 24 H 26 BrN 3 03.
Description Nicergoline occurs as white to light yellow,
crystals or crystalline powder.
It is soluble in acetonitrile, in ethanol (99.5) and in acetic
anhydride, and practically insoluble in water.
It is gradually colored to light brown by light.
Melting point: about 136°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Nicergoline in ethanol (99.5) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Nicer-
goline as directed in the potassium bromide disk method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: +5.2- +6.2° (after drying,
0.5 g, ethanol (95), 10 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Nicergoline according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Related substances — Dissolve 25 mg of Nicergoline in
25 mL of acetonitrile, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add acetoni-
trile to make exactly 100 mL. Pipet 10 mL of this solution,
add acetonitrile to make exactly 50 mL, and use this solution
as the standard solution. Perform the test with exactly 20 11L
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak, having the
relative retention time of about 0.5 with respect to nicergo-
line, is not larger than 4 times the peak area of nicergoline
from the standard solution, and the area of the peak other
than nicergoline and other than the peak mentioned above is
not larger than 2.5 times the peak area of nicergoline from
914
Nicergoline Powder / Official Monographs
JP XV
the standard solution. The peak which area is larger than the
peak area of nicergoline from the standard solution is not
more than two peaks, and the total area of the peaks other
than the peak of nicergoline is not larger than 7.5 times the
peak area of nicergoline from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 288 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 7.0 with triethylamine. To 350 mL
of this solution add 350 mL of methanol and 300 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
nicergoline is about 25 minutes.
Time span of measurement: About 2 times as long as the
retention time of nicergoline beginning after the solvent
peak.
System suitability —
Test for required detectability: To 1 mL of the sample solu-
tion add acetonitrile to make exactly 50 mL, and use this so-
lution as the solution for system suitability test. Pipet 5 mL
of the solution for system suitability test, and add acetonitrile
to make exactly 100 mL. Confirm that the peak area of nicer-
goline obtained with 20 /uL of this solution is equivalent to 3
to 7% of that with 20 /uL of the solution for system suitability
test.
System performance: When the procedure is run with 20
fiL of the sample solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of nicergoline are not less than 8000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nicergoline is not more than 4.0%.
Loss on drying <2.41> Not more than 0.5% (2 g, in vacuum,
60°C, 2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Nicergoline, previ-
ously dried, add 10 mL of acetic anhydride, and warm to dis-
solve. After cooling, add 40 mL of nitrobenzene, and titrate
<2.50> with 0.1 mol/L perchloric acid VS until the color of
the solution changes to blue-green from red through a blue-
purple (indicator: 10 drops of neutral red TS). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.22 mg of C 24 H 26 BrN 3 03
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Nicergoline Powder
Nicergoline Powder contains not less than 95.0%
and not more than 105.0% of the labeled amount of
nicergoline (Cz^eBrNjOj: 484.39).
Method of preparation Prepare as directed under Powders,
with Nicergoline.
Identification Vigorously shake for 10 minutes a quantity
of Nicergoline Powder, equivalent to 10 mg of Nicergoline
according to the labeled amount, with 20 mL of diluted
ethanol (4 in 5), and centrifuge for 10 minutes. To 2 mL of
the supernatant liquid add ethanol (99.5) to make 100 mL.
Determine the absorption spectrum of this solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>; it ex-
hibits maxima between 226 nm and 230 nm, and between 286
nm and 290 nm.
Purity Related substances — Perform the test with 20 /xh of
the sample solution obtained in the Assay as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine each peak area by the automatic in-
tegration method, and calculate the amount of substances
other than nicergoline by the area percentage method: the
total amount of them is not more than 2.0%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of nicergoline after the solvent peak.
System suitability —
Test for required detectability: To 1 mL of the standard so-
lution obtained in the Assay add a mixture of acetonitrile and
water (17:3) to make 50 mL, and use this solution as the solu-
tion for system suitability test. Pipet 5 mL of the solution for
system suitability test, add the mixture of acetonitrile and
water (17:3) to make exactly 100 mL. Confirm that the peak
area of nicergoline obtained with 20 fiL of this solution is
equivalent to 3 to 7% of that with 20 /xL of the solution for
system suitability test.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of nicergoline is not more than 1.5%.
Uniformity of dosage unit <6.02> The Nicergoline Powder
in single-unit container meets the requirement of the Mass
variation test.
Particle size <6.03> It meets the requirements of Powders.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Weigh accurately a quantity of Nicergoline Powder, e-
quivalent to about 5 mg of nicergoline (C 2 4H 26 BrN 3 3 ), and
perform the test at 50 revolutions per minute according to the
Paddle method using 900 mL of 2nd fluid for dissolution test
JPXV
Official Monographs / Nicergoline Tablets 915
as the dissolution medium. Withdraw 20 mL or more of the
dissolution medium 15 minutes after starting the test, and
filter through a laminated polyester fiver filter. Discard the
first 10 mL of the filtrate, and use the subsequent filtrate as
the sample solution. Separately, weigh accurately about 50
mg of nicergoline for assay, previously dried in vacuum at 60
°C for 2 hours, and dissolve in 0.1 mol/L hydrochloric acid
TS to make exactly 50 mL. Pipet 5 mL of this solution, and
add 2nd fluid for dissolution test to make exactly 100 mL.
Pipet 10 mL of this solution, add 2nd fluid for dissolution
test to make exactly 100 mL, and use this solution as the stan-
dard solution. Determine the absorbances at 225 nm, A Ti and
^4si> and at 250 nm, A T2 and A$ 2 , of the sample solution and
the standard solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate in 15 minutes
is not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
nicergoline (C 24 H 26 BrN 3 3 )
= (fT s /W T )x{(^ T1 -^ T2 )/(^ sl -^ S2 )}x(l/C)x9
W s : Amount (mg) of nicergoline for assay
W T : Amount (g) of sample
C: Labeled amount (mg) of nicergoline (C 24 H 26 BrN 3 3 ) in
lg
Assay Weigh accurately a quantity of Nicergoline Powder,
equivalent to about 20 mg of nicergoline (C 24 H 26 BrN 3 3 ),
add exactly 20 mL of a mixture of acetonitrile and water
(17:3), vigorously shake for 10 minutes, centrifuge for 10
minutes, and use the supernatant liquid as the sample solu-
tion. Separately, weigh accurately about 20 mg of nicergoline
for assay, previously dried in vacuum at 60°C for 2 hours,
dissolve in exactly 20 mL of the mixture of acetonitrile and
water (17:3), and use this solution as the standard solution.
Perform the test with exactly 20 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of nicergoline.
Amount (mg) of nicergoline (C 24 H 26 BrN 3 3 )
= ^ s x(A T /A s )
W s : Amount (mg) of nicergoline for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 288 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 7.0 with triethylamine. To 350 mL
of this solution add 350 mL of methanol and 300 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
nicergoline is about 25 minutes.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of nicergoline are not less than 8000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nicergoline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Nicergoline Tablets
Nicergoline Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of nicer-
goline (C 2 4H 26 BrN 3 3 : 484.39).
Method of preparation Prepare as directed under Tablets,
with Nicergoline.
Identification Take a quantity of powdered Nicergoline
Tablets, equivalent to 10 mg of Nicergoline according to the
labeled amount, add 20 mL of ethanol (99.5), shake
vigorously for 10 minutes, and filter through a 0.45-//m pore-
size membrane filter. To 2 mL of the filtrate add ethanol
(99.5) to make 100 mL. Determine the absorption spectrum
of this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 226 nm
and 230 nm, and between 286 nm and 290 nm.
Purity Related substances — Perform the test with 20 /xh of
the sample solution obtained in the Assay as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine each peak area by the automatic in-
tegration method, and calculate the amount of substances
other than nicergoline by the area percentage method: the
total amount of them is not more than 2.0%.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of nicergoline beginning after the solvent
peak.
System suitability —
Test for required detectability: To 1 mL of the standard so-
lution obtained in the Assay add a mixture of acetonitrile and
water (17:3) to make 50 mL, and use this solution as the solu-
tion for system suitability test. Pipet 5 mL of the solution for
system suitability test, add the mixture of acetonitrile and
water (17:3) to make exactly 100 mL. Confirm that the peak
area of nicergoline obtained with 20 /uL of this solution is
equivalent to 3 to 7% of that with 20 /xL of the solution for
system suitability test.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 /iL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of nicergoline is not more than 1.5%.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
916
Niceritrol / Official Monographs
JP XV
To 1 tablet of Nicergoline Tablets add exactly 25 mL of
diluted ethanol (4 in 5), disperse to fine particles with the aid
of ultrasonic wave, and shake for 5 minutes. Centrifuge this
solution for 10 minutes, pipet exactly 4 mL of the super-
natant liquid, add diluted ethanol (4 in 5) to make exactly 25
mL, and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of nicergoline for assay, previ-
ously dried in vacuum at 60°C for 2 hours, and dissolve in ex-
actly 25 mL of diluted ethanol (4 in 5). Pipet 4 mL of this so-
lution, add diluted ethanol (4 in 5) to make exactly 50 mL,
and use this solution as the standard solution. Determine the
absorbances at 288 nm, A Tl and A sl , and at 340 nm, A T2 and
A S2 , of the sample solution and the standard solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of nicergoline (C 24 H 26 BrN 3 03)
= W s x {(A Tl -A T2 )/(A si -A S2 )} x (1/2)
W s : Amount (mg) of nicergoline for assay
Dissolution Being specified separately.
Assay Weigh accurately the mass of not less than 20 Nicer-
goline Tablets, and powder. Weigh accurately a portion of
the powder, equivalent to about 20 mg of nicergoline
(C 24 H 26 BrN 3 3 ), add exactly 20 mL of a mixture of acetoni-
trile and water (17:3), vigorously shake for 10 minutes, cen-
trifuge for 10 minutes, and use the supernatant liquid as the
sample solution. Separately, weigh accurately about 20 mg of
nicergoline for assay, previously dried in vacuum at 60°C for
2 hours, dissolve in exactly 20 mL of the mixture of acetoni-
trile and water (17:3), and use this solution as the standard
solution. Perform the test with exactly 20 /xL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of nicergo-
line.
Amount (mg) of nicergoline (C 24 H 26 BrN 3 3 ) = W s X (A T /A S )
W s : Amount (mg) of nicergoline for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 288 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 7.0 with triethylamine. To 350 mL
of this solution add 350 mL of methanol and 300 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
nicergoline is about 25 minutes.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of nicergoline are not less than 8000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nicergoline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Niceritrol
--tzU r-P-JU
C 2 9H 24 N 4 8 : 556.52
Pentaerythritol tetranicotinate
[5868-05-3]
Niceritrol, when dried,
of C 29 H 24 N 4 8 .
contains not less than 99.0%
Description Niceritrol occurs as a white to pale yellowish
white powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in chloroform, soluble in /V,iV-dimethyl-
formamide, very slightly soluble in ethanol (95), and practi-
cally insoluble in water and in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Niceritrol in 0. 1 mol/L hydrochloric acid TS (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Niceritrol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 162 - 165 °C
Purity (1) Chloride <1.03>— To 2.0 g of Niceritrol add 50
mL of water, and warm at 70°C for 20 minutes, while shak-
ing occasionally. After cooling, filter, and to 25 mL of the
filtrate add 6 mL of dilute nitric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 1.0 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.036%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Niceritrol according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Niceritrol according to Method 3, and perform the test.
Use 10 mL of a solution of magnesium nitrate hexahydrate in
ethanol (95) (1 in 10) (not more than 2 ppm).
(4) Pyridine — Dissolve 0.5 g of Niceritrol in N,N-
dimethylformamide to make exactly 10 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 0.1 g of pyridine, and add A^/V-dimethylformamide to
make exactly 100 mL. Pipet 1 mL of this solution, add N,N-
JPXV
Official Monographs / Nicomol 917
dimethylformamide to make exactly 100 mL, then pipet 0.5
mL of this solution, add iY,/V-dimethylformamide to make
exactly 10 mL, and use this solution as the standard solution.
Perform the test with 2 /uL each of the sample solution and
standard solution as directed under Gas Chromatography
<2.02> according to the following conditions. Determine each
peak area of pyridine in both solutions: the peak area of pyri-
dine from the sample solution is not larger than the peak area
of pyridine from the standard solution.
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A column 3 mm in inside diameter and 3 m in
length, packed with polyethylene glycol 20M for gas chro-
matography coated at the ratio of 10% on acid-treated and
silanized siliceous earth for gas chromatography (150 to 180
/um in particle diameter).
Column temperature: A constant temperature of about
160°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
pyridine is about 2 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, the number of theoretical steps of the peak of pyridine
is not less than 1500 steps.
System repeatability: When the test is repeated 6 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
pyridine is not more than 3.0%.
(5) Free acids — Transfer about 1 g of Niceritrol, weighed
accurately, to a separator, dissolve in 20 mL of chloroform,
and extract with 20 mL and then 10 mL of water while shak-
ing well. Combine the whole extracts, and titrate <2.50> with
0.01 mol/L sodium hydroxide VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination, make
any necessary correction, and calculate the amount of free
acid by the following equation: it is not more than 0.1%.
Each mL of 0.01 mol/L sodium hydroxide VS
= 1.231 mg of C 6 H 5 N0 2
(6) Related substances — Dissolve 0.10 g of Niceritrol in
10 mL of chloroform, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add chlo-
roform to make exactly 20 mL. Pipet exactly 2 mL of this so-
lution, add chloroform to make exactly 20 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform and ethanol (95) (4:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Niceritrol, previously
dried, add exactly 25 mL of 0.5 mol/L sodium hydroxide VS,
boil gently for 20 minutes under a reflux condenser with a
carbon dioxide absorber (soda lime). After cooling, titrate
<2.50> immediately the excess sodium hydroxide with 0.5
mol/L hydrochloric acid VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination.
Each mL of 0.5 mol/L sodium hydroxide VS
= 69.57 mg of C 29 H 2 4N 4 8
Containers and storage Containers — Well-closed contain-
ers.
Nicomol
-zi^E-JU
C34H32N4CV 640.64
(2-Hydroxycyclohexane-l,l,3,3-tetrayl)tetramethyl
tetranicotinate [27959-26-8]
Nicomol, when dried, contains not less than 98.0%
of C 3 4H3 2 N 4 9 .
Description Nicomol occurs as a white, crystalline powder.
It is odorless and tasteless.
It is soluble in chloroform, and practically insoluble in
water, in ethanol (95) and in diethyl ether.
It dissolves in dilute hydrochloric acid and in dilute nitric
acid.
Identification (1) Mix 0.01 g of Nicomol with 0.02 g of 1-
chloro-2,4-dinitrobenzene, add 2 mL of dilute ethanol, heat
in a water bath for 5 minutes, cool, and add 4 mL of potassi-
um hydroxide-ethanol TS: a dark red color develops.
(2) Dissolve 0.1 g of Nicomol in 5 mL of dilute
hydrochloric acid, and add 5 drops of Reinecke salt TS: a
light red precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Nicomol in 1 mol/L hydrochloric acid TS (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of
Nicomol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 181 - 185°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Nicomol in 10 mL of 1 mol/L hydrochloric acid TS: the solu-
tion is clear and colorless.
(2) Acidity— To 1.0 g of Nicomol add 50 mL of freshly
boiled and cooled water, shake for 5 minutes, filter, and to 25
918
Nicomol Tablets / Official Monographs
JP XV
mL of the filtrate add 0.60 mL of 0.01 mol/L sodium
hydroxide VS and 2 drops of phenolphthalein TS: a red color
develops.
(3) Chloride <1.03>— Dissolve 0.6 g of Nicomol in 15 mL
of dilute nitric acid, and add water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 0.40 mL of 0.01 mol/L
hydrochloric acid VS add 15 mL of dilute nitric acid and
water to make 50 mL (not more than 0.024%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Nicomol
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(5) Arsenic </.//> — Prepare the test solution with 1.0 g
of Nicomol according to Method 3, and perform the test (not
more than 2 ppm).
(6) Related substances — Dissolve 0.20 g of Nicomol in 20
mL of chloroform, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add chloroform
to make exactly 20 mL. Pipet 2 mL of this solution, add chlo-
roform to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
dichloromethane, ethanol (95), acetonitrile and ethyl acetate
(5:3:1:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 .5 g of Nicomol, previously
dried, add exactly 40 mL of 0.5 mol/L sodium hydroxide VS,
and boil gently under a reflux condenser connected to a car-
bon dioxide absorption tube (soda lime) for 10 minutes. Af-
ter cooling, titrate <2.50> immediately the excess sodium
hydroxide with 0.25 mol/L sulfuric acid VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion.
Each mL of 0.5 mol/L sodium hydroxide VS
= 80.08 mg of C34H32N4O9
Containers and storage Containers — Tight containers.
Nicomol Tablets
-zi^E-JU^
Nicomol Tablets contain not less than 95% and not
more than 105% of the labeled amount of nicomol
(C3 4 H3 2 N 4 9 : 640.64).
Method of preparation Prepare as directed under Tablets,
with Nicomol.
equivalent to 0.5 g of Nicomol according to the labeled
amount, add 20 mL of chloroform, shake, and filter.
Evaporate the filtrate on a water bath to dryness. Proceed
with the residue as directed in the Identification (1) and (2)
under Nicomol.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Nicomol Tablets at 75
revolutions per minute according to the Paddle method using
900 mL of the 1st fluid for dissolution test. Take 20 mL or
more of the dissolved solution 60 minutes after starting the
test, and filter through a membrane filter with a pore size not
exceeding 0.8 /xm. Discard the first 10 mL of the filtrate, pipet
2 mL of the subsequent, add the 1st fluid for dissolution test
to make exactly 25 mL, and use this solution as the sample
solution. Separately, weigh accurately about 0.1 g of nicomol
for assay, previously dried at 105°C for 4 hours, dissolve in
the 1st fluid for dissolution test to make exactly 100 mL, then
pipet 2 mL of this solution, add the 1st fluid for dissolution
test to make exactly 100 mL, and use this solution as the stan-
dard solution. Determine the absorbances, A T and A s , of the
sample solution and standard solution at 262 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>. The dis-
solution rate of Nicomol Tablets in 60 minutes is not less than
75%.
Dissolution rate (%) with respect to the labeled
amount of nicomol (C34H32N4O9)
= W s x (A T /A S ) x (1/ C) x 225
W s : Amount (mg) of nicomol for assay
C: Labeled amount (mg) of nicomol (C34H32N4O9) in 1
tablet
Assay Weigh accurately not less than 20 Nicomol Tablets
and powder. Weigh accurately a portion of the powder,
equivalent to about 1 g of nicomol (C34H32N4O9), add 100 mL
of 1 mol/L hydrochloric acid TS, shake well, add water to
make exactly 500 mL, and filter. Discard the first 50 mL of
the filtrate, pipet 2 mL of the subsequent filtrate, add 50 mL
of 1 mol/L hydrochloric acid TS and water to make exactly
250 mL, and use this solution as the sample solution.
Separately, weigh accurately about 80 mg of nicomol for
assay, previously dried at 105°C for 4 hours, dissolve in 50
mL of 1 mol/L hydrochloric acid TS, and add water to make
exactly 100 mL. Pipet 2 mL of this solution, add 20 mL of 1
mol/L hydrochloric acid TS and water to make exactly 100
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and the
standard solution at 262 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>.
Amount (mg) of nicomol (C34H32N4O9)
= W s x (Aj/A s ) x (25/2)
W s : Amount (mg) of nicomol for assay
Containers and storage Containers — Tight containers.
Identification To a portion of powdered Nicomol Tablets,
JPXV
Official Monographs / Nicotinamide 919
Nicorandil
— =l^>vJU
C 8 H 9 N 3 4 : 211.17
7V-[2-(Nitrooxy)ethyl]pyridine-3-carboxamide
[65141-46-0]
Nicorandil contains not less than 98.5% and not
more than 101.0% of C 8 H 9 N 3 4 , calculated on the
anhydrous basis.
Description Nicorandil occurs as white crystals.
It is freely soluble in methanol, in ethanol (99.5) and in
acetic acid (100), soluble in acetic anhydride, and sparingly
soluble in water.
Melting point: about 92°C (with decomposition).
Identification Determine the infrared absorption spectrum
of Nicorandil as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity (1) Sulfate <1.14> — Dissolve 2.0 g of Nicorandil in
20 mL of dilute ethanol, add 1 mL of dilute hydrochloric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution with
0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of dilute
ethanol and 1 mL of dilute hydrochloric acid, and dilute with
water to make 50 mL (not more than 0.010%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Nicoran-
dil according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(3) Related substances — Dissolve 20 mg of Nicorandil in
10 mL of the mobile phase, and use this solution as the
sample solution. Perform the test with 10 juL of the sample
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine each
peak area by the automatic integration method: the peak area
of iV-(2-hydroxyethyl)isonicotinamide nitric ester, having the
relative retention time of about 0.86 with respect to nicoran-
dil, is not more than 0.5% of the peak area of nicorandil, the
area of all other peaks is less than 0.1%, and the sum area of
the peaks other than nicorandil and Af-(2-hydrox-
yethyl)isonicotinamide nitric ester is not more than 0.25% of
the total peak area.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, tetrahydrofuran,
triethylamine and trifluoroacetic acid (982:10:5:3).
Flow rate: Adjust the flow rate so that the retention time of
nicorandil is about 18 minutes.
Time span of measurement: About 3 times as long as the
retention time of nicorandil beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, add the mobile phase to make exactly
500 mL, and use this solution as the solution for system
suitability test. Pipet 1 mL of the solution for system suitabil-
ity test, and add the mobile phase to make exactly 20 mL.
Confirm that the peak area of nicorandil obtained with 10 fiL
of this solution is equivalent to 2 to 8% of that with 10 [iL of
the solution for system suitability test.
System performance: Dissolve 10 mg of ./V-(2-hydrox-
yethyl)isonicotinamide nitric ester in the mobile phase to
make 100 mL. To 1 mL of this solution add 10 mL of the
sample solution. When the procedure is run with this solution
under the above operating conditions, A r -(2-hydrox-
yethyl)isonicotinamide nitric ester and nicorandil are eluted
in this order with the resolution between these peaks being
not less than 3.0.
System repeatability: When the test is repeated 6 times with
10 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of nicorandil is not more than 1.5%.
Water <2.48> Not more than 0.1% (2 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Nicorandil, dissolve
in 30 mL of a mixture of acetic anhydride and acetic acid
(100) (7:3), and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determination
in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 21.12 mg of C 8 H 9 N 3 4
Containers and storage Containers — Tight containers.
Storage — At a temperature between 2°C and 8°C.
Nicotinamide
NHj
Pyridine-3-carboxamide [98-92-0]
Nicotinamide, when dried, contains not less than
98.5% and not more than 102.0% of C 6 H 6 N 2 0.
Description Nicotinamide occurs as white crystals or crys-
talline powder. It is odorless, and has a bitter taste.
Nicotinamide is freely soluble in water and in ethanol (95),
and slightly soluble in diethyl ether.
Identification (1) Mix 5 mg of Nicotinamide with 0.01 g
of l-chloro-2,4-dinitrobenzene, heat gently for 5 to 6 se-
conds, and fuse the mixture. Cool, and add 4 mL of potassi-
920
Nicotinic Acid / Official Monographs
JP XV
um hydroxide-ethanol TS: a red color is produced.
(2) To 0.02 g of Nicotinamide add 5 mL of sodium
hydroxide TS, and boil carefully: the gas evolved turns
moistened red litmus paper blue.
(3) Dissolve 0.02 g of Nicotinamide in water to make
1000 mL. Determine the absorption spectrum of the solultion
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Nicotinamide Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
pH <2.54> Dissolve 1.0 g of Nicotinamide in 20 mL of
water: the pH of this solution is between 6.0 and 7.5.
Melting point <2.60> 128-131°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Nicotinamide in 20 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Take 0.5 g of Nicotinamide, and
perform the test. Prepare the control solution with 0.30 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Take 1.0 g of Nicotinamide, and per-
form the test. Prepare the control solution with 0.40 mL of
0.005 mol/L sulfuric acid VS (not more than 0.019%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Nicotinamide according to Method 1, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
(5) Readily carbonizable substances <1.15> — Take 0.20 g
of Nicotinamide, and perform the test. The solution has no
more color than Matching Fluid A.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 25 mg each of Nicotinamide
and Nicotinamide Reference Standard, both previously
dried, dissolve separately in 3 mL of water, and add the mo-
bile phase to make them exactly 100 mL. Pipet 8 mL each of
these solutions, and add the mobile phase to make exactly 50
mL. Pipet 5 mL each of these solutions, add exactly 5 mL of
the internal standard solution, and use these solutions as the
sample solution and the standard solution, respectively. Per-
form the test with 20 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of nicotinamide to
that of the internal standard.
Amount (g) of nicotinamide (C 6 H 6 N 2 0) = W s x (Q T /Q S )
W s : Amount (mg) of dried Nicotinamide Reference Stan-
dard
Internal standard solution — A solution of nicotinic acid (1 in
1250).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1 g of sodium 1-heptane sulfonate
in water to make 1000 mL. To 700 mL of this solution add
300 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
nicotinamide is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, nicotinic acid and nicotinamide are eluted in this order
with the resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of nicotinamide to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Nicotinic Acid
-zj^>m
C 6 H 5 N0 2 : 123.11
Pyridine-3-carboxylic acid
CQ>H
[59-67-6]
Nicotinic Acid, when dried, contains not less than
99.5% of C 6 H 5 N0 2 .
Description Nicotinic Acid occurs as white crystals or crys-
talline powder. It is odorless, and has a slightly acid taste.
It is sparingly soluble in water, slightly soluble in ethanol
(95), and very slightly soluble in diethyl ether.
It dissolves in sodium hydroxide TS and in sodium car-
bonate TS.
Identification (1) Triturate 5 mg of Nicotinic Acid with
0.01 g of l-chloro-2,4-dinitrobenzene, and fuse the mixture
by gentle heating for 5 to 6 seconds. Cool, and add 4 mL of
potassium hydroxide-ethanol TS: a dark red color is
produced.
(2) Dissolve 0.02 g of Nicotinic Acid in water to make
1000 mL. Determine the absorption spectrum of the solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Nicotinic Acid Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
pH <2.54> Dissolve 0.20 g of Nicotinic Acid in 20 mL of
water: the pH of this solution is between 3.0 and 4.0.
Melting point <2.60> 234 - 238 C C
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Nicotinic Acid in 20 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Perform the test with 0.5 g of Nico-
JPXV
Official Monographs / Nicotinic Acid Injection
921
tinic Acid. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14> — Dissolve 1.0 g of Nicotinic Acid in 3
mL of dilute hydrochloric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.40 mL of 0.005 mol/L sul-
furic acid VS and 3 mL of dilute hydrochloric acid, and dilute
with water to make 50 mL (not more than 0.019%).
(4) Nitro compounds — Dissolve 1.0 g of Nicotinic Acid
in 8 mL of sodium hydroxide TS, and add water to make 20
mL: the solution has no more color than Matching Fluid A.
(5) Heavy metals <1.07> — Proceed with 1.0 g of Nicotinic
Acid according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
Loss on drying <2.4I> Not more than 0.5% (1 g, 105 °C, 1
hour).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Nicotinic Acid,
previously dried, dissolve in 50 mL of water, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 5
drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.31 mg of C 6 H 5 N0 2
Containers and storage Containers — Well-closed contain-
ers.
Nicotinic Acid Injection
-3 *>£&**«
Nicotinic Acid Injection is an aqueous solution for
injection.
It contains not less than 95% and not more than
110% of the labeled amount of nicotinic acid
(C 6 H 5 N0 2 : 123.11).
Method of preparation Prepare as directed under Injec-
tions, with Nicotinic Acid. It may contain Sodium Carbonate
or Sodium Hydroxide as a solubilizer.
Description Nicotinic Acid Injection is a clear, colorless
liquid.
pH: 5.0-7.0
Identification (1) To a volume of Nicotinic Acid Injec-
tion, equivalent to 0.1 g of Nicotinic Acid according to the la-
beled amount, add 0.3 mL of dilute hydrochloric acid, and
evaporate on a water bath to 2 mL. After cooling, collect the
crystals formed, wash with small portions of ice-cold water
until the last washing shows no turbidity on the addition of
silver nitrate TS, and dry at 105°C for 1 hour: the crystals
melt <2.60> between 234°C and 238°C. With the crystals,
proceed as directed in the Identification (1) under Nicotinic
Acid.
(2) Dissolve 0.02 g of the dried crystals obtained in (1) in
water to make 1000 mL, and determine the absorption spec-
trum as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 261 nm and 263 nm,
and a minimum between 235 nm and 239 nm. Separately, de-
termine the absorbances of this solution, A x and A 2 , at each
wavelength of maximum and minimum absorption, respec-
tively: the ratio A 2 /A l is between 0.35 and 0.39.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Nicotinic Acid Injec-
tion, equivalent to about 0.1 g of nicotinic acid (C 6 H 5 N02),
and add the mobile phase to make exactly 100 mL. Pipet 10
mL of this solution, add exactly 10 mL of the internal stan-
dard solution, then add the mobile phase to make 100 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 0.1 g of Nicotinic Acid Reference
Standard, previously dried at 105°C for 1 hour, and dissolve
in the mobile phase to make exactly 100 mL. Pipet 10 mL of
this solution, add exactly 10 mL of the internal standard so-
lution, then add the mobile phase to make 100 mL, and use
this solution as the standard solution. Perform the test with
10 [iL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, g T and Q s ,
of the peak area of nicotinic acid to that of the internal stan-
dard.
Amount (mg) of nicotinic acid (C 6 H 5 N0 2 )
= W S X(Q J /Q S )
W s : Amount (mg) of Nicotinic Acid Reference Standard
Internal standard solution — A solution of caffeine in the
mobile phase (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 260 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 1.1 g of sodium 1-octane sulfonate
in a mixture of 0.05 mol/L sodium dihydrogenphosphate TS,
pH 3.0 and methanol (4:1) to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
caffeine is about 9 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, nicotinic acid and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of nicotinic acid to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
922
Nifedipine / Official Monographs
JP XV
Nifedipine
-7i-/ti>
H,C N CH 3
TT
H3C'
<x
I
o
NO,
C 17 H 18 N 2 6 : 346.33
Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-l ,4-
dihydropyridine-3,5-dicarboxylate [21829-25-4]
Nifedipine contains not less than 98.0% and not
more than 102.0% of C 17 H 18 N 2 6 , calculated on the
dried basis.
Description Nifedipine occurs as a yellow, crystalline pow-
der. It is odorless and tasteless.
It is freely soluble in acetone and in dichloromethane, spar-
ingly soluble in methanol, in ethanol (95) and in acetic acid
(100), slightly soluble in diethyl ether, and practically insolu-
ble in water.
It is affected by light.
Identification (1) Dissolve 0.05 g of Nifedipine in 5 mL of
ethanol (95), and add 5 mL of hydrochloric acid and 2 g of
zinc powder. Allow to stand for 5 minutes, and filter. Per-
form the test with the filtrate as directed under Qualitative
Tests <].09> for primary aromatic amines: a red-purple color
develops.
(2) Determine the absorption spectrum of a solution of
Nifedipine in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Nifedipine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 172 - 175 °C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Nifedipine in 5 mL of acetone: the solution is clear and yel-
low.
(2) Chloride <I.03>— To 2.5 g of Nifedipine add 12 mL of
dilute acetic acid and 13 mL of water, and heat to boil. After
cooling, filter, and discard the first 10 mL of the filtrate. To 5
mL of the subsequent filtrate add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution with
0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.021%).
(3) Sulfate <1.14>— To 4 mL of the filtrate obtained in (2)
add 1 mL of dilute hydrochloric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 0.45 mL of 0.005 mol/L
sulfuric acid VS (not more than 0.054%).
(4) Heavy metals <1.07>— Proceed with 2.0 g of Nifedi-
pine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Nifedipine according to Method 3, and perform the test
(not more than 2 ppm).
(6) Basic substances — The procedure should be per-
formed under protection from direct sunlight in light-
resistant vessels. Dissolve 5.0 g of Nifedipine in 80 mL of a
mixture of acetone and acetic acid (100) (5:3), and titrate
<2.50> with 0.02 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction. Not more than 1.9 mL of 0.02 mol/L
perchloric acid VS is consumed.
(7) Dimethyl-2,6-dimethyl-4-(2-nitrosophenyl)-3 ,5-
pyridinedicarboxylate — The procedure should be performed
under protection from direct sunlight in light-resistant ves-
sels. Dissolve 0.15 g of Nifedipine in dichloromethane to
make exactly 10 mL, and use this solution as the sample solu-
tion. Separately, dissolve 10 mg of dimethyl 2,6-dimethyl-4-
(2-nitrosophenyl)-3,5-pyridine-dicarboxylate for thin-layer
chromatography in exactly 10 mL of dichloromethane.
Measure exactly 1 mL of this solution, add dichloromethane
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uh each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of cyclohexane and ethyl
acetate (3:2) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spot from the sample solution, corresponding to
that from the standard solution, is not more intense than the
spot from the standard solution.
Loss on drying <2.41>
2 hours).
Not more than 0.5% (0.5 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay The procedure should be performed under protec-
tion from direct sunlight in light-resistant vessels. Weigh ac-
curately about 0.12 g of Nifedipine, and dissolve in methanol
to make exactly 200 mL. Measure exactly 5 mL of this solu-
tion, and add methanol to make exactly 100 mL. Determine
the absorbance A of this solution at the wavelength of maxi-
mum absorption at about 350 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 17 H 18 N 2 6
= (A/ 142.3)* 40,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Nilvadipine 923
Nilvadipine
and enantiomer
C 19 H 19 N 3 6 : 385.37
3-Methyl 5-(l-methylethyl) (4i?S)-2-cyano-6-methyl-
4-(3-nitrophenyl)-l,4-dihydropyridine-3,5-dicarboxylate
[75530-68-6]
Nilvadipine contains not less than 98.0% and not
more than 102.0% of C 19 H 19 N 3 6 .
Description Nilvadipine occurs as a yellow crystalline
powder.
It is freely soluble in acetonitrile, soluble in methanol,
sparingly soluble in ethanol (99.5), and practically insoluble
in water.
A solution of Nilvadipine in acetonitrile (1 in 20) shows no
optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Nilvadipine in ethanol (99.5) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Nilvadipine Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Nilvadipine as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the
spectrum of Nilvadipine Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2. 60> 1 67 - 1 7 1 °C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Nilvadipine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Related substances — Dissolve 20 mg of Nilvadipine in
20 mL of acetonitrile, and use this solution as the sample so-
lution. Perform the test with 5 /xL of the sample solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area by the
automatic integration method, and calculate the amount of
them by the area percentage method: the amount of each
related substance is not more than 0.3%, and the total of
them is not more than 0.5%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fxm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of phosphate buffer solution,
pH 7.4, methanol and acetonitrile (32:27:18).
Flow rate: Adjust the flow rate so that the retention time of
nilvadipine is about 12 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of nilvadipine beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 1 mL of the sample
solution, add acetonitrile to make exactly 100 mL, and use
this solution as the solution for system suitability test. Pipet 1
mL of the solution for system suitability test, and add
acetonitrile to make exactly 10 mL. Confirm that the peak
area of nilvadipine obtained from 5 /xL of this solution is
equivalent to 7 to 13% of that obtained from 5 /xL of the
solution for system suitability test.
System performance: When the procedure is run with 5 juL
of the solution for system suitability test under the above
operating conditions, the number of theoretical plates and
the symmetry factor of the peak of nilvadipine is not less than
3300 and not more than 1.3, respectively.
System repeatability: Pipet 1 mL of the solution for system
suitability test, and add acetonitrile to make exactly 10 mL.
When the test is repeated 6 times with 5 /xL of this solution
under the above operating conditions, the relative standard
deviation of the peak area of nilvadipine is not more than
1.5%.
Loss on drying <2.41> Not more than 0.1% (1 g, 105°C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 25 mg each of Nilvadipine
and Nilvadipine Reference Standard, dissolve in methanol to
make exactly 25 mL. Pipet 10 mL each of these solutions,
add exactly 20 mL of the internal standard solution, 20 mL
of water and methanol to make 100 mL, and use these solu-
tions as the sample solution and the standard solution, re-
spectively. Perform the test with 5 /uL each of the sample so-
lution and standard solution as directed under the Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Q T and Q s , of the peak area
of nilvadipine to that of the internal standard.
Amount (mg) of C 19 H I9 N 3 6 = W s x (Q T /Q 5 )
W s : Amount (mg) of Nilvadipine Reference Standard
Internal standard solution — A solution of acenaphthene in
methanol (1 in 200).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.5 g of diammonium hydrogen
phosphate in 1000 mL of water, add 10 mL of tetrabutylam-
monium hydoxide TS, adjust the pH to 7.0 with diluted phos-
924
Nilvadipine Tablets / Official Monographs
JP XV
phoric acid (1 in 10), and add 900 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
nilvadipine is about 12 minutes.
System suitability—
System performance: When the procedure is run with 5 iiL
of the standard solution under the above operating condi-
tions, nilvadipine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
5 iiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of nilvadipine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Nilvadipine Tablets
Nilvadipine Tablets contain not less than 93.0% and
not more than 107.0% of the labeled amount of
nilvadipine (C 19 H 19 N 3 6 : 385.37).
Method of preparation Prepare as directed under Tablets,
with Nilvadipine.
Identification To a quantity of powdered Nilvadipine
Tablets, equivalent to 1 mg of Nilvadipine according to the
labeled amount, add 100 mL of ethanol (99.5), shake for 10
minutes, centrifuge, and use the supernatant liquid as the
sample solution. Determine the absorption spectrum of the
sample solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 239
nm and 243 nm and a maximum having a broad-ranging ab-
sorption between 371 nm and 381 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Nilvadipine Tablets add KmL of a mixture
of acetonitrile and water (7:3) so that each mL of the solution
contains about 0.2 mg of nilvadipine (Ci9H 19 N 3 6 ) according
to the labeled amount, add exactly FmL of the internal stan-
dard solution, and disperse the particles with the aid of ultra-
sonic waves. Centrifuge for 10 minutes, and use the super-
natant liquid as the sample solution. Separately, weigh ac-
curately about 20 mg of Nilvadipine Reference Standard, dis-
solve in the mixture of acetonitrile and water (7:3) to make
exactly 20 mL. Pipet 5 mL of this solution, add exactly 25
mL of the internal standard solution and the mixture of
acetonitrile and water (7:3) to make 50 mL, and use this
solution as the standard solution. Proceed as directed in the
Assay.
Amount (mg) of nilvadipine (C 19 H 19 N 3 6 )
= W S X(Q T /Q S )X(V/100)
W s : Amount (mg) of Nilvadipine Reference Standard
Internal standard solution — A solution of acenaphthene in
acetonitrile (1 in 500).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Nilvadipine Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of water as the dissolution medium. Withdraw 20
mL or more of the dissolution medium 30 minutes after start-
ing the test, and filter through a membrane filter with a pore
size not exceeding 0.5 Lira. Discard the first 10 mL of the
filtrate, pipet 10 mL of the subsequent filtrate, add exactly 1
mL of methanol, and use this solution as the sample solution.
Separately, weigh accurately an amount of Nilvadipine
Reference Standard, equivalent to 10 times the labeled
amount of Nilvadipine Tablets, and dissolve in methanol to
make exactly 50 mL. Pipet 5 mL of this solution, and add
methanol to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add exactly 10 mL of water, and use this solution as the
standard solution. Perform the test with exactly 20 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
nilvadipine: the dissolution rate in 30 minutes is not less than
85%.
Dissolution rate (%) with respect to the labeled amount of
nilvadipine (Ci9H 19 N 3 6 )
= W s x(Aj/A s )x(l/C)x9
W s : Amount (mg) of Nilvadipine Reference Standard
C: Labeled amount (mg) of nilvadipine (C 1 9H I9 N 3 6 ) in 1
tablet
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 242 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 fim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of phosphate buffer solution, pH
7.4, methanol and acetonitrile (7:7:6)
Flow rate: Adjust the flow rate so that the retention time of
nilvadipineis about 5 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of nilvadipine are not less than 2000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nilvadipine is not more than 1.5%.
Assay Weigh accurately not less than 20 Nilvadipine
Tablets, and powder. Weigh accurately an amount of the
powder, equivalent to about 5 mg of nilvadipine
(Ci9H 19 N 3 6 ), add 10 mL of a mixture of acetonitrile and
water (7:3) and exactly 25 mL of the internal standard solu-
tion, shake for 15 minutes, and add the mixture of acetoni-
trile and water (7:3) to make 50 mL. Centrifuge, and use the
supernatant liquid as the sample solution. Separately, weigh
accurately about 20 mg of Nilvadipine Reference Standard,
dissolve in the mixture of acetonitrile and water (7:3) to make
exactly 20 mL. Pipet 5 mL of this solution, add exactly 25
mL of the internal standard solution and the mixture of
JPXV
Official Monographs / Nitrazepam
925
acetonitrile and water (7:3) to make 50 mL, and use this
solution as the standard solution. Perform the test with 5 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Q T and Q s , of
the peak area of nilvadipine to that of the internal standard.
Amount (mg) of nilvadipine (Ci9H I9 N 3 6 )
= ^sX(e T /Gs)x(l/4)
W s : Amount (mg) of Nilvadipine Reference Standard
Internal standard solution — A solution of acenaphthene in
acetonitrile (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.5 g of diammonium hydrogen
phosphate in 1000 mL of water, add 10 mL of tetrabutylam-
monium hydoxide TS, adjust the pH to 7.0 with diluted phos-
phoric acid (1 in 10), and add 900 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
nilvadipine is about 12 minutes.
System suitability —
System performance: When the procedure is run with 5 juL
of the standard solution under the above operating condi-
tions, nilvadipine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of nilvadipine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Nitrazepam
QjHuNjCv 281.27
7-Nitro-5-phenyl-l,3-dihydro-2//-l,4-benzodiazepin-2-one
[146-22-5]
Nitrazepam, when dried, contains not less than
99.0% of QjHuNAi.
Description Nitrazepam occurs as white to yellow crystals
or crystalline powder. It is odorless.
It is freely soluble in acetic acid (100), soluble in acetone
and in chloroform, slightly soluble in methanol, in ethanol
(95) and in ethanol (99.5), very slightly soluble in diethyl
ether, and practically insoluble in water.
Melting point: about 227°C (with decomposition).
Identification (1) To 3 mL of a solution of Nitrazepam in
methanol (1 in 500) add 0.1 mL of sodium hydroxide TS: a
yellow color is produced.
(2) To 0.02 g of Nitrazepam add 15 mL of dilute
hydrochloric acid, boil for 5 minutes, cool, and filter: the
filtrate responds to the Qualitative Tests <1.09> for primary
aromatic amines.
(3) Neutralize 0.5 mL of the filtrate obtained in (2) with
sodium hydroxide TS, add 2 mL of ninhydrin TS, and heat
on a water bath: a purple color is produced.
(4) Determine the absorption spectrum of a solution of
Nitrazepam in ethanol (99.5) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Nitrazepam in 20 mL of acetone: the solution is clear and
pale yellow to light yellow in color.
(2) Heavy metals <1.07> — Proceed with 1 .0 g of Nitrazep-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Nitrazepam according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.25 g of Nitrazepam in
a 10 mL of mixture of methanol and chloroform (1:1), and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add a mixture of methanol and chloroform
(1:1) to make exactly 20 mL, pipet 2 mL of this solution, add
a mixture of methanol and chloroform (1:1) to make exactly
50 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of nitromethane and ethyl acetate
(17:3) to a distance of about 10 cm, and air-dry the plate. Ex-
amine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Nitrazepam, previ-
ously dried, and dissolve in 40 mL of acetic acid (100). Titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.13 mg of C 15 H U N303
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
926
Nitrendipine / Official Monographs
JP XV
Nitrendipine
- hU>vtf>
H 3 C
C 18 H 20 N 2 O 6 : 360.36
3-Ethyl 5-methyl (4.RS)-2,6-dimethyl-4-(3-nitrophenyl)-
l,4-dihydropyridine-3,5-dicarboxylate
[39562-70-4]
Nitrendipine, when dried, contains not less than
98.5% and not more than 101.0% of C 18 H 20 N 2 O 6 .
Description Nitrendipine occurs as a yellow crystalline
powder.
It is soluble in acetonitrile, sparingly soluble in methanol
and in ethanol (99.5), and practically insoluble in water.
It is gradually colored to brownish yellow by light.
A solution of Nitrendipine in acetonitrile (1 in 50) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Nitrendipine in methanol (1 in 80,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Nitrendipine as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 157-161°C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Nitrendipine according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Related substances — Conduct this procedure rapidly
using light-resistant vessels. Dissolve 40 mg of Nitrendipine
in 5 mL of acetonitrile, add the mobile phase to make 25 mL,
and use this solution as the sample solution. Pipet 1 mL of
this solution, add the mobile phase to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test immediately with exactly 10 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Determine each peak area by the automatic integration
method, and calculate the amount of related substances by
the following equation: the amount of a related substance,
having the relative retention time of about 0.8 with respect to
nitrendipine, is not more than 1.0%, a related substance,
having the relative retention time of about 1.3, is not more
than 0.25%, and other related substances are not more than
0.2%, respectively. The total amount of the substances other
than nitrendipine is not more than 2.0%.
Amount (%) of related substance =A T /A S
A T : Each peak area other than nitrendipine obtained from
the sample solution
^4 S : Peak area of nitrendipine obtained from the standard
solution
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, tetrahydrofuran and
acetonitrile (14:6:5).
Flow rate: Adjust the flow rate so that the retention time of
nitrendipine is about 12 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of nitrendipine beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 10 mL.
Confirm that the peak area of nitrendipine obtained with 10
/xL of this solution is equivalent to 14 to 26% of that with 10
/xL of the standard solution.
System performance: Dissolve 10 mg of Nitrendipine and 3
mg of propyl parahydroxybenzoate in 5 mL of acetonitrile,
and add the mobile phase to make 100 mL. When the proce-
dure is run with 5 fiL of this solution under the above operat-
ing conditions, propyl parahydroxybenzoate and nitrendi-
pine are eluted in this order with the resolution between these
peaks being not less than 6.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nitrendipine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Nitrendipine, previ-
ously dried, dissolve in 60 mL of a solution of sulfuric acid in
ethanol (99.5) (3 in 100), add 50 mL of water, and titrate
<2.50> with 0.1 mol/L serium (IV) tetraammonium sulfate
VS until the red-orange color of the solution vanishes (indica-
tor: 3 drops of 1,10-phenanthroline TS). Perform a blank de-
termination in the same manner, and make any necessary
correction.
Each mL of 0.1 mol/L serium (IV) tetraammonium sulfate
VS
= 18.02 mg of C 18 H 20 N 2 O 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Nitrendipine Tablets
927
Nitrendipine Tablets
Nitrendipine Tablets contain not less than 93.0%
and not more than 107.0% of the labeled amount of
nitrendipine (C 18 H 2 oN 2 6 : 360.36).
Method of preparation Prepare as directed under Tablets,
with Nitrendipine.
Identification Shake a quantity of powdered Nitrendipine
Tablets, equivalent to 5 mg of Nitrendipine according to the
labeled amount, with 70 mL of methanol, then add methanol
to make 100 mL, and centrifuge. To 5 mL of the supernatant
liquid add methanol to make 20 mL, and determine the ab-
sorption spectrum of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 234 nm and 238 nm, and between 350 nm and 354
nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Conduct this procedure using light-resistant vessels. To 1
tablet of Nitrendipine Tablets add 15 mL of diluted acetoni-
trile (4 in 5), stir until the tablet is completely disintegrated,
and further stir for 10 minutes. Add diluted acetonitrile (4 in
5) to make exactly 20 mL, and centrifuge. Pipet KmL of the
supernatant liquid, equivalent to about 1 mg of nitrendipine
(C 18 H 2 oN 2 6 ), add exactly 5 mL of the internal standard solu-
tion, then add diluted acetonitrile (4 in 5) to make 25 mL, and
use this solution as the sample solution. Proceed as directed
in the Assay.
Amount (mg) of nitrendipine (Ci 8 H 20 N 2 O 6 )
= W s x(Q T /Q s )x(UV)*(\/5)
W s : Amount (mg) of nitrendipine for assay
Internal standard solution— A solution of propyl para-
hydroxybenzoate in diluted acetonitrile (4 in 5) (1 in 10,000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Conduct this procedure using light-resistant vessels. Per-
form the test with 1 tablet of Nitrendipine Tablets at 100
revolutions per minute according to the Paddle method, us-
ing 900 mL of the dissolution medium (a solution of polysor-
bate 80 (3 in 5000) for a 5-mg tablet, and a solution of poly-
sorbate 80 (3 in 2000) for a 10-mg tablet). Withdraw 20 mL or
more of the dissolution medium 45 minutes after starting the
test, and filter through a membrane filter with a pore size not
exceeding 0.45 Lira. Discard the first 10 mL of the filtrate,
pipet the subsequent KmL, add the dissolution medium to
make exactly V mL so that each mL contains about 5.6 ii% of
nitrendipine (C 18 H 20 N 2 O 6 ) according to the labeled amount,
and use this solution as the sample solution. Separately,
weigh accurately about 28 mg of nitrendipine for assay,
previously dried at 105°C for 2 hours, dissolve in methanol to
make exactly 100 mL, then pipet 5 mL of this solution, and
add the dissolution medium to make exactly 50 mL. Pipet 5
mL of this solution, add the dissolution medium to make ex-
actly 25 mL, and use this solution as the standard solution.
Perform the test with exactly 20 iiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of nitrendipine.
The dissolution rate in 45 minutes is not less than 70%.
Dissolution rate (%) with respect to the labeled amount of
nitrendipine (C 18 H 20 N 2 O 6 )
= W s x (A T /A S ) x (V'/V) x (1/C) x 18
W s : Amount (mg) of nitrendipine for assay
C: Labeled amount (mg) of nitrendipine (C 18 H 20 N 2 O 6 ) in 1
tablet
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 356 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 Lira in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, tetrahydrofuran and
acetonitrile (14:6:5)
Flow rate: Adjust the flow rate so that the retention time of
nitrendipine is about 9 minutes.
System suitability —
System performance: When the procedure is run with 20
liL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of nitrendipine are not less than 5000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nitrendipine is not more than 2.0%.
Assay Conduct this procedure using light-resistant vessels.
To 20 tablets of Nitrendipine Tablets add 150 mL of diluted
acetonitrile (4 in 5), stir until the tablets completely disinte-
grate, and stir for further 10 minutes. Add diluted acetoni-
trile (4 in 5) to make exactly 200 mL, and centrifuge. Pipet a
volume of the supernatant liquid, equivalent to about 2 mg
of nitrendipine (C 18 H 20 N 2 O 6 ), add exactly 10 mL of the inter-
nal standard solution and diluted acetonitrile (4 in 5) to make
50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.1 g of nitrendipine for
assay, previously dried at 105°C for 2 hours, and dissolve in
diluted acetonitrile (4:5) to make exactly 200 mL. Pipet 4 mL
of this solution, add exactly 10 mL of the internal standard
solution and diluted acetonitrile (4:5) to make 50 mL, and use
this solution as the standard solution. Perform the test with
10 iiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Q T and
Qs, of the peak area of nitrendipine to that of the internal
standard.
Amount (mg) of nitrendipine (C 18 H 20 N 2 O 6 )
= ^ s x(g T /g s )x(l/50)
W s : Amount (mg) of nitrendipine for assay
Internal standard solution — A solution of propyl para-
hydroxybenzoate in diluted acetonitrile (4 in 5) (1 in 10,000).
928
Nitrogen / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about 25°
C.
Mobile phase: A mixture of water, tetrahydrofuran and
acetonitrile (14:6:5)
Flow rate: Adjust the flow rate so that the retention time of
nitrendipine is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the internal standard and nitrendipine are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
nitrendipine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Nitrogen
N 2 : 28.01
Nitrogen contains not less than 99.5 vol% of N 2 .
Description Nitrogen is a colorless gas and is odorless.
Nitrogen (1 mL) dissolves in 65 mL of water and in 9 mL
of ethanol (95) at 20°C and at a pressure of 101.3 kPa.
Nitrogen (1000 mL) at 0°C and at a pressure of 101.3 kPa
weighs about 1.251 g.
It is inert and does not support combustion.
Identification The flame of a burning wood splinter is extin-
guished immediately in an atmosphere of Nitrogen.
Purity Carbon dioxide — Maintain the containers of Nitro-
gen at a temperature between 18°C and 22°C for more than 6
hours before the test, and correct the volume to be at 20°C
and 101.3 kPa.
Pass 1000 mL of Nitrogen into 50 mL of barium hydroxide
TS in a Nessler tube during 15 minutes through a delivery
tube with an orifice approximately 1 mm in diameter, keeping
the end of the tube at a distance of 2 mm from the bottom of
the Nessler tube: any turbidity produced does not exceed that
produced in the following control solution.
Control solution: To 50 mL of barium hydroxide TS in a
Nessler tube add 1 mL of a solution of 0.1 g of sodium
hydrogen carbonate in 100 mL of freshly boiled and cooled
water.
Assay Collect the sample as directed under Purity. In-
troduce 1.0 mL of Nitrogen into a gas-measuring tube or
syringe for gas chromatography from a metal cylinder with a
pressure-reducing valve, through a directly connected poly-
vinyl chloride tube. Perform the test with this solution as
directed under Gas Chromatography <2.02> according to the
following conditions. Measure the peak area A T of oxygen.
Separately, introduce 1.0 mL of oxygen into the gas mixer,
add carrier gas to make exactly 100 mL, mix thoroughly, and
use this as the standard gas mixture. Proceed with 1.0 mL of
this mixture in the same manner under Nitrogen, and meas-
ure the peak area A s of oxygen.
Amount (vol%) of N 2 = 100- (A T /A S )
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with zeolite for gas chro-
matography (250 to 350 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium
Flow rate: Adjust the flow rate so that the retention time of
oxygen is about 3 minutes.
Selection of column: Introduce 1.0 mL of oxygen into the
gas mixer, add Nitrogen to make 100 mL, and mix thorough-
ly. Proceed with 1.0 mL of this mixture under the above
operating conditions. Use a column giving well-resolved
peaks of oxygen and nitrogen in this order.
System repeatability: Repeat the test 5 times according to
the above conditions with the standard gas mixture. Relative
standard deviation of peak area of oxygen is not more than
2.0%.
Containers and storage Containers — Metal cylinders.
Storage— Not exceeding 40°C.
Nitroglycerin Tablets
Nitroglycerin Tablets contain not less than 80% and
not more than 120% of the labeled amount of
nitroglycerin (C 3 H 5 N 3 9 : 227.09).
Method of preparation Prepare as directed under Tablets,
with nitroglycerin.
Identification (1) Weigh a quantity of powdered
Nitroglycerin Tablets, equivalent to 6 mg of nitroglycerin
(C3H5N3O9) according to the labeled amount, shake
thoroughly with 12 mL of diethyl ether, filter, and use the
filtrate as the sample solution. Evaporate 5 mL of the sample
solution, dissolve the residue in 1 to 2 drops of sulfuric acid,
and add 1 drop of diphenylamine TS: a deep blue color de-
velops.
(2) Evaporate 5 mL of the sample solution obtained in
(1), add 5 drops of sodium hydroxide TS, heat over a low
flame, and concentrate to about 0.1 mL. Cool, heat the
residue with 0.02 g of potassium hydrogen sulfate: the odor
of acrolein is perceptible.
Purity Free nitrate ion — Transfer an accurately measured
quantity of powdered Nitroglycerin Tablets, equivalent to 20
mg of nitroglycerin (C3H5N3O9) according to the labeled
amount, to a separator, add 40 mL of isopropylether and 40
mL of water, shake for 10 minutes, and allow the layers to
JPXV
Official Monographs / Nitrous Oxide 929
separate. Collect the aqueous layer, add 40 mL of
isopropylether, shake for 10 minutes, collect the aqueous lay-
er, filter, and use the filtrate as the sample solution. Separate-
ly, transfer 10 mL of Standard Nitric Acid Solution to a sepa-
rator, add 30 mL of water and 40 mL of the isopropyl ether
layer of the first extraction of the sample solution, shake for
10 minutes, continue the procedure in the same manner as the
sample solution, and use the solution so obtained as the stan-
dard solution. Transfer 20 mL each of the sample solution
and the standard solution to Nessler tubes, respectively,
shake well with 30 mL of water and 0.06 g of Griess-
Romijin's nitric acid reagent, allow to stand for 30 minutes,
and observe the tubes horizontally: the sample solution has
no more color than the standard solution.
Uniformity fo dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirements of the
Content uniformity test.
Transfer 1 tablet of Nitroglycerin Tablets to a glass-stop-
pered centrifuge tube, and add exactly V mL of acetic acid
(100) to provide a solution containing about 30,ug of
nitroglycerin (C3H5N3O9) per ml. Shake vigorously for 1
hour, and after disintegrating the tablet, centrifuge, and use
the supernatant liquid as the sample solution. When the
tablet does not disintegrate during this procedure, transfer 1
tablet of Nitroglycerin Tablets to a glass-stoppered centrifuge
tube, wet the tablet with 0.05 mL of acetic acid (100), and
grind down it with a glass rod. While rinsing the glass rod,
add acetic acid (100) to make exactly FmL of a solution con-
taining about 30 /xg of nitroglycerin (C3H5N3O9) per ml.
Shake for 1 hour, centrifuge, and use the supernatant liquid
as the sample solution. Separately, weigh accurately about 90
mg of potassium nitrate, previously dried at 105 °C for 4
hours, dissolve in 5 mL of water, and add acetic acid (100) to
make exactly 100 mL. Pipet 5 mL of the solution, add acetic
acid (100) to make exactly 100 mL, and use this solution as
the standard solution. Measure exactly 2 mL each of the sam-
ple solution and the standard solution, add 2 mL each of sali-
cylic acid TS shake, allow to stand for 15 minutes, and add 10
mL each of water. Render the solution alkaline with about 12
mL of a solution of sodium hydroxide (2 in 5) while cooling
in ice, and add water to make exactly 50 mL. Perform the test
with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using a solution, prepared with 2
mL of acetic acid (100) in the same manner, as the blank. De-
termine the absorbances, A T and A s , of the subsequent solu-
tions of the sample solution and the standard solution at 410
nm, respectively.
Amount (mg) of nitroglycerin (C3H5N3O9)
= W s x (A T /A S ) x (F/2000) x 0.7487
WS: Amount (mg) of potassium nitrate
Calculate the average content from the contents of 10
tablets: it meets the requirements of the test when each con-
tent deviates from the average content by not more than
25%. When there is 1 tablet showing a deviation exceeding
25% and not exceeding 30%, determine the content of an ad-
ditional 20 tablets in the same manner. Calculate the 30 devi-
ations from the new average of all 30 tablets: it meets the re-
quirements of the test when 1 tablet may deviate from the
average content by between 25% and 30%, but no tablet
deviates by more than 30%.
Disintegration <6.09> It meets the requirement, provided
that the time limit of the test is 2 minutes, and the use of the
disks is omitted.
Assay Weigh accurately and disintegrate, by soft pressing,
not less than 20 Nitroglycerin Tablets. Weigh accurately a
portion of the powder, equivalent to about 3.5 mg of
nitroglycerin (C3H5N3O9), add exactly 50 mL of acetic acid
(100), shake for 1 hour, filter, and use this filtrate as the sam-
ple solution. Separately, weigh accurately about 90 mg of
potassium nitrate, previously dried at 105°C for 4 hours, dis-
solve in 5 mL of water, and add acetic acid (100) to make ex-
actly 100 mL. Pipet 10 mL of the solution, add acetic acid
(100) to make exactly 100 mL, and use this solution as the
standard solution. Measure exactly 2 mL each of the sample
solution and the standard solution, to each solution add 2
mL of salicylic acid TS, shake, allow to stand for 15 minutes,
and add 10 mL of water. Render the solution alkaline with
about 12 mL of a solution of sodium hydroxide (2 in 5) while
cooling in ice, and add water to make exactly 50 mL. Per-
form the test with these solutions as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using a solution, pre-
pared with 2 mL of acetic acid (100) in the same manner, as
the blank. Determine the absorbances, A T and A s , of the sub-
sequent solutions of the sample solution and the standard so-
lution at 410 nm, respectively.
Amount (mg) of nitroglycerin (C3H5N3O9)
= W s x (Aj/A s ) x (1/20) x 0.7487
W s : Amount (mg) of potassium nitrate
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 20°C.
Nitrous Oxide
N 2 0: 44.01
Nitrous Oxide contains not less than 97.0 vol% of
N 2 0.
Description Nitrous Oxide is a colorless gas at room tem-
perature and at atmospheric pressure, and is odorless.
1 mL of Nitrous Oxide dissolves in 1.5 mL of water and in
0.4 mL of ethanol (95) at 20°C and at a pressure of 101.3
kPa. It is soluble in diethyl ether and in fatty oils.
1000 mL of Nitrous Oxide at 0°C and at a pressure of
101.3 kPa weighs about 1 .96 g.
Identification (1) A glowing splinter of wood held in Ni-
trous Oxide: it bursts into flame immediately.
(2) Transfer 1 mL each of Nitrous Oxide and nitrous
oxide directly from metal cylinders with a pressure-reducing
valve to gas measuring tubes or syringes for gas chro-
matography, using a polyvinyl chloride induction tube. Per-
form the test with these gases as directed under Gas Chro-
matography <2.02> according to the conditions of the Assay:
the retention time of the main peak from Nitrous Oxide coin-
cides with that of nitrous oxide.
Purity Maintain the containers of Nitrous Oxide between
18°C and 22°C for more than 6 hours before the test, and
930
Noradrenaline / Official Monographs
JP XV
correct the volume at 20°C and at a pressure of 101.3 kPa.
(1) Acidity or alkalinity — To 400 mL of freshly boiled
and cooled water add 0.3 mL of methyl red TS and 0.3 mL of
bromothymol blue TS, and boil for 5 minutes. Transfer 50
mL of this solution to each of three Nessler tubes marked A,
B and C. Add 0.10 mL of 0.01 mol/L hydrochloric acid VS
to tube A, 0.20 mL of 0.01 mol/L hydrochloric acid VS to
tube B, stopper each of the tubes, and cool. Pass 100 mL of
Nitrous Oxide through the solution in tube A for 15 minutes,
employing delivery tube with an orifice approximately 1 mm
in diameter and extending to within 2 mm of the bottom of
the Nessler tube: the color of the solution in tube A is not
deeper orange-red than that of the solution in tube B and not
deeper yellow-green than that of the solution in tube C.
(2) Carbon dioxide — Pass 1000 mL of Nitrous Oxide
through 50 mL of barium hydroxide TS in a Nessler tube, in
the same manner as directed in (1): any turbidity produced
does not exceed that produced in the following control solu-
tion.
Control solution: To 50 mL of barium hydroxide TS in a
Nessler tube add 1 mL of a solution of 0.1 g of sodium
hydrogen carbonate in 100 mL of freshly boiled and cooled
water.
(3) Oxidizing substances — Transfer 15 mL of potassium
iodide-starch TS to each of two Nessler tubes marked A and
B, add 1 drop of acetic acid (100) to each of the tubes, shake,
and use these as solution A and solution B, respectively. Pass
2000 mL of Nitrous Oxide through solution A for 30 minutes
in the same manner as directed in (1): the color of solution A
is the same as that of the stoppered, untreated solution B.
(4) Potassium permanganate-reducing substance — Pour
50 mL of water into each of two Nessler tubes marked A and
B, add 0.10 mL of 0.02 mol/L potassium permanganate VS
to each of the tubes, and use these as solution A and solution
B, respectively. Pass 1000 mL of Nitrous Oxide through solu-
tion A in the manner as directed in (1): the color of solution
A is the same as that of solution B.
(5) Chloride <1.03> — Pour 50 mL of water into each of
two Nessler tubes marked A and B, add 0.5 mL of silver ni-
trate TS to each of the tubes, shake, and use these as solution
A and solution B, respectively. Pass 1000 mL of Nitrous
Oxide through solution A in the same manner as directed in
(1): the turbidity of solution A is the same as that of solution
B.
(6) Carbon monoxide — Introduce 5.0 mL of Nitrous
Oxide into a gas-cylinder or a syringe for gas chro-
matography from a metal cylinder holding gas under pressure
and fitted with a pressure-reducing valve, through a directly
connected polyvinyl tube. Perform the test with this accord-
ing to the Gas Chromatography <2.02> under the following
conditions: no peak is observed at the same retention time as
that of carbon monoxide.
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with 300 to 500 fim zeolite for
gas chromatography (0.5 nm in pore size).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
carbon monoxide is about 20 minutes.
Selection of column: To 0.1 mL each of carbon monoxide
and air in a gas mixer add carrier gas to make 100 mL, and
mix well. Proceed with 5.0 mL of the mixed gas under the
above operating conditions. Use a column giving well-
resolved peaks of oxygen, nitrogen and carbon monoxide in
this order.
Detection sensitivity: Adjust the sensitivity so that the peak
height of carbon monoxide obtained from 5.0 mL of the mix-
ed gas used in the selection of column is about 10 cm.
Assay Withdraw Nitrous Oxide as directed in the Purity.
Introduce 1.0 mL of Nitrous Oxide into a gas-measuring
tube or syringe for gas chromatography from a metal cylin-
der under pressure through a pressure-reducing valve and a
directly connected polyvinyl tube. Perform the test with this
solution as directed under Gas Chromatography <2.02> ac-
cording to the following conditions, and determine the peak
area A T of air. Separately, introduce 3.0 mL of nitrogen into
a gas mixer, add carrier gas to make exactly 100 mL, mix
thoroughly, and use this as the standard mixed gas. Proceed
with 1.0 mL of this mixture as directed in the case of Nitrous
Oxide, and determine the peak area A s of nitrogen in the
same manner.
Amount (vol%) of N 2 = 100 - 3 x (A T /A S )
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with silica gel for gas chro-
matography (300 to 500 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
nitrogen is about 2 minutes.
Selection of column: To 3.0 mL of nitrogen in a gas mixer
add Nitrous Oxide to make 100 mL, and mix well. Proceed
with 1 .0 mL of the mixed gas under the above operating con-
ditions. Use a column giving well-resolved peaks of nitrogen
and nitrous oxide in this order.
System repeatability: Repeat the test five times with the
standard mixed gas under the above operating conditions: the
relative standard deviation of the peak area of nitrogen is not
more than 2.0%.
Containers and storage Containers-
Storage — Not exceeding 40°C.
Noradrenaline
Norepinephrine
H OH
■Metal cylinders.
and enantiomer
C 8 H„N0 3 : 169.18
4-[(l/?S)-2-Amino-l-hydroxyethyl]benzene-l,2-diol
[51-41-2]
Noradrenaline, when dried, contains not less than
JPXV
Official Monographs / Noradrenaline Injection 931
98.0% of ^/-norepinephrine (C 8 H„N0 3 ).
Description Noradrenaline occurs as a white to light brown
or slightly reddish brown, crystalline powder.
It is freely soluble in acetic acid (100), very slightly soluble
in water, and practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
It gradually changes to brown by air and by light.
Identification (1) Determine the absorption spectrum of a
solution of Noradrenaline in 0.1 mol/L hydrochloric acid TS
(3 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Noradrenaline, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Noradrenaline in 10 mL of 0.1 mol/L hydrochloric acid
TS, and add water to make 100 mL: the solution is clear and
colorless.
(2) Arterenone — Dissolve 50 mg of Noradrenaline in 0.01
mol/L hydrochloric acid TS to make exactly 100 mL. Deter-
mine the absorbance of the solution at 310 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>: it is not
more than 0.1.
(3) Adrenaline — Dissolve 10.0 mg of Noradrenaline in
2.0 mL of diluted acetic acid (100) (1 in 2). Pipet 1 mL of this
solution, add water to make 10 mL, then mix with 0.3 mL of
a solution of sodium nitrite (1 in 100), and allow to stand for
1 minute: the solution has no more color than the following
control solution.
Control solution: Dissolve 2.0 mg of Adrenaline Bitartrate
Reference Standard and 90 mg of Noradrenaline Bitartrate
Reference Standard in water to make exactly 10 mL. Measure
exactly 1 mL of this solution, add 1.0 mL of diluted acetic
acid (100) (1 in 2) and water to make 10 mL, and proceed in
the same manner.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
silica gel, 18 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Noradrenaline,
previously dried, dissolve in 50 mL of acetic acid for nona-
queous titration by warming, if necessary, and titrate <2.50>
with 0.1 mol/L perchloric acid VS until the color of the solu-
tion changes from blue-purple through blue to blue-green (in-
dicator: 2 drops of crystal violet TS). Perform a blank deter-
mination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.92 mg of C 8 H u N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant, under nitrogen atmosphere, and
in a cold place.
Noradrenaline Injection
Noradrenaline Hydrochloride Injection
Norepinephrine Hydrochloride Injection
Norepinephrine Injection
Noradrenaline Injection is an aqueous solution for
injection.
It contains not less than 90% and not more than
110% of the labeled amount of ^/-noradrenaline
(C 8 H„N0 3 : 169.18).
Method of preparation Dissolve Noradrenaline in 0.01 mol
/L hydrochloric acid TS, and prepare as directed under Injec-
tions.
Description Norepinephrine Injection is a clear, colorless
liquid.
It gradually becomes a pale red color by light and by air.
pH: 2.3-5.0
Identification Transfer a volume of Noradrenaline Injec-
tion, equivalent to 1 mg of Noradrenaline according to the la-
beled amount, to each of two test tubes A and B, and add
1 mL of water to each tube. Add 10 mL of potassium hydro-
gen phthalate buffer solution, pH 3.5, to A, and 10 mL of
phosphate buffer solution, pH 6.5, to B. To each of these so-
lutions add 1.0 mL of iodine TS, allow to stand for 5
minutes, and add 2.0 mL of sodium thiosulfate TS: no color
or a pale red color develops in test tube A, and a deep red-
purple color develops in test tube B.
Purity (1) Arterenone — Measure a volume of Noradrena-
line Injection, equivalent to 10 mg of Noradrenaline accord-
ing to the labeled amount, add water to make exactly 20 mL,
and determine the absorbance of this solution at 310 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
the absorbance is not more than 0.10.
(2) Adrenaline — Measure a volume of Noradrenaline In-
jection, equivalent to 5 mg of Noradrenaline according to the
labeled amount, add 1 mL of diluted acetic acid (100) (1 in 2)
and water to make exactly 10 mL, and proceed as directed in
the Purity (3) under Noradrenaline.
Extractable volume <6.05> It meets the requirement.
Assay Pipet a volume of Noradrenaline Injection, equiva-
lent to about 5 mg of (//-noradrenaline (C 8 H u N0 3 ), add
water to make exactly 25 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Noradrenaline Bitartrate Reference Standard, previously
dried in a desiccator (in vacuum, silica gel) for 24 hours, dis-
solve in water to make exactly 25 mL, and use this solution as
the standard solution. Piper 5 mL each of the sample solu-
tion and the standard solution, add 0.2 mL each of starch
TS, then add iodine TS dropwise with swirling until a persis-
tent blue color is produced. Add 2 mL of iodine TS, and
shake. Adjust the pH of the solution to 6.5 with 0.05 mol/L
disodium hydrogenphosphate TS, add 10 mL of phosphate
buffer solution, pH 6.5, and shake. Immediately after allow-
ing to stand for 3 minutes, add sodium thiosulfate TS drop-
932
Norethisterone / Official Monographs
JP XV
wise until a red-purple color develops, then add water to
make exactly 50 mL. Determine the absorbances, A T and A s ,
of the subsequent solutions of the sample solution and the
standard solution at 515 nm within 5 minutes as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of (//-noradrenaline (C 8 H u N0 3 )
= W s x(A T /As)x 0.5016
W s : Amount (mg) of Noradrenaline Bitartrate Reference
Standard
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
Norethisterone
-Hermetic containers,
yjHfXfQ>
=CH
C 20 H 26 O 2 : 298.42
17-Hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one
[68-22-4]
Norethisterone, when dried, contains not less than
97.0% and not more than 103.0% of C 20 H 26 O 2 .
Description Norethisterone occurs as a white to pale yel-
lowish white, crystalline powder. It is odorless.
It is soluble in chloroform, sparingly soluble in ethanol
(95) and in tetrahydrofuran, slightly soluble in diethyl ether,
and very slightly soluble in water.
It is affected by light.
Identification (1) To 2 mg of Norethisterone add 2 mL of
sulfuric acid: the solution shows a red-brown color and a yel-
low-green fluorescence. Add 10 mL of water to this solution
cautiously: a yellow color develops and a yellow-brown
precipitate is formed.
(2) To 25 mg of Norethisterone add 3.5 mL of a solution
of 0.05 g of hydroxylammonium chloride and 0.05 g of anhy-
drous sodium acetate trihydrate in 25 mL of methanol. Heat
under a reflux condenser on a water bath for 5 hours, cool,
and add 15 mL of water. Collect the precipitate formed, wash
with 1 to 2 mL of water, recrystallize from methanol, and dry
in a desiccator (in vacuum, silica gel) for 5 hours: the crystals
melt <2.60> between 112°C and 118°C.
Optical rotation <2.49> [a]™: -23- -27° (after drying,
0.25 g, chloroform, 25 mL, 200 mm).
Melting point <2.60> 203 - 209°C
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.2 g of Norethisterone,
previously dried, dissolve in 40 mL of tetrahydrofuran, add
10 mL of a solution of silver nitrate (1 in 20), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 29.84 mg of C 20 H 26 O 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Norfloxacin
CCfeH
C 16 H 18 FN 3 3 : 319.33
l-Ethyl-6-fluoro-4-oxo-7-(piperazin-l-yl)-
l,4-dihydroquinoline-3-carboxylic acid [70458-96-7]
Norfloxacin, when dried, contains not less than
99.0% of C 16 H 18 FN 3 3 .
Description Norfloxacin occurs as a white to pale yellow
crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
ethanol (99.5) and in acetone, very slightly soluble in
methanol, and practically insoluble in water.
It dissolves in dilute hydrochloric acid TS and in sodium
hydroxide TS.
It is hygroscopic.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Norfloxacin in a solu-
tion of sodium hydroxide (1 in 250) to make 100 mL. To 5
mL of this solution add a solution of sodium hydroxide (1 in
250) to make 100 mL. Determine the absorption spectrum of
this solution as directed under Ultraviolet-visible Spec-
trophotometry <2.44>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Dissolve a suitable amount of Norfloxacin in a suita-
ble amount of acetone, evaporate the acetone under reduced
pressure, and dry the residue. Determine the infrared absorp-
tion spectrum of the residue so obtained as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Purity (1) Sulfate <I.I4> — Dissolve 1.0 g of Norfloxacin
in 7 mL of 0.5 mol/L sodium hydroxide TS and 23 mL of
water, and add 1 drop of phenolphthalein TS. Add gradually
diluted hydrochloric acid (1 in 3) to this solution until the red
color disappears, then add 0.5 mL of dilute hydrochloric
acid, and cool in ice for 30 minutes. Filter through a glass
filter (G4), and wash the residue with 10 mL of water. Com-
bine the filtrate and the washing, and add 1 mL of dilute
hydrochloric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control so-
lution as follows. To 0.50 mL of 0.005 mol/L sulfuric acid
JPXV
Official Monographs / Norgestrel 933
VS add 7 mL of 0.5 mol/L sodium hydroxide TS and 1 drop
of phenolphthalein TS, add diluted hydrochloric acid (1 in 3)
until the red color disappears, then add 1.5 mL of dilute
hydrochloric acid, 1 or 2 drops of bromophenol blue TS and
water to make 50 mL (not more than 0.024%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Norflox-
acin according to Method 2, and perform the test. Prepare
the control solution with 3.0 mL of Standard Lead Solution
(not more than 15 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Norfloxacin according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve 0.10 g
of Norfloxacin in 50 mL of a mixture of methanol and ace-
tone (1:1), and use this solution as the sample solution. Pipet
1 mL of the sample solution, add a mixture of methanol and
acetone (1:1) to make exactly 100 mL. Pipet 2 mL of this so-
lution, add a mixture of methanol and acetone (1:1) to make
exactly 10 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 20 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography (5-7
fim in particle diameter). Develop with a mixture of
methanol, chloroform, toluene, diethylamine and water
(20:20:10:7:4) to a distance of about 9 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm and 366 nm): the number of the spot other than the prin-
cipal spot from the sample solution is not more than 2 and
they are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Norfloxacin, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.93 mg of C 16 H 18 FN 3 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Norgestrel
™CH
of C 21 H 28 2 .
Description Norgestrel occurs as white crystals or crystal-
line powder.
It is soluble in tetrahydrofuran and in chloroform, sparing-
ly soluble in ethanol (95), slightly soluble in diethyl ether, and
practically insoluble in water.
Identification (1) Dissolve 1 mg of Norgestrel in 2 mL of
ethanol (95), and add 1 mL of sulfuric acid: a red-purple
color develops. With this solution, examine under ultraviolet
light (main wavelength: 365 nm): the solution shows a red-
orange fluorescence.
(2) Determine the infrared absorption spectrum of Nor-
gestrel, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 206 - 212°C
Purity (1) Heavy metals <1.07> — Take 1.0 g of Norgestrel,
heat gently to carbonize, cool, add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10), and
ignite the ethanol to burn. After cooling, add 1 mL of sulfur-
ic acid, proceed with this solution according to Method 4,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 30 mg of Norgestrel in
5 mL of chloroform, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add chloroform to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of dichloromethane and
ethyl acetate (2:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 0.2 g of Norgestrel, previ-
ously dried, dissolve in 40 mL of tetrahydrofuran, add 10 mL
of a solution of silver nitrate (1 in 20), and titrate <2.50> with
0.1 mol/L sodium hydroxide VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L sodium hydroxide VS
= 31.25 mg of C 21 H 28 2
Containers and storage Containers — Well-closed contain-
ers.
C 21 H 28 2 : 312.45
13-Ethyl-17-hydroxy-18,19-dinor-17a-pregn-4-en-20-yn-
3-one [6533-00-2]
Norgestrel, when dried, contains not less than 98.0%
934
Norgestrel and Ethinylestradiol Tablets / Official Monographs
JP XV
Norgestrel and Ethinylestradiol
Tablets
Norgestrel and Ethinylestradiol Tablets contain not
less than 90% and not more than 110% of the labeled
amount of norgestrel (C 2 iH280 2 : 312.45) and ethinyles-
tradiol (C 2 oH 24 2 : 296.40).
Method of preparation Prepare as directed under Tablets,
with Norgestrel and Ethinylestradiol.
Identification (1) Weigh a quantity of Norgestrel and
Ethinylestradiol Tablets, equivalent to 10 mg of Norgestrel
according to the labeled amount, previously powdered, add
10 mL of chloroform, shake for 10 minutes, and filter. To 2
mL of the filtrate add 6 mL of sodium hydroxide TS, shake
vigorously, and centrifuge. Take 1 mL of the chloroform lay-
er, evaporate on a water bath to dryness, dissolve the residue
in 2 mL of ethanol (95), and add 1 mL of sulfuric acid: a red-
purple color develops. Examine under ultraviolet light (main
wavelength: 365 nm): this solution shows a red-orange
fluorescence (norgestrel).
(2) Take 1 mL of the filtrate obtained in (1), evaporate on
a water bath to dryness, add 1 mL of boric acid-methanol
buffer solution to the residue, shake, and cool in ice. Add 1
mL of ice-cold diazo TS, shake, add 1 mL of sodium
hydroxide TS, and shake: a red-orange color develops
(ethinylestradiol).
(3) Use the filtrate obtained in (1) as the sample solution.
Separately, dissolve 10 mg of Norgestrel Reference Standard
and 1 mg of Ethinylestradiol Reference Standard, respective-
ly, in 10 mL of chloroform, and use these solutions as the
standard solution (1) and the standard solution (2). Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 20 /uL each of the sample solu-
tion and standard solutions (1) and (2) on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of 1,2-dichloroethane, methanol and water (368:32:1) to
a distance of about 10 cm, and air-dry the plate. Spray evenly
a solution of /?-toluenesulfonate in ethanol (95) (1 in 5) on the
plate, and heat at 105°C for 5 minutes. Examine under
ultraviolet light (main wavelength: 365nm): two spots from
the sample solution show the similar color tone and Ri value
to each spot from the standard solutions (1) and (2).
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Add 2 mL of diluted methanol (7 in 10) to 1 tablet of Nor-
gestrel and Ethinylestradiol Tablets, add exactly 2 mL of the
internal standard solution, shake for 20 minutes, and cen-
trifuge. Filter the supernatant liquid through a membrane
filter with pore size of not more than 0.2 fim, and use this
filtrate as the sample solution. Separately, weigh accurately
quantities of Norgestrel Reference Standard and of
Ethinylestradiol Reference Standard, equivalent to 100 times
each of the labeled amounts, dissolve in diluted methanol (7
in 10) to make exactly 200 mL. Pipet 2 mL of this solution,
add exactly 2 mL of the internal standard solution, and use
this solution as the standard solution. Perform the test with
20 fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Calculate the ratios, g Ta and Q Tb ,
of the peak areas of norgestrel and ethinylestradiol to the
peak area of the internal standard of the sample solution and
also the ratios, gsa and Q sb , of the peak areas of norgestrel
and ethinylestradiol to the peak area of the internal standard
of the standard solution.
Amount (mg) of norgestrel (C2iH 28 2 )
= ^SaX(e T a/Gsa)X (1/100)
Amount (mg) of ethinylestradiol (C20H24O2)
= ^s b x(eTb/Gsb)x (1/100)
W Sa : Amount (mg) of Norgestrel Reference Standard
W S b' Amount (mg) of Ethinylestradiol Reference
Standard
Internal standard solution — A solution of diphenyl in diluted
methanol (7 in 10) (1 in 50,000).
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
Proceed as directed in the system suitability in the Assay.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Norgestrel and Ethinyles-
tradiol Tablets at 50 revolutions per minute according to the
Paddle method, using 900 mL of water as the test solution.
Take 50 mL or more of the dissolved solution 45 minutes af-
ter starting the test, and membrane filter through a mem-
brane filter with a pore size not exceeding 0.8 [im. Discard the
first 10 mL of the filtrate, transfer exactly 30 mL of the subse-
quent into a chromatography column [prepared by packing
0.36 g of octadecylsilanized silica gel for pretreatment (55 to
105 /xm in particle diameter) in a tube about 1 cm in inside di-
ameter]. After washing the column with 15 mL of water, e-
lute with 3 mL of methanol, and evaporate the effluent on a
water bath to dryness at about 40°C with the aid of a current
air. Dissolve the residue in exactly 2 mL of diluted methanol
(7 in 10), and use this solution as the sample solution.
Separately, weigh accurately about 25 mg of Norgestrel
Reference Standard and about 2.5 mg of Ethinylestradiol
Reference Standard dissolve in diluted methanol (7 in 10) to
make exactly 100 mL, then pipet 3 mL of this solution, add
diluted methanol (7 in 10) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 50 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the peak
areas, A Ta and A Tb , of norgestrel and ethinylestradiol from
the sample solution, and the peak areas, y4 Sa and A sb , of nor-
gestrel and ethinylestradiol from the standard solution.
The dissolution rate of Norgestrel and Ethinylestradiol
Tablets in 45 minutes is not less than 70%.
Dissolution rate (%) with respect to the labeled amount
of norgestrel (C 2 iH 28 02)
= ^SaX04 Ta A4sa)X(l/C a )X(9/5)
Dissolution rate (%) with respect to the labeled amount
of ethinylestradiol (C20H24O2)
JPXV
Official Monographs / Nortriptyline Hydrochloride
935
= ^sb x (A n /A sb ) x (l/C b ) x (9/5)
W Sa : Amount (mg) of Norgestrel Reference Standard
W sb : Amount (mg) of Ethinylestradiol Reference Standard
C a : Labeled amount (mg) of norgestrel (C21H28O2) in 1
tablet
C b : Labeled amount (mg) of ethinylestradiol (C20H24O2) in
1 tablet
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
Proceed as directed in the system suitability in the Assay.
Assay Weigh accurately not less than 20 Norgestrel and
Ethinylestradiol Tablets, and powder. Weigh accurately a
portion of the powder, equivalent to about 1 mg of norgestrel
(C21H28O2), add 4 mL of diluted methanol (7 in 10), add ex-
actly 4 mL of the internal standard solution, shake for 20
minutes, and centrifuge. Filter the supernatant liquid
through a membrane filter with pore size of not more than 0.2
/um, and use this filtrate as the sample solution. Separately,
weigh accurately about 50 mg of Norgestrel Reference Stan-
dard and about 5 mg of Ethinylestradiol Reference Standard,
and dissolve in diluted methanol (7 in 10) to make exactly 200
mL. Pipet 4 mL of this solution, add exactly 4 mL of the in-
ternal standard solution, and use this solution as the standard
solution. Perform the test with 20 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Calculate the ratios, Q Ta and Q Tb , of the peak areas of nor-
gestrel and ethinylestradiol to the peak area of the internal
standard of the sample solution and also the ratios, Q Sa and
Q sb , of the peak areas of norgestrel and ethinylestradiol to
the peak area of the internal standard of the standard solu-
tion.
Amount (mg) of norgestrel (C21H28O2)
= ^Sa><(GTa/eSa)X(l/50)
Amount (mg) of ethinylestradiol (C20H24O2)
= ^sbX(Q Tb /esb)x(l/50)
W S!i : Amount (mg) of Norgestrel Reference Standard
W sb : Amount (mg) of Ethinylestradiol Reference Stand-
ard
Internal standard solution — A solution of diphenyl in diluted
methanol (7 in 10) (1 in 50,000).
Operating conditions —
Detector: Norgestrel — An ultraviolet absorption photome-
ter (wavelength: 241 nm).
Ethinylestradiol — A fluorophotometer (excitation wave-
length: 281 nm, fluorescence wavelength: 305 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 [im in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetonitrile and water (11:9).
Flow rate: Adjust the flow rate so that the retention time of
norgestrel is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, ethinylestradiol, norgestrel and the internal standard
are eluted in this order, and the resolution between the peaks
of norgestrel and the internal standard is not less than 8.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of ethinylestradiol and norgestrel to that of the in-
ternal standard are not more than 1.0%, respectively.
Containers and storage Containers — Tight containers.
Nortriptyline Hydrochloride
•HCI
C 19 H 2I N.HC1: 299.84
3-(10,ll-Dihydro-5//-dibenzo[a,rf]cyclohepten-5-
ylidene)-/V-methylpropylamine monohydrochloride
[894-71-3]
Nortriptyline Hydrochloride, when dried, contains
not less than 98.5% of C 19 H 21 N.HC1.
Description Nortriptyline Hydrochloride occurs as a white
to yellowish white, crystalline powder. It is odorless, or has a
faint, characteristic odor.
It is freely soluble in acetic acid (100) and in chloroform,
soluble in ethanol (95), sparingly soluble in water, and practi-
cally insoluble in diethyl ether.
The pH of a solution of Nortriptyline Hydrochloride (1 in
100) is about 5.5.
Melting point: 215 - 220°C
Identification (1) To 5 mL of a solution of Nortriptyline
Hydrochloride (1 in 100) add 1 mL of bromine TS: the color
of the test solution disappears.
(2) To 5 mL of a solution of Nortriptyline Hydrochloride
(1 in 100) add 1 to 2 drops of a solution of quinhydrone in
methanol (1 in 40): a red color gradually develops.
(3) Determine the absorption spectrum of a solution of
Nortriptyline Hydrochloride (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of Nor-
triptyline Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(5) A solution of Nortriptyline Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Nortriptyline Hydrochloride in 10 mL of water: the solu-
tion is clear and colorless to very light yellow.
936
Noscapine / Official Monographs
JP XV
(2) Heavy metals <1.07> — Proceed with 1.0 g of Nortrip-
tyline Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Nortriptyline Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.50 g of Nortriptyline
Hydrochloride in 20 mL of chloroform, and use this solution
as the sample solution. Pipet 2 mL of the sample solution,
and add chloroform to make exactly 100 mL. Pipet 5 mL of
this solution, add chloroform to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 4 /uL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of cyclohexane, methanol and diethylamine
(8:1:1) to a distance of about 15 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Nortriptyline
Hydrochloride, previously dried, dissolve in 5 mL of acetic
acid (100), add 50 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 29.98 mg of d 9 H 21 N.HCl
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Noscapine
Narcotine
SXt)tf>
eh*
C 22 H 23 N0 7 : 413.42
(3S)-6,7-Dimethoxy-3-[(5.R)-4-methoxy-
6-methyl-5,6,7,8-tetrahydro[l,3]dioxolo[4,5-g]isoquinolin-
5-yl]isobenzofuran-l(3//)-one [128-62-1]
Noscapine, when dried, contains not less than 98.5%
of C 22 H 23 N0 7 .
Description Noscapine occurs as white crystals or crystal-
line powder. It is odorless and tasteless.
It is very soluble in acetic acid (100), slightly soluble in
ethanol (95) and in diethyl ether, and practically insoluble in
water.
Identification (1) Determine the absorption spectrum of a
solution of Noscapine in methanol (1 in 20,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Noscapine, previously dried, as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [ a ]^°: +42 - +48° (after drying, 0.5
g, 0.1 mol/L hydrochloric acid TS, 25 mL, 100 nm).
Melting point <2.60> 174 - 177°C
Purity (1) Chloride <1.03> — Dissolve 0.7 g of Noscapine
in 20 mL of acetone, add 6 mL of dilute nitric acid and water
to make 50 mL, and perform the test with this solution. Pre-
pare the control solution as follows: To 0.4 mL of 0.01 mol/
L hydrochloric acid add 20 mL of acetone, 6 mL of dilute
nitric acid and water to make 50 mL (not more than 0.02%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Nosca-
pine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Morphine — Dissolve 10 mg of Noscapine in 1 mL of
water and 5 mL of l-nitroso-2-naphthol TS with shaking,
add 2 mL of a solution of potassium nitrate (1 in 10), and
warm at 40°C for 2 minutes. Add 1 mL of a solution of sodi-
um nitrite (1 in 5000), and warm at 40°C for 5 minutes. After
cooling, shake the solution with 10 mL of chloroform, cen-
trifuge, and collect the aqueous layer: the solution so ob-
tained has no more color than a pale red.
(4) Related substances — Dissolve 0.7 g of Noscapine in
50 mL of acetone, and use this solution as the sample solu-
tion. Pipet 5 mL of the sample solution, add acetone to make
exactly 50 mL. Pipet 5 mL of this solution, add acetone to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of acetone, toluene, ethanol (99.5) and ammonia so-
lution (28) (60:60:9:2) to a distance of about 10 cm, and air-
dry the plate. Spray evenly dilute bismuth subnitrate-potassi-
um iodide TS for spray on the plate: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Noscapine, previ-
ously dried, dissolve in 30 mL of acetic acid (100) and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 3 drops
of crystal violet TS). Perform a blank determination, and
JPXV
Official Monographs / Nystatin 937
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 41.34 mg of C 22 H 23 N0 7
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Noscapine Hydrochloride Hydrate
Narcotine Hydrochloride
• HCI • X H;0
C 22 H 23 N0 7 .HCl.xH 2
(3S)-6,7-Dimethoxy-3-[(5.R)-4-methoxy-
6-methyl-5,6,7,8-tetrahydro[l,3]dioxolo[4,5-g]isoquinolin-
5-yl]isobenzofuran-l(3//)-one monohydrochloride hydrate
[912-60-7, anhydride]
Noscapine Hydrochloride Hydrate, when dried, con-
tains not less than 98.0% of noscapine hydrochloride
C 22 H 23 N0 7 .HC1: 449.88.
Description Noscapine Hydrochloride Hydrate occurs as
colorless or white crystals or crystalline powder. It is odor-
less, and has a bitter taste.
It is freely soluble in water, in acetic acid (100), and in acet-
ic anhydride, soluble in ethanol (95), and practically insolu-
ble in diethyl ether.
Identification (1) To 1 mg of Noscapine Hydrochloride
Hydrate add 1 drop of formaldehyde-sulfuric acid TS: a pur-
ple color, changing to yellow-brown, is produced.
(2) To 1 mg of Noscapine Hydrochloride Hydrate add 1
drop of a solution of ammonium vanadate (V) in sulfuric
acid (1 in 200): an orange color is produced.
(3) Dissolve 0.02 g of Noscapine Hydrochloride Hydrate
in 1 mL of water, and add 3 drops of sodium acetate TS: a
white, flocculent precipitate is produced.
(4) Dissolve 1 mg of Noscapine Hydrochloride Hydrate
in 1 mL of diluted sulfuric acid (1 in 35), shake with 5 drops
of a solution of disodium chlomotropate dihydrate (1 in 50),
and add 2 mL of sulfuric acid dropwise: a purple color is
produced.
(5) Dissolve 0.1 g of Noscapine Hydrochloride Hydrate
in 10 mL of water, make the solution alkaline with ammonia
TS, and shake with 10 mL of chloroform. Separate the chlo-
roform layer, wash with 5 mL of water, and filter. Distil most
of the filtrate on a water bath, add 1 mL of ethanol (99.5),
and evaporate to dryness. Dry the residue at 105°C for 4
hours: the residue so obtained melts <2.60> between 174°C
and 177°C.
(6) Make a solution of Noscapine Hydrochloride Hy-
drate (1 in 50) alkaline with ammonia TS, and filter the
precipitate. Acidify the filtrate with dilute nitric acid: the so-
lution responds to the Qualitative Tests <1.09> (2) for chlo-
ride.
Purity Morphine — Dissolve 10 mg of Noscapine
Hydrochloride Hydrate in 1 mL of water, add 5 mL of 1-
nitroso-2-naphthol TS and 2 mL of a solution of potassium
nitrate (1 in 10), and warm at 40°C for 2 minutes. Add 1 mL
of a solution of sodium nitrite (1 in 5000), and warm at 40°C
for 5 minutes. After cooling, shake the mixture with 10 mL
of chloroform, centrifuge, and separate the aqueous layer:
the solution so obtained has no more color than a pale red
color.
Loss on drying <2.41> Not more than 9.0% (0.5 g, 120°C,
4 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately about 0.5 g of Noscapine
Hydrochloride Hydrate, previously dried, dissolve in 50 mL
of a mixture of acetic anhydride and acetic acid (100) (7:3),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 44.99 mg of C 22 H 23 N0 7 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Nystatin
Nystatin is a mixture of polyene macrolide sub-
stances having antifungal activity produced by the
growth of Streptomyces noursei.
It contains not less than 4600 units (potency) per mg,
calculated on the dried basis. The potency of Nystatin
is expressed as the unit of nystatin (C 47 H 75 NO n :
926.09), and one unit corresponds to 0.27 fig of nysta-
tin (C 47 H 75 NO n ).
Description Nystatin occurs as a white to light yellow-
brown powder.
It is soluble in formamide, sparingly soluble in methanol,
slightly soluble in ethanol (95), and very slightly soluble in
water.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 1 mg of Nystatin in 5 mL of
water and 1 mL of sodium hydroxide TS, heat for 2 minutes,
and cool. To this solution add 3 mL of a solution of 4-
aminoacetophenone in methanol (1 in 200) and 1 mL of
hydrochloric acid: a red-purple color develops.
(2) To 10 mg of Nystatin add 50.25 mL of a mixture of
diluted methanol (4 in 5) and sodium hydroxide TS (200:1),
heat at not exceeding 50°C to dissolve, then add diluted
methanol (4 in 5) to make 500 mL. Determine the absorption
spectrum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of Nys-
938
Ofloxacin / Official Monographs
JP XV
tatin Reference Standard prepared in the same manner as the
sample solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
Purity Heavy metals <1.07> — Proceed with 1.0 g of Nysta-
tin according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
Loss on drying <2.41> Not more than 5.0% (0.3 g, in vacu-
um, 60°C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Saccharomyces cerevisiae ATCC 9763
(ii) Culture medium — Use the medium 2) Medium for
test organism [12] under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Use a light-resistant container.
Weigh accurately an amount of Nystatin Reference Standard
equivalent to about 60,000 units, previously dried at 40°C for
2 hours in vacuum (not more than 0.67 kPa), dissolve in for-
mamide to make a solution of 3000 units per mL, and use this
solution as the standard stock solution. Keep the standard
stock solution at 5°C or below and use within 3 days. Take
exactly a suitable amount of the standard stock solution be-
fore use, add phosphate buffer solution, pH 6.0 to make solu-
tions so that each mL contains 300 units and 150 units, and
use these solutions as the high concentration standard solu-
tion and low concentration standard solution, respectively.
(iv) Sample solutions — Use a light-resistant container.
Weigh accurately an amount of Nystatin equivalent to about
60,000 units, dissolve in formamide to make a solution of
3000 units per mL, and use this solution as the sample stock
solution. Take exactly a suitable amount of the sample stock
solution, add phosphate buffer solution, pH 6.0 to make so-
lutions so that each mL contains 300 units and 150 units, and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Ofloxacin
C0 2 H
and enantiomer
C 18 H 20 FN 3 O4: 361.37
(3RS )-9-Fluoro-3-m ethyl- 1 0-(4-methylpiperazin-
l-yl)-7-oxo-2,3-dihydro-7//-pyrido[l,2,3-tfe]-
[l,4]benzooxazine-6-carboxylic acid [82419-36-1]
Ofloxacin, when dried, contains not less than
99.0% and not more than 101.0% of ofloxacin
(C 18 H 20 FN 3 O 4 ).
Description Ofloxacin occurs as pale yellowish white to
light yellowish white, crystals or crystalline powder.
It is freely soluble in acetic acid (100), slightly soluble in
water, and very slightly soluble in acetonitrile and in ethanol
(99.5).
A soluton of Ofloxacin in sodium hydroxide TS (1 in 20)
does not show optical rotation.
It is changed in color by light.
Melting point: about 265°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ofloxacin in 0.1 mol/L hydrochloric acid TS (1 in
150,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Oflox-
acin as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Ofloxacin according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Related substances — Conduct this procedure without
exposure to light. Dissolve 10 mg of Ofloxacin in 50 mL of a
mixture of water and acetonitrile (6:1), and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
and add a mixture of water and acetonitrile (6:1) to make ex-
actly 20 mL. Pipet 1 mL of this solution, add a mixture of
water and acetonitrile (6:1) to make exactly 10 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak other than ofloxacin obtained from the sample solution
is not more than 0.4 times the peak area of ofloxacin from the
standard solution, and the total area of the peaks other than
ofloxacin from the sample solution is not more than the peak
area from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 294 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
45°C.
Mobile phase: Dissolve 7.0 g of sodium perchlorate mono-
hydrate and 4.0 g of ammonium acetate in 1 300 mL of water,
adjust the pH to 2.2 with phosphoric acid, and add 240 mL
of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
ofloxacin is about 20 minutes.
Time span of measurement: About 1.8 times as long as the
retention time of ofloxacin beginning after the solvent peak.
System suitability —
Test for required detectability: Measure 1 mL of the stan-
dard solution, and add a mixture of water and acetonitrile
JPXV
Official Monographs / Powdered Opium
939
(6:1) to make exactly 20 mL. Confirm that the peak area of
ofloxacin obtained from 10 /xh of this solution is equivalent
to 4 to 6% of that from 10 /xL of the standard solution.
System performance: To 0.5 mL of the sample solution
add 1 mL of a solution of ofloxacin demethyl substance in a
mixture of water and acetonitrile (6:1) (1 in 20,000) and a
mixture of water and acetonitrile (6:1) to make 100 mL.
When the procedure is run with 10 iiL of this solution under
the above operating conditions, ofloxacin demethyl substance
and ofloxacin are eluted in this order with the resolution be-
tween these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ofloxacin is not more than 2.0%.
Loss on drying <2.41> Not less than 0.2% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Ofloxacin, previous-
ly dried, dissolve in 100 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 36.14 mg of C I8 H 20 FN 3 O4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Olive Oil
Oleum Olivae
Olive Oil is the fixed oil obtained by expression from
the ripe fruit of Olea europaea Linne (Oleaceae).
Description Olive Oil is a light yellow oil. It has a faint
odor, which is not rancid, and has a bland taste.
It is miscible with diethyl ether, with petroleum diethyl
ether and with carbon disulfide.
It is slightly soluble in ethanol (95).
The whole or a part of it congeals between 0°C and 6°C.
Congealing point of the fatty acids: 17 - 26°C
Specific gravity <1.13>
d\\: 0.908-0.914
Acid value <1.13> Not more than 1.0.
Saponification value <7.73> 186 - 194
Unsaponifiable matters <1.13> Not more than 1.5%.
Iodine value <7.73> 79-88
Purity (1) Drying oil— Mix 2 mL of Olive Oil with 10 mL
of diluted nitric acid (1 in 4), add 1 g of powdered sodium ni-
trite little by little with thorough shaking, and allow to stand
in a cold place for 4 to 10 hours: the mixture congeals to a
white solid.
(2) Peanut oil — Weigh exactly 1 .0 g of Olive Oil, dissolve
in 60 mL of sulfuric acid-hexane-methanol TS, boil for 2.5
hours on a water bath under a reflux condenser, cool, trans-
fer to a separator, and add 100 mL of water. Wash the flask
with 50 mL of petroleum ether, add the washing to the sepa-
rator, shake, allow to stand, and separate the petroleum ether
layer. Extract the water layer with another 50 mL of petrole-
um ether, and combine the petroleum ether layer with the
former petroleum ether solution. Wash the petroleum ether
solution repeatedly with 20-mL portions of water until the
washings show no more acidity to methyl orange TS. Then
add 5 g of anhydrous sodium sulfate, shake, filter, wash an-
hydrous sodium sulfate with two 10-mL portions of petrole-
um ether, filter the washings using the former separator,
combine the filtrates, distil the petroleum ether on a water
bath, passing nitrogen. Dissolve the residue in acetone to
make exactly 20 mL, and use this solution as the sample solu-
tion. Separately, dissolve 0.067 g of methyl behenate in ace-
tone to make exactly 50 mL. Pipet 2 mL of this solution, add
acetone to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with exactly 2 fxh each of
the sample solution and standard solution as directed under
Gas Chromatography <2.02> according to the following con-
ditions. Measure the peak heights, H T and H s , of methyl be-
henate of respective solutions: H T is not higher than H s .
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with silanized siliceous earth
for gas chromatography (150 to 180 /am in particle diameter),
coated with polyethylene glycol 20 mol/L in a ratio of 5%.
Column temperature: A constant temperature of about
220°C.
Carrier gas: Nitrogen.
Flow rate: Adjust the flow rate so that the retention time of
methyl behenate is about 18 minutes.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of methyl behenate obtained from 2 /uL
of the standard solution is 5 to 10 mm.
Containers and storage Containers — Tight containers.
Powdered Opium
Opium Pulveratum
Powdered Opium is a homogeneous powder of opi-
um obtained from Papaver somniferum Linne
(Papaveraceae). Starch or Lactose Hydrate may be ad-
ded.
Powdered Opium contains not less than 9.5% and
not more than 10.5% of morphine (C 17 H 19 N0 3 :
285.34).
Description Powdered Opium occurs as a yellow-brown to
dark brown powder.
Identification (1) To 0.1 g of Powdered Opium add 5 mL
of diluted ethanol (7 in 10), dissolve by treating with ultra-
sonic waves for 10 minutes, and add diluted ethanol (7 in 10)
to make 10 mL. Filter this solution, and use the filtrate as the
sample solution. Separately, dissolve 25 mg of Morphine
Hydrochloride Hydrate, 12 mg of Codeine Phosphate Hy-
940
Diluted Opium Powder / Official Monographs
JP XV
drate, 2 mg of Papaverine Hydrochloride, and 12 mg of
Noscapine Hydrochloride Hydrate separately in 25 mL of
diluted ethanol (7 in 10), and use these solutions as the stan-
dard solution (1), the standard solution (2), the standard so-
lution (3) and the standard solution (4), respectively. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot lO^L each of the sample solu-
tion and standard solutions on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
acetone, toluene, ethanol (99.5) and ammonia water (28)
(20:20:3:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly Dragendorff's TS for spraying on the
plate: each spot from the sample solution shows the same
color tone and Rf value of each spot obtained from the stan-
dard solution (1), the standard solution (2), the standard so-
lution (3), and the standard solution (4) (morphine, codeine,
papaverine and noscapine), respectively.
(2) To 0.1 g of Powdered Opium add 5 mL of water, and
shake the mixture for 5 minutes. Filter, to the filtrate add 1
mL of a solution of hydroxylammonium chloride (3 in 10)
and 1 drop of iron (III) chloride TS, and shake: a red-brown
color is produced. To this solution add immediately 5 mL of
diethyl ether, and shake: the diethyl ether layer has no red-
purple color (meconic acid).
Loss on drying <2.41> Not more than 8.0% (1 g, 105°C,
5 hours).
Assay Place about 5 g of Powdered Opium, accurately
weighed, in a mortar, and triturate it with exactly 10 mL of
water. Add 2 g of calcium hydroxide and exactly 40 mL of
water, and stir the mixture for 20 minutes. Filter, and shake
30 mL of the filtrate with 0.1 g of magnesium sulfate hepta-
hydrate for 1 minute. To the mixture add 0.3 g of calcium
hydroxide, shake for 1 minute, and allow to stand for 1 hour.
Filter, place 20 mL of the filtrate, exactly measured, in a
glass-stoppered flask, and add 10 mL of diethyl ether and 0.3
g of ammonium chloride. Shake vigorously with caution.
When crystals begin to separate out, shake for 30 minutes
with a mechanical shaker, and set aside overnight at a tem-
perature of 5°C to 10°C. Decant the diethyl ether layer and
filter first, and then the water layer through filter paper 7 cm
in diameter. Wash the adhering crystals in the flask with three
5-mL portions of water saturated with diethyl ether, and
wash the crystals on the filter paper with each of these wash-
ings. Wash the top of the glass-stoppered flask and the upper
part of the filter paper with final 5 mL of water saturated with
diethyl ether. Transfer the crystals and the filter paper to a
beaker. Dissolve the crystals remaining in the glass-stoppered
flask with the aid of 15 mL of 0.05 mol/L sulfuric acid VS,
accurately measured, and pour the solution into the beaker.
Wash the glass-stoppered flask with four 5-mL portions of
water, and add the washings to the solution in the beaker. Ti-
trate <2.50> the excess sulfuric acid with 0.1 mol/L sodium
hydroxide VS (indicator: 4 drops of methyl red-methylene
blue TS).
Each mL of 0.05 mol/L sulfuric acid VS
= 28.53 mg of C 17 H I9 N0 3
Containers and storage Containers — Tight containers.
Diluted Opium Powder
Diluted Opium Powder contains not less than 0.90%
and not more than 1.10 % of morphine
(C 17 H 19 N0 3 : 285.34).
Method of preparation
Powdered Opium
Starch or a suitable diluent
100 g
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients. Lactose Hydrate should not be used.
Description Diluted Opium Powder occurs as a light brown
powder.
Identification (1) Proceed with 1 g of Diluted Opium
Powder as directed in the Identification (1) under Powdered
Opium.
(2) Proceed with 1 g of Diluted Opium Powder as direct-
ed in the Identification (2) under Powdered Opium.
Assay Place about 50 g of Diluted Opium Powder, ac-
curately weighed, in a glass-stoppered flask, and stir with 250
mL of dilute ethanol in a water bath at 40°C for 1 hour.
Filter the mixture through a glass filter (G3). Transfer the
residue on the filter to the first glass-stoppered flask, and add
50 mL of dilute ethanol. Stir the mixture in a water bath at 40
°C for 10 minutes, and filter through the same glass filter.
Repeat the extraction with three 50-mL portions of dilute
ethanol. Evaporate the combined filtrate in a mortar to dry-
ness on a water bath. Add 10 mL of ethanol (99.5) to the
residue, evaporate to dryness again, and, after cooling,
triturate it with exactly 10 mL of water. Proceed with this so-
lution as directed in Assay under Powdered Opium.
Each mL of 0.05 mol/L sulfuric acid VS
= 28.53 mg of C I7 H 19 N0 3
Containers and storage Containers — Tight containers.
Opium Tincture
7^>1->*r
Opium Tincture contains not less than 0.93 w/v%
and not more than 1.07 w/v% of morphine
(C 17 H 19 N0 3 : 285.34).
Method of preparation
Powdered Opium
35 vol% Ethanol
100 g
a sufficient quantity
To make
1000 mL
Prepare as directed under Tinctures, with the above in-
gredients. May be prepared with an appropriate quantity of
Ethanol and Purified Water in place of 35 vol% Ethanol.
Description Opium Tincture is a dark red-brown liquid.
JPXV
Official Monographs / Opium Alkaloids Hydrochlorides
941
It is affected by light.
Identification (1) To 1 mL of Opium Tincure add diluted
ethanol (7 in 10) to make 10 mL, filter, and use the filtrate as
the sample solution. Proceed as directed in the Identification
(1) under Powdered Opium.
(2) Evaporate 1 mL of Opium Tincture to dryness on a
water bath, and proceed with the residue as directed in the
Identification (2) under Powdered Opium.
Alcohol number <7.07> Not less than 3.5 (Method 1).
Assay Evaporate 50 mL of Opium Tincture, accurately
measured, on a water bath to dryness. Add 10 mL of ethanol
(99.5) to the residue, evaporate to dryness again, cool, and
triturate with exactly 10 mL of water. Proceed with this solu-
tion as directed in the Assay under Powdered Opium.
Each mL of 0.05 mol/L sulfuric acid VS
= 28.53 mg of C 17 H 19 N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Opium Alkaloids Hydrochlorides
Opium Alkaloids Hydrochlorides consist of the
hydrochlorides of some of the main alkaloids obtained
from opium.
It contains not less than 47.0% and not more than
52.0% of morphine (C 17 H 19 N0 3 : 285.34), and not less
than 35.0% and not more than 41.0% of other opium
alkaloids.
Description Opium Alkaloids Hydrochlorides occur as a
white to light brown powder.
It is soluble in water, and slightly soluble in ethanol (99.5).
It is colored by light.
Identification (1) Dissolve 0.1 g of Opium Alkaloids
Hydrochlorides in 10 mL of diluted ethanol (1 in 2), and use
this solution as the sample solution. Separately, dissolve
60 mg of Morphine Hydrochloride Hydrate, 40 mg of Nosca-
pine Hydrochloride Hydrate, 10 mg of Codein Phosphate
Hydrate and 10 mg of Papaverine Hydrochloride in 10 mL
each of diluted ethanol (1 in 2), and use these solutions as the
standard solutions (1), (2), (3) and (4), respectively. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 20 /uL each of the sample solu-
tion and standard solutions on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of acetone, toluene, ethanol (99.5)
and ammonia solution (28) (20:20:3:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): each spot from the sample solu-
tion is the same in color tone and Ri value with the corre-
sponding spot from the standard solutions (1), (2), (3) and (4)
(morphine, noscapine, codeine and papaverine).
(2) A solution of Opium Alkaloids Hydrochlorides (1 in
50) responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Opium Alkaloids Hydrochlo-
rides in 50 mL of water: the pH of the solution is between 3.0
and 4.0.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Opium Alkaloids Hydrochlorides in 10 mL of water: the so-
lution is clear, and its absorbance <2.24> at 420 nm is not
more than 0.20.
(2) Meconic acid — Dissolve 0.1 g of Opium Alkaloids
Hydrochlorides in 2 mL of water, and pour into a polyethy-
lene column 1 cm in inside diameter, packed with about 0.36
g of aminopropylsilanized silica gel for pretreatment (55 -
105 /xm in particle diameter) and previously washed through
with 5 mL of water. Then, wash the column with 5 mL of
water, 5 mL of methanol and 10 mL of 0.1 mol/L
hydrochloric acid in this order, then elute with 2 mL of 1 mol
/L hydrochloric acid, and use the eluate as the test solution.
To the test solution add 2 mL of dilute sodium hydroxide TS
and 1 drop of iron (III) chloride TS: no red color develops.
Loss on drying <2.41> Not more than 6.0% (0.5 g, 120°C,
8 hours).
Residue on ignition <2.44> Not more than 0.5% (0.5 g).
Assay Weigh accurately about 0.1 g of Opium Alkaloids
Hydrochlorides, and dissolve in water to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 60 mg of morphine hydrochloride for
assay, dissolve in water to make exactly 50 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the peak areas of
morphine, codeine, papaverine, thebaine, narceine and
noscapine, A Ti , A T2 , A T} , A T4 , A T5 and A T6 , from the sample
solution, and the peak area of morphine, A s , from the
standard solution.
Amount (mg) of morphine (C I7 H 19 N0 3 )
= W s x(A Ti /A s )x 0.8867
Amount (mg) of other opium alkaloids
= W S
x {(A T2 + 0.29A T , + 0.20A T4 + 0A9A T5 +A T6 )/A S }
x 0.8867
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
The relative retention time of codine, papaverine, the-
baine, narceine and noscapine with respect to morphine
obtained under the following operating conditions are as
follows.
Component
Relative retention time
codeine
1.1
papaverine
1.9
thebaine
2.5
narceine
2.8
noscapine
3.6
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
942
Opium Alkaloids Hydrochlorides Injection / Official Monographs
JP XV
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: Dissolve 60 mg of Morphine
Hydrochloride Hydrate, 10 mg of Codeine Phosphate Hy-
drate, 10 mg of Papaverine Hydrochloride and 40 mg of
Noscapine Hydrochloride Hydrate in water to make 50 mL.
When the procedure is run with 20 liL of this solution under
the above operating conditions, morphine, codeine, papaver-
ine and noscapine are eluted in this order with the complete
separation between these peaks and with the resolution be-
tween the peaks of morphine and codeine being not less than
1.5.
System repeatability: When the test is repeated 6 times with
20 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
morphine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Opium Alkaloids Hydrochlorides
Injection
7^>7;i^n-f l*£K£&*f*
Opium Alkaloids Hydrochlorides Injection is an
aqueous solution for injection.
It contains not less than 0.90 w/v% and not more
than 1.10 w/v% of morphine (C 17 H 19 N0 3 : 285.34).
Method of preparation
Opium Alkaloids Hydrochlorides 20 g
Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Opium Alkaloids Hydrochlorides Injection is a
clear, colorless or light brown liquid.
It is affected by light.
pH: 2.5-3.5
Identification To 1 mL of Opium Alkaloids Hydrochlorides
Injection add 1 mL of ethanol (99.5), mix, and use this solu-
tion as the sample solution, and proceed as directed in the
Identification (1) under Opium Alkaloids Hydrochlorides.
Extractable volume <6.05> It meets the requirements.
Assay Pipet 2 mL of Opium Alkaloids Hydrochlorides In-
jection, add exactly 10 mL of the internal standard solution
and water to make 50 mL, and use this solution as the sample
solution. Proceed as directed in the Assay (1) under Opium
Alkaloids Hydrochlorides.
Amount (mg) of morphine (Ci 7 H 19 N0 3 )
= W s x(Q T /Qs)x 0.8867
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrine
hydrochloride (1 in 500).
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Opium Alkaloids and Atropine
Injection
7^>7JU^P-f K-7 r- nt?>>±£t>£
Opium Alkaloids and Atropine Injection is an aque-
ous solution for injection.
It contains not less than 0.90 w/v% and not more
than 1.10 w/v% of morphine (C 17 H 19 N0 3 : 285.34),
and not less than 0.027 w/v% and not more than 0.033
w/v% of atropine sulfate Hydrate [(C 17 H23N03) 2 .
H 2 S0 4 .H 2 0: 694.84].
Method of preparation
Opium Alkaloids Hydrochlorides 20 g
Atropine Sulfate Hydrate 0.3 g
Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Opium Alkaloids and Atropine Injection is a
colorless or light brown, clear liquid.
It is affected by light.
pH: 2.5-3.5
Identification (1) To 1 mL of Opium Alkaloids and Atro-
pine Injection add 1 mL of ethanol (99.5), mix, and use this
solution as the sample solution. Proceed with the sample so-
lution as directed in the Identification (1) under Opium
Alkaloids Hydrochlorides.
(2) To 2 mL of Opium Alkaloids and Atropine Injection
add 2 mL of ammonia TS, extract with 10 mL of diethyl
ether, and filter the diethyl ether layer. Evaporate the filtrate
on a water bath to dryness, add 1 mL of ethanol (99.5) to the
residue, and heat to dissolve. Allow to stand this solution in
an ice water for 30 minutes with occasional shaking. After
crystals are formed, use the supernatant liquid as the sample
solution. Separately, dissolve 0.03 g of Atropine Sulfate
Reference Standard in 100 mL of water, proceed with 2 mL
of this solution in the same manner as for the sample solu-
tion, and use a solution so obtained as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of methanol and ammonia water (28) (200:3) to a distance of
about 10 cm, and air-dry the plate. Spray evenly Dragendor-
ff's TS for spraying on the plate: a spot of about 0.2 Rf value
among the several spots from the sample solution and an
JPXV
Official Monographs / Opium Alkaloids and Scopolamine Injection
943
orange colored spot from the standard solution show the
same color tone, and have the same Ri value (atropine).
Extractable volume <6.05> It meets the requirements.
Assay (1) Morphine — Pipet 2 mL of Opium Alkaloids
and Atropine Injection, add exactly 10 mL of the internal
standard solution, then add water to make 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of morphine hydrochloride for assay,
dissolve in exactly 10 mL of the internal standard solution,
then add water to make 50 mL, and use this solution as the
standard solution. Perform the test with 20 ^L each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, Q T and Qs, of the peak area
of morphine to that of the internal standard.
Amount (mg) of morphine (C17LL9NO3)
= W s x(Q T /Q s )x 0.8867
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of ethylefrine
hydrochloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, morphine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 2.0%.
(2) Atropine sulfate hydrate — Pipet 2 mL of Opium
Alkaloids and Atropine Injection, add exactly 2 mL of the in-
ternal standard solution, and add 10 mL of diluted dilute
hydrochloric acid (1 in 10). Shake this solution with two
10-mL portions of dichloromethane. Remove the
dichloromethane layer, to the water layer add 2 mL of am-
monia TS, immediately add 20 mL of dichloromethane,
shake vigorously, filter the dichloromethane extract through
filter paper on which 5 g of anhydrous sodium sulfate is
placed, and evaporate the filtrate to dryness under reduced
pressure. To the residue add 0.5 mL of 1,2-dichloromethane
and 0.5 mL of bis-trimethylsilylacetamide, stopper tightly,
warm in a water bath at 60°C for 15 minutes, and use this so-
lution as the sample solution. Separately, weigh accurately
about 30 mg of Atropine Sulfate Reference Standard (deter-
mine previously loss on drying <2.41> in the same manner as
directed under Atropine Sulfate Hydrate), and dissolve in
water to make exactly 100 mL. Pipet 2 mL of this solution,
and add exactly 2 mL of the internal standard solution. Pro-
ceed with this solution in the same manner as directed for the
sample solution, and use this solution as the standard solu-
tion. Perform the test with 2 /uL each of the sample solution
and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of atro-
pine to that of the internal standard.
Amount (mg) of atropine sulfate hydrate
[(C 17 H 23 N0 3 )2.H 2 S04.H 2 0]
= W s x(Q T /Q s )x (1/50) x 1.027
W s : Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
Internal standard solution — A solution of homatropine
hydrobromide (1 in 4000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 1.5
m in length, packed with 180 to 250 /um siliceous earth for gas
chromatography coated in 1 to 3% with 50% phenyl-methyl
silicone polymer for gas chromatography.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
atropine is about 5 minutes.
System suitability —
System performance: When the procedure is run with 2 /iL
of the standard solution under the above operating condi-
tions, the internal standard and atropine are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 5 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of atropine to that of the internal standard is not
more than 2.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Opium Alkaloids and Scopolamine
Injection
Opium Alkaloids and Scopolamine Injection is an
aqueous solution for injection.
It contains not less than 1.80 w/v% and not more
than 2.20 w/v% of morphine (C 17 H 19 N0 3 : 285.34) and
not less than 0.054 w/v% and not more than 0.066
w/v% of scopolamine hydrobromide hydrate
(C 17 H 21 N0 4 .HBr.3H 2 0: 438.31).
944
Opium Alkaloids and Scopolamine Injection / Official Monographs
JP XV
Method of preparation
Opium Alkaloids Hydrochlorides 40 g
Scopolamine Hydrobromide Hydrate 0.6 g
Water for Injection a sufficient quantity
To make
1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Opium Alkaloids and Scopolamine Injection is
a clear, colorless to light brown liquid.
It is affected by light.
pH: 2.5-3.5
Identification (1) To 1 mL of Opium Alkaloids and
Scopolamine Injection add 1 mL of water and 2 mL of
ethanol (99.5), mix, and use this solution as the sample solu-
tion. Proceed with the sample solution as directed in the
Identification (1) under Opium Alkaloids Hydrochlorides.
(2) To 1 mL of Opium Alkaloids and Scopolamine Injec-
tion add 1 mL of water and 2 mL of ammonia TS, extract
with 10 mL of diethyl ether, and filter the diethyl ether layer.
Evaporate the filtrate on a water bath to dryness, add 1 mL of
ethanol (99.5) to the residue, and heat to dissolve. Allow to
stand this solution in an ice water for 30 minutes with oc-
casional shaking. After crystals are formed, use the super-
natant liquid as the sample solution. Separately, dissolve 0.03
g of Scopolamine Hydrobromide Reference Standard in 100
mL of water. To 2 mL of this solution add 2 mL of ammonia
TS, proceed with this solution in the same manner as for the
sample solution, and use a solution so obtained as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10 /xL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of methanol and ammonia water (28) (200:3)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly Dragendorff's TS for spraying on the plate: a spot of
about 0.7 Rf value among the several spots from the sample
solution and an orange colored spot from the standard solu-
tion show the same color tone, and have the same Rf value
(scopolamine).
Extractable volume <6.05> It meets the requirements.
Assay (1) Morphine — Pipet 1 mL of Opium Alkaloids
and Scopolamine Injection, add 10 mL of the internal stan-
dard solution and water to make 50 mL, and use this solution
as the sample solution. Separately, weigh accurately about 25
mg of morphine hydrochloride for assay, dissolve in exactly
10 mL of the internal standard solution, add water to make
50 mL, and use this solution as the standard solution. Per-
form the test with 20 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g T and Q s , of the peak area of morphine to that
of the internal standard.
Amount (mg) of morphine (C 17 H 19 N0 3 )
= W s x(Q T /Q s )x 0.8867
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrin
hydrochloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, morphine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 2.0%.
(2) Scopolamine hydrobromide hydrate — Pipet 2 mL of
Opium Alkaloids and Scopolamine Injection, and add exact-
ly 2 mL of the internal standard solution. To this solution
add 10 mL of diluted dilute hydrochloric acid (1 in 10), and
shake with two 10-mL portions of dichloromethane. Remove
the dichloromethane layer, to the water layer add 2 mL of
ammonia TS, add immediately 20 mL of dichloromethane,
shake vigorously, filter the dichloromethane extract through
a filter paper on which 5 g of anhydrous sodium sulfate is
placed, and evaporate the filtrate to dryness under reduced
pressure. To the residue add 0.5 mL of 1,2-dichloroethane
and 0.5 mL of bis-trimethyl silyl acetamide, stopper tightly,
warm in a water bath at 60°C for 15 minutes, and use this so-
lution as the sample solution. Separately, weigh accurately
about 60 mg of Scoporamine Hydrobromide Reference Stan-
dard (determine previously its loss on drying <2.41> in the
same manner as directed under Scopolamine Hydrobromide
Hydrate), and dissolve in water to make exactly 100 mL.
Pipet 2 mL of this solution, add exactly 2 mL of the internal
standard solution. Proceed with this solution in the same
manner as for the sample solution, and use thus obtained so-
lution as the standard solution. Perform the test with 2,mL
each of the sample solution and standard solution as directed
under Gas Chromatography <2.02> according to the follow-
ing conditions, and calculate the ratios, Q T and Q s , of the
peak area of scopolamine to that of the internal standard.
Amount (mg) of scopolamine hydrobromide hydrate
(C 17 H 21 N0 4 .HBr.3H 2 0)
= W s x (Qj/Qs) x (1/50) x 1 .1406
W s : Amount (mg) of Scopolamine Hydrobromide Refer-
ence Standard, calculated on the dried basis
Internal standard solution — A solution of homatropine
hydrobromide (1 in 4000).
Operating conditions —
JPXV
Official Monographs / Weak Opium Alkaloids and Scopolamine Injection
945
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 1.5
m in length, packed with 180 to 250 /xm siliceous earth for gas
chromatography coated in 1 to 3% with 50% phenyl-methyl
silicone polymer for gas chromatography.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
scopolamine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, the internal standard and scopolamine are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 5 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of scopolamine to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Weak Opium Alkaloids and
Scopolamine Injection
Weak Opium Alkaloids and Scopolamine Injection
is an aqueous solution for injection.
It contains not less than 0.90 w/v% and not more
than 1.10 w/v% of morphine (C 17 H 19 N0 3 : 285.34) and
not less than 0.027 w/v% and not more than 0.033
w/v% of scopolamine hydrobromide hydrate
(C 17 H 21 N0 4 .HBr.3H 2 0: 438.31).
Method of preparation
Opium Alkaloids Hydrochlorides 20 g
Scopolamine Hydrobromide Hydrate 0.3 g
Water for Injection a sufficient quantity
To make
1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Weak Opium Alkaloids and Scopolamine In-
jection is a clear, colorless or light brown liquid.
It is affected by light.
pH: 2.5-3.5
Identification (1) To 1 mL of Opium Alkaloids and
Scopolamine Injection add 1 mL of ethanol (99.5), mix, and
use this solution as the sample solution. Proceed with the
sample solution as directed in the Identification (1) under
Opium Alkaloids Hydrochlorides.
(2) To 2 mL of Weak Opium Alkaloids and Scopolamine
Injection add 2 mL of ammonia TS, extract with 10 mL of
diethyl ether, and filter the diethyl ether layer. Evaporate the
filtrate on a water bath to dryness, add 1 mL of ethanol (99.5)
to the residue, and heat to dissolve. Allow to stand this solu-
tion in an ice water for 30 minutes with occasional shaking.
After crystals are formed, use the supernatant liquid as the
sample solution. Separately, dissolve 0.03 g of Scopolamine
Hydrobromide Reference Standard in 100 mL of water, pro-
ceed with 2 mL of this solution in the same manner as for the
sample solution, and use a solution so obtained as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of methanol and ammonia water (28) (200:3)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly Dragendorff's TS for spraying on the plate: a spot of
about 0.7 Rf value among the several spots from the sample
solution and an orange colored spot from the standard solu-
tion show the same color tone, and have the same Rf value
(scopolamine).
Extractable volume <6.05> It meets the requirements.
Assay (1) Morphine — Pipet 2 mL of Weak Opium
Alkaloids and Scopolamine Injection, add exactly 10 mL of
the internal standard solution and water to make 50 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of morphine hydrochloride for assay,
dissolve in exactly 10 mL of the internal standard solution,
add water to make 50 mL, and use this solution as the stan-
dard solution. Perform the test with 20 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Q T and Q s , of the peak area of
morphine to that of the internal standard.
Amount (mg) of morphine (C 17 H 19 N0 3 )
= W s x(Q T xQ s )x 0.8867
W s : Amount (mg) of morphine hydrochloride for assay,
calculated on the anhydrous basis
Internal standard solution — A solution of etilefrin
hydrochloride (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in
500 mL of diluted phosphoric acid (1 in 1000), and adjust the
pH to 3.0 with sodium hydroxide TS. To 240 mL of this solu-
tion add 70 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
morphine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, morphine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
946
Orange Oil / Official Monographs
JP XV
conditions, the relative standard deviation of the ratios of the
peak area of morphine to that of the internal standard is not
more than 2.0%.
(2) Scopolamine hydrobromide hydrate — Pipet 4 mL of
Weak Opium Alkaloids and Scopolamine Injection, and add
exactly 2 mL of the internal standard solution. To this solu-
tion add 10 mL of diluted dilute hydrochloric acid (1 in 10),
and shake with two 10-mL portions of dichloromethane. Re-
move the dichloromethane layer, to the water layer add 2 mL
of ammonia TS, add immediately 20 mL of
dichloromethane, shake vigorously, filter the
dichloromethane extract through a filter paper on which 5 g
of anhydrous sodium sulfate is placed, and evaporate the
filtrate to dryness under reduced pressure. To the residue add
0.5 mL of 1,2-dichloroethane and 0.5 mL of bis-trimethyl
silyl acetamide, stopper tightly, warm in a water bath at 60°C
for 15 minutes, and use this solution as the sample solution.
Separately, weigh accurately about 60 mg of Scoporamine
Hydrobromide Reference Standard (separately determine its
loss on drying <2.41> in the same manner as directed under
Scopolamine Hydrobromide Hydrate), and dissolve in water
to make exactly 100 mL. Pipet 2 mL of this solution, add ex-
actly 2 mL of the internal standard solution. Proceed with
this solution in the same manner as for the sample solution,
and use so obtained solution as the standard solution. Per-
form the test with 2 /xL each of the sample solution and stan-
dard solution as directed under Gas Chromatography <2.02>
according to the following conditions, and calculate the ra-
tios, Qt and Q s , of the peak area of scopolamine to that of
the internal standard.
Amount (mg) of scopolamine hydrobromide hydrate
(C 17 H 21 N0 4 .HBr.3H 2 0)
= W s x(Q T / Qs) x (1/50) x 1.1406
W s : Amount (mg) of Scopolamine Hydrobromide Refer-
ence Standard, calculated on the dried basis
Internal standard solution — A solution of homatropine
hydrobromide (1 in 4000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 1.5
m in length, packed with 180 to 250 /xm siliceous earth for gas
chromatography coated in 1 to 3% with 50% phenyl-methyl
silicone polymer for gas chromatography.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen or helium
Flow rate: Adjust the flow rate so that the retention time of
scopolamine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 2//L
of the standard solution under the above operating condi-
tions, the internal standard and scopolamine are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 5 times with
2 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of scopolamine to that of the internal standard is
not more than 2.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Orange Oil
Oleum Aurantii
Orange Oil is the essential oil obtained by expression
from the peel of the edible fruit of Citrus species
(Rutaceae).
Description Orange Oil is a yellow to yellow-brown liquid.
It has a characteristic, aromatic odor, and a slightly bitter
taste.
It is miscible with an equal volume of ethanol (95) with tur-
bidity.
Refract
Optical rotation <2.49> a™: +85 - +99° (100 mm).
Specific gravity <7.75> df : 0.842 - 0.848
Purity Heavy metals <1.07> — Proceed with 1.0 mL of
Orange Oil according to Method 2, and perform the test. Pre-
pare the control solution with 4.0 mL of Standard Lead Solu-
tion (not more than 40 ppm).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Orciprenaline Sulfate
2 and enantiomer
(C„H 17 N03) 2 .H 2 S0 4 : 520.59
5-{(l7?S)-l-Hydroxy-2-[(l-methylethyl)amino]ethyl}benzene-
1,3-diolhemisulfate [5874-97-5]
Orciprenaline Sulfate contains not less than 98.5%
of (C 11 H 17 N03)2.H 2 S04, calculated on the dried basis.
Description Orciprenaline Sulfate occurs as white crystals
or crystalline powder.
It is freely soluble in water, slightly soluble in ethanol (95)
and in acetic acid (100), and practically insoluble in diethyl
ether.
A solution of Orciprenaline Sulfate (1 in 20) shows no opti-
cal rotation.
Melting point: about 220°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Orciprenaline Sulfate in 0.01 mol/L hydrochloric
acid TS (1 in 10,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Or-
JPXV
Official Monographs / Oxapium Iodide
947
ciprenaline Sulfate, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) A solution of Orciprenaline Sulfate (1 in 100)
responds to the Qualitative Tests <1.09> for sulfate.
pH <2.54>Dissolve 1.0 g of Orciprenaline Sulfate in 10 mL
of water: the pH of this solution is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Orciprenaline Sulfate in 10 mL of water: the solution is clear,
and has no more color than the following control solution.
Control solution: To 3 mL of Matching Fluid T add 1 mL
of diluted hydrochloric acid (1 in 40).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Or-
ciprenaline Sulfate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Orciprenalone — Dissolve 0.200 g of Orciprenaline
Sulfate in 0.01 mol/L hydrochloric acid TS to make exactly
20 mL. Perform the test with this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: the absorbance
at 328 nm is not more than 0.075.
Loss on drying <2.41> Not more than 1.5% (1 g, in vacuum,
105 °C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Orciprenaline Sul-
fate, dissolve in 100 mL of acetic acid (100) by warming on a
water bath, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 52.06 mg of (C„H 17 N03)2.H 2 S0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxapium Iodide
C 22 H 34 IN0 2 : 471.42
1 -(2-Cyclohexyl-2-phenyl- 1 , 3-dioxolan-4-ylm ethyl)- 1 -
methylpiperidinium iodide [6577-41-9]
Oxapium Iodide, when dried, contains not less than
98.5% of C 22 H 3 4lN0 2 .
Description Oxapium Iodide occurs as a white, crystalline
powder.
It is soluble in acetonitrile, in methanol and in ethanol (95),
slightly soluble in water, in acetic anhydride and in acetic acid
(100), and practically insoluble in diethyl ether.
A solution of Oxapium Iodide in methanol (1 in 100) does
not show optical rotation.
Identification (1) Determine the infrared absorption spec-
trum of Oxapium Iodide, previously dried, as directed in the
paste method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(2) Dissolve 0.1 g of Oxapium Iodide in 10 mL of
methanol, and add 2 mL of dilute nitric acid and 2 mL of sil-
ver nitrate TS: a greenish yellow precipitate is formed.
Melting point <2.60> 198 - 203°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Oxapium Iodide according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.05 g of Oxapium
Iodide in 1 00 mL of a mixture of water and acetonitrile (1:1),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add a mixture of water and acetonitrile
(1 : 1) to make exactly 50 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 50 iiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions. Determine each peak area of each solution by the au-
tomatic integration method: the total area of the peaks other
than the peak of oxapium from the sample solution is not
larger than the area of the peak from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of 20°C to
30°C.
Mobile phase: To 57 mL of acetic acid (100) and 139 mL of
triethylamine add water to make 1000 mL. To 50 mL of this
solution add 500 mL of acetonitril, 10 mL of dilute acetic
acid and 440 mL of water.
Flow rate: Adjust the flow rate so that the retention time of
oxapium is about 4 minutes.
Selection of column: Dissolve 0.05 g of Oxapium Iodide
and 3 mg of benzophenone in 100 mL of the mobile phase.
Proceed with 20 juL of this solution under the above operat-
ing conditions, and calculate the resolution. Use a column
giving elution of oxapium and benzophenone in this order
with the resolution between these peaks being not less than 5.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of oxapium obtained from 50 iiL of the
standard solution composes 5 to 15% of the full scale.
Time span of measurement: About 6 times as long as the
retention time of oxapium beginning after the peak of iodide
ion.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Oxapium Iodide,
previously dried, dissolve in 50 mL of a mixture of acetic an-
948
Oxaprozin / Official Monographs
JP XV
hydride and acetic acid (100) (9:1), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration, platinum
electrode). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 47.14 mg of C 22 H 34 IN0 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxaprozin
CO ? H
C 18 H 15 N0 3 : 293.32
3-(4,5-Diphenyloxazol-2-yl)propanoic acid
[21256-18-8]
Oxaprozin, when dried, contains not less than
98.5% of C 18 H 15 N0 3 .
Description Oxaprozin occurs as a white to yellowish white
crystalline powder.
It is sparingly soluble in methanol and in ethanol (95),
slightly soluble in diethyl ether, and practically insoluble in
water.
It is gradually affected by light.
Identification Determine the infrared absorption spectrum
of Oxaprozin, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Absorbance <2.24> E^ (285 nm): 455 - 495 (after drying,
10 mg, methanol, 1000 mL).
Melting point <2.60> 161 - 165 °C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Oxaprozin according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Oxaprozin according to Method 3, and perform the test
(not more than 1 ppm).
(3) Related substances — Dissolve 0.10 g of Oxaprozin in
10 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 100 mL, and use this solution as the standard
solution (1). Pipet 5 mL, 3 mL and 1 mL of this solution, add
methanol to each to make exactly 10 mL, and use these solu-
tions as the standard solutions (2), (3) and (4), respectively.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solutions (1), (2), (3) and (4) on a plate
of silica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and acetic acid (100) (99:1) to a distance of about 15
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the total intensity of the spots
other than the principal spot from the sample solution is not
more than 1.0% calculated on the basis of intensities of the
spots from the standard solutions (1), (2), (3) and (4).
Loss on drying <2.41>
2 hours).
Not more than 0.3% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.5 g of Oxaprozin, previ-
ously dried, dissolve in 50 mL of ethanol (95), and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 29.33 mg of d 8 H 15 N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxazolam
7|-*H*V7A
CH :i
C 18 H 17 C1N 2 2 : 328.79
10-Chloro-2-methyl-llb-phenyl-2, 3,7,1 lb-
tetrahydro[l,3]oxazolo[3,2-rf][l,4]benzodiazepin-6(5//)-one
[24143-17-7]
Oxazolam, when dried, contains not less than 99.0%
of C 18 H 17 C1N 2 2 .
Description Oxazolam occurs as white crystals or crystal-
line powder.
It is odorless and tasteless.
It is freely soluble in acetic acid (100), soluble in 1,4-di-
oxane and in dichloromethane, slightly soluble in ethanol
(95) and in diethyl ether, and practically insoluble in water.
It dissolves in dilute hydrochloric acid.
It gradually changes in color by light.
Melting point: about 187°C (with decomposition).
Identification (1) Dissolve 0.01 g of Oxazolam in 10 mL
of ethanol (95) by heating, and add 1 drop of hydrochloric
acid: a light yellow color develops, and the solution shows a
yellow-green fluorescence under ultraviolet light (main
wavelength: 365 nm). Add 1 mL of sodium hydroxide TS to
this solution: the color and fluorescence of this solution dis-
appear immediately.
(2) Dissolve 0.01 g of Oxazolam in 5 mL of dilute
hydrochloric acid by heating in a water bath for 10 minutes.
After cooling, 1 mL of this solution responds to the Qualita-
tive Tests <1.09> for primary aromatic amines.
(3) Place 2 g of Oxazolam in a 200-mL flask, add 50 mL
of ethanol (95) and 25 mL of 6 mol/L hydrochloric acid TS,
JPXV
Official Monographs / Oxethazaine
949
and boil under a reflux condenser for 5 hours. After cooling,
neutralize with a solution of sodium hydroxide (1 in 4), and
extract with 30 mL of dichloromethane. Dehydrate with 3 g
of anhydrous sodium sulfate, filter, and evaporate the
dichloromethane of the filtrate. Dissolve the residue in 20 mL
of methanol by heating on a water bath, and cool immediate-
ly in an ice bath. Collect the crystals, and dry in vacuum at
60°C for 1 hour: the crystals melt <2.60> between 96°C and
100°C.
(4) Determine the absorption spectrum of a solution of
Oxazolam in ethanol (95) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(5) Proceed with Oxazolam as directed under Flame
Coloration Test <1.04> (2), and perform the test: a green
color appears.
Absorbance <2.24> E{°^ (246 nm): 410 - 430 (after drying,
1 mg, ethanol (95), 100 mL).
Purity (1) Chloride <1.03>— To 1.0 g of Oxazolam add 50
mL of water, allow to stand for 1 hour with occasional shak-
ing, and filter. To 25 mL of this filtrate add 6 mL of dilute
nitric acid and water to make 50 mL, and perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not
more than 0.014%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Oxazol-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Place 1.0 g of Oxazolam in a Kjel-
dahl flask, add 5 mL of sulfuric acid and 5 mL of nitric acid,
and heat gently. Repeat the addition of 2 to 3 mL of nitric
acid at times, and continue to heat until a colorless to light
yellow solution is obtained. After cooling, add 15 mL of
saturated ammonium oxalate monohydrate solution, heat the
solution until dense white fumes are evolved, and evaporate
to a volume of 2 to 3 mL. After cooling, dilute with water to
10 mL, and perform the test with this solution as the test so-
lution (not more than 2 ppm).
(4) Related substances — Dissolve 0.05 g of Oxazolam in
10 mL of dichloromethane, and use this solution as the
sample solution. Pipet 1 mL of this solution, add
dichloromethane to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /uL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Immediately air-dry, develop the plate with
a mixture of toluene and acetone (8:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.65 g of Oxazolam, previ-
ously dried, dissolve in 100 mL of a mixture of acetic acid
(100) and 1,4-dioxane (1:1). Titrate <2.50> with 0.1 mol/L
perchloric acid VS until the color of the solution changes
from purple through blue to blue-green (indicator: 2 drops of
crystal violet TS). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.88 mg of C I8 H 17 C1N 2 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxethazaine
Oxetacaine
OH;
CH:
H3C CH3 q I q H3C CHa
CH
2,2' -(2-Hydroxyethylimino)bis [N-(l , 1 -dimethyl-2-
phenylethyi)-7V-methylacetamide] [126-27-2]
Oxethazaine, when dried, contains not less than
98.5% of C 28 H 41 N 3 03.
Description Oxethazaine occurs as a white to pale yellowish
white, crystalline powder.
Identification (1) Determine the absorption spectrum of a
solution of Oxethazaine in ethanol (95) (1 in 2500) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Ox-
ethazaine as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibits
similar intensities of absorption at the same wave numbers.
Melting point <2.60> 101 - 104 C C
Purity (1) Chloride <1.03> — Dissolve 1 .0 g of Oxethazaine
in 20 mL of ethanol (95), add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 0.30 mL
of 0.01 mol/L hydrochloric acid VS, 20 mL of ethanol (95), 6
mL of dilute nitric acid and water to make 50 mL (not more
than 0.011%).
(2) Heavy metals <1.07>— Proceed with 2.0 g of Ox-
ethazaine according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Related substances — Dissolve 0.40 g of Oxethazaine
in 10 mL of ethanol (95), and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add ethanol (95)
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 [iL each of
950
Oxprenolol Hydrochloride / Official Monographs
JP XV
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of isopropylether, tetra-
hydrofuran, methanol and ammonia solution (28) (24:10:5:1)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
(4) 2-Aminoethanol — To 1.0 g of Oxethazaine add
methanol to make exactly 10 mL, then add 0.1 mL of a solu-
tion of l-fluoro-2,4-dinitrobenzene in methanol (1 in 25),
shake well, and heat at 60°C for 20 minutes: the solution has
no more color than the following control solution.
Control solution: To 0.10 g of 2-aminoethanol add
methanol to make exactly 200 mL, pipet 1 mL of this solu-
tion, and add methanol to make exactly 10 mL. Proceed as
directed above.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.9 g of Oxethazaine, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 2 drops
of crystal violet TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 46.76 mg of C 28 H 41 N 3 03
Containers and storage Containers — Tight containers.
Oxprenolol Hydrochloride
7t-<7X-fls/U-)U£mt&
•HCI
and enantiomer
C 15 H 23 N0 3 .HC1: 301.81
(2jRS)-l-[2-(Allyloxy)phenoxy]-3-
(l-methylethyl)aminopropan-2-ol monohydrochloride
[6452-73-9]
Oxprenolol Hydrochloride, when dried, contains not
less than 98.5% of C 15 H 2 3N0 3 .HC1.
Description Oxprenolol Hydrochloride occurs as a white,
crystalline powder.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), slightly soluble in acetic anhydride, and
practically insoluble in diethyl ether.
Identification (1) To 2 mL of a solution of Oxprenolol
Hydrochloride (1 in 100) add 1 drop of copper (II) sulfate TS
and 2 mL of sodium hydroxide TS: a blue-purple color de-
velops. To this solution add 1 mL of diethyl ether, shake
well, and allow to stand: a red-purple color develops in the
diethyl ether layer, and a blue-purple color develops in the
water layer.
(2) To 3 mL of a solution of Oxprenolol Hydrochloride
(1 in 150) add 3 drops of Reinecke salt TS: a light red
precipitate is formed.
(3) Determine the infrared absorption spectrum of Ox-
prenolol Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Oxprenolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Oxprenolol Hydrochloride in
10 mL of water: the pH of this solution is between 4.5 and
6.0.
Melting point <2.60> 107 - 110°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Oxprenolol Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07>— Proceed with 2.0 g of Ox-
prenolol Hydrochloride according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than lOppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Oxprenolol Hydrochloride according to Method 3, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.25 g of Oxprenolol
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 4 mL of the sample solution, and add
water to make exactly 100 mL. Pipet 5 mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 iuL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate in a developing chamber
saturated with ammonia vapor with a mixture of chloroform
and methanol (9:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 80°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Oxprenolol
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.18 mg of C 15 H 23 N0 3 .HC1
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Oxycodone Hydrochloride Hydrate
951
Oxybuprocaine Hydrochloride
Benoxinate Hydrochloride
,CH 3
C 17 H 28 N 2 3 .HC1: 344.88
2-(Diethylamino)ethyl 4-amino-3-butyloxybenzoate
monohydrochloride [5987-82-6]
Oxybuprocaine Hydrochloride, when dried, con-
tains not less than 99.0% of C 17 H 28 N 2 3 .HC1.
Description Oxybuprocaine Hydrochloride occurs as white
crystals or crystalline powder. It is odorless, and has a saline
taste. It exhibits anesthetic properties when placed on the
tongue.
It is very soluble in water, freely soluble in ethanol (95) and
in chloroform, and practically insoluble in diethyl ether.
The pH of a solution of Oxybuprocaine Hydrochloride (1
in 10) is between 5.0 and 6.0.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Oxybuprocaine
Hydrochloride in 1 mL of dilute hydrochloric acid and 4 mL
of water. This solution responds to the Qualitative Tests
<1.09> for primary aromatic amines.
(2) Dissolve 0.1 g of Oxybuprocaine Hydrochloride in 8
mL of water, and add 3 mL of ammonium thiocyanate TS:
an oily substance is produced. Rub the inner surface of the
container with a glass rod: white crystals are formed. Collect
the crystals so obtained, recrystallize from water, and dry in a
desiccator (in vacuum, phosphorus (V) oxide) for 5 hours:
the crystals melt <2.60> between 103°C and 106°C.
(3) Determine the absorption spectrum of a solution of
Oxybuprocaine Hydrochloride (1 in 100,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(4) A solution of Oxybuprocaine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 158 - 162°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Oxybuprocaine Hydrochloride in 10 mL of water: the solu-
tion is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Oxyb-
uprocaine Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.25 g of Oxyb-
uprocaine Hydrochloride in 10 mL of chloroform, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, and add chloroform to make exactly 20 mL. Pipet 1
mL of this solution, add chloroform to make exactly 50 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, ethanol (95) and formic acid (7:2:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly 4-
dimethylaminobenzaldehyde TS for spraying on the plate:
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Oxybuprocaine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.49 mg of C 17 H 28 N 2 3 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Oxycodone Hydrochloride Hydrate
H,C-
• HCI ■ 3HjO
C 18 H 21 N0 4 .HC1.3H 2 0: 405.87
(5i?)-4,5-Epoxy-14-hydroxy-3-methoxy-17-
methylmorphinan-6-one monohydrochloride trihydrate
[124-90-3, anhydride]
Oxycodone Hydrochloride Hydrate contains not less
than 98.0% of C 18 H 21 N0 4 .HC1 (mol. wt.: 351.83), cal-
culated on the anhydrous basis.
Description Oxycodone Hydrochloride Hydrate occurs as a
white, crystalline powder.
It is freely soluble in water, in methanol and in acetic acid
(100), sparingly soluble in ethanol (95), slightly soluble in
acetic anhydride, and practically insoluble in diethyl ether.
The pH of a solution dissolved 1.0 g of Oxycodone
Hydrochloride Hydrate in 10 mL of water is between 3.8 and
5.8.
It is affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Oxycodone Hydrochloride Hydrate (1 in 10,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
952
Compound Oxycodone Injection / Official Monographs
JP XV
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Oxyc-
odone Hydrochloride Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Oxycodone Hydrochloride Hydrate (1
in 50) responds to the Qualitative Tests <1.09> (2) for chlo-
ride.
Optical rotation <2.49> [ a £°: - 140 - - 149° (0.5 g, calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Oxycodone Hydrochloride Hydrate in 10 mL of water: the
solution is clear and colorless.
(2) Morphine — Dissolve 10 mg of Oxycodone
Hydrochloride Hydrate in 1 mL of water, add 5 mL of 1-
nitroso-2-naphthole TS and 2 mL of a solution of potassium
nitrate (1 in 10), and warm at 40°C for 2 minutes. To this so-
lution add 1 mL of a solution of sodium nitrite (1 in 5000),
and warm at 40°C for 5 minutes. After cooling, add 10 mL of
chloroform, shake, centrifuge, and separate the water layer:
the color of the solution is not more intense than a pale red.
(3) Codeine — Dissolve 10 mg of Oxycodone Hydrochlo-
ride Hydrate in 5 mL of sulfuric acid, add 1 drop of iron (III)
chloride TS, and warm: no blue color is produced. Add 1
drop of nitric acid: no red color develops.
(4) Thebaine — Dissolve 0.10 g of Oxycodone Hydrochlo-
ride Hydrate in 2 mL of diluted hydrochloric acid (1 in 10),
and heat the solution in a water bath for 25 minutes. After
cooling, add 0.5 mL of 4-aminoantipyrine hydrochloride TS
and 0.5 mL of a solution of potassium hexacyanoferrate (III)
(1 in 100), and shake. Then shake the solution with 2 mL of
ammonia TS and 3 mL of chloroform: no red color develops
in the chloroform layer.
Water <2.48> 12 - 15% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.5 g of Oxycodone
Hydrochloride Hydrate, dissolve in 50 mL of a mixture of a-
cetic anhydride and acetic acid (100) (7:3), and titrate <2.50>
with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 35.18 mg of C 18 H 21 N0 4 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Compound Oxycodone Injection
Compound Hycodenone Injection
«***•> 3 r*>a#t*
Compound Oxycodone Injection is an aqueous solu-
tion for injection.
It contains not less than 0.74 w/v% and not more
than 0.86 w/v% of oxycodone hydrochloride hydrate
(C 18 H 21 N0 4 .HC1.3H 2 0: 405.87), and not less than 0.18
w/v% and not more than 0.22 w/v% of hydrocotar-
nine hydrochloride hydrate (C 12 H 15 N03.HC1.H 2 0:
275.73).
Method of preparation
Oxycodone Hydrochloride Hydrate 8 g
Hydrocotarnine Hydrochloride Hydrate 2 g
Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Compound Oxycodone Injection is a clear,
colorless to pale yellow liquid.
It is affected by light.
pH: 2.5-4.0
Identification (1) To 1 mL of Compound Oxycodone In-
jection add 1 mL of 2,4-dinitrophenylhydrazine-ethanol TS:
a yellow precipitate is formed (oxycodone).
(2) Evaporate 1 mL of Compound Oxycodone Injection
on a water bath. Dissolve the residue in 2 mL of sulfuric acid:
a yellow color is produced. Heat the solution: it changes to
red, and then to deep orange-red (hydrocotarnine).
(3) Evaporate 1 mL of Compound Oxycodone Injection
on a water bath. Dissolve the residue in 3 mL of sulfuric acid,
add 2 drops of a solution of tannic acid in ethanol (95) (1 in
20), and allow to stand: a deep green color is produced
(hydrocotarnine).
Extractable volume <6.05> It meets the requirement
Assay Pipet 2 mL of Compound Oxycodone Injection, add
exactly 10 mL of the internal standard solution, and use this
solution as the sample solution. Separately, weigh accurately
about 0.4 g of oxycodone hydrochloride for assay and about
0.1 g of hydrocotarnine hydrochloride for assay previously
dried at 105°C for 3 hours, and dissolve in water to make ex-
actly 50 mL. Pipet 2 mL of this solution, add exactly 10 mL
of the internal standard solution, and use this solution as the
standard solution. Perform the test with 10 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions. Calculate the ratios, g Ta and Q Tb , of the peak area of
oxycodone and hydrocotarnine to that of the internal stan-
dard from the sample solution, and the ratios, gsa and Q sb ,
of the peak area of oxycodone and hydrocotarnine to that of
the internal standard from the standard solution.
Amount (mg) of oxycodone hydrochloride hydrate
(C 18 H 21 N0 4 .HC1.3H 2 0)
= W Sa x(Q Ta /Q s J xi. 1536 x (1/25)
Amount (mg) of hydrocotarnine hydrochloride hydrate
(C 12 H 15 N0 3 .HC1.H 2 0)
= W sb x (Q Jb /Q sb ) x 1 .0699 x (1/25)
W Sa : Amount (mg) of oxycodone hydrochloride for assay,
calculated on the anhydrous basis
W sb : Amount (mg) of hydrocotarnine hydrochloride for
assay
Internal standard solution — Dissolve 0.02 g of phenacetin in
10 mL of ethanol (95), and add water to make 100 mL.
JPXV
Official Monographs / Compound Oxycodone and Atropine Injection
953
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized polyvinyl alcohol gel polymer for liquid chro-
matography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 500 mL of 0.05 mol/L disodium hydro-
gen phosphate TS add 0.05 mol/L sodium dihydrogen phos-
phate TS, and adjust the pH to 8.0. To 300 mL of this solu-
tion add 200 mL of acetonitrile, and mix.
Flow rate: Adjust the flow rate so that the retention time of
oxycodone is about 8 minutes.
Selection of column: Proceed with 10 /xL of the standard
solution under the above operating conditions, and use a
column giving elution of the internal standard, oxycodone
and hydrocotarnine in this order, with complete separation
of these peaks.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Compound Oxycodone and
Atropine Injection
Hycoato Injection
Wi't^'y =i H > ■ 7 r- n tf >alt;tt
Compound Oxycodone and Atropine Injection is an
aqueous solution for injection.
It contains not less than 0.74 w/v% and not more
than 0.86 w/v% of oxycodone hydrochloride hydrate
(C 18 H 21 N0 4 .HC1.3H 2 0: 405.87), not less than 0.18 w/
v% and not more than 0.22 w/v% of hydrocotarnine
hydrochloride hydrate (C 12 H 15 N0 3 .HC1.H 2 0: 275.73),
and not less than 0.027 w/v% and not more than 0.033
w/v% of atropine sulfate hydrate [(C 17 H 23 N0 3 ) 2 .
H 2 S0 4 .H 2 0: 694.83].
Method of preparation
Oxycodone Hydrochloride Hydrate 8 g
Hydrocotarnine Hydrochloride Hydrate 2 g
Atropine Sulfate Hydrate 0.3 g
Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description Compound Oxycodone and Atropine Injection
is a colorless or pale yellow, clear liquid.
It is affected by light.
pH: 2.5 -4.0
Identification (1) To 1 mL of Compound Oxycodone and
Atropin Injection add 1 mL of 2,4-dinitrophenylhydrazine-
ethanol TS: a yellow precipitate is formed (oxycodone).
(2) Evaporate 1 mL of Compound Oxycodone and
Atropin Injection on a water bath, and dissolve the residue in
2 mL of sulfuric acid: a yellow color is produced. Heat the
solution: it changes to red, and then to deep orange-red
(hydrocotarnine).
(3) Evaporate 1 mL of Compound Oxycodone and
Atropin Injection on a water bath. Dissolve the residue in 3
mL of sulfuric acid, add 2 drops of a solution of tannic acid
in ethanol (95) (1 in 20), and allow to stand: a deep green
color is produced (hydrocotarnine).
(4) To 1 mL of Compound Oxycodone and Atropine In-
jection add 0.5 mL of 2,4-dinitrophenylhydrazine-ethanol
TS, and allow to stand for 1 hour. Centrifuge, and add ace-
tone to the supernatant liquid until no more precipitate is
produced. Allow to stand for 20 minutes, and centrifuge. To
the supernatant liquid add potassium hydroxide TS until the
liquid is light purple. Shake the liquid with 5 mL of
dichloromethane, and separate the dichloromethane layer.
Take 0.5 mL of the dichloromethane layer, and evaporate to
dryness on a water bath. Add 5 drops of fuming nitric acid to
the residue, and evaporate to dryness on a water bath. Cool,
dissolve the residue in 1 mL of A^Af-dimethylformamide, and
add 6 drops of tetraethylammonium hydroxide TS: a red-
purple color is produced (atropine).
Extractable volume <6.05> It meets the requirement.
Assay (1) Oxycodone hydrochloride hydrate and
hydrocotarnine hydrochloride hydrate — Pipet 2 mL of Com-
pound Oxycodone and Atropine Injection, add exactly 10
mL of the internal standard solution, and use this solution as
the sample solution. Separately, weigh accurately about 0.4 g
of oxycodone hydrochloride for assay and about 0.1 g of
hydrocotarnine hydrochloride for assay previously dried at
105 °C for 3 hours, and dissolve in water to make exactly 50
mL. Pipet 2 mL of this solution, add exactly 10 mL of the in-
ternal standard solution, and use this solution as the standard
solution. Perform the test with 1 /xh each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions.
Calculate the ratios, Q Ta and Q sb , of the peak area of oxyco-
done and hydrocotarnine to that of the internal standard
from the sample solution, and the ratios, Q Sa and Q sb , of the
peak area of oxycodone and hydrocotarnine to that of the in-
ternal standard from the standard solution.
Amount (mg) of oxycodone hydrochloride hydrate
(C 18 H 21 N0 4 .HC1.3H 2 0)
= W Sa x(g Ta /e Sa ) xl. 1536 x (1/25)
Amount (mg) of hydrocotarnine hydrochloride
hydrate (C 12 H 15 N0 3 .HC1.H 2 0)
= W sb x (Q lb /Q sb ) x 1.0699 x (1/25)
Wsz'. Amount (mg) of oxycodone hydrochloride for assay,
calculated on the anhydrous basis
W sb : Amount (mg) of hydrocotarnine hydrochloride for
assay
Internal standard solution — Dissolve 0.02 g of phenacetin in
10 mL of ethanol (95), and add water to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized polyvinyl alcohol gel polymer for liquid chro-
matography (5 /um in particle diameter).
954 Oxydol / Official Monographs
JP XV
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 500 mL of 0.05 mol/L disodium
hydrogenphosphate TS add 0.05 mol/L sodium dihydrogen-
phosphate TS, and adjust the pH to 8.0. To 300 mL of this
solution add 200 mL of acetonitrile, and mix.
Flow rate: Adjust the flow rate so that the retention time of
oxycodone hydrochloride is about 8 minutes.
Selection of column: Proceed with 10 /xL of the standard
solution under the above operating conditions, and use a
column giving elution of the internal standard, oxycodone
and hydrocotarine in this order with complete separation of
these peaks.
(2) Atropine sulfate hydrate — Pipet 2 mL of Compound
Oxycodone and Atropine Injection, and add exactly 2 mL of
the internal standard solution. To this solution add 10 mL of
diluted dilute hydrochloric acid (1 in 10) and 2 mL of ammo-
nia TS, immediately add 20 mL of dichloromethane, shake
vigorously, filter the dichloromethane layer through filter
paper on which 5 g of anhydrous sodium sulfate is placed,
and evaporate the filtrate to dryness under reduced pressure.
To the residue add 0.5 mL of 1 ,2-dichloromethane and 0.5
mL of bis-trimethylsilylacetamide, stopper tightly, warm in a
water bath at 60°C for 15 minutes, and use this solution as
the sample solution. Separately, weigh accurately about 30
mg of Atropine Sulfate Reference Standard (separately deter-
mine its loss on drying <2.41> in the same manner as directed
under Atropine Sulfate Hydrate), and dissolve in water to
make exactly 100 mL. Pipet 2 mL of this solution, and add
exactly 2 mL of the internal standard solution. Proceed with
this solution in the same manner as directed for the sample
solution, and use so obtained solution as the standard solu-
tion. Perform the test with 2//L each of the sample solution
and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, Q T and Q s , of the peak area of atro-
pine to that of the internal standards.
Amount (mg) of atropine sulfate hydrate
[(C 17 H 2 ,N0 3 ) 2 .H 2 S0 4 .H 2 0]
= W s x(Q T /Q s )x (1/50) x 1.027
fV s : Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
Internal standard solution — A solution of homatropine
hydrobromide (1 in 4000).
Operating conditions —
Detector: A hydrogen fiame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 1.5 m in length, packed with 180- to 250-//m
siliceous earth for gas chromatography coated with 1 to 3%
of 50% phenyl-methylsilicone polymer.
Column temperature: A constant temperature of about
210°C.
Carrier gas: Nitrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
atropine is about 5 minutes.
Selection of column: Proceed with 2 fiL of the standard so-
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of the internal
standard and atropine in this order with the resolution be-
tween these peaks being not less than 3.
and colored containers may be used.
Storage — Light-resistant.
Oxydol
Oxydol contains not less than 2.5 w/v% and not
more than 3.5w/v% of hydrogen peroxide (H 2 2 :
34.01). It contains suitable stabilizers.
Description Oxydol occurs as a clear, colorless liquid. It is
odorless or has an odor resembling that of ozone.
It gradually decomposes upon standing or upon vigorous
agitation.
It rapidly decomposes when in contact with oxidizing sub-
stances as well as reducing substances.
It, when alkalized, decomposes with effervescence.
It is affected by light.
pH: 3.0-5.0
Specific gravity d 2 2 ° : about 1.01
Identification 1 mL of Oxydol responds to the Qualitative
Tests <1.09> for peroxide.
Purity (1) Acidity— To 25.0 mL of Oxydol add 2 drops of
phenolphthalein TS and 2.5 mL of 0.1 mol/L sodium
hydroxide VS: a red color develops.
(2) Heavy metals <1.07>— To 5.0 mL of Oxydol add 20
mL of water and 2 mL of ammonia TS, evaporate on a water
bath to dryness, dissolve the residue in 2 mL of dilute acetic
acid by heating, add water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution with 2 mL of dilute acetic acid, 2.5 mL of Stan-
dard Lead Solution and water to make 50 mL (not more than
5 ppm).
(3) Arsenic <7.77>— To 1.0 mL of Oxydol add 1 mL of
ammonia TS, evaporate on a water bath to dryness, take the
residue, prepare the test solution according to Method 1, and
perform the test (not more than 2 ppm).
(4) Organic stabilizer — Extract 100 mL of Oxydol with
50-mL, 25-mL and 25-mL portions of a mixture of chlo-
roform and diethyl ether (3:2) successively, combine the ex-
tracts in a tared vessel, and evaporate the combined extract
on a water bath. Dry the residue over silica gel to constant
mass: the mass of the residue is not more than 50 mg.
(5) Nonvolatile residue — Evaporate 20.0 mL of Oxydol
on a water bath to dryness, and dry the residue at 105 °C for 1
hour: the mass of the residue is not more than 20 mg.
Assay Pipet 1.0 mL of Oxydol, transfer it to a flask con-
taining 10 mL of water and 10 mL of dilute sulfuric acid, and
titrate <2.50> with 0.02 mol/L potassium permanganate VS.
Each mL of 0.02 mol/L potassium permanganate VS
= 1.701 mg of H 2 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 30°C.
Containers and storage Containers — Hermetic containers,
JPXV
Official Monographs / Oxygen 955
Oxygen
2 : 32.00
Oxygen contains not less than 99.5 v/v% of 2 .
Description Oxygen is a colorless gas, and is odorless.
1 mL of Oxygen dissolves in 32 mL of water, and in 7 mL
of ethanol (95) at 20°C and at a pressure of 101.3 kPa.
1000 mL of Oxygen at 0°C and at a pressure of 101 .3 kPa
weighs about 1.429 g.
Identification (1) Put a glowing splinter of wood into
Oxygen: it bursts into flame immediately.
(2) Transfer 1 mL each of Oxygen and oxygen directly
from metal cylinders with a pressure-reducing valve to gas-
measuring tubes or syringes for gas chromatography, using a
polyvinyl chloride induction tube. Perform the test with these
gases as directed under Gas Chromatography <2.02> accord-
ing to the conditions of Purity (5): the retention time of prin-
cipal peak from Oxygen coincides with that of oxygen.
Purity Keep the containers of Oxygen between 18°C and
22°C for not less than 6 hours before carrying out the follow-
ing tests, and calculate the volume to be used with reference
to the gas at 20°C and at 101.3 kPa.
(1) Acidity or alkalinity— To 400 mL of freshly boiled
and cooled water add 0.3 mL of methyl red TS and 0.3 mL of
bromothymol blue TS, and boil for 5 minutes. Transfer 50
mL of this solution to each of three Nessler tubes marked A,
B and C. Add 0.10 mL of 0.01 mol/L hydrochloric acid VS
to tube A, 0.20 mL of 0.01 mol/L hydrochloric acid VS to
tube B, stopper each of the tubes, and cool. Pass 1000 mL of
Oxygen through the solution in tube A for 15 minutes, em-
ploying delivery tube with an orifice approximately 1 mm in
diameter and extending to within 2 mm of the bottom of the
Nessler tube: the color of the solution in tube A is not deeper
orange-red than that of the solution in tube B and not deeper
yellow-green than that of the solution in tube C.
(2) Carbon dioxide — Pass 1000 mL of Oxygen through
50 mL of barium hydroxide TS in a Nessler tube, in the same
manner as directed in (1): any turbidity produced does not ex-
ceed that of the following control solution.
Control solution: To 50 mL of barium hydroxide TS in a
Nessler tube add 1 mL of a solution of 0.1 g of sodium
hydrogen carbonate in 100 mL of freshly boiled and cooled
water.
(3) Oxidizing substances — Transfer 15 mL of potassium
iodide-starch TS to each of two Nessler tubes marked A and
B, add 1 drop of acetic acid (100) to each of the tubes, mix,
and use these as solution A and solution B, respectively. Pass
2000 mL of Oxygen through solution A for 30 minutes in the
same manner as directed in (1): the color of solution A is the
same as that of the stoppered, untreated solution B.
(4) Chloride <1.03> — Pour 50 mL of water into each of
two Nessler tubes marked A and B, add 0.5 mL of silver ni-
trate TS to each of the tubes, mix, and use these as solution A
an solution B, respectively. Pass 1000 mL of Oxygen through
solution A in the same manner as directed in (1): the turbidity
of solution A is the same as that of solution B.
(5) Nitrogen — Introduce 1.0 mL of Oxygen into a gas-
measuring tube or syringe for gas chromatography from a
metal hermetic container under pressure through a pressure-
reducing valve and a directly connected polyvinyl tube. Per-
form the test as directed under Gas Chromatography <2.02>
according to the following conditions, and determine the
peak area A T of nitrogen. Introduce 0.50 mL of nitrogen into
the gas mixer, draw carrier gas into the mixer to make exactly
100 mL, and allow to mix thoroughly. Perform the test in the
same manner with 1.0 mL of this mixture as directed above,
and determine the peak area A s of nitrogen: A T is not larger
than A s .
Operating conditions —
Detector: A thermal-conductivity detector.
Column: A column about 3 mm in inside diameter and
about 3 m in length, packed with 250- to 355-//m zeolite for
gas chromatography (0.5 mm).
Column temperature: A constant temperature of about
50°C.
Carrier gas: Hydrogen or helium.
Flow rate: Adjust the flow rate so that the retention time of
nitrogen is about 5 minutes.
Selection of column: Introduce 0.5 mL of nitrogen into a
gas mixer, add Oxygen to make 100 mL, and mix well. Pro-
ceed with 1.0 mL of the mixture under the above operating
conditions. Use a column giving well-resolved peaks of Oxyg-
en and nitrogen in this order.
Assay (i) Apparatus — The apparatus is shown diagram-
matically in the accompanying figure. A is a 100-mL gas
buret having a two-way stopcock a, b - c, d - e and e - f are
graduated in 0.1 mL, and c - d is graduated in 2 mL. A is
properly connected with a leveling tube B by a thick rubber
tube. Fill ammonium chloride-ammonia TS up to the middle
of A and B. Place in the absorption ball g of the gas pipette C
a coil of copper wire, not more than 2 mm in diameter, which
extends to the uppermost portion of the bulb, add 125 mL of
ammonium chloride-ammonia TS, and stopper with a rubber
stopper i. Connect C with A using the thick rubber tube.
(ii) Procedure — Open a, set B downward and draw the
liquid in g to the stopcock opening a. Then close a. Open a to
the intake tube h, and fill A and h with ammonium chloride-
ammonia TS by lifting B. Close a, connect h with a container
of Oxygen, open a, set B downward and measure accurately
100 mL of Oxygen. Open a toward C, and transfer the Oxyg-
en to g by lifting B. Close a, and rock C gently for 5 minutes.
Open a, draw the residual gas back into A by setting B down-
ward, and measure the volume of the residual gas. Repeat the
procedure until the volume of residual gas is constant, and
designate this as C(mL). With fresh ammonium chloride-am-
monia TS in C, repeat the procedure at least four times, and
measure the volume of residual gas. Calculate V and the
volume of Oxygen used as the sample with reference to the
gas at 20°C and at 101.3 kPa.
Volume (mL) of oxygen (0 2 )
= calculated volume of the sample (mL)
— calculated volume of V (mL)
Containers and storage Containers — Metal cylinders.
Storage— Not exceeding 40°C.
956 Oxymetholone / Official Monographs
JP XV
b-c: calibrated in 0.1 mL
c-d: calibrated in 2 mL
d-e: calibrated in 0.1 mL
c-f. calibrated in 0.1 mL
The graduations are
marked with red line
b-f: =100 mL
Optical rotation <2.49> [a]™: +34
g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 175 - 182°C
■38° (after drying, 0.2
Oxymetholone
7t-+'>y r- P>
C 21 H 32 3 : 332.48
17/?-Hydroxy-2-hydroxymethylene-17a-methyl-5a-
androstan-3-one [434-07-1]
Oxymetholone, when dried, contains not less than
97.0% and not more than 103.0% of C 21 H 32 3 .
Description Oxymetholone occurs as a white to pale yellow-
ish white, crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in 1,4-dioxane,
sparingly soluble in methanol, in ethanol (95) and in acetone,
slightly soluble in diethyl ether, and practically insoluble in
water.
It is gradually colored and decomposed by light.
Identification (1) Dissolve 2 mg of Oxymetholone in 1 mL
of ethanol (95), and add 1 drop of iron (III) chloride TS: a
purple color develops.
(2) Dissolve 0.01 g of Oxymetholone in methanol to
make 50 mL. To 5 mL of the solution add 5 mL of sodium
hydroxide-methanol TS and methanol to make 50 mL. Deter-
mine the absorption spectrum of the solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Oxymetholone as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Oxymetholone in 25 mL of 1,4-dioxane: the solution is clear,
and shows a colorless to pale yellow color.
(2) Related subslances — Dissolve 50 mg of Oxymetho-
lone in 5 mL of chloroform, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add chlo-
roform to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
juL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography, and air-dry
the spot. Develop immediately the plate with a mixture of tol-
uene and ethanol (99.5) (49:1) to a distance of about 12 cm,
and air-dry the plate. Spray evenly vanillin-sulfuric acid TS
on the plate, and heat at 100°C for 3 to 5 minutes: any spot
other than the principal spot and starting point obtained
from the sample solution is not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 40 mg of Oxymetholone,
previously dried, and dissolve in methanol to make exactly 50
mL. Pipet 5 mL of this solution, and add methanol to make
exactly 50 mL. To exactly measured 5 mL of this solution
add 5 mL of sodium hydroxide-methanol TS and methanol
to make exactly 50 mL. Determine the absorbance A of this
solution at the wavelength of maximum absorption at about
315 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using a solution, prepared by adding methanol
to 5 mL of sodium hydroxide-methanol TS to make 50 mL,
as the blank.
Amount (mg) of C 2 iH 32 3
= (.4/541) x 50,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxytetracycline Hydrochloride
N-!a
HO CI+jHOH H N-CH 3
HsC
C 22 H 24 N 2 9 .HC1: 496.89
(4S,4a.R,5S,5afl,6S,12aS>4-Dimethylamino-
3,5,6,10,12,12a-hexahydroxy-6-methyl-l,ll-
dioxo-l,4,4a,5,5a,6,ll,12a-octahydrotetracene-2-
carboxamide monohydrochloride [2058-46-0]
Oxytetracycline Hydrochloride is the hydrochloride
JPXV
Official Monographs / Oxytetracycline Hydrochloride
957
of a tetracycline substance having antibacterial activity
produced by the growth of Streptomyces rimosus.
It contains not less than 880 fig (potency) and not
more than 945 fig (potency) per mg, calculated on the
dried basis. The potency of Oxytetracycline
Hydrochloride is expressed as mass (potency) of oxyt-
etracycline (C 2 2H 24 N 2 9 : 460.43).
Description Oxytetracycline Hydrochloride occurs as yel-
low, crystals or crystalline powder.
It is freely soluble in water, and slightly soluble in ethanol
(99.5).
Identification (1) Determine the absorption spectrum of a
solution of Oxytetracycline Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Oxytetracycline Hydrochloride Reference Standard prepared
in the same manner as the sample solution: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Dissolve 20 mg of Oxytetracycline Hydrochloride in 3
mL of water, and add 1 drop of silver nitrate TS: a white tur-
bidity is produced.
Optical rotation <2.49> [a]o- - 188 - -200° (0.25 g calcu-
lated on the dried basis, 0.1 mol/L hydrochloric acid, 25 mL,
100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Oxytetracycline Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.5 mL of
Standard Lead Solution (not more than 50 ppm).
(2) Related substances — Dissolve 20 mg of Oxytetracy-
cline Hydrochloride in 0.01 mol/L hydrochloric acid TS to
make exactly 25 mL, and use this solution as the sample solu-
tion. Separately, dissolve 20 mg of 4-epioxytetracycline in
0.01 mol/L hydrochloric acid TS to make exactly 25 mL, and
use this solution as 4-epioxytetracycline stock solution.
Separately, dissolve 20 mg of tetracycline hydrochloride in
0.01 mol/L hydrochloric acid TS to make exactly 25 mL, and
use this solution as tetracycline hydrochloride stock solution.
Separately, dissolve 8 mg of jS-apooxytetracycline in 5 mL of
0.01 mol/L sodium hydroxide TS, add 0.01 mol/L
hydrochloric acid TS to make exactly 100 mL, and use this
solution as /?-apooxytetracycline stock solution. Pipet 1 mL
of 4-epioxytetracycline stock solution, 4 mL of tetracycline
hydrochloride stock solution and 40 mL of jS-apooxytetracy-
cline stock solution, add 0.01 mol/L hydrochloric acid TS to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with exactly 20 fiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the peak areas of 4-epioxytetracycline and
tetracycline obtained from the sample solution are not more
than each of the peak area obtained from the standard solu-
tion, and the total area of the peaks, a-apooxytetracycline
having the relative retention time of about 2.1 with respect to
oxytetracycline, /?-apooxytetracycline and the peaks, which
appear between a-apooxytetracycline and /?-apooxytetracy-
cline, is not more than the peak area of /?-apooxytetracycline
from the standard solution. The peak area of 2-acetyl-2-
decarboxamide oxytetracycline, which appears after the prin-
cipal peak, obtained from the sample solution is not more
than 4 times the peak area of 4-epioxytetracycline from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with styrene-divinylben-
zene copolymer for liquid chromatography (8 ftm in particle
diameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase A: Mix 60 mL of 0.33 mol/L potassium di-
hydrogen phosphate TS, 100 mL of a solution of
tetrabutylammonium hydrogensulfate (1 in 100), 10 mL of a
solution of disodium dihydrogen ethylenediamine tetra-
acetate dihydrate (1 in 2500) and 200 mL of water, and adjust
the pH to 7.5 with 2 mol/L sodium hydroxide TS. To this so-
lution add 30 g of Z-butanol and water to make 1000 mL.
Mobile phase B: Mix 60 mL of 0.33 mol/L potassium
dihydrogen phosphate TS, 50 mL of a solution of
tetrabutylammonium hydrogensulfate (1 in 100), 10 mL of a
solution of disodium dihydrogen ethylenediamine tetra-
acetate dihydrate (1 in 2500) and 200 mL of water, and adjust
the pH to 7.5 with 2 mol/L sodium hydroxide TS. To this so-
lution add 100 g of ^-butanol and water to make 1000 mL.
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-20
20-35
70-
10
10
20
30
90
90
80
Flow rate: l.OmL/min
Time span of measurement: About 3.5 times as long as the
retention time of oxytetracycline beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 1 mL of 4-
epioxytetracycline stock solution, and add 0.01 mol/L
hydrochloric acid TS to make exactly 200 mL. Pipet 4 mL of
this solution, and add 0.01 mol/L hydrochloric acid TS to
make exactly 20 mL. Confirm that the peak area of 4-epiox-
ytetracycline obtained from 20 fiL of this solution is equiva-
lent to 14 to 26% of that from 20 fiL of the standard solution.
System performance: Dissolve 8 mg of a-apooxytetracy-
cline in 5 mL of 0.01 mol/L sodium hydroxide TS, add 0.01
mol/L hydrochloric acid TS to make 100 mL, and use this so-
lution as a-apooxytetracycline stock solution. Mix 3 mL of
the sample solution, 2 mL of 4-epioxytetracycline stock solu-
tion, 6 mL of tetracycline hydrochloride stock solution, 6 mL
of /3-apooxytetracycline stock solution and 6 mL of a-apoox-
ytetracycline stock solution, and add 0.01 mol/L hydrochlor-
ic acid TS to make 50 mL. When the procedure is run with 20
fiL of this solution under the above operating conditions, 4-
epioxytetracycline, oxytetracycline, tetracycline, a-apoox-
ytetracycline and /3-apooxytetracycline are eluted in this ord-
er with the resolutions between the peaks, 4-epioxytetracy-
cline and oxytetracycline, oxytetracycline and tetracycline,
and a-apooxytetracycline and /?-apooxytetracycline being not
less than 4, not less than 5 and not less than 4, respectively,
958
Oxytocin / Official Monographs
JP XV
and the symmetry coefficient of the peak of oxytetracycline is
not more than 1.3.
System repeatability: Pipet 1 mL of 4-epioxytetracycline
stock solution, and add 0.01 mol/L hydrochloric acid TS to
make exactly 200 mL. When the test is repeated 6 times with
20 /xL of this solution under the above operating conditions,
the relative standard deviation of the peak area of 4-epiox-
ytetracycline is not more than 2.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Weigh accurately an amount of Oxytetracycline
Hydrochloride and Oxytetracycline Hydrochloride Reference
Standard, equivalent to about 50 mg (potency), and dissolve
each in diluted hydrochloric acid (1 in 100) to make exactly 50
mL. Pipet 5 mL each of these solutions, add diluted
methanol (3 in 20) to make exactly 50 mL, and use these solu-
tions as the sample solution and the standard solution. Per-
form the test with exactly 20 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of oxytetracycline.
Amount [/xg (potency)] of oxytetracycline (C22H24N2O9)
= ^s x G4tA4s)x1000
fV s : Amount [mg (potency)] of Oxytetracycline
Hydrochloride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 263 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with strongly acidic ion
exchange resin for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 3.402 g of potassium dihydrogen
phosphate and 9.306 g of disodium dihydrogen ethylenedia-
mine tetraacetate dihydrate in 700 mL of water, add 300 mL
of methanol, and adjust the pH to 4.5 with dilute hydrochlor-
ic acid.
Flow rate: Adjust the flow rate so that the retention time of
oxytetracycline is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
[iL of the standard solution under the above operating condi-
tions, the theoretical plates and the symmetrical coefficient of
the peak of oxytetracycline are not less than 1000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
oxytetracycline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Oxytocin
Cys -Tyr - lie - G In - Asn - Cys - Pro - Leu - G ly - NH ?
C43H 66 N 12 1 2S 2 : 1007.19
[50-56-6]
Oxytocin is a synthetic peptide having the property
of causing the contraction of uterine smooth muscle.
It contains not less than 540 oxytocin Units and not
more than 600 oxytocin Units per mg, calculated on the
dehydrated and de-acetic acid basis.
Description Oxytocin occurs as a white powder.
It is very soluble in water, and freely soluble in ethanol
(99.5).
It dissolves in hydrochloric acid TS.
The pH of a solution prepared by dissolving 0.10 g of
Oxytocin in 10 mL of freshly boiled and cooled water is be-
tween 4.0 and 6.0.
It is hygroscopic.
Identification Determine the absorption spectrum of a
solution of Oxytocin (1 in 2000) as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
Constituent amino acids Put about 1 mg of Oxytocin in a
test tube for hydrolysis, add 6 mol/L hydrochloric acid TS to
dissolve, replace the air in the tube with Nitrogen, seal the
tube under reduced pressure, and heat at 110 to 115°C for
16 hours. After cooling, open the tube, evaporate the
hydrolyzate to dryness under reduced pressure, add 2 mL of
0.02 mol/L hydrochloric acid TS to dissolve the residue, and
use this solution as the sample solution. Separately, weigh ac-
curately about 27 mg of L-aspartic acid, about 24 mg of
L-threonine, about 21 mg of L-serine, about 29 mg of
L-glutamic acid, about 23 mg of L-proline, about 15 mg of
glycine, about 18 mg of L-alanine, about 23 mg of L-valine,
about 48 mg of L-cystine, about 30 mg of methionine, about
26 mg of L-isoleucine, about 26 mg of L-leucine, about 36 mg
of L-tyrosine, about 33 mg of phenylalanine, about 37 mg of
L-lysine hydrochloride, about 42 mg of L-histidine
hydrochloride monohydrate and about 42 mg of L-arginine
hydrochloride, dissolve them in 10 mL of 1 mol/L
hydrochloric acid TS, and add water to make exactly 100 mL.
Pipet 5 mL of this solution, add water to make exactly 20
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the respective molar ratios with respect to leucine: 0.95 - 1.05
for aspartic acid, 0.95 - 1.05 for glutamic acid, 0.95 - 1.05
for proline, 0.95 - 1.05 for glycine, 0.80 - 1.10 for isoleucine,
0.80 - 1.05 for tyrosine and 0.80 - 1.05 for cystine, and not
more than 0.01 each for others.
Operating conditions —
Detector: A visible spectrophotometer (wavelength: 440
nm and 570 nm).
JP XV
Official Monographs / Oxytocin 959
Column: A stainless steel column 4.6 mm in inside diameter
and 8 cm in length, packed with strongly acidic ion-exchange
resin for liquid chromatography (sodium type) composed
with a sulfonated polystyrene copolymer (3 /um in particle
diameter).
Column temperature: A constant temperature of about
57°C.
Chemical reaction bath temperature: A constant tempera-
ture of about 130°C.
Color developing time: About 1 minute.
Mobile phase: Prepare mobile phases A, B and C accord-
ing to the following table.
Mobile phase
B
C
Citric acid mono-
hydrate
Trisodium citrate
dihydrate
Sodium chloride
Ethanol (99.5)
Benzyl alcohol
Thiodiglycol
Lauromacrogol
solution (1 in 4)
Capryric acid
Water
19.80 g
22.00 g
6.10g
6.19g
7.74 g
26.67 g
5.66 g
7.07 g
54.35 g
260.0 mL
20.0 mL
—
—
—
5.0mL
5.0 mL
5.0 mL
—
4.0 mL
4.0 mL
4.0 mL
0.1 mL 0.1 mL 0.1 mL
a sufficient a sufficient a sufficient
amount amount amount
Total amount
2000 mL 1000 mL
1000 mL
PH
3.3
3.2
4.9
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A, B and C as directed in the following
table.
Time after
injection of
sample (min)
Mobile
phase
A (vol%)
Mobile
phase
B (vol%)
Mobile
phase
C (vol%)
0-9
100
9-25
100
25-61
100^
0^ 100
61 -80
100
Reaction reagent: Mix 407 g of lithium acetate dihydrate,
245 mL of acetic acid (100) and 801 mL of l-methoxy-2-
propanol, add water to make 2000 mL, stir for more than 10
minutes while passing Nitrogen, and use this solution as Solu-
tion A. Separately, to 1957 mL of l-methoxy-2-propanol add
77 g of ninhydrin and 0.134 g of sodium borohydride, stir for
more than 30 minutes while passing Nitrogen, and use this so-
lution as Solution B. Mix Solution A and Solution B before
use.
Flow rate of mobile phase: About 0.26 mL per minute.
Flow rate of reaction reagent: About 0.3 mL per minute.
System suitability —
System performance: When the procedure is run with
20 /xL of the standard solution under the above operating
conditions, aspartic acid, threonine, serine, glutamic acid,
proline, glycine, alanine, valine, cystine, methionine, isoleu-
cine, leucine, tyrosine, phenylalanine, lysine, histidine and
arginine are eluted in this order with the resolutions between
the peaks of threonine and serine, glycine and alanine, and
isoleucine and leucine being not less than 1.5, 1.4 and 1.2, re-
spectively.
System repeatability: When the test is repeated 3 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviations of the peak area
of aspartic acid, proline, valine and arginine are not more
than 2.0%, respectively.
Purity (1) Acetic acid — Weigh accurately about 15 mg of
Oxytocin, dissolve in the internal standard solution to make
exactly 10 mL, and use this solution as the sample solution.
Separately, weigh accurately about 1 g of acetic acid (100),
add the internal standard solution to make exactly 100 mL.
Pipet 2 mL of this solution, add the internal standard
solution to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with 10 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following
conditions, and calculate the ratios, Q T and Q s , of the peak
area of acetic acid to that of the internal standard: the
amount of acetic acid is not less than 6.0% and not more
than 10.0%.
Amount (%) of acetic acid (C 2 H 4 2 )
= (W s /Wj)x(Q T /Q s )x(U 10)
W s : Amount (mg) of acetic acid (100)
W T : Amount (mg) of the sample
Internal standard solution — A solution of propionic acid in
the mobile phase (1 in 10,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 0.7 mL of phosphoric acid add 900 mL
of water, adjust the pH to 3.0 with 8 mol/L sodium
hydroxide TS, and add water to make 1000 mL. To 950 mL
of this solution add 50 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
acetic acid is about 3 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating
conditions, acetic acid and propionic acid are eluted in this
order with the resolution between these peaks being not less
than 14.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of acetic acid to that of the internal standard is not
more than 2.0%.
(2) Related substances — Dissolve 25 mg of Oxytocin in
100 mL of the mobile phase A, and use this solution as the
sample solution. Perform the test with 50 /xL of the sample
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, determine each peak
area by the automatic integration method, and calculate the
amount of them by the area percentage method: the amount
960
Oxytocin Injection / Official Monographs
JP XV
of each peak other than Oxytocin is not more than 1.5%, and
the total of them is not more than 5.0%.
Operating conditions —
Detector, column, column temperature, mobile phase,
flowing of mobile phase, and flow rate: Proceed as directed in
the operating conditions in the Assay.
Time span of measurement: About 2.5 times as long as the
retention time of oxytocin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, add the mobile phase A to make exactly
100 mL, and use this solution as the solution for system
suitability test. Pipet 1 mL of the solution for system suitabil-
ity test, and add the mobile phase A to make exactly 10 mL.
Confirm that the peak area of oxytocin obtained from 50 /xL
of this solution is equivalent to 5 to 15% of that from 50 /uL
of the solution for system suitability test.
System performance: Dissolve an adequate amount of
oxytocin and vasopressin in the mobile phase A, so that each
mL contains about 0.1 mg each of them. When the procedure
is run with 50 /uL of this solution under the above operating
conditions, vasopressin and oxytocin are eluted in this order
with the resolution between these peaks being not less than
14, and the symmetry factor of the peak of oxytocin is not
more than 1.5.
System repeatability: When the test is repeated 6 times with
50 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of oxytocin is not more than 2.0%.
Water <2.48> Not more than 5.0% (50 mg, coulometric
titration).
Assay Weigh accurately an amount of Oxytocin, equivalent
to about 13,000 Units, dissolve in the mobile phase A to
make exactly 100 mL, and use this solution as the sample so-
lution. Separately, dissolve 1 bottle of the Oxytocin Refer-
ence Standard in the mobile phase A to make a known con-
centration solution containing each mL contains about 130
Units, and use this solution as the standard solution. Perform
the test with exactly 25 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of oxytocin.
Units per mg of Oxytocin, calculated on the dehydrated and
de-acetic acid basis
= (W S /Wj) x (A T /A S ) x 100
W s : Units per mL of the standard solution
W T : Amount (mg) of sample, calculated on the dehydrated
and de-acetic acid basis
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 15.6 g of sodium dihydrogen
phosphate dihydrate in 1000 mL of water.
Mobile phase B: A mixture of water and acetonitrile (1:1).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following ta-
ble.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-30
70^40
30^60
30-30.1
40^70
60^30
30.1 -45
70
30
Flow rate: About 1.0 mL per minute.
System suitability —
System performance: Dissolve 2 mg each of oxytocin and
vasopressin in 20 mL of the mobile phase A. When the proce-
dure is run with 25 fxL of this solution under the above oper-
ating conditions, vasopressin and oxytocin are eluted in this
order with the resolution between these peaks being not less
than 14, and the symmetry factor of the peak of oxytocin is
not more than 1.5.
System repeatability: When the test is repeated 6 times with
25 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
oxytocin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage— At 2 to 8°C.
Oxytocin Injection
^"+-> r-v>alt;i£
Oxytocin Injection is an aqueous solution for injec-
tion.
It contains not less than 90.0% and not more than
110.0% of the labeled oxytocin Units.
Method of preparation Prepare as directed under Injec-
tions, with Oxytocin.
Description Oxytocin Injection is a colorless, clear liquid.
pH <2.54> 2.5-4.5
Bacterial endotoxins <4.01> Less than 10 EU/oxytocin
Unit.
Extractable volume <6.05> It meets the requirement
Foreign insoluble matter <6.06> Perform the test according
to the Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Measure exactly a portion of Oxytocin Injection ac-
cording to the labeled Units, dilute with the diluent so that
each mL contains about 1 Unit, and use this solution as the
sample solution. Separately, dissolve 1 bottle of Oxytocin
Reference Standard in the mobile phase A to make exactly 20
mL. Pipet a suitable volume of this solution, dilute with the
diluent to make a known concentration solution so that each
JPXV
Official Monographs / Pancreatin
961
mL contains about 1 Unit, and use this solution as the stan-
dard solution. Perform the test with exactly 100 fiL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the peak areas, A T and A s , of
oxytocin.
Units per mL of Oxytocin Injection
= W s x(A 1 /A s )x(b/a)
W s : Units per mL of the standard solution
a: Volume (mL) of sample
b: Total volume of the sample solution prepared by dilut-
ing with the diluent
Diluent: Dissolve 5 g of chlorobutanol, 1.1 g of sodium
acetate trihydrate, 5 g of acetic acid (100) and 6
mL of ethanol (99.5) in water to make 1000 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 15.6 g of sodium dihydrogen
phosphate dihydrate in 1000 mL of water.
Mobile phase B: A mixture of water and acetonitrile (1:1).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following ta-
ble.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-30
70^40
30^60
30-30.1
40^70
60^30
30.1 -45
70
30
Flow rate: About 1.0 mL per minute.
System suitability —
System performance: Dissolve 2 mg each of oxytocin and
vasopressin in 100 mL of the mobile phase A. When the
procedure is run with 100 fiL of this solution under the above
operating conditions, vasopressin and oxytocin are eluted in
this order with the resolution between these peaks being not
less than 14, and the symmetry factor of the peak of oxytocin
is not more than 1.5.
System repeatability: When the test is repeated 6 times with
100 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
oxytocin is not more than 2.0%.
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Pancreatin
Pancreatin is a substance containing enzymes pre-
pared from the pancreas of edible animals, mostly the
hog, and has amylolytic, proteolytic and lipolytic activ-
ities.
It contains not less than 2800 starch saccharifying
activity units, not less than 28,000 proteolytic activity
units, and not less than 960 lipolytic activity units per
g-
It is usually diluted with suitable excipients.
Description Pancreatin occurs as a white to light yellow
powder. It has a characteristic odor.
Purity (1) Rancidity — Pancreatin has no unpleasant or
rancid odor and is tasteless.
(2) Fat— Add 20 mL of diethyl ether to 1 .0 g of Pancrea-
tin, extract with occasional shaking for 30 minutes, and filter.
Wash the residue with 10 mL of diethyl ether, combine the
washing with the filtrate, evaporate the diethyl ether, and dry
the residue at 105 °C for 2 hours: the mass of the residue does
not exceed 20 mg.
Loss on drying <2.41> Not more than 4.0% (1 g, in vacuum,
phosphorus (V) oxide, 24 hours).
Residue on ignition <2.44> Not more than 5% (1 g).
Assay (1) Starch digestive activity <4.03>
(i) Substrate solution — Use potato starch TS for amylo-
lytic activity test, prepared by adding 10 mL of phosphate
buffer solution for pancreatin instead of 10 mL of 1 mol/L a-
cetic acid-sodium acetate buffer solution, pH 5.0.
(ii) Sample solution — Weigh accurately about 0.1 g of
Pancreatin, add a suitable amount of ice-cold water, stir, and
add ice-cold water to make exactly 100 mL. Pipet 10 mL of
this solution, and add ice-cold water to make exactly 100 mL.
(iii) Procedure — Proceed as directed in (i) Measurement
of starch saccharifying activity of (1) Assay for starch diges-
tive activity under Digestion Test..
(2) Protein digestive activity <4.03>
(i) Substrate solution — Use the substrate solution 2
described in (2) Assay for protein digestive activity under
Digestion Test after adjusting the pH to 8.5.
(ii) Sample solution — Weigh accurately about 0.1 g of
Pancreatin, add a suitable amount of ice-cold water, stir, and
add ice-cold water to make exactly 200 mL.
(iii) Procedure — Proceed as directed in (2) Assay for pro-
tein digestive activity under Digestion Test, using trichloroa-
cetic acid TS B as the precipitation reagent.
(3) Fat digestive activity <4.03>
(i) Emulsifier — Prepare with 18 g of polyvinyl alcohol I
and 2 g of polyvinyl alcohol II as directed in (3) Assay for fat
digestive activity under Digestion Test.
(ii) Substrate solution — Use the substrate solution
described in (3) Assay for fat digestive activity under the
Digestion Test.
(iii) Sample solution — Weigh accurately about 0.1 g of
Pancreatin, add a suitable amount of ice-cold water, stir, and
962
Pancuronium Bromide / Official Monographs
JP XV
add ice-cold water to make exactly 100 mL.
(iv) Procedure — Proceed as directed in (3) Assay for fat
digestive activity under Digestion Test, using phosphate
buffer solution, pH 8.0, as the buffer solution.
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
Pancuronium Bromide
2Br
C 35 H 60 Br 2 N 2 O 4 : 732.67
1 , 1 ' -(3a, 1 7/?-Diacetoxy-5a-androstan-2/?, 1 6/?-diyl)bis( 1 -
methylpiperidinium) dibromide [15500-66-0]
Pancuronium Bromide contains not less than 98.0%
and not more than 102.0% of C 3 5H 60 Br2N 2 O4, calculat-
ed on the dehydrated basis.
Description Pancuronium Bromide occurs as a white crys-
talline powder.
It is very soluble in water, and freely soluble in ethanol (95)
and in acetic anhydride.
It is hygroscopic.
Identification (1) Determine the infrared absorption spec-
trum of Pancuronium Bromide as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) A solution of Pancuronium Bromide (1 in 100)
responds to the Qualitative Tests <1.09> (1) for bromide.
Optical rotation <2.49> [ a ]g>: +38 - +42° (0.75 g calculat-
ed on the dehydrated basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution of Pancuronium Bromide
(1 in 100) is between 4.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Pancuronium Bromide in 10 mL of water: the solution is
clear and colorless.
(2) Related substances — Dissolve 50 mg of Pancuronium
Bromide in 5 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of this solution, add ethanol (95)
to make exactly 100 mL, and use this solution as the standard
solution (1). Separately, weigh exactly 5 mg of dacuronium
bromide for thin-layer chromatography, add ethanol (95) to
make exactly 25 mL, and use this solution as the standard so-
lution (2). Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 2 fiL each of
the sample solution and standard solutions (1) and (2) on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of 2-propanol, acetonitrile and a solu-
tion of sodium iodide (1 in 5) (17:2:1) to a distance of about
12 cm, and air-dry the plate. Spray evenly a solution of sodi-
um nitrite in methanol (1 in 100) on the plate, allow to stand
for 2 minutes, and spray evenly potassium bismuth iodide TS
on the plate: a spot from the sample solution, corresponding
to that from the standard solution (2), has no more color
than that from the standard solution (2), and the spots other
than the principal spot and the above mentioned spot from
the sample solution have no more color than the spot from
the standard solution (1).
Water <2.48> Not more than 8.0% (0.3 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Pancuronium
Bromide, dissolve in 50 mL of acetic anhydride by warming,
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 36.63 mg of C 3 5H 6 oBr 2 N 2 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Panipenem
/\ — <%A
HO -J
H3C
C0 2 H f ^\ P H s
H H
C 15 H 21 N 3 04S: 339.41
(5i?,6S)-6-[(U?)-l-Hydroxyethyl]-3-[(3S)-l-
(l-iminoethyl)pyrrolidin-3-ylsulfanyl] -7-oxo- 1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
[87726-17-8]
Panipenem contains not less than 900 fig (potency)
and not more than 1010 fig (potency) per mg, calculat-
ed on the anhydrous and desolvent basis. The potency
of Panipenem is expressed as mass (potency) of
panipenem (Q5H21N3O4S).
Description Panipenem occurs as a white to light yellow,
crystalline powder or mass.
It is very soluble in water, freely soluble in methanol,
slightly soluble in ethanol (99.5), and practically insoluble in
diethyl ether.
It is hygroscopic.
It deliquesces in the presence of moisture.
Identification (1) Dissolve 0.02 g of Panipenem in 2 mL
of water, add 1 mL of hydroxylammonium chloride-ethanol
TS, allow to stand for 3 minutes, add 1 mL of acidic ammo-
nium iron (III) sulfate TS, and shake: a red-brown color de-
velops.
(2) Determine the absorption spectrum of a solution of
Panipenem in 0.02 mol/L 3-(/V-morpholino)propanesulfonic
acid buffer solution, pH 7.0 (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
JPXV
Official Monographs / Panipenem
963
maximum between 296 nm and 300 nm.
(3) Determine the infrared absorption spectrum of
Panipenem as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 1760 cm -1 , 1676 cm -1 ,
1632 cm -1 , 1588 cm -1 , 1384 cm -1 and 1249 cm -1 .
Absorbance <2.24> E\ 0/ ° m (298 nm): 280 - 310 (50 mg calcu-
lated on the anhydrous and desolvent basis, 0.02 mol/L 3-(iV-
morpholino)propanesulfonic acid buffer solution, pH 7.0,
2500 mL).
Optical rotation <2.49> [ a ] 2 £: +55 - +65° (0.1 g, calculat-
ed on the anhydrous and desolvent basis, 0.1 mol/L 3-(N-
morpholino)propanesulfonic acid buffer solution, pH 7.0, 10
mL, 100 mm).
pH <2.54> Dissolve 0.5 g of Panipenem in 10 mL of water:
the pH of the solution is between 4.5 and 6.5.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Panipen-
em according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Residual solvents <2.46> — Weigh accurately about 0.2
g of Panipenem, transfer to a 20-mL narrow-mouthed cylin-
drical glass bottle, add exactly 2 mL of the internal standard
solution and 2 mL of water to dissolve, seal tightly a rubber
stopper with aluminum cap, and use this solution as the sam-
ple solution. Separately, pipet 15 mL of ethanol (99.5) and 3
mL of acetone, add water to make exactly 200 mL. Pipet 1
mL and 2 mL of this solution, and add water to them to make
exactly 20 mL. Transfer exactly 2 mL each of these solutions
to a 20-mL narrow-mouthed cylindrical glass bottle, add ex-
actly 2 mL of the internal standard solution, seal tightly a
rubber stopper with aluminum cap, and use these solutions as
the standard solution (1) and the standard solution (2). Shake
gently in a water bath at a constant room temperature, and
allow to stand for 30 minutes. Perform the test with 1 mL of
the sample gas in each container as directed under Gas Chro-
matography <2.02> according to the following condition. Cal-
culate the ratios, Q T a and Q Tb , of the peak area of ethanol
and acetone to that of the internal standard from the sample
solution, the ratios, Qsai and Qsm, of the peak area of
ethanol and acetone to that of the internal standard from the
standard solution (1), and the ratios, Qsa2 and Q S bi, of the
peak area of ethanol and acetone to that of the internal stan-
dard from the standard solution (2). Calculate the amount of
the ethanol and acetone by the following formula: ethanol is
not more than 5.0% and acetone is not more than 1.0%.
Amount (%) of ethanol in Panipenem
= 15 x 0.79 x {(Q Ta + Q Sa2 - 26 Sal )/2(e Sa2 - Q Sal )}
x (1/1000) x(l00/W)
W: Amount (g) of Panipenem
Amount (%) of acetone in Panipenem
= 3 x 0.79 x {(g Tb + g sb2 - 2e SM )/2(Q sb2 - Q shl )}
x (1/1000) x(100/H0
W: amount (g) of Panipenem
0.79: Specific gravity (o^o) of ethanol (99.5) and acetone
Internal standard solution — A solution of 1-propanol (1 in
400).
Operating conditions —
Detector: Hydrogen flame-ionization detector
Column: A glass column 1 mm in inside diameter and 40 m
in length, coated with porous polymer bead for gas chro-
matography.
Column temperature: A constant temperature of about
140°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
1-propanol is about 6 minutes.
System suitability —
System performance: When the procedure is run with 1 mL
of the gas of the standard solution (2) under the above oper-
ating conditions, ethanol, acetone and the internal standard
are eluted in this order with the resolution between ethanol
and acetone being not less than 4.
System repeatability: When the test is repeated 6 times with
1 mL of the gas of the standard solution (2) under the above
operating conditions, the relative standard deviation of the
ratios of the peak area of ethanol to that of the internal stan-
dard is not more than 5.0%.
(4) Related substances — Being specified separately.
Water <2.48> Weigh accurately about 0.5 g of Panipenem,
transfer to a 15-mL narrow-mouthed cylindrical glass bottle,
add exactly 2 mL of the internal standard solution to dis-
solve, seal tightly a rubber stopper with aluminum cap, and
use this solution as the sample solution. Separately, weigh ac-
curately 2 g of water, and add the internal standard solution
to make exactly 100 mL. Pipet 5 mL and 10 mL of this solu-
tion, add the internal standard solution to make exactly 20
mL, and use these solutions as the standard solution (1) and
the standard solution (2). Perform the test with 1 /uL of the
sample solution and standard solutions (1) and (2) as directed
under Gas Chromatography <2.02> according to the follow-
ing condition, and calculate the ratios, Q T , Q sl and Q S2 of the
peak area of water to that of the internal standard. Calculate
the amount of water by the following formula: water is not
more than 5.0%.
Amount of water (%)
= ( W s / W T ) x {(Q T + Q S2 -2Q sl )/2(Q S2 -Q sl )}
x (1/100) x 100
W s : Amount (g) of weighed water
W T ; Amount (g) of Panipenem
Internal standard solution — A solution of acetonitrile in
methanol (1 in 100).
Operating conditions —
Detector: A thermal conductivity detector
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with porous ethylvinylbenzene-divinylben-
zene copolymer for gas chromatography (150 to 180//m in
particle diameter).
Column temperature: A constant temperature of about
125°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
acetonitrile is about 8 minutes.
System suitability —
System performance: When the procedure is run with 1 fiL
of the standard solution (2) under the above operating condi-
tions, water, methanol, and the internal standard are eluted
964
Pantethine / Official Monographs
JP XV
in this order with the resolution between water and internal
standard being not less than 10.
System repeatability: When the test is repeated 6 times with
1 [iL of the standard solution (2) under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of water to that of the internal standard is not more
than 5.0%.
Residue on ignition Being specified separately.
Bacterial endotoxins <4.0J> Less than 0.15 EU/mg (poten-
cy).
Assay Weigh accurately an amount of Panipenem and
Panipenem Reference Standard, equivalent to about 0.1 g
(potency), dissolve separately in 0.02 mol/L 3-(7V-mor-
pholino)propanesulfonic acid buffer solution, pH 7.0 to
make exactly 100 mL. Pipet 5 mL each of these solutions,
add exactly 5 mL of the internal standard solution, add 0.02
mol/L 3-(/V-morpholino)propanesulfonic acid buffer solu-
tion, pH 7.0 to make 20 mL, and use these solutions as the
sample solution and standard solution. Perform the test wi-
thin 30 minutes after preparation of the solutions with 10 liL
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and calculate the ratios, Qt and Q s , of the
peak area of panipenem to that of the internal standard.
Amount [fig (potency)] of panipenem (C15H21N3O4S)
= ^sX(Gt/Qs)x1000
W s : Amount [mg (potency)] of Panipenem Reference
Standard
Internal standard solution — A solution of sodium p-
styrenesulfonate in 0.02 mol / L 3-(/V-morpholino)
propanesulfonic acid buffer solution, pH 7.0 (1 in 1000).
Operating, conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silicone polymer coated silica gel for liquid chromatography
(5 11m in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.02 mol/L 3-(A r -mor-
pholino)propanesulfonic acid buffer solution, pH 8.0 and
acetonitrile (50:1).
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, panipenem and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 11L of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of panipenem to that of the internal standard is not
more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Not exceeding — 10°C.
Pantethine
J\'>7f>
H 3 C CH 3 9
r-O
N
H OH H
H OH
-orirW 1 ^
H3C CH 30
C 22 H 42 N 4 8 S 2 : 554.72
Bis(2-{3-[(2fl)-2,4-dihydroxy-3,3-
dimethylbutanoylamino]propanoylamino} ethyl) disulfide
[16816-67-4]
Pantethine is an aqueous solution containing 80% of
pantethine.
Pantethine contains not less than 98.0% of pan-
tethine (C 2 2H42N 4 8 S2), calculated on the anhydrous
basis.
Description Pantethine is a clear, colorless to pale yellow
viscous liquid.
It is miscible with water, with methanol and with ethanol
(95).
It is decomposed by light.
Identification (1) To 0.7 g of Pantethine add 5 mL of so-
dium hydroxide TS, shake, and add 1 to 2 drops of copper
(II) sulfate TS: a blue-purple color develops.
(2) To 0.7 g of Pantethine add 3 mL of water, shake, add
0.1 g of zinc powder and 2 mL of acetic acid (100), and boil
for 2 to 3 minutes. After cooling, add 1 to 2 drops of sodium
pentacyanonitrosylferrate (III) TS: a red-purple color de-
velops.
(3) To 1.0 g of Pantethine add 500 mL of water, and
shake. To 5 mL of this solution add 3 mL of 1 mol/L
hydrochloric acid TS, and heat on a water bath for 30
minutes. After cooling, add 7 mL of a solution of hydrox-
ylammonium chloride in sodium hydroxide TS (3 in 140), and
allow to stand for 5 minutes. Add 3 drops of 2,4-
dinitrophenol TS, and add 1 mol/L hydrochloric acid TS
dropwise until the solution has no color, and then add 1 mL
of iron (III) chloride TS: a red-purple color develops.
Optical rotation <2.49> [ a ]g>: +15.0- +18.0° (1 g calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Pantethine according to Method 1 , and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 2.0 g
of Pantethine according to Method 3, and perform the test
(not more than 1 ppm).
(3) Related substances — Dissolve 0.6 g of Pantethine in
10 mL of water, and use this solution as the sample solution.
Pipet 2 mL of the sample solution, add water to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 2 11L each of the sample so-
lution and standard solution on a plate of silica gel for thin-
JP XV
Official Monographs / Papaverine Hydrochloride Injection
965
layer chromatography. Develop the plate with 2-butanone
saturated with water to a distance of about 10 cm, and air-dry
the plate. Allow the plate to stand for about 10 minutes in
iodide vapor: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
(4) Mercapto compounds — To 1.5 g of Pantethine add 20
mL of water, shake, add 1 drop of ammonia TS and 1 to 2
drops of sodium pentacyanonitrosylferrate (III) TS: a red
color is not developed.
Water <2.48>
titration).
18-22% (0.2 g, volumetric titration, direct
Residue on Ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately about 0.3 g of Pantethine, add
water to make exactly 20 mL. Transfer exactly 5 mL of this
solution in an iodine bottle, and add exactly 25 mL of 0.05
mol/L bromine VS and 100 mL of water. Add 5 mL of dilut-
ed sulfuric acid (1 in 5) rapidly, stopper tightly immediately,
and warm at 40 to 50°C for 15 minutes with occasional shak-
ing. After cooling, carefully add 5 mL of a solution of potas-
sium iodide (2 in 5), then immediately stopper tightly, shake,
add 100 mL of water and titrate <2.50> the liberated iodine
with 0.1 mol/L sodium thiosulfate VS (indicator: 2 mL of
starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 5.547 mg of C 22 H 42 N 4 8 S 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at a temperature not exceeding
10°C.
Papaverine Hydrochloride
a°a°^u >^kWk
• HO
C 20 H 21 NO 4 .HCl: 375.85
6,7-Dimethoxy-l-(3,4-dimethoxybenzyl)isoquinoline
monohydrochloride [61-25-6]
Papaverine Hydrochloride, when dried, contains not
less than 98.5% of C 20 H 21 NO 4 .HCl.
Description Papaverine Hydrochloride occurs as white
crystals or crystalline powder.
It is sparingly soluble in water and in acetic acid (100),
slightly soluble in ethanol (95), and practically insoluble in
acetic anhydride and in diethyl ether.
The pH of a solution of Papaverine Hydrochloride (1 in
50) is between 3.0 and 4.0.
Identification (1) To 1 mg of Papaverine Hydrochloride
add 1 drops of formaldehyde-sulfuric acid TS: a colorless to
light yellow-green color is produced, and it gradually changes
to deep red, then to brown.
(2) Dissolve 0.02 g of Papaverine Hydrochloride in 1 mL
of water, and add 3 drops of sodium acetate TS: a white
precipitate is produced.
(3) Dissolve 1 mg of Papaverine Hydrochloride in 3 mL
of acetic anhydride and 5 drops of sulfuric acid, heat in a
water bath for 1 minute, and examine under ultraviolet light
(main wavelength: 365 nm): the solution shows a yellow-
green fluorescence.
(4) Dissolve 0.1 g of Papaverine Hydrochloride in 10 mL
of water, make alkaline with ammonia TS, and shake with 10
mL of diethyl ether. Draw off the diethyl ether layer, wash
with 5 mL of water, and filter. Evaporate the filtrate on a
water bath, and dry the residue at 105 °C for 3 hours: the
residue so obtained melts <2.60> between 145 C C and 148°C.
(5) Alkalify a solution of Papaverine Hydrochloride (1 in
50) with ammonia TS, and filter the precipitate. Acidify the
filtrate with dilute nitric acid: the solution responds to
Qualitative Tests <1.09> (2) for chloride.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Papaverine Hydrochloride in 10 mL of water: the solution
is clear and colorless.
(2) Morphine — Dissolve 10 mg of Papaverine
Hydrochloride in 1 mL of water, add 5 mL of l-nitroso-2-
naphthol TS and 2 mL of a solution of potassium nitrate (1 in
10), and warm at 40°C for 2 minutes. Add 1 mL of a solution
of sodium nitrate (1 in 5000), and warm at 40°C for 5
minutes. After cooling, shake the mixture with 10 mL of
chloroform, centrifuge, and separate the aqueous layer: the
solution so obtained has no more color than a pale red color.
(3) Readily carbonizable substances <].15> — Perform the
test with 0.12 g of Papaverine Hydrochloride: the solution
has no more color than Matching Fluid S or P.
Loss on drying <2.41>
4 hours).
Not more than 1.0% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Papaverine
Hydrochloride, previously dried, dissolve in 100 mL of a
mixture of acetic anhydride and acetic acid (100) (7:3) by
warming, cool, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 37.59 mg of C 20 H 21 NO 4 .HCl
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Papaverine Hydrochloride
Injection
a°a°-^ u >±£i^a#j«
Papaverine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of papaverine hydrochlo-
ride (C 20 H 21 NO 4 .HCl: 375.85).
966
Paraffin / Official Monographs
JP XV
Method of preparation Prepare as directed under Injec-
tions, with Papaverine Hydrochloride.
Description Papaverine Hydrochloride Injection is a clear,
colorless liquid.
pH: 3.0-5.0
Identification (1) To 1 mL of Papaverine Hydrochloride
Injection add 3 drops of sodium acetate TS: a white
precipitate is produced.
(2) Dilute a volume of Papaverine Hydrochloride Injec-
tion, equivalent to 0.1 g of Papaverine hydrochloride accord-
ing to the labeled amount, with water to 10 mL, render the
solution alkaline with ammonia TS, and shake with 10 mL of
diethyl ether. Draw off the diethyl ether layer, wash with 5
mL of water, and filter. Evaporate the filtrate on a water bath
to dryness, and dry the residue at 105 °C for 3 hours: the
residue so obtained melts <2.60> between 145°C and 148 C C.
(3) Proceed with 1 mg each of the residue obtained in (2)
as directed in the Identification (1) and (3) under Papaverine
Hydrochloride.
(4) Alkalify 2 mL of Papaverine Hydrochloride Injection
with ammonia TS, filter the precipitate off, and acidity the
filtrate with dilute nitric acid: the solution responds to
Qualitative Tests <1.09> (2) for chloride.
Extractable volume <6.05> It meets the requirement.
Assay Dilute an exactly measured volume of Papaverine
Hydrochloride Injection, equivalent to about 0.2 g of
papaverine hydrochloride (C 2 oH 2 iN0 4 .HCl), with water to 10
mL, render the solution alkaline with ammonia TS, and ex-
tract with 20-mL, 15-mL, 10-mL and 10-mL portions of chlo-
roform. Combine the extracts, wash with 10 mL of water,
and re-extract the washings with two 5-mL portions of chlo-
roform. Combine all the chloroform extracts, and distil the
chloroform on a water bath. Dissolve the residue in 30 mL of
acetic acid (100), and titrate <2.50> with 0.05 mol/L perchlor-
ic acid VS (indicator: 2 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 18.79 mg of C 20 H 21 NO 4 .HCl
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
(2) Heat 0.5 g of Paraffin with 0.5 g of sulfur with shak-
ing carefully: the odor of hydrogen sulfide is perceptible.
Melting point <2.60> 50 - 75°C (Method 2).
Purity (1) Acidity or alkalinity — Boil 10.0 g of Paraffin
with 10 mL of hot water and 1 drop of phenolphthalein TS in
a water bath for 5 minutes, and shake vigorously: a red color
is not produced. Add 0.20 mL of 0.02 mol/L sodium
hydroxide VS to this solution, and shake: a red color is
produced.
(2) Heavy metals <1.07> — Ignite 2.0 g of Paraffin in a
crucible, first moderately until charred, then between 450°C
and 550°C to ash. Cool, add 2 mL of hydrochloric acid, and
evaporate on a water bath to dryness. To the residue add 2
mL of dilute acetic acid and water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 2.0 mL of Standard Lead
Solution add 2 mL of dilute acetic acid and water to make 50
mL (not more than lOppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Paraffin according to Method 3, and perform the test (not
more than 2 ppm).
(4) Sulfur compounds — To 4.0 g of Paraffin add 2 mL of
ethanol (99.5), further add 2 drops of a clear saturated solu-
tion of lead (II) oxide in a solution of sodium hydroxide (1 in
5), and heat for 10 minutes at 70°C with occasional shaking:
no dark brown color develops in the aqueous layer.
(5) Readily carbonizable substances — Melt 5.0 g of
Paraffin placed in a Nessler tube at a temperature near the
melting point. Add 5 mL of sulfuric acid for readily car-
bonizable substances, and warm at 70°C for 5 minutes in a
water bath. Remove the tube from the water bath, immedi-
ately shake vigorously and vertically for 3 seconds, and warm
for 1 minute in a water bath at 70 °C. Repeat this procedure
five times: the color of the sulfuric acid layer is not darker
than that of the following control solution.
Control solution: Add 1.5 mL of Cobaltous Chloride
Colorimetric Stock Solution, 0.5 mL of Cupric Sulfate
Colorimetric Stock Solution and 5 mL of liquid paraffin to
3.0 mL of Ferric Chloride Colorimetric Stock Solution, and
shake vigorously.
Containers and storage Containers — Well-closed contain-
ers.
Paraffin
A77 -f >
Liquid Paraffin
Paraffin is a mixture of solid hydrocarbons obtained
from petroleum.
Description Paraffin occurs as a colorless or white, more or
less transparent, crystalline mass. It is odorless and tasteless.
Paraffin is sparingly soluble in diethyl ether and practically
insoluble in water, in ethanol (95) and in ethanol (99.5).
Specific gravity d 2 ° : about 0.92 (proceed as directed in the
Specific gravity (2) under Fats and Fatty Oils <1.13>).
Identification (1) Heat Paraffin strongly in a porcelain
dish, and ignite: it burns with a bright flame and the odor of
paraffin vapor is perceptible.
Liquid Paraffin is a mixture of liquid hydrocarbons
obtained from petrolatum.
Tocopherols of a suitable form may by added at a
concentration not exceeding 0.001% as a stabilizer.
Description Liquid Paraffin is a colorless, transparent, oily
liquid, nearly free from fluorescence. It is odorless and taste-
less.
It is freely soluble in diethyl ether, very slightly soluble in
ethanol (99.5), and practically insoluble in water and in
ethanol (95).
Boiling point: above 300°C.
Identification (1) Heat Liquid Paraffin strongly in a por-
JPXV
Official Monographs / Light Liquid Paraffin
967
celain dish, and fire: it burns with a bright flame and the odor
of paraffin vapor is perceptible.
(2) Heat 0.5 of Liquid Paraffin with 0.5 g of sulfur with
shaking carefully: the odor of hydrogen sulfide is perceptible.
Specific gravity <2.56> ctf : 0.860 - 0.890
Viscosity <2.53> Not less than 37 mm 2 /s (Method 1,
37.8°C).
Purity (1) Odor — Transfer a suitable amount of Liquid
Paraffin to a small beaker, and heat on a water bath: a for-
eign odor is not perceptible.
(2) Acidity or alkalinity — Shake vigorously 10 mL of
Liquid Paraffin with 10 mL of hot water and 1 drop of
phenolphthalein TS: no red color develops. Shake this solu-
tion with 0.20 mL of 0.02 mol/L sodium hydroxide: a red
color develops.
(3) Heavy metals <1.07> — Ignite 2.0 g of Liquid Paraffin
in a crucible, first moderately until charred, then between 450
°C and 550°C to ash. Cool, add 2 mL of hydrochloric acid,
and evaporate on a water bath to dryness. To the residue add
2 mL of dilute acetic acid and water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 2.0 mL of Standard Lead
Solution add 2 mL of dilute acetic acid and water to make 50
mL (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Liquid Paraffin, according to Method 3 except that after
addition of 10 mL of a solution of magnesium nitrate hexa-
hydrate in ethanol (95) (1 in 50), add 1.5 mL of hydrogen
peroxide (30), fire to burn, and perform the test (not more
than 2 ppm).
(5) Solid paraffin — Transfer 50 mL of Liquid Paraffin,
previously dried at 105°C for 2 hours, to a Nessler tube, and
cool in ice water for 4 hours: the turbidity produced, if any, is
not deeper than that of the following control solution.
Control solution: To 1.5 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 50
mL, add 1 mL of silver nitrate TS, and allow to stand for 5
minutes.
(6) Sulfur compounds — Prepare a saturated solution of
lead (II) oxide in a solution of sodium hydroxide (1 in 5), and
mix 2 drops of this clear solution with 4.0 mL of Liquid
Paraffin and 2 mL of ethanol (99.5). Heat at 70°C for 10
minutes with frequent shaking, and cool: no dark brown
color develops.
(7) Polycyclic aromatic hydrocarbons — Take 25 mL of
Liquid Paraffin by a 25-mL measuring cylinder, transfer to a
100-mL separator, and wash out the cylinder with 25 mL of
hexane for ultraviolet-visible spectrophotometry. Combine
the washings with the liquid in the separator, and shake
vigorously. Shake this solution vigorously for 2 minutes with
5.0 mL of dimethylsulfoxide for ultraviolet-visible spec-
trophotometry, and allow to stand for 15 minutes. Transfer
the lower layer to a 50-mL separator, add 2 mL of hexane for
ultraviolet-visible spectrophotometry, shake vigorously for 2
minutes, and allow to stand for 2 minutes. Transfer the lower
layer to a 10-mL glass-stoppered centrifuge tube, and cen-
trifuge between 2500 revolutions per minute and 3000 revolu-
tions per minute for about 10 minutes, and use the clear solu-
tion obtained as the sample solution. Transfer 25 mL of
hexane for ultraviolet-visible spectrophotometry to another
50-mL separator, shake vigorously for 2 minutes with 5.0 mL
of dimethylsulfoxide for ultraviolet-visible spectrophotomet-
ry, and allow to stand for 2 minutes. Transfer the lower layer
to a 10-mL glass-stoppered centrifuge tube, centrifuge be-
tween 2500 revolutions per minute and 3000 revolutions per
minute for about 10 minutes, and use the clear solution thus
obtained as a control solution. Immediately determine the
absorbance of the sample solution using the control solution
as the blank as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: not more than 0.10 at the wavelength
region between 260 nm and 350 nm.
(8) Readily carbonizable substances — Transfer 5 mL of
Liquid Paraffin to a Nessler tube, and add 5 mL of sulfuric
acid for readily carbonizable substances. After heating in a
water bath for 2 minutes, remove the tube from the water
bath, and immediately shake vigorously and vertically for 5
seconds. Repeat this procedure four times: the Liquid
Paraffin layer remains unchanged in color, and the sulfuric
acid layer has no more color than the following control solu-
tion.
Control solution: Mix 3.0 mL of Ferric Chloride Colori-
metric Stock Solution with 1.5 mL of Cobaltous Chloride
Colorimetric Stock Solution and 0.50 mL of Cupric Sulfate
Colorimetric Stock Solution.
Containers and storage Containers — Tight containers.
Light Liquid Paraffin
t3fti/jM°7 7>f >
Light Liquid Paraffin is a mixture of hydrocarbons
obtained from petroleum.
Tocopherols of a suitable form may be added at a
concentration not exceeding 0.001% as a stabilizer.
Description Light Liquid Paraffin is a clear, colorless oily
liquid, nearly free from fluorescence. It is odorless and taste-
less.
It is freely soluble in diethyl ether, and practically insoluble
in water and in ethanol (95).
Boiling point: above 300°C
Identification (1) Heat Light Liquid Paraffin strongly in a
porcelain dish, and fire: it burns with a bright flame and the
odor of paraffin vapor is perceptible.
(2) Heat 0.5 of Light Liquid Paraffin with 0.5 g of sulfur
with shaking carefully: the odor of hydrogen sulfide is per-
ceptible.
Specific gravity <2.56> clf : 0.830 - 0.870
Viscosity <2.53> Less than 37 mm 2 /s (Method 1, 37.8°C).
Purity (1) Odor — Transfer a suitable amount of Light
Liquid Paraffin to a small beaker, and heat on a water bath:
no foreign odor is perceptible.
(2) Acidity or alkalinity — Shake vigorously 10 mL of
Light Liquid Paraffin with 10 mL of hot water and 1 drop of
phenolphthalein TS: no red color develops. Shake this solu-
tion with 0.20 mL of 0.02 mol/L sodium hydroxide: a red
color develops.
(3) Heavy metals <1.07> — Ignite 2.0 g of Light Liquid
Paraffin in a crucible, first moderately until charred, then be-
tween 450°C and 550°C to ash. Cool, add 2 mL of
968
Paraformaldehyde / Official Monographs
JP XV
hydrochloric acid, and evaporate on a water bath to dryness.
To the residue add 2 mL of dilute acetic acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution as follows: to 2.0
mL of Standard Lead Solution add 2 mL of dilute acetic acid
and water to make 50 mL (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Light Liquid Paraffin according to Method 3, and perform
the test (not more than 2 ppm).
(5) Solid paraffin — Transfer 50 mL of Light Liquid
Paraffin, previously dried at 105°C for 2 hours, to a Nessler
tube, and cool in ice water for 4 hours: the turbidity
produced, if any, is not deeper than that of the following
control solution.
Control solution: To 1.5 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 50
mL, add 1 mL of silver nitrate TS, and allow to stand for 5
minutes.
(6) Sulfur compounds — Prepare a saturated solution of
lead (II) oxide in a solution of sodium hydroxide (1 in 5), and
mix 2 drops of this clear solution with 4.0 mL of Light Liquid
Paraffin and 2 mL of ethanol (99.5). Heat at 70°C for 10
minutes with frequent shaking, and cool: no dark brown
color develops.
(7) Polycyclic aromatic hydrocarbons — Take 25 mL of
Light Liquid Paraffin by a 25-mL measuring cylinder, trans-
fer to a 100-mL separator, and wash out the cylinder with 25
mL of hexane for ultraviolet-visible spectrophotometry.
Combine the washings with the liquid in the separator, and
shake vigorously. Shake this solution vigorously for 2
minutes with 5.0 mL of dimethylsulf oxide for ultraviolet-
visible spectrophotometry, and allow to stand for 15 minutes.
Transfer the lower layer to a 50-mL separator, add 2 mL of
hexane for ultraviolet-visible spectrophotometry, shake
vigorously for 2 minutes, and allow to stand for 2 minutes.
Transfer the lower layer to a glass-stoppered 10-mL cen-
trifuge tube, and centrifuge between 2500 revolutions per
minute and 3000 revolutions per minute for about 10
minutes, and use the clear solution so obtained as the sample
solution. Separately, transfer 25 mL of hexane for ultrav-
iolet-visible spectrophotometry to a 50-mL separator, add 5.0
mL of dimethylsulfoxide for ultraviolet-visible spectrophoto-
metry, shake vigorously for 2 minutes, and allow to stand for
2 minutes. Transfer the lower layer to a glass-stoppered
10-mL centrifuge tube, centrifuge between 2500 revolutions
per minute and 3000 revolutions per minute for about 10
minutes, and use the clear solution so obtained as a control
solution. Immediately determine the absorbance of the sam-
ple solution using the control solution as the blank as directed
under Ultraviolet-visible Spectrophotometry <2.24>: not
more than 0. 10 at the wavelength region between 260 nm and
350 nm.
(8) Readily carbonizable substances — Transfer 5 mL of
Light Liquid Paraffin to a Nessler tube, and add 5 mL of sul-
furic acid for readily carbonizable substances. After heating
in a water bath for 2 minutes, remove the tube from the water
bath, and immediately shake vigorously and vertically for 5
seconds. Repeat this procedure four times: the liquid paraffin
layer remains unchanged in color, and sulfuric acid layer has
no more color than the following control solution.
Control solution: Mix 3.0 mL of Ferric Chloride Colori-
metric Stock Solution with 1.5 mL of Cobaltous Chloride
Colorimetric Stock Solution and 0.50 mL of Cupric Sulfate
Colorimetric Stock Solution.
Containers and storage Containers — Tight containers.
Paraformaldehyde
(CH 2 0)„
Poly(oxymethylene)
[30525-89-4]
Paraformaldehyde contains not less than 95.0% of
CH 2 Or 30.03.
Description Paraformaldehyde occurs as a white powder. It
has a slight odor of formaldehyde, but a very strong irritating
odor is perceptible when it is heated.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in hot water, in hot dilute hydrochloric acid, in
sodium hydroxide TS and in ammonia TS.
It sublimes at about 100°C.
Identification (1) Dissolve 0.1 g of Paraformaldehyde in 5
mL of ammonia TS, add 5 mL of silver nitrate TS, shake,
and add 3 mL of a solution of sodium hydroxide (1 in 10): a
mirror of metallic silver is immediately formed on the sides of
the container.
(2) Add a solution of 0.04 g of salicylic acid in 5 mL of
sulfuric acid to 0.02 g of Paraformaldehyde, and warm slow-
ly: a persistent, dark red color is produced.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Paraformaldehyde in 10 mL of ammonia TS: the solution
is clear and colorless.
(2) Acidity or alkalinity — To 0.5 g of Paraformaldehyde
add 10 mL of water, shake vigorously for 1 minute, and
filter: the filtrate is neutral.
(3) Chloride <1.03> — Dissolve 1.5 g of Paraformaldehyde
in 75 mL of water and 7.5 mL of sodium carbonate TS,
evaporate on a water bath to dryness, and ignite at about
500°C. Dissolve the residue in 15 mL of water, filter, if neces-
sary, neutralize with diluted nitric acid (3 in 10), and add 6
mL of dilute nitric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 0.25 mL of 0.01 mol/L
hydrochloric acid VS add 7.5 mL of sodium carbonate TS, a
volume of diluted nitric acid (3 in 10) required for neutraliza-
tion of the sample, 6 mL of dilute nitric acid and water to
make 50 mL (not more than 0.006%).
(4) Sulfate <1.14> — Dissolve 1.5 g of Paraformaldehyde
in 45 mL of water and 4.5 mL of sodium carbonate TS,
evaporate on a water bath to dryness, and ignite at abut
500°C. Dissolve the residue in 15 mL of water, filter, if neces-
sary, neutralize the diluted hydrochloric acid (3 in 5), and
boil for 5 minutes. After cooling, add 1 mL of dilute
hydrochloric acid and water to make 50 mL. Perform the test
using this solution as the test solution. Prepare the control so-
lution as follows: to 4.5 mL of sodium carbonate TS add an
equal volume of diluted hydrochloric acid (3 in 5) for the neu-
tralization of the sample and 15 mL of water, and boil for 5
minutes. After cooling, add 0.35 mL of 0.005 mol/L sulfuric
acid VS, 1 mL of dilute hydrochloric acid and water to make
JPXV
Official Monographs / Parnaparin Sodium
969
50 mL (not more than 0.011%).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 50 mg of Paraformaldehyde, ac-
curately weighed, in 10 mL of potassium hydroxide TS in an
iodine flask. Add 40 mL of water and an exactly measured 50
mL of 0.05 mol/L iodine VS, stopper, and allow to stand for
5 minutes. Then add 5 mL of dilute hydrochloric acid, stop-
per immediately, allow to stand for 15 minutes, and titrate
<2.50> the excess iodine with 0.1 mol/L sodium thiosulfate
VS (indicator: 1 mL of starch TS). Perform a blank determi-
nation.
Each mL of 0.05 mol/L iodine VS = 1.501 mg of CH 2
Containers and storage Containers — Tight containers.
Dental Paraformaldehyde Paste
Method of preparation
35 g
Paraformaldehyde, finely powdered
Procaine Hydrochloride, finely
powdered 35 g
Hydrous Lanolin a sufficient quantity
To make 100 g
Prepare as directed under Ointments, with the above in-
gredients.
Description Dental Paraformaldehyde Paste is yellowish
white in color. It has a characteristic odor.
Identification (1) To 0.15 g of Dental Paraformaldehyde
Paste add 20 mL of diethyl ether and 20 mL of 0.5 mol/L so-
dium hydroxide TS, shake well, separate the water layer, and
dilute with water to make 100 mL. To 1 mL of this solution
add 10 mL of acetylacetone TS, and heat on a water bath for
10 minutes: a yellow color is produced (paraformaldehyde).
(2) To the diethyl ether layer obtained in (1) add 5 mL of
dilute hydrochloric acid and 20 mL of water, shake well, and
separate the water layer: the solution responds to Qualitative
Tests <1.09> for primary aromatic amines (procaine
hydrochloride).
(3) To 0.15 g of Dental Paraformaldehyde Paste add 25
mL of diethyl ether and 25 mL of water, shake, separate the
water layer, filter, and use the filtrate as the sample solution.
Seperately, dissolve 0.01 g of procaine hydrochloride in 5 mL
of water, and use this solution as standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 5 /xL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, ethanol (99.5) and am-
monia solution (28) (50:5:1) to a distance of about 10 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): spots from the sample solution and
standard solution show the same Ri value.
Containers and storage Containers — Tight containers.
Parnaparin Sodium
R'.R 3 . R J = SO,Na or H
R 2 = SQjNa or — ^
CHj
R 6 = C0 2 Na. R 6 = H
or
R 5 = H, R 6 = C0 2 Na
n = 4-21
Parnaparin Sodium is a low-molecular heparin sodi-
um obtained by depolymerization, with hydrogen
peroxide and with copper (II) acetate, of heparins sodi-
um from the healthy edible porcine intestinal mucosa.
The mass-average molecular mass ranges between 4500
and 6400.
The potency is not less than 70 low-molecular-mass-
heparin units and not more than 95 low-molecular -
mass-heparin units of anti-factor Xa activity per milli-
gram calculated with reference of the dried substance.
Description Parnaparin Sodium occurs as a white or light
yellow powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
It is hygroscopic.
Identification (1) Mix 0.1 mL of a solution of Parnaparin
Sodium (1 in 20) and 10 mL of a solution of tritoluidine blue
O (1 in 100,000), and shake the mixture: the blue color of so-
lution immediately changes to purple.
(2) A solution of Parnaparin Sodium (1 in 20) responds
to Qualitative Tests <1.09> for sodium salt.
pH <2.54> Dissolve 0. 1 g of Parnaparin Sodium in 10 mL of
water: the pH of this solution is between 6.0 and 8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Parnaparin Sodium in 10 mL of water: the solution is clear
and colorless or pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Parnapa-
rin Sodium according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 8.0% (0.2 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours)
Molecular mass Calculate the molecular mass of Parnapa-
rin Sodium by the following methods: The mass-average
molecular mass ranges between 4500 and 6400.
(i) Creation of calibration curve Weigh 20 mg of low-
molecular mass heparin for calibration of molecular mass,
and dissolve it in 2.0 mL of the mobile phase as the standard
970
Parnaparin Sodium / Official Monographs
JP XV
solution. Perform the test with 50,mL of the standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the peak
height, H uv , in chromatogram obtained by the ultraviolet ab-
sorption photometer, and determine the peak height, HRI, in
chromatogram obtained by the differential refractometer.
Calculate the ratio of H uv to H Rl , H Rl /H uv , at each peak.
Assume the molecular mass in the 4th peak from the low
molecular mass in chromatogram obtained by the ultraviolet
absorption photometer as 2400, and make the calculation of
the standard coefficient from dividing 2400 by the H Rl /H uv at
the corresponding peak. Make the calculation to multiply the
Hri/H uv at each peak by the standard coefficient, and deter-
mine the molecular mass of each peak by the calculation.
Prepare the calculation curve by plotting the logarithm of
molecular masses at each peak on the vertical axis and the
retention time on the chromatogram obtained by the differen-
tial refractometer on the horizontal axis.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 234 nm) and a differential refractometer.
Column: Connect two stainless steel columns which are 7.5
mm in inside diameter and 30 cm in length, and are packed
with porous silica gel for liquid chromatography; one
column, the molecular mass of limited size exclusion is about
500,000; the other, the molecular mass of limited size exclu-
sion is about 100,000. Connect a pump, the about 500,000-
molecular mass of limited size exclusion column, the about
100,000-molecular mass of limited size exclusion column, the
ultraviolet absorption photometer and the differential refrac-
tometer in this order.
Column temperature; A constant temperature of about
40°C.
Mobile phase: Dissolve 28.4 g of sodium sulfate anhydride
in 1000 mL of water, and 5.0 with 0.05 mol/L sulfuric acid
TS.
Flow rate: 0.5 mL/min
System suitability —
System performance: When the procedure is run with 50
[iL of the standard solution under the above operating condi-
tions, confirm that more than ten peaks in chromatogram ob-
tained as directed under either the Ultraviolet-visible Spec-
trophotometry, or the Differential Refractometry are ob-
served.
System repeatability: When the tests repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, relative standard deviation of the 4th peak height
in chromatogram (H vv and H R1 ) is not more than 3.0%.
(ii) Determination of molecular mass Dissolve the 20 mg
of Parnaparin Sodium with 2.0 mL of mobile phase, and use
this solution as the sample solution. Perform the test with 50
[iL of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions. Di-
vide the main peak observed between 30 min and 45 min to 30
sec-interval fractions, and determine the strength of differen-
tial refractometer of each 30 sec-interval fraction. Determine
the molecular mass of each fraction using the calibration
curve and the retention time of each fraction. Determine the
mean of molecular mass in the entire peak using the strength
of differential refractometer and the molecular mass in every
fractions.
Mean molecular mass of parnaparin sodium
= Z(n r M^/Znj
«,: The differential refractometer strength of fraction i in
the main peak of chromatogram
M,: Molecular mass of fraction i in main peak
Sn t : Sum of differential refractometer strength in the each
fraction between 1500 and 10,000 molecular mass in
the main peak
Operating conditions —
Detector: A differential refractometer.
Column, column temperature, mobile phase, and flow
rate: Proceed as directed in (i) Creation of calibration curve.
System suitability —
Proceed as directed in (i) Creation of calibration curve.
Distribution of molecular mass The molecular mass of Par-
naparin Sodium is calculated as directed in the determination
of molecular mass and the distribution of molecular mass is
determined by the following equation: the molecular mass of
not less than 80% parnaparin sodium is between 1500 and
10,000.
Distribution of molecular mass (%)
= (Zn/Zn.) x 100
«,: The differential refractometer strength of fraction i in
the main peak of chromatogram
Snf. Sum of differential refractometer strength in the each
fraction between 1500 and 10,000 molecular mass in
the main peak
The degree of sulfate ester Dissolve 0.5 g of Parnaparin So-
dium with 10 mL water. Treat the solution with 5 mL of a
strongly basic ion exchange resin, and subsequently with 10
mL of a strongly acidic ion exchange resin. Dilute the solu-
tion with water to 50 mL, and titrate <2.50> with 0.1 mol/L
Sodium hydroxide VS (potentiometric titration). Determine
the degree of sulfate ester of Parnaparin Sodium from the
equivalence point by the following equation; it is between 2.0
and 2.4.
The degree of sulfate ester
= the first equivalence point (mL)/[the second
equivalence point (mL) - first equivalence point (mL)]
Total nitrogen Weigh accurately about 0.10 g of Parnapa-
rin Sodium which is dried, and perform the test as directed
under Nitrogen Determination <1.08>: it contains not less
than 1.9% and not more than 2.3% of nitrogen (N: 14.01).
Anti-factor Ha activity When the potency of anti-factor Ha
activity is determined according to the following method, it
contains not less than 35 and not more than 60 low-
molecular-mass-heparin unit per milligram calculated with
reference to the dried substance.
(i) Standard solution Dissolve low-molecular weight
heparin standard with isotonic sodium chloride solution to
make solutions which contain 0.1, 0.2 and 0.3 low-molecular-
mass-heparin unit (anti-factor Ha activity) in 1 mL, respec-
tively.
(ii) Sample solution Weigh accurately about 50 mg of
Parnaparin Sodium, and dissolve it with isotonic sodium
chloride solution to adjust the solution which contains 4//g
parnaparin sodium in 1 mL.
(iii) Procedure To each plastic tube add 0.10 mL of the
sample solution and the standard solution, separately. To
JPXV
Official Monographs / Peanut Oil 971
each tube add 0.10 mL of human normal plasma and mix it,
and incubate at 37 ± 1°C accurately for 1 min. Next, to each
test tube add 0.10 mL of activated thromboplastin-time assay
solution, which is pre-warmed at 37 ± 1 °C, and after the mix-
ing incubate accurately for 5 min at 37 ± 1°C. Then, to each
tube add 0.10 mL of sodium calcium solution (277 in
100,000) which is pre-warmed at 37 ± 1 °C, mix it, start a stop
watch simultaneously, and permit to stand at the same tem-
perature. Determine the time for the first appearance of fibrin
clot.
(iv) Calculation Determine the low-molecular-mass-
heparin unit (anti-factor Ha activity) of the sample solution
from calibration curve obtained plots of clotting times for
each standard solution; calculate the low-molecular-mass-
heparin unit (anti-factor Ha activity) for 1 mg of parnaparin
sodium as following equation.
The low-molecular-mass-heparin unit (anti-factor Ha activi-
ty) for 1 mg of parnaparin sodium
= the low-molecular-mass-heparin unit (anti-factor Ha
activity) in 1 mL of sample solution x (b/a)
a: Amount (mg) of Parnaparin Sodium taken
b: The total volume (mL) in which Parnaparin Sodium has
been dissolved with isotonic sodium chloride solution
for the preparation of sample solution.
The ratio of anti-factor Xa activity to anti-factor Ha activity
Divide the anti-factor Xa activity, obtained in the Assay, by
the anti-factor Ha activity which has been obtained from the
test according to the method of anti-factor Ha activity; the
ratio of anti-factor Xa activity to anti-factor Ha activity is be-
tween 1.5 and 2.5.
Assay
(i) Standard solution Dissolve low-molecular-mass-hepa-
rin for calculation of molecular mass in isotonic sodium chlo-
ride solution to make solutions which contain 0.4, 0.6 and 0.8
low-molecular-mass-heparin units, (anti-factor Xa activity)
in 1 mL, respectively.
(ii) Sample solution Weigh accurately about 50 mg of
Parnaparin Sodium, and dissolve it in isotonic sodium chlo-
ride solution to make a solution which contains 7 fig par-
naparin sodium in 1 mL.
(iii) Procedure To each plastic tube add 0.10 mL of either
the sample solution or the standard solution, separately. Sub-
sequently to the every tubes add 0.70 mL of Tris-buffered so-
lution (pH 8.4), 0.10 mL of anti-thrombin III solution, and
0.10 mL of normal human plasma, and mix them. To
another plastic tube transfer 0.20 mL of these solutions,
separately, and incubate for accurate 3 minutes at 37 ± 1°C.
Next, to each tube add 0.10 mL of facter Xa TS and mix it,
permit to stand 37 ± 1°C accurately for 30 seconds, and im-
mediately add 0.20 mL of chromogenic synthetic substrate
solution (3 in 4000) and mix it, and subsequently incubate ac-
curately for 3 min at 37 ± 1 °C. To each test tube add 0.30 mL
of diluted acetic acid (100) solution (1 in 2) to stop the reac-
tion. Separately, to plastic tube add 0.10 mL of isotonic sodi-
um chloride solution, 0.70 mL of Tris-buffered solution (pH
8.4), 0.10 mL of anti-thrombin solution, and 0.10 mL of nor-
mal human plasma to every tubes, and mix well. To another
plastic tube transfer 0.2 mL of the solution, separately, and
add both 0.30 mL of water and 0.30 mL of diluted acetic acid
(100) (1 in 2). Determine the absorbance of both the sample
solution and the standard solution at 405 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24> using a so-
lution obtained from this solution as the blank.
(iv) Calculation method Determine the low-molecular-
mass unit (anti-factor Xa activity) of the sample solution us-
ing the calibration curve prepared from the absorbance of the
standard solutions and their logarithmic concentrations, and
calculate the low-molecular-mass unit (anti-factor Xa activi-
ty) in 1 mg of Parnaparin Sodium.
Low-molecular-mass-heparin unit (anti-factor Xa activity) in
1 mg of Parnaparin Sodium
= the low-molecular-mass-heparin unit (anti-factor Xa
activity) in 1 mL of the sample solution x (b/a)
a: Amount (mg) of Parnaparin Sodium taken
b: The total volume (mL) in which Parnaparin Sodium has
been dissolved with isotonic sodium chloride solution
for the preparation of sample solution.
Container and Storage
Container — Well-closed containers .
Peanut Oil
Oleum Arachidis
Peanut Oil is the fixed oil obtained from the seeds of
Arachis hypogaea Linne (Leguminosae).
Description Peanut Oil is a pale yellow, clear oil. It is odor-
less or has a slight odor. It has a mild taste.
It is miscible with diethyl ether and with petroleum ether.
It is slightly soluble in ethanol (95).
Specific gravity d 2 2 \: 0.909 - 0.916
Congealing point of the fatty acids: 22 - 33 °C
Identification Saponify 5 g of Peanut Oil by boiling with
2.5 mL of sodium hydroxide solution (3 in 10) and 12.5 mL
of ethanol (95). Evaporate the ethanol, dissolve the residue in
50 mL of hot water, and add dilute hydrochloric acid in ex-
cess until the free fatty acids separate as an oily layer. Cool
the mixture, remove the separated fatty acids, and dissolve
them in 75 mL of diethyl ether. To the diethyl ether solution
add a solution of 4 g of lead (II) acetate trihydrate in 40 mL
of ethanol (95), and allow the mixture to stand for 18 hours.
Filter the supernatant liquid, transfer the precipitate to the
filter with the aid of diethyl ether, and filter by suction. Place
the precipitate in a beaker, heat it with 40 mL of dilute
hydrochloric acid and 20 mL of water until the oily layer is
entirely clear, cool, and decant the water layer. Boil the fatty
acids with 50 mL of diluted hydrochloric acid (1 in 100).
When the solution prepared by dissolving 0.1 g of the fatty
acids in 10 mL of ethanol (95) is not darkened by the addition
of 2 drops of sodium sulfide TS, allow the fatty acids to
solidify, and press them between dry filter papers to exclude
moisture. Dissolve the solid fatty acid in 25 mL of diluted
ethanol (9 in 10) with the aid of gentle heat, and then cool to
15°C to crystallize the fatty acids. Recrystallize them from
diluted ethanol (9 in 10) and dry in a desiccator (phosphorus
(V) oxide, in vacuum) for 4 hours: the melting point <1.13> of
the dried crystals is between 73 °C and 76°C.
972 Penbutolol Sulfate / Official Monographs
JP XV
Acid value <I.I3> Not more than 0.2.
Saponification value <1.13> 188-196
Unsaponifiable matters <1.13> Not more than 1.5%.
Iodine value <I.13> 84 - 103
Containers and storage Containers — Tight containers.
Penbutolol Sulfate
H OH
• H 2 SOi
(C 18 H 29 N0 2 ) 2 .H 2 S0 4 : 680.94
^SJ-S-P-Cyclopentylphenoxy)-!-
(1,1 -dimethylethyl)aminopropan-2-ol hemisulf ate
[38363-32-5]
Penbutolol Sulfate, when dried, contains not less
than 98.5% of (C 18 H 29 N0 2 ) 2 .H 2 S0 4 .
Description Penbutolol Sulfate occurs as a white crystalline
powder.
It is very soluble in acetic acid (100), freely soluble in
methanol, sparingly soluble in ethanol (95), slightly soluble in
water, and practically insoluble in acetic anhydride and in
diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Penbutolol Sulfate in methanol (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Pen-
butolol Sulfate, previously dried, as directed in the paste
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Dissolve 0.1 g of Penbutolol Sulfate in 25 mL of
water by warming, and cool: this solution responds to
Qualitative Tests <1.09> for sulfate.
Optical rotation <2.49> [a]™: -23 - -25° (after drying, 0.2
g, methanol, 20 mL, 100 mm).
Melting point <2. 60> 2 1 3 - 2 1 7 °C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Penbutolol Sulfate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Penbutolol Sulfate according to Method 4, and perform
the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.8 g of Penbutolol
Sulfate in 10 mL of methanol, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 [iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of 2-
propanol, ethanol (95) and ammonia solution (28) (85:12:3)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41>
3 hours).
Not more than 0.5% (0.5 g, 105 °C,
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.8 g of Penbutolol Sulfate,
previously dried, dissolve in 50 mL of a mixture of acetic an-
hydride and acetic acid (100) (7:3), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 68.09 mg of (C 18 H 29 N0 2 ) 2 .H 2 S0 4
Containers and storage Containers — Well-closed contain-
ers.
Pentazocine
9'/~/>
H^C^ ^CHj
and enantiomer
285.42
(2RS,6RS, 1 1 RS)-6, 1 1 -Dimethyl-
3-(3-methylbut-2-en-l-yl)-l,2,3,4,5,6-hexahydro-
2,6-methano-3-benzoazocin-8-ol
[359-83-1]
Pentazocine, when dried,
99.0% of C 19 H 27 NO.
contains not less than
Description Pentazocine occurs as a white to pale yellowish
white, crystalline powder. It is odorless.
It is freely soluble in acetic acid (100) and in chloroform,
soluble in ethanol (95), sparingly soluble in diethyl ether and
practically insoluble in water.
Identification (1) To 1 mg of Pentazocine add 0.5 mL of
formaldehyde-sulfuric acid TS: a deep red color is produced,
and it changes to grayish brown immediately.
(2) Dissolve 5 mg of Pentazocine in 5 mL of sulfuric acid,
add 1 drop of iron (III) chloride TS, and heat in a water bath
for 2 minutes: the color of the solution changes from light
yellow to deep yellow. Shake the solution with 1 drop of
nitric acid: the solution remains yellow in color.
JPXV
Official Monographs / Pentobarbital Calcium
973
(3) Determine the absorption spectrum of a solution of
Pentazocine in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
Absorbance <2.24> E
i%
(278 nm): 67.5-71.5 (after
drying, 0.1 g, 0.01 mol/L hydrochloric acid TS, 1000 mL).
Melting point <2.60> 150 - 158°C
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Pentazocine in 20 mL of 0.1 mol/L hydrochloric acid TS:
the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Pentazo-
cine according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pentazocine according to Method 3, and perform the test
with a solution of magnesium nitrate hexahydrate in ethanol
(95) (1 in 10) (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Pentazocine
in 10 mL of chloroform, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add chloroform
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of chloroform, methanol and isopropylamine
(94:3:3) to a distance of about 13 cm, and air-dry the plate.
Allow to stand for 5 minutes in iodine vapor: any spot other
than the principal spot from the sample solution is not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 5 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Pentazocine, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 2 drops
of crystal violet TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.54 mg of C 19 H 27 NO
Containers and storage Containers — Well-closed contain-
ers.
Pentobarbital Calcium
<> hM'JUe'^-JUJUUv^A
H CHao
Ca= +
and enantiomer
C 22 H34CaN 4 6 : 490.61
Monocalcium bis[5-ethyl-5-[(l/?5')-l-methylbutyl]-4,6-
dioxo-l,4,5,6-tetrahydropyrimidin-2-olate]
[76-74-4, Pentobarbital]
Pentobarbital Calcium contains not less than 98.0%
and not more than 102.0% of C22H 3 4CaN 4 6 , calculat-
ed on the dried basis.
Description Pentobarbital Calcium occurs as a white pow-
der.
It is sparingly soluble in water, slightly soluble in ethanol
(95), and practically insoluble in acetonitrile.
A solution of Pentobarbital Calcium (1 in 100) shows no
optical rotation.
Identification (1) Determine the infrared absorption spec-
trum of Pentobarbital Calcium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) To 1 g of Pentobarbital Calcium add 5 mL of ethanol
(95) and 5 mL of dilute hydrochloric acid, dissolve by warm-
ing with shaking, shake with 5 mL of dilute hydrochloric acid
and 10 mL of water, allow to cool, and filter. To the filtrate
add 1 drop of methyl red TS, and add ammonia TS until a
slight yellow color develops: the solution responds to
Qualitative Tests <1.09> (1), (2) and (3) for calcium salt.
Purity (1) Chloride <1.03>— To 1.0 g of Pentobarbital
Calcium add 5 mL of ethanol (95) and 2.5 mL of dilute nitric
acid, dissolve by warming with shaking, cool, add water to
make 50 mL, shake well, and filter. Discard the first 10 mL of
the filtrate, and to the subsequent 15 mL add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows: To 0.30 mL of 0.01 mol/L hydrochloric acid VS
add 1.5 mL of ethanol (95), 6 mL of dilute nitric acid and
water to make 50 mL (not more than 0.035%).
(2) Heavy metals <1.07>— To 2.0 g of Pentobarbital Cal-
cium add 5 mL of ethanol (95) and 5 mL of dilute
hydrochloric acid, dissolve by warming with shaking, cool,
add water to make 80 mL, shake well, and filter. Discard the
first 10 mL of the filtrate, to the subsequent 40 mL add 1 drop
of phenolphthalein TS, add dropwise ammonia TS until a
pale red color develops, and add 2 mL of dilute acetic acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: To 2.5 mL of ethanol (95) add 2.5 mL of dilute
hydrochloric acid and water to make 30 mL. Add 1 drop of
phenolphthalein TS, add dropwise ammonia TS until a pale
red color develops, then add 2.0 mL of Standard Lead Solu-
tion, 2 mL of dilute acetic acid and water to make 50 mL (not
more than 20 ppm).
(3) Related substances — Dissolve 10 mg of Pentobarbital
Calcium in 100 mL of water, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the areas of each peak by the automatic
integration method: the area of any peak other than the peak
of pentobarbital from the sample solution is not bigger than
3/10 of the peak area of pentobarbital from the standard so-
974
Pentoxyverine Citrate / Official Monographs
JP XV
lution, and the total of these peak area is not bigger than the
peak area of pentobarbital from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of pentobarbital beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 2 mL of the standard so-
lution, add water to make exactly 20 mL, and confirm that
the peak area of pentobarbital obtained from 20 /uL of this
solution is equivalent to 5 to 15% of that of pentobarbital ob-
tained from 20 iiL of the standard solution.
System performance: Proceed as directed in the system
performance in the Assay.
System repeatability: When the test is repeated 6 times with
20 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of pentobarbital is not more than 5%.
Loss on drying <2.41>
5 hours).
Not more than 7.0% (1 g, 105°C,
Assay Weigh accurately about 20 mg of Pentobarbital Cal-
cium, dissolve in 5 mL of water, add exactly 5 mL of the in-
ternal standard solution and water to make 50 mL. To 5 mL
of this solution add water to make 20 mL. To 2 mL of this so-
lution add water to make 20 mL, and use this solution as the
sample solution. Separately, weigh accurately about 18 mg of
Pentobarbital Reference Standard, previously dried at 105 °C
for 2 hours, dissolve in 10 mL of acetonitrile, add exactly 5
mL of the internal standard solution and water to make 50
mL. To 5 mL of this solution add water to make 20 mL. To 2
mL of this solution add water to make 20 mL, and use this
solution as the standard solution. Perform the test with 20 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, g T and g s , of the
peak area of pentobarbital to that of the internal standard.
Amount (mg) of C22H 3 4CaN 4 6
= W s x(Q T /Q s )x 1.0841
W s : Amount (mg) of Pentobarbital Reference Standard
Internal standard solution — Dissolve 0.2 g of isopropyl para-
hydroxybenzoate in 20 mL of acetonitorile, and add water to
make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.36 g of potassium dihydrogen-
phosphate in 1000 mL of water, and adjust to pH 4.0 with
diluted phosphoric acid (1 in 10). To 650 mL of this solution
add 350 mL of acetonitorile.
Flow rate: Adjust the flow rate so that the retention time of
pentobarbital is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, pentobarbital and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of pentobarbital to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Pentoxyverine Citrate
Carbetapentane Citrate
Carbetapentene Citrate
HO CO^H
C 2 „H 31 N0 3 .C 6 H 8 7 : 525.59
2-[2-(Diethylamino)ethoxy]ethyl
1-phenylcyclopentanecarboxylate monocitrate
[23142-01-0]
Pentoxyverine Citrate, when dried, contains not less
than 98.5% of C 20 H 31 NO 3 .C 6 H 8 O 7 .
Description Pentoxyverine Citrate occurs as a white, crys-
talline powder.
It is very soluble in acetic acid (100), freely soluble in water
and in ethanol (95), and practically insoluble in diethyl ether.
Identification (1) Dissolve 0.1 g of Pentoxyverine Citrate
in 10 mL of water, and add 10 mL of Reinecke salt TS: a light
red precipitate is formed.
(2) Determine the infrared absorption spectrum of Pen-
toxyverine Citrate, previously dried, as directed in the paste
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) A solution of Pentoxyverine Citrate (1 in 10) responds
to Qualitative Tests <1.09> (1) and (2) for citrate.
Melting point <2.60> 92 - 95 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Pentoxyverine Citrate in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1. 07>— Proceed with 2.0 g of Pentox-
yverine Citrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pentoxyverine Citrate according to Method 3, and per-
form the test (not more than 2 ppm).
JPXV
Official Monographs / Peplomycin Sulfate 975
(4) Related substances — Dissolve 0.20 g of Pentoxyverine
Citrate in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
ethanol (95) to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
15 fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Immediate-
ly after air-drying, develop the plate with a mixture of chlo-
roform, methanol, ethyl acetate and ammonia solution (28)
(25:10:10:1) to a distance of about 10 cm, and air-dry the
plate. Allow to stand in iodine vapor for 10 minutes: the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Pentoxyverine Ci-
trate, previously dried, dissolve in 30 mL of acetic acid (100),
add 30 mL of acetic anhydride, and titrate <2.50> with 0.1
mol/L of perchloric acid VS until the color of the solution
changes from purple through blue-green to green (indicator:
3 drops of crystal violet TS). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 52.56 mg of C 2 „H 31 N0 3 .C 6 H 8 7
Containers and storage Containers — Well-closed contain-
ers.
Peplomycin Sulfate
•<^nyf
H NH;
1 II hV"" s h h li
H H 1.P*H H?
H CH 3
HjSOj
H CH 3
C 61 H 88 N 18 21 S 2 .H 2 S0 4 : 1571.67
N 1 - {3-[(l S)-(l -Phenylethyi)amino]propyl] bleomycinamide
monosulfate [70384-29-1]
Peplomycin Sulfate is the sulfate of a substance hav-
ing antitumor activity produced by the growth of
Streptomyces verticillus.
It contains not less than 865 fig (potency) and not
more than 1010 fig (potency) per mg, calculated on the
dried basis. The potency of Peplomycin Sulfate is
expressed as mass (potency) of peplomycin
(C 61 H 88 N 18 21 S 2 : 1473.59).
Description Peplomycin Sulfate occurs as a white to light
yellowish white powder.
It is freely soluble in water, and practically insoluble in
ethanol (95).
It is hygroscopic.
Identification (1) To 4 mg of Peplomycin Sulfate add
5 fiL of copper (II) sulfate TS, and dissolve in water to make
100 mL. Determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Dissolve 10 mg each of Peplomycin Sulfate and
Peplomycin Sulfate Reference Standard in 6 mL of water,
add 0.5 mL of a solution of copper (II) sulfate pentahydrate
(1 in 125), and use these solutions as the sample solution and
the standard solution. Perform the test with 10 fiL each of
these solutions as directed under Liquid Chromatography
<2.01> according to the following conditions: the retention
time of the principal peak obtained from the sample solution
is the same as that from the standard soution.
Operating conditions —
Detector, column, column temperature, mobile phase
stock solution, mobile phase A, mobile phase B, flowing of
the mobile phase, and flow rate: Proceed as directed in the
operating conditions in the Purity (3).
(3) A solution of Peplomycin Sulfate (1 in 200) responds
to Qualitative Tests <1.09> (1) and (2) for sulfate.
Optical rotation <2.49> [a]™: -2- -5° (0.1 g calculated
on the dried basis, 0.1 mol/L phosphate buffer solution, pH
5.3, 10 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Peplomycin Sulfate in 20 mL of water is between 4.5 and
6.0.
Purity (1) Clarity and color of solution — Dissolve 80 mg
of Peplomycin Sulfate in 4 mL of water: the solution is clear
and colorless.
(2) Copper — Dissolve exactly 75 mg of Peplomycin Sul-
fate in exactly 10 mL of diluted nitric acid (1 in 100), and use
this solution as the sample solution. Separately, to 5.0 mL of
Standard Copper Stock Solution add diluted nitric acid (1 in
100) to make exactly 100 mL. To 3.0 mL of this solution add
diluted nitric acid (1 in 100) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
the sample solution and the standard solution as directed un-
der Atomic Absorption Spectrophotometry <2.23> according
to the following conditions: the absorbance of the sample so-
lution is not more than that of the standard solution (not
more than 200 ppm).
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Copper hollow cathode lamp
Wavelength: 324.8 nm
(3) Related substances — Dissolve about 10 mg of
Peplomycin Sulfate in 6 mL of water, add 0.5 mL of a solu-
tion of copper (II) sulfate pentahydrate (1 in 125), and use
this solution as the sample solution. Perform the test with 10
fiL of the sample solution as directed under Liquid Chro-
976 Peplomycin Sulfate / Official Monographs
JP XV
matography <2.01> according to the following conditions.
Determine the areas of the peaks, appeared after the peak of
copper sulfate, by the automatic integration method, and cal-
culate the amounts of them by the area percentage method:
the total amount of the peaks other than peplomycin is not
more than 7.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase stock solution: Dissolve 0.96 g of sodium 1-
pentanesulfonate and 1.86 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in 1000 mL of water
and 5 mL of acetic acid (100), and adjust the pH to 4.3 with
ammonia TS.
Mobile phase A: A mixture of mobile phase stock solution
and methanol (9:1).
Mobile phase B: A mixture of mobile phase stock solution
and methanol (3:2).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase Mobile phase
A (vol%) B (vol%)
0-60
60-75
100^0
0^ 100
100
Flow rate: 1.2 mL per minute.
Time span of measurement: As long as 20 minutes after
elution of peplomycin beginning after the peak of copper sul-
fate.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, add water to make exactly 10 mL, and
use this solution as the solution for system suitability test.
Pipet 1 mL of the solution for system suitability test, and add
water to make exactly 10 mL. Confirm that the peak area of
peplomycin obtained from 10 /xL of this solution is
equivalent to 7 to 13% of that from 10 /uL of the solution for
system suitability test.
System performance: When the procedure is run with 10
/uL of the sample solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of peplomycin are not less than 30,000 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of
peplomycin is not more than 2.0%.
Loss on drying <2.41> Not more than 3.0% (60 mg, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours). Handle the sam-
ple avoiding absorption of moisture.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Mycobacterium smegmatis ATCC
607
(ii) Agar media for seed and base layer, and for transfer-
ring test organism
Glycerin 10.0 g
Peptone 10.0 g
Meat extract 10.0 g
Sodium chloride 3.0 g
Agar 15.0 g
Water 1000 mL
Mix all the ingredients and adjust the pH of the solution
with sodium hydroxide TS so that it will be 6.9 to 7.1 after
sterilization.
(iii) Liquid medium for suspending test organism
Glycerin 10.0 g
Peptone 10.0 g
Meat extract 10.0 g
Sodium chloride 3.0 g
Water 1000 mL
Mix all the ingredients and adjust the pH of the solution
with sodium hydroxide TS so that it will be 6.9 to 7.1 after
sterilization.
(iv) Preparation of agar medium of seeded layer — Inocu-
late the test organism onto the slant of the agar medium for
transferring test organism, and incubate the slant at 27°C for
40 to 48 hours. Inoculate the subcultured test organism into
100 mL of the liquid medium for suspending test organism,
incubate at 25 to 27 °C for 5 days while shaking, and use this
suspension as the suspension of the test organism. Keep the
suspension of the test organism at a temperature of not ex-
ceeding 5°C and use within 14 days. Add 0.5 mL of the sus-
pension of the test organism in 100 mL of the Agar medium
for seed layer previously kept at 48°C, mix thoroughly, and
use this as the agar medium of seeded layer.
(v) Preparation of cylinder-agar plate — Proceed as
directed in 7. Preparation of cylinder-agar plates under the
Microbial Assay for Antibiotics with the exception of the
amounts of the agar medium for base layer and the agar
medium of seeded layer to put in the Petri dish, which are 5.0
mL and 8.0 mL, respectively.
(vi) Standard solutions — Weigh accurately an amount of
Peplomycin Sulfate Reference Standard, equivalent to about
20 mg (potency), dissolve in 0.1 mol/L phosphate buffer so-
lution, pH 6.8 to make exactly 100 mL, and use this solution
as the standard stock solution. Keep the standard stock solu-
tion at a temperature not exceeding 5°C, and use within 15
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solu-
tion, pH 6.8 to make solutions so that each mL contains 4//g
(potency) and 2//g (potency), and use these solutions as the
high concentration standard solution and low concentration
standard solution, respectively.
(vii) Sample solutions — Weigh accurately an amount of
Peplomycin Sulfate, equivalent to about 20 mg (potency),
and dissolve in 0.1 mol/L phosphate buffer solution, pH 6.8
to make exactly 100 mL. Take exactly a suitable amount of
this solution, add 0.1 mol/L phosphate buffer solution, pH
6.8 to make solutions so that each mL contains 4//g (potency)
and 2,wg (potency), and use these solutions as the high con-
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Tight containers
JPXV
Official Monographs / Perphenazine Tablets
977
Perphenazine
<)\,-?*i-p>
C 21 H 26 C1N 3 0S: 403.97
2- {4- [3-(2-Chloro- 1 0//-phenothiazin- 1 0-yl)propyl]piperazin-
l-yl}ethanol [58-39-9]
Perphenazine, when dried,
98.5% of C 21 H 26 ClN 3 OS.
contains not less than
Description Perphenazine occurs as white to light yellow
crystals or crystalline powder. It is odorless, and has a bitter
taste.
It is freely soluble in methanol and in ethanol (95), soluble
in acetic acid (100), sparingly soluble in diethyl ether, and
practically insoluble in water.
It dissolves in dilute hydrochloric acid.
It is gradually colored by light.
Identification (1) Dissolve 5 mg of Perphenazine in 5 mL
of sulfuric acid: a red color, changing to deep red-purple
upon warming, is produced.
(2) Dissolve 0.2 g of Perphenazine in 2 mL of methanol,
add this solution to 10 mL of a warm solution of 2,4,6-
trinitrophenol in methanol (1 in 25), and allow to stand for 4
hours. Collect the crystals, wash with a small volume of
methanol, and dry at 105°C for 1 hour: the crystals so ob-
tained melt <2.60> between 237°C and 244°C (with decompo-
sition).
(3) Determine the absorption spectrum of a solution of
Perphenazine in 0.1 mol/L hydrochloric acid TS (1 in
200,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum 1 or the spectrum of a solution of Perphenazine
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths. Separately, to 10 mL of the
solution add 10 mL of water. Determine the absorption spec-
trum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum 2 or the spectrum of a solution of
Perphenazine Reference Standard prepared in the same man-
ner as the sample solution: both spectra exhibit similar inten-
sities of absorption at the same wavelengths.
(4) Perform the test with Perphenazine as directed under
Flame Coloration Test <1.04> (2): a green color appears.
Melting point <2.60> 95 - 100°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Perphenazine according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Perform the test in the current
of nitrogen in light-resistant containers under the protection
from sunlight. Dissolve 0.10 g of Perphenazine in 10 mL of
ethanol (95), and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add ethanol (95) to
make exactly 10 mL. Pipet 1 mL of this solution, add ethanol
(95) to make exactly 20 mL, and use this solution as the stan-
dard solution. Perform the test with these solution as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol
and 1 mol/L ammonia TS (5:1) to a distance of about 12 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): any spot other than the principal spot
from the sample solution is not more intense than that from
the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 65 °C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Perphenazine,
previously dried, dissolve in 50 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from purple through blue-pur-
ple to blue-green (indicator: 3 drops of crystal violet TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 20.20 mg of C 21 H 26 ClN 3 OS
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Perphenazine Tablets
<)17 i1--/>§£
Perphenazine Tablets contain not less than 90% and
not more than 110% of the labeled amount of per-
phenazine (C 2l H 26 ClN 3 OS: 403.97).
Method of preparation
with Perphenazine.
Prepare as directed under Tablets,
Identification (1) Shake well a quantity of powdered Per-
phenazine Tablets, equivalent to 25 mg of Perphenazine ac-
cording to the labeled amount, with 10 mL of methanol, and
filter. Evaporate 2 mL of the filtrate on a water bath to dry-
ness. With the residue, proceed as directed in the Identifica-
tion (1) under Perphenazine.
(2) Add 5 mL of the filtrate obtained in the Identification
(1) to 10 mL of a warm solution of 2,4,6-trinitrophenol in
methanol (1 in 25), and proceed as directed in the Identifica-
tion (2) under Perphenazine.
(3) Determine the absorption spectrum of the filtrate ob-
tained in the Assay as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum between 309
nm and 313 nm. Add 30 mL of methanol to another 10 mL
of the filtrate, and determine the absorption spectrum: it ex-
hibits a maximum between 256 nm and 260 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
978
Perphenazine Maleate / Official Monographs
JP XV
Disintegrate 1 Perphenazine Tablet by shaking with 5 mL
of water, shake well with 70 mL of methanol, and add
methanol to make exactly 100 mL. Centrifuge this solution,
pipet x mL of the supernatant liquid, add methanol to make
exactly FmL of a solution containing about A fig of per-
phenazine (C 2 iH 26 ON 3 OS) in each ml, and use this solution
as the sample solution. Separately, weigh accurately about 10
mg of perphenazine for assay, previously dried in vacuum
over phosphorus (V) oxide at 65 °C for 4 hours, dissolve in
methanol to make exactly 250 mL. Pipet 5 mL of this solu-
tion, add methanol to make exactly 50 mL, and use this solu-
tion as the standard solution. Determine the absorbances, A T
and A s , of the sample solution and the standard solution at
258 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>.
Amount (mg) of perphenazine (C 21 H 26 C1N 3 0S)
= W s x (A T /A S ) x (K/25) x ( 1 /x)
W s : Amount (mg) of perphenazine for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Perphenazine Tablets at
100 revolutions per minute according to the Paddle method,
using 900 mL of 2nd fluid for dissolution test as the dissolu-
tion medium. Take 30 mL or more of the dissolved solution
90 minutes after start of the test, and filter through a mem-
brane filter with pore size of not more than 0.8 //m. Discard
the first 10 mL of the filtrate, and use the subsequent filtrate
as the sample solution. Separately, weigh accurately about 10
mg of Perphenazine Reference Standard, previously dried in
vacuum with phosphorus (V) oxide at 65 C C for 4 hours, dis-
solve in 5 mL of 0.1 mol/L hydrochloric acid TS, and add
2nd fluid for dissolution test to make exactly 250 mL. Pipet 5
mL of this solution, add 2nd fluid for dissolution test to make
exactly 50 mL, and use this solution as the standard solution.
Determine the absorbances, A T and A s , of the sample solu-
tion and standard solution at 255 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>. The dissolu-
tion rate of Perphenazine Tablets in 90 minutes is not less
than 70%.
Dissolution rate (%) with respect to the
labeled amount of perphenazine (C 21 H 26 C1N 3 0S)
= fF s x(,4 T A4 s )x(l/C)x36
W s : Amount (mg) of Perphenazine Reference Standard
C: Labeled amount (mg) of perphenazine (C 2 iH 2 6ClN 3 OS)
in 1 tablet
Assay Weigh accurately and powder not less than 20 Per-
phenazine Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 4 mg of perphenazine
(C 21 H 26 CIN 3 OS), add 70 mL of methanol, shake well, and
add methanol to make exactly 100 mL. Filter the solution,
and discard the first 20 mL of the filtrate. Pipet 5 mL of the
subsequent filtrate, add methanol to make exactly 50 mL,
and use this solution as the sample solution. Weigh accurate-
ly about 10 mg of perphenazine for assay, previously dried in
vacuum over phosphorus (V) oxide at 65 °C for 4 hours, and
dissolve in methanol to make exactly 250 mL. Pipet 5 mL of
this solution, add methanol to make exactly 50 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and the standard
solution at 258 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of perphenazine (C 2I H 26 ClN 3 OS)
= W s x(A T /A s )x (2/5)
W 5 : Amount (mg) of perphenazine for assay
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Perphenazine Maleate
CO;H
COjH
C 2l H 26 ClN 3 OS.2C 4 H 4 4 : 636.11
2-{4-[3-(2-Chlorophenothiazin-10-yl)propyl]piperazin-
l-yl}ethanol dimaleate [58-39-9, Perphenazine]
Perphenazine Maleate, when dried, contains not less
than 98.0% of C 21 H 26 C1N 3 0S.2C4H 4 04.
Description Perphenazine Maleate occurs as a white to light
yellow powder. It is odorless.
It is sparingly soluble in acetic acid (100), slightly soluble in
water and in ethanol (95), and practically insoluble in chlo-
roform.
It dissolves in dilute hydrochloric acid.
It is gradually colored by light.
Melting point: about 175°C (with decomposition).
Identification (1) Dissolve 8 mg of Perphenazine Maleate
in 5 mL of sulfuric acid: a red color is produced, which
becomes deep red-purple on warming.
(2) Dissolve 0.3 g of Perphenazine Maleate in 3 mL of di-
lute hydrochloric acid, add 2 mL of water and 3 mL of am-
monia solution (28), shake, and extract with three 10-mL
portions of chloroform. [Reserve the aqueous layer, and use
for test (5)]. Evaporate the combined chloroform extracts on
a water bath to dryness, dissolve the residue in 20 mL of
methanol, and pour into 10 mL of a warm solution of 2,4,6-
trinitrophenol in methanol (1 in 25). Allow to stand for 4
hours, collect the crystals, wash with a small amount of
methanol, and dry at 105°C for 1 hour: the crystals melt
<2.60> between 237°C and 244°C (with decomposition).
(3) Determine the absorption spectrum of a solution of
Perphenazine Maleate (1 in 20,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum 1: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Separately, to 10 mL of the solution add 30 mL of water. De-
termine the absorption spectrum of the solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum 2: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(4) Perform the test with Perphenazine Maleate as direct-
ed under Flame Coloration Test <1.04> (2): a green color ap-
JPXV
Official Monographs / Perphenazine Maleate Tablets
979
pears.
(5) Evaporate the aqueous layer reserved in (2) to dry-
ness. To the residue add 1 mL of dilute sulfuric acid and 5
mL of water, and extract with four 25-mL portions of diethyl
ether. Combine the diethyl ether extracts, and evaporate in a
water bath at about 35°C with the aid of a current of air: the
residue melts <2.60> between 128°C and 136°C.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
perphenazine maleate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Perphenazine Maleate according to Method 3, and per-
form the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Perphenazine Male-
ate, previously dried, dissolve in 70 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from purple through blue to
blue-green (indicator: 3 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 31.81 mg of C 21 H 26 C1N 3 0S.2C 4 H 4 4
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Perphenazine Maleate Tablets
<)17 ity>?l^f >lgi£i£
Perphenazine Maleate Tablets contain not less than
93% and not more than 107% of the labeled amount of
perphenazine maleate (C21H26CIN3OS.2C4H4O4:
636.11).
Method of preparation Prepare as directed under Tablets,
with Perphenazine Maleate.
Identification (1) Shake a quantity of powdered Per-
phenazine Maleate Tablets, equivalent to 0.04 g of Perphena-
zine Maleate according to the labeled amount, with 3 mL of
dilute hydrochloric acid and 30 mL of water, centrifuge, filter
the supernatant solution, add 3 mL of ammonia solution (28)
to the filtrate, and extract with three 10-mL portions of chlo-
roform. [Reserve the aqueous layer, and use for test (4).]
Wash the combined chloroform extracts with two 5-mL por-
tions of water, and separate the chloroform layer. Evaporate
6 mL of the chloroform solution on a water bath to dryness.
Proceed with the residue as directed in the Identification (1)
under Perphenazine Maleate.
(2) Evaporate 20 mL of the chloroform solution obtained
in (1) on a water bath to dryness, dissolve the residue in 20
mL of methanol, and filter, if necessary. Warm the filtrate,
add 5 mL of a warm solution of 2,4,6-trinitrophenol in
methanol (1 in 25), allow to stand for 4 hours, and proceed as
directed in the Identification (2) under Perphenazine Maleate.
(3) To 2 mL of the filtrate obtained in the Assay add
water to make 50 mL. Determine the absorption spectrum of
the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 253 nm
and 257 nm and between 303 nm and 313 nm.
(4) Filter, if necessary, the aqueous layer reserved in (1),
evaporate the filtrate to make about 5 mL, add 2 mL of dilute
sulfuric acid, and extract with two 10-mL portions of diethyl
ether. Combine the diethyl ether extracts, evaporate on a
water bath to dryness, dissolve the residue in 5 mL of sulfuric
acid TS, and add 1 to 2 drops of potassium permanganate
TS: the red color of potassium permanganate TS fades im-
mediately.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Disintegrate 1 tablet of Perphenazine Maleate Tablets by
shaking with 15 mL of 0.1 mol/L hydrochloric acid TS,
shake vigorously with 50 mL of methanol, add water to make
exactly 100 mL, and centrifuge. Pipet xmL of the super-
natant liquid, add water to make exactly FmL of a solution
containing about 6 n% of perphenazine maleate (C 21 H 26 QN 3
OS.2C 4 H 4 4 ) in each ml, and use this solution as the sample
solution. Separately, weigh accurately 30 mg of perphenazine
maleate for assay, previously dried at 105°C for 3 hours, dis-
solve in 15 mL of 0.1 mol/L hydrochloric acid TS and 50 mL
of methanol, and add water to make exactly 100 mL. Pipet 5
mL of this solution, add 3 mL of 0.1 mol/L hydrochloric
acid TS, 10 mL of methanol and water to make exactly 250
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and the
standard solution at 255 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using water as the blank.
Amount (mg) of perphenazine maleate
(C 21 H 26 C1N 3 0S.2C 4 H 4 4 )
= W s x (A T /A S ) x (V'/V) x (1/50)
W s : Amount (mg) of perphenazine maleate for assay
Assay Weigh accurately and powder not less than 20 Per-
phenazine Maleate Tablets. Weigh accurately a portion of the
powder, equivalent to about 40 mg of perphenazine maleate
(C 21 H 26 C1N 3 0S.2C 4 H 4 4 ), shake well with 15 mL of 1 mol/L
hydrochloric acid TS and 50 mL of methanol, add water to
make exactly 100 mL, and filter. Discard the first 20 mL of
the filtrate, measure exactly 5 mL of the subsequent filtrate,
add water to make exactly 250 mL, and use this solution as
the sample solution. Separately, weigh accurately about
40 mg of perphenazine maleate for assay, previously dried at
105°C for 3 hours, dissolve in a mixture of 15 mL of 1 mol/L
hydrochloric acid TS and 50 mL of methanol, and add water
to make exactly 100 mL. Pipet 5 mL of this solution, add
water to make exactly 250 mL, and use this solution as the
standard solution. Determine the absorbances, A T and ^4 S , of
the sample solution and the standard solution at 255 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using water as the blank.
Amount (mg) of perphenazine maleate
(C 21 H 26 ClN 3 OS.2C 4 H 4 4 )
= W s x(A T /A s )
W s : Amount (mg) of perphenazine maleate for assay
Containers and storage Containers — Tight containers.
980
Adsorbed Purified Pertussis Vaccine / Official Monographs
JP XV
Storage — Light-resistant.
Adsorbed Purified Pertussis
Vaccine
Adsorbed Purified Pertussis Vaccine is a liquid for
injection prepared by adding an aluminum salt to a liq-
uid containing the protective antigen of Bordetella per-
tussis to make the antigen insoluble.
It conforms to the requirements of Adsorbed Puri-
fied Pertussis Vaccine in the Minimum Requirements
for Biological Products.
Description Adsorbed Purified Pertussis Vaccine forms a
homogeneous, white turbidity on shaking.
Pethidine Hydrochloride
Operidine
CH a ■ HCI
C 15 H 21 N0 2 .HC1: 283.79
Ethyl 1 -methyl-4-phenylpiperidine-4-carboxylate
monohydrochloride [50-75-5]
Pethidine Hydrochloride, when dried, contains not
less than 98.0% of C 15 H 21 N0 2 .HC1.
Description Pethidine Hydrochloride occurs as a white,
crystalline powder.
It is very soluble in water and in acetic acid (100), freely
soluble in ethanol (95), sparingly soluble in acetic anhydride,
and practically insoluble in diethyl ether.
The pH of a solution dissolved 1.0 g of Pethidine
Hydrochloride in 20 mL of water is between 3.8 and 5.8.
Identification (1) Determine the absorption spectrum of a
solution of Pethidine Hydrochloride (1 in 2000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Pethi-
dine Hydrochloride, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Pethidine Hydrochloride (1 in 50)
responds to Qualitative Tests <1.09> (2) for chloride.
Melting point <2.60> 187 - 189°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Pethidine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.20 g of Pethi-
dine Hydrochloride. Prepare the control solution with 1.0
mL of 0.005 mol/L sulfuric acid VS (not more than 0.240%).
(3) Related substances — Dissolve 0.05 g of Pethidine
Hydrochloride in 20 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area obtained from both solutions by the automatic integra-
tion method: the total area of the peaks other than that of
pethidine from the sample solution is not larger than the peak
area of perthidine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 257 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.0 g of sodium lauryl sulfate in
1000 mL of diluted phosphoric acid (1 in 1000), adjust the pH
to 3.0 with sodium hydroxide TS, and to 550 mL of this solu-
tion add 450 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
pethidine is about 7 minutes.
Time span of measurement: About 2 times as long as the
retention time of pethidine beginning after the solvent peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of pethidine obtained from 20 /uL
of this solution is equivalent to 5 to 15% of that of pethidine
obtained from 20 /xL of the standard solution.
System performance: To 2 mL each of the sample solution
and a solution of isoamyl parahydroxybenzoate in the mobile
phase (1 in 50,000) add the mobile phase to make 10 mL.
When the procedure is run with 20 fiL of this solution accord-
ing to the above operating conditions, pethidine and isoamyl
parahydroxybenzoate are eluted in this order with the resolu-
tion between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
pethidine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.5 g of Pethidine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
JPXV
Official Monographs / White Petrolatum
981
Each mL of 0.1 mol/L perchloric acid VS
= 28.38 mg of C 15 H 21 N0 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Pethidine Hydrochloride Injection
Operidine Injection
Pethidine Hydrochloride Injection is an aqueous so-
lution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of pethidine hydrochlo-
ride (C 15 H 21 N0 2 .HC1: 283.79).
Method of preparation Prepare as directed under Injec-
tions, with Pethidine Hydrochloride.
Description Pethidine Hydrochloride Injection is a clear,
colorless liquid.
It is affected by light.
pH 4.0-6.0
Identification Take a volume of Pethidine Hydrochloride
Injection equivalent to 0.1 g of Pethidine Hydrochloride ac-
cording to the labeled amount, and add water to make 200
mL. Determine the absorption spectrum of this solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 250 nm and 254 nm, between 255
nm and 259 nm, and between 261 nm and 265 nm.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Pethidine Hydrochlo-
ride Injection, equivalent to about 0.1 g of pethidine
hydrochloride (C I5 H 21 N0 2 .HC1) according to the labeled
amount, add exactly 10 mL of the internal standard solution,
and add the mobile phase to make 50 mL. To 5 mL of this so-
lution add the mobile phase to make 20 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 0.1 g of pethidine hydrochloride for assay, previously
dried at 105 °C for 3 hours, add exactly 10 mL of the internal
standard solution, and add the mobile phase to make 50 mL.
To 5 mL of this solution add the mobile phase to make 20
mL, and use this solution as the standard solution. Perform
the test with 20 /xL of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of pethidine to that of the inter-
nal standard.
Amount (mg) of pethidine hydrochloride (C 15 H 21 N0 2 .HC1)
= W S X(Q T /Q S )
W s : Amount (mg) of pethidine hydrochloride for assay
Internal standard solution — A solution of isoamyl para-
hydroxybenzoate in the mobile phase (1 in 12,500).
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 257 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [im in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.0 g of sodium lauryl sulfate in
1000 mL of diluted phosphoric acid (1 in 1000), adjust the pH
to 3.0 with sodium hydroxide TS, and to 550 mL of this solu-
tion add 450 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
pethidine is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, pethidine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 2.0.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of pethidine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers-
and colored containers may be used.
Storage — Light-resistant.
•Hermetic containers,
Hydrophilic Petrolatum
IM^ir U >
Method of preparation
White Beeswax
80 g
Stearyl Alcohol or Cetanol
30 g
Cholesterol
30 g
White Petrolatum
a sufficient quantity
To make 1000 g
Melt and mix Stearyl Alcohol or Cetanol, White Beeswax
and White Petrolatum on a water bath. Add Cholesterol, and
melt completely by stirring. Stop warming, and stir until the
mixture congeals.
Description Hydrophilic Petrolatum is white in color. It has
a slight, characteristic odor.
When mixed with an equal volume of water, it retains the
consistency of ointment.
Containers and storage Containers — Tight containers.
White Petrolatum
White Petrolatum is a decolorized and purified mix-
ture of hydrocarbons obtained from petroleum.
Description White Petrolatum is a white to pale yellow,
homogeneous, unctuous mass. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95) and in
ethanol (99.5).
982
Yellow Petrolatum / Official Monographs
JP XV
It dissolves in diethyl ether making a clear liquid or
producing slight insoluble substances.
It becomes a clear liquid when warmed.
Melting point <2.60> 38 - 60°C (Method 3).
Purity (1) Color — Melt White Petrolatum by warming,
and pour 5 mL of it into a test tube, and keep the content in a
liquid condition: the liquid has no more color than the fol-
lowing control solution, when observed transversely from
side against a white background.
Control solution: Add 3 .4 mL of water to 1 .6 mL of Ferric
Chloride Colorimetric Stock Solution.
(2) Acidity or alkalinity — To 35.0 g of White Petrolatum
add 100 mL of hot water, shake vigorously for 5 minutes,
and then draw off the aqueous layer. Treat the White Petrola-
tum layer in the same manner using two 50-mL portions of
hot water. To the combined aqueous layer add 1 drop of
phenolphthalein TS, and boil: no red color is produced. Fur-
ther add 2 drops of methyl orange TS: no red color is
produced.
(3) Heavy metals <1.07> — Proceed with 1.0 g of White
Petrolatum according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of White Petrolatum, according to Method 3, and perform
the test. Add 10 mL of a solution of magnesium nitrate hexa-
hydrate in ethanol (95) (1 in 50), then add 1.5 mL of hydro-
gen peroxide (30), and fire to burn (not more than 2 ppm).
(5) Sulfur compound — To 4.0 g of White Petrolatum add
2 mL of ethanol (99.5) and 2 drops of sodium hydroxide so-
lution (1 in 5) saturated with lead (II) oxide, warm the mix-
ture for 10 minutes at about 70°C with frequent shaking, and
allow to cool: no dark color is produced.
(6) Organic acids — To 100 mL of dilute ethanol add 1
drop of phenolphthalein TS, and titrate with 0.01 mol/L so-
dium hydroxide VS, until the color of the solution changes to
light red. Mix this solution with 20.0 g of White Petrolatum,
and boil for 10 minutes under a reflux condenser. Add 2 to 3
drops of phenolphthalein TS to the mixture and 0.40 mL of
0.1 mol/L sodium hydroxide VS with vigorous shaking: the
color of the solution remains red.
(7) Fats and fatty oils or resins — To 10.0 g of White
Petrolatum add 50 mL of sodium hydroxide solution (1 in 5),
and boil for 30 minutes under a reflux condenser. Cool the
mixture, separate the aqueous layer, and filter, if necessary.
To the aqueous layer add 200 mL of dilute sulfuric acid:
neither oily matter nor precipitate is produced.
Residue on ignition <2.44> Not more than 0.05% (2g).
Containers and storage Containers — Tight containers.
Yellow Petrolatum
It is slightly soluble in ethanol (95), and practically insolu-
ble in water.
It dissolves in diethyl ether, in petroleum benzine and in
turpentine oil, making a clear liquid or producing slight in-
soluble substances.
It becomes a yellow, clear liquid with slight fluorescence
when warmed.
Melting point <2.60> 38 - 60°C (Method 3).
Purity (1) Color — Melt Yellow Petrolatum by warming,
and pour 5 mL of it into a test tube, and keep the content in a
liquid condition: the liquid has no more color than the fol-
lowing control solution, when observed transversely from
side against a white background.
Control solution: To 3.8 mL of Ferric Chloride Stock CS
add 1.2 mL of Cobaltous Chloride Stock CS.
(2) Acidity or alkalinity — To 35.0 g of Yellow Petrola-
tum add 100 mL of hot water, shake vigorously for 5
minutes, and then draw off the aqueous layer. Treat the Yel-
low Petrolatum layer in the same manner using two 50-mL
portions of hot water. To the combined aqueous layer add 1
drop of phenolphthalein TS, and boil: no red color is
produced. Further add 2 drops of methyl orange TS: no red
color is produced.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Yellow
Petrolatum according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Yellow Petrolatum, according to Method 3, and perform
the test. Add 10 mL of a solution of magnesium nitrate hexa-
hydrate in ethanol (95) (1 in 50), then add 1.5 mL of hydro-
gen peroxide (30), and fire to burn (not more than 2 ppm).
(5) Sulfur compound — To 4.0 g of Yellow Petrolatum
add 2 mL of ethanol (99.5) and 2 drops of sodium hydroxide
solution (1 in 5) saturated with lead (II) oxide, warm the mix-
ture for 10 minutes at about 70°C with frequent shaking, and
allow to cool: no dark color is produced.
(6) Organic acids — To 100 mL of dilute ethanol add 1
drop of phenolphthalein TS, and titrate with 0.01 mol/L so-
dium hydroxide VS, until the color of the solution changes to
light red. Mix this solution with 20.0 g of Yellow Petrolatum,
and boil for 10 minutes under a reflux condenser. Add 2 to 3
drops of phenolphthalein TS to the mixture and 0.40 mL of
0.1 mol/L sodium hydroxide VS with vigorous shaking: the
color of the solution remains red.
(7) Fats and fatty oils or resins — To 10.0 g of Yellow
Petrolatum add 50 mL of sodium hydroxide solution (1 in 5),
and boil for 30 minutes under a reflux condenser. Cool the
mixture, separate the aqueous layer, and filter, if necessary.
To the aqueous layer add 200 mL of dilute sulfuric acid:
neither oily matter nor precipitate is produced.
Residue on ignition <2.44> Not more than 0.05% (2g).
Containers and storage Containers — Tight containers.
Yellow Petrolatum is a purified mixture of hydrocar-
bons obtained from petroleum.
Description Yellow Petrolatum occurs as a yellow,
homogeneous, unctuous mass, It is odorless and tasteless.
Petroleum Benzin
5>4-^
^ ~s s
Petroleum Benzin is a mixture of low-boiling point
JPXV
Official Monographs / Phenethicillin Potassium
983
hydrocarbons from petroleum.
Description Petroleum Benzin occurs as a colorless, clear,
volatile liquid. It shows no fluorescence. It has a chracteristic
odor.
It is miscible with ethanol (99.5) and with diethyl ether.
It is practically insoluble in water.
It is very flammable.
Specific gravity df : 0.65 - 0.71
Purity (1) Acid — Shake vigorously 10 mL of Petroleum
Benzin with 5 mL of water for 2 minutes, and allow to stand:
the separated aqueous layer does not change moistened blue
litmus paper to red.
(2) Sulfur compounds and reducing substances — To 10
mL of Petroleum Benzin add 2.5 mL of ammonia-ethanol TS
and 2 to 3 drops of silver nitrate TS, and warm the mixture at
about 50°C for 5 minutes, protected from light: no brown
color develops.
(3) Fatty oil and sulfur compounds — Drop and evaporate
10 mL of Petroleum Benzin in small portions on odorless
filter paper spread on a previously warmed glass plate: no
spot or no foreign odor is perceptible.
(4) Benzene — Warm 5 drops of Petroleum Benzin with 2
mL of sulfuric acid and 0.5 mL of nitric acid for about 10
minutes, allow to stand for 30 minutes, transfer the mixture
to a porcelain dish, and dilute with water: no odor of
nitrobenzene is perceptible.
(5) Residue on evaporation — Evaporate 140 mL of
Petroleum Benzin on a water bath to dryness, and heat the
residue at 105°C to constant mass: the mass is not more than
1 mg.
(6) Readily carbonizable substances — Shake vigorously 5
mL of Petroleum Benzin with 5 mL of sulfuric acid for readi-
ly carbonizable substances for 5 minutes in a Nessler tube,
and allow to stand: the sulfuric acid layer has no more color
than Matching Fluid A.
Distilling range <2.57> 50 - 80°C, not less than 90 vol%.
Containers and storage Containers — Tight containers.
Storage — Remote from fire, and not exceeding 30°C.
Phenethicillin Potassium
7i^y'J >*)U^7A
H CO E K
H H
*rV
CH 3
arid epjmer at C
C 17 H 19 KN 2 5 S: 402.51
Monopotassium (2S,5i?,6i?)-3,3-dimethyl-7-oxo-6-
[(2RS )-2-phenoxypropanoylamino] -4-thia- 1 -
azabicyclo[3.2.0]heptane-2-carboxylate [132-93-4]
Phenethicillin Potassium contains not less than 1400
units and not more than 1480 units per mg, calculated
on the dried basis. The potency of Phenethicillin
Potassium is expressed as unit based on the amount of
phenethicillin potassium (C 17 H 19 KN 2 05S). One unit of
Phenethicillin Potassium is equivalent to 0.68 jug of
phenethicillin potassium (CnH^K^OsS).
Description Phenethicillin Potassium occurs as a white to
light yellowish white crystalline powder.
It is freely soluble in water, and slightly soluble in ethanol
(99.5).
Identification (1) Determine the absorption spectrum of a
solution of Phenethicillin Potassium (1 in 5000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Phenethicillin Potassium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Phenethicillin Potassium responds to Qualitative
Tests <1.09> (1) for potassium salt.
Optical rotation <2.49> [ a ]g>: +217 - +244° (1 g calculated
on the dried basis, phosphate TS, 100 mL, 100 mm).
L-a-Phenethicillin potassium Dissolve about 50 mg of
Phenethicillin Potassium in the mobile phase to make exactly
50 mL, and use this solution as the sample solution. Perform
the test with 10 /xL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine the peak areas, A D and A L , of D-a-
phenethicillin and L-a-phenethicillin by the automatic in-
tegration method: A L /(A D + A L ) is between 0.50 and 0.70.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Adjust the pH of a mixture of a solution of
diammonium hydrogen phosphate (1 in 150) and acetonitrile
(41:10) to 7.0 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
L-a-phenethicillin is about 25 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the sample solution under the above operating condi-
tions, D-a-phenethicillin and L-a-phenethicillin are eluted in
this order with the resolution between these peaks being not
less than 1.5.
System repeatability: When the test is repeated 6 times with
10 fxh of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of l-
a-phenethicillin is not more than 2.0%.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Phenethicillin Potassium according to Method 2, and per-
form the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Phenethicillin Potassium according to Method 4 and, per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Phenethicillin
984
Phenobarbital / Official Monographs
JP XV
Potassium in 50 mL of the mobile, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
the mobile phase to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the total of the peak areas
other than D-a-phenethicillin and L-a-phenethicillin obtained
from the sample solution is not more than 5 times the total of
the peak areas of D-a-phenethicillin and L-a-phenethicillin
from the standard solution.
Operating conditions-
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the L-a-Phenethicillin potassium.
Time span of measurement: About 1.5 times as long as the
retention time of L-a-phenethicillin.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mobile phase to make ex-
actly 10 mL. Confirm that the peak area of L-a-phenethicillin
obtained from 10,mL of this solution is equivalent to 14 to
26% of that from 10,mL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the L-a-Phenethicillin
potassium.
Loss on drying <2.41> Not more than 1.0% (0.1 g, in vacu-
um, 60°C, 3 hours).
Assay Weigh accurately an amount of Phenethicillin Potas-
sium and dried L-Phenethicillin Potassium Reference Stan-
dard, equivalent to about 40,000 units, dissolve each in phos-
phate buffer solution, pH 6.0 to make exactly 20 mL, and use
these solutions as the sample solution and standard solution,
respectively. Pipet 2 mL each of these solutions in 100-mL
glass-stoppered flasks, add 2.0 mL of sodium hydroxide TS
to them, and allow to stand for exactly 15 minutes. To them
add 2.0 mL of diluted hydrochloric acid (1 in 10) and exactly
10 mL of 0.005 mol/L iodine VS, and allow them to stand
for exactly 15 minutes. Add 0.2 - 0.5 mL of starch TS, and
titrate <2.50> with 0.01 mol/L sodium thiosulfate VS until the
color of the solution disappears. Separately, to exactly 2 mL
each of the sample solution and standard solution add exactly
10 mL of 0.005 mol/L iodine VS, then proceed in the same
manner as above without allowing to stand for 15 minutes as
a blank determination, and make any necessary correction.
Determine the volumes, V T and V s , of 0.005 mol/L iodine VS
consumed in the sample solution and standard solution.
Amount (unit) of C 17 H 19 KN 2 5 S = W S X(V T /V S )
W s : Amount (unit) of L-Phenethicillin Potassium Refer-
ence Standard
Containers and storage Containers — Well-closed contain-
ers.
Phenobarbital
7x/A"JHi'^-JU
C 12 H, 2 N 2 3 : 232.24
5-Ethyl-5-phenylpyrimidine-2,4,6(l//,3//,5//)-trione
[50-06-6]
Phenobarbital, when dried, contains not less than
99.0% of C 12 H 12 N 2 3 .
Description Phenobarbital occurs as white crystals or crys-
talline powder. It is odorless, and has a bitter taste.
It is very soluble in A^TV-dimethylformamide, freely solu-
ble in ethanol (95), in acetone and in pyridine, soluble in
diethyl ether, and very slightly soluble in water.
It dissolves in sodium hydroxide TS and in ammonia TS.
The pH of a saturated solution of Phenobarbital is be-
tween 5.0 and 6.0.
Identification (1) Boil 0.2 g of Phenobarbital with 10 mL
of sodium hydroxide TS: the gas evolved changes moistened
red litmus paper to blue.
(2) Dissolve 0.1 g of Phenobarbital in 5 mL of diluted
pyridine (1 in 10), shake the solution with 0.3 mL of copper
(II) sulfate TS, and allow to stand for 5 minutes: a light red-
purple precipitate is produced. Shake the mixture with 5 mL
of chloroform: the chloroform layer remains colorless. Dis-
solve 0.1 g of Phenobarbital in a mixture of 2 to 3 drops of
ammonia-ammonium chloride buffer solution, pH 10.7, and
5 mL of diluted pyridine (1 in 10), then add 5 mL of chlo-
roform and 0.3 mL of copper (II) sulfate TS: a light red-pur-
ple precipitate is produced in the water layer. Shake again:
the chloroform layer remains colorless.
(3) Shake 0.4 g of Phenobarbital with 0. 1 g of anhydrous
sodium carbonate and 4 mL of water, and add a solution of
0.3 g of 4-nitrobenzyl chloride in 7 mL of ethanol (95). Heat
on a water bath for 30 minutes under a reflux condenser, and
allow to stand for 1 hour. Filter the crystals, wash with 7 mL
of sodium hydroxide TS, then with a small amount of water,
recrystallize from a mixture of ethanol (95) and chloroform
(1:1), and dry at 105°C for 30 minutes: the crystals melt
<2.60> between 181°C and 185°C.
(4) Dissolve 0.1 g of Phenobarbital in 2 mL of sulfuric
acid, shake the solution with 5 to 6 mg of potassium nitrate,
and allow to stand for 10 minutes: a yellow to yellow-brown
color develops.
Melting point <2.60> 175 - 179°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Phenobarbital in 5 mL of sodium hydroxide TS: the solution
is clear and colorless.
(2) Chloride <7.03>— Dissolve 0.30 g of Phenobarbital in
20 mL of acetone, and add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: take
JPXV
Official Monographs / Phenol
985
0.30 mL of 0.01 mol/L hydrochloric acid VS, 20 mL of ace-
tone and 6 mL of dilute nitric acid, and add water to make 50
mL (not more than 0.035%).
(3) Sulfate <1.14>— Dissolve 0.40 g of Phenobarbital in
20 mL of acetone, and add 1 mL of dilute hydrochloric acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: take 0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL
of acetone, and 1 mL of dilute hydrochloric acid, and add
water to make 50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Phenobarbital according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
solution (not more than 20 ppm).
(5) Phenylbarbituric acid — Boil 1.0 g of Phenobarbital
with 5 mL of ethanol (95) for 3 minutes: the solution is clear.
(6) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Phenobarbital. The solution has not more
color than Matching Fluid A.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Phenobarbital,
previously dried, dissolve in 50 mL of A^JV-dimethylfor-
mamide, and titrate <2.50> with 0.1 mol/L potassium
hydroxide-ethanol VS until the color of the solution change
from yellow to yellow-green (indicator: 1 mL of alizarin yel-
low GG-thymolphthalein TS). Perform a blank determina-
tion using a mixture of 50 mL of A^N-dimethylformamide
and 22 mL of ethanol (95), and make any necessary correc-
tion.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 23.22 mg of C 12 H 12 N 2 3
Containers and storage Containers — Well-closed contain-
ers.
10% Phenobarbital Powder
Phenobarbital Powder
10% Phenobarbital Powder contains not less than
9.3% and not more than 10.7% of phenobarbital
(C 12 H 12 N 2 3 : 232.24).
Method of preparation
Phenobarbital
Starch, Lactose Hydrate or
their mixture
100 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification Shake thoroughly 5 g of 10% Phenobarbital
Powder with 20 mL of hexane, and filter. Collect the residue,
and dry on a water bath, then extract with four 30-mL por-
tions of chloroform. Filter the combined chloroform ex-
tracts, and evaporate the filtrate to dryness. Dry the residue
at 105°C for 1 hour: the residue so obtained melts <2.60> be-
tween 174°C and 179°C. With the residue, proceed as direct-
ed in the Identification (1) and (2) under Phenobarbital.
Assay Weigh accurately about 10 g of 10% Phenobarbital
Powder, transfer to a glass-stoppered flask, and add exactly
100 mL of a mixture of chloroform and ethanol (95) (10:1).
Stopper tightly, shake, and allow to stand for 30 minutes.
Transfer the mixture to a glass-stoppered centrifuge tube,
and centrifuge. Measure exactly 50 mL of the supernatant
liquid, evaporate on a water bath to dryness, dissolve the
residue in 50 mL of 7V,7V-dimethylformamide, and proceed as
directed in the Assay under Phenobarbital.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 23.22 mg of C 12 H I2 N 2 3
Containers and storage Containers — Well-closed contain-
ers.
Phenol
Carbolic Acid
7i/-ll
OH
C 6 H 6 0: 94.11
Phenol [108-95-2]
Phenol contains not less than 98.0%, of C 6 H e O.
Description Phenol occurs as colorless to slightly red crys-
tals or crystalline masses. It has a characteristic odor.
It is very soluble in ethanol (95) and in diethyl ether, and
soluble in water.
Phenol (10 g) is liquefied by addition of 1 mL of water.
The color changes gradually through red to dark red by
light or air.
It cauterizes the skin, turning it white.
Congealing point: about 40°C
Identification (1) Add 1 drop of iron (III) chloride TS to
10 mL of a solution of Phenol (1 in 100): a blue-purple color
develops.
(2) Add bromine TS dropwise to 5 mL of a solution of
Phenol (1 in 10,000): a white precipitate is produced, which
at first dissolves with shaking, but becomes permanent as ex-
cess of the reagent is added.
Purity (1) Clarity and color of solution and acidity or
alkalinity — Dissolve 1.0 g of Phenol in 15 mL of water: the
solution is clear, and neutral or only faintly acid. Add 2
drops of methyl orange TS: no red color develops.
(2) Residue on evaporation — Weigh accurately about 5 g
of Phenol, evaporate on a water bath, and dry the residue at
105 °C for 1 hour: the mass is not more than 0.05% of the
mass of the sample.
Assay Dissolve about 1.5 g of Phenol, accurately weighed,
in water to make exactly 1000 mL. Transfer exactly 25 mL of
this solution to an iodine flask, add exactly 30 mL of 0.05
986
Phenol for Disinfection / Official Monographs
JP XV
mol/L bromine VS, then 5 mL of hydrochloric acid, and im-
mediately stopper the flask. Shake the flask repeatedly for 30
minutes, allow to stand for 15 minutes, then add 7 mL of
potassium iodide TS, at once stopper the flask, and shake
well. Add 1 mL of chloroform, stopper the flask, and shake
thoroughly. Titrate <2.50> the liberated iodine with 0.1 mol/
L sodium thiosulfate VS (indicator: 1 mL of starch TS). Per-
form a blank determination.
Each mL of 0.05 mol/L bromine VS
= 1.569 mg of C 6 H 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Phenol for Disinfection
Carbolic Acid for Disinfection
Phenol for Disinfection contains not less than 95.0%
of phenol (C 6 H 6 0: 94.11).
Description Phenol for Disinfection occurs as colorless to
slightly red crystals, crystalline masses, or liquid containing
these crystals. It has a characteristic odor.
It is very soluble in ethanol (95) and in diethyl ether, and
freely soluble in water.
Phenol for Disinfection (10 g) is liquefied by addition of 1
mL of water.
It cauterizes the skin, turning it white.
Congealing point: about 30°C
Identification (1) To 10 mL of a solution of Phenol for
Disinfection (1 in 100) add 1 drop of iron (III) chloride TS: a
blue-purple color is produced.
(2) To 5 mL of a solution of Phenol for Disinfection (1 in
10,000) add bromine TS dropwise: a white precipitate is
formed, and it dissolves at first upon shaking but becomes
permanent as excess of the reagent is added.
Purity (1) Clarity of solution — Dissolve 1.0 g of Phenol
for Disinfection in 15 mL of water: the solution is clear.
(2) Residue on evaporation — Weigh accurately about 5 g
of Phenol for Disinfection, evaporate on a water bath, and
dry the residue at 105°C for 1 hour: the mass is not more than
0.10% of the mass of the sample.
Assay Dissolve about 1 g of Phenol for Disinfection, ac-
curately weighed, in water to make exactly 1000 mL. Pipet 25
mL of the solution into an iodine flask, add exactly 30 mL of
0.05 mol/L bromine VS and 5 mL of hydrochloric acid, stop-
per immediately, shake for 30 minutes and allow to stand for
15 minutes. Add 7 mL of potassium iodide TS, stopper im-
mediately, shake well, and titrate <2.50> the liberated iodine
with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of
starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 1.569 mg of QH 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Liquefied Phenol
Liquefied Carbolic Acid
;M7iy-Jl
Liquefied Phenol is Phenol maintained in a liquid
condition by the presence of 10% of Water or Purified
Water.
It contains not less than 88.0% of phenol (C 6 H e O:
94.11)
Description Liquefied Phenol is a colorless or slightly red-
dish liquid. It has a characteristic odor.
It is miscible with ethanol (95), with diethyl ether and with
glycerin.
A mixture of equal volumes of Liquefied Phenol and glyce-
rin is miscible with water.
The color changes gradually to dark red on exposure to
light or air.
It cauterizes the skin, turning it white.
Specific gravity df : about 1.065
Identification (1) Add 1 drop of iron (III) chloride TS to
10 mL of a solution of Liquefied Phenol (1 in 100): a blue-
purple color develops.
(2) Add bromine TS dropwise to 5 mL of a solution of
Liquefied Phenol (1 in 10,000): a white precipitate is
produced, which at first dissolves with shaking, but becomes
permanent as excess of the reagent is added.
Boiling point <2.57> Not more than 182°C.
Purity (1) Clarity and color of solution and acidity or
alkalinity — Dissolve 1.0 g of Liquefied Phenol in 15 mL of
water: the solution is clear, and neutral or only faintly acid.
Add 2 drops of methyl orange TS: no red color develops.
(2) Residue on evaporation — Weigh accurately about 5 g
of Liquefied Phenol, evaporate on a water bath, and dry the
residue at 105°C for 1 hour: the mass is not more than 0.05%
of the mass of the sample.
Assay Dissolve about 1.7 g of Liquefied Phenol, accurately
weighed, in a water to make exactly 1000 mL. Transfer exact-
ly 25 mL of this solution to an iodine flask, add exactly 30
mL of 0.05 mol/L bromine VS, then 5 mL of hydrochloric
acid, and immediately stopper the flask. Shake the flask
repeatedly for 30 minutes, allow to stand for 15 minutes, then
add 7 mL of potassium iodide TS, at one stopper the flask
tightly, and shake well. Add 1 mL of chloroform, stopper the
flask, and shake thoroughly. Titrate <2.50> the liberated io-
dine with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL
of starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 1.569 mg of C 6 H 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Phenolated Water for Disinfection
987
Dental Phenol with Camphor
ftM7i/-Jl-^>7Jl
Method of preparation
Phenol
d- or tf/-Camphor
35.
65,
To make 100 g
Melt Phenol by warming, add (/-Camphor or dl- Camphor,
and mix.
Description Dental Phenol with Camphor is a colorless or
light red liquid. It has a characteristic odor.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
roform layer, and use this solution as the sample solution.
Separately, dissolve 0.01 g of phenol in 5 mL of chloroform,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, ethanol (99.5) and ammonia solution (28) (50:5:1) to
a distance of about 10 cm, and air-dry the plate. Allow the
plate to stand in iodine vapor: the spots obtained from the
sample solution and the standard solution show the same Rf
value.
Containers and storage Containers — Tight containers.
Phenolated Water
7x/-JU7k
Phenol and Zinc Oxide Liniment
7i/-JU
•u-y>h
Method of preparation
Liquefied Phenol
22 mL
Powdered Tragacanth
20 g
Carmellose Sodium
30 g
Glycerin
30 mL
Zinc Oxide
100 g
Purified Water
a sufficient quantity
To make
1000 g
Mix Liquefied Phenol, Glycerin and Purified Water, add
Powdered Tragacanth in small portions by stirring, and allow
the mixture to stand overnight. To the mixture add Carmel-
lose Sodium in small portions by stirring to make a pasty
mass, add Zinc Oxide in small portions, and prepare the lini-
ment as directed under Liniments. Less than 5 g of Powdered
Tragacanth or Carmellose Sodium can be replaced by each
other to make 50 g in total.
Description Phenol and Zinc Oxide Liniment is a white,
pasty mass. It has a slight odor of phenol.
Identification (1) Shake well 1 g of Phenol and Zinc Oxide
Liniment with 10 mL of diethyl ether, and filter. To the
filtrate add 10 mL of dilute sodium hydroxide TS, shake well,
and separate the water layer. To 1 mL of the water layer add
1 mL of sodium nitrite TS and 1 mL of dilute hydrochloric
acid, shake, and add 3 mL of sodium hydroxide TS: a yellow
color develops (phenol).
(2) Place 1 g of Phenol and Zinc Oxide Liniment in a por-
celain crucible, heat gradually raising the temperature until
the content is charred, and then ignite it strongly: a yellow
color develops, and disappears on cooling. To the residue
add 10 mL of water and 5 mL of dilute hydrochloric acid,
shake well, and filter. To the filtrate add 2 to 3 drops of
potassium hexacyanoferrate (II) TS: a white precipitate is
produced (zinc oxide).
(3) Shake 0.5 g of Phenol and Zinc Oxide Liniment with
1 mL of water and 5 mL of chloroform, separate the chlo-
Phenolated Water contains not less than 1.8 w/v%
and not more than 2.3 w/v% of phenol (C 6 H 6 0:
94.11).
Method of preparation
Liquefied Phenol
Water or Purified Water
22 mL
a sufficient quantity
To make 1000 mL
Mix the above ingredients.
Description Phenolated Water is a colorless, clear liquid,
having the odor of phenol.
Identification (1) Add 1 drop of iron (III) chloride TS to
10 mL of Phenolated Water: a blue-purple color develops.
(2) To 5 mL of a solution of Phenolated Water (1 in 200)
add bromine TS dropwise: a white precipitate is formed, and
it dissolves at first upon shaking but becomes permanent as
excess of the reagent is added.
Assay Take exactly 2 mL of Phenolated Water into an io-
dine flask, add 25 mL of water, then add exactly 40 mL of
0.05 mol/L bromine VS and 5 mL of hydrochloric acid, stop-
per immediately, shake for 30 minutes, and allow to stand for
15 minutes. Add 7 mL of potassium iodide TS, stopper tight-
ly at once, shake well, and titrate <2.50> the liberated iodine
with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of
starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 1.569 mg of C 6 H 6
Containers and storage Containers — Tight containers.
Phenolated Water for Disinfection
Phenolated Water for Disinfection contains not less
than 2.8 w/v% and not more than 3.3 w/v% of phenol
(C 6 H 6 0: 94.11).
Method of preparation
988
Phenolsulfonphthalein Injection / Official Monographs
JP XV
Phenol for Disinfection
Water or Purified Water
31 g
a sufficient quantity
To make 1000 mL
Mix the above ingredients.
Description Phenolated Water for Disinfection is a clear,
colorless liquid, having the odor of phenol.
Identification (1) Add 1 drop of iron (III) chloride TS to
10 mL of Phenolated Water for Disinfection: a blue-purple
color develops.
(2) Proceed with 5 mL of a solution of Phenolated Water
for Disinfection (1 in 200) as directed in the Identification (2)
under Phenol for Disinfection.
Assay Take exactly 5 mL of Phenolated Water for Disin-
fection, add water to make exactly 100 mL, then pipet 25 mL
of the solution into an iodine flask, and proceed as directed in
the Assay under Phenol for Disinfection.
Each mL of 0.05 mol/L bromine VS
= 1.569 mg of C 6 H 6
Containers and storage Containers — Tight containers.
Phenolsulfonphthalein
C 19 H 14 5 S: 354.38
2-[Bis(4-hydroxyphenyl)methyliumyl]benzenesulfonate
[143-74-8]
Phenolsulfonphthalein, when dried, contains not
less than 98.0% of C 19 H 14 O s S.
Description Phenolsulfonphthalein occurs as a vivid red to
dark red, crystalline powder.
It is very slightly soluble in water and in ethanol (95).
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 5 mg of Phenolsulfonphthalein
in 2 to 3 drops of sodium hydroxide TS, add 2 mL of 0.05
mol/L bromine VS and 1 mL of dilute sulfuric acid, shake
well, and allow to stand for 5 minutes. Render the solution
alkaline with sodium hydroxide TS: a deep blue-purple color
develops.
(2) Dissolve 0.01 g of Phenolsulfonphthalein in diluted
sodium carbonate TS (1 in 10) to make 200 mL. To 5 mL of
this solution add diluted sodium carbonate TS (1 in 10) to
make 100 mL. Perform the test with this solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
Purity (1) Insoluble substances — To about 1 g of Phenol-
sulfonphthalein, accurately weighed, add 20 mL of a solution
of sodium hydrogen carbonate (1 in 40). Allow the mixture to
stand for 1 hour with frequent shaking, dilute with water to
100 mL, and allow to stand for 24 hours. Collect the insolu-
ble substances using a tared glass filter (G4), wash with 25 mL
of a solution of sodium hydrogen carbonate (1 in 100) and
with five 5-mL portions of water, and dry at 105°C for 1
hour: the mass of the residue is not more than 0.2%.
(2) Related substances — Dissolve 0.10 g of Phenolsul-
fonphthalein in 5 mL of dilute sodium hydroxide TS, and use
this solution as the sample solution. Pipet 0.5 mL of this so-
lution, add dilute sodium hydroxide TS to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 10 /xL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate with a mixture of ?-amyl alcohol, acetic acid (100) and
water (4:1:1) to a distance of about 15 cm, and air-dry the
plate. After allowing the plate to stand in an ammonia vapor,
examine under ultraviolet light (main wavelength: 254 nm):
the spots other than the principal spot from the sample solu-
tion are not more intense than the spot from the standard so-
lution.
Loss on drying <2.41> Not more than 1.0% (1 g, silica gel,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.15 g of Phenolsul-
fonphthalein, previously dried, transfer to an iodine flask,
dissolve in 30 mL of a solution of sodium hydroxide (1 in
250), and add water to make 200 mL. Add exactly measured
50 mL of 0.05 mol/L bromine VS, add 10 mL of hydrochlor-
ic acid to the solution quickly, and stopper immediately. Al-
low the mixture to stand for 5 minutes with occasional shak-
ing, add 7 mL of potassium iodide TS, stopper again immedi-
ately, and shake gently for 1 minute. Titrate <2.50> the liber-
ated iodine with 0.1 mol/L sodium thiosulfate VS (indicator:
1 mL of starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 4.430 mg of C 19 H 14 5 S
Containers and storage Containers — Well-closed contain-
ers.
Phenolsulfonphthalein Injection
Phenolsulfonphthalein Injection is an aqueous solu-
tion for injection.
It contains not less than 0.54 w/v% and not more
than 0.63 w/v% of phenolsulfonphthalein (C 19 H 14 5 S:
354.38).
Method of preparation
Phenolsulfonphthalein
Sodium Chloride
Sodium Bicarbonate
(or Sodium Hydroxide)
Water for Injection
6g
9g
1.43 g
(0.68 g)
a sufficient quantity
To make 1000 mL
JPXV
Official Monographs / L-Phenylalanine
989
Prepare as directed under Injections, with the above in-
gredients.
Description Phenolsulfonphthalein Injection is a clear,
orange-yellow to red liquid.
Identification To 1 mL of Phenolsulfonphthalein Injection
add 2 to 3 drops of sodium hydroxide TS, and proceed as
directed in the Identification (1) under Phenolsul-
fonphthalein.
pH <2.54> 6.0 - 7.6
Extractable volume <6.05> It meets the requirement.
Sensitivity To 1.0 mL of Phenolsulfonphthalein Injection
add 5 mL of water. To 0.20 mL of this solution add 50 mL of
freshly boiled and cooled water and 0.40 mL of 0.01 mol/L
sodium hydroxide VS: a deep red-purple color develops, and
it changes to light yellow on the addition of 0.40 mL of 0.005
mol/L sulfuric acid VS.
Assay Pipet 5 mL of Phenolsulfonphthalein Injection, and
add a solution of anhydrous sodium carbonate (1 in 100) to
make exactly 250 mL. Pipet 5 mL of this solution, add a solu-
tion of anhydrous sodium carbonate (1 in 100) to make exact-
ly 200 mL, and use this solution as the sample solution.
Separately, weigh accurately about 30 mg of phenolsul-
fonphthalein for assay, previously dried in a desiccator (silica
gel) for 4 hours, and dissolve in a solution of anhydrous sodi-
um carbonate (1 in 100) to make exactly 250 mL. Pipet 5 mL
of this solution, add a solution of anhydrous sodium car-
bonate (1 in 100) to make exactly 200 mL, and use this solu-
tion as the standard solution. Determine the absorbances, A T
and A s , of the sample solution and standard solution at 559
nm as directed under Ultraviolet-visible Spectrophotometry
<2.24>.
Amount (mg) of phenolsulfonphthalein (C I9 H 14 5 S)
= W s x(A T /A s )
W s : Amount (mg) of phenolsulfonphthalein for assay
Containers and storage Containers — Hermetic containers.
CQ?H
L-Phenylalanine
l-7i-JU7^->
H NHj
C 9 H„N0 2 : 165.19
(25)-2-Amino-3-phenylpropanoic acid [63-91-2]
L-Phenylalanine, when dried, contains not less than
98.5% of C 9 H„N0 2 .
Description L-Phenylalanine occurs as white crystals or
crystalline powder. It is odorless or has a faint characteristic
odor, and has a slightly bitter taste.
It is freely soluble in formic acid, sparingly soluble in
water, and practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Phenylalanine, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Optical rotation <2.49> [a]™: -33.0
drying, 0.5 g, water, 25 mL, 100 mm).
■35.5° (after
pH <2.54> Dissolve 0.20 g of L-Phenylalanine in 20 mL of
water: the pH of this solution is between 5.3 and 6.3.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
L-Phenylalanine in 10 mL of 1 mol/L hydrochloric acid TS:
the solution is clear and colorless.
(2) Chloride <7.0?>— Perform the test with 0.5 g of L-
Phenylalanine. Prepare the control solution with 0.30 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of l-
Phenylalanine. Prepare the control solution with 0.35 mL of
0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Phenylalanine. Prepare the control solution with 5.0 mL of
Standard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Dissolve 1.0 g of L-Phenylala-
nine in 40 mL of water and 2 mL of dilute acetic acid by
warming, cool, and add water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 2 mL of dilute acetic acid and water to make 50 mL (not
more than 20 ppm).
(6) Arsenic <1.11> — Dissolve 1.0 g of L-Phenylalanine in
5 mL of dilute hydrochloric acid and 15 mL of water, and
perform the test with this solution as the test solution (not
more than 2 ppm).
(7) Related substances — Dissolve 0.10 g of L-Phenylala-
nine in 25 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, and add water to
make exactly 50 mL. Pipet 5 mL of this solution, add water
to make exactly 20 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-butanol, water and acetic acid (100) (3:1:1) to a
distance of about 10 cm, and dry the plate at 80°C for 30
minutes. Spray evenly a solution of ninhydrin in acetone (1 in
50) on the plate, and heat at 80°C for 5 minutes: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.17 g of L-Phenylalanine,
previously dried, and dissolve in 3 mL of formic acid, add 50
mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.52 mg of C 9 H„N0 2
Containers and storage Containers — Tight containers.
990
Phenylbutazone / Official Monographs
JP XV
Phenylbutazone
7i=JU7?7>
4-Butyl-l ,2-diphenylpyrazolidine-3,5-dione [50-33-9]
Phenylbutazone, when dried, contains not less than
99.0% of C 19 H 20 N 2 O 2 .
Description Phenylbutazone occurs as a white to slightly
yellowish white, crystalline powder. It is odorless, and is at
first tasteless but leaves a slightly bitter aftertaste.
It is freely soluble in acetone, soluble in ethanol (95) and in
diethyl ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Identification (1) To 0.1 g of Phenylbutazone add 1 mL
of acetic acid (100) and 1 mL of hydrochloric acid, and heat
on a water bath under a reflux condenser for 30 minutes. Add
10 mL of water, and cool with ice water. Filter, and to the
filtrate add 3 to 4 drops of sodium nitrite TS. To 1 mL of this
solution add 1 mL of 2-naphthol TS and 3 mL of chlo-
roform, and shake: a deep red color develops in the chlo-
roform layer.
(2) Dissolve 1 mg of Phenylbutazone in 10 mL of dilute
sodium hydroxide TS, and dilute with water to make 100 mL.
Determine the absorption spectrum of the solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
Melting point <2.60> 104 - 107 °C
Purity (1) Clarity of solution — Dissolve 1.0 g of Phenyl-
butazone in 20 mL of sodium hydroxide solution (2 in 25),
and allow to stand at 25 ± 1°C for 3 hours: the solution is
clear. Determine the absorbance of this solution at 420 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it is not more than 0.05.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Phenyl-
butazone according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of phenylbutazone, according to Method 3, and perform the
test (not more than 2 ppm).
(4) Readily carbonizable substances — Dissolve 1.0 g of
Phenylbutazone in 20 mL of sulfuric acid, and allow to stand
at 25 ± 1°C for exactly 30 minutes: the solution is clear. De-
termine the absorbance of this solution at 420 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it is not
more than 0.10.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Phenylbutazone,
previously dried, dissolve in 25 mL of acetone, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS until the solu-
tion shows a blue color which persists for 15 seconds (indica-
tor: 5 drops of bromothymol blue TS). Perform a blank de-
termination with a mixture of 25 mL of acetone and 16 mL of
water, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 30.84 mg of C 19 H 20 N 2 O 2
Containers and storage Containers — Tight containers.
Phenylephrine Hydrochloride
7i = l/7U>*»Ji
H OH
•HCI
C 9 H 13 N0 2 .HC1: 203.67
(1 R )- 1 -(3-Hydroxyphenyl)-2-methylaminoethanol
monohydrochloride [61-76-7]
Phenylephrine Hydrochloride, when dried, contains
not less than 98.0% and not more than 102.0% of
C 9 H 13 N0 2 .HC1.
Description Phenylephrine Hydrochloride occurs as white
crystals or crystalline powder. It is odorless, and has a bitter
taste.
It is very soluble in water, freely soluble in ethanol (95),
and practically insoluble in diethyl ether.
The pH of a solution of Phenylephrine Hydrochloride (1 in
100) is between 4.5 and 5.5.
Identification (1) To 1 mL of a solution of Phenylephrine
Hydrochloride (1 in 100) add 1 drop of copper (II) sulfate TS
and 1 mL of a solution of sodium hydroxide (1 in 5): a blue
color is produced. To the solution so obtained add 1 mL of
diethyl ether, and shake vigorously: no blue color develops in
the diethyl ether layer.
(2) To 1 mL of a solution of Phenylephrine Hydrochlo-
ride (1 in 100) add 1 drop of iron (III) chloride TS: a persis-
tent purple color is produced.
(3) Dissolve 0.3 g of Phenylephrine Hydrochloride in 3
mL of water, add 1 mL of ammonia TS, and rub the inner
side of the test tube with a glass rod: a precipitate is
produced. Collect the precipitate, wash with a few drops of
ice-cold water, and dry at 105°C for 2 hours: it melts <2.60>
between 170°C and 177°C.
(4) A solution of Phenylephrine Hydrochloride (1 in 100)
responds to Qualitative Tests <1.09> (2) for chloride.
Optical rotation <2.49> [«]£>: -42.0- -47.5° (after
drying, 0.5 g, water, 10 mL, 100 mm).
Melting point <2.60> 140 - 145 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
JPXV
Official Monographs / Phenytoin
991
Phenylephrine Hydrochloride in 10 mL of water: the solution
is clear and colorless.
(2) Sulfate <1.I4>— Take 0.5 g of Phenylephrine
Hydrochloride, and perform the test. Prepare the control so-
lution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not
more than 0.048%).
(3) Ketone — Dissolve 0.20 g of Phenylephrine
Hydrochloride in 1 mL of water, and add 2 drops of sodium
pentacyanonitrosylferrate (III) TS, 1 mL of sodium
hydroxide TS and then 0.6 mL of acetic acid (100): the solu-
tion has no more color than the following control solution.
Control solution: Prepare as directed above without
Phenylephrine Hydrochloride.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.1 g of Phenylephrine
Hydrochloride, previously dried, dissolve in 40 mL of water
contained in an iodine flask, add exactly measured 50 mL of
0.05 mol/L bromine VS, then add 5 mL of hydrochloric acid,
and immediately stopper tightly. Shake the mixture, and al-
low to stand for 15 minutes. To this solution add 10 mL of
potassium iodide TS carefully, stopper tightly immediately,
shake thoroughly, allow to stand for 5 minutes, and titrate
<2.50> with 0.1 mol/L sodium thiosulfate VS (indicator: 1
mL of starch TS). Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 3.395 mg of C 9 H 13 N0 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Phenytoin
Diphenylhydantoin
7i- M >
C 15 H 12 N 2 2 : 252.27
5,5-Diphenylimidazolidine-2,4-dione
[57-41-0]
Phenytoin, when dried, contains not less than 99.0%
of C 15 H 12 N 2 2 .
Description Phenytoin occurs as a white, crystalline powder
or granules. It is odorless and tasteless.
It is sparingly soluble in ethanol (95) and in acetone, slight-
ly soluble in diethyl ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Melting point: about 296°C (with decomposition).
Identification (1) Dissolve 0.02 g of Phenytoin in 2 mL of
ammonia TS, and add 5 mL of silver nitrate TS: a white
precipitate is produced.
(2) Boil a mixture of 0.01 g of Phenytoin, 1 mL of am-
monia TS and 1 mL of water, and add dropwise 2 mL of a
mixture prepared from 50 mL of a solution of copper (II) sul-
fate pentahydrate (1 in 20) and 10 mL of ammonia TS: a red,
crystalline precipitate is produced.
(3) Heat 0.1 g of Phenytoin with 0.2 g of sodium
hydroxide, and fuse: the gas evolved turns moistened red lit-
mus paper blue.
(4) Add 3mL of chlorinated lime TS to 0.1 g of
phenytoin, shake for 5 minutes, and dissolve the oily
precipitate in 15 mL of hot water. After cooling, add 1 mL of
dilute hydrochloric acid dropwise, then add 4 mL of water.
Filter the white precipitate thus obtained, wash with water,
and press it with dry filter paper to remove the accompanying
water. Dissolve the precipitate with 1 mL of chloroform, add
5 mL of diluted ethanol (9 in 10), and rub the inner surface of
the flask to produce a white, crystalline precipitate. Collect
the precipitate, wash with ethanol (95), and dry: the melting
point <2.60> is between 165 °C and 169°C.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Phenytoin in 10 mL of 0.2 mol/L sodium hydroxide VS:
the solution is clear and colorless. Then heat the solution: no
turbidity is produced. Cool, and mix the solution with 5 mL
of acetone: the solution is clear and colorless.
(2) Acidity or alkalinity — Shake 2.0 g of phenytoin with
40 mL of water for 1 minute, filter, and perform the follow-
ing tests using this filtrate as the sample solution.
(i) To 10 mL of the sample solution add 2 drops of
phenolphthalein TS: no color develops. Then add 0.15 mL of
0.01 mol/L sodium hydroxide VS: a red color develops.
(ii) To 10 mL of the sample solution add 0.30 mL of 0.01
mol/L hydrochloric acid VS and 5 drops of methyl red TS: a
red to orange color develops.
(3) Chloride <1.03>— Dissolve 0.30 g of Phenytoin in 30
mL of acetone, and add 6 mL of dilute nitric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution from 30 mL of ace-
tone, 0.60 mL of 0.01 mol/L hydrochloric acid VS and 6 mL
of dilute nitric acid, and add water to 50 mL (not more than
0.071%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Phenytoin according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Phenytoin, previ-
ously dried, dissolve in 40 mL of ethanol (95) with the aid of
gentle heating, add 0.5 mL of thymolphthalein TS immedi-
ately, and titrate with 0.1 mol/L sodium hydroxide VS until a
light blue color develops. Then add 1 mL of pyridine, 5 drops
of phenolphthalein TS and 25 mL of silver nitrate TS, and ti-
trate <2.50> with 0.1 mol/L sodium hydroxide VS until a light
red color, which persists for 1 minute, develops.
Each mL of 0.1 mol/L sodium hydroxide VS
= 25.23 mg of C 15 H 12 N 2 2
Containers and storage Containers — Well-closed contain-
ers.
992
Phenytoin Powder / Official Monographs
JP XV
Phenytoin Powder
Diphenylhydantoin Powder
Phenytoin Powder contains not less than 95% and
not more than 105% of the labeled amount of
phenytoin (C 15 H 12 N 2 2 : 252.27).
Method of preparation
with Phenytoin.
Prepare as directed under Powders,
Identification Weigh a portion of Phenytoin Powder,
equivalent to 0.3 g of Phenytoin according to the labeled
amount, stir well with two 100-mL portions of diethyl ether,
and extract. Combine the diethyl ether extracts, and filter.
Evaporate the filtrate on a water bath to dryness, and proceed
with the residue as directed in the Identification under
Phenytoin.
Assay Weigh accurately Phenytoin Powder, equivalent to
about 0.5 g of phenytoin (QsH^NjC^), add exactly 100 mL
of ethanol (95), stir for 30 minutes, and centrifuge. Pipet 50
mL of the supernatant liquid, add 0.5 mL of
thymolphthalein TS, titrate with 0.1 mol/L sodium
hydroxide VS until a light blue color develops, then add 1 mL
of pyridine, 5 drops of phenolphthalein TS and 12.5 mL of
silver nitrate TS, and titrate <2.50> with 0.1 mol/L sodium
hydroxide VS until a light red color persists for 1 minute.
Each mL of 0.1 mol/L sodium hydroxide VS
= 25.23 mg of C 15 H, 2 N 2 2
Containers and storage Containers — Well-closed contain-
ers.
Phenytoin Tablets
Diphenylhydantoin Tablets
Phenytoin Tablets contain not less than 95% and not
more than 105% of the labeled amount of phenytoin
(C 15 H 12 N 2 2 : 252.27).
Method of preparation
with Phenytoin.
Prepare as directed under Tablets,
Identification Proceed with the residue obtained in the
Assay as directed in the Identification under Phenytoin.
Assay Weigh accurately and powder not less than 20
Phenytoin Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 0.3 g of phenytoin (C 15 H 12 N 2 2 ),
transfer to a separator, and add 1 mL of dilute hydrochloric
acid and 10 mL of water. Extract with 100 mL of diethyl
ether, then with four 25-mL portions of diethyl ether. Com-
bine the extracts, and evaporate the diethyl ether. Dry the
residue at 105 °C for 2 hours, and weigh it as the mass of
phenytoin (C 15 H 12 N 2 2 ).
Containers and storage Containers — Well-closed contain-
ers.
Phenytoin Sodium for Injection
Diphenylhydantoin Sodium for Injection
ONa
C 15 H u N 2 Na0 2 : 274.25
Monosodium 5 ,5-diphenyl-4-oxoimidazolidin-2-olate
[630-93-3]
Phenytoin Sodium for Injection is a preparation for
injection which is dissolved before use.
When dried, it contains not less than 98.5% of
phenytoin sodium (C 15 H 11 N 2 Na0 2 ), and contains not
less than 92.5% and not more than 107.5% of the la-
beled amount of phenytoin sodium (C 15 H n N 2 Na0 2 ).
Method of preparation
tions.
Prepare as directed under Injec-
Description Phenytoin Sodium for Injection occurs as
white crystals or crystalline powder. It is odorless.
It is soluble in water and in ethanol (95), and practically in-
soluble in chloroform and in diethyl ether.
The pH of a solution of Phenytoin Sodium for Injection (1
in 20) is about 12.
It is hygroscopic.
A solution of Phenytoin Sodium for Injection absorbs car-
bon dioxide gradually when exposed to air, and a crystalline
precipitate of phenytoin is produced.
Identification (1) With the residue obtained in the Assay,
proceed as directed in the Identification under Phenytoin.
(2) Ignite 0.5 g of Phenytoin Sodium for Injection, cool,
and dissolve the residue in 10 mL of water: the solution
changes red litmus paper to blue, and responds to Qualitative
Tests <1.09> (1) for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Phenytoin Sodium for Injection in 20 mL of freshly boiled
and cooled water in a glass-stoppered test tube: the solution is
clear and colorless. If any turbidity is produced, add 4.0 mL
of 0.1 mol/L sodium hydroxide VS: the solution becomes
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Phenytoin Sodium for Injection according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
Loss on drying <2.41>
4 hours).
Not more than 2.5% (1 g, 105 °C,
Assay Weigh accurately the content of not less than 10
preparations of Phenytoin Sodium for Injection, transfer
about 0.3 g of the content, previously dried and accurately
weighed, to a separator, dissolve in 50 mL of water, add 10
JPXV
Official Monographs / Phytonadione
993
mL of dilute hydrochloric acid, and extract with 100 mL of
diethyl ether, then with four 25-mL portions of diethyl ether.
Combine the diethyl ether extracts, and evaporate on a water
bath. Dry the residue at 105°C for 2 hours, and weigh it as
the mass of phenytoin (C 15 H I2 N 2 02: 252.27).
Amount (mg) of phenytoin sodium (Ci 5 H u N 2 Na02)
= amount (mg) of phenytoin (C 15 H 12 N 2 2 ) X 1.0871
Containers and storage Containers — Hermetic containers.
Phytonadione
Phytomenadione
Vitamin Ki
H CHg H CHg
C 3 iH 46 2 : 450.70
2-Methyl-3-[(2£ , ,7/?, 1 1 R )-3 ,7, 1 1 , 1 5-tetramethylhexadec-
2-en- 1 -yl] - 1 ,4-naphthoquinone [84-80-0]
Phytonadione contains not less than 97.0% and not
more than 102.0% of C 31 H 46 2 .
Description Phytonadione is a clear yellow to orange-yel-
low, viscous liquid.
It is miscible with isooctane.
It is soluble in ethanol (99.5), and practically insoluble in
water.
It decomposes gradually and changes to a red-brown by
light.
Specific gravity rf 2 „: about 0.967
Identification (1) Determine the absorption spectrum of a
solution of Phytonadione in isooctane (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum 1:
both spectra exhibit similar intensities of absorption at the
same wavelengths. Separately, determine the absorption
spectrum of a solution of Phytonadione in isooctane (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum 2: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Phytonadione as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Refractive index <2.45> n™\ 1.525 - 1.529
Purity (1) Ratio of absorbances — Determine the absor-
bances, A lt A 2 and A } , of a solution of Phytonadione in
isooctane (1 in 100,000) at 248.5 nm, 253.5 nm and 269.5 nm,
respectively, as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: the ratio A 2 /A\ is between 0.69 and
0.73, and the ratio A 2 /A 3 is between 0.74 and 0.78. Deter-
mine the absorbances, A 4 and A s , of a solution of
Phytonadione in isooctane (1 in 10,000) at 284.5 nm and 326
nm, respectively: the ratio A 4 /A 5 is between 0.28 and 0.34.
(2) Heavy metals < 1.07 > — Carbonize 1.0 g of
Phytonadione by gentle heating. Cool, add 10 mL of a solu-
tion of magnesium nitrate hexahydrate in ethanol (95) (1 in
10), and ignite the ethanol to burn. Cool, add 1 mL of sulfur-
ic acid, proceed according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Menadione — Dissolve 20 mg of Phytonadione in 0.5
mL of a mixture of water and ethanol (95) (1:1), add 1 drop
of a solution of 3-methyl-l-phenyl-5-pyrazolone in ethanol
(95) (1 in 20) and 1 drop of ammonia solution (28), and allow
to stand for 2 hours: no blue-purple color develops.
Isomer ratio Conduct this procedure rapidly and without
exposure to light. Dissolve 30 mg of Phytonadione in 50 mL
of the mobile phase. To 4 mL of this solution add the mobile
phase to make 25 mL. To 10 mL of this solution add the mo-
bile phase to make 25 mL, and use this solution as the sample
solution. Perform the test with 50 iiL of the sample solution
as directed under Liquid Chromatograph <2.01> according to
the following conditions, and determine the peak areas of Z-
isomer and E-isomer, A TZ and A TE : At Z /(A jz +At E ) is be-
tween 0.05 and 0.18.
Operating conditions —
Proceed as directed in the operating conditions in the As-
say.
System suitability —
System performance: When the procedure is run with 50
iiL of the sample solution under the above operating condi-
tions, Z-isomer and is-isomer are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
50 /uL of the sample solution under the above operating con-
ditions, the relative standard deviation of the total area of the
peaks of Z-isomer and ii-isomer is not more than 2.0%.
Assay Conduct this procedure rapidly and without ex-
posure to light. Weigh accurately about 30 mg each of
Phytonadione and Phytonadione Reference Standard, and
dissolve each in the mobile phase to make exactly 50 mL.
Pipet 4 mL each of these solutions, and add the mobile phase
to make exactly 25 mL. To exactly 10 mL each of these solu-
tion add exactly 7 mL of the internal standard solution and
the mobile phase to make 25 mL, and use these as the sample
solution and standard solution, respectively. Perform the test
with 50 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the ratios, Qj and
Q s , of the total area of the peaks of Z-isomer and .E-isomer to
the peak area of the internal standard.
Amount (mg) of C 3 ,H 46 2 = W s x(Q T /Q s )
W s : Amount (mg) of Phytonadione Reference Standard
Internal standard solution — A solution of cholesterol benzo-
ate in the mobile phase (1 in 400).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with porous silica gel for
994
Pilocarpine Hydrochloride / Official Monographs
JP XV
liquid chromatography (5 /urn in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of hexane and n-amyl alcohol
(4000 : 3).
Flow rate: Adjust the flow rate so that the retention time of
the peak of Zs-isomer of phytonadione is about 25 minutes.
System suitability-
System performance: When the procedure is run with 50
/uL of the standard solution under the above operating condi-
tions, the internal standard, Z-isomer and is-isomer are elut-
ed in this order with the resolution between the peaks of Z-
isomer and Zs-isomer being not less than 1.5.
System repeatability: When the test is repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
total area of the peaks of Z-isomer and ii-isomer to the peak
area of the internal standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at a cold place.
Pilocarpine Hydrochloride
¥nt>)\,\d>i%mt&
C„H 16 N 2 2 .HC1: 244.72
(3 S,4R )-3-Ethyl-4-(l-methyl-l i/-imidazol-5-ylmethyl)-
4,5-dihydrofuran-2(3//)-one monohydrochloride
[54-71-7]
Pilocarpine Hydrochloride, when dried,
not less than 99.0% of C„H 16 N 2 2 .HC1.
contains
Description Pilocarpine Hydrochloride occurs as colorless
crystals or white powder. It is odorless, and has a slightly bit-
ter taste.
It is very soluble in acetic acid (100), freely soluble in
water, in methanol and in ethanol (95), soluble in acetic an-
hydride, and practically insoluble in diethyl ether.
The pH of a solution of Pilocarpine Hydrochloride (1 in
10) is between 3.5 and 4.5.
It is hygroscopic.
It is affected by light.
Identification (1) Dissolve 0.1 g of Pilocarpine
Hydrochloride in 5 mL of water, add 1 drop of dilute nitric
acid, 1 mL of hydrogen peroxide TS, 1 mL of chloroform
and 1 drop of a potassium dichromate solution (1 in 300),
and shake the mixture vigorously: a violet color develops in
the chloroform layer while no color or a light yellow color is
produced in the aqueous layer.
(2) To 1 mL of a solution of Pilocarpine Hydrochloride
(1 in 20) add 1 mL of dilute nitric acid and 2 to 3 drops of sil-
ver nitrate TS: a white precipitate or opalescence is produced.
Melting point <2.60> 200 - 203 °C
Purity (1) Sulfate — Dissolve 0.5 g of Pilocarpine
Hydrochloride in 20 mL of water, and use this solution as the
sample solution. To 5.0 mL of the sample solution add 1 mL
of dilute hydrochloric acid and 0.5 mL of barium chloride
TS: no turbidity is produced.
(2) Nitrate — To 2.0 mL of the sample solution obtained
in (1) add 2 mL of iron (II) sulfate TS, and superimpose the
mixture upon 4 mL of sulfuric acid: no dark brown color de-
velops at the zone of contact.
(3) Related substances — Dissolve 0.3 g of Pilocarpine
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, methanol and ammonia
TS (85:14:2) to a distance of about 13 cm, and dry the plate at
105°C for 10 minutes. Cool, and spray evenly bismuth potas-
sium iodide TS on the plate: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
(4) Readily carbonizable substances <].15> — Take 0.25 g
of Pilocarpine Hydrochloride, and perform the test: the solu-
tion has no more color than Matching Fluid B.
Loss on drying <2.41> Not more than 3.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.5% (0.1 g).
Assay Weigh accurately about 0.5 g of Pilocarpine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.47 mg of C„H 16 N 2 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Pimaricin
Natamycin
tfvU->>
H OH
H H
C33H 47 N0 13 : 665.73
(IR*,3S*,5R*,1R*,8EA2R*,14E,16E,18E,20E,22R*,
24S *,25R *,26S *)-22-(3-Amino-3 ,6-dideoxy-/?-D-
mannopyranosyloxy)- 1 ,3 ,26-trihydroxy- 1 2-methyl- 1 0-oxo-
6,ll,28-trioxatricyclo[22.3.1.0 5 . 7 ]octacosa-8,14,16,18,20-
JPXV
Official Monographs / Pindolol
995
pentaene-25-carboxylic acid [7681-93-8]
Pimaricin is a polyene macrolide substance having
antifungal activity produced by the growth of Strep-
tomyces natalensis.
It contains not less than 900 fig (potency) and not
more than 1020 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Pimaricin is expressed
as mass (potency) of pimaricin (C33H47NO13).
Description Pimaricin occurs as white to yellowish white
crystalline powder.
It is slightly soluble in methanol and in acetic acid (100),
and practically insoluble in water and in ethanol (99.5).
Identification (1) To 3 mg of Pimaricin add 1 mL of
hydrochloric acid, and mix: a blue-purple color appears.
(2) Dissolve 5 mg of Pimaricin in a solution of acetic acid
(100) in methanol (1 in 100) to make 1000 mL. Determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Pimaricin Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Optical rotation <2.49> [a]g>: +243 - +259° (0.1 g, acetic
acid (100), 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Pimaricin according to Method 4, and perform the test. Pre-
pare the control solution with 3 .0 mL of Standard Lead Solu-
tion (not more than 30 ppm).
(2) Related substances — Dissolve 20 mg of Pimaricin in
methanol to make 100 mL, and use this solution as the sam-
ple solution. Perform the test with 10 fiL of the sample solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the total
area of the peaks other than pimaricin by the automatic in-
tegration method: not more than 4.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 303 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.0 g of ammonium acetate in 1000
mL of a mixture of water, methanol and tetrahydrofuran
(47:44:2).
Flow rate: Adjust the flow rate so that the retention time of
pimaricin is about 10 minutes.
Time span of measurement: About 3 times as long as the
retention time of pimaricin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, add methanol to make exactly 100 mL,
and use this solution as the solution for system suitability
test. Pipet 1 mL of the solution for system suitability test,
and add methanol to make exactly 10 mL. Confirm that the
peak area of pimaricin obtained from 10 ^L of this solution
is equivalent to 7 to 13% of that from 10 fiL of the solution
for system suitability test.
System performance: When the procedure is run with
10 fiL of the solution for system suitability test under the
above operating conditions, the number of theoretical plates
and the symmetry factor of the peak of pimaricin are not less
than 1500 and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of pimaricin is not more than 2.0%.
Water <2.48> Between 6.0% and 9.0% (0.2 g, volumetric
titration, direct titration).
Assay Weigh accurately an amount of Pimaricin and
Pimaricin Reference Standard, equivalent to about 25 mg
(potency), and dissolve each in methanol to make exactly 100
mL. Pipet 2 mL each of these solution, add a solution of
acetic acid (100) in methanol (1 in 100) to make exactly 100
mL, and use these solutions as the sample solution and stan-
dard solution. Determine the absorbances at 295.5 nm, A Tl
and^4 sl , at 303 nm, A T2 andA S2 , and at 311 nm, A T3 and/4 S3 ,
of the sample solution and standard solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount [fig (potency)] of C33H47NO13
A-T7
We X
A„ -
A s , + A
x 1000
si ~ -«S3
W s : Amount [mg (potency)] of Pimaricin Reference
Standard
Containers and storage Containers — Tight containers.
Storage — Light resistant.
Pindolol
b°> KP-JU
H OH
C 14 H 20 N 2 O 2 : 248.32
(2i?S>l-(l//-Indol-4-yloxy)-
3-(l-methylethyl)aminopropan-2-ol
and enantiomer
[13523-86-9]
Pindolol, when dried, contains not less than 98.5%
of C 14 H 20 N 2 O 2 .
Description Pindolol occurs as a white, crystalline powder.
It has a slight, characteristic odor.
It is sparingly soluble in methanol, slightly soluble in
ethanol (95), and practically insoluble in water and in diethyl
ether.
It dissolves in dilute sulfuric acid and in acetic acid (100).
Identification (1) To 1 mL of a solution of Pindolol in
methanol (1 in 10,000) add 1 mL of a solution of l-(4-
pyridyl)-pyridinium chloride hydrochloride (1 in 1000) and 1
mL of sodium hydroxide TS, then add 1 mL of hydrochloric
acid: a blue to blue-purple color, changing to red-purple, is
produced.
996
Pipemidic Acid Hydrate / Official Monographs
JP XV
(2) Dissolve 0.05 g of Pindolol in 1 mL of dilute sulfuric
acid, and add 1 mL of Reinecke salt TS: a light red
precipitate is produced.
(3) Determine the absorption spectrum of a solution of
Pindolol in methanol (1 in 50,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectrum of Pin-
dolol, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Absorbance <2.24> E\ 0/ ° m (264 nm): 333 - 350 (10 mg,
methanol, 500 mL).
Melting point <2.60> 169 - 173 °C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Pindolol in 10 mL of acetic acid (100), and observe immedi-
ately: the solution is clear, and has no more color than the
following control solution.
Control solution: Measure accurately 4 mL of Matching
Fluid A, add exactly 6 mL of water, and mix.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Pindolol
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pindolol according to Method 3, and perform the test (not
more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Pindolol in 10
mL of methanol, and use this solution as the sample solution.
Pipet 2 mL of the sample solution, and add methanol to
make exactly 100 mL. Pipet 5 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of chloroform, acetone and isopropylamine
(5:4:1) to a distance of about 12 cm, and air-dry the plate.
Spray evenly diluted sulfuric acid (3 in 5) and a sodium nitrite
solution (1 in 50) on the plate: the spots other than the prin-
cipal spot from the sample solution are not more intense than
the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Pindolol, previously
dried, dissolve in 80 mL of methanol, and titrate <2.50> with
0.1 mol/L hydrochloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L hydrochloric acid VS
= 24.83 mg of C 14 H 20 N 2 O 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Pipemidic Acid Hydrate
b°<s KBTkftgg
HOjC
CH 3 ^
N N N
• 3H 2
C 14 H 17 N 5 3 .3H 2 0: 357.36
8-Ethyl-5-oxo-2-(piperazin-l-yl)-
5,8-dihydropyrido[2,3-rf]pyrimidine-
6-carboxylic acid trihydrate
[51940-44-4, anhydride]
Pipemidic Acid Hydrate, when dried, contains not
less than 98.5% and not more than 101.0% of
pipemidic acid (Ci4H 17 N 5 3 : 303.32).
Description Pipemidic Acid Hydrate occurs as a pale yel-
low, crystalline powder.
It is freely soluble in acetic acid (100), very slightly soluble
in water and in ethanol (99.5).
It dissolves in sodium hydroxide TS.
It is gradually colored by light.
Melting point: about 250°C (with decomposition).
Identification (1) Dissolve 0.1 g of Pipemidic Acid Hy-
drate in 20 mL of sodium hydroxide TS, and dilute with
water to make 200 mL. To 1 mL of the solution add water to
make 100 mL. Determine the absorption spectrum of the
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Pipemidic Acid Hydrate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Purity (1) Chloride <1.03> — Dissolve 1.0 g of Pipemidic
Acid Hydrate in 35 mL of water and 10 mL of sodium
hydroxide TS, shake well with 15 mL of dilute nitric acid,
and filter through a glass filter (G3). To 30 mL of the filtrate
add 6 mL of dilute nitric acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 0.30 mL of 0.01 mol/L
hydrochloric acid VS add 5 mL of sodium hydroxide TS,
13.5 mL of dilute nitric acid and water to make 50 mL (not
more than 0.021%).
(2) Sulfate <1.14>— Dissolve 1.0 g of Pipemidic Acid Hy-
drate in 35 mL of water and 10 mL of sodium hydroxide TS,
shake well with 15 mL of dilute hydrochloric acid, and filter
through a glass filter (G3). To 30 mL of the filtrate add water
to make 50 mL. Perform the test using this solution as the
test solution. Prepare the control solution as follows: to 0.50
mL of 0.005 mol/L sulfuric acid VS add 5 mL of sodium
hydroxide TS, 7.5 mL of dilute hydrochloric acid and water
to make 50 mL (not more than 0.048%).
JPXV
Official Monographs / Piperacillin Sodium 997
(3) Heavy metals <1.07> — Proceed with 2.0 g of Pipemid-
ic Acid Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pipemidic Acid Hydrate according to Method 3, and per-
form the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Pipemidic
Acid Hydrate in 10 mL of diluted acetic acid (100) (1 in 20),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add diluted acetic acid (100) (1 in 20) to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform, methanol,
formic acid and triethylamine (25:15:5:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.4I> 14.5 - 16.0% (1 g, 105 °C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Pipemidic Acid Hy-
drate, previously dried, dissolve in 40 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.33 mg of C 14 H 17 N 5 3
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Piperacillin Sodium
CH 3
C 23 H 26 N 5 Na0 7 S: 539.54
Monosodium (2S,5R,6R)-6- {(2i?)-2-[(4-ethyl-2,3-
dioxopiperazine-l-carbonyl)amino]-2-phenylacetylamino}-
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-
carboxylate [59703-84-3]
Piperacillin Sodium contains not less than 863 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Piperacillin Sodium is expressed as
mass (potency) of piperacillin (C23H27N5O7S: 517.55).
Description Piperacillin Sodium occurs as a white powder
or mass.
It is very soluble in water, freely soluble in methanol and in
ethanol (95), and practically insoluble in acetonitrile.
Identification (1) Determine the infrared absorption spec-
trum of Piperacillin Sodium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) Piperacillin Sodium responds to Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [ a ]™: + 175 - + 190° (0.8 g calculated
on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Piperacillin Sodium in 4 mL of
water: the pH of the solution is between 5.0 and 7.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Piperacillin Sodium in 10 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Piper-
acillin Sodium according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Piperacillin Sodium according to Method 4, and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve 0.1 g of Piperacillin So-
dium in 50 mL of the mobile phase A, and use this solution as
the sample solution. Pipet 1 mL of this solution, add the mo-
bile phase A to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 20 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the areas of each peak by the
automatic integration method: the area of the peak of am-
picillin appeared at the retention time of about 7 minutes
from the sample solution is not larger than 1/2 of that of
piperacillin from the standard solution, the total area of
related compounds 1 appeared at the retention times of about
17 minutes and about 21 minutes is not larger than 2 times of
the peak area of piperacillin from the standard solution, the
peak area of related compound 2 appeared at the retention
time of about 56 minutes is not larger than that of piperacillin
from the standard solution, and the total area of the peaks
other than piperacillin is not larger than 5 times of the peak
area of piperacillin from the standard solution. The peak
areas of ampicillin, related compounds 1 and related com-
pound 2 are used after multiplying by their relative response
factor, 1.39, 1.32 and 1.11, respectively.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of water, acetonitrile and 0.2
mol/L potassium dihydrogenphosphate (45:4:1).
Mobile phase B: A mixture of acetonitrile, water and 0.2
mol/L potassium dihydrogenphosphate (25:24:1).
Flowing of the mobile phase: Control the gradient by mix-
998
Piperacillin Sodium for Injection / Official Monographs
JP XV
ing the mobile phases A and B as directed in the following
table.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0- 7
7-13
13-41
41 -56
56-60
100
100^83 0^17
83 17
83^20 17^80
20 80
Flow rate: 1.0 mL per minute. The retention time of piper-
acillin is about 33 minutes.
Time span of measurement: About 1.8 times as long as the
retention time of piperacillin beginning after the solvent
peak.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of piperacillin are not less than 1 5 ,000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 3 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of piperacillin is not more than 2.0%.
Water <2.48> Not more than 1.0% (3 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Piperacillin Sodium,
equivalent to about 0.1 g (potency), and dissolve in water to
make exactly 100 mL. To exactly 5 mL of this solution add
exactly 5 mL of the internal standard solution, and use this
solution as the sample solution. Separately, weigh accurately
an amount of Piperacillin Reference Standard, equivalent to
about 0.1 g (potency), and dissolve in the mobile phase to
make exactly 100 mL. Pipet 5 mL of this solution, add ex-
actly 5 mL of the internal standard solution, and use this so-
lution as the standard solution. Perform the test with 5 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and calculate the ratios, Q T and Q s , of the
peak height of piperacillin to that of the internal standard.
Amount \jug (potency)] of piperacillin (C23H27N5O7S)
= ^ s x(e T /es)xiooo
W s : Amount [mg (potency)] of Piperacillin Reference
Standard
Internal standard solution — A solution of acetanilide in the
mobile phase (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 60.1 g of acetic acid (100) and 101.0 g of
triethylamine add water to make exactly 1000 mL. To 25 mL
of this solution add 25 mL of dilute acetic acid and 210 mL of
acetonitrile, and add water to make exactly 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
piperacillin is about 5 minutes.
System suitability —
System performance: When the procedure is run with 5 ftL
of the standard solution under the above operating condi-
tions, the internal standard and piperacillin are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak height of piperacillin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Piperacillin Sodium for Injection
Piperacillin Sodium for Injection is a preparation
for injection which is dissolved before use.
It contains not less than 93.0% and not more than
107.0% of the labeled amount of piperacillin
(C 23 H 27 N 5 7 S: 517.55).
Method of preparation Prepare as directed under Injec-
tions, with Piperacillin Sodium.
Description Piperacillin Sodium for Injection is a white
powder or masses.
Identification Proceed as directed in the Identification un-
der Piperacillin Sodium.
pH <2.54> The pH of a solution prepared by dissolving an
amount of Piperacillin Sodium for Injection, equivalent to
1.0 g (potency) of Piperacillin Sodium, in 4 mL of water is
5.0-7.0.
Purity (1) Clarity and color of solution — Dissolve an
amount of Piperacillin Sodium for Injection, equivalent to
4.0 g (potency) of Piperacillin Sodium according to the la-
beled amount, in 17 mL of water: the solution is clear and
colorless.
(2) Related substances — Proceed as directed in the Purity
(4) under Piperacillin Sodium.
Water <2.48> Not more than 1.0% (3 g, volumetric titra-
tion, direct titration).
Bacterial endotoxins <4.01> Less than 0.04 EU/mg (poten-
cy).
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
JPXV
Official Monographs / Piperazine Phosphate Hydrate
999
filtration method: it meets the requirement.
Assay Weigh accurately the mass of the contents of not less
than 10 Piperacillin Sodium for Injection. Weigh accurately
an amount of the contents, equivalent to about 20 mg (poten-
cy) of Piperacillin Sodium according to the labeled amount,
dissolve in water to make exactly 20 mL. Pipet 5 mL of this
solution, add exactly 5 mL of the internal standard solution,
and use this solution as the sample solution. Separately,
weigh accurately about 20 mg (potency) of Piperacillin Refer-
ence Standard, and dissolve in the mobile phase to make ex-
actly 20 mL. Pipet 5 mL of this solution, add exactly 5 mL of
the internal standard solution, and use this solution as the
standard solution. Proceed as directed in the Assay under
Piperacillin Sodium.
Amount [mg (potency)] of piperacillin (C23H27N5O7S)
= W s x(Qt/Q s )
W s : Amount [mg (potency)] of Piperacillin Reference
Standard
Internal standard solution-
mobile phase (1 in 5000).
-A solution of acetonitrile in the
Containers and storage Containers — Hermetic containers.
Polyethylene or polypropylene containers for aqueous injec-
tions may be used.
20) is between 5.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Piperazine Adipate in 30 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Pipera-
zine Adipate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
Loss on drying <2.41>
4 hours).
Not more than 0.5% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Piperazine Adipate,
previously dried, dissolve in a mixture of 20 mL of acetic acid
for nonaqueous titration and 40 mL of acetone for nonaque-
ous titration, and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the red-purple color of the solution changes to
blue-purple (indicator: 6 drops of bromocresol green-methyl-
rosaniline chloride TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 11.61 mg of C4H 10 N 2 .C 6 H 10 O4
Containers and storage Containers — Well-closed contain-
ers.
Piperazine Adipate
Piperazine Phosphate Hydrate
o
HO;C'
C 4 H 10 N 2 .C 6 H 10 O 4 : 232.28
Piperazine hexanedioate [142-88-1]
Piperazine Adipate, when dried, contains not less
than 98.5% of C 4 H 10 N 2 .C 6 H 10 O4.
Description Piperazine Adipate occurs as a white, crystal-
line powder. It is odorless, and has a slightly acid taste.
It is soluble in water and in acetic acid (100), and practical-
ly insoluble in ethanol (95), in acetone and in diethyl ether.
Melting point: about 250°C (with decomposition).
Identification (1) Dissolve 0.5 g of Piperazine Adipate in
10 mL of water, add 1 mL of hydrochloric acid, and extract
with two 20-mL portions of diethyl ether. Combine the
diethyl ether extracts, evaporate to dryness on a water bath,
and dry the residue at 105°C for 1 hour: the melting point
<2.60> is between 152°C and 155°C.
(2) To 3 mL of a solution of Piperazine Adipate (1 in 1 00)
add 3 drops of Reinecke salt TS: a light red precipitate is
formed.
(3) Determine the infrared absorption spectrum of Piper-
azine Adipate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> The pH of a solution of Piperazine Adipate (1 in
o
• H 3 PO, * H 2
C 4 H 10 N 2 .H 3 PO 4 .H 2 O: 202.15
Piperazine monophosphate monohydrate
[18534-18-4]
Piperazine Phosphate Hydrate contains not less than
98.5% of piperazine phosphate (C 4 H 10 N2.H3PO 4 :
184.13), calculated on the anhydrous basis.
Description Piperazine Phosphate Hydrate occurs as white
crystals or crystalline powder. It is odorless, and has a slight-
ly acid taste.
It is soluble in formic acid, sparingly soluble in water, very
slightly soluble in acetic acid (100), and practically insoluble
in methanol, in ethanol (95) and in diethyl ether.
It dissolves in dilute hydrochloric acid.
Melting point: about 222°C (with decomposition).
Identification (1) To 3 mL of a solution of Piperazine
Phosphate Hydrate (1 in 100) add 3 drops of Reinecke salt
TS:a light red precipitate is formed.
(2) Determine the infrared absorption spectrum of Piper-
azine Phosphate Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Piperazine Phosphate Hydrate (1 in
100) responds to Qualitative Tests <1.09> (1) and (3) for phos-
1000 Piperazine Phosphate Tablets / Official Monographs
JP XV
phate.
pH <2.54> Dissolve 1.0 g of Piperazine Phosphate Hydrate
in 100 mL of water: the pH of the solution is between 6.0 and
6.5.
Purity (1) Chloride <1.03> — To 0.5 g of Piperazine Phos-
phate Hydrate add 6 mL of dilute nitric acid and water to
make 50 mL. Use this solution as the test solution, and per-
form the test. Prepare the control solution with 0.25 mL of
0.01 mol/L hydrochloric acid (not more than 0.018%).
(2) Heavy metals <1.07> — To 2.0 g of Piperazine Phos-
phate Hydrate add 5 mL of dilute hydrochloric acid, 30 mL
of water and 2 mL of dilute acetic acid, and dissolve. Add so-
dium hydroxide TS, adjust the pH of the solution to 3.3, and
add water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Dissolve 2.0 g of Piperazine Phos-
phate Hydrate in 5 mL of dilute hydrochloric acid, and use
this solution as the test solution. Perform the test (not more
than 1 ppm).
(4) Related substances — Dissolve 50 mg of Piperazine
Phosphate Hydrate in 10 mL of water, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
/uL each of the sample solution and standard solution on a
plate of cellulose for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, acetone, ammonia solu-
tion (28) and ethanol (99.5) (8:3:3:2) to a distance of about 13
cm, and air-dry the plate. Spray evenly 4-dimethylaminocin-
namaldehyde TS, and allow to stand for 15 minutes: the
spots other than the principal spot and the spot on the start-
ing line from the sample solution are not more intense than
the spot from the standard solution.
Water <2..
8.0 - 9.5% (0.3 g, direct titration).
Assay Weigh accurately about 0.15 g of Piperazine Phos-
phate Hydrate, dissolve in 10 mL of formic acid, add 60 mL
of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 9.207 mg of C 4 H 10 N 2 .H 3 PO 4
Containers and storage Containers — Well-closed contain-
ers.
Piperazine Phosphate Tablets
£< ! 7i» l )>WMIfi
Piperazine Phosphate Tablets contain not less than
95% and not more than 105% of the labeled amount of
piperazine phosphate hydrate (C4HjoN2.H3PO4.H2O:
202.15).
Method of preparation Prepare as directed under Tablets,
with Piperazine Phosphate Hydrate.
Identification Take a quantity of Piperazine Phosphate
Tablets equivalent to 0.1 g of Piperazine Phosphate Hydrate
according to the labeled amount, previously powdered, add
10 mL of water, shake while warming for 10 minutes, allow
to cool, and filter. To 3 mL of the filtrate add 3 drops of
Reinecke salt TS: a light red precipitate is formed.
Disintegration <6.09> It meets the requirement. The time
limit of the test is 10 minutes.
Assay Weigh accurately not less than 20 Piperazine Phos-
phate Tablets, and powder. Weigh accurately a quantity of
the powder, equivalent to about 0.15 g of piperazine phos-
phate hydrate (C 4 H 10 N 2 .H 3 PO 4 .H 2 O) according to the la-
beled amount. Add 5 mL of formic acid, shake for 5 minutes,
centrifuge, and collect the supernatant liquid. To the residue
add 5 mL of formic acid, shake for 5 minutes, centrifuge,
and collect the supernatant liquid. Repeat twice the same
procedure with 5 mL each of acetic acid (100), combine all
the supernatant liquids, add 50 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 10.107 mg of C 4 H I0 N 2 .H 3 PO4.H 2 O
Containers and storage Containers — Tight containers.
Pirarubicin
tfvJUfv-V
H,C
C 32 H 37 N0 12 : 627.64
(2S,4S)-4-{3-Amino-2,3,6-trideoxy-4-0-[(2i?)-3,4,5,6-
tetrahydro-2//-pyran-2-yl] -a-L-/yxo-hexopyranosyloxy j -
2,5,12-trihydroxy-2-hydroxyacetyl-7-methoxy-l,2,3,4-
tetrahydrotetracene-6,ll-dione [72496-41-4]
Pirarubicin is a derivative of daunorubicin.
It contains not less than 950 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Pirarubicin is expressed as mass (potency) of pirarubi-
cin (C 3 2H3 7 N0 12 ).
Description Pirarubicin occurs as a red-orange crystalline
powder.
It is soluble in chloroform, very slightly soluble in acetoni-
trile, in methanol and in ethanol (99.5), and practically in-
soluble in water.
Identification (1) Dissolve 10 mg of Pirarubicin in 80 mL
of methanol and 6 mL of diluted hydrochloric acid (1 in
5000), and add water to make 100 mL. To 10 mL of this solu-
tion add diluted methanol (4 in 5) to make 100 mL. Deter-
JPXV
Official Monographs / Pirenoxine 1001
mine the absorption spectrum of this solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of a solution of Pirarubicin Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Dissolve 5 mg each of Pirarubicin and Pirarubicin
Reference Standard in 5 mL of chloroform, and use these so-
lutions as the sample solution and standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /uL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
chloroform and methanol (5:1) to a distance of about 10 cm,
and air-dry the plate. Examine the spots with the necked eye:
the principal spot obtained from the sample solution and the
spot from the standard solution show a red-orange color and
the same Rf value.
Optical rotation <2.49> [a]™: + 195 - +215° (10 mg, chlo-
roform, 10 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 10 mg
of Pirarubicin in 10 mL of 0.01 mol/L hydrochloric acid TS:
the solution is clear and red.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Pirarubi-
cin according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Related substances — Dissolve 10 mg of Pirarubicin in
20 mL of the mobile phase, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add the mo-
bile phase to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with exactly 20 liL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the peak area of doxorubicin, hav-
ing the relative retention time of about 0.45 with respect to
pirarubicin, and the area of the peak, having the relative
retention time of about 1.2 with respect to pirarubicin, ob-
tained from the sample solution are not more than the peak
area of pirarubicin from the standard solution, respectively,
and the sum of the areas of the peaks, having the relative
retention times of about 1.9 and about 2.0 with respect to
pirarubicin, from the sample solution is not more than 5
times the peak area of pirarubicin from the standard solu-
tion. For these calculations, use the peak area for doxorubi-
cin after multiplying by the relative response factor 0.94 and
the area for the two peaks, having the relative retention times
of about 1.9 and about 2.0, after multiplying by their relative
response factors, 1.09, respectively.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of pirarubicin.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mobile phase to make ex-
actly 10 mL. Confirm that the peak area of pirarubicin ob-
tained from 20 iiL of this solution is equivalent to 14 to 26%
of that from 20 liL of the standard solution.
System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay.
Water <2.48> Not more than 2.0% (0.1 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Pirarubicin and
Pirarubicin Reference Standard, equivalent to about 10 mg
(potency), and dissolve in the mobile phase to make exactly
10 mL. Pipet 5 mL of these solutions, add exactly 5 mL of
the internal standard solution, and use these solutions as the
sample solution and standard solution. Perform the test with
20 /uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Qt and
Qs, of the peak area of pirarubicin to that of the internal
standard.
Amount [iig (potency)] of C32H37NO12
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Pirarubicin Reference
Standard
Internal standard solution — A solution of 2-naphthol in the
mobile phase (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 11m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of 0.05 mol/L ammonium for-
mate buffer solution, pH 4.0 and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
pirarubicin is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
liL of the standard solution under the above operating condi-
tions, pirarubicin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 9.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of pirarubicin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Pirenoxine
t°U/+v>
C 16 H 8 N 2 5 : 308.25
l-Hydroxy-5-oxo-5//-pyrido[3,2-a]phenoxazine-3-
carboxylic acid [1043-21-6]
1002 Pirenzepine Hydrochloride Hydrate / Official Monographs
JP XV
Pirenoxine, when dried, contains not less than
98.0% of C 16 H 8 N 2 5 .
Description Pirenoxine occurs as a yellow-brown powder.
It is odorless, and has a slightly bitter taste.
It is very slightly soluble in dimethylsulfoxide, and practi-
cally insoluble in water, in acetonitrile, in ethanol (95), in
tetrahydrofuran and in diethyl ether.
Melting point: about 250°C (with decomposition).
Identification (1) Dissolve 2 mg of Pirenoxine in 10 mL of
phosphate buffer solution, pH 6.5, add 5 mL of a solution of
L-ascorbic acid (1 in 50), and shake vigorously: a dark purple
precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Pirenoxine in phosphate buffer solution, pH 6.5 (1 in
200,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Pirenoxine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Pirenoxine according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 10 mg of Pirenoxine in
50 mL of the mobile phase, and use this solution as the sam-
ple solution. Pipet 3 mL of the sample solution, add the mo-
bile phase to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with exactly 5 /uL each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine each peak area of both solu-
tions by the automatic integration method: the total area of
the peaks other than pirenoxine is not larger than the peak
area of pirenoxine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 1.39 g of tetra M-butylammonium
chloride and 4.5 g of disodium hydrogen phosphate dodeca-
hydrate in 1000 mL of water, and adjust the pH to 6.5 with
phosphoric acid. To 700 mL of this solution add 200 mL of
acetonitrile and 30 mL of tetrahydrofuran, and mix.
Flow rate: Adjust the flow rate so that the retention time of
pirenoxine is about 10 minutes.
Time span of measurement: About 3 times as long as the
retention time of pirenoxine.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 30 mL.
Confirm that the peak area of pirenoxine obtained from 5 fiL
of this solution is equivalent to 5 to 8% of that of pirenoxine
obtained from 5 jXL of the standard solution.
System performance: Dissolve 3 mg of Pirenoxine and
16 mg of methyl parahydroxybenzoate in 100 mL of the
mobile phase. When the procedure is run with 5 fiL of this so-
lution under the above operating conditions, pirenoxine and
methyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
pirenoxine is not more than 1.0%.
Loss on drying <2.41> Not more than 1.5% (0.5 g, in vacu-
um, 80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of Pirenoxine, previ-
ously dried, dissolve in 140 mL of dimethylsulfoxide by heat-
ing on a water bath. After cooling, add 30 mL of water, and
titrate <2.50> immediately with 0.02 mol/L sodium hydroxide
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.02 mol/L sodium hydroxide VS
= 6.165 mg of C 16 H 8 N 2 5
Containers and storage Containers — Tight containers.
Pirenzepine Hydrochloride Hydrate
if U >M if >±M@g±£7kfttl
• 2HCI ■ H 2
C 19 H 21 N 5 2 .2HC1.H 2 0: 442.34
1 1 - [(4-Methylpiperazin- 1 -yl)acetyl] -5 , 1 1 -dihydro-6//-
pyrido[2,3-6][l,4]benzodiazepin-6-one dihydrochloride
monohydrate [29868-97-1, anhydride]
Pirenzepine Hydrochloride Hydrate contains not
less than 98.5% and not more than 101.0% of pirenze-
pine hydrochloride (C 19 H 21 N 5 2 .2HC1: 424.32), calcu-
lated on the anhydrous basis.
Description Pirenzepine Hydrochloride Hydrate occurs as a
white to pale yellow crystalline powder.
It is freely soluble in water and in formic acid, slightly solu-
ble in methanol, and very slightly soluble in ethanol (99.5).
The pH of a solution by dissolving 1 g of Pirenzepine
Hydrochloride Hydrate in 10 mL of water is between 1.0 and
2.0.
Melting point: about 245°C (with decomposition).
It is gradually colored by light.
Identification (1) Determine the absorption spectrum of a
JPXV
Official Monographs / Piroxicam 1003
solution of Pirenzepine Hydrochloride Hydrate (1 in 40,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Piren-
zepine Hydrochloride Hydrate as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) A solution of Pirenzepine Hydrochloride Hydrate (1
in 50) responds to Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — A solution
obtained by dissolving 1.0 g of Pirenzepine Hydrochloride
Hydrate in 10 mL of water is clear and not more color than
that of the following control solution.
Control solution: To 1.2 mL of Matching fluid for color F
add 8.8 mL of diluted hydrochloric acid (1 in 40).
(2) Heavy metals <1.07> — Proceed with 2.0 g of Pirenze-
pine Hydrochloride Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 0.3 g of Pirenzepine
Hydrochloride Hydrate in 10 mL of water. To 1 mL of this
solution add 5 mL of methanol and the mobile phase A to
make 10 mL, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add 5 mL of
methanol and the mobile phase A to make exactly 10 mL.
Pipet 1 mL of this solution, add 5 mL of methanol and the
mobile phase A to make exactly 10 mL, and use this solution
as the standard solution. Perform the test with exactly 10,mL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
pirenzepine is not more than 3/10 times the peak area of
pirenzepine from the standard solution, and the total area of
the peaks other than pirenzepine is not more than 3/5 times
the peak area of pirenzepine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 283 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase A: Dissolve 2 g of sodium lauryl sulfate in
900 mL of water, adjust the pH to 3.2 with acetic acid (100),
and add water to make 1000 mL.
Mobile phase B: Methanol
Mobile phase C: Acetonitrile
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A, B and C as directed in the fol-
lowing table.
Time after injection Mobile phase Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%) C (vol%)
0-15
15-
55 ^ 25
25
30
30
15 ^ 45
45
Flow rate: Adjust the flow rate so that the retention time of
pirenzepine is about 8 minutes.
Time span of measurement: About 2 times as long as the
retention time of pirenzepine beginning after the solvent
peak.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add 5 mL of methanol and the mobile phase A
to make exactly 10 mL. Confirm that the peak area of piren-
zepine obtained from 10 /xL of this solution is equivalent to 7
to 13% of that from 10,mL of the standard solution.
System performance: Dissolve 0.1 g of phenylpiperazine
hydrochloride in 10 mL of methanol. Mix 1 mL of this
solution and 1 mL of the sample solution, and add 5 mL of
methanol and the mobile phase A to make 10 mL. When the
procedure is run with 10 fiL of this solution under the above
operating conditions, pirenzepine and phenylpiperazine are
eluted in this order with the resolution between these peaks
being not less than 5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
pirenzepine is not more than 2.0%.
Water <2.48> Not less than 3.5% and not more than 5.0%
(0.3 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Pirenzepine
Hydrochloride Hydrate, dissolve in 2 mL of formic acid, add
60 mL of acetic anhydride, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 14.14 mg of C I9 H 21 N 5 2 .2HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Piroxicam
tin+v^A
QsHoNjCmS: 331.35
4-Hydroxy-2-methy WV-(pyridin-2-yl)-2//- 1 ,2-
benzothiazine-3-carboxamide 1,1 -dioxide [36322-90-4]
Piroxicam contains not less than 98.5% and not
1004 Pivmecillinam Hydrochloride / Official Monographs
JP XV
more than 101.0% of QsHn^C^S, calculated on the
dried basis.
Description Piroxicam occurs as a white to pale yellow crys-
talline powder.
It is sparingly soluble in acetic anhydride, slightly soluble
in acetonitrile, in methanol and in ethanol (99.5), very slight-
ly soluble in acetic acid (100), and practically insoluble in
water.
Melting point: about 200°C (with decomposition).
Identification (1) Dissolve 0.1 g of Piroxicam in a mixture
of methanol and 0.5 mol/L hydrochloric acid TS (490:1) to
make 200 mL. To 1 mL of this solution add the mixture of
methanol and 0.5 mol/L hydrochloric acid TS (490:1) to
make 100 mL. Determine the absorption spectrum of this so-
lution as directed under Ultraviolet-visible Spectrophotomet-
ry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Pirox-
icam as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers. If any
difference appears between the spectra, dissolve the sample
with dichloromethane, evaporate the solvent, dry the residue
on a water bath, and perform the test.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Piroxicam according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 75 mg of Piroxicam in
50 mL of acetonitrile for liquid chromatography, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add acetonitrile for liquid chromatography to make
exactly 10 mL. Pipet 1 mL of this solution, add acetonitrile
for liquid chromatography to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak other than piroxicam is not larger than the peak area of
piroxicam with the standard solution, and the total area of
the peaks other than piroxicam is not larger than 2 times the
peak area of piroxicam with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0 and acetonitrile for liquid
chromatography (3:2).
Flow rate: Adjust the flow rate so that the retention time of
piroxicam is about 10 minutes.
Time span of measurement: About 5 times as long as the
retention time of piroxicam beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add acetonitrile for liquid chromatography to
make exactly 20 mL. Confirm that the peak area of piroxicam
obtained with 20 [iL of this solution is equivalent to 17.5 to
32.5% of that with 20 /iL of the standard solution.
System performance: When the procedure is run with
20 /uL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry factor of the peak of piroxicam are not less than 6000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
piroxicam is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.25 g of Piroxicam, dis-
solve in 60 mL of a mixture of acetic anhydride and acetic
acid (100) (1:1), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation in the same manner, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 33.14 mg of C^H^C^S
Containers and storage Containers — Tight containers.
Pivmecillinam Hydrochloride
tf It v V i-Ai^m^
o
o o
CH 3 CH3 * HCr
C 21 H33N 3 05S.HC1: 476.03
2,2-Dimethylpropanoyloxymethyl (2S,5i?,6i?)-6-[(azepan-
l-ylmethylene)amino]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate monohydrochloride
[32887-03-9]
Pivmecillinam Hydrochloride contains not less than
630 fig (potency) and not more than 710 fig (potency)
per mg, calculated on the anhydrous basis. The poten-
cy of Pivmecillinam Hydrochloride is expressed as
mass (potency) of mecillinam (Q5H23N3O3S: 325.43).
Description Pivmecillinam Hydrochloride occurs as a white
to yellowish white crystalline powder.
It is very soluble in methanol and in acetic acid (100), freely
soluble in water and in ethanol (99.5), and soluble in acetoni-
trile.
Identification (1) Determine the infrared absorption spec-
trum of Pivmecillinam Hydrochloride as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
JPXV
Official Monographs / Live Oral Poliomyelitis Vaccine 1005
Reference Spectrum or the spectrum of Pivmecillinam
Hydrochloride Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
(2) Dissolve 0.5 g of Pivmecillinam Hydrochloride in
10 mL of water, and add 1 mL of dilute nitric acid and 1 drop
of silver nitrate TS: a white precipitate is formed.
Optical rotation <2.49> [a]™: + 200 - + 220° (1 g calculated
on the anhydrous basis, water, 100 mL, 100 mm).
Purity (1) Heavy metals <1.07> — To 1.0 g of Pivmecillin-
am Hydrochloride in a crucible add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10), fire
the ethanol to burn, and heat gradually to incinerate. If a car-
bonized substance remains, moisten with a small amount of
nitric acid, and ignite to incinerate. Cool, add 3 mL of
hydrochloric acid to the residue, dissolve by warming on a
water bath, and heat to dryness. To the residue add 10 mL of
water, and dissolve by warming on a water bath. After cool-
ing, adjust the pH to 3 to 4 with ammonia TS, add 2 mL of
dilute acetic acid, filter if necessary, and wash the crucible
and the filter with 10 mL of water. Put the filtrate and the
washings to a Nessler tube, add water to make 50 mL, and
use this solution as the test solution. Prepare the control solu-
tion in the same manner as the test solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Pivmecillinam Hydrochloride according to Method 4, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Pivmecillinam
Hydrochloride in 4.0 mL of a mixture of acetonitrile and
acetic acid (100) (97:3), and use this solution as the sample
solution. Separately, dissolve 2.0 mg of Pivmecillinam
Hydrochloride Reference Standard in 4.0 mL of water, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 2//L of the standard solution on a
plate of silica gel for thin-layer chromatography, allow to
stand for 30 minutes, then spot 2 iiL of the sample solution
on the plate. Immediately, develop the plate with a mixture
of acetone, water and acetic acid (100) (10:1:1) to a distance
of about 12 cm, and air-dry the plate. Allow the plate to
stand for 10 minutes in iodine vapor: the spot from the sam-
ple solution appeared at the position corresponding to the
spot obtained from the standard solution is not larger and
not more intense than the spot from the standard solution,
and any spot other than the principal spot and the above spot
is not observable.
Water <2.48> Not more than 1.0% (0.25 g, coulometric
titration).
Assay Weigh accurately an amount of Pivmecillinam
Hydrochloride and Pivmecillinam Hydrochloride Reference
Standard, equivalent to about 20 mg (potency), dissolve in a
suitable amount of the mobile phase, add exactly 10 mL of
the internal standard solution and the mobile phase to make
100 mL, and use these solutions as the sample solution and
standard solution, respectively. Perform the test with 10 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Q T and Q s , of
the peak area of pivmecillinam to that of the internal stan-
dard.
Amount [fig (potency)] of mecillinam (Q5H23N3O3S)
= W s x (Q T /Q S ) x 1000
W s : Amount [mg (potency)] of Pivmecillinam Hydro-
chloride Reference Standard
Internal standard solution — A solution of diphenyl in the
mobile phase (1 in 12,500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 30 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (lO^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.771 g of ammonium acetate in
about 900 mL of water, adjust the pH to 3.5 with acetic acid
(100), and add water to make 1000 mL. To 400 mL of this so-
lution add 600 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
pivmecillinam is about 6.5 minutes.
System suitability —
System performance: When the procedure is run with 10
11L of the standard solution under the above operating condi-
tions, pivmecillinam and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of pivmecillinam to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Live Oral Poliomyelitis Vaccine
Live Oral Poliomyelitis Vaccine contains live at-
tenuated poliovirus of type I, II and III.
Monovalent or bivalent product may be prepared, if
necessary.
Live Oral Poliomyelitis Vaccine conforms to the re-
quirements of Live Oral Poliomyelitis Vaccine in the
Minimum Requirements for Biological Products.
Description Live Oral Poliomyelitis Vaccine is a light yel-
low-red to light red, clear liquid.
1006 Polymixin B Sulfate / Official Monographs
JP XV
Polymixin B Sulfate
r
R— Dbu-Th r-Dbu- Dbu- Dbu- D- Phe-Leu-Dbu-Dbij-Thi
— ' ' X H 2 SO<
Polymyxin B ( : R = 6-Methyloctanoic acid
Dbu = L-er,y-DiaminobLJtyricacial
Polymyxin E 2 '■
R = &-M ethyl heplanoic acid
Dbu = i-ff.y-Diaminobutyric acid
Polymixin B Sulfate is the sulfate of a mixture of
peptide substances having antibacterial activity pro-
duced by the growth of Bacillus polymyxa.
It contains not less than 6500 units per mg, calculat-
ed on the dried basis. The potency of Polymixin B
Sulfate is expressed as mass unit of polymixin B
(C 5 5.5 6 H9 6 .9 g N 16 13 ). One unit of Polymixin B Sulfate is
equivalent to 0.129,ug of polymixin B sulfate
(C 55 . 56 H 96 . 98 N 16 13 .1-2H 2 S0 4 ).
Description Polymixin B Sulfate occurs as a white to yel-
low-brown powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) To 5 mL of a solution of Polymixin B
Sulfate (1 in 10) add 5 mL of a solution of sodium hydroxide
(1 in 10), add 5 drops of a solution of copper (II) sulfate pen-
tahydrate (1 in 100) while shaking: a purple color develops.
(2) Transfer 5 mg each of Polymixin B Sulfate and Poly-
mixin B Sulfate Reference Standard separately into two glass
stoppered test tubes, add 1 mL of diluted hydrochloric acid (1
in 2), stopper the tube, heat at 135°C for 5 hours, then heat
to dryness on a water bath, and keep the heating until no
more hydrochloric acid odor is evolved. Dissolve the residue
in 0.5 mL of water, and use these solutions as the sample so-
lution and standard solution (1). Separately, dissolve 20 mg
each of L-leucine, L-threonine, phenylalanine and L-serine
separately in 10 mL of water, and use these solutions as the
standard solutions (2), (3), (4) and (5), respectively. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 3 /xL each of the sample solu-
tion, the standard solutions (1), (2), (3), (4) and (5) on a plate
of silica gel for thin-layer chromatography, and expose the
plate to a saturated vapor of the developing solvent for 15
hours. Develop the plate with a mixture of phenol and water
(3:1) to a distance of about 13 cm while without exposure to
light, and dry the plate at 110°C for 5 minutes. Spray evenly
ninhydrin-acetic acid TS on the plate, and heat at 1 10°C for 5
minutes: Rf value of each spot obtained from the sample so-
lution is the same with Rf value of the corresponding spots
from the standard solution (1). Each of the spots from the
sample solution appears at the position corresponding to
each of the spots from the standard solutions (2), (3) and (4),
but not appears at the position corresponding to the spot
from the standard solution (5).
(3) A solution of Polymixin B Sulfate (1 in 20) responds
to the Qualitative Tests <1.09> for sulfate.
Optical rotation <2.49> [ a ]g>: -78 - -90° (0.5 g calculated
on the dried basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Polymixin B Sulfate in 50 mL of water is between 5.0 and
7.0.
Phenylalanine Weigh accurately about 0.375 g of Polymix-
in B Sulfate, dissolve in 0.1 mol/L hydrochloric acid TS to
make exactly 100 mL. Determine absorbances, A it A 2 , A it A 4
and A 5 , of this solution at 252 nm, at 258 nm, at 264 nm, at
280 nm and at 300 nm, respectively, as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and calculate the
amount of phenylalanine by the following equation: the
amount of phenylalanine calculated on the dried basis is not
less than 9.0% and not more than 12.0%.
Amount (%) of phenylalanine
= {(.42-0.5A + 0. 5A 3 -l.8A A + 0.SA 5 )/W T } x 9.4787
W T : Amount (g) of the sample, calculated on the dried
basis
Purity Heavy metals <1.07> — Proceed with 1.0 g of Poly-
mixin B Sulfate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 6.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.75% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Escherichia coli NIHJ
(ii) Agar media for seed and base layer
Peptone 10.0 g
Meat extract 3.0 g
Sodium chloride 30.0 g
Agar 20.0 g
Water 1000 mL
Mix all the ingredients, and sterilize. Adjust the pH <2.54>
of the solution so that it will be 6.5 to 6.6 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Polymixin B Sulfate Reference Standard, equivalent to about
200,000 units, dissolve in phosphate buffer solution, pH 6.0
to make exactly 20 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at not ex-
ceeding 5°C and use within 14 days. Take exactly a suitable
amount of the standard stock solution before use, add phos-
phate buffer solution, pH 6.0 to make solutions so that each
mL contains 4000 units and 1000 units, and use these solu-
tions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Polymixin B Sulfate, equivalent to about 200,000 units, and
dissolve in phosphate buffer solution, pH 6.0 to make exactly
20 mL. Take exactly a suitable amount of this solution, add
phosphate buffer solution, pH 6.0 to make solutions so that
each mL contains 4000 units and 1000 units, and use these so-
lutions as the high concentration sample solution and low
concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Potash Soap 1007
Polyoxyl 40 Stearate
Polyoxyl 40 Stearate is the monostearate of conden-
sation polymers of ethylene oxide represented by the
formula H(OCH 2 CH 2 )„OCOC 17 H 35 , in which n is ap-
proximately 40.
Description Polyoxyl 40 Stearate occurs as a white to light
yellow, waxy solid or powder. It is odorless or has a faint fat-
like odor.
It is soluble in water, in ethanol (95) and in diethyl ether.
Congealing point <2.42> 39.0 - 44.0°C
Congealing point of the fatty acid <1.13> Not below 53 °C.
Acid value <1.13> Not more than 1.
Saponification value <1.13> 25-35
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Polyoxyl 40 Stearate in 20 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Polyoxyl
40 Stearate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.67 g
of Polyoxyl 40 Stearate, according to Method 3, and perform
the test (not more than 3 ppm).
Residue on ignition <2.44> Not more than 0.1% (1 g)
Containers and storage Containers — Tight containers.
Polysorbate 80
*°U yjL.-i- h 80
Polysorbate 80 is a polyoxyethylene ether of anhy-
drous sorbitol, partially esterified with oleic acid.
Description Polysorbate 80 is a colorless or orange-yellow,
viscous liquid, having a faint, characteristic odor and a
warm, slightly bitter taste.
It is miscible with methanol, with ethanol (95), with warm
ethanol (95), with pyridine and with chloroform.
It is freely soluble in water and slightly soluble in diethyl
ether.
The pH of a solution of Polysorbate 80 (1 in 20) is between
5.5 and 7.5.
Identification (1) To 5 mL of a solution of Polysorbate 80
(1 in 20) add 5 mL of sodium hydroxide TS, boil for 5
minutes, cool, and acidify with dilute hydrochloric acid: the
solution is opalescent.
(2) To 5 mL of a solution of polysorbate 80 (1 in 20) add
2 to 3 drops of bromine TS: the color of the test solution is
discharged.
(3) Mix 6 mL of Polysorbate 80 with 4 mL of water at an
ordinary, or lower than ordinary, temperature: a jelly-like
mass is produced.
(4) To 10 mL of a solution of Polysorbate 80 (1 in 20) add
5 mL of ammonium thiocyanate-cobaltous nitrate TS, shake
well, add 5 mL of chloroform, shake, and allow to stand: a
blue color develops in the chloroform layer.
Viscosity <2.53> 345 - 445 mm 2 /s (Method 1, 25 °C).
Specific gravity <7.75> d%: 1.065 - 1.095
Acid value <1.13> Not more than 2.0.
Saponification value <1.13> 45 - 55
Iodine value <1.13> 19-24 Use chloroform instead of cy-
clohexane, and titrate without using an indicator, until the
yellow color of iodine disappears.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Polysorbate 80 according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Polysorbate 80 according to Method 3, and perform the
test (not more than 2 ppm).
Water <2.48> Not more than 3.0% (1 g, back titration).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Containers and storage Containers — Tight containers.
Potash Soap
Potash Soap contains not less than 40.0% as fatty
acids.
Method of preparation
Fixed oil
Potassium Hydroxide
Water or Purified Water
470 mL
a sufficient quantity
a sufficient quantity
To make 1000 g
Dissolve Potassium Hydroxide, in required quantity for
saponification, in Water or Purified Water, add this solution
to fixed oil, previously warmed, add a sufficient quantity of
Ethanol if necessary, stir thoroughly, heat in a water bath,
and continue the saponification. After complete saponifica-
tion, add Water or Purified Water to make 1000 g.
Description Potash Soap occurs as a yellow-brown, trans-
parent, unctuous, soft mass, having a characteristic odor.
It is freely soluble in water and in ethanol (95).
Purity Silicic acid and alkalinity — Dissolve 10 g of Potash
Soap in 30 mL of ethanol (95), and add 0.50 mL of 1 mol/L
hydrochloric acid VS: no turbidity is produced. Add 1 drop
of phenolphthalein TS to this solution: no red color develops.
Assay Weigh accurately about 5 g of Potash Soap, dissolve
in 100 mL of hot water, and transfer to a separator. Acidify
the mixture with dilute sulfuric acid, and cool. Extract the so-
lution with 50-mL, 40-mL, and 30-mL portions of diethyl
ether. Wash the combined diethyl ether extracts with 10-mL
portions of water until the washing contains no acid. Trans-
1008 Potassium Bromide / Official Monographs
JP XV
fer the diethyl ether solution to a tared flask, evaporate
diethyl ether on a water bath at a temperature as low as possi-
ble. Dry the residue at 80°C to constant mass, and weigh as
fatty acids.
Containers and storage Containers — Tight containers.
Potassium Bromide
Skill) U *A
previously dried, and dissolve in 50 mL of water. Add 10 mL
of dilute nitric acid and exactly measured 50 mL of 0.1
mol/L silver nitrate VS, and titrate <2.50> the excess silver ni-
trate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2
mL of ammonium iron (III) sulfate TS). Perform a blank de-
termination.
Each mL of 0.1 mol/L silver nitrate VS
= 11.90 mg of KBr
Containers and storage Containers — Tight containers.
KBr: 119.00
Potassium Bromide,
than 99.0% of KBr.
when dried, contains not less
Potassium Canrenoate
Description Potassium Bromide occurs as colorless or white
crystals, granules or crystalline powder. It is odorless.
It is freely soluble in water and in glycerin, soluble in hot
ethanol (95), and slightly soluble in ethanol (95).
Identification A solution of Potassium Bromide (1 in 10)
responds to Qualitative Tests <1.09> for potassium salt and
for bromide.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Bromide in 3 mL of water: the solution is clear
and colorless.
(2) Alkalinity — Dissolve 1.0 g of Potassium Bromide in
10 mL of water, add 0.10 mL of 0.05 mol/L sulfuric acid VS
and 1 drop of phenolphthalein TS, heat to boiling, and cool:
no color develops.
(3) Chloride — Make a calculation from the result ob-
tained in the Assay: not more than 84.5 mL of 0.1 mol/L sil-
ver nitrate VS is consumed for 1 g of Potassium Bromide.
(4) Sulfate <1.14> — Proceed with 2.0 g of Potassium
Bromide, and perform the test. Prepare the control solution
with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than
0.024%).
(5) Iodide — Dissolve 0.5 g of Potassium Bromide in 10
mL of water, add 2 to 3 drops of iron (III) chloride TS and 1
mL of chloroform, and shake: no red-purple to purple color
develops in the chloroform layer.
(6) Bromate — Dissolve 1.0 g of Potassium Bromide in 10
mL of freshly boiled and cooled water, and add 0.1 mL of
potassium iodide TS, 1 mL of starch TS and 3 drops of dilute
sulfuric acid. Shake the mixture gently, and allow to stand
for 5 minutes: no blue color develops.
(7) Heavy metals <1.07> — Proceed with 2.0 g of Potassi-
um Bromide according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(8) Barium — Dissolve 0.5 g of Potassium Bromide in 10
mL of water, add 0.5 mL of dilute hydrochloric acid and 1
mL of potassium sulfate TS, and allow to stand for 10
minutes: no turbidity is produced.
(9) Arsenic </.//> — Prepare the test solution with 1.0 g
of Potassium Bromide according to Method 1, and perform
the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, 110°C,
4 hours).
Assay Weigh accurately about 0.4 g of Potassium Bromide,
co 2 K
396.56
Monopotassium 17-hydroxy-3-oxo-17a-pregna-4,6-diene-
21-carboxylate [2181-04-6]
Potassium Canrenoate, when dried, contains not less
than 98.0% and not more than 102.0% of
C22H29KO4.
Description Potassium Canrenoate occurs as a pale yellow-
ish white to pale yellow-brown, crystalline powder.
It is freely soluble in water, soluble in methanol, sparingly
soluble in ethanol (95), and practically insoluble in chlo-
roform and in diethyl ether.
Identification (1) Dissolve 2 mg of Potassium Canrenoate
in 2 drops of sulfuric acid: an orange color develops. Observe
under ultraviolet light (main wavelength: 365 nm): the solu-
tion shows a yellow-green fluorescence. Add 1 drop of acetic
anhydride to this solution: the color of the solution changes
to red.
(2) Determine the absorption spectrum of a solution of
Potassium Canrenoate in methanol (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of Potas-
sium Canrenoate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) The solution of Potassium Canrenoate (1 in 10)
responds to Qualitative Tests <1.09> (1) for potassium salt.
Optical rotation <2.49> [a]™: -71 - -76° (after drying, 0.2
g, methanol, 20 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Potassium Canrenoate in 20
mL of water: the pH of this solution is between 8.4 and 9.4.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Potassium Canrenoate in 5 mL of water: the solution is clear,
JPXV
Official Monographs / Potassium Chloride 1009
and shows a pale yellow to light yellow color.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Potassi-
um Canrenoate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Potassium Canrenoate according to Method 3, and per-
form the test (not more than 2 ppm).
(4) Canrenone — Place 0.40 g of Potassium Canrenoate in
a glass-stoppered centrifuge tube, cool in ice-water to a tem-
perature not higher than 5°C, add 6 mL of boric acid-potas-
sium chloride-sodium hydroxide buffer solution, pH 10.0,
being cooled to a temperature not higher than 5°C to dis-
solve, and add 8 mL of water being cooled to a temperature
not higher than 5°C. Add exactly 10 mL of chloroform, al-
low to stand for 3 minutes at a temperature not higher than
5°C, shake vigorously for 2 minutes, and centrifuge. Drain
off the water layer, collect 5 mL of the chloroform layer,
transfer to a glass-stoppered centrifuge tube containing 3 mL
of boric acid-potassium chloride-sodium hydroxide buffer so-
lution, pH 10.0, cooled to a temperature not higher than
5°C, and 4 mL of water cooled to a temperature not higher
than 5°C, shake for 1 minute, and centrifuge. Drain off the
water layer, pipet 2 mL of the chloroform layer, and add
chloroform to make exactly 10 mL. Determine the absor-
bance of this solution at 283 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it is not more than
0.67.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Assay Weigh accurately about 0.2 g of Potassium Canreno-
ate, previously dried, dissolve in 75 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Use a solution of saturated potassium
chloride-acetic acid (100) as the internal liquid.). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 39.66 mg of C 22 H 29 K0 4
Containers and storage Containers — Tight containers.
Potassium Carbonate
K 2 C0 3 : 138.21
Potassium Carbonate, when dried, contains not less
than 99.0% of K 2 C0 3 .
Description Potassium Carbonate occurs as white granules
or powder. It is odorless.
It is very soluble in water, and practically insoluble in
ethanol (95).
A solution of Potassium Carbonate (1 in 10) is alkaline.
It is hygroscopic.
Identification A solution of Potassium Carbonate (1 in 10)
responds to Qualitative Tests <1.09> for potassium salt and
for carbonate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Carbonate in 20 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07> — Dissolve 1.0 g of Potassium
Carbonate in 2 mL of water and 6 mL of dilute hydrochloric
acid, and evaporate to dryness on a water bath. Dissolve the
residue in 35 mL of water and 2 mL of dilute acetic acid, di-
lute with water to 50 mL, and perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: evaporate 6 mL of dilute hydrochloric acid on a water
bath to dryness, add 2 mL of dilute acetic acid and 2.0 mL of
Standard Lead Solution to dryness, and dilute with water to
50 mL (not more than 20 ppm).
(3) Sodium — Dissolve 1.0 g of Potassium Carbonate in
20 mL of water, and perform the test as directed under Flame
Coloration Test <1.04> (1): no persisting yellow color is
produced.
(4) Arsenic <1.11> — Prepare the test solution with 0.5 g
of Potassium Carbonate, according to Method 1, and per-
form the test (not more than 4 ppm).
Loss on drying <2.41> Not more than 1.0% (3 g, 180°C,
4 hours).
Assay Dissolve about 1.5 g of Potassium Carbonate, previ-
ously dried and accurately weighed, in 25 mL of water, titrate
with 0.5 mol/L sulfuric acid VS until the blue color of the so-
lution changes to yellow-green, boil cautiously, then cool,
and titrate <2.50> until a greenish yellow color develops (indi-
cator: 2 drops of bromocresol green TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 69.10 mg of K 2 C0 3
Containers and storage Containers — Tight containers.
Potassium Chloride
KC1: 74.55
Potassium Chloride, when dried, contains not less
than 99% of KC1.
Description Potassium Chloride occurs as colorless or
white crystals or crystalline powder. It is odorless, and has a
saline taste.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
A solution of Potassium Chloride (1 in 10) is neutral.
Identification A solution of Potassium Chloride (1 in 50)
responds to Qualitative Tests <1.09> for potassium salt and
for chloride.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Chloride in 5 mL of water: the solution is clear
and colorless.
(2) Acidity and alkalinity — Dissolve 5.0 g of Potassium
Chloride in 50 mL of freshly boiled and cooled water, and
add 3 drops of phenolphthalein TS: no red color develops.
Then add 0.50 mL of 0.01 mol/L sodium hydroxide VS: a
red color develops.
(3) Bromide — Dissolve 1.0 g of Potassium Chloride in
1010 Potassium Clavulanate / Official Monographs
JP XV
water to make 100 mL. To 5 mL of the solution add 3 drops
of dilute hydrochloric acid and 1 mL of chloroform, and add
3 drops of sodium toluensulfonchloramide TS dropwise
while shaking: no yellow to yellow-red color develops in the
chloroform layer.
(4) Iodide — Dissolve 0.5 g of Potassium Chloride in 10
mL of water, add 3 drops of iron (III) chloride TS and 1 mL
of chloroform, shake, allow to stand for 30 minutes, and
shake again: no red-purple to purple color develops in the
chloroform layer.
(5) Heavy metals <1.07> — Proceed with 4.0 g of Potassi-
um Chloride according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 5 ppm).
(6) Calcium and magnesium — Dissolve 0.20 g of Potassi-
um Chloride in 20 mL of water, add 2 mL of ammonia TS, 2
mL of ammonium oxalate TS and 2 mL of disodium
hydrogenphosphate TS, and then allow to stand for 5
minutes: no turbidity is produced.
(7) Sodium — Dissolve 1.0 g of Potassium Chloride in 20
mL of water, and perform the Flame Coloration Test <1.04>
(1): no persistent, yellow color develops.
(8) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Potassium Chloride according to Method 1, and perform
the test (not more than 2 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 130°C,
2 hours).
Assay Weigh accurately about 0.2 g of Potassium Chloride,
previously dried, dissolve in 50 mL of water, and titrate
<2.50> with 0.1 mol/L silver nitrate VS while shaking
vigorously (indicator: 3 drops of fluorescein sodium TS).
Each mL of 0.1 mol/L silver nitrate VS = 7.455 mg of KC1
Containers and storage Containers — Tight containers.
Potassium Clavulanate
<7^y^>mt> i )i>^
H.
C 8 H 8 KN0 5 : 237.25
Monopotassium (2i?,5i?)-3-[(lZ)-2-hydroxyethylidene]-7-
oxo-4-oxa-l-azabicyclo[3.2.0]heptane-2-carboxylate
[61177-45-5]
Potassium Clavulanate is the potassium salt of a
substance having /^-lactamase inhibiting activity pro-
duced by the growth of Streptomyces clavuligerus.
It contains not less than 810 /ug (potency) and not
more than 860 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Potassium
Clavulanate is expressed as mass (potency) of clavular-
ic acid (QH 9 N0 5 : 199.16).
Description Potassium Clavulanate occurs as a white to
light yellowish white, crystalline powder.
It is very soluble in water, soluble in methanol, and slightly
soluble in ethanol (95).
It is hygroscopic.
Identification (1) To 1 mL of a solution of Potassium
Clavulanate (1 in 50,000) add 5 mL of imidazole TS, and
warm in a water bath at 30°C for 12 minutes. After cooling,
determine the absorption spectrum of this solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Potas-
sium Clavulanate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Potassium Clavulanate responds to Qualitative Tests
<1.09> (1) for potassium salt.
Optical rotation <2.49> [«]£?: + 53 - +63° (0.5 g calculated
on the anhydrous basis, water, 50 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Potassium Clavulanate according to Method 2, and perform
the test. Prepare the control solution with 4.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Potassium Clavulanate according to Method 3, and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Potassium
Clavulanate in 10 mL of the mobile phase A, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase A to make exactly 100 mL, and
use this solution as the standard solution. Perform the test
with exactly 20 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of each
peak other than clavulanic acid from the sample solution is
not more than the peak area of clavulanic acid from the stan-
dard solution, and the total area of the peaks other than
clavulanic acid from the sample solution is not more than 2
times of the peak area of clavulanic acid from the standard
solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fan in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase A: Adjust the pH of 0.05 mol/L sodium di-
hydrogen phosphate TS to 4.0 with phosphoric acid.
Mobile phase B: A mixture of the mobile phase A and
methanol (1:1).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
JPXV
Official Monographs / Potassium Guaiacolsulfonate 1011
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0- 4
4-15
15-25
100
100^0
0^ 100
100
Flow rate: 1.0 mL per minute.
Time span of measurement: About 6 times as long as the
retention time of clavulanic acid.
System suitability —
Test for required detectability: Pipet 1 mL of the standard
solution, and add the mobile phase A to make exactly 10 mL.
Confirm that the peak area of clavulanic acid obtained from
20 /xL of this solution is equivalent to 7 to 13% of that from
20 /xL of the standard solution.
System performance: Dissolve 10 mg each of Potassium
Clavulanate and Amoxycillin in 100 mL of the mobile phase
A. When the procedure is run with 20 fiL of this solution un-
der the above operating conditions, clavulanic acid and
amoxycillin are eluted in this order with the resolution be-
tween these peaks being not less than 8 and the number of
theoretical plates of the peak of clavulanic acid is not less
than 2500.
System repeatability: When the test is repeated 3 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
clavulanic acid is not more than 2.0%.
Water <2.48> Not more than 1.5% (5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Potassium
Clavulanate and Lithium Clavulanate Reference Standard,
equivalent to about 12.5 mg (potency), dissolve each in 30
mL of water, add exactly 5 mL of the internal standard solu-
tion and water to make 50 mL, and use these solutions as the
sample solution and standard solution, respectively. Perform
the test with 5 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of clavularic acid to that of the
internal standard.
Amount \p.g (potency)] of clavularic acid (C 8 H 9 N0 5 )
= ^sX(G T /es)xiooo
W s : Amount [mg (potency)] of Lithium Clavulanate
Reference Standard
Internal standard solution — Dissolve 0.3 g of sulfanilamide
in 30 mL of methanol, and add water to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.36 g of sodium acetate trihydrate
in 900 mL of water, adjust to pH 4.5 with diluted acetic acid
(31) (2 in 5), and add 30 mL of methanol and water to make
1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
clavularic acid is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 [iL
of the standard solution under the above operating condi-
tions, clavularic acid and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of clavularic acid to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Potassium Guaiacolsulfonate
£*7-y=l-,IU7JU*>®*) U ^7A
ex
'CH 3
C 7 H 7 K0 5 S: 242.29
Monopotassium 4-hydroxy-3-methoxybenzenesulfonate
[1321-14-8]
Potassium Guaiacolsulfonate contains not less than
98.5% of C7H7KO5S, calculated on the anhydrous ba-
sis.
Description Potassium Guaiacolsulfonate occurs as white
crystals or crystalline powder. It is odorless or has a slight,
characteristic odor and a slightly bitter taste.
It is freely soluble in water and in formic acid, soluble in
methanol, and practically insoluble in ethanol (95), in acetic
anhydride and in diethyl ether.
Identification (1) To 10 mL of a solution of Potassium
Guaiacolsulfonate (1 in 100) add 2 drops of iron (III) chlo-
ride TS: a blue-purple color develops.
(2) Dissolve 0.25 g of Potassium Guaiacolsulfonate in
water to make 500 mL, and to 10 mL of this solution add
phosphate buffer solution, pH 7.0, to make 100 mL. Deter-
mine the absorption spectrum of this solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(3) A solution of Potassium Guaiacolsulfonate (1 in 10)
responds to Qualitative Tests <1.09> for potassium salt.
pH <2.54> Dissolve 1 .0 g of Potassium Guaiacolsulfonate in
20 mL of water: the pH of the solution is between 4.0 and
5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Guaiacolsulfonate in 20 mL of water: the solution
is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.8 g of Potas-
sium Guaiacolsulfonate . Prepare the control solution with
0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
1012 Potassium Hydroxide / Official Monographs
JP XV
0.030%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Potassi-
um Guaiacolsulfonate according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Potassium Guaiacolsulfonate according to Method 1, and
perform the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.20 g of Potassium
Guaiacolsulfonate in 200 mL of mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 5 juL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area obtained from these solutions by the automatic integra-
tion method: the total area of peaks other than the peak of
potassium guaiacolsulfonate from the sample solution is not
larger than the peak area of potassium guaiacolsulfonate
from the standard solution.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 279 nm).
Column: A stainless steel column 4 mm in inside diameter
and 20 to 25 cm in length, packed with dimethyl-
aminopropylsilanized silica gel, (5 to 10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogenphosphate VS and methanol (20:1).
Flow rate: Adjust the flow rate so that the retention time of
potassium guaiacolsulfonate is about 10 minutes.
Selection of column: Weigh 50 mg each of potassium
guaiacolsulfonate and guaiacol, and dissolve in 50 mL of the
mobile phase. Proceed with 5 /xL of this solution under the
above operating conditions, and calculate the resolution. Use
a column giving elution of guaiacol and potassium guaiacol-
sulfonate in this order with the resolution of these peaks
being not less than 4.
Detection sensitivity: Adjust the sensitivity so that the peak
height of potassium guaiacolsulfonate from 5 /xL of the stan-
dard solution is not less than 10mm.
Time span of measurement: About twice as long as the
retention time of potassium guaiacolsulfonate.
Water <2. 48> 3 . - 4 . 5 % (0 . 3 g , direct titration) .
Assay Weigh accurately about 0.3 g of Potassium Guaia-
colsulfonate, dissolve in 2.0 mL of formic acid, add 50 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.23 mg of C 7 H 7 K0 5 S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Potassium Hydroxide
jkmtt) u ^ a
KOH: 56.11
Potassium Hydroxide contains not less than 85.0%
of KOH.
Description Potassium Hydroxide occurs as white fused
masses, in small pellets, in flakes, in sticks and in other
forms. It is hard and brittle, and shows a crystalline fracture.
It is freely soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
It rapidly absorbs carbon dioxide in air.
It deliquesces in the presence of moisture.
Identification (1) A solution of Potassium Hydroxide (1
in 500) is alkaline.
(2) A solution of Potassium Hydroxide (1 in 25) responds
to Qualitative Tests <1.09> for potassium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Hydroxide in 20 mL of water: the solution is clear
and colorless.
(2) Chloride <1.03>— Dissolve 2.0 g of Potassium
Hydroxide in water, and add water to make 100 mL. To 25
mL of the solution add 8 mL of dilute nitric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.7 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.050%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Potassium
Hydroxide in 5 mL of water, add 7 mL of dilute hydrochloric
acid, and evaporate on a water bath to dryness. Dissolve the
residue in 35 mL of water, 2 mL of dilute acetic acid and 1
drop of ammonia TS, add water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: evaporate 7 mL of dilute
hydrochloric acid on a water bath to dryness, dissolve the
residue in 2 mL of dilute acetic acid and 3.0 mL of Standard
Lead Solution, and add water to make 50 mL (not more than
30 ppm).
(4) Sodium — Dissolve 0.10 g of Potassium Hydroxide in
10 mL of dilute hydrochloric acid, and perform the test as
directed under Flame Coloration Test <1.04> (1): no persis-
tent yellow color develops.
(5) Potassium carbonate — The amount of potassium car-
bonate (K 2 C0 3 : 138.21) is not more than 2.0% when calculat-
ed by the following equation using B (mL) obtained in the
Assay.
Amount of potassium carbonate (mg)= 138.21 xB
Assay Weigh accurately about 1.5 g of Potassium
Hydroxide, and dissolve in 40 mL of freshly boiled and
cooled water. Cool the solution to 15°C, add 2 drops of
phenolphthalein TS, and titrate <2.50> with 0.5 mol/L sulfur-
ic acid VS until the red color of the solution disappears.
Record the amount A (mL) of 0.5 mol/L sulfuric acid VS
consumed, then add 2 drops of methyl orange TS, and titrate
<2.50> again with 0.5 mol/L sulfuric acid VS until the solu-
tion changes to a persistent light red color. Record the
JPXV
Official Monographs / Potassium Permanganate 1013
amount B (mL) of 0.5 mol/L sulfuric acid VS consumed.
Calculate the amount KOH from the amount, A (mL) — B
(mL).
Each mL of 0.5 mol/L sulfuric acid VS
= 56.11 mg of KOH
Containers and storage Containers — Tight containers.
Potassium Iodide
KI: 166.00
Potassium Iodide, when dried, contains not less than
99.0% of KI.
Description Potassium Iodide occurs as colorless or white
crystals, or a white crystalline powder.
It is very soluble in water, soluble in ethanol (95), and prac-
tically insoluble in diethyl ether.
It is slightly deliquescent in moist air.
Identification A solution of Potassium Iodide (1 in 20)
responds to Qualitative Tests <1.09> for potassium salt and
for iodide.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Potassium Iodide in 2 mL of water: the solution is clear and
colorless.
(2) Alkalinity — Dissolve 1.0 g of Potassium Iodide in 10
mL of freshly boiled and cooled water, and add 0.50 mL of
0.005 mol/L sulfuric acid and 1 drop of phenolphthalein TS:
no color develops.
(3) Chloride, bromide and thiosulfate — Dissolve 0.20 g
of Potassium Iodide in 5 mL of ammonia TS, add 15.0 mL of
0.1 mol/L silver nitrate VS, shake for 2 to 3 minutes, and
filter. To 10 mL of the filtrate, add 15 mL of dilute nitric
acid: no brown color develops. The solution has no more tur-
bidity than that of the following control solution.
Control solution: To 0.30 mL of 0.01 mol/L hydrochloric
acid VS add 2.5 mL of ammonia TS, and 7.5 mL of 0.1 mol/
L silver nitrate VS and 15 mL of dilute nitric acid.
(4) Nitrate, nitrite and ammonium — Place 1 .0 g of Potas-
sium Iodide in a 40-mL test tube, and add 5 mL of water, 5
mL of sodium hydroxide TS and 0.2 g of aluminum wire. In-
sert the absorbent cotton in the mouth of the test tube, and
place a piece of moistened red litmus paper on it. Heat the
test tube carefully on a water bath for 15 minutes: the gas
evolved does not turn red litmus paper to blue.
(5) Cyanide — Dissolve 0.5 g of Potassium Iodide in 10
mL of water. To 5 mL of this solution add 1 drop of iron (II)
sulfate TS and 2 mL of sodium hydroxide TS, warm, then
add 4 mL of hydrochloric acid: no green color develops.
(6) Iodate — Dissolve 0.5 g of Potassium Iodide in 10 mL
of freshly boiled and cooled water, and add 2 drops of dilute
sulfuric acid and 1 drop of starch TS: no blue color develops
immediately.
(7) Heavy metals <1.07> — Proceed with 2.0 g of Potassi-
um Iodide according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(8) Barium — Dissolve 0.5 g of Potassium Iodide in 10 mL
of water, add 1 mL of dilute sulfuric acid, and allow to stand
for 5 minutes: no turbidity is produced.
(9) Sodium — Dissolve 1.0 g of Potassium Iodide in 10
mL of water, and perform the Flame Coloration Test (1)
<1.04>: a yellow color develops, but does not persist.
(10) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Potassium Iodide according to Method 1, and perform the
test (not more than 5 ppm).
Loss on drying <2.41> Not more than 1.0% (2 g, 105°C,
4 hours).
Assay Weigh accurately about 0.5 g of Potassium Iodide,
previously dried, in an iodine flask, dissolve in 10 mL of
water, add 35 mL of hydrochloric acid and 5 mL of chlo-
roform, and titrate <2.50> with 0.05 mol/L potassium iodate
VS with shaking until the red-purple color of the chloroform
layer disappears. The end point is reached when the red-pur-
ple color does not reappear in the chloroform layer within 5
minutes after the layer has been decolorized.
Each mL of 0.05 mol/L potassium iodate VS
= 16.60 mg of KI
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Potassium Permanganate
®v>;r;>miU^A
KMn0 4 : 158.03
Potassium Permanganate, when dried, contains not
less than 99.0% of KMn0 4 .
Description Potassium Permanganate occurs as dark pur-
ple crystals and has a metallic luster.
It is soluble in water.
A solution of Potassium Permanganate (1 in 1000) has a
slightly sweet, astringent taste.
Identification A solution of Potassium Permanganate (1 in
100) responds to Qualitative Tests <1.09> for permanganate.
Purity (1) Water-insoluble substances — Dissolve 2.0 g of
Potassium Permanganate, previously powdered, in 200 mL
of water. Filter the insoluble substances through a tared glass
filter (G4), wash with water until the last washing shows no
color, and dry at 105 °C for 2 hours: the mass of the residue is
not more than 4 mg.
(2) Arsenic <1.11> — Dissolve 0.40 g of Potassium Per-
manganate in 10 mL of water, add 1 mL of sulfuric acid, add
hydrogen peroxide (30) dropwise until the solution remains
colorless, and evaporate on a sand bath nearly to dryness.
Dissolve the residue in 5 mL of water, and perform the test
with this solution as the test solution: the color produced is
not more intense than the following standard color.
Standard color: To 10 mL of water add 1 mL of sulfuric
acid and the same volume of hydrogen peroxide (30) as used
for the preparation of the test solution. Evaporate the solu-
tion on a sand bath nearly to dryness, add 2.0 mL of Stan-
dard Arsenic Solution and water to make 5 mL, and carry out
1014 Potassium Sulfate / Official Monographs
JP XV
the test with this solution in the same manner as the test solu-
tion (not more than 5 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
18 hours).
Assay Weigh accurately about 0.6 g of Potassium Perman-
ganate, previously dried, dissolve in water to make exactly
200 mL, and use this solution as the sample solution. Pipet 25
mL of 0.05 mol/L oxalic acid VS into a 500-mL conical flask,
add 200 mL of diluted sulfuric acid (1 in 20), and keep at a
temperature between 30°C and 35°C. Transfer the sample so-
lution to a buret. Add quickly 23 mL of the sample solution
from the buret to the flask while shaking gently, and then al-
low the flask to stand until the red color disappears. Warm
the mixture to a temperature between 55°C and 60°C, and
continue the titration <2. 50> slowly until the red color persists
for 30 seconds.
Each mL of 0.05 mol/L oxalic acid VS
= 3.161 mg of KM11O4
Containers and storage Containers — Tight containers.
Potassium Sulfate
mm* u oa
K 2 S0 4 : 174.26
Potassium Sulfate, when dried, contains not less
than 99.0% of K 2 S0 4 .
Description Potassium Sulfate occurs as colorless crystals
or a white, crystalline powder. It has a slightly saline, some-
what bitter taste.
It is soluble in water and practically insoluble in ethanol
(95).
Identification A solution of Potassium Sulfate (1 in 20)
responds to Qualitative Tests <1.09> for potassium salt and
for sulfate.
Purity (1) Clarity and color of solution, and acid or
alkali — Dissolve 1.0 g of Potassium Sulfate in 20 mL of
water: the solution is clear, colorless and neutral.
(2) Chloride <1.03>— Perform the test with 0.5 g of
Potassium Sulfate. Prepare the control solution with 0.40
mL of 0.01 mol/L hydrochloric acid VS (not more than
0.028%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Potassi-
um Sulfate according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(4) Sodium — Dissolve 1.0 g of Potassium Sulfate in 20
mL of water, and perform the test as directed under Flame
Coloration Test <1.04> (1): no persistent yellow color de-
velops.
(5) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Potassium Sulfate according to Method 1, and perform
the test (not more than 5 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, 110°C,
4 hours).
Assay Weigh accurately about 0.5 g of Potassium Sulfate,
previously dried, boil with 200 mL of water and 1.0 mL of
hydrochloric acid, and add gradually 8 mL of boiling barium
chloride TS. Heat the mixture on a water bath for 1 hour,
collect the precipitate, and wash the precipitate with water
until the last washing shows no opalescence on the addition
of silver nitrate TS. Dry, heat strongly to constant mass be-
tween 500°C and 600°C by raising the temperature gradually,
and weigh as barium sulfate (BaS0 4 : 233.39).
Amount (mg) of K 2 S0 4
= amount (mg) of barium sulfate (BaS0 4 ) x 0.7466
Containers and storage Containers — Well-closed contain-
ers.
Potato Starch
Amylum Solani
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (♦ ♦).
Potato Starch consists of starch granules derived
from the tuber of Solarium tuberosum Linne
(Solanaceae).
♦Description Potato Starch occurs as a white powder.
It is practically insoluble in water and in ethanol (99.5).*
Identification (1) Under a microscope, Potato Starch,
preserved in a mixture of water and glycerin (1:1), appears as
unevenly ovoid or pyriform simple grains usually 30 - 100
/um, often more than 100 ^m in diameter, or spherical simple
grains 10-35//m in diameter, rarely 2- to 4-compound
grains; ovoid or pyriform simple grains with eccentric hilum,
spherical simple grains with non-centric or slightly eccentric
hilum; striation distinct in all grains; a black cross, its inter-
section point on hilum, is observed when grains are put be-
tween two nicol prisms fixed at right angle to each other.
(2) To 1 g of Potato Starch add 50 mL of water, boil for
1 minute, and allow to cool: a subtle white-turbid, pasty
liquid is formed.
(3) To 1 mL of the pasty liquid obtained in (2) add 0.05
mL of diluted iodine TS (1 in 10): an orange-red to deep blue
color is formed, and the color disappears by heating.
pH <2.54> Put 5.0 g of Potato Starch in a non-metal vessel,
add 25.0 mL of freshly boiled and cooled water, mix gently
for 1 minute, and allow to stand for 15 minutes: the pH of
the solution is between 5.0 and 8.0.
Purity (1) Iron— To 1.5 g of Potato Starch add 15 mL of
2 mol/L hydrochloric acid TS, mix, filter, and use the filtrate
as the test solution. To 2.0 mL of Standard Iron Solution add
water to make 20 mL, and use as the control solution. Put 10
mL each of the test solution and the control solution in test
tubes, add 2 mL of a solution of citric acid (2 in 10) and 0.1
mL of mercapto acetic acid, and mix. Alkalize with ammonia
solution (28) to litmus paper, add water to make 20 mL, and
mix. Transfer 10 mL each of these solutions into test tubes,
allow to stand for 5 minutes, and compare the color of these
solutions against a white background: the color of the test so-
JPXV
Official Monographs / Povidone 1015
A; Boiling flask (!>00 ml-)
B: Funnel (100 mL)
C: Condenser
D: Test-lube
E:Tap
The figures arc in mm.
lution is not darker than that of the control solution (not
more than 10 ppm).
(2) Oxidizing substances — To 4.0 g of Potato Starch add
50.0 mL of water, shake for 5 minutes, and centrifuge. To
30.0 mL of the supernatant liquid add 1 mL of acetic acid
(100) and 0.5 to 1.0 g of potassium iodide, shake, and allow
to stand for 25 to 30 minutes at a dark place. Add 1 mL of
starch TS, and titrate <2.50> with 0.002 mol/L sodium
thiosulfate VS until the color of the solution disappears. Per-
form a blank determination and make any necessary correc-
tion: the volume of 0.002 mol/L sodium thiosulfate VS con-
sumed is not more than 1.4 mL (not more than 20 ppm, cal-
culated as hydrogen peroxide).
(3) Sulfur dioxide —
(i) Apparatus Use as shown in the figure.
(ii) Procedure Introduce 150 mL of water into the
boiling flask, close the tap of the funnel, and pass carbon
dioxide through the whole system at a rate of 100 ±5 mL per
minute. Pass cooling water through the condenser, and place
10 mL of hydrogen peroxide-sodium hydroxide TS in the
test-tube. After 15 minutes, remove the funnel without inter-
rupting the stream of carbon dioxide, and introduce through
the opening into the flask about 25 g of Potato Starch,
accurately weighed, with the aid of 100 mL of water. Apply
tap grease to the outside of the connection part of the funnel,
and load the funnel. Close the tap of the funnel, pour 80 mL
of 2 mol/L hydrochloric acid TS into the funnel, open the tap
to introduce the hydrochloric acid into the flask, and close
the tap while several mL of the hydrochloric acid remains, in
order to avoid losing sulfur dioxide. Place the flask in a water
bath, and heat the mixture for 1 hour. Transfer the contents
of the test-tube with the aid of a little water to a wide-necked
conical flask. Heat in a water bath for 15 minutes, and cool.
Add 0.1 mL of bromophenol blue TS, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS until the color changes from
yellow to violet-blue lasting for at least 20 seconds. Perform a
blank determination and make any necessary correction. Cal-
culate the amount of sulfur dioxide by applying the following
formula: it is not more than 50 ppm.
Amount (ppm) of sulfur dioxide = ( VI W) x 1000x3.203
W: Amount (g) of the sample
V: Amount (mL) of 0.1 mol/L sodium hydroxide VS
consumed
Loss on drying <2.41> Not more than 20.0% (1 g, 130°C, 90
minutes).
Residue on ignition <2.44> Not more than 0.6% (1 g).
♦Containers and storage Containers — Well-closed contain-
ers. »
Povidone
Polyvidone
Polyvinylpyrrolidone
7fc° £ K >
(C 6 H 9 NO) n
Poly[(2-oxopyrrolidin-l-yl)ethylene] [9003-39-8]
Povidone is a chain polymer of 1 -vinyl -2-pyrroli-
done. It contains not less than 11.5% and not more
than 12.8%o of nitrogen (N: 14.01), calculated on the
anhydrous basis.
It has a nominal K-value of not less than 25 and not
more than 90. The nominal K-value is shown on the
label.
Description Povidone occurs as a white to slightly yellowish
fine powder. It is odorless or has a faint, characteristic odor.
It is freely soluble in water, in methanol and in ethanol
(95), slightly soluble in acetone, and practically insoluble in
diethyl ether.
It is hygroscopic.
Identification Determine the infrared absorption spectrum
of Povidone, previously dried at 105 °C for 6 hours, as direct-
ed in the potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum or the spectrum of Povidone Refer-
ence Standard previously dried at 105°C for 6 hours: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
pH <2.54> Dissolve 1.0 g of Povidone in 20 mL of water:
the pH of this solution is between 3.0 and 5.0 for Povidone
having the nominal K-value of 30 or less, and between 4.0
and 7.0 for Povidone having the nominal K-value exceeding
30.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Povidone in 20 mL of water: the solution is clear and color-
less to pale yellow, or pale red.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Povidone
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(3) Aldehydes — Weigh accurately about 1.0 g of Povi-
done and dissolve in 0.05 mol/L pyrophosphate buffer solu-
1016 Povidone / Official Monographs
JP XV
tion, pH 9.0 to make exactly 100 mL. Stopper, heat at 60°C
for 60 minutes, allow to cool to room temperature, and use
this solution as the sample solution. Separately, dissolve
0.100 g of freshly distilled acetaldehyde in water previously
cooled to 4°C to make exactly 100 mL. Allow to stand at 4°C
for about 20 hours, pipet 1 mL of this solution, add 0.05
mol/L pyrophosphate buffer solution, pH 9.0 to make exact-
ly 100 mL, and use this solution as the standard solution.
Measure 0.5 mL each of the sample solution, standard solu-
tion and water (for blank test), transfer to separate cells, add
2.5 mL of 0.05 mol/L pyrophosphate buffer solution, pH
9.0, and 0.2 mL of /^-nicotinamide adenine dinucleotide TS
to each of these cells, mix and stopper tightly. Allow to stand
for 2 to 3 minutes at 22 ± 2°C, and perform the test with
these solutions as directed under Ultraviolet-visible Spec-
trophotometry <2.24> using water as the control solution. De-
termine the absorbances, A Tl , A sl and A m of the subsequent
solutions of the sample solution, the standard solution and
water at 340 nm. Add 0.05 mL of aldehyde dehydrogenase
solution to each of the cells, mix and stopper tightly. Allow
to stand for 5 minutes at 22 ± 2°C. Determine the absor-
bances, A T2 , A S2 and A m of these solutions in the same man-
ner as above: the content of aldehydes is not more than 500
ppm (expressed as acetaldehyde).
Content (ppm) of aldehydes expressed as acetaldehyde
i) ~ (A B2 ~ A Bi )
~ Asi) — {Am - A Bi )
.1000
W (A,
W: Amount (g) of povidone, calculated on the anhydrous
basis.
(4) l-Vinyl-2-pyrrolidone — Weigh accurately about 0.25
g of Povidone, dissolve in diluted methanol (1 in 5) to make
exactly 10 mL, and use this solution as the sample solution.
Separately, dissolve 50 mg of l-vinyl-2-pyrrolidone in
methanol to make exactly 100 mL. Pipet 1 mL of this solu-
tion and add methanol to make exactly 100 mL. Pipet 5 mL
of this solution, add diluted methanol (1 in 5) to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 50 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of l-vinyl-2-pyr-
rolidone in each solution: the content of l-vinyl-2-pyrroli-
done is not more than 10 ppm.
Content (ppm) of l-vinyl-2-pyrrolidone
= (2.5/W)x(A T /A s )
W: Amount (g) of Povidone, calculated on the anhydrous
basis.
Operating conditions —
Detector: An ultraviolet spectrophotometer (detection
wavelength: 254 nm).
Column: Stainless steel columns about 4 mm in inside di-
ameter and about 25 mm in length, and about 4 mm in inside
diameter and about 250 mm in length, packed with octyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter), and use them as a guard column and a separa-
tion column, respectively.
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and methanol (4:1).
Flow rate: Adjust the flow rate so that the retention time of
l-vinyl-2-pyrrolidone is about 10 minutes.
Selection of column: Dissolve 0.01 g of l-vinyl-2-pyrroli-
done and 0.5 g of vinyl acetate in 100 mL of methanol. To 1
mL of this solution add diluted methanol (1 in 5) to make 100
mL. Proceed with 50 /xL of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of l-vinyl-2-pyrrolidone and vinyl
acetate in this order with the resolution between these peaks
being not less than 2.0.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of l-vinyl-2-pyrrolidone obtained from
50,mL of the standard solution is between 10 mm and 15 mm.
System repeatability: When the test is repeated 6 times with
the standard solution under the above operating conditions,
the relative standard deviation of obtained peak areas of 1-
vinyl-2-pyrrolidone is not more than 2%.
Washing of the guard column: After each test with the
sample solution, wash away the polymeric material of Povi-
done from the guard column by passing the mobile phase
through the column backwards for about 30 minutes at the
same flow rate as applied in the test.
(5) Peroxides — Weigh exactly an amount of Povidone,
equivalent to 4.0 g calculated on the anhydrous basis, dis-
solve in water to make exactly 100 mL, and use this solution
as the sample solution. To 25 mL of the sample solution add
2 mL of titanium (III) chloride-sulfuric acid TS, and mix.
Allow to stand for 30 minutes, and perform the test with this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, using a solution prepared by adding 2 mL of 13
% sulfuric acid to 25 mL of the sample solution as a blank:
the absorbance of the subsequent solution of the sample solu-
tion at 405 nm is not more than 0.35 (not more than 400 ppm,
expressed as hydrogen peroxide).
(6) Hydrazine — Transfer 2.5 g of Povidone to a 50-mL
centrifuge tube, add 25 mL of water, and stir to dissolve.
Add 500 //L of a solution of salicylaldehyde in methanol (1 in
20), stir and warm at 60°C for 15 minutes in a water bath. Al-
low to cool, add 2.0 mL of toluene, stopper tightly, shake
vigorously for 2 minutes, centrifuge, and use the upper layer
of the mixture as the sample solution. Separately, dissolve
0.09 g of salicylaldazine in toluene to make exactly 100 mL.
Pipet 1 mL of this solution, add toluene to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 10 /xL each of the sample solu-
tion and standard solution on a plate coated with a 0.25-mm
layer of dimethylsilanized silica gel with fluorescent indicator
for thin-layer chromatography. Develop the plate with a mix-
ture of methanol and water (2:1) to a distance of about three-
fourths of the length of the plate, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 365 nm): the
Ri value of the fluorescent spot from the standard solution is
about 0.3, and the fluorescence of the spot from the sample
solution corresponding to the spot from the standard solu-
tion is not more intense than that of the spot from the stan-
dard solution (not more than 1 ppm).
Water <2.48> Not more than 5.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
K-value Weigh accurately an amount of Povidone, equiva-
lent to 1.00 g calculated on the anhydrous basis, and dissolve
in water to make exactly 100 mL, allow to stand for 60
JPXV
Official Monographs / Povidone-Iodine 1017
minutes, and use this solution as the sample solution. Per-
form the test with the sample solution and with water at 25 °C
as directed in Method 1 under Viscosity Determination
<2.53>, and calculate the K-value by the following formula.
1.5 log /? rd -l 7300 clog >y le i + (c+ 1.5 clog >? re i) 2
0.15 + 0.003 c 0.15 c + 0. 003 c 2
c: Mass (g) of Povidone in 100 mL of the solution, calcu-
lated on the anhydrous basis.
ri Kl : Kinematic viscosity of the sample solution relative to
that of water.
The K-value of Povidone is not less than 90% and not
more than 108% of the nominal K-value.
Assay Weigh accurately about 0.1 g of Povidone, and place
in a Kjeldahl flask. Add 5 g of a powdered mixture of 33 g of
potassium slfate, 1 g of copper (II) sulfate pentahydrate and
1 g of titanium (IV) oxide, and wash down any adhering sam-
ple from the neck of the flask with a small amount of water.
Add 7 mL of sulfuric acid allowing to flow down the inside
wall of the flask. Heat the flask gradually over a free flame
until the solution has a clear, yellow-green color and the in-
side wall of the flask is free from a carbonaceous material,
and then heat for further 45 minutes. After cooling, add cau-
tiously 20 mL of water, cool the solution, and connect the
flask to the distillation apparatus previously washed by pass-
ing steam through it. To the absorption flask add 30 mL of a
solution of boric acid (1 in 25), 3 drops of bromocresol green-
methyl red TS and sufficient water to immerse the lower end
of the condenser tube. Add 30 mL of a solution of sodium
hydroxide (2 in 5) through the funnel, rinse cautiously the
funnel with 10 ml of water, immediately close the clamp at-
tached to the rubber tube, then start the distillation with
steam to get 80 to 100 mL of the distillate. Remove the ab-
sorption flask from the lower end of the condenser tube, rins-
ing the end part with a small quantity of water, and titrate
<2.50> the distillate with 0.025 mol/L sulfuric acid VS until
the color of the solution changes from green through pale
grayish blue to pale grayish red-purple. Perform a blank de-
termination in the same manner, and make any necessary
correction.
Each mL of 0.025 mol/L sulfuric acid VS
= 0.700 mg of N
Containers and storage Containers — Tight containers.
Povidone-Iodine
*° tr H > a - K
(C 6 H 9 NO) n .xI
Poly[(2-oxopyrrolidin-l-yl)ethylene] iodine [25655-41-8]
Povidone-Iodine is a complex of iodine with 1-vinyl-
2-pyrrolidone polymer.
It contains not less than 9.0% and not more than
12.0% of available iodine (I: 126.90), and not less than
9.5% and not more than 11.5% of nitrogen (N: 14.01),
calculated on the dried basis.
Description Povidone-Iodine occurs as a dark red-brown
powder. It has a faint, characteristic odor.
It is freely soluble in water and in ethanol (99.5).
The pH of a solution obtained by dissolving 1.0 g of
Povidone-Iodine in 100 mL of water is between 1.5 and 3.5.
Identification (1) To 10 mL of diluted starch TS (1 in 10)
add 1 drop of a solution of Povidone-Iodine (1 in 10): a deep
blue color develops.
(2) To 1 mL of a solution of Povidone-Iodine (1 in 100)
add 1 mL of sodium thiosulfate TS, and add 1 mL of ammo-
nium thiocyanate-cobaltous nitrate TS and 2 drops of 1 mol/
L hydrochloric acid TS: a blue color develops, and a blue
precipitate is gradually formed.
Purity (1) Clarity and color of solution — Dissolve 0.30 g
of Povidone-Iodine in 100 mL of water: the solution is clear
and brown.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Povidone-Iodine according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Povidone-Iodine according to Method 4, and perform the
test (not more than 2 ppm).
(4) Iodide ion — Weigh accurately about 0.5 g of
Povidone-Iodine, dissolve in 100 mL of water, and add sodi-
um hydrogensulfite TS until the color of iodine completely
disappears. To this solution add exactly 25 mL of 0.1 mol/L
silver nitrate VS, shake well with 10 mL of nitric acid, titrate
<2.50> the excess silver nitrate with 0.1 mol/L ammonium
thiocyanate VS until the solution develops a red-brown color,
and calculate the total amount of iodine (indicator: 1 mL of
ammonium iron (III) sulfate TS). Perform a blank determi-
nation.
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 12.69 mg of I
Obtain the amount of iodide ion, calculated on the dried
basis, by deducting the amount (%) of available iodine from
the total amount (%) of iodine: it is not more than 6.6%.
Loss on drying <2.41> Not more than 8.0% (1 g, 100°C,
3 hours).
Residue on ignition <2.44> Not more than 0.05% (5 g).
Assay (1) Available iodine — Weigh accurately about 0.5 g
of Povidone-Iodine, dissolve in 30 mL of water, and titrate
<2.50> with 0.02 mol/L sodium thiosulfate VS (indicator: 2
mL of starch TS).
Each mL of 0.02 mol/L sodium thiosulfate VS
= 2.538 mg of I
(2) Nitrogen — Weigh accurately about 20 mg of
Povidone-Iodine, and perform the test as directed under
Nitrogen Determination <1.08>.
Containers and storage Containers — Tight containers.
1018 Pranoprofen / Official Monographs
JP XV
Pranoprofen
and enantiomer
C 15 H 13 N0 3 : 255.27
(2jRS)-2-(1 OH-9-Oxa- 1 -azaanthracen-6-yl)propanoic acid
[52549-17-4]
Pranoprofen, when dried, contains not less than
98.5% of C 15 H 13 N03.
Description Pranoprofen occurs as a white to pale yellow-
ish white crystalline powder.
It is freely soluble in A^TV-dimethylformamide, soluble in
acetic acid (100), sparingly soluble in methanol, slightly solu-
ble in acetonitrile, in ethanol (95) and in acetic anhydride,
very slightly soluble in diethyl ether, and practically insoluble
in water.
A solution of Pranoprofen in A^iV-dimethylformamide (1
in 30) shows no optical rotation.
Identification (1) Dissolve 0.02 g of Pranoprofen in 1
mol/L hydrochloric acid TS to make 100 mL, and dilute 10
mL of the solution with water to make 100 mL. Determine
the absorption spectrum of the solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Pranoprofen as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Melting point <2.60> 186 - 190°C
Purity (1) Chloride < 1.03 > —Dissolve 0.5 g of
Pranoprofen in 40 mL of methanol, and 6 mL of dilute nitric
acid, and add water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows. To 0.30 mL of 0.01 mol/L hydrochloric acid add
40 mL of methanol, 6 mL of dilute nitric acid and water to
make 50 mL (not more than 0.021%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Pranoprofen according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of the Standard
Lead Solution (not more than 10 ppm).
(3) Related Substances — Dissolve 50 mg of Pranoprofen
in 50 mL of the mobile phase, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add the
mobile phase to make exactly 200 mL, and use this solution
as the standard solution. Perform the test with exactly 10 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine each peak area from both solu-
tions by the automatic integration method: the each area of
the peaks other than the peak of pranoprofen from the sam-
ple solution is not larger than the peak area of pranoprofen
from the standard solution, and the total peak area of them is
not larger than twice of the peak area of pranoprofen from
the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column about 6 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 7.02 g of sodium perchlorate
monohydrate in 1000 mL of water, and adjust the pH to 2.5
with perchloric acid. To 2 volumes of this solution add 1
volume of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
pranoprofen is about 10 minutes.
Selection of column: Dissolve 4 mg each of Pranoprofen
and ethyl parahydroxybenzoate in 200 mL of the mobile
phase. Proceed with 10 /xL of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of pranoprofen and ethyl parahydrox-
ybenzoate in this order with the resolution between these
peaks being not less than 2.1.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of pranoprofen from 10 /uL of the stan-
dard solution is between 10 mm and 20 mm.
Time span of measurement: About three times as long as
the retention time of pranoprofen.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Pranoprofen, previ-
ously dried, dissolve in 70 mL of a mixture of acetic anhy-
dride and acetic acid (100) (7:3), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 25.53 mg of C 15 H 13 N0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Pravastatin Sodium
C 23 H 35 Na0 7 : 446.51
Monosodium (3i?,5i?)-3,5-dihydroxy-
7- {( 1 S,2S,6S, 85, 8ai?)-6-hy droxy-2-m ethyl-
JPXV
Official Monographs / Pravastatin Sodium 1019
8-[(2S)-2-methylbutanoyloxy]-
l,2,6,7,8,8a-hexahydronaphthalen-l-yl}heptanoate
[81131-70-6]
Pravastatin Sodium contains not less than 98.5%
and not more than 101.0% of C23H 35 Na07, calculated
on the anhydrous basis and corrected on the amount of
residual solvent.
Description Pravastatin Sodium occurs as a white to yel-
lowish white, powder or crystalline powder.
It is freely soluble in water and in methanol, and soluble in
ethanol (99.5).
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Pravastatin Sodium (1 in 100,000) as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Pravastatin Sodium as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>: it ex-
hibits absorption at the wave numbers of about 2970 cm -1 ,
2880 cm" 1 , 1727 cm" 1 and 1578 cm" 1 .
(3) Dissolve 50 mg of Pravastatin Sodium in 5 mL of
methanol, and use this solution as the sample solution.
Separately, dissolve 24 mg of Pravastatin 1,1,3,3-
tetramethylbutylammonium Reference Standard in 2 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 2 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (99.5) and a-
cetic acid (100) (80:16:1) to a distance of about 8 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the color tone and the Rf value of the
principal spot with the sample solution are not different with
them of the spot with the standard solution.
(4) A solution of Pravastatin Sodium (1 in 10) responds
to Qualitative Tests <1.09> (1) for sodium salt.
Optical rotation <2.49>: + 153 - + 159° (0.1 g calculated on
the anhydrous basis and corrected on the amount of residual
solvent, water, 20 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Pravastatin Sodium in 20 mL of freshly boiled and cooled
water is between 7.2 and 8.2.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Pravastatin Sodium according to Method 2, and perform the
test. Prepare the control solution with 1.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.10 g of Pravastatin
Sodium in 100 mL of a mixture of water and methanol (1 1 :9),
and use this solution as the sample solution. Pipet 10 mL of
the sample solution, add the mixture of water and methanol
(11:9) to make exactly 100 mL. Pipet 5 mL of this solution,
add the mixture of water and methanol (1 1 :9) to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 10 iiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine each peak area by the automatic integration
method: the area of the peak other than pravastatin is not
larger than 1/5 times the peak area of pravastatin from the
standard solution, and the total area of the peaks other than
pravastatin is not larger than the peak area of pravastatin
from the standard solution. Keep the sample solution and the
standard solution at not over thanl5°C.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2.5 times as long as the
retention time of pravastatin beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add a mixture of water and methanol (11:9) to
make exactly 50 mL. Confirm that the peak area of pravasta-
tin obtained with 10/iL of this solution is equivalent to 7 to
13% of that with 10 /uL of the standard solution.
System performance: Dissolve 5 mg of Pravastatin Sodium
in 50 mL of the mixture of water and methanol (1 1 :9). When
the procedure is run with 10 iiL of this solution under the
above operating conditions, the number of theoretical plates
and the symmetry factor of the peak of pravastatin are not
less than 3500 and not more than 1.6, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
pravastatin is not more than 2.0%.
(3) Residual solvent — Being specified separately.
Water <2.48> Not more than 4.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately about 0. 1 g of Pravastatin Sodium,
and dissolve in a mixture of water and methanol (11:9) to
make exactly 100 mL. Pipet 10 mL of this solution, add ex-
actly 10 mL of the internal standard solution and the mixture
of water and methanol (11:9) to make 100 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 30 mg of Pravastatin 1,1,3,3-tetramethylbutylamine
Reference Standard (previously determine the water with 0.5
g by direct titration in volumetric titration) dissolve in the
mixture of water and methanol (11:9) to make exactly 25 mL.
Proceed with exactly 10 mL of this solution in the same man-
ner for the preparation of the sample solution, and use the
solution so obtained as the standard solution. Perform the
test with 10 fiL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qt and Q s , of the peak area of pravastatin to that of the in-
ternal standard.
Amount (mg) of C 2 3H3 5 Na07
= W s X(0r/ft)x4x 1.0518
W s : Amount (mg) of Pravastatin 1,1,3,3-tetramethyl-
butylamine Reference Standard, calculated on the an-
hydrous basis
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in the mixture of water and methanol (11:9) (3 in
4000).
1020 Prazepam / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 238 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water, methanol, acetic acid
(100) and triethylamine (550:450:1:1).
Flow rate: Adjust the flow rate so that the retention time of
pravastatin is about 21 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the internal standard and pravastatin are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of pravastatin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Prazepam
■/7-riA°A
^ ,
C 19 H 17 C1N 2 0: 324.80
7-Chloro-l-(cyclopropylmethyl)-5-phenyl-l,3-dihydro-
2//-l,4-benzodiazepin-2-one [2955-38-6]
Prazepam, when dried, contains not less than 98.5%
of C 19 H 17 C1N 2 0.
Description Prazepam occurs as white to light yellow crys-
tals or crystalline powder. It is odorless.
It is freely soluble in acetone, soluble in acetic anhydride,
sparingly soluble in ethanol (99.5) and in diethyl ether, and
practically insoluble in water.
Identification (1) Dissolve 0.01 g of Prazepam in 3 mL of
sulfuric acid, and observe under ultraviolet light (main
wavelength: 365 nm): the solution shows a grayish blue
fluorescence.
(2) Dissolve 0.01 g of Prazepam in 1000 mL of a solution
of sulfuric acid in ethanol (99.5) (3 in 1000). Determine the
absorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Prazepam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) Perform the Flame Coloration Tests <1.04> (2) with
Prazepam: a green color appears.
Melting point <2.60> 145 - 148°C
Purity (1) Chloride <1.03>— To 1.0 g of Prazepam add 50
mL of water, allow to stand for 1 hour with occasional shak-
ing, and filter. To 20 mL of the filtrate add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.036%).
(2) Sulfate <1.14>— To 20 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.40 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Prazep-
am according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Prazepam according to Method 3, and perform the test
(not more than 2 ppm).
(5) Related substances — Dissolve 0.40 g of Prazepam in
10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, and add acetone to
make exactly 20 mL. Pipet 1 mL of this solution, add acetone
to make exactly 25 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and acetone
(9:1) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.20% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Prazepam, previ-
ously dried, dissolve in 60 mL of acetic anhydride, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.48 mg of C 19 H 17 C1N 2
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Prednisolone 1021
Prazepam Tablets
Prazepam Tablets contain not less than 93% and not
more than 107% of the labeled amount of prazepam
(C 19 H 17 C1N 2 0: 324.80).
Method of preparation
with Prazepam.
Prepare as directed under Tablets,
Identification (1) To a quantity of powdered Prazepam
Tablets, equivalent to 0.05 g of Prazepam according to the la-
beled amount, add 25 mL of acetone, shake well, and filter.
Take 5 mL of the filtrate, evaporate on a water bath to dry-
ness, and dissolve the residue in 3 mL of sulfuric acid. With
this solution, proceed as directed in the Identification (1) un-
der Prazepam.
(2) To a quantity of powdered Prazepam Tablets, equiva-
lent to 0.02 g of Prazepam according to the labeled amount,
add 200 mL of a solution of sulfuric acid in ethanol (99.5) (3
in 1000), shake well, and filter. To 5 mL of the filtrate add a
solution of sulfuric acid in ethanol (99.5) (3 in 1000) to make
50 mL, and determine the absorption spectrum as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits maxima between 241 nm and 245 nm, between 283 nm
and 287 nm and between 363 nm and 367 nm, and minima
between 263 nm and 267 nm and between 334 nm and 338
nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Proceed with 1 tablet of Prazepam Tablets according to the
Basket method, using 900 mL of 0. 1 mol/L hydrochloric acid
TS as the test solution at 100 rotations per minute. 30 minutes
after starting the test, separate 20 mL or more of the dis-
solved solution, and filter with a membrane filter with pore
size not more than 0.8 //m. Discard the first 10 mL of the
filtrate, measure exactly the subsequent FmL of the filtrate,
add 0.1 mol/L hydrochloric acid TS to make exactly V mL
so that each mL of this solution might contain about 5 n% of
prazepam (C 19 H 17 C1N 2 0) according to the labeled amount,
and use this solution as the sample solution. Separately,
weigh accurately about 5 mg of prazepam for assay, previ-
ously dried at 105 C C for 2 hours, add 200 mL of 0.1 mol/L
hydrochloric acid TS and dissolve with shaking, or by
ultrasonication if necessary, add 0.1 mol/L hydrochloric acid
TS to make exactly 1000 mL and use this solution as the stan-
dard solution. Determine the absorbances, A T and A s , of the
sample solution and the standard solution at 240 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate of Prazepam Tablets during 30 minutes
is not less than 80%.
Dissolution rate (%) of prazepam
(C 19 H 17 C1N 2 0) to the labeled amount
= W s x(A T /A s ) x (V'/V) x (90/C)
W s : Amount (mg) of prazepam for assay.
C: Labeled amount (mg) of prazepam (C 19 H 17 C1N 2 0) in
each tablet.
Assay Weigh accurately not less than 20 Prazepam Tablets,
and powder. Weigh accurately a quantity of the powder,
equivalent to about 50 mg of prazepam (Ci 9 H 17 ClN 2 0), add
30 mL of acetone, shake well, centrifuge, and separate the su-
pernatant. Repeat the same procedure twice with 30 mL each
of acetone, combine all the supernatants, and evaporate on a
water bath to dryness. Dissolve the residue in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.02 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.02 mol/L perchloric acid VS
= 6.496 mg of C I9 H 17 C1N 2
Containers and storage Containers — Tight containers.
Prednisolone
7"Ur* = '/n>
C 21 H 28 5 : 360.44
1 1/?, 17,21-Trihydroxypregna-l ,4-diene-3 ,20-dione
[50-24-8]
Prednisolone, when dried, contains not less than
97.0% and not more than 102.0% of C 21 H 28 5 .
Description Prednisolone occurs as a white, crystalline
powder.
It is soluble in methanol and in ethanol (95), slightly solu-
ble in ethyl acetate and in chloroform, and very slightly solu-
ble in water.
Melting point: about 235°C (with decomposition).
Identification (1) To 2 mg of Prednisolone add 2 mL of
sulfuric acid, and allow to stand for 2 to 3 minutes: a deep
red color, without fluorescence, develops. To this solution
add 10 mL of water cautiously: the color disappears and a
gray, flocculent precipitate is formed.
(2) Determine the infrared absorption spectrum of Pred-
nisolone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Prednisolone Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers. If any difference appears
between the spectra, dissolve Prednisolone and Prednisolone
Reference Standard in ethyl acetate, respectively, then
evaporate the ethyl acetate to dryness, and repeat the test on
the residues.
Optical rotation <2.49> [ a ] 2 D °: + 113 - +119° (after drying,
0.2 g, ethanol (95), 20 mL, 100 mm).
Purity (1) Selenium — To 0.10 g of Prednisolone add 0.5
mL of a mixture of perchloric acid and sulfuric acid (1:1) and
2 mL of nitric acid, and heat on a water bath until no more
brown gas evolves and the solution becomes to be a light yel-
1022 Prednisolone Tablets / Official Monographs
JP XV
low clear solution. After cooling, add 4 mL of nitric acid to
this solution, then add water to make exactly 50 mL, and use
this solution as the sample solution. Separately, pipet 3 mL
of Standard Selenium Solution, add 0.5 mL of a mixture of
perchloric acid and sulfuric acid (1:1) and 6 mL of nitric acid,
then add water to make exactly 50 mL, and use this solution
as the standard solution. Perform the test with the sample so-
lution and standard solution as directed under Atomic Ab-
sorption Spectrophotometry <2.23> according to the follow-
ing conditions, and determine constant absorbances, A T and
^4 S , obtained on a recorder after rapid increasing of the ab-
sorption: A T is smaller than A s (not more than 30 ppm).
Perform the test by using a hydride generating system and
a thermal absorption cell.
Lamp: A selenium hollow cathode lamp
Wavelength: 196.0 nm
Temperature of sample atomizer: When an electric furnace
is used, about 1000°C.
Carrier gas: Nitrogen or argon
(2) Related substances — Dissolve 20 mg of Prednisolone
in exactly 2 mL of a mixture of methanol and chloroform
(1:1), and use this solution as the sample solution. Separate-
ly, dissolve 20 mg of hydrocortisone and 10 mg of predniso-
lone acetate each in a mixture of methanol and chloroform
(1:1) to make exactly 100 mL, and use these solutions as the
standard solution (1) and standard solution (2). Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 iXL each of the sample solution
and standard solutions (1) and (2) on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of acetone, toluene and diethylamine (55:45:2) to a distance
of about 15 cm, and air-dry the plate (do not dip the filter
paper in the developing vessel). Spray evenly alkaline blue
tetrazolium TS on the plate: the spots from the sample solu-
tion corresponding to those from the standard solutions (1)
and (2) are not more intense than the spots from the standard
solutions (1) and (2), and no spots other than the principal
spot, hydrocortisone and prednisolone acetate appear from
the sample solution.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 25 mg each of Prednisolone and
Prednisolone Reference Standard, previously dried and ac-
curately weighed, in 50 mL of methanol, add exactly 25 mL
of the internal standard solution to each, and add methanol
to make 100 mL. To 1 mL each of these solutions add the
mobile phase to make 10 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
20 /xL each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, Qt and Q s , of the peak area of pred-
nisolone to that of the internal standard.
Amount (mg) of C2iH 28 5
= W S X(Q,/Q S )
W s : Amount (mg) of Prednisolne Reference Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in methanol (1 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 247 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with fiuorosilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and methanol (13:7).
Flow rate: Adjust the flow rate so that the retention time of
prednisolone is about 15 minutes.
System suitability —
System performance: Dissolve 25 mg of Prednisolone and
25 mg of hydrocortisone in 100 mL of methanol. To 1 mL of
this solution add the mobile phase to make 10 mL. When the
procedure is run with 20 /xh of this solution under the above
operating conditions, hydrocortisone and prednisolone are
eluted in this order with the resolution between these peaks
being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of prednisolone to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Prednisolone Tablets
Prednisolone Tablets contain not less than 90% and
not more than 110% of the labeled amount of pred-
nisolone (C 2 iH 28 5 : 360.44).
Method of preparation Prepare as directed under Tablets,
with Prednisolone.
Identification (1) Weigh a quantity of powdered Pred-
nisolone Tablets, equivalent to 0.05 g of Prednisolone ac-
cording to the labeled amount, add 10 mL of chloroform,
shake for 15 minutes, and filter. Evaporate the filtrate on a
water bath to dryness. Dry the residue at 105 °C for 1 hour,
and proceed as directed in the Identification (1) under Pred-
nisolone.
(2) Determine the infrared absorption spectra of the
residue obtained in (1) and Prednisolone Reference Standard,
previously dried, as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>: both spec-
tra exhibit similar intensities of absorption at the same wave
numbers. If any difference appears, dissolve the sample and
the Reference Standard in ethyl acetate, evaporate to dry-
ness, and repeat the test on the residues.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Transfer 1 tablet of Prednisolone Tablets to a volumetric
flask, and shake with 10 mL of water until the tablet is disin-
tegrated. Add 50 mL of methanol, shake for 30 minutes, and
add methanol to make exactly 100 mL. Centrifuge this solu-
tion, pipetxmL of the supernatant liquid, and add methanol
to make exactly KmL to provide a solution that contains
about 10 //g of prednisolone (C21H28O5) per ml, and use this
solution as the sample solution. Separately, weigh accurately
JPXV
Official Monographs / Prednisolone Acetate 1023
about 10 mg of Prednisolone Reference Standard, previously
dried at 105°C for 3 hours, dissolve in 10 mL of water and 50
mL of methanol, and add methanol to make exactly 100 mL.
Pipet 5 mL of this solution, add methanol to make exactly 50
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution at 242 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>.
Amount (mg) of prednisolone (C 2 iH 18 5 )
= W s x (A T /A S ) x (K/100) x (1/x)
W s : Amount (mg) of Prednisolone Reference Standard
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Predonisolone Tablets at
100 revolutions per minute according to the Paddle method
using 900 mL of water as the test solution. Twenty minutes
after the start of the test, take 20 mL or more of the dissolved
solution, and filter through a membrane filter with pore size
of 0.8 /um or less. Discard the first 10 mL of the filtrate, and
use the subsequent filtrate as the sample solution. Weigh ac-
curately about 10 mg of Prednisolone Reference Standard,
previously dried at 105°C for 3 hours, and dissolve in ethanol
(95) to make exactly 100 mL. Pipet 5 mL of this solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and ^4 S , of
the sample solution and standard solution at the maximum
wavelength at about 242 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>. The dissolution rate of
Prednisolone Tablets after 20 minutes should be not less than
70%.
Dissolution rate (%) with respect to
the labeled amount of prednisolone (C2iH 28 5 )
= W s x(A T /A s )x(45/Q
W s : Amount (mg) of Prednisolone Reference Standard.
C: Labeled amount (mg) of prednisolone (C21H28O5) in 1
tablet.
Assay Weigh accurately and powder not less than 20 Pred-
nisolone Tablets using an agate mortar. Weigh accurately a
portion of the powder, equivalent to about 5 mg of predniso-
lone (C21H28O5), add 1 mL of water, and shake gently. Add
exactly 5 mL of the internal standard solution and 15 mL of
methanol, and shake vigorously for 20 minutes. To 1 mL of
this solution add the mobile phase to make 10 mL, and filter
through a membrane filter with pore size of 0.45 ^m. Discard
the first 3 mL of the filtrate, and use the subsequent filtrate as
the sample solution. Separately, weigh accurately about 25
mg of Prednisolone Reference Standard, previously dried at
105 °C for 3 hours, dissolve in 50 mL of methanol, add exact-
ly 25 mL of the internal standard solution, and add methanol
to make 100 mL. To 1 mL of this solution add the mobile
phase to make 10 mL, and use this solution as the standard
solution. Proceed as directed in the Assay under Predniso-
lone with these solutions.
Amount (mg) of prednisolone (C2iH 28 5 )
= W s x(Qt/Q s )x(U5)
W s : Amount (mg) of Prednisolone Reference Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in methanol (1 in 2000).
Containers and storage Containers — Tight containers.
Prednisolone Acetate
CK
H H,C ~\
^■■OH
H^iC 1 V
C 2 3H 30 O 6 : 402.48
ll/?,17,21-Trihydroxypregna-l,4-diene-3,20-dione
21-acetate [52-21-1]
Prednisolone Acetate, when dried, contains not less
than 96.0% and not more than 102.0% of C 23 H 3 o0 6 .
Description Prednisolone Acetate occurs as a white, crys-
talline powder.
It is slightly soluble in methanol, in ethanol (95), in ethanol
(99.5), and in chloroform, and practically insoluble in water.
Melting point: about 235°C (with decomposition).
Identification (1) To 2 mg of Prednisolone Acetate add 2
mL of sulfuric acid, and allow to stand for 2 to 3 minutes: a
deep red color, without fluorescence, develops. To this solu-
tion add 10 mL of water cautiously: the color disappears and
a gray, flocculent precipitate is formed.
(2) Determine the infrared absorption spectra of Pred-
nisolone Acetate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum in a range between 4000
cm -1 and 650 cm -1 with the Infrared Reference Spectrum or
the spectrum of previously dried Prednisolone Acetate Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers. If any difference ap-
pears, dissolve the sample and the Reference Standard in
ethanol (99.5), respectively, evaporate to dryness, and repeat
the test on the residues.
Optical rotation <2.49> [a]o- + 128
70 mg, methanol, 20 mL, 100 mm).
137° (after drying,
Purity Related substanes — Dissolve 0.20 g of Prednisolone
Acetate in exactly 10 mL of a mixture of chloroform and
methanol (9:1), and use this solution as the sample solution.
Separately, dissolve 20 mg each of prednisolone, cortisone
acetate and hydrocortisone acetate in exactly 10 mL of a mix-
ture of chloroform and methanol (9:1). Pipet 1 mL of this so-
lution, add a mixture of chloroform and methanol (9:1) to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of dichloromethane, diethyl
ether, methanol and water (385:75:40:6) to a distance of
about 15 cm, and air-dry the plate. Examine under ultraviolet
light (wavelength: 254 mm): the spots from the sample solu-
tion corresponding to those from the standard solution are
1024 Prednisolone Succinate / Official Monographs
JP XV
not more intense than the spots from the standard solution,
and any spot from the sample solution other than the prin-
cipal spot and the spots from prednisolone, cortisone acetate
and hydrocortisone acetate does not appear.
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Dissolve about 10 mg each of Prednisolone Acetate
and Prednisolone Acetate Reference Standard, previously
dried and accurately weighed, in 60 mL each of methanol,
add exactly 2 mL each of the internal standard solution, then
add methanol to make 100 mL, and use these solutions as the
sample solution and standard solution. Perform the text with
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, Q T and Q s ,
of the peak height of prednisolone acetate to that of the inter-
nal standard.
Amount (mg) of C 23 H 30 O 6 = W s x (g T /g s )
W s : Amount (mg) of Prednisolone Acetate Reference
Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in methanol (3 in 1000).
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
prednisolone acetate is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, prednisolone acetate and the internal standard are elut-
ed in this order with the resolution between these peaks being
not less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of prednisolone acetate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Prednisolone Succinate
"/U r i -'/P>=l/\'7lgiXxJU
C 25 H 32 8 : 460.52
ll/?,17,21-Trihydroxypregna-l,4-diene-3,20-dione
21-(hydrogen succinate) [2920-86-7]
Prednisolone Succinate, when dried, contains not
less than 91.0% and not more than 103. 0% of
C25H320 8 .
Description Prednisolone Succinate occurs as a white, fine,
crystalline powder. It is odorless.
It is freely soluble in methanol, soluble in ethanol (95), and
very slightly soluble in water and in diethyl ether.
Melting point: about 205°C (with decomposition).
Identification (1) To 2 mg of Prednisolone Succinate add
2 mL of sulfuric acid, and allow to stand for 2 to 3 minutes: a
deep red color, without fluorescence, develops. To this solu-
tion add 10 mL of water cautiously: the color disappears and
a gray, flocculent precipitate is formed.
(2) Determine the infrared absorption spectrum of Pred-
nisolone Succinate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Prednisolone Succinate Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Optical rotation <2.49> [ a ]o- + 114 -
67 mg, methanol, 10 mL, 100 mm).
120° (after drying,
Purity Related substances — Dissolve 0.10 g of Predniso-
lone Succinate in methanol to make exactly 10 mL, and use
this solution as the sample solution. Separately, dissolve 30
mg of prednisolone in methanol to make exactly 10 mL.
Pipet 1 mL of the solution, add methanol to make exactly 10
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 5 /ah of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate and ethanol (95) (2:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine the
plate under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 6 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 10 mg each of Prednisolone
JPXV
Official Monographs / Prednisolone Sodium Succinate for Injection 1025
Succinate and Prednisolone Succinate Reference Standard,
previously dried, and dissolve each in methanol to make
exactly 100 mL. Pipet 5 mL each of these solutions, add
methanol to make exactly 50 mL, and use these solutions as
the sample solution and standard solution. Determine the ab-
sorbances, A T and A s , of the sample solution and standard
solution at 242 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of C25H 32 8
= W s x(A T /A s )
W s : Amount (mg) of Prednisolone Succinate Reference
Standard
Containers and storage Containers — Tight containers.
Prednisolone Sodium Succinate
for Injection
CO,M
C 25 H 31 Na0 8 : 482.50
Monosodium 1 1/?,1 7,21 -trihydroxypregna-l,4-diene-3, 20-
dione 21-succinate [1715-33-9]
Prednisolone Sodium Succinate for Injection is a
preparation for injection which is dissolved before
used.
It contains not less than 72.4% and not more than
83.2% of prednisolone sodium succinate (C 25 H 31 Na0 8 ),
and the equivalent of not less than 90% and not more
than 110% of the labeled amount of prednisolone
(C 21 H 28 O s : 360.44).
The amount should be stated as the amount of pred-
nisolone (C 2 iH 2 80 5 ).
Method of preparation Prepare as directed under Injec-
tions, with Prednisolone Succinate and Dried Sodium Car-
bonate or Sodium Hydroxide.
It contains a suitable buffer agent.
Description Prednisolone Sodium Succinate for Injection
occurs as a white powder or porous, friable mass.
It is freely soluble in water.
It is hygroscopic.
Identification (1) To 2 mg of Prednisolone Sodium Suc-
cinate for Injection add 2 mL of sulfuric acid, and allow to
stand for 2 to 3 minutes: a deep red color, without fluores-
cence, develops. To this solution add 10 mL of water cauti-
ously: the color disappears and a gray, flocculent precipitate
is formed.
(2) Dissolve 0.01 g of Prednisolone Sodium Succinate for
Injection in 1 mL of methanol, add 1 mL of Fehling's TS,
and heat: an orange to red precipitate is formed.
(3) Dissolve 0.1 g of Prednisolone Sodium Succinate for
Injection in 2 mL of sodium hydroxide TS, allow to stand for
10 minutes, and filter. Add 1 mL of dilute hydrochloric acid
to the filtrate, shake, and filter if necessary. Adjust the solu-
tion with diluted ammonia TS (1 in 10) to a pH of about 6,
and add 2 to 3 drops of iron (III) chloride TS: a brown
precipitate is formed.
(4) Prednisolone Sodium Succinate for Injection
responds to the Qualitative Tests <1.09> (1) for sodium salt.
pH <2.54> Dissolve 1 .0 g of Prednisolone Sodium Succinate
for Injection in 40 mL of water: the pH of the solution is be-
tween 6.5 and 7.2.
Purity Clarity and color of solution — Dissolve 0.25 g of
Prednisolone Sodium Succinate for Injection in 10 mL of
water: the solution is clear and colorless.
Loss on drying <2.41> Not more than 2.0% (0.15 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Assay Take a quantity of sealed containers of Prednisolone
Sodium Succinate for Injection, equivalent to about 0.1 g of
prednisolone (C21H28O5), and dissolve the contents in a suita-
ble amount of diluted methanol (1 in 2), and transfer to a
100-mL volumetric flask. Wash each container with diluted
methanol (1 in 2), collect the washings in the volumetric flask,
and add diluted methanol (1 in 2) to make volume. Pipet 4
mL of this solution, add diluted methanol (1 in 2) to make ex-
actly 50 mL. Pipet 5 mL of this solution, add exactly 5 mL of
the internal standard solution, mix, and use this solution as
the sample solution. Separately, weigh accurately about 25
mg of Prednisolone Succinate Reference Standard, previous-
ly dried in a desiccator for 6 hours (in vacuum, phosphorus
(V) oxide, 60°C), dissolve in methanol to make exactly 25
mL. Pipet 5 mL of this solution, add diluted methanol (1 in
2) to make exactly 50 mL. Pipet 5 mL of this solution, add
exactly 5 mL of the internal standard solution, mix, and use
this solution as the standard solution. Perform the test with
10 /uL of the sample solution and standard solution as direct-
ed under Liquid Chromatography according <2.01> to the fol-
lowing conditions, and calculate the ratios, g T and Q s , of the
peak area of prednisolone succinate to that of the internal
standard.
Amount (mg) of prednisolone sodium succinate
(C 25 H 31 Na0 8 )
= W s x (Q T /Q S ) x 5 x 1 .0477
Amount (mg) of prednisolone (C21H28O5)
= W s x (Qj/Qs) x 5 x 0.7827
W s : Amount (mg) of Prednisolone Succinate Reference
Standard
Internal standard solution — A solution of propyl para-
hydroxybenzoate in diluted methanol (1 in 2) (1 in 25,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 0.32 g of tetra M-butylammonium
bromide, 3.22 g of disodium hydrogen phosphate dodacahy-
1026 Primidone / Official Monographs
JP XV
drate and 6.94 g of potassium dihydrogen phosphate in 1000
mL of water. To 840 mL of this solution add 1160 mL of
methanol.
Flow rate: Adjust the flow rate so that the retention time of
prednisolone succinate is about 15 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, prednisolone succinate and the internal standard are
eluted in this order with the resolution between these peaks
being not less than 6.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of prednisolone succinate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Primidone
H
0-... A...
w-
C 12 H 14 N 2 2 : 218.25
5-Ethyl-5-phenyl-2,3-dihyropyrimidine-4,6(l//,5//)-dione
[125-33-7]
Primidone, when dried, contains not less than
98.5% of C 12 H 14 N 2 2 .
Description Primidone occurs as a white, crystalline pow-
der or granules. It is odorless and has a slightly bitter taste.
It is soluble in 7V,7V-dimethylformamide, sparingly soluble
in pyridine, slightly soluble in ethanol (95), very slightly solu-
ble in water, and practically insoluble in diethyl ether.
Identification (1) Heat 0.5 g of Primidone with 5 mL of
diluted sulfuric acid (1 in 2): the odor of formaldehyde is per-
ceptible.
(2) Mix 0.2 g of Primidone with 0.2 g of anhydrous sodi-
um carbonate, and heat: the gas evolved changes moistened
red litmus paper to blue.
Melting point <2.60> 279 - 284°C
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Primidone in 10 mL of 7V,./V-dimethylformamide: the solu-
tion is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Primi-
done according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) 2-Ethyl-2-phenylmalonediamide — Dissolve 0.10 g of
Primidone in 2 mL of pyridine, add exactly 2 mL of the inter-
nal standard solution, then add 1 mL of bis-trimethyl silyl
acetamide, shake well, and heat at 100°C for 5 minutes.
Cool, add pyridine to make 10 mL, and use this solution as
the sample solution. Separately, dissolve 50 mg of 2-ethyl-2-
phenylmalonediamide in pyridine to make exactly 100 mL.
Pipet 2 mL of this solution, add exactly 2 mL of the internal
standard solution, proceed in the same manner as Primidone,
and use this solution as the standard solution. Perform the
test with 2 fiL of the sample solution and standard solution as
directed under Gas Chromatography <2.02> according to the
following conditions, and calculate the ratios, Q T and Q s , of
the peak area of 2-ethyl-2-phenylmalonediamide to that of
the internal standard: Q T is not more than Q s .
Internal standard solution — A solution of stearylalcohol in
pyridine (1 in 2000).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 150
cm in length, packed with siliceous earth for gas chro-
matography (125 to 150 /am in particle diameter) coated with
50% phenyl-methyl silicon polymer for gas chromatography
at the ratio of 3%.
Column temperature: A constant temperature of about
195°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
stearylalcohol is about 10 minutes.
System suitability —
System performance: When the procedure is run with 2 ftL
of the standard solution under the above operating condi-
tion, 2-ethyl-2-phenylmalonediamide and the internal stan-
dard are eluted in this order with the resolution between these
peaks being not less than 3.
System repeatability: When the test is repeated 5 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of 2-ethyl-2-phenylmalonediamide to that of the
internal standard is not more than 1.5%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 20 mg each of Primidone
and Primidone Reference Standard, previously dried, dis-
solve each in 20 mL of ethanol (95) by warming, and after
cooling, add ethanol (95) to make exactly 25 mL, and use
these solutions as the sample solution and standard solution,
respectively. Determine the absorbance, A,, of the sample so-
lution and the standard solution at the wavelength of maxi-
mum absorption at about 257 nm, and the absorbances, A 2
and A 3 , at the wavelength of minimum absorption at about
254 nm and at about 261 nm, as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using ethanol (95) as the
blank.
Amount (mg) of C^H^^C^
= W s x {(2A,-A 2 -A 3 ) T /(2A I -A 2 -A 3 ) S }
W s : Amount (mg) of Primidone Reference Standard
where, (2^4, — A 2 — A 3 ) T is the value from the sample solu-
tion, and (2^4, — A 2 — A 3 ) s is from the standard solution.
Containers and storage Containers — Tight containers.
JPXV
Official Monographs / Probenecid Tablets 1027
Probenecid
H,G
CO;H
Assay Weigh accurately about 0.5 g of Probenecid, previ-
ously dried, and dissolve in 50 mL of neutralized ethanol. Ti-
trate <2.50> with 0.1 mol/L sodium hydroxide VS (indicator:
3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 28.54 mg of C 13 H 19 N0 4 S
Containers and storage Containers — Well-closed contain-
ers.
C 13 H 19 N0 4 S: 285.36
4-(Dipropylaminosulfonyl)benzoic acid [57-66-9]
Probenecid Tablets
Probenecid, when dried,
98.0% of C 13 H 19 N0 4 S.
contains not less than "/n-K^-v HIS;
Description Probenecid occurs as white crystals or crystal-
line powder. It is odorless, and has a slightly bitter taste, fol-
lowed by unpleasant bitter.
Probenecid is sparingly soluble in ethanol (95), slightly
soluble in diethyl ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS and in ammonia TS.
Melting point: 198 - 200°C
Identification (1) Heat Probenecid strongly: the odor of
sulfur dioxide is perceptible.
(2) Determine the absorption spectrum of a solution of
Probenecid in ethanol (95) (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Probenecid Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Purity (1) Acidity— To 2.0 g of Probenecid add 100 mL
of water, heat on a water bath with occasional shaking for 30
minutes, cool, and filter. To the filtrate add 1 drop of
phenolphthalein TS and 0.50 mL of 0.1 mol/L sodium
hydroxide VS: a red color develops.
(2) Chloride <1.03>— To 1.0 g of Probenecid add 100 mL
of water and 1 mL of nitric acid, and heat on a water bath
with occasional shaking for 30 minutes. After cooling, add, if
necessary, water to make 100 mL, and filter. Perform the test
using 50 mL of the filtrate as the test solution. Prepare the
control solution with 0.30 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.021%).
(3) Sulfate <1.14>— To 1.0 g of Probenecid add 100 mL
of water and 1 mL of hydrochloric acid, and heat on a water
bath with occasional shaking for 30 minutes. After cooling,
add, if necessary, water to make 100 mL, and filter. Perform
the test using 50 mL of the filtrate as the test solution. Pre-
pare the control solution with 0.40 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.038%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of
Probenecid according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Probenecid according to Method 3, and perform the test
(not more than 2 ppm).
Loss on drying <2.41>
4 hours).
Not more than 0.5% (1 g, 105°C,
Probenecid Tablets contain not less than 95% and
not more than 105% of the labeled amount of
probenecid (C 13 H 19 N0 4 S: 285.36).
Method of preparation
with Probenecid.
Prepare as directed under Tablets,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Identification (1) Weigh a quantity of powdered
Probenecid Tablets, equivalent to 0.5 g of Probenecid ac-
cording to the labeled amount, add 50 mL of ethanol (95) and
1 mL of 1 mol/L hydrochloric acid TS, shake, and filter.
Evaporate the filtrate on a water bath to about 20 mL. After
cooling, collect produced crystals, recrystallize with 50 mL of
dilute ethanol, and dry at 105°C for 4 hours: it melts <2.60>
between 198°C and 200 C C. With the crystals so obtained,
proceed as directed in the Identification (1) under
Probenecid.
(2) Determine the absorption spectrum of a solution of
the dried crystals obtained in (1) in ethanol (95) (1 in 50,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits maxima between 224 nm and 226 nm and
between 247 nm and 249 nm, and a minimum between 234
nm and 236 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: It meets the requirement.
Perform the test with 1 tablet of Probenecid Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of 2nd fluid for dissolution test as the test solu-
tion. Take 30 mL or more of the dissolved solution 30
minutes after start of the test, and filter through a membrane
filter with pore size of not more than 0.8 fim. Discard the first
10 mL of the filtrate, pipet the subsequent FmL, add 2nd
fluid for dissolution test to make exactly V mL so that each
mL contains about 14 /ng of probenecid (Ci 3 H 19 N0 4 S) ac-
cording to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 70 mg of
Probenecid Reference Standard, previously dried at 105°C
for 4 hours, and dissolve in 2nd fluid for dissolution test to
make exactly 100 mL. Pipet 1 mL of this solution, add 2nd
fluid for dissolution test to make exactly 50 mL, and use this
solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 244 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>. The dissolution rate of Probenecid
Tablets in 30 minutes is not less than 80%.
Dissolution rate (%) with respect to the
1028 Procainamide Hydrochloride / Official Monographs
JP XV
labeled amount of probenecid (Ci 3 H 19 N0 4 S)
= W s x(A T /A s )x(V'/V)x(l/C)xn
W s : Amount (mg) of Probenecid Reference Standard.
C: Labeled amount (mg) of probenecid (Ci 3 H 19 N0 4 S) in 1
tablet.
Assay Weigh accurately, and powder not less than 20
Probenecid Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 0.15 g of probenecid (Ci3H I9 N0 4 S),
add 200 mL of ethanol (95) and 5 mL of 1 mol/L hydrochlor-
ic acid TS, and heat on a water-bath at 70°C for 30 minutes
with occasional shaking. After cooling, add ethanol (95) to
make exactly 250 mL, and filter. Discard the first 20 mL of
the filtrate. To 5 mL of the subsequent filtrate, exactly meas-
ured, add 5 mL of 0.1 mol/L hydrochloric acid TS, dilute
with ethanol (95) to exactly 250 mL, and use this solution as
the sample solution. Separately, weigh accurately about
0.15 g of Probenecid Reference Standard, previously dried at
105 °C for 4 hours, dissolve in 5 mL of 1 mol/L hydrochloric
acid TS, and add ethanol (95) to make exactly 250 mL. Pipet
5 mL of the solution, add 5 mL of 0.1 mol/L hydrochloric
acid TS and ethanol (95) to make exactly 250 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 248 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using a solution prepared by mixing 5
mL of 0.1 mol/L hydrochloric acid TS and ethanol (95) to
make exactly 250 mL as the blank.
Amount (mg) of probenecid (C 13 H 19 N0 4 S)
= W s x(A T /A s )
W s : Amount (mg) of Probenecid Reference Standard
Containers and storage Containers — Well-closed contain-
ers.
Procainamide Hydrochloride
•HCI
C 13 H 21 N 3 O.HCl: 271.79
4-Amino-/V-(2-diethylaminoethyi)benzamide
monohydrochloride [614-39-1]
Procainamide Hydrochloride, when dried, contains
not less than 98.0% of C 13 H 21 N 3 O.HCl.
Description Procainamide Hydrochloride occurs as a white
to light yellow, crystalline powder. It is odorless.
It is very soluble in water, freely soluble in methanol, in
acetic acid (100) and in ethanol (95), slightly soluble in acetic
anhydride, and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) Dissolve 1 g of Procainamide
Hydrochloride in 10 mL of water, add 10 mL of sodium
hydroxide TS, and extract with two 10-mL portions of a mix-
ture of diethyl ether and chloroform (1:1). Combine the ex-
tracts, add calcium chloride for drying, and dry the extracts
for 30 minutes. Decant the solution into a small flask, add 5
mL of pyridine, and slowly add dropwise 1 mL of benzoyl
chloride. Heat the mixture on a water bath for 30 minutes,
add 20 mL of a mixture of diethyl ether and chloroform
(1:1), shake, and pour the mixture into 100 mL of sodium
hydroxide TS, then shake. Separate the organic solvent layer,
wash it with 20 mL of water, cool to 10°C, and allow the
crystals to separate. Collect the separated crystals, recrystal-
lize from 10 mL of dilute ethanol, and dry at 105°C for 1
hour: the crystals so obtained melt <2.60> between 180°C and
187°C.
(2) Dissolve 0.01 g of Procainamide Hydrochloride in 1
mL of dilute hydrochloric acid and 4 mL of water: the solu-
tion responds to the Qualitative Tests <1.09> for primary aro-
matic amines.
(3) A solution of Procainamide Hydrochloride (1 in 20)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1 .0 g of Procainamide Hydrochloride in
10 mL of water: the pH of this solution is between 5.0 and
6.5.
Melting point <2.60> 165 - 169 C C
Purity (1) Clarity of solution — Dissolve 1 .0 g of
Procainamide Hydrochloride in 10 mL of water: the solution
is clear.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Procainamide Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Procainamide Hydrochloride according to Method 1, and
perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Procainamide
Hydrochloride in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Then spot
10,mL each of a solution of ammonia solution (28) in
methanol (11 in 50) on each of the above spots. Develop the
plate with a mixture of chloroform, methanol and ammonia
solution (28) (700:300:7) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 4-dimethylaminobenzaldehyde
TS for spray on the plate: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.30% (2 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Assay Weigh accurately about 0.5 g of Procainamide
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 27.18 mg of C 13 H 21 N 3 O.HCl
JPXV
Official Monographs / Procainamide Hydrochloride Tablets 1029
Containers and storage Containers — Tight containers.
Procainamide Hydrochloride
Injection
Procainamide Hydrochloride Injection is an aque-
ous solution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of procainamide
hydrochloride (C 13 H 21 N 3 0.HC1: 271.79).
Method of preparation Prepare as directed under Injec-
tions, with Procainamide Hydrochloride.
Description Procainamide Hydrochloride Injection is a
clear, colorless or light yellow liquid.
pH: 4.0-6.0
Identification (1) Proceed with a volume of Procainamide
Hydrochloride Injection, equivalent to 1 g of Procainamide
Hydrochloride according to the labeled amount, as directed
in the Identification (1) under Procainamide Hydrochloride.
(2) Dilute a volume of Procainamide Hydrochloride In-
jection, equivalent to 0.01 g of Procainamide Hydrochloride
according to the labeled amount, with 1 mL of dilute
hydrochloric acid and water to 5 mL: the solution responds
to the Qualitative Tests <1.09> for primary aromatic amines.
(3) Procainamide Hydrochloride Injection responds to
the Qualitative Tests <1.09> (2) for chloride.
Assay Dilute an accurately measured volume of
Procainamide Hydrochloride Injection, equivalent to about
0.5 g of procainamide hydrochloride (Ci 3 H 21 N 3 O.HCl), with
5 mL of hydrochloric acid and water to 50 mL, cool to 15°C,
and titrate <2.50> with 0.1 mol/L sodium nitrite VS (poten-
tiometric titration method or amperometric titration).
Each mL of 0.1 mol/L sodium nitrite VS
= 27.18 mg of C 13 H 21 N 3 0/HC1
Containers and storage Containers — Hermetic containers.
Procainamide Hydrochloride
Tablets
Procainamide Hydrochloride Tablets contain not
less than 95% and not more than 105% of the labeled
amount of procainamide hydrochloride
(C 13 H 21 N 3 O.HCl: 271.79).
Method of preparation Prepare as directed under Tablets,
with Procainamide Hydrochloride.
Identification (1) Shake a quantity of powdered
Procainamide Hydrochloride Tablets, equivalent to 1.5 g of
Procainamide Hydrochloride according to the labeled
amount, with 30 mL of water, filter, and use the filtrate as the
sample solution. To 20 mL of the sample solution add 10 mL
of sodium hydroxide TS, and proceed as directed in the Iden-
tification (1) under Procainamide Hydrochloride.
(2) To 0.2 mL of the sample solution obtained in (1) add
1 mL of dilute hydrochloric acid and 4 mL of water: the solu-
tion responds to the Qualitative Tests <1.09> for primary aro-
matic amines.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Procainamide Hydrochlo-
ride Tablets at 50 revolutions per minute according to the
Paddle method, using 900 mL of water as the test solution.
Take 30 mL or more of the dissolved solution 30 minutes af-
ter start of the test, and filter through a membrane filter with
pore size of not more than 0.8 fim. Discard the first 10 mL of
the filtrate, pipet the subsequent KmL, add 2nd fluid for dis-
solution test to make exactly V mL so that each mL contains
about 7 fig of procainamide hydrochloride (Ci 3 H 2 iN 3 O.HCl)
according to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 0.125 g
of procainamide hydrochloride for assay, previously dried at
105°C for 4 hours, and dissolve in water to make exactly 1000
mL. Pipet 5 mL of this solution, add 2nd fluid for dissolution
test to make exactly 100 mL, and use this solution as the stan-
dard solution. Determine the absorbances, A T and A s , of the
sample solution and standard solution at 278 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>. The dis-
solution rate of Procainamide Hydrochloride Tablets in 30
minutes is not less than 80%.
Dissolution rate (%) with respect to the labeled amount
of procainamide hydrochloride (C 13 H 2I N 3 O.HCl)
= W s x(A T /A s )x(V'/V)x(l/C)x4.5
W s : Amount (mg) of procainamide hydrochloride for as-
say.
C: Labeled amount (mg) of procainamide hydrochloride
(C 13 H 21 N 3 O.HCl) in 1 tablet.
Assay Weigh accurately and powder not less than 20
Procainamide Hydrochloride Tablets. Weigh accurately a
portion of the powder, equivalent to about 0.5 g of
procainamide hydrochloride (C 13 H 2 iN 3 O.HCl), stir well with
25 mL of 1 mol/L hydrochloric acid TS, centrifuge, and
separate the supernatant liquid. Wash the residue with four
10-mL portions of 1 mol/L hydrochloric acid VS in the same
manner. Add 10 mL of a solution of potassium bromide (3 in
10) to the combined supernatant liquid, cool to below 15°C,
and titrate <2.50> with 0.1 mol/L sodium nitrite VS (poten-
tiometric titration or amperometric titration).
Each mL of 0.1 mol/L sodium nitrite VS
= 27.18 mg of C n H 21 N 3 O.HCl
Containers and storage Containers — Tight containers.
1030 Procaine Hydrochloride / Official Monographs
JP XV
Procaine Hydrochloride
-fu-hi >£K£
H 2 N
■HCI
C 13 H 20 N 2 O 2 .HCl: 272.77
2-(Diethylamino)ethyl 4-aminobenzoate monohydrochloride
[51-05-8]
Procaine Hydrochloride, when dried, contains not
less than 99.0% of C 13 H 20 N 2 O 2 .HCl.
Description Procaine Hydrochloride occurs as white crys-
tals or crystalline powder.
It is very soluble in water, soluble in ethanol (95), and prac-
tically insoluble in diethyl ether.
Identification (1) Determine the absorption spectrum of a
solution of Procaine Hydrochloride (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Procaine Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Procaine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> The pH of a solution prepared by dissoluing 1.0
g of Procaine Hydrochloride in 20 mL of water is between 5.0
and 6.0.
Melting point <2.60> 155 - 158°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Procaine Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Procaine
Hydrochloride according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Related substances — To 1 .0 g of Procaine Hydrochlo-
ride add 5 mL of ethanol (95), dissolve by mixing well, add
water to make exactly 10 mL, and use this solution as the
sample solution. Separately, dissolve 10 mg of 4-aminoben-
zoic acid in ethanol (95) to make exactly 20 mL, then pipet 1
mL of this solution, add 4 mL of ethanol (95) and water to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of dibutyl ether, w-hexane
and acetic acid (100) (20:4:1) to a distance of about 10 cm,
and air-dry the plate. After drying the plate more at 105°C
for 10 minutes, examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution. The principal spot from the sam-
ple solution stays at the origin.
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Procaine
Hydrochloride, previously dried, dissolve in 5 mL of
hydrochloric acid and 60 mL of water, add 10 mL of a solu-
tion of potassium bromide (3 in 10), cool to below 15°C, and
titrate <2.50> with 0.1 mol/L sodium nitrite VS (potentiomet-
ric titration or amperometric titration).
Each mL of 0.1 mol/L sodium nitrite VS
= 27.28 mg of C n H 20 N 2 O 2 .HCl
Containers and storage Containers — Well-closed contain-
ers.
Procaine Hydrochloride Injection
^n7]^>M^ilt«
Procaine Hydrochloride Injection is an aqueous so-
lution for injection.
It contains not less than 95% and not more than
105% of the labeled amount of procaine hydrochloride
(C 13 H 2 oN 2 2 .HCl: 272.77).
Method of preparation Prepare as directed under Injec-
tions, with Procaine Hydrochloride.
Description Procaine Hydrochloride Injection is a clear,
colorless liquid.
Identification (1) To a volume of Procaine Hydrochloride
Injection, equivalent to 0.01 g of Procaine Hydrochloride ac-
cording to the labeled amount, add water to make 1000 mL.
Determine the absorption spectrum of this solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits maxima between 219 nm and 223 nm, and between 289
nm and 293 nm.
(2) Procaine Hydrochloride Injection responds to the
Qualitative Tests <1.09> (2) for chloride.
pH <2.54> 3.3-6.0
Extractable volume <6.05> It meets the requirement.
Assay To an exactly measured volume of Procaine
Hydrochloride Injection, equivalent to about 20 mg of
procaine hydrochloride (Ci 3 H 20 N 2 O 2 .HCl), add the mobile
phase to make exactly 20 mL. Pipet 5 mL of this solution,
add exactly 5 mL of the internal standard solution and the
mobile phase to make 20 mL, and use this solution as the
sample solution. Separately, weigh accurately about 50 mg of
procaine hydrochloride for assay, previously dried in a desic-
cator (silica gel) for 4 hours, dissolve in the mobile phase to
make exactly 50 mL. Pipet 5 mL of this solution, add exactly
5 mL of the internal standard solution and the mobile phase
to make 20 mL, and use this solution as the standard solu-
JPXV
Official Monographs / Procarbazine Hydrochloride 1031
tion. Perform the test with 5 fiL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, Q T and Q s , of the peak area of
procaine hydrochloride to that of the internal standard.
Amount (mg) of procaine hydrochloride
(C I3 H 20 N 2 O 2 .HCl)
= W s x(Q T /Q s )x(2/5)
W s : Amount (mg) of procaine hydrochloride for assay
Internal standard solution — A solution of caffeine in the mo-
bile phase (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 6 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 jum in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Adjust the pH of 0.05 mol/L potassium di-
hydrogen phosphate TS to 3.0 with phosphoric acid, and add
an amount of sodium 1-pentane sulfonate to make a solution
so that containing 0.1%. To 800 mL of this solution add 200
mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
procaine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, procaine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 8.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of procaine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Hermetic containers.
Procarbazine Hydrochloride
f^i
O CH;
AnA
H
H
H*\ ,n^
A^J
1
H
CH 3
■HCI
C 12 H 19 N 3 O.HCl: 257.76
A f -(l-Methylethyl)-4-[(2-methylhydrazino)methyl]benzamide
monohydrochloride [366-70-1]
Procarbazine Hydrochloride, when dried, contains
not less than 98.5% and not more than 101.0% of
C 12 H 19 N 3 O.HCl.
Description Procarbazine Hydrochloride occurs as white to
light yellowish white crystals or crystalline powder.
It is freely soluble in water, and slightly soluble in ethanol
(99.5).
It dissolves in dilute hydrochloric acid.
Melting point: about 223°C (with decomposition).
Identification (1) Dissolve 0.01 g of Procarbazine
Hydrochloride in 1 mL of diluted copper (II) sulfate TS (1 in
10), and add 4 drops of sodium hydroxide TS: a green
precipitate is formed immediately, and the color changes
from green through yellow to orange.
(2) Determine the absorption spectrum of a solution of
Procarbazine Hydrochloride in 0.1 mol/L hydrochloric acid
TS (1 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Procarbazine Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Procarbazine Hydrochloride (1 in 20)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 0.10 g of Procarbazine Hydrochloride
in 10 mL of water: the pH of this solution is between 3.0 and
5.0.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Procarbazine Hydrochloride according to Method 4, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 50 mg of Procarbazine
Hydrochloride in 5.0 mL of a solution of L-cysteine
hydrochloride in diluted methanol (7 in 10) (1 in 200), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add a solution of L-cysteine hydrochloride in dilut-
ed methanol (7 in 10) (1 in 200) to make exactly 50 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Immerse slowly, by inclining, a plate of
silica gel with fluorescent indicator for thin-layer chro-
matography in a solution of L-cysteine hydrochloride in dilut-
ed methanol (7 in 10) (1 in 200), allow to stand for 1 minute,
lift the plate from the solution, dry it in cold wind for 10
minutes, then dry in warm wind for 5 minutes, and then dry
at 60°C for 5 minutes. After cooling, spot 5 /uL each of the
sample solution and standard solution on the plate. Develop
the plate with a mixture of methanol and ethyl acetate (1 : 1) to
a distance of about 12 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): not more
than 1 spot other than the principal spot and the spot of the
starting point from the sample solution appears, and is not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.15 g of Procarbazine
Hydrochloride, previously dried, place in a glass-stoppered
flask, dissolve in 25 mL of water, add 25 mL of hydrochloric
acid, and cool to room temperature. To this solution add 5
mL of chloroform, and titrate <2.50>, while shaking, with
0.05 mol/L potassium iodate VS until the purple color of the
1032 Procaterol Hydrochloride Hydrate / Official Monographs
JP XV
chloroform layer disappears. The end point is reached when
the red-purple color of the chloroform layer no more reap-
pears within 5 minutes after the purple color disappeared.
Each mL of 0.05 mol/L potassium iodate VS
= 8.592 mg of C 12 H 19 N 3 0.HC1
Containers and storage Containers — Tight containers.
Procaterol Hydrochloride Hydrate
• HCI
CH 3 and enantiomer
C 16 H 22 N 2 3 .HC1.V 2 H 2 0: 335.83
8-Hydroxy-5-{(l/?S,25i?)-l-hydroxy-
2-[(l-methylethyl)amino]butyl]}quinolin-2(l//)-one
monohydrochloride hemihydrate [62929-91-3, anhydride]
Procaterol Hydrochloride Hydrate contains not
less than 98.5% of procaterol hydrochloride
(C 16 H 22 N 2 3 .HC1: 326.82), calculated on the anhy-
drous basis.
Description Procaterol Hydrochloride Hydrate occurs as
white to pale yellowish white crystals or crystalline powder.
It is soluble in water, in formic acid and in methanol,
slightly soluble in ethanol (95), and practically insoluble in
diethyl ether.
The pH of a solution of Procaterol Hydrochloride Hydrate
(1 in 100) is between 4.0 and 5.0.
It is gradually colored by light.
Melting point: about 195 °C (with decomposition).
The solution of Procaterol Hydrochloride Hydrate (1 in
20) shows no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Procaterol Hydrochloride Hydrate (7 in
1,000,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Procaterol Hydrochloride Hydrate as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Procaterol Hydrochloride Hydrate (1 in
50) responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Procaterol Hydrochloride Hydrate in 30 mL of water: the so-
lution is clear, and has no more color than the following con-
trol solution.
Control solution: To 3.0 mL of Ferric Chloride Stock CS
add water to make 50 mL.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Procaterol Hydrochloride Hydrate according to Method 2,
and perform the test. Prepare the control solution with 2.0
mL of Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 0.10 g of Procaterol
Hydrochloride Hydrate in 100 mL of diluted methanol (1 in
2), and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add diluted methanol (1 in 2) to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with exactly 2 iuL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of these solutions by the au-
tomatic integration method: the total area of the peaks other
than procaterol from the sample solution is not larger than
the peak area of procaterol from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 0.87 g of sodium 1-pentanesul-
fonate in 1000 mL of water. To 760 mL of this solution add
230 mL of methanol and 10 mL of glacial acetic acid.
Flow rate: Adjust the flow rate so that the retention time of
procaterol is about 15 minutes.
Selection of column: Dissolve 20 mg each of Procaterol
Hydrochloride Hydrate and threoprocaterol hydrochloride in
100 mL of diluted methanol (1 in 2). To 15 mL of this solu-
tion add diluted methanol (1 in 2) to make 100 mL. Proceed
with 2 /uL of this solution under the above operating condi-
tions, and calculate the resolution. Use a column giving elu-
tion of procaterol and threoprocaterol in this order with the
resolution of these peaks being not less than 3.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of procaterol obtained from 2 juL of the
standard solution is not less than 10 mm.
Time span of measurement: 2.5 times as long as the reten-
tion time of procaterol beginning after the solvent peak.
Water <2.48> 2.5 - 3.3% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Procaterol
Hydrochloride Hydrate, add 2 mL of formic acid, dissolve
by warming, and add exactly 15 mL of 0.1 mol/L perchloric
acid VS. Add 1 mL of acetic anhydride, heat on a water bath
for 30 minutes, cool, add 60 mL of acetic anhydride, and ti-
trate <2.50> the excess perchloric acid with 0.1 mol/L sodium
acetate VS (potentiometric titration). Perform a blank deter-
mination.
Each mL of 0.1 mol/L perchloric acid VS
= 32.68 mg of C 16 H 22 N 2 3 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
JPXV
Official Monographs / Prochlorperazine Maleate Tablets 1033
Prochlorperazine Maleate
^n^nMT/>7l/'( >m)k
r?
-CH 3
c
CO^H
"CO ? H
C 20 H 2 4ClN3S.2C4H 4 O4: 606.09
2-Chloro-10-[3-(4-methylpiperazin-l-yl)propyl]-
10//-phenothiazine dimaleate [84-02-6]
Prochlorperazine Maleate, when dried, contains not
less than 98.0% of QoH^ClNjS^I^CV
Description Prochlorperazine Maleate occurs as a white to
light yellow powder. It is odorless, and has a slightly bitter
taste.
It is slightly soluble in acetic acid (100), very slightly solu-
ble in water and in ethanol (95), and practically insoluble in
diethyl ether.
It gradually acquires a red tint by light.
Melting point: 195 -203°C (with decomposition).
Identification (1) Dissolve 5 mg of Prochlorperazine
Maleate in 5 mL of sulfuric acid: a red color develops, which
darkens slowly on standing. Warm a half of the solution: the
color changes to red-purple. To the remainder add 1 drop of
potassium dichromate TS: a green-brown color develops,
which changes to brown on standing.
(2) Boil 0.5 g of Prochlorperazine Maleate with 10 mL of
hydrobromic acid under a reflux condenser for 10 minutes.
After cooling, add 100 mL of water, and filter through glass
filter (G4). Wash the residue with three 10-mL portions of
water, and dry at 105 °C for 1 hour: it melts <2.60> between
195 °C and 198 °C (with decomposition).
(3) Dissolve 0.2 g of Prochlorperazine Maleate in 5 mL
of a solution of sodium hydroxide (1 in 10), and extract with
three 3-mL portions of diethyl ether [reserve the aqueous lay-
er, and use for test (4)]. Evaporate the combined diethyl ether
extracts on a water bath to dryness, dissolve the residue in 10
mL of methanol by warming, and pour into 30 mL of a solu-
tion of 2,4,6-trinitrophenol in methanol (1 in 75), previously
warmed to 50°C. Allow to stand for 1 hour, collect the crys-
tals, wash with a small amount of methanol, and dry at
105°C for 1 hour: the crystals melt <2.60> between 252°C and
258°C (with decomposition).
(4) To the aqueous layer reserved in (3) add boiling chips,
and heat on a water bath for 10 minutes. Cool, add 2 mL of
bromine TS, heat on a water bath for 10 minutes, and heat
the solution to boil. After cooling, add 2 drops of this solu-
tion to 3 mL of a solution of resorcinol in sulfuric acid (1 in
300), and heat on a water bath for 15 minutes: a red-purple
color is produced.
Purity Heavy metals <1.07> — Proceed with 1.0 g of
Prochlorperazine Maleate according to Method 2, and per-
form the test. Prepare the control solution with 1.0 mL of
Standard Lead Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, 105 C C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Prochlorperazine
Maleate, previously dried, dissolve in 60 mL of acetic acid
(100) while stirring and warming. Cool, and titrate <2.50>
with 0.05 mol/L perchloric acid VS until the color of the so-
lution changes from orange to green (indicator: 0.5 mL of p-
naphtholbenzein TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 15.15 mg of C 20 H 2 4ClN3S.2C4H 4 O4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Prochlorperazine Maleate Tablets
Prochlorperazine Maleate Tablets contain not less
than 95% and not more than 105% of the labeled
amount of prochlorperazine maleate
(C 20 H 2 4ClN 3 S.2C 4 H 4 O 4 : 606.09).
Method of preparation Prepare as directed under Tablets,
with Prochlorperazine Maleate.
Identification (1) Weigh a quantity of powdered
Prochlorperazine Maleate Tablets, equivalent to 5 mg of
Prochlorperazine Maleate according to the labeled amount,
add 15 mL of acetic acid (100), shake, and filter. To 5 mL of
the filtrate add 3 mL of sulfuric acid, and shake: a light red
color develops. To this solution add 1 drop of potassium
dichromate TS: a green-brown color is produced and changes
to brown on standing.
(2) Weigh a quantity of powdered Prochlorperazine
Maleate Tablets, equivalent to 0.08 g of Prochlorperazine
Maleate according to the labeled amount, add 15 mL of
methanol and 1 mL of dimethylamine, shake, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 0.08 g of Prochlorperazine Maleate Reference Stan-
dard in 15 mL of a mixture of methanol and dimethylamine
(15:1), and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 [iL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of 1-
butanol and ammonia TS (15:2) to a distance of about 10 cm,
and air-dry the plate. Spray evenly palladium (II) chloride TS
on the plate: the spots obtained from the sample solution and
standard solution show a red-purple color, and has the same
Rf value.
(3) To a quantity of powdered Prochlorperazine Maleate
Tablets, equivalent to 0.04 g of Prochlorperazine Maleate ac-
cording to the labeled amount, add 10 mL of 1 mol/L
hydrochloric acid TS and 20 mL of diethyl ether, shake, and
centrifuge. Transfer the diethyl ether layer to a separator,
wash with 5 mL of 0.05 mol/L sulfuric acid TS, and
evaporate on a water bath to dryness. Dissolve the residue in
1034 Progesterone / Official Monographs
JP XV
5 mL of sulfuric acid TS, filter, if necessary, and add 1 to 2
drops of potassium permanganate TS: the red color of the
test solution is discharged immediately.
Assay Weigh accurately and powder not less than 20
Prochlorperazine Maleate Tablets using an agate mortar.
Weigh accurately a portion of the powder, equivalent to
about 16 mg of prochlorperazine maleate (C 2 oH 24 ClN 3 S.2C 4
H4O4), transfer to a glass-stoppered centrifuge tube, add ex-
actly 25 mL of a mixture of 7V,Af-dimethylformamide and
dimethylamine (100:1), stopper tightly, shake vigorously for
15 minutes, and centrifuge. Use the supernatant liquid as the
sample solution. Separately, weigh accurately about 64 mg of
Prochlorperazine Maleate Reference Standard, previously
dried in a desiccator (in vacuum, silica gel) for 4 hours, dis-
solve in a mixture of A^Af-dimethylformamide and
dimethylamine (100:1) to make exactly 100 mL, and use this
solution as the standard solution. Pipet 4 mL each of the
sample solution and the standard solution into glass-stop-
pered centrifuge tubes, add exactly 10 mL of boric acid-
potassium chloride-sodium hydroxide buffer solution, pH
9.0, and 20 mL of cyclohexane, stopper tightly, and cen-
trifuge after shaking vigorously for 5 minutes. Pipet 10 mL
each of the cyclohexane layer of these solutions into glass-
stoppered centrifuge tubes, add exactly 20 mL of palladium
(II) chloride TS and 5 mL of iV,./V-dimethylformamide, stop-
per tightly, and centrifuge after shaking vigorously for 15
minutes. Determine the absorbances, A T and A s , of the water
layers obtained from the sample solution and the standard
solution at 495 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using palladium (II) chloride TS as the
blank.
Amount (mg) of prochlorperazine maleate
(C 2 „H 24 C1N3S.2C4H40 4 )
= W s x(A T /A s )X (1/4)
W s : Amount (mg) of Prochlorperazine Maleate Reference
Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Progesterone
C 21 H 30 O 2 : 314.46
Pregn-4-ene-3,20-dione [57-83-0]
Progesterone, when dried, contains not less than
97.0% and not more than 103.0% of C 21 H 30 O 2 .
Description Progesterone occurs as white crystals or crys-
talline powder. It is odorless.
It is soluble in methanol, in ethanol (95), in ethanol (99.5)
and in 1,4-dioxane, sparingly soluble in diethyl ether, and
practically insoluble in water.
Identification (1) To 0.05 g of progesterone add a solution
of 0.05 g of hydroxylammonium chloride and 0.05 g of anhy-
drous sodium acetate in 5 mL of ethanol (95). Boil for 2
hours under a reflux condenser, evaporate the ethanol to 3
mL, and add 10 mL of water. Filter by suction, and wash the
precipitate on the filter with a small amount of water.
Recrystallize from dilute ethanol, and dry at 105°C for 1
hour: the dried crystals melt <2.60> between 235 °C and
240°C.
(2) Determine the infrared absorption spectrum of
Progesterone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Progesterone Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers. If any difference appears
between the spectra, dissolve Progesterone and Progesterone
Reference Standard in ethanol (95), respectively, then
evaporate the ethanol to dryness, and repeat the test on the
residues.
Optical rotation <2.49> [a£°: + 174 - + 182° (after drying,
0.2 g, 1,4-dioxane, 10 mL, 100 mm).
Melting point <2.60> 128 - 133°C or 120 - 122°C
Purity Related substances — Dissolve 80 mg of Progester-
one in 2 mL of methanol, and use this solution as the sample
solution. Pipet 1 mL of this solution, add methanol to make
exactly 100 mL, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 //L each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of chloroform and diethyla-
mine (19:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 10 mg of Progesterone,
previously dried, and dissolve in ethanol (99.5) to make ex-
actly 100 mL. To 5 mL of this solution, exactly measured,
add ethanol (99.5) to make exactly 50 mL, and determine the
absorbance A at the wavelength of maximum absorption at
about 241 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of C 21 H 30 O 2 =(.4/540) x 10,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Progesterone Injection
-fn?X7-n>&ttfe
Progesterone Injection is an oily solution for injec-
JPXV
Official Monographs / Proglumide 1035
tion.
It contains not less than 90% and not more than
110% of the labeled amount of progesterone
(C 21 H 30 O 2 : 314.46).
Method of preparation Prepare as directed under Injec-
tions, with Progesterone.
Description Progesterone Injection is a clear, colorless to
pale yellow, oily liquid.
Identification Transfer a volume of Progesterone Injection,
equivalent to 0.02 g of Progesterone according to the labeled
amount, to a separator. Add 40 mL of hexane, and mix
thoroughly, then extract with three 20-mL portions of diluted
ethanol (99.5) (9 in 10). Evaporate the combined extracts on a
water bath to dryness. Add 75 mg of 2,4-dinitrophenylhydra-
zine and 30 mL of ethanol (95) to the residue, and boil for 15
minutes under a reflux condenser. Add 1 mL of hydrochloric
acid, and heat for 15 minutes. Cool, and collect the
precipitate on a glass filter (G4). Wash the precipitate with
five 10-mL portions of hexane and three 5-mL portions of
ethanol (95). Then wash with diluted hydrochloric acid (1 in
20) until the washings become colorless, and dry at 105°C for
3 hours: the residue melts <2.60> between 269°C and 275°C.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Progesterone Injection,
equivalent to about 50 mg of progesterone (C21H30O2), and
dissolve in chloroform to make exactly 100 mL. To exactly
measured 3 mL of this solution add chloroform to make ex-
actly 50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 50 mg of Progesterone
Reference Standard, previously dried in a desiccator (in vacu-
um, phosphorus (V) oxide) for 4 hours, and prepare the stan-
dard solution in the same manner as directed for the prepara-
tion of the sample solution. Pipet 5 mL each of the sample
solution and standard solution, add exactly measured 10 mL
of isoniazid TS and methanol to make exactly 20 mL, respec-
tively. Allow to stand for 45 minutes, and perform the test
with these solutions as directed under Ultraviolet-visible
Spectrophotometry <2.24>, using a solution, prepared with 5
mL of chloroform in the same manner, as the blank. Deter-
mine the absorbances, A T and A s , of the subsequent solu-
tions of the sample solution and the standard solution at 380
nm.
Amount (mg) of progesterone (C21H30O2)
= W s x(A T /A s )
W s : Amount (mg) of Progesterone Reference Standard
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Proglumide
CO.H
and enantiomer
C 18 H2 6 N 2 4 : 334.41
(4i?S)-4-Benzoylamino-/V, 7V-dipropylglutaramic acid
[6620-60-6]
Proglumide, when dried, contains not less than
98.5% of C 18 H 26 N 2 4 .
Description Proglumide occurs as white crystals or crystal-
line powder.
It is freely soluble in methanol, soluble in ethanol (95),
sparingly soluble in diethyl ether, and very slightly soluble in
water.
A solution of Proglumide in methanol (1 in 10) shows no
optical rotation.
Identification (1) Put 0.5 g of Proglumide in a round bot-
tom tube, add 5 mL of hydrochloric acid, seal the tube, and
heat the tube carefully at 120°C for 3 hours. After cooling,
open the tube, filter the content to collect crystals separated
out, wash the crystals with 50 mL of water, and dry at 100°C
for 1 hour: the melting point <2.60> of the crystals is between
121 °C and 124°C.
(2) Determine the infrared absorption spectrum of
Proglumide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Absorbance <2.24> E[f m (225 nm): 384 - 414 (after drying,
4 mg, methanol, 250 mL)
Melting point <2.60> 148 - 150 C C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Proglumide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.U>— To 1.0 g of Proglumide add lOmLof
a solution of magnesium nitrate hexahydrate in ethanol (95)
(1 in 10) and 1.5 mL of hydrogen peroxide (30), burn the
ethanol, and prepare the test solution according to Method 3,
and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Proglumide in
5 mL of methanol, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add methanol to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of cyclohexane, ethyl
1036 Promethazine Hydrochloride / Official Monographs
JP XV
acetate, acetic acid (100) and methanol (50:18:5:4) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.10% (1 g, reduced
pressure, phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.16 g of Proglumide, previ-
ously dried, dissolve in 40 mL of methanol, add 10 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 33.44 mg of C 18 H 26 N 2 4
Containers and storage Containers — Well-closed contain-
ers.
Promethazine Hydrochloride
-fu/-9if>i§mt&
N
H
CH 3
■HCI
and enantiomer
C 17 H 20 N 2 S.HC1: 320.88
(2jRS)-Af,iV-Dimethyl- 1 -(1 0//-phenothiazin- 1 0-yl)propan-
2-ylamine monohydrochloride [55-55-5]
Promethazine Hydrochloride, when dried, contains
not less than 98.0% of C 17 H 20 N 2 S.HC1.
Description Promethazine Hydrochloride occurs as a white
to light yellow powder.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), sparingly soluble in acetic anhydride, and
practically insoluble in diethyl ether.
It is gradually colored by light.
A solution of Promethazine Hydrochloride (1 in 25) shows
on optical rotation.
Melting point: about 223 °C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Promethazine Hydrochloride (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Promethazine Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Dissolve 0.5 g of Promethazine Hydrochloride in 5
mL of water, add 2 mL of ammonia TS, and filter. To 5 mL
of the filtrate add dilute nitric acid to make acidic: the solu-
tion responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> The pH of a solution of Promethazine
Hydrochloride (1 in 10) is between 4.0 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Promethazine Hydrochloride in 10 mL of water, protecting
from direct sunlight: the solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Promethazine Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Perform the test under the pro-
tection from sunlight. Dissolve 0.10 g of Promethazine
Hydrochloride in exactly 5 mL of ethanol (95), and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add ethanol (95) to make exactly 200 mL, and use this
solution as the standard solution (1). Separately, dissolve 20
mg of isopromethazine hydrochloride for thin-layer chro-
matography in ethanol (95) to make exactly 100 mL, and use
this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solutions (1) and (2) on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of methanol and diethylamine (19:1)
to a distance of about 12 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
from the sample solution corresponding to the spots from the
standard solution (2) are not more intense than the spot from
the standard solution (2), and any spot other than the prin-
cipal spot from the sample solution is not more intense than
the spot from the standard solution (1).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Promethazine
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 32.09 mg of C, 7 H 20 N 2 S.HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Propantheline Bromide
Br
C 2 3H 30 BrNO 3 : 448.39
N-Methyl-A r ,Af-bis(l-methylethyl)-2-[(9//-xanthen-
9-ylcarbonyl)oxy]ethylaminium bromide [50-34-0]
JPXV
Official Monographs / Propranolol Hydrochloride 1037
Propantheline Bromide, when dried, contains not
less than 98.0% and not more than 102.0% of
CzsHsoBrNOj.
Description Propantheline Bromide occurs as a white to
yellowish white, crystalline powder. It is odorless and has a
very bitter taste.
It is very soluble in water, in ethanol (95), in acetic acid
(100) and in chloroform, soluble in acetic anhydride, and
practically insoluble in diethyl ether.
The pH of a solution of Propantheline Bromide (1 in 50) is
between 5.0 and 6.0.
Melting point: about 161 °C (with decomposition, after
drying).
Identification (1) To 5 mL of a solution of Propantheline
Bromide (1 in 20) add 10 mL of sodium hydroxide TS, heat to
boil for 2 minutes. Cool to 60°C, and add 5 mL of dilute
hydrochloric acid. After cooling, collect the precipitates, and
wash with water. Recrystallize from dilute ethanol, and dry
at 105°C for 1 hour: the crystals melt <2.60> between 217°C
and 222°C.
(2) Dissolve 0.01 g of the crystals obtained in (1) in 5 mL
of sulfuric acid: a vivid yellow to yellow-red color develops.
(3) To 5 mL of a solution of Propantheline Bromide (1 in
10) add 2 mL of dilute nitric acid: this solution responds to
the Qualitative Tests <1.09> (1) for bromide.
Purity Xanthene-9-carboxylic acid and xanthone — Dissolve
10 mg of Propantheline Bromide in exactly 2 mL of chlo-
roform, and use this solution as the sample solution.
Separately, dissolve 1.0 mg of xanthene-9-carboxylic acid
and 1.0 mg of xanthone in exactly 40 mL of chloroform, and
use this solution as the standard solution. Perform the test
immediately with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 25 /uL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography, and air-dry the
plate for 10 minutes. Develop the plate with a mixture of 1,2-
dichloroethane, methanol, water and formic acid (56:24:1:1)
to a distance of about 12 cm, and air-dry the plate. Examine
under ultraviolet light: the spots from the sample solution
corresponding to the spots from the standard solution are not
more intense than those from the standard solution.
Loss on drying <2.41> Not more than 0.5% (2 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Propantheline
Bromide, previously dried, dissolve in 50 mL of a mixture of
acetic anhydride and acetic acid (100) (7:3), and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 44.84 g of C 23 H 30 BrNO3
Containers and storage Containers — Well-closed contain-
ers.
Propranolol Hydrochloride
H OH
• HCI
and enantiomer
C 16 H 2 ,N0 2 .HC1: 295.80
(2i?S>l-(l-Methylethyl)amino-3-(naphthalen-
l-yloxy)propan-2-ol monohydrochloride [318-98-9]
Propranolol Hydrochloride, when dried, contains
not less than 99.0% and not more than 101.0% of
C 16 H 21 N0 2 .HC1.
Description Propranolol Hydrochloride occurs as a white,
crystalline powder.
It is freely soluble in methanol, soluble in water and in
acetic acid (100), and sparingly soluble in ethanol (99.5).
A solution of Propranolol Hydrochloride in methanol (1 in
40) shows no optical rotation.
It is gradualy colored to yellowish white to light brown by
light.
Identification (1) Determine the absorption spectrum of a
solution of Propranolol Hydrochloride in methanol (1 in
50,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths
(2) Determine the infrared absorption spectrum of
Propranolol Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Propranolol Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> The pH of a solution prepared by dissolving 0.5
g of Propranolol Hydrochloride in 50 mL of water is 5.0 -
6.0.
Melting point <2.60> 163 - 166°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Propranolol Hydrochloride in 20 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Propranolol Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 20 mg of Propranolol
Hydrochloride in 10 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 2 mL of the sample solu-
tion, and add the mobile phase to make exactly 100 mL.
Pipet 1 mL of this solution, add the mobile phase to make ex-
actly 10 mL, and use this solution as the standard solution.
Perform the test with exactly 20 /uL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
1038 Propranolol Hydrochloride Tablets / Official Monographs
JP XV
and determine each peak area by the automatic integration
method: the area of the peak other than propranolol is not
larger than 1/2 times the peak area of propranolol from the
standard solution, and the total area of the peaks other than
the peak of propranolol is not larger than 2 times the peak
area of propranolol from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 292 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.6 g of sodium lauryl sulfate and
0.31 g of tetrabutylammonium phosphate in 450 mL of
water, add 1 mL of sulfuric acid and 550 mL of acetonitrile
for liquid chromatography, and adjust to pH 3.3 with 2
mol/L sodium hydroxide TS.
Flow rate: Adjust the flow rate so that the retention time of
propranolol is about 4 minutes.
Time span of measurement: About 5 times as long as the
retention time of propranolol.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add the mobile phase to make ex-
actly 20 mL. Confirm that the peak area of propranolol ob-
tained with 20 /xL of this solution is equivalent to 17 to 33%
of that with 20 /xL of the standard solution.
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of propranolol is not less than 3000 and not
more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
propranolol is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Propranolol
Hydrochloride, previously dried, dissolove in 50 mL of a
mixture of acetic anhydride and acetic acid (100) (7:3), and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (potentiomet-
ric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.58 mg of C 16 H 21 N0 2 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Propranolol Hydrochloride Tablets
-fu^^JU-ili^M^M
Propranolol Hydrochloride Tablets contain not
less than 95.0% and not more than 105.0% of the
labeled amount of propranolol hydrochloride
(C 16 H 21 N0 2 .HC1: 295.80).
Method of preparation Prepare as directed under Tablets,
with Propranolol Hydrochloride.
Identification Determine the absorption spectrum of the
sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>; it exhibits max-
ima between 288 nm and 292 nm, and between 317 nm and
321 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Propranolol Hydrochloride Tablets add 20
mL of water, and shake until the tablet is completely disin-
tegrated. Add 50 mL of methanol, shake vigorously for 10
minutes, then add methanol to make exactly 100 mL, and
filter. Discard the first 20 mL of the filtrate, pipet KmL of the
subsequent filtrate, add methanol to make exactly V mL so
that each mL contains about 20 /xg of propranolol hydrochlo-
ride (C 16 H 21 N0 2 .HC1), and use this solution as the sample so-
lution. Separately, weigh accurately about 50 mg of
propranolol hydrochloride for assay, previously dried at
105 °C for 4 hours, and dissolve in methanol to make exactly
50 mL. Pipet 2 mL of this solution, add methanol to make
exactly 100 mL, and use this solution as the standard solu-
tion. Determine the absorbances, A T and A s , of the sample
solution and the standard solution at 290 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of propranolol hydrochloride
(C 16 H 21 N0 2 .HC1)
= W s x (A T /A S ) x (V'/V) x (1/25)
W s : Amount (mg) of propranolol hydrochloride for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Propranolol Hydrochlo-
ride Tablets at 50 revolutions per minute according to the
Paddle method, using 900 mL of water as the dissolution
medium. Withdraw 20 mL or more of the dissolution medi-
um 15 minutes after starting the test, and filter through a
membrane filter with pore size of not more than 0.45 //m.
Discard the first 10 mL of the filtrate, pipet KmL of the sub-
sequent filtrate, add water to make exactly V mL so that
each mL contains about 10 /xg of propranolol hydrochloride
(C 16 H 21 N0 2 .HC1) according to the labeled amount, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of propranolol hydrochloride for assay,
previously dried at 105°C for 4 hours, and dissolve in water
to make exactly 50 mL. Pipet 1 mL of this solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and ^4 S , of
the sample solution and standard solution at 290 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>.
The dissolution rate in 15 minutes is not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
propranolol hydrochloride (C 16 H 21 N0 2 .HC1)
= W s x (A T /A S ) x (V'/V)* (1/C) x 18
W s : Amount (mg) of propranolol hydrochloride for assay
C: Labeled amount (mg) of propranolol hydrochloride
JPXV
Official Monographs / Propylene Glycol 1039
(C 16 H 21 N0 2 .HC1) in 1 tablet
Assay Weigh accurately the mass of not less than 20
Propranolol Hydrochloride Tablets, and powder. Weigh ac-
curately a portion of the powder, equivalent to about 20 mg
of propranolol hydrochloride (C 16 H 2 iN0 2 .HCl), add 60 mL
of methanol, shake for 10 minutes, and add methanol to
make exactly 100 mL. Filter, discard the first 20 mL of the
filtrate, pipet 10 mL of the subsequent filtrate, add methanol
to make exactly 100 mL, and use this solution as the sample
solution. Separately, weigh accurately about 50 mg of
propranolol hydrochloride for assay, previously dried at
105 °C for 4 hours, and dissolve in methanol to make exactly
50 mL. Pipet 2 mL of this solution, add methanol to make
exactly 100 mL, and use this solution as the standard solu-
tion. Determine the absorbances, A T and A s , of the sample
solution and the standard solution at 290 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of propranolol hydrochloride
(C 16 H 21 N0 2 .HC1)
= W s x(A T /A s )x(2/5)
W s : Amount (mg) of propranolol hydrochloride for assay
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Propyl Parahydroxybenzoate
/\°75r*y$lfi7n tiJL-
■ ;.' ; '
HO
^-\^-CHj
Propyl 4-hydroxybenzoate [94-13-3]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Propyl Parahydroxybenzoate contains not less than
98.0% and not more than 102.0% of C 10 H 12 O 3 .
♦Description Propyl Parahydroxybenzoate occurs as color-
less crystals or a white, crystalline powder.
It is freely soluble in ethanol (95) and in acetone, and very
slightly soluble in water. »
Identification (1) The melting point <2.60> of Propyl
Parahydroxybenzoate is between 96°C and 99°C.
*(2) Determine the infrared absorption spectrum of
Propyl Parahydroxybenzoate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.*
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Propyl Parahydroxybenzoate in 10 mL of ethanol (95): the
solution is clear and not more intensely colored than the fol-
lowing control solution.
Control solution: To 5.0 mL of cobalt (II) chloride colori-
metric stock solution, 12.0 mL of iron (III) chloride colori-
metric stock solution and 2.0 mL of cupper (II) sulfate colori-
metric stock solution add water to make 1000 mL.
(2) Acidity — Dissolve 0.20 g of Propyl Parahydroxyben-
zoate in 5 mL of ethanol (95), add 5 mL of freshly boiled and
cooled water and 0.1 mL of bromocresol green-sodium
hydroxide-ethanol TS, then add 0.1 mL of 0.1 mol/L sodium
hydroxide VS: the solution shows a blue color.
*(3) Heavy metals <1.07> — Dissolve 1.0 g of Propyl
Parahydroxybenzoate in 25 mL of acetone, add 2 mL of di-
lute acetic acid and water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 2.0 mL of Standard Lead Solution
add 25 mL of acetone, 2 mL of dilute acetic acid, and water
to make 50 mL (not more than 20 ppm).»
(4) Related substances — Dissolve 0.10 g of Propyl Para-
hydroxybenzoate in 10 mL of acetone, and use this solution
as the sample solution. Pipet 0.5 mL of the sample solution,
add acetone to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 2
/uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
methanol, water and acetic acid (100) (70:30:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spot other than the
principal spot is not more intense than the spot obtained with
the standard solution.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 .0 g of Propyl Parahydrox-
ybenzoate add exactly 20 mL of 1 mol/L VS, heat at about 70
°C for 1 hour, and immediately cool in ice. Titrate <2.50> the
excess sodium hydroxide with 0.5 mol/L sulfuric acid VS up
to the second equivalent point (potentiometric titration). Per-
form a blank determination.
Each mL of 1 mol/L sodium hydroxide VS
= 180.2 mg of C 10 H I2 O 3
♦Containers and storage Containers — Well-closed contain-
ers. ♦
Propylene Glycol
-fa t°u><yu =i-ju
H ;! C
H OH
and enantiomer
C 3 H 8 2 : 76.09
(2i?S>Propane-l,2-diol [57-55-6]
Description Propylene Glycol is a clear, colorless, viscous
liquid. It is odorless, and has a slightly bitter taste.
It is miscible with water, with methanol, with ethanol (95)
and with pyridine.
It is freely soluble in diethyl ether.
It is hygroscopic.
Identification (1) Mix 2 to 3 drops of Propylene Glycol
with 0.7 g of triphenylchloromethane, add 1 mL of pyridine,
1040 Propylthiouracil / Official Monographs
JP XV
and heat under a reflux condenser on a water bath for 1 hour.
After cooling, dissolve the mixture in 20 mL of acetone by
warming, shake with 0.02 g of activated charcoal, and filter.
Concentrate the filtrate to about 10 mL, and cool. Collect the
separated crystals, and dry in a desiccator (silica gel) for 4
hours: the crystals melt <2.60> between 174°C and 178°C.
(2) Heat gently 1 mL of Propylene Glycol with 0.5 g of
potassium hydrogen sulfate: a characteristic odor is evolved.
Specific gravity <2.56> d™: 1.035 - 1.040
Purity (1) Acidity — Mix 10.0 mL of Propylene Glycol
with 50 mL of freshly boiled and cooled water, and add 5
drops of phenolphthalein TS and 0.30 mL of 0.1 mol/L sodi-
um hydroxide VS: the solution has a red color.
(2) Chloride <1.03>— Perform the test with 2.0 g of
Propylene Glycol. Prepare the control solution with 0.40 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.007%).
(3) Sulfate <1.14>— Perform the test with 10.0 g of
Propylene Glycol. Prepare the control solution with 0.40 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.002%).
(4) Heavy metals <1.07> — Perform the test with 5.0 g of
Propylene Glycol according to Method 1. Prepare the control
solution with 2.5 mL of Standard Lead Solution (not more
than 5 ppm).
(5) Arsenic </.//> — Prepare the test solution with 1.0 g
of Propylene Glycol according to Method 1, and perform the
test (not more than 2 ppm).
(6) Glycerin — Heat 1.0 g of Propylene Glycol with 0.5 g
of potassium hydrogen sulfate and evaporate to dryness: no
odor of acrolein is perceptible.
Water <2.48> Not more than 0.5% (2 g, direct titration).
Residue on ignition <2.44> Weigh accurately about 20 g of
Propylene Glycol in a tared crucible, and heat to boiling.
Stop heating, and immediately ignite to burn. Cool, moisten
the residue with 0.2 mL of sulfuric acid, and heat strongly
with care to constant mass: the mass of the residue is not
more than 0.005%.
Distilling range <2.57> 184 - 189°C, not less than 95 vol%.
Containers and storage Containers — Tight containers.
Propylthiouracil
-fa tfju^Tl-^vJU
H
H-,C
C 7 H 10 N 2 OS: 170.23
6-Propyl-2-thiouracil [51-52-5]
Propylthiouracil, when dried, contains not less than
98.0% of C 7 H 10 N 2 OS.
Description Propylthiouracil occurs as a white powder. It is
odorless, and has a bitter taste.
It is sparingly soluble in ethanol (95), and very slightly
soluble in water and in diethyl ether.
It dissolves in sodium hydroxide TS and in ammonia TS.
Identification (1) Shake well 0.02 g of Propylthiouracil
with 7 mL of bromine TS for 1 minute, and heat until the
color of bromine TS disappears. Cool, filter, and add 10 mL
of barium hydroxide TS to the filtrate: a white precipitate is
produced. The color of the precipitate does not turn purple
within 1 minute.
(2) To 5 mL of a hot saturated solution of
Propylthiouracil add 2 mL of a solution of sodium pentac-
yanoammine ferroate (II) w-hydrate (1 in 100): a green color
develops.
Melting point <2.60> 218 - 221 °C
Purity (1) Sulfate <1.14> — Triturate Propylthiouracil fine-
ly in a mortar. To 0.75 g of the powder add 25 mL of water,
heat for 10 minutes on a water bath, cool, filter, and wash the
residue with water until the volume of the filtrate becomes 30
mL. To 10 mL of the filtrate add 1 mL of dilute hydrochloric
acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.077%).
(2) Thiourea — Dissolve 0.30 g of Propylthiouracil in 50
mL of water by heating under a reflux condenser for 5
minutes, cool, and filter. To 10 mL of the filtrate add 3 mL of
ammonia TS, shake well, and add 2 mL of silver nitrate TS:
the solution has no more color than the following control so-
lution.
Control solution: Weigh exactly 60 mg of thiourea, and
dissolve in water to make exactly 100 mL. Pipet 1 mL of this
solution, add water to make exactly 100 mL, and proceed
with 10 mL of this solution in the same manner.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Propylthiouracil,
previously dried, and add 30 mL of water. Add 30 mL of 0.1
mol/L sodium hydroxide VS from a burette, heat to boil, and
dissolve by stirring. Wash down the solid adhering to the wall
of the flask with a small amount of water, and add 50 mL of
0.1 mol/L silver nitrate VS with stirring. Boil gently for 5
minutes, add 1 to 2 mL of bromothymol blue TS, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS until a persistent
blue-green color develops. Determine the total volume of 0.1
mol/L sodium hydroxide VS consumed.
Each mL of 0.1 mol/L sodium hydroxide VS
= 8.512 mg of C 7 H 10 N 2 OS
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Propylthiouracil Tablets
^ntfju^Tt-^vvJUSS
Propylthiouracil Tablets contain not less than 93%
and not more than 107% of the labeled amount of
propylthiouracil (C 7 H 10 N 2 OS: 170.23).
Method of preparation Prepare as directed under Tablets,
JPXV
Official Monographs / Protamine Sulfate 1041
with Propylthiouracil.
Identification To a quantity of powdered Propylthiouracil
Tablets, equivalent to 0.3 g of Propylthiouracil according to
the labeled amount, add 5 mL of ammonia TS, allow to
stand for 5 minutes with occasional shaking, add 10 mL of
water, and centrifuge. To the supernatant liquid add acetic
acid (31), collect the precipitate produced, recrystallize from
water, and dry at 105 C C for 1 hour: it melts <2.60> between
218°C and 221 °C. Proceed with the residue as directed in the
Identification under Propylthiouracil.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Propylthiouracil Tablets
at 75 revolutions per minute according to the Paddle method,
using 900 mL of 2nd fluid for dissolution test as the test solu-
tion. Take 20 mL or more of the dissolved solution 30
minutes after starting the test, and filter through a membrane
filter with pore size of not more than 0.8 fim. Discard the first
10 mL of the filtrate, pipet 5 mL of the subsequent filtrate,
add 2nd fluid for dissolution test to make exactly 50 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of propylthiouracil for assay, previous-
ly dried at 105°C for 3 hours, and dissolve in 2nd fluid for
dissolution test to make exactly 1000 mL. Pipet 5 mL of this
solution, add 2nd fluid for dissolution test to make exactly 50
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution at 274 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rate of
Propylthiouracil Tablets in 30 minutes should be not less
than 80%.
Dissolution rate (%) with respect to the
labeled amount of propylthiouracil (C 7 H I0 N 2 OS)
= W s x (Aj/As) x (1/ C) x 90
fV s : Amount (mg) of propylthiouracil for assay.
C: Labeled amount (mg) of propylthiouracil (C 7 H 10 N 2 OS)
in 1 tablet.
Assay Weigh accurately and powder not less than 20
Propylthiouracil Tablets. Weigh accurately a portion of the
powder, equivalent to about 0.3 g of propylthiouracil
(C 7 H 10 N 2 OS), transfer to a Soxhlet extractor, and extract
with 100 mL of acetone for 4 hours. Evaporate the acetone
extract by warming on a water bath to dryness. To the residue
add 30 mL of water, and proceed as directed in the Assay un-
der Propylthiouracil.
Each mL of 0.1 mol/L sodium hydroxide VS
= 8.512 mg of C 7 H 10 N 2 OS
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Protamine Sulfate
Protamine Sulfate is the sulfate of protamine pre-
pared from the mature spermary of fish belonging to
the family Salmonidae and others.
Description Protamine Sulfate occurs as a white to light
grayish yellow powder.
It is sparingly soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
The pH of a solution of Protamine Sulfate (1 in 100) is be-
tween 4.0 and 7.0.
Identification (1) Dissolve 1 mg of Protamine Sulfate in 2
mL of water, add 5 drops of a solution prepared by dissolv-
ing 0.1 g of 1-naphthol in 100 mL of diluted ethanol (7 in 10)
and 5 drops of sodium hypochlorite TS, then add sodium
hydroxide TS until the solution becomes alkaline: a vivid red
color develops.
(2) Dissolve 5 mg of Protamine Sulfate in 1 mL of water
by warming, add 1 drop of a solution of sodium hydroxide (1
in 10) and 2 drops of copper (II) sulfate TS: a red-purple
color develops.
(3) An aqueous solution of Protamine Sulfate (1 in 20)
responds to the Qualitative Tests <1.09> for sulfate.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Protamine Sulfate in 10 mL of water: the solution is clear
and colorless.
(2) Nitrogen — Weigh accurately about 10 mg of Prota-
mine Sulfate, previously dried at 105°C to constant mass,
and perform the test as directed under Nitrogen Determina-
tion <1.08>: not more than 0.255 mg of nitrogen (N: 14.01) is
found for each mg of Protamine Sulfate.
Potency as antiheparin (i) Sample solution — Dissolve
20.0 mg of Protamine Sulfate in isotonic sodium chloride so-
lution to make exactly 20 mL.
(ii) Heparin sodium standard solution — Dissolve 10.0 mg
of Heparin Sodium Reference Standard in isotonic sodium
chloride solution to make a standard solution containing ex-
actly 0.7 mg per ml.
(iii) Sulfated whole blood — Place 250 mL of fresh bovine
blood in a wide-mouthed stoppered polyethylene bottle con-
taining 50 mL of a solution of sodium sulfate decahydrate (9
in 50), and store between 1°C and 4°C. Remove any clotted
substance before use.
(iv) Thrombokinase extract — To 1.5 g of acetone-dried
cattle brain add 60 mL of water, extract at 50°C for 10 to 15
minutes, and centrifuge for 2 minutes at 1500 revolutions per
minute. To the supernatant add cresol to make 0.3% as a
preservative, and store between 1°C and 4°C. The potency of
this solution will be maintained for several days.
(v) Procedure — To one of 10 clean, glass-stoppered test
tubes, 13 mm in inside diameter and 150 mm in length, trans-
fer 1.30 mL of isotonic sodium chloride solution and 0.20
mL of thrombokinase extract, then add exactly 1 mL of sul-
fated whole blood, stopper the tube, mix the contents by in-
verting once, and note the time on a stop watch. When the
solid clot which is formed at the bottom of the tube does not
fall on inverting the tube, designate this time as the control
clotting time. Adjust appropriately the volume of throm-
bokinase extract so that the control clotting time is between 2
and 3 minutes. To the nine remaining tubes add 0.50 mL of
the sample solution and the same volume of thrombokinase
extract as was used in the previous measurement of the con-
trol clotting time, pipet into the tubes 0.43 mL, 0.45 mL, 0.47
mL, 0.49 mL, 0.50 mL, 0.51 mL, 0.53 mL, 0.55 mL and 0.57
mL of the heparin sodium standard solution, respectively,
1042 Protamine Sulfate Injection / Official Monographs
JP XV
and make the volume in each tube up to 1.50 mL by adding
isotonic sodium chloride solution. Add finally 1.0 mL of sul-
fated whole blood, stopper, mix the contents by inverting
once, and determine the clotting times with a stop watch. The
estimated ratio, v/V, is between 0.85 and 1.15, where v is the
volume of the heparin sodium standard solution and Fis the
volume of the sample solution in that tube in which the clot-
ting time is most nearly the same as the control clotting time.
Containers and storage Containers — Tight containers.
Prothionamide
-fnttTZ K
Protamine Sulfate Injection
C 9 H 12 N 2 S: 180.27
2-Propylpyridine-4-carbothioamide
[14222-60-7]
■7n*$>9HHiai**
Protamine Sulfate Injection is an aqueous solution
for injection.
The amount of Protamine Sulfate should be labeled.
Method of preparation Prepare as directed under Injec-
tions, with Protamine Sulfate.
Description Protamine Sulfate Injection is a colorless liq-
uid. It is odorless or has the odor of preservatives.
Identification (1) Dilute a volume of Protamine Sulfate
Injection, equivalent to 1 mg of Protamine Sulfate according
to the labeled amount, with water to make 2 mL, and proceed
as directed in the Identification (1) under Protamine Sulfate.
(2) Dilute a volume of Protamine Sulfate Injection,
equivalent to 5 mg of Protamine Sulfate according to the la-
beled amount, with water to make 1 mL, and proceed as
directed in the Identification (2) under Protamine Sulfate.
(3) Protamine Sulfate Injection responds to the Qualita-
tive Tests <1.09> for sulfate.
pH <2.54> 5.0-7.0
Purity Nitrogen — Transfer an exactly measured volume of
Protamine Sulfate Injection, equivalent to about 10 mg of
Protamine Sulfate according to the labeled amount, to a
Kjeldahl flask, and evaporate on a water bath with the aid of
a current of air to dryness. Perform the test as directed under
Nitrogen Determination <1.08>: 0.225 to 0.255 mg of nitro-
gen (N: 14.01) is found for each mg of the labeled amount of
Protamine Sulfate.
Extractable volume <6.05> It meets the requirement.
Potency as antiheparin Proceed as directed in the Potency
as antiheparin under Protamine Sulfate, but use the follow-
ing sample solution.
Sample solution: Dilute an exactly measured volume of
Protamine Sulfate Injection, equivalent to 20.0 mg of Prota-
mine Sulfate according to the labeled amount, with isotonic
sodium chloride solution to make exactly 20 mL.
Containers and storage Containers — Hermetic containers.
Prothionamide, when dried, contains not less than
98.0% of C 9 H 12 N 2 S.
Description Prothionamide occurs as yellow crystals or
crystalline powder. It has a slight, characteristic odor.
It is freely soluble in methanol and in acetic acid (100),
soluble in ethanol (95), slightly soluble in diethyl ether, and
practically insoluble in water.
It dissolves in dilute hydrochloric acid and in dilute sulfuric
acid.
Identification (1) Mix 0.05 g of Prothionamide with 0.1 g
of l-chloro-2,4-dinitrobenzene, transfer about 10 mg of this
mixture to a test tube, and heat for several seconds over a
small flame until the mixture is fused. Cool, and add 3 mL of
potassium hydroxide-ethanol TS: a red to orange-red color
develops.
(2) Place 0.5 g of Prothionamide in a 100-mL beaker,
and dissolve in 20 mL of sodium hydroxide TS by heating
while shaking occasionally: the gas evolved turns a moistened
red litmus paper to blue. Boil gently, and evaporate the solu-
tion to 3 to 5 mL. After cooling, add gradually 20 mL of
acetic acid (100), and heat on a water bath: the gas evolved
darkens moistened lead (II) acetate paper. Evaporate the so-
lution on a water bath to 3 to 5 mL with the aid of a current
of air, cool, add 10 mL of water, and mix well. Filter the
crystals by suction, recrystallize from water immediately, and
dry in a desiccator (in vacuum, silica gel) for 6 hours: the
crystals melt <2.60> between 198 C C and 203°C (with decom-
position).
Melting point <2.60> 142 - 145 °C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Prothionamide in 20 mL of ethanol (95): the solution is clear,
and shows a yellow color.
(2) Acidity — Dissolve 3.0 g of Prothionamide in 20 mL
of methanol with warming. Add 100 mL of water to the solu-
tion, cool in an ice water bath with agitation, and remove any
precipitate by filtration. Allow 80 mL of the filtrate to cool to
room temperature, and add 0.8 mL of cresol red TS and 0.20
mL of 0.1 mol/L sodium hydroxide VS: a red color develops.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Pro-
thionamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.6 g
of Prothionamide according to Method 3, and perform the
test. To the test solution add 10 mL of a solution of magnesi-
um nitrate hexahydrate in ethanol (95) (1 in 50), then add 1.5
mL of hydrogen peroxide (30), and ignite to burn (not more
JPXV
Official Monographs / Protirelin 1043
than 3.3 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 80°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Prothionamide,
previously dried, dissolve in 50 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from orange-red to dark oran-
ge-brown (indicator: 2 mL of /?-naphtholbenzein TS). Per-
form a blank determination.
Each mL of 0.1 mol/L perchloric acid VS
= 18.03 mg of C 9 H 12 N 2 S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Protirelin
"V- NH =
JrWb
°=U " o
C 16 H 22 N 6 4 : 362.38
5-Oxo-L-prolyl-L-histidyl-L-prolinamide [24305-27-9]
Protirelin contains not less than 98.5%
C 16 H 2 2N 6 4 , calculated on the dehydrated basis.
of
Description Protirelin occurs as a white powder.
It is freely soluble in water, in methanol, in ethanol (95)
and in acetic acid (100).
It is hygroscopic.
Identification (1) Take 0.01 g of Protirelin in a test tube
made of hard glass, add 0.5 mL of 6 mol/L hydrochloric acid
TS, seal the upper part of the tube, and heat carefully at
110°C for 5 hours. After cooling, open the seal, transfer the
contents into a beaker, and evaporate on a water bath to dry-
ness. Dissolve the residue in 1 mL of water, and use this solu-
tion as the sample solution. Separately, dissolve 0.08 g of l-
glutamic acid, 0.12 g of L-histidine hydrochloride monohy-
drate and 0.06 g of L-proline in 20 mL of water, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water, acetic acid (100) and pyridine (4:1:1:1) to a distance of
about 12 cm, and dry the plate at 100°C for 30 minutes.
Spray evenly a solution of ninhydrin in acetone (1 in 50) on
the plate, and heat at 80°C for 5 minutes: the three spots ob-
tained from the sample solution show the same color and the
same Rt value as each corresponding spots obtained from the
standard solution.
(2) Determine the infrared absorption spectrum of Pro-
tirelin, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Optical rotation <2.49> [a]g>: -66.0 - -69.0° (0.1 g calcu-
lated on the dehydrated basis, water, 20 mL, 100 mm).
pH <2.54> Dissolve 0.20 g of Protirelin in 10 mL of water:
the pH of this solution is between 7.5 and 8.5.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Protirelin in 10 mL of water: the solution is clear and
colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Protire-
lin according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Related substances — Dissolve 0.20 g of Protirelin in
10 mL of water, and use this solution as the sample solution.
Pipet 1 mL of this solution, add water to make exactly 200
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 5 [iL each of the sample solu-
tion and standard solution on a plate (1) of silica gel for thin-
layer chromatography, and spot 5 [iL of the sample solution
on a plate (2) of silica gel for thin-layer chromatography. De-
velop the plates with a mixture of 1-butanol, water, pyridine
and acetic acid (100) (4:2:1:1) to a distance of about 12 cm,
and dry the plates at 100°C for 30 minutes. Spray evenly a
mixture of a solution of sulfanilic acid in 1 mol/L
hydrochloric acid TS (1 in 200) and a solution of sodium ni-
trite (1 in 20) (1:1) on the plate (1), and air-dry the plates.
Successively spray evenly a solution of sodium carbonate
decahydrate (1 in 10) on it: the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution. Spray evenly a solution of
ninhydrin in acetone (1 in 50) on the plate (2), and heat at 80°
C for 5 minutes: no colored spot appears.
Water <2.48> Not more than 5.0% (0.1 g, direct titration).
Residue on ignition <2.44> Not more than 0.3% (0.2 g).
Assay Weigh accurately about 70 mg of Protirelin dissolve
in 50 mL of acetic acid (100), and titrate <2.50> with 0.02
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination, and make any necessary correction.
Each mL of 0.02 mol/L perchloric acid VS
= 7.248 mg of C 16 H 22 N 6 4
Containers and storage Containers — Tight containers.
1044 Protirelin Tartrate Hydrate / Official Monographs
JP XV
Protirelin Tartrate Hydrate
v- NH?
Oh
HO^C
C0 2 H
• HjO
C 16 H 22 N 6 4 .C 4 H 6 6 .H 2 0: 530.49
5-Oxo-L-prolyl-L-histidyl-L-prolinamide monotartrate
monohydrate [24305-27-9, Protirelin]
Protirelin Tartrate Hydrate, calculated on the anhy-
drous basis, contains not less than 98.5% of protirelin
tartrate (C 16 H 22 N 6 4 .C 4 H 6 6 : 512.48).
Description Protirelin Tartrate Hydrate occurs as white to
pale yellowish white crystals or crystalline powder.
It is freely soluble in water, sparingly soluble in acetic acid
(100), and practically insoluble in ethanol (95) and in diethyl
ether.
Melting point: about 187°C (with decomposition).
Identification (1) To 1 mL of a solution of Protirelin Tar-
trate Hydrate (1 in 1000) add 2 mL of a solution of 4-
nitrobenzene diazonium fluoroborate (1 in 2000) and 2 mL of
boric acid-potassium chloride-sodium hydroxide buffer solu-
tion, pH 9.0: a red color develops.
(2) Dissolve 0.03 g of Protirelin Tartrate Hydrate in 5 mL
of sodium hydroxide TS, add 1 drop of copper (II) sulfate
TS: a purple color develops.
(3) To 0.20 g of Protirelin Tartrate Hydrate add 5.0 mL
of 6 mol/L hydrochloric acid TS, and boil for 7 hours under
a reflux condenser. After cooling, evaporate 2.0 mL of this
solution on a water bath to dryness, dissolve the residue in
2.0 mL of water and use this solution as the sample solution.
Separately, dissolve 22 mg of L-glutamic acid, 32 mg of l-
histidine hydrochloride (monohydrate) and 17 mg of L-pro-
line in 2.0 mL of 0.1 mol/L hydrochloric acid TS by heating,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 2 /uL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, water, acetic acid (100) and pyridine (4:1:1:1) to a
distance of about 12 cm, and dry at 100°C for 30 minutes.
Spray evenly a solution of ninhydrin in acetone (1 in 50) on
the plate, and dry at 80°C for 5 minutes: the three spots ob-
tained from the sample solution show, respectively, the same
color and the same Rf value as the corresponding spot from
the standard solution.
(4) A solution of Protirelin Tartrate Hydrate (1 in 40)
responds to the Qualitative Tests <1.09> for tartrate.
Optical rotation <2.49> [a]™: ~ 50.0 - - 53.0° (0.5 g calcu-
lated on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Protirelin Tartrate Hydrate in
100 mL of water: the pH of this solution is between 3.0 and
4.0.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Protirelin Tartrate Hydrate in 10 mL of water: the solu-
tion is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Protire-
lin Tartrate Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Take 1.0 g of Protirelin Tartrate Hy-
drate in a porcelain crucible. Add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10), ig-
nite the ethanol, and heat gradually to incinerate. If a car-
bonized material still remains in this method, moisten with a
small quantity of nitric acid, and ignite to incinerate. After
cooling, add 10 mL of dilute hydrochloric acid, heat on a
water bath to dissolve the residue, use this solution as the test
solution, and perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.60 g of Protirelin
Tartrate Hydrate in 10 mL of water, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
(1) of silica gel for thin-layer chromatography. Spot 5 /uL of
the sample solution on a plate (2) of silica gel for thin-layer
chromatography. Develop the plates with a mixture of chlo-
roform, methanol and ammonia solution (28) (6:4:1) to a dis-
tance of about 10 cm, and dry at 100°C for 30 minutes. Spray
evenly a mixture of a solution of sulfanilic acid in 1 mol/L
hydrochloric acid TS (1 in 200) and a solution of sodium ni-
trite (1 in 20) (1:1) on the plate (1), and air-dry the plate.
Then, spray evenly a solution of sodium carbonate decahy-
drate (1 in 10) on the plate: the spots other than the principal
spot from the sample solution are not more intense than
those from the standard solution in color. On the other hand,
spray evenly a solution of ninhydrin in acetone (1 in 50) on
the plate (2), and dry at 80°C for 5 minutes: no colored spot
is obtained.
Water <2.48> Not more than 4.5% (0.2 g, direct titration).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 0.5 g of Protirelin Tartrate
Hydrate, dissolve in 80 mL of acetic acid (100) by warming,
cool, and titrate <2.50> with 0.1 mol/L perchloric acid VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 51.25 mg of C 16 H 22 N 6 4 .C 4 H 6 6
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Pyrantel Pamoate 1045
Pullulan
(Ci8H 30 Oi 5 ) n
Poly[6)-a-D-glucopyranosyl-(l->4)-a-D-
glucopyranosyl-(l -> 4)-a-D-glucopyranosyl-(l -» ]
[905 7-02- 7]
Pullulan is a neutral simple polysaccharide produced
by the growth of Aureobasidium pullulans. It has a
chain structure of repeated a- 1,6 binding of mal-
totriose composed of three glucoses in a- 1,4 binding.
Description Pullulan occurs as a white powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Identification (1) Dissolve 10 g of Pullulan in 100 mL of
water with stirring by adding in small portions: a viscous so-
lution is produced.
(2) Mix 10 mL of the viscous solution obtained in (1) with
0.1 mL of pullulanase TS, and allow to stand: the solution
loses its viscosity.
(3) To 10 mL of a solution of Pullulan (1 in 50) add 2 mL
of macrogol 600: a white precipitate is formed immediately.
Viscosity <2.53> Take exactly 10.0 g of Pullulan, previously
dried, dissolve in water to make exactly 100 g, and perform
the test at 30±0.1°C as directed in Method 1: the kinematic
viscosity is between 100 and 180mm 2 /s.
pH <2.54> Dissolve 1.0 g of Pullulan in 10 mL of freshly
boiled and cooled water: the pH is between 4.5 and 6.5.
Purity (1) Heavy metals <1.07> — Proceed with 4.0 g of
Pullulan according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 5 ppm).
(2) Nitrogen — Weigh accurately about 3 g of Pullulan,
previously dried, and perform the test as directed under
Nitrogen Determination <1.08>: the amount of nitrogen (N:
14.01) is not more than 0.05%. Use 12 mL of sulfuric acid for
the decomposition, and add 40 mL of a solution of sodium
hydroxide (2 in 5).
(3) Monosaccharide and oligosaccharides — Dissolve 0.8
g of Pullulan, previously dried, in 100 mL of water, and
designate this solution as the sample stock solution. To 1 mL
of the sample stock solution add 0.1 mL of potassium chlo-
ride saturated solution, and shake vigorously with 3 mL of
methanol. Centrifuge, and use the supernatant liquid as the
sample solution. Separately, pipet 1 mL of the sample stock
solution, add water to make exactly 50 mL, and use this solu-
tion as the standard solution. Pipet 0.2 mL each of the sam-
ple solution, the standard solution and water, transfer them
gently to each test tube containing 5 mL of a solution of an-
throne in diluted sulfuric acid (3 in 4) (1 in 500) and cooling in
ice water, stir immediately, then heat at 90°C for 10 minutes,
and cool immediately. Perform the test with these solutions
so obtained as directed under Ultraviolet-visible Spectrophto-
metry <2.24> using water as a blank, and determine the absor-
bances at 620 nm, A T , A s and A B : the amount of monosac-
charide and oligosaccharides is not more than 10.0%.
Amount (%) of monosaccharide and oligosaccharides
= {(^ T -^ B )/(^ S -^1 B )}X8.2
Loss on drying <2.41> Not more than 6.0% (1 g, in vacuum,
90°C, 6 hours).
Residue on ignition <2.44> Not more than 0.3% (2 g).
Containers and storage Containers — Well-closed contain-
ers.
Pyrantel Pamoate
GO = H
594.68
1 -Methyl-2- [( l£)-2-(thien-2-yl)vinyl] -1,4,5,6-
tetrahydropyrimidine mono [4,4' -methylenebis(3-
hydroxy-2-naphthoate)] (1/1) [22204-24-6]
Pyrantel Pamoate, when dried, contains not less
than 98.0% of C n H 14 N 2 S.C 2 3H 16 6 .
Description Pyrantel Pamoate occurs as a light yellow to
yellow, crystalline powder. It is odorless and tasteless.
It is sparingly soluble in 7V,iV-dimethylformamide, very
slightly soluble in methanol and in ethanol (95), and practi-
cally insoluble in water, in ethyl acetate and in diethyl ether.
Melting point: 256 - 264°C (with decomposition).
Identification (1) To 0.05 g of Pyrantel Pamoate add 10
mL of methanol and 1 mL of a mixture of hydrochloric acid
and methanol (1:1), and shake vigorously: a yellow
precipitate is produced. Filter the solution, and use the
filtrate as the sample solution. Use the precipitate for the test
(2). To 0.5 mL of the sample solution add 1 mL of a solution
of 2,3-indolinedione in sulfuric acid (1 in 1000): a red color
develops.
(2) Collect the precipitate obtained in the test (1), wash
with methanol, and dry at 105 °C for 1 hour. To 0.01 g of the
dried precipitate add 10 mL of methanol, shake well, and
filter. To 5 mL of the filtrate add 1 drop of iron (III) chloride
TS: a green color develops.
(3) Dissolve 0.1 g of Pyrantel Pamoate in 50 mL of N,N-
dimethylformamide, and add methanol to make 200 mL. To
2 mL of the solution add a solution of hydrochloric acid in
methanol (9 in 1000) to make 100 mL. Determine the absorp-
tion spectrum of the solution as directed under Ultraviolet-
1046 Pyrazinamide / Official Monographs
JP XV
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectrum of
Pyrantel Pamoate, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Purity (1) Chloride <7.0?>— To 1.0 g of Pyrantel Pamoate
add 10 mL of dilute nitric acid and 40 mL of water, and heat
on a water bath with shaking for 5 minutes. After cooling,
add water to make 50 mL, and filter. To 20 mL of the filtrate
add 2 mL of dilute nitric acid and water to make 50 mL. Pro-
ceed the test using this solution as the test solution. Prepare
the control solution with 0.40 mL of 0.01 mol/L hydrochlor-
ic acid VS (not more than 0.036%).
(2) Sulfate <1.14>— To 0.75 g of Pyrantel Pamoate add 5
mL of dilute hydrochloric acid and water to make 100 mL,
and heat on a water bath for 5 minutes with shaking. After
cooling, add water to make 100 mL, and filter. To 20 mL of
the filtrate add water to make 50 mL. Proceed the test using
this solution as the test solution. Prepare the control solution
with 0.45 mL of 0.005 mol/L sulfuric acid VS (not more than
0.144%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Pyrantel
Pamoate according to Method 2, and perform the test. Pre-
pare the control solution with 3 .0 mL of Standard Lead Solu-
tion (not more than 30 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pyrantel Pamoate according to Method 3, and perform
the test (not more than 2 ppm).
(5) Related substances — The procedure should be per-
formed under protection from direct sunlight in light-
resistant vessels. Dissolve 0.10 g of Pyrantel Pamoate in 10
mL of /V,/V-dimethylformamide, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
A^/V-dimethylformamide to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 ixL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, water and acetic acid (100) (3:1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the spot of pyrantel and the spot of pamoic acid from
the sample solution are not more intense than the spot of
pyrantel (Ri value: about 0.3) from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.3% (1 g).
Assay Weigh accurately about 0.5 g of Pyrantel Pamoate,
previously dried, add 25 mL of chloroform and 25 mL of so-
dium hydroxide TS, shake for 15 minutes, and extract. Ex-
tract further with two 25-mL portions of chloroform. Filter
each extract through 5 g of anhydrous sodium sulfate on a
pledget of absorbent cotton. Combine the chloroform ex-
tracts, add 30 mL of acetic acid (100), and titrate <2.50> with
0.1 mol/L perchloric acid VS (indicator: 2 drops of crystal
violet TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 59.47 mg of C„H 14 N 2 S.C 23 H 16 6
Containers and storage Containers — Tight containers.
Pyrazinamide
b°7-/1-* K
Cr*
C5H5N3O: 123.11
Pyrazine-2-carboxamide [98-96-4]
Pyrazinamide, when dried, contains not less than
99.0% and not more than 101.0% of C 5 H 5 N 3 0.
Description Pyrazinamide occurs as white crystals or
crystalline powder.
It is sparingly soluble in water and in methanol, and slight-
ly soluble in ethanol (99.5) and in acetic anhydride.
Identification (1) Determine the absorption spectrum of a
solution of Pyrazinamide in 0.1 mol/L hydrochloric acid TS
(1 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Pyrazinamide, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 188 - 193°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Pyrazinamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.10 g of Pyrazinamide
in 10 mL of methanol, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, add methanol to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 20 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of 1-butanol, water and a-
cetic acid (100) (3:1:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spot other than the principal spot
obtained from the sample solution is not more intense than
the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.1 g of Pyrazinamide,
JP XV
Official Monographs / Pyridoxine Hydrochloride 1047
previously dried, dissolve in 50 mL of acetic anhydride, and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 12.31 mg of C5H5N3O
Containers and storage Containers — Well-closed contain-
ers.
Pyridostigmine Bromide
tru Kxf L ys>IMk»
a
A
Er
,-CHa
N
I
CH-,
C 9 H 13 BrN 2 2 : 261.12
3-Dimethylcarbamoyloxy-l-methylpyridinium bromide
[101-26-8]
Pyridostigmine Bromide, when dried,
less than 98.5% of C 9 H 13 BrN 2 2 .
contains not
Description Pyridostigmine Bromide occurs as a white,
crystalline powder. It is odorless or has a slightly characteris-
tic odor.
It is very soluble in water, freely soluble in ethanol (95) and
in acetic acid (100), and practically insoluble in diethyl ether.
The pH of a solution of Pyridostigmine Bromide (1 in 10)
is between 4.0 and 6.0.
It is deliquescent.
Identification (1) Dissolve 0.02 g of Pyridostigmine
Bromide in 10 mL of water, add 5 mL of Reinecke salt TS: a
light red precipitate is produced.
(2) To 0.1 g of Pyridostigmine Bromide add 0.6 mL of
sodium hydroxide TS: the unpleasant odor of dimethylamine
is perceptible.
(3) Determine the absorption spectrum of a solution of
Pyridostigmine Bromide in 0.1 mol/L hydrochloric acid TS
(1 in 30,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(4) A solution of Pyridostigmine Bromide (1 in 50)
responds to the Qualitative Tests <1.09> for Bromide.
Melting point <2.60> 153 - 157°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Pyridostigmine Bromide in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Pyridostigmine Bromide according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Pyridostigmine Bromide according to Method 1, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Pyridostig-
mine Bromide in 10 mL of ethanol (95), and use this solution
as the sample solution. Pipet 2 mL of the sample solution,
and add ethanol (95) to make exactly 10 mL. Pipet 1 mL of
this solution, add ethanol (95) to make exactly 25 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of methanol, chloroform and ammonium
chloride TS (5:4:1) to a distance of about 12 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution in color.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
phosphorus (V) oxide, 100°C, 5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Pyridostigmine
Bromide, previously dried, dissolve in 10 mL of acetic acid
(100), add 40 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 26.11 mg of C 9 H 13 BrN 2 2
Containers and storage Containers — Hermetic containers.
Pyridoxine Hydrochloride
Vitamin B 6
IiX
,OH ■ HCt
C 8 H„N0 3 .HC1: 205.64
4,5-Bis(hydroxymethyl)-2-methylpyridin-3-ol
monohydrochloride [58-56-0]
Pyridoxine Hydrochloride, when dried, contains not
less than 98.0% and not more than 101.0% of
C 8 H„N0 3 .HC1.
Description Pyridoxine Hydrochloride occurs as a white to
pale yellow, crystalline powder.
It is freely soluble in water, slightly soluble in ethanol
(99.5), and practically insoluble in acetic anhydride and in
acetic acid (100).
It is gradually affected by light.
Melting point: about 206°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Pyridoxine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum or the spectrum of a
1048 Pyridoxine Hydrochloride Injection / Official Monographs
JP XV
solution of Pyridoxine Hydrochloride Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Pyridoxine Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Pyridoxine Hydrochloride Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(3) A solution of Pyridoxine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of Pyridoxine Hydrochloride in 50 mL of water is between
2.5 and 3.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Pyridoxine hydrochloride in 20 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Pyridoxine Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 30 ppm).
(3) Related substances — Dissolve 1.0 g of Pyridoxine
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 2.5 mL of the sample solution, and
add water to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add water to make exactly 10 mL, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 2//L each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, and
air-dry the plate. Develop the plate with a mixture of acetone,
tetrahydrofuran, hexane and ammonia solution (28)
(65:13:13:9) to a distance of about 10cm, and air-dry the
plate. Spray evenly a solution of sodium carbonate in diluted
ethanol (99.5) (3 in 10) (1 in 20) on the plate. After air-drying,
spray evenly a solution of 2,6-dibromo-iV-chloro-l,4-benzo-
quinone monoimine in ethanol (99.5) (1 in 1000) on the plate,
and air-dry: the spot other than the principal spot obtained
from the sample solution is not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, in vacu-
um, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Pyridoxine
Hydrochloride, previously dried, add 5 mL of acetic acid
(100) and 5 mL of acetic anhydride, dissolve by gentle boil-
ing, cool, add 30 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 20.56 mg of C 8 H„N0 3 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Pyridoxine Hydrochloride Injection
Vitamin B 6 Injection
Pyridoxine Hydrochloride Injection is an aqueous
solution for injection.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of pyridoxine
hydrochloride (C 8 H„N0 3 .HC1: 205.64).
Method of preparation Prepare as directed under Injec-
tions, with Pyridoxine Hydrochloride.
Description Pyridoxine Hydrochloride Injection is a color-
less or pale yellow, clear liquid.
It is gradually affected by light.
pH: 3.0-6.0
Identification (1) To a volume of Pyridoxine Hydrochlo-
ride Injection, equivalent to 0.05 g of Pyridoxine Hydrochlo-
ride according to the labeled amount, add 0.1 mol/L
hydrochloric acid TS to make 100 mL. To 2 mL of this solu-
tion add 0.1 mol/L hydrochloric acid TS to make 100 mL.
Determine the absorption spectrum of this solution as direct-
ed under Ultraviolet-visible spectrophotometry <2.24>: it ex-
hibits a maximum between 288 nm and 292 nm.
(2) To a volume of Pyridoxine Hydrochloride Injection,
equivalent to 0.01 g of Pyridoxine Hydrochloride according
to the labeled amount, add water to make 10 mL, and use this
solution as the sample solution. Separately, dissolve 0.01 g of
Pyridoxine Hydrochloride Reference Standard in 10 mL of
water, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 2 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography, and air-dry the plate. Develop the
plate with a mixture of acetone, tetrahydrofuran, hexane and
ammonia solution (28) (65:13:13:9) to a distance of about 10
cm, and air-dry the plate. Spray evenly a solution of sodium
carbonate in diluted ethanol (99.5) (3 in 10) (1 in 20) on the
plate. After air-drying, spray evenly a solution of 2,6-
dibromo-A f -chloro-l,4-benzoquinone monoimine in ethanol
(99.5) (1 in 1000) on the plate: the spots obtained from the
sample solution and the standard solution are blue in color
and have the same Rf value.
Bacterial endotoxins <4.01> Less than 3.0 EU/mg.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Pyridoxine Hydrochlo-
ride Injection, equivalent to about 20 mg of pyridoxine
hydrochloride (C 8 H U N03.HC1), dilute with water, if necessa-
ry, and add water to make exactly 100 mL. Pipet 25 mL of
this solution, add water to make exactly 200 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 0.1 g of Pyridoxine Hydrochloride Reference Stan-
dard, previously dried in a desiccator (in vacuum, silica gel)
for 4 hours, and dissolve in water to make exactly 100 mL.
Pipet 5 mL of this solution, add water to make exactly 200
mL, and use this solution as the standard solution. Pipet 1
JPXV
Official Monographs / Pyrrolnitrin 1049
mL each of the sample solution and standard solution, add
2.0 mL of barbital buffer solution, 9.0 mL of 2-propanol and
2.0 mL of a freshly prepared solution of 2,6-dibromo-iV-
chloro-l,4-benzoquinone monoimine in ethanol (95) (1 in
4000), shake well, add 2-propanol to make exactly 25 mL,
and allow to stand for 90 minutes. Determine the absor-
bances, A T and A s , of the subsequent sample solution and
subsequent standard solution, respectively, at 650 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using a solution, prepared in the same manner with 1 mL of
water, as the blank.
Amount (mg) of pyridoxine hydrochloride
(C 8 N U N0 3 .HC1)
= ^ s x(,4 T A4 s )x(l/5)
W s : Amount (mg) of Pyridoxine Hydrochloride Reference
Standard
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Pyrrolnitrin
tin-jL-- h U >
Pyroxylin
Pyroxylin is a nitric acid ester of cellulose. It is usual-
ly moistened with 2-propanol or some other solvent.
Description Pyroxylin occurs as a white cotton-like sub-
stance or white flakes.
It is freely soluble in acetone, and very slightly soluble in
diethyl ether.
Upon heating or exposure to light, it is decomposed with
the evolution of nitrous acid vapors.
Identification Ignite Pyroxylin: it burns very rapidly with a
luminous flame.
Purity (1) Clarity of solution — Dissolve 1.0 g of Pyroxy-
lin, previously dried at 80°C for 2 hours, in 25 mL of a mix-
ture of diethyl ether and ethanol (95) (3:1): the solution is
clear.
(2) Acidity — Shake 1.0 g of Pyroxylin, previously dried
at 80°C for 2 hours, with 20 mL of water for 10 minutes: the
filtrate is neutral.
(3) Water-soluble substances — Evaporate 10 mL of the
filtrate obtained in (2) on a water bath to dryness, and dry at
105 °C for 1 hour: the mass of the residue is not more than 1.5
mg.
(4) Residue on ignition — Weigh accurately about 2 g of
Pyroxylin, previously dried at 80°C for 2 hours, and moisten
with 10 mL of a solution of castor oil in acetone (1 in 20) to
gelatinize the sample. Ignite the contents to carbonize the
sample, heat strongly at about 500°C for 2 hours, and allow
to cool over silica gel: the amount of the residue is not more
than 0.30%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, packed loosely, remote from
fire, and preferably in a cold place.
C 10 H 6 Cl 2 N 2 O 2 : 257.07
3-Chloro-4-(3-chloro-2-nitrophenyl)pyrrole [1018- 71-9 ]
Pyrrolnitrin contains not less than 970 fig (potency)
and not more than 1020 fig (potency) per mg,
calculated on the dried basis. The potency of Pyrrol-
nitrin is expressed as mass (potency) of pyrrolnitrin
(C 10 H 6 Cl 2 N 2 O 2 ).
Description Pyrrolnitrin occurs as yellow to yellow-brown,
crystals or crystalline powder.
It is freely soluble in methanol and in ethanol (95), and
practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Pyrrolnitrin in ethanol (95) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Pyrrolnitrin Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Pyr-
rolnitrin as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Pyrrolnitrin Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Melting point <2.60> 124 - 128 C C
Purity Related substances — Dissolve 0.10 g of Pyrrolnitrin
in 10 mL of methanol, and use this solution as the sample so-
lution. Pipet 1 mL of the sample solution, and add methanol
to make exactly 100 mL. Pipet 3 mL of this solution, add
methanol to make exactly 10 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of xylene, ethyl acetate and formic acid
(18:2:1) to a distance of about 10 cm, and dry the plate at
80°C for 30 minutes. Spray evenly diluted sulfuric acid (1 in
3) on the plate, and heat at 100°C for 30 minutes: the spot
other than the principal spot obtained from the sample solu-
tion is not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Conduct this procedure using light-resistant vessels.
1050 Quinidine Sulfate Hydrate / Official Monographs
JP XV
Weigh accurately an amount of Pyrrolnitrin and Pyrrolnitrin
Reference Standard, equivalent to about 50 mg (potency),
dissolve in diluted acetonitrile (3 in 5) to make exactly 50 mL.
Pipet 10 mL each of these solutions, add exactly 10 mL of the
internal standard solution, add diluted acetonitrile (3 in 5) to
make 100 mL, and use these solutions as the sample solution
and standard solution. Perform the test with 5 ,«L each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak
area of pyrrolnitrin to that of the internal standard.
Amount [/ug (potency)] of CioH 6 2 N 2 2
= ^ s x(g T /g s )xl000
W s : Amount [mg (potency)] of Pyrrolnitrin Reference
Standard
Internal standard solution — A solution of benzyl benzoate in
diluted acetonitrile (3 in 5) (3 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octylsilanized silica gel for
liquid chromatography (5 /um in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
pyrrolnitrin is about 9 minutes.
System suitability —
System performance: When the procedure is run with 5 fiL
of the standard solution under the above operating condi-
tions, pyrrolnitrin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratios of the
peak area of pyrrolnitrin to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Quinidine Sulfate Hydrate
■V
*>BiEK£*»ai
• H 2 S0 4 ■ 2HjO
(C 20 H 24 N 2 O 2 ) 2 .H 2 SO 4 .2H 2 O: 782.94
(95')-6'-Methoxycinchonan-9-ol hemisulfate
monohydrate [6591-63-5]
Quinidine Sulfate Hydrate, when dried, contains not
less than 98.5% of quinidine sulfate [(C 20 H 24^02)2.1^2
S0 4 : 746.91].
Description Quinidine Sulfate Hydrate occurs as white
crystals. It is odorless, and has a very bitter taste.
It is freely soluble in ethanol (95) and in boiling water,
sparingly soluble in water, and practically insoluble in diethyl
ether. Quinidine Sulfate Hydrate, previously dried, is freely
soluble in chloroform.
It darkens gradually by light.
Optical rotation [a]™'- +275 - +287° (after drying, 0.5 g,
0.1 mol/L hydrochloric acid VS, 25 mL, 100 mm).
Identification (1) Dissolve 0.01 g of Quinidine Sulfate Hy-
drate in 10 mL of water and 2 to 3 drops of dilute sulfuric
acid: a blue fluorescence is produced.
(2) To 5 mL of an aqueous solution of Quinidine Sulfate
Hydrate (1 in 1000) add 1 to 2 drops of bromine TS, then add
1 mL of ammonia TS: a green color develops.
(3) To 5 mL of an aqueous solution of Quinidine Sulfate
Hydrate (1 in 100) add 1 mL of silver nitrate TS, stir with a g-
lass rod, and allow to stand for a short interval: a white
precipitate is produced, and it dissolves on addition of nitric
acid.
(4) Dissolve 0.4 g of Quinidine Sulfate Hydrate in 20 mL
of water and 1 mL of dilute hydrochloric acid: the solution
responds to the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 1.0 g of Quinidine Sulfate Hydrate in
100 mL of freshly boiled and cooled water: the pH of this so-
lution is between 6.0 and 7.0.
Purity (1) Chloroform-ethanol-insoluble substances —
Warm 2.0 g of Quinidine Sulfate Hydrate with 15 mL of a
mixture of chloroform and ethanol (99.5) (2:1) at about 50°C
for 10 minutes. After cooling, filter through a tared glass
filter (G4) by gentle suction. Wash the residue with five
10-mL portions of a mixture of chloroform and ethanol
(99.5) (2:1), and dry at 105°C for 1 hour: the mass of the
residue is not more than 2.0 mg.
(2) Related substances — Dissolve 20 mg of Quinidine
Sulfate Hydrate in the mobile phase to make exactly 100 mL,
and use this solution as the sample solution. Separately, dis-
solve 25 mg of cinchonine in the mobile phase to make exact-
ly 100 mL. Pipet 2 mL of this solution, add the mobile phase
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 50 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of the sample solution by the
automatic integration method, and calculate their amount by
the area percentage method: the amount of dihydroquinidine
sulfate is not more than 15.0%, and those of quinine sulfate
and dihydroquinine sulfate are not more than 1.0%. The
total area of the peaks other than the principal peak and the
above peaks is not larger than the peak area of cinchonine
from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A column about 4 mm in inside diameter and
about 25 cm in length, packed with octadecylsilanized silica
gel (10 /xm in particle diameter).
Temperature: Room temperature.
Mobile phase: A mixture of water, acetonitrile, methane-
JPXV
Official Monographs / Quinine Ethyl Carbonate 1051
sulfonic acid TS and a solution of diethylamine (1 in 10)
(43:5:1:1).
Flow rate: Adjust the flow rate so that the retention time of
quinidine is about 10 minutes.
Selection of column: Dissolve 0.01 g each of Quinidine Sul-
fate Hydrate and quinine sulfate in 5 mL of methanol, and
add the mobile phase to make 50 mL. Proceed with 50 fiL of
this solution under the above operating conditions, and cal-
culate the resolution. Use a column giving elution of quini-
dine, quinine, dihydroquinidine and dihydroquinine in this
order with a resolution between quinidine and quinine and
that between quinine and dihydroquinidine being not less
than 1.2, respectively.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of cinchonine obtained from 50 [iL of
the standard solution is between 5 mm and 10 mm.
Time span of measurement: About twice as long as the
retention time of quinidine beginning after the solvent peak.
(3) Readily carbonizable substances <1.15> — Take 0.20 g
of Quinidine Sulfate Hydrate and perform the test: the solu-
tion has no more color than Matching fluid M.
Loss on drying <2.41> Not more than 5.0% (1 g, 130 °C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Quinidine Sulfate
Hydrate, previously dried, dissolve in 20 mL of acetic acid
(100), and add 80 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS until the color of the solu-
tion changes from purple through blue to blue-green (indica-
tor: 3 drops of crystal violet TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.90 mg of (C 2 „H 24 N 2 2 ) 2 .H 2 S0 4
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Quinine Ethyl Carbonate
^--^ji^-iii^m^xTJi
H S C^
C 23 H 28 N 2 4 : 396.48
Ethyl (8S,9i?)-6'-methoxycinchonan-9-yl carbonate
[83-75-0]
Quinine Ethyl Carbonate contains not less than
98.5% of C23H28N2O4, calculated on the dehydrated
basis.
Description Quinine Ethyl Carbonate occurs as white crys-
tals. It is odorless, and tasteless at first but slowly develops a
bitter taste.
It is very soluble in methanol, freely soluble in ethanol (95)
and in ethanol (99.5), soluble in diethyl ether, and practically
insoluble in water.
It dissolves in dilute hydrochloric acid.
Identification (1) Determine the absorption spectrum of a
solution of Quinine Ethyl Carbonate in methanol (1 in
20,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
(2) Determine the infrared absorption spectrum of Qui-
nine Ethyl Carbonate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Optical rotation <2.49> [ a ] 2 D °: -42.2 - -44.0° (0.5 g, calcu-
lated on the dehydrated basis, methanol, 50 mL, 100 mm).
Melting point <2.60> 91 - 95°C
Purity (1) Chloride <1.03>— Dissolve 0.30 g of Quinine
Ethyl Carbonate in 10 mL of dilute nitric acid and 20 mL of
water. To 5 mL of the solution add 2 to 3 drops of silver ni-
trate TS: no color develops.
(2) Sulfate <1.14>— Dissolve 1.0 g of Quinine Ethyl Car-
bonate in 5 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 1.0 mL of 0.005 mol
/L sulfuric acid VS, 5 mL of dilute hydrochloric acid and
water to make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Quinine
Ethyl Carbonate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than lOppm).
(4) Related substances — Dissolve 20 mg of Quinine Ethyl
Carbonate in the mobile phase to make exactly 100 mL, and
use this solution as the sample solution. Separately, dissolve
25 mg of quinine sulfate in the mobile phase to make exactly
100 mL. Pipet 2 mL of this solution, add mobile phase to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of these solutions by the au-
tomatic integration method, and calculate the amount of a
main impurity in the sample solution which appears at about
1.2 times of the retention time of quinine ethyl carbonate by
the area percentage method: it is not more than 10.0%. The
total peak area other than the principal peak and above men-
tioned peak from the sample solution is not larger than the
peak area of Quinine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.2 g of sodium 1-octanesulfonate
in 1000 mL of a mixture of water and methanol (1 : 1), and ad-
1052 Quinine Hydrochloride Hydrate / Official Monographs
JP XV
just to pH 3.5 with diluted phosphoric acid (1 in 20).
Flow rate: Adjust the flow rate so that the retention time of
the peak of quinine ethyl carbonate is about 20 minutes.
Selection of column: Dissolve 5 mg each of Quinine Ethyl
Carbonate and quinine sulfate in the mobile phase to make
50 mL. Proceed with lO^L of this solution under the above
operating conditions, and calculate the resolution. Use a
column giving elution of quinine, dihydroquinine, quinine
ethyl carbonate and the main impurity of quinine ethyl car-
bonate in this order with the resolution between the peaks of
quinine and dihydroquinine being not less than 2.7, and be-
tween the peaks of quinine and quinine ethyl carbonate being
not less than 5.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of quinine obtained from 10 /uL of the
standard solution is between 5 mm and 10 mm.
Time span of measurement: About 2 times as long as the
retention time of quinine ethyl carbonate.
Water <2.48> Not more than 3.0% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Quinine Ethyl Car-
bonate, dissolve in 60 mL of acetic acid (100), add 2 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 19.82 mg of C 23 H 28 N 2 4
Containers and storage Containers — Well-closed contain-
ers.
Quinine Hydrochloride Hydrate
*--*£K£*5frjai
• HCI«2HjO
C 20 H 24 N 2 O 2 .HC1.2H 2 O: 396.91
(85,9i?)-6'-Methoxycinchonan-9-ol monohydrochloride
dihydrate [6119-47-7]
Quinine Hydrochloride Hydrate, when dried, con-
tains not less than 98.5% of quinine hydrochloride
(C 20 H 24 N 2 O 2 .HCl: 360.88).
Description Quinine Hydrochloride Hydrate occurs as
white crystals. It is odorless, and has a very bitter taste.
It is very soluble in ethanol (99.5), freely soluble in acetic
acid (100), in acetic anhydride and in ethanol (95), soluble in
water, and practically insoluble in diethyl ether. Quinine
Hydrochloride Hydrate, previously dried, is freely soluble in
chloroform.
It gradually changes to brown by light.
Identification (1) A solution of Quinine Hydrochloride
Hydrate (1 in 50) shows no fluorescence. To 1 mL of the solu-
tion add 100 mL of water and 1 drop of dilute sulfuric acid: a
blue fluorescence is produced.
(2) To 5 mL of a solution of Quinine Hydrochloride Hy-
drate (1 in 1000) add 1 to 2 drops of bromine TS and 1 mL of
ammonia TS: a green color develops.
(3) To 5 mL of a solution of Quinine Hydrochloride Hy-
drate (1 in 50) add 1 mL of dilute nitric acid and 1 mL of sil-
ver nitrate TS: a white precipitate is produced. Collect the
precipitate, and add an excess of ammonia TS: it dissolves.
Optical rotation <2.49> [«]£>: -245 - -255° (after drying,
0.5 g, 0.1 mol/L hydrochloric acid VS, 25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Quinine Hydrochloride Hy-
drate in 100 mL of freshly boiled and cooled water: the pH of
this solution is between 6.0 and 7.0.
Purity (1) Sulfate <1.14>— Perform the test with 1.0 g of
Quinine Hydrochloride Hydrate. Prepare the control solu-
tion with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more
than 0.048%).
(2) Barium — Dissolve 0.5 g of Quinine hydrochloride
Hydrate in 10 mL of water by warming, and add 1 mL of di-
lute sulfuric acid: no turbidity is produced.
(3) Chloroform-ethanol-insoluble substances — Warm 2.0
g of Quinine Hydrochloride Hydrate with 15 mL of a mixture
of chloroform and ethanol (99.5) (2:1) at 50°C for 10
minutes. After cooling, filter through a tared glass filter (G4)
by gentle suction. Wash the residue with five 10-mL portions
of a mixture of chloroform and ethanol (99.5) (2:1), dry at
105 °C for 1 hour, and weigh: the mass of the residue so ob-
tained is not more than 2.0 mg.
(4) Related substances — Dissolve 20 mg of Quinine
Hydrochloride Hydrate in the mobile phase to make exactly
100 mL, and use this solution as the sample solution.
Separately, dissolve 25 mg of cinchonidine in the mobile
phase to make exactly 100 mL. Pipet 2 mL of this solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 50 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine each peak area of the
sample solution by the automatic integration method, and
calculate the amount of dihydroquinine hydrochloride by the
area percentage method: it is not more than 10.0%. The total
area of the peaks other than the main peak and the above
peaks is not larger than the peak area of cinchonidine from
the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 25 cm in length, packed with octadecyl-
silanized silica gel (10 /um in particle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of water, acetonitrile,
methanesulfonic acid TS and a solution of diethylamine (1 in
10) (43:5:1:1).
Flow rate: Adjust the flow rate so that the retention time of
quinine is about 10 minutes.
Selection of column: Dissolve 10 mg each of Quinine
Hydrochloride and quinidine sulfate in 5 mL of methanol,
and add the mobile phase to make 50 mL. Proceed with 50
/xL of this solution under the above operating conditions. Use
JPXV
Official Monographs / Quinine Sulfate Hydrate 1053
a column giving elution of quinidine, quinine, dihydroquini-
dine and dihydroquinine in this order with the resolution be-
tween quinidine and quinine, and that between quinine and
dihydroquinidine being not less than 1.2, respectively.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of cinchonidine from 50 iiL of the stan-
dard solution is between 5 mm and 10 mm.
Time span of measurement: About twice as long as the
retention time of quinine beginning after the solvent peak.
(5) Readily carbonizable substances <1.15> — Perform the
test with 0.25 g of Quinine Hydrochloride Hydrate. The solu-
tion has no more color than Matching Fluid M.
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C,
5 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Quinine Hydrochlo-
ride Hydrate, previously dried, dissolve in 100 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3) by warm-
ing, cool, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 18.04 mg of C 20 H 24 N 2 O 2 .HCl
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Quinine Sulfate Hydrate
4A
■ H 2 S0 4 ■ 2H30
(C 20 H 24 N 2 O 2 ) 2 .H 2 SO 4 .2H 2 O: 782.94
(8S , ,9i?)-6'-Methoxycinchonan-9-ol hemisulfate
monohydrate [6119-70-6]
Quinine Sulfate Hydrate contains not less than
98.5%, of quinine sulfate [(C 20 H 2 4N 2 O 2 ) 2 .H 2 SO 4 :
746.91], calculated on the dried basis.
Description Quinine Sulfate Hydrate occurs as white crys-
tals or crystalline powder. It is odorless, and has a very bitter
taste.
It is freely soluble in acetic acid (100), slightly soluble in
water, in ethanol (95), in ethanol (99.5) and in chloroform,
and practically insoluble in diethyl ether.
It gradually changes to brown by light.
Identification (1) Determine the absorption spectrum of a
solution of Quinine Sulfate Hydrate (1 in 20,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Qui-
nine Sulfate Hydrate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) To 0.4 g of Quinine Sulfate Hydrate add 20 mL of
water and 1 mL of dilute hydrochloric acid: the solution
responds to the Qualitative Tests <1.09> for sulfate.
Optical rotation <2.49> [«]£>: -235 - -245°(after drying,
0.5 g, 0.1 mol/L hydrochloric acid VS, 25 mL, 100 mm).
pH <2.54> Shake 2.0 g of Quinine Sulfate Hydrate in 20 mL
of freshly boiled and cooled water, and filter: the pH of this
filtrate is between 5.5 and 7.0.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Quinine Sulfate Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Chloroform-ethanol-insoluble substances — Warm 2.0
g of Quinine Sulfate Hydrate with 15 mL of a mixture of
chloroform and ethanol (99.5) (2:1) at 50°C for 10 minutes.
After cooling, filter through a tared glass filter (G4) by gentle
suction. Wash the residue with five 10-mL portions of a mix-
ture of chloroform and ethanol (99.5) (2:1), dry at 105°C for
1 hour, and weigh: the mass of the residue is not more than
2.0 mg.
(3) Related substances — Dissolve 20 mg of Quinine Sul-
fate Hydrate in the mobile phase to make exactly 100 mL,
and use this solution as the sample solution. Separately, dis-
solve 25 mg of cinchonidine in the mobile phase to make ex-
actly 100 mL. Pipet 2 mL of this solution, add the mobile
phase to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 50 11L each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area from the sample
solution by the automatic integration method, and calculate
the amount of dihydroquinine sulfate by the area percentage
method: it is not more than 5%. The total area of the peaks
other than the main peak and the above peak is not larger
than the peak area of cinchonidine from the standard solu-
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 235 nm).
Column: A column about 4 mm in inside diameter and
about 25 cm in length, packed with octadecylsilanized silica
gel (10 //m in particle diameter).
Temperature: Room temperature
Mobile phase: A mixture of water, acetonitrile, methane
sulfonic acid TS and a solution of diethylamine (1 in 10)
(43:5:1:1).
Flow rate: Adjust the flow rate so that the retention time of
quinine is about 10 minutes.
Selection of column: Dissolve 0.01 g each of Quinine Sul-
fate Hydrate and quinidine sulfate in 5 mL of methanol, and
add the mobile phase to make 50 mL. Proceed with 50 iiL of
this solution under the above operating conditions, and cal-
culate the resolution. Use a column giving elution of quini-
1054 Freeze-dried Inactivated Tissue Culture Rabies Vaccine / Official Monographs
JP XV
dine, quinine, dihydroquinidine and dihydroquinine in this
order with the resolution between quinidine and quinine and
that between quinine and dihydroquinidine being not less
than 1.2.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of cinchonidine obtained from 50 /xh of
the standard solution is between 5 mm and 10 mm.
Time span of measurement: About twice as long as the
retention time of quinine beginning after the solvent peak.
Loss on drying <2.41> 3.0% - 5.0% (1 g, 105°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Quinine Sulfate Hy-
drate, dissolve in 20 mL of acetic acid (100), add 80 mL of
acetic anhydride, and titrate <2.50> with 0.1 mol/L perchloric
acid VS until the color of the solution changes from purple
through blue to blue-green (indicator: 2 drops of crystal vio-
let TS). Perform a blank determination, and make any neces-
sary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.90 mg of (C 20 H 24 N 2 O 2 ) 2 .H 2 SO 4
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Freeze-dried Inactivated Tissue
Culture Rabies Vaccine
Freeze-dried Inactivated Tissue Culture Rabies Vac-
cine is a dried preparation containing inactivated rabies
virus.
It conforms to the requirements of Freeze-dried In-
activated Tissue Culture Rabies Vaccine in the Mini-
mum Requirements of Biologic Products.
Description Freeze-dried Inactivated Tissue Culture Rabies
Vaccine becomes a colorless or light yellow-red clear liquid
on addition of solvent.
Ranitidine Hydrochloride
• HCI
and geometrical isomer at C*"
Ci3H 22 N 4 3 S.HCl: 350.86
(\EZ)-N- {2-[( {5-[(Dimethylamino)methyl]furan-
2-yl} methyl)sulfanyl]ethyl} -N '-methyl-2-nitroethene-
1,1 -diamine monohydrochloride [66357-59-3]
Ranitidine Hydrochloride, when dried, contains not
less than 97.5% and not more than 102.0% of
C 13 H 22 N40 3 S.HC1.
Description Ranitidine Hydrochloride occurs as a white to
pale yellow, crystalline or fine granular powder.
It is very soluble in water, freely soluble in methanol, and
slightly soluble in ethanol (99.5).
It is hygroscopic.
It is gradually colored by light.
Melting point: about 140°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Ranitidine Hydrochloride (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Ranitidine Hydrochloride Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Ranitidine Hydrochloride as directed in the paste method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
previously dried Ranitidine Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Ranitidine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Ranitidine Hydrochloride in 100 mL of water is between
4.5 and 6.0.
Purity (1) Clarity and color of solution — A solution of
Ranitidine Hydrochloride (1 in 10) is clear and pale yellow to
light yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Raniti-
dine Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Ranitidine Hydrochloride according to Method 4, and per-
form the test (not more than 2 ppm).
(4) Related substances — Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve 0.22 g
of Ranitidine Hydrochloride in methanol to make exactly 10
mL, and use this solution as the sample solution. Pipet 0.5
mL of the sample solution, add methanol to make exactly 100
mL, and use this solution as the standard solution (1). Pipet 6
mL, 4 mL, 2 mL and 1 mL of the standard solution (1), add
to each methanol to make exactly 10 mL, and use these solu-
tions as the standard solution (2), the standard solution (3),
the standard solution (4) and the standard solution (5), re-
spectively. Separately, dissolve 12.7 mg of ranitidinediamine
in methanol to make exactly 10 mL, and use this solution as
the standard solution (6). Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /iL each of the sample solution and standard solu-
tions (1), (2), (3), (4) and (5) on a plate of silica gel for thin-
layer chromatography. Separately, spot 10 /uL of the sample
solution on the plate, then spot lO^L of the standard solu-
tion (6) on the spotted position of the sample solution. Imme-
diately develop the plate with a mixture of ethyl acetate, 2-
propanol, ammonia solution (28) and water (25:15:5:1) to a
distance of about 15 cm, and air-dry the plate. Allow the
plate to stand in iodine vapor until the spot from the standard
solution (5) appears: the spot obtained from the standard so-
JP XV
Official Monographs / Reserpine 1055
lution (6) is completely separated from the principal spot
from the sample solution. The spot having Ri value of about
0.7 from the sample solution is not more intense than the spot
from the standard solution (1), the spots other than the prin-
cipal spot and the spot of RiO.l from the sample solution are
not more intense than the spot from the standard solution
(2), and the total amount of these related substances, calcu-
lated by comparison with the spots from the standard solu-
tions (1), (2), (3), (4) and (5), is not more than 1.0%.
Loss on drying <2.41> Not more than 0.75% (1 g, in vacu-
um, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 20 mg of Ranitidine
Hydrochloride and Ranitidine Hydrochloride Reference
Standard, previously dried, dissolve each in the mobile phase
to make exactly 200 mL, and use these solutions as the sam-
ple solution and the standard solution. Perform the test with
exactly 10 fiL each of the sample solution and the standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of ranitidine.
Amount (mg) of C 13 H 22 N 4 3 S.HC1 = W s x (A T /A S )
W s : Amount (mg) of Ranitidine Hydrochloride Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 322 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 20 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and diluted 0.5
mol/L ammonium acetate TS (1 in 5) (17:3).
Flow rate: Adjust the flow rate so that the retention time of
ranitidine is about 5 minutes.
System suitability —
System performance: Dissolve 20 mg of Ranitidine
Hydrochloride and 5 mg of benzalphthalide in 200 mL of the
mobile phase. When the procedure is run with 10 fiL of this
solution under the above operating conditions, ben-
zalphthalide and ranitidine are eluted in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ranitidine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Rape Seed Oil
Oleum Rapae
Rape Seed Oil is the fixed oil obtained from the seed
of Brassica campestris Linne subsp. napus Hooker fil.
et Anderson var. nippo-oleifera Makino (Cruciferae).
Description Rape Seed Oil is a clear, pale yellow, slightly
viscous oil. It is odorless or has a slight odor and a mild taste.
It is miscible with diethyl ether and with petroleum diethyl
ether. It is slightly soluble in ethanol (95).
Specific gravity d\\: 0.906 - 0.920
Acid value <1.13> Not more than 0.2.
Saponification value <1.13> 169 - 195
Unsaponiftable matters <1.13> Not more than 1.5%.
Iodine value <7.75> 95 - 127
Containers and storage Containers — Tight containers.
Reserpine
U-tzJUti>
C 33 H 40 N 2 O 9 : 608.68
Methyl (3S, 1 6S, 1 1R, 1 SR,20R)- 1 1 , 1 7-dimethoxy- 1 8-
(3,4,5-trimethoxybenzoyloxy)yohimban-16-carboxylate
[50-55-5]
Reserpine, when dried, contains not less than 96.0%
of C 33 H4oN 2 9 .
Description Reserpine occurs as white to pale yellow crys-
tals or crystalline powder.
It is freely soluble in acetic acid (100) and in chloroform,
slightly soluble in acetonitrile, very slightly soluble in ethanol
(95), and practically insoluble in water and in diethyl ether.
It is affected by light.
Identification (1) To 1 mg of Reserpine add 1 mL of vanil-
lin-hydrochloric acid TS, and warm: a vivid red-purple color
develops.
(2) Determine the absorption spectrum of a solution of
Reserpine in acetonitrile (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Reserpine Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Reser-
pine, previously dried, as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of previously dried Reserpine Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
1056 Reserpine Injection / Official Monographs
JP XV
Optical rotation <2.49> [ a ]o- -114
0.25 g, chloroform, 25 mL, 100 mm).
127° (after drying,
Purity Related substances — Conduct this procedure
without exposure to daylight, using light-resistant vessels.
Dissolve 50 mg of Reserpine in 50 mL of acetonitrile, and use
this solution as the sample solution. Pipet 3 mL of the sample
solution, add acetonitrile to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area from these solutions by the automatic integration
method: the total area of all peaks other than reserpine peak
from the sample solution is not larger than the peak area of
reserpine from the standard solution.
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Assay.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate, pH 3.0 and acetonitrile (13:7).
Flow rate: Adjust the flow rate so that the retention time of
reserpine is about 20 minutes.
Time span of measurement: About twice as long as the
retention time of reserpine.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add acetonitorile to make exactly 50 mL. Con-
firm that the peak area of reserpine obtained from 10 /uL of
this solution is equivalent to 3 to 5% of that of reserpine ob-
tained from 10 /xL of the standard solution.
System performance: Dissolve 0.01 g of Reserpine and 4
mg of butyl parahydroxybenzoate in 100 mL of acetonitrile.
To 5 mL of this solution add acetonitrile to make 50 mL.
When the procedure is run with 20 /xL of this solution accord-
ing to the operating conditions in the Assay, reserpine and
butyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
reserpine is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (0.2 g, in vacu-
um, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.2 g).
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately about 10
mg each of Reserpine and Reserpine Reference Standard,
previously dried, and dissolve each in acetonitrile to make ex-
actly 100 mL. Pipet 5 mL each of these solutions, add exactly
10 mL of the internal standard solution, 5 mL of acetonitrile
and water to make 50 mL, and use these solutions as the sam-
ple solution and the standard solution, respectively. Perform
the test with 20 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and g s , of the peak area of reserpine to that of the inter-
nal standard.
Amount (mg) of C 3 3H 40 N 2 O 9 = W s x(Q T /Qs)
W s : Amount (mg) of Reserpine Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 50,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate, pH 3.0 and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
reserpine is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, reserpine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 2.0.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of reserpine to that of the internal standard is not
more than 2.0%.
Containers and Storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Reserpine Injection
Hr;utr>&*f*
Reserpine Injection is an aqueous solution for injec-
tion. It contains not less than 90% and not more than
110% of the labeled amount of reserpine (C33H40N2O9:
608.68).
Method of preparation Prepare as directed under Injec-
tions, with Reserpine.
Description Reserpine Injection is a clear, colorless or pale
yellow liquid.
pH: 2.5-4.0
Identification Measure a volume of Reserpine Injection,
equivalent to 1.5 mg of Reserpine according to the labeled
amount, add 10 mL of diethyl ether, shake for 10 minutes,
and take the aqueous layer. If necessary, add 10 mL of
diethyl ether to the aqueous layer, and shake for 10 minutes
to repeat the process. To the aqueous layer add water to
make 50 mL, and determine the absorption spectrum of this
solution as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: it exhibits a maximum between 265 nm and 269
nm.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Reserpine Injection,
equivalent to about 4 mg of reserpine (C33H4 N 2 O9).
Separately, weigh accurately about 4 mg of Reserpine Refer-
ence Standard, previously dried in vacuum at 60°C for 3
JPXV
Official Monographs / Reserpine Tablets 1057
hours. Transfer them to separate separator, add 10 mL each
of water and 5 mL each of ammonia TS, and extract with one
20-mL portion of chloroform, then with three 10-mL por-
tions of chloroform with shaking vigorously. Combine the
chloroform extracts, wash with two 50-mL portions of dilut-
ed hydrochloric acid (1 in 1000), and combine the washings.
Then wash the chloroform extract with two 50-mL portions
of a solution of sodium hydrogen carbonate (1 in 100), and
combine the all washings. Extract the combined washing with
two 10-mL portions of chloroform, and combine the wash-
ings with the former chloroform extract. Transfer the chlo-
roform solution to a 100-mL volumetric flask through a
pledget of absorbent cotton previously wetted with chlo-
roform, wash with a small amount of chloroform, dilute with
chloroform to make 100 mL, and use these solutions as the
sample solution and the standard solution, respectively. De-
termine the absorbances, A T and A s , of the sample solution
and the standard solution, respectively, at 295 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of reserpine (C33H40N2O9)
= W s x(A T /A s )
W s : Amount (mg) of Reserpine Reference Standard
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
0.1% Reserpine Powder
Reserpine Powder
U-teJHf>gc0.1 o /o
0.1% Reserpine Powder contains not less than
0.09% and not more than 0.11% of reserpine
(C 3 3H 40 N 2 O 9 : 608.68).
Method of preparation
Reserpine
Lactose Hydrate
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Identification To 0.4 g of 0.1% Reserpine Powder add 20
mL of acetonitrile, shake for 30 minutes, and centrifuge. De-
termine the absorption spectrum of the supernatant liquid as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits maxima between 265 nm and 269 nm, and between
294 nm and 298 nm.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately a quanti-
ty of 0.1% Reserpine Powder, equivalent to about 0.5 mg of
reserpine (C33H40N2O9), disperse in 12 mL of water, add ex-
actly 10 mL of the internal standard solution and 10 mL of
acetonitrile, and dissolve by warming at 50°C for 15 minutes,
then add water to make 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Reserpine Reference Standard, previously dried at 60°C in
vacuum for 3 hours, dissolve in acetonitrile to make exactly
100 mL. Pipet 5 mL of this solution, add exactly 10 mL of
the internal standard solution, 5 mL of acetonitrile and water
to make 50 mL, and use this solution as the standard solu-
tion. Proceed with the sample solution and standard solution
as directed in the Assay under Reserpine.
Amount (mg) of reserpine (C33H40N2O9)
= ^sX(e T /Gs)x(l/20)
W s : Amount (mg) of Reserpine Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 50,000).
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Reserpine Tablets
Reserpine Tablets contain not less than 90% and not
more than 110% of the labeled amount of reserpine
(C 33 H4oN 2 9 : 608.68).
Method of preparation Prepare as directed under Tablets,
with Reserpine.
Identification Take a portion of powdered Reserpine
Tablets, equivalent to 0.4 mg of Reserpine according to the
labeled amount, add 20 mL of acetonitrile, shake for 30
minute, and centrifuge. Determine the absorption spectrum
of the supernatant liquid as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 265
nm and 269 nm, and between 294 nm and 298 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Conduct this procedure without exposure to daylight, us-
ing light-resistant vessels. To one tablet of Reserpine Tablets
add 2 mL of water, disintegrate by warming at 50°C for 15
minutes while shaking. After cooling, add exactly 2 mL of
the internal standard solution per 0.1 mg of reserpine
(C33H4 N 2 O 9 ) according to the labeled amount, add 2 mL of
acetonitrile, warm at 50°C for 15 minutes while shaking, and
after cooling, add water to make 10 mL. Centrifuge the solu-
tion, and use the supernatant liquid as the sample solution.
Separately, weigh accurately about 10 mg of Reserpine Refer-
ence Standard, previously dried at 60°C in vacuum for 3
hours, dissolve in acetonitrile to make exactly 100 mL. Pipet
5 mL of this solution add exactly 10 mL of the internal stan-
dard solution, 5 mL of acetonitrile and water to make 50 mL,
and use this solution as the standard solution. Proceed with
the sample solution and standard solution as directed in the
Assay under Reserpine.
Amount (mg) of reserpine (C33H40N2O9)
= W s x(Q J /Q s )x(C/W)
W s : Amount (mg) of Reserpine Reference Standard
C: Labeled amount (mg) of reserpine in each tablet.
Internal standard solution — A solution of butyl parahydrox-
1058 Retinol Acetate / Official Monographs
JP XV
ybenzoate in acetonitrile (1 in 50,000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Take 1 tablet of Reserpine Tablets, and perform the test at
100 revolutions per minute with 500 mL of a solution of poly-
sorbate 80 (1 in 20,000) in diluted dilute acetic acid (1 in 200)
as the test solution according to the Paddle method. Take 20
mL or more of the dissolved solution 30 minutes after start-
ing the dissolution test, filter through a filter laminated with
polyester fibers, discard the first 10 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Separately, dry
Reserpine Reference Standard at 60°C in vacuum for 3
hours, weigh accurately an amount 100 times the labeled
amount, dissolve in 1 mL of chloroform and 80 mL of
ethanol (95), and add a solution of polysorbate 80 in diluted
dilute acetic acid (1 in 200) (1 in 20,000) to make exactly 200
mL. Pipet 1 mL of this solution, add a solution of polysor-
bate 80 in diluted dilute acetic acid (1 in 200) (1 in 20,000) to
make exactly 250 mL, and use this solution as the standard
solution. Pipet 5 mL each of the sample solution and stan-
dard solution, transfer to glass-stoppered brown test tubes T
and S, respectively, add exactly 5 mL each of ethanol (99.5),
shake well, add exactly 1 mL each of diluted vanadium (V)
oxide (1 in 2), shake vigorously, and allow to stand for 30
minutes. Perform the test with these solutions as directed un-
der Fluorometry <2.22>, and determine the intensity of
fluorescence, F T and F s , at the wavelength of excitation at 400
nm and at the wavelength of fluorescence at 500 nm. Dissolu-
tion rate of Reserpine Tablets after 30 minutes should be not
less than 70%.
Dissolution rate (%) to the labeled
amount of reserpine (C33H40N2O9)
= ^ s x(F T /F s )x(l/C)
W s : Amount (mg) of Reserpine Reference Standard.
C: Labeled amount (mg) of reserpine (C 3 3H4 N 2 C)9) in each
tablet.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately and pow-
der not less than 20 Reserpine Tablets. Weigh accurately a
quantity of the powder, equivalent to about 0.5 mg of reser-
pine (C 3 3H4 N 2 O9), add 3 mL of water, and warm at 50°C for
15 minutes while shaking. After cooling, add exactly 10 mL
of the internal standard solution, 10 mL of acetonitrile and
warm at 50°C for 15 minutes while shaking. After cooling,
add water to make 50 mL, centrifuge, and use the super-
natant liquid as the sample solution. Separately, weigh ac-
curately about 10 mg of Reserpine Reference Standard,
previously dried at 60°C in vacuum for 3 hours, and dissolve
in acetonitrile to make exactly 100 mL. Pipet 5 mL of this so-
lution, add exactly 10 mL of the internal standard solution, 5
mL of acetonitrile and water to make 50 mL, and use this so-
lution as the standard solution. Proceed with the sample solu-
tion and standard solution as directed in Assay under Reser-
pine.
Amount (mg) of reserpine (C33H40NO9)
= W s x(Q T /Q s )x (1/20)
W s : Amount (mg) of Reserpine Reference Standard
Internal standard solution — A solution of butyl parahydrox-
ybenzoate in acetonitrile (1 in 50,000).
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Retinol Acetate
Vitamin A Acetate
C 22 H 32 2 : 328.49
(2£,4£,6£,8£)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-
l-en-l-yl)nona-2,4,6,8-tetraen-l-yl acetate [127-47-9]
Retinol Acetate is synthetic retinol acetate or syn-
thetic retinol acetate diluted with fixed oil.
It contains not less than 2,500,000 Vitamin A Units
per gram.
A suitable antioxidant may be added.
Retinol Acetate contains not less than 95.0% and not
more than 105.0% of the labeled Units.
Description Retinol Acetate occurs as pale yellow to yellow-
red crystals or an ointment-like substance, and has a faint,
characteristic odor, but has no rancid odor.
It is freely soluble in petroleum ether, soluble in ethanol
(95), and practically insoluble in water.
It is decomposed by air and by light.
Identification Dissolve Retinol Acetate and Retinol Acetate
Reference Standard, equivalent to 15,000 Units, in 5 mL of
petroleum ether, and use these solutions as the sample solu-
tion and standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop with a mixture of cyclohexane and diethyl ether (12:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly antimony (III) chloride TS: the principal spot ob-
tained from the sample solution is the same in color tone and
Rf value with the blue spot from the standard solution.
Purity (1) Acid value <1.13> — Take exactly 5.0 g of
Retinol Acetate, and perform the test: not more than 2.0.
(2) Peroxide — Weigh accurately about 5 g of Retinol
Acetate, transfer in a 250-mL glass-stoppered conical flask,
add 50 mL of a mixture of acetic acid (100) and isooctane
(3:2), and gently mix to dissolve completely. Replace the air
of the inside gradually with about 600 mL of Nitrogen, then
add 0.1 mL of saturated potassium iodide TS under a current
of Nitrogen. Immediately stopper tightly, and mix with a
swirling motion for 1 minute. Add 30 mL of water, stopper
tightly, and shake vigorously for 5 to 10 seconds. Titrate
<2.50> this solution with 0.01 mol/L sodium thiosulfate VS
until the blue color of the solution disappears after addition
of 0.5 mL of starch TS near the end point where the solution
is a pale yellow color. Calculate the amount of peroxide by
the following formula: not more than 10 meq/kg.
JPXV
Official Monographs / Riboflavin 1059
Amount (meq/kg) of peroxide = ( V/ W) x 10
V: Volume (mL) of 0.01 mol/L sodium thiosulfate VS
consumed
W: Amount (g) of the sample
Assay Proceed as directed in Method 1-1 under Vitamin A
Assay <2.55>.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled, or under
Nitrogen atmosphere, and in a cold place.
Retinol Palmitate
Vitamin A Palmitate
H3C CH 3 CH 3 CH 3
oVl.
CH 3
7
CH 3
C 36 H 60 O 2 : 524.86
(2£,4£,6£,8£)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-
l-en-l-yl)nona-2,4,6,8-tetraen-l-yl palmitate [79-81-2]
Retinol Palmitate is a synthetic retinol palmitate or a
synthetic retinol palmitate diluted with fixed oil, and
contains not less than 1,500,000 Vitamin A Units in
each gram.
It may contain a suitable antioxidant.
Retinol Palmitate contains not less than 95.0% and
not more than 105.0% of the labeled Units.
Description Retinol Palmitate occurs as a light yellow to
yellow-red, ointment-like or an oily substance. It has a faint,
characteristic odor, but has no rancid odor.
It is very soluble in petroleum ether, slightly soluble in
ethanol (95), and practically insoluble in water.
It is decomposed by air and by light.
Identification Dissolve Retinol Palmitate and Retinol
Palmitate Reference Standard, equivalent to 15,000 Units, in
5 mL of petroleum ether, and use these solutions as the sam-
ple solution and standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 ,uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop with a mixture of cyclohexane and
diethyl ether (12:1) to a distance of about 10 cm, and air-dry
the plate. Spray evenly antimony (III) chloride TS: the prin-
cipal spot obtained from the sample solution is the same in
color tone and Rf value with the blue spot from the standard
solution.
Purity (1) Acid value <1.13> — Take exactly 5.0 g of
Retinol Palmitate, and perform the test: not more than 2.0.
(2) Peroxide — Weigh accurately about 5 g of Retinol
Palmitate, transfer in a 250-mL glass-stoppered conical flask,
add 50 mL of a mixture of acetic acid (100) and isooctane
(3:2), and gently mix to dissolve completely. Replace the air
of the inside gradually with about 600 mL of Nitrogen, then
add 0.1 mL of saturated potassium iodide TS under a current
of Nitrogen. Immediately stopper tightly, and mix with a
swirling motion for 1 minute. Add 30 mL of water, stopper
tightly, and shake vigorously for 5 to 10 seconds. Titrate
<2.50> this solution with 0.01 mol/L sodium thiosulfate VS
until the blue color of the solution disappears after addition
of 0.5 mL of starch TS near the end point where the solution
is a pale yellow color. Calculate the amount of peroxide by
the following formula: not more than 10 meq/kg.
Amount (meq/kg) of peroxide =(V/W)x 10
V: Volume (mL) of 0.01 mol/L sodium thiosulfate VS
W: Amount (g) of the sample
Assay Proceed as directed in Method 1-1 under the Vitamin
A Assay <2.55>.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled, or under
Nitrogen atmosphere, and in a cold place.
Riboflavin
Vitamin B 2
H3Cv ^tW°
K 3 C-W-N^Y NH
C 17 H 20 N 4 O 6 : 376.36
7,8-Dimethyl-10-[(2S,3S,4/?)-2,3,4,5-
tetrahydroxypentyl]benzo [g] pteridine-2 ,4(3//, 1 0// )-dione
[83-88-5]
Riboflavin, when dried, contains not less than 98.0%
of C 17 H 20 N 4 O 6 .
Description Riboflavin occurs as yellow to orange-yellow
crystals. It has a slight odor.
It is very slightly soluble in water, practically insoluble in
ethanol (95), in acetic acid (100), and in diethyl ether.
It dissolves in sodium hydroxide TS.
A saturated solution of Riboflavin is neutral.
It is decomposed by light.
Melting point: about 290°C (with decomposition).
Identification (1) A solution of Riboflavin (1 in 1 00,000) is
light yellow-green in color and has an intense yellow-green
fluorescence. The color and fluorescence of the solution dis-
appear upon the addition of 0.02 g of sodium hydrosulfite to
5 mL of the solution, and reappear on shaking the mixture in
air. This fluorescence disappears upon the addition of dilute
hydrochloric acid or sodium hydroxide TS.
(2) To 10 mL of a solution of Riboflavin (1 in 100,000)
placed in a glass-stoppered test tube add 1 mL of sodium
hydroxide TS, and after illumination with a fluorescence
lamp of 10 to 30 watts at 20-cm distance for 30 minutes be-
tween 20°C and 40°C, acidify with 0.5 mL of acetic acid (31),
1060 Riboflavin Powder / Official Monographs
JP XV
and shake with 5 mL of chloroform: the chloroform layer
shows a yellow-green fluorescence.
(3) Determine the absorption spectrum of a solution of
Riboflavin in phosphate buffer solution, pH 7.0 (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Rivoflavin Refer-
ence Standard prepared in the same manner as the sample so-
lution: both spectra exhibit similar intensities of absorption
at the same wavelengths.
Optical rotation <2.49> [a]™: -128- -142° Weigh ac-
curately about 0.1 g of dried Riboflavin, dissolve in exactly 4
mL of dilute sodium hydroxide TS, add 10 mL of freshly
boiled and cooled water, add exactly 4 mL of aldehyde-free
alcohol while shaking, add freshly boiled and cooled water to
make exactly 20 mL, and determine the rotation in a 100-mm
cell within 30 minutes after preparing the solution.
Purity Lumiflavin — Shake 25 mg of Riboflavin with 10 mL
of ethanol-free chloroform for 5 minutes, and filter: the
filtrate has no more color than the following control solution.
Control solution: To 2.0 mL of l/60mol/L potassium
dichromate VS add water to make 1000 mL.
Loss on drying <2.41> Not more than 1.5% (0.5 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately about
15 mg of Riboflavin, previously dried, dissolve in 800 mL of
diluted acetic acid (100) (1 in 400) by warming, cool, add
water to make exactly 1000 mL, and use this solution as the
sample solution. Dry Riboflavin Reference Standard at
105 °C for 2 hours, weigh accurately about 15 mg, dissolve in
800 mL of diluted acetic acid (100) (1 in 400) by warming,
cool, add water to make exactly 1000 mL, and use this solu-
tion as the standard solution. Perform the test with the sam-
ple solution and standard solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
blank, and determine the absorbances, A T and A s , at 445 nm.
Add 0.02 g of sodium hydrosulfite to 5 mL of each solution,
shake until decolorized, and immediately measure the absor-
bances, At' and A s ' , of the solutions.
Amount (mg) of C 17 H2oN 4 6
= W s x{(A J -A J ')/(A s -A s ')}
W s : Amount (mg) of Riboflavin Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Riboflavin Powder
Vitamin B 2 Powder
Riboflavin Powder contains not less than 95% and
not more than 115% of the labeled amount of
riboflavin (C 17 H 20 N 4 O 6 : 376.36).
Method of preparation Prepare as directed under Powders,
with Riboflavin.
Identification Shake a portion of Riboflavin Powder,
equivalent to 1 mg of Riboflavin according to the labeled
amount, with 100 mL of water, filter, and proceed with the
filtrate as directed in the Identification (1) and (2) under
Riboflavin.
Purity Rancidity — Riboflavin Powder is free from any un-
pleasant or rancid odor or taste.
Assay The procedure should be performed under protec-
tion from direct sunlight and in light-resistant vessels. Weigh
accurately Riboflavin Powder equivalent to about 15 mg of
riboflavin (C 17 H2oN 4 6 ), add 800 mL of diluted acetic acid
(100) (1 in 400), and extract by warming for 30 minutes with
occasional shaking. Cool, dilute with water to make exactly
1000 mL, and filter through a glass filter (G4). Use this filtrate
as the sample solution, and proceed as directed in the Assay
under Riboflavin.
Amount (mg) of riboflavin (C 17 H 2 oN 4 6 )
= W s x{(A J -A T ')/(A s -A s ')}
W s : Amount (mg) of Riboflavin Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Riboflavin Butyrate
U*'77t':
iJLXTll
H,C
r*ir
Cri,
CH 3
,«AA N
NH
C33H44N4O10: 656.72
(2i?,3S,4S)-5-(7,8-Dimethyl-2,4-dioxo-3,4-
dihydrobenzo[g]pteridin-10(2//)-yi)pentan-l,2,3,4-tetrayl
tetrabutanoate [752-56-7]
Riboflavin Butyrate, when dried, contains not less
than 98.5% of C33H44N4O10.
Description Riboflavin Butyrate occurs as orange-yellow
crystals or crystalline powder. It has a slight, characteristic
odor and a slightly bitter taste.
It is freely soluble in methanol, in ethanol (95) and in chlo-
roform, slightly soluble in diethyl ether, and practically in-
soluble in water.
It is decomposed by light.
Identification (1) A solution of Riboflavin Butyrate in
ethanol (95) (1 in 100,000) shows a light yellow-green color
with a strong yellowish green fluorescence. To the solution
add dilute hydrochloric acid or sodium hydroxide TS: the
fluorescence disappears.
(2) Dissolve 0.01 g of Riboflavin Butyrate in 5 mL of
JPXV
Official Monographs / Riboflavin Sodium Phosphate 1061
ethanol (95), add 2 mL of a mixture of a solution of hydrox-
ylammonium chloride (3 in 20) and a solution of sodium
hydroxide (3 in 20) (1:1), and shake well. To this solution add
0.8 mL of hydrochloric acid and 0.5 mL of iron (III) chloride
TS, and add 8 mL of ethanol (95): a deep red-brown color de-
velops.
(3) Determine the absorption spectrum of the sample so-
lution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
Melting point <2.60> 146 - 150°C
Purity (1) Chloride — Dissolve 2.0 g of Riboflavin
Butyrate in 10 mL of methanol, and add 24 mL of dilute
nitric acid and water to make 100 mL. After shaking well, al-
low to stand for 10 minutes, filter, discard the first 10 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. To 25 mL of the sample solution add water to make 50
mL, then add 1 mL of silver nitrate TS, and allow to stand
for 5 minutes: the turbidity of the solution is not thicker than
that of the following control solution.
Control solution: To 25 mL of the sample solution add 1
mL of silver nitrate TS, allow to stand for 10 minutes, and
filter. Wash the precipitate with four 5-mL portions of water,
and combine the washings with the filtrate. To this solution
add 0.30 mL of 0.01 mol/L hydrochloric acid VS and water
to make 50 mL, add 1 mL of water, and mix (not more than
0.021%).
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Riboflavin Butyrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Free acid — To 1 .0 g of Riboflavin Butyrate add 50 mL
of freshly boiled and cooled water, shake, and filter. To 25
mL of the filtrate add 0.50 mL of 0.01 mol/L sodium
hydroxide VS and 2 drops of phenolphthalein TS: the solu-
tion shows a red color.
(4) Related substances — Dissolve 0.10 g of Riboflavin
Butyrate in 10 mL of chloroform, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
chloroform to make exactly 50 mL. Pipet 5 mL of this solu-
tion, add chloroform to make exactly 20 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform and 2-propanol (9:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately about 40
mg of Riboflavin Butyrate, previously dried, dissolve in
ethanol (95) to make exactly 500 mL, and pipet 10 mL of this
solution, add ethanol (95) to make exactly 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of Riboflavin Reference Standard,
previously dried at 105°C for 2 hours, dissolve in 150 mL of
diluted acetic acid (100) (2 in 75) by warming, and after cool-
ing, add water to make exactly 500 mL. Pipet 5 mL of this so-
lution, add ethanol (95) to make exactly 50 mL, and use this
solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 445 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of C33H44N4O10
= W s x (Aj/A s ) x 1 .7449 x ( 1 /2)
W s : Amount (mg) of Riboflavin Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Riboflavin Sodium Phosphate
Riboflavin Phosphate
Vitamin B 2 Phosphate Ester
'J 71*77 fcf>U >igxXxJU1-r- 'J7A
.,0-poaHNa
C l7 H 2 oN 4 Na0 9 P: 478.33
Monosodium (2i?,3S,4S)-5-(7,8-dimethyl-2,4-dioxo-3,4-
dihydrobenzo[g]pteridin-10(2//)-yi)-2,3,4-trihydroxypentyl
monohydrogenphosphate [J 30-40-5]
Riboflavin Sodium Phosphate contains not less than
92% of CnHjo^NaOgP, calculated on the anhydrous
basis.
Description Riboflavin Sodium Phosphate is a yellow to
orange-yellow, crystalline powder. It is odorless, and has a
slightly bitter taste.
It is soluble in water, and practically insoluble in ethanol
(95), in chloroform and in diethyl ether.
It is decomposed on exposure to light.
It is very hygroscopic.
Identification (1) A solution of Riboflavin Sodium Phos-
phate (1 in 100,000) is light yellow-green in color and has an
intense yellow-green fluorescence. The color and fluorescence
of the solution disappear upon the addition of 0.02 g of sodi-
um hydrosulfite to 5 mL of the solution, and reappear on
shaking the mixture in air. This fluorescence disappears upon
the addition of dilute hydrochloric acid or sodium hydroxide
TS.
(2) To 10 mL of a solution of Riboflavin Sodium Phos-
phate (1 in 100,000) placed in a glass-stoppered test tube add
1 mL of sodium hydroxide TS, and after illumination with a
fluorescence lamp of 10 to 30 watts at 20-cm distance for 30
1062 Riboflavin Sodium Phosphate Injection / Official Monographs
JP XV
minutes between 20°C and 40°C, acidify with 0.5 mL of acet-
ic acid (31), and shake with 5 mL of chloroform: the chlo-
roform layer shows a yellow-green fluorescence.
(3) Determine the absorption spectrum of a solution of
Riboflavin Sodium Phosphate in phosphate buffer solution,
pH 7.0, (1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(4) To 0.05 g of Riboflavin Sodium Phosphate add 10 mL
of nitric acid, evaporate on a water bath to dryness, and ig-
nite. Boil the residue with 10 mL of nitric acid (1 in 50) for 5
minutes, after cooling, neutralize this solution with ammonia
TS, and filter, if necessary: the solution responds to the
Qualitative Tests <1.09> for sodium salt and phosphate.
Optical rotation <2.49> [a]™'- +38 - +43° (0.3 g, calculat-
ed on the anhydrous basis, 5 mol/L hydrochloric acid TS, 20
mL, 100 mm).
pH <2.54> Dissolve 0.20 g of Riboflavin Sodium Phosphate
in 20 mL of water: the pH of the solution is between 5.0 and
6.5.
Purity (1) Clarity and color of solution — Dissolve 0.20 g
of Riboflavin Sodium Phosphate in 10 mL of water: the solu-
tion is clear and yellow to orange-yellow in color.
(2) Lumiflavin — To 35 mg of Riboflavin Sodium Phos-
phate add 10 mL of ethanol-free chloroform, and shake for 5
minutes, then filter: the filtrate has no more color than the
control solution.
Control solution: To 3.0 mL of 1/60 mol/L potassium
dichromate VS add water to make 1000 mL.
(3) Free phosphoric acid — Weigh accurately about 0.4 g
of Riboflavin Sodium Phosphate, dissolve in water to make
exactly 100 mL, and use this solution as the sample solution.
Measure exactly 5 mL each of the sample solution and Phos-
phoric Acid Standard Solution, transfer to separate 25-mL
volumetric flasks, add 2.5 mL of hexaammonium hep-
tamolybdate-sulfuric acid TS and 1 mL of l-amino-2-
naphthol-4-sulfonic acid TS to each of these flasks, mix, and
add water to make 25 mL. Allow to stand for 30 minutes at
20 ± 1 °C, and perform the test with these solutions as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, using
a solution prepared with 5 mL of water in the same manner as
ablank. Determine the absorbances, ^4 T and A s , of the subse-
quent solutions of the sample solution and standard phos-
phoric acid solution at 740 nm: the free phosphoric acid con-
tent is not more than 1.5%.
Content (%) of free phosphoric acid (H 3 P0 4 )
= (A T /A S )X(1/W)X257.S
W: Amount (mg) of Riboflavin Sodium Phosphate calcu-
lated on the anhydrous basis.
Water <2.48> Place 25 mL of a mixture of methanol for
Karl Fischer method and ethylene glycol for Karl Fischer
method (1:1) in a dry flask for titration, and titrate with water
determination TS to the end point. Weigh accurately about
0.1 g of Riboflavin Sodium Phosphate, place quickly into the
flask, add a known excess volume of Karl Fischer TS, mix for
10 minutes, and perform the test: the water content is not
more than 10.0%.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. To about 0.1 g of
Riboflavin Sodium Phosphate, accurately weighed, dissolve
in diluted acetic acid (100) (1 in 500) to make exactly 1000
mL, then pipet 10 mL of this solution, and add diluted acetic
acid (100) (1 in 500) to make exactly 50 mL. Use this solution
as the sample solution. Separately, dry Riboflavin Reference
Standard at 105°C for 2 hours, weigh accurately about 15
mg, dissolve in 800 mL of diluted acetic acid (100) (1 in 400)
by warming, cool, add water to make exactly 1000 mL, and
use this solution as the standard solution. Perform the test
with the sample solution and standard solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using
water as the blank, and determine the absorbances, A T and A
s , at 445 nm. Add 0.02 g of sodium hydrosulfite to 5 mL of
each solution, shake until decolorized, and immediately
measure the absorbances, A T ' and A s ' , of the solutions.
Amount (mg) of C 17 H2oN 4 Na09P
= W s x {(A T -Aj')/(A S -A s ')} x 1.2709X5
W s : Amount (mg) of Riboflavin Reference Standard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Riboflavin Sodium Phosphate
Injection
Riboflavin Phosphate Injection
Vitamin B 2 Phosphate Ester Injection
Riboflavin Sodium Phosphate Injection is an aque-
ous solution for injection.
It contains not less than 95% and not more than
120% of the labeled amount of riboflavin (C 17 H 2 oN 4 6 :
376.36).
The concentration of Riboflavin Sodium Phosphate
Injection should be stated as the amount of riboflavin
(C 17 H 20 N 4 O 6 ).
Method of preparation Prepare as directed under Injec-
tions, with Riboflavin Sodium Phosphate.
Description Riboflavin Sodium Phosphate Injection is a
clear, yellow to orange-yellow liquid.
pH: 5.0-7.0
Identification (1) To a measured volume of Riboflavin So-
dium Phosphate Injection, equivalent to 1 mg of Riboflavin
according to the labeled amount, add water to make 100 mL,
and proceed with this solution as directed in the Identification
(1) and (2) under Riboflavin Sodium Phosphate.
(2) To a measured volume of Riboflavin Sodium Phos-
phate Injection, equivalent to 0.05 g of Riboflavin according
to the labeled amount, and evaporate on a water bath to dry-
ness. Proceed with this residue as directed in the Identifica-
tion (4) under Riboflavin Sodium Phosphate.
Extractable volume <6.05> It meets the requirement.
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. To an accurately measured
JPXV
Official Monographs / Ribostamycin Sulfate 1063
volume of Riboflavin Sodium Phosphate Injection, equiva-
lent to about 15 mg of riboflavin (C 17 H2oN 4 6 ), add diluted a-
cetic acid (100) (1 in 500) to make exactly 1000 mL, and use
this solution as the sample solution. Proceed as directed in
the Assay under Riboflavin Sodium Phosphate.
Amount (mg) of Riboflavin (C 17 H2oN 4 6 )
= W s x{(A T -Aj')/(A s -A s ')}
W s : Amount (mg) of Riboflavin Reference Standard
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Ribostamycin Sulfate
i)Xx9v{'»m&i§.
■ JfHjSOj
C17-H34N4O10.XH2SO4
2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(l -> 4)-
[/?-D-ribofuranosyl-(l -> 5)]-2-deoxy-D-streptamine sulfate
[53797-35-6]
Ribostamycin Sulfate is the sulfate of an
aminoglycoside substance having antibacterial activity
produced by the growth of Streptomyces ribosidificus.
It contains not less than 680 /ug (potency) and not
more than 780 fig (potency) per mg, calculated on the
dried basis. The potency of Ribostamycin Sulfate is
expressed as mass (potency) of ribostamycin
(C 17 H 34 N 4 O 10 : 454.47).
Description Ribostamycin Sulfate occurs as a white to yel-
lowish white powder.
It is freely soluble in water, and practically insoluble in
ethanol (95).
Identification (1) Dissolve 20 mg of Ribostamycin Sulfate
in 2 mL of phosphate buffer solution, pH 6.0, add 1 mL of
ninhydrin TS, and boil: a blue-purple color develops.
(2) Dissolve 0.12 g each of Ribostamycin Sulfate and
Ribostamycin Sulfate Reference Standard in 20 mL of water,
and use these solutions as the sample solution and standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 fiL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
solution of potassium dihydrogen phosphate (3 in 40) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
0.2% ninhydrin-water saturated 1-butanol TS, and heat at
100°C for 10 minutes: the principal spots obtained from the
sample solution and standard solution show a purple-brown
color and the same Ri value.
(3) To 2 mL of a solution of Ribostamycin Sulfate (1 in 5)
add 1 drop of barium chloride TS: a white turbidity is pro-
duced.
Optical rotation <2.49> [a]o- +42- +49° (after drying,
0.25 g, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Ribostamycin Sulfate in 20 mL of water is between 6.0 and
8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Ribostamycin Sulfate in 5 mL of water: the solution is clear,
and colorless or pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Ribostamycin Sulfate according to Method 1, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 30ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ribostamycin Sulfate according to Method 1, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.12 g of Ribostamycin
Sulfate in water to make exactly 20 mL, and use this solution
as the sample solution. Pipet 5 mL of the sample solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
fiL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a solution of potassium dihydrogen phosphate (3
in 40) to a distance of about 10 cm, and air-dry the plate.
Spray evenly 0.2% ninhydrin-water saturated 1-butanol TS
on the plate, and heat at 100°C for 10 minutes: the spot other
than the principal spot obtained from the sample solution is
not more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 5.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under <4.02> Microbial Assay for An-
tibiotics according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er.
(iii) Standard solutions — Weigh accurately an amount of
Ribostamycin Sulfate Reference Standard, previously dried,
equivalent to about 20 mg (potency), dissolve in diluted phos-
phate buffer solution, pH 6.0 (1 in 2) to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5 to 15 °C and use within 20 days.
Take exactly a suitable amount of the standard stock solution
before use, add 0.1 mol/L phosphate buffer solution, pH 8.0
to make solutions so that each mL contains 20 fig (potency)
and 5 fig (potency), and use these solutions as the high con-
centration standard solution and low concentration standard
solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Ribostamycin Sulfate, equivalent to about 20 mg (potency),
1064 Rifampicin / Official Monographs
JP XV
and dissolve in water to make exactly 50 mL. Take exactly a
suitable amount of this solution, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 20 fig (potency) and 5 fig (potency), and use these so-
lutions as the high concentration sample solution and low
concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Rifampicin
U7r>tfv>
H a C H H CH 3
"■CH*
C4 3 H 58 N 4 12 : 822.94
(25, \2Z,UE, 165,17s, 18/?, \9R, 20R, 215,22/?, 23S,24£>
5,6,9,17,19-Pentahydroxy-23-methoxy-
2,4, 12,16, 18,20,22-heptamethyl-8-(4-methylpiperazin-l-
yliminomethyl)- 1 , 1 1 -dioxo- 1 ,2-dihydro-2 ,7-
(epoxypentadeca [1 ,11,1 3]trienimino)naphtho [2, 1 -b] furan-
21-yl acetate [13292-46-1]
Rifampicin is a derivative of a substance having an-
tibacterial activity produced by the growth of Strep-
tomyces mediterranei.
It contains not less than 970 fig (potency) and not
more than 1020 fig (potency) per mg, calculated on the
dried basis. The potency of Rifampicin is expressed as
mass (potency) of rifampicin (C 4 3H5 8 N40 12 ).
Description Rifampicin occurs as orange-red to red-brown,
crystals or crystalline powder.
It is slightly soluble in water, in acetonitrile, in methanol
and in ethanol (95).
Identification (1) To 5 mL of a solution of Rifampicin in
methanol (1 in 5000) add 0.05 mol/L phosphate buffer solu-
tion, pH 7.0 to make 100 mL. Determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum or the spectrum of a solution of
Rifampicin Reference Standard prepared in the same manner
as the sample solution: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Rifampicin as directed in the potassium bromide disk method
under the Infrared Spectrophotometry <2.25>, and compare
the spectrum with the Reference Spectrum or the spectrum of
Rifampicin Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Rifampicin according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Rifampicin according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Perform the test immediately af-
ter preparing of the sample and standard solutions. Dissolve
0. 10 g of Rifampicin in 50 mL of acetonitrile, and use this so-
lution as the sample stock solution. Pipet 5 mL of the sample
stock solution, add citric acid-phosphate-acetonitrile TS to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, pipet 1 mL of the sample stock solution,
and add acetonitrile to make exactly 100 mL. Pipet 5 mL of
this solution, add citric acid-phosphate-acetonitrile TS to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with exactly 50 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the area of the peak appeared at the relative
retention time of about 0.7 with respect to rifampicin from
the sample solution is not more than 1.5 times the peak area
of rifampicin from the standard solution, the area of the
peak other than rifampicin and the peak mentioned above
from the sample solution is not more than the peak area of
rifampicin from the standard solution, and the total area of
the peaks other than rifampicin and the peak mentioned
above from the sample solution is not more than 3.5 times the
peak area of rifampicin from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of rifampicin beginning after the peak of the
solvent.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add citric acid-phosphate-acetoni-
trile TS to make exactly 20 mL. Confirm that the peak area of
rifampicin obtained from 50 fiL of this solution is equivalent
to 7 to 13% of that from 50 fiL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
50 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
rifampicin is not more than 2.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Rifampicin and
Rifampicin Reference Standard, equivalent to about 40 mg
(potency), and dissolve each in acetonitrile to make exactly
200 mL. Pipet 10 mL each of these solutions, add citric acid-
phosphate-acetonitrile TS to make exactly 100 mL, and use
these solutions as the sample solution and standard solution.
Perform the test with exactly 50 fiL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of rifampicin.
Amount [fig (potency)] of C 4 3H5 8 N40 12
JPXV
Official Monographs / Rifampicin Capsules 1065
= ^ s x(-4t/-4s)x1000
W s : Amount [mg (potency)] of Rifampicin Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 10 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 4.2 g of citric acid monohydrate
and 1.4 g of sodium perchlorate in 1000 mL of a mixture of
water, acetonitrile and phosphate buffer solution, pH 3.1
(11:7:2).
Flow rate: Adjust the flow rate so that the retention time of
rifampicin is about 8 minutes.
System suitability —
System performance: To 5 mL of a solution of Rifampicin
in acetonitrile (1 in 5000) add 1 mL of a solution of butyl
parahydroxybenzoate in acetonitrile (1 in 5000) and citric
acid-phosphate-acetonitrile TS to make 50 mL. When the
procedure is run with 50 /uL of this solution under the above
operating conditions, butyl parahydroxybenzoate and rifam-
picin are eluted in this order with the resolution between these
peaks being not less than 1.5.
System repeatability: When the test is repeated 5 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
rifampicin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Rifampicin Capsules
Rifampicin Capsules contain not less than 93.0%
and not more than 105.0 % of rifampicin
(C 43 H 58 N 4 12 : 822.94).
Method of preparation Prepare as directed under Capsules,
with Rifampicin.
Identification Dissolve an amount of the content of Rifam-
picin Capsules, equivalent to 20 mg (potency) of Rifampicin
according to the labeled amount, in methanol to make 100
mL, and filter. To 5 mL of the filtrate add 0.05 mol/L phos-
phate buffer solution, pH 7.0 to make 100 mL. Determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 234 nm and 238 nm, between 252 nm and 256
nm, between 331 nm and 335 nm, and between 472 nm and
476 nm.
Purity Related substances — Perform the test quickly after
the sample solution and the standard solution are prepared.
Open the capsules of not less than 20 Rifampicin Capsules,
carefully take out the content, weigh accurately, and powder.
Weigh accurately a portion of the powder, equivalent to
about 20 mg (potency) of Rifampicin according to the labeled
amount, and dissolve in acetonitrile to make exactly 10 mL.
Pipet 2 mL of this solution, add a mixture of acetonitrile and
methanol (1:1) to make exactly 20 mL, and use this solution
as the sample solution. Separately, weigh accurately about 20
mg (potency) of Rifampicin Reference Standard, and dis-
solve in acetonitrile to make exactly 10 mL. Pipet 2 mL of
this solution, and add the mixture of acetonitrile and
methanol (1 : 1) to make exactly 20 mL. Pipet 1 mL of this so-
lution, add the mixture of acetonitrile and methanol (1:1) to
make exactly 50 mL, and use this solution as the standard so-
lution. Perform the test with exactly 20 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the amount of the peaks of quinone substance
and TV-oxide substance, having the relative retention time of
about 0.5 and about 1.2 with respect to rifampicin, respec-
tively, are not more than 4.0% and not more than 1.5%, re-
spectively. The amount of the peak other than the peaks men-
tioned above is not more than 1.0%, and the total amount of
these related substances is not more than 2.0%. For these cal-
culations, use the areas of the peaks of the quinone substance
and TV-oxide substance after multiplying by their relative
response factors, 1.24 and 1.16, respectively.
Amount (mg) of quinone substance
= (W s /Wj)x(A T!i /A s )x2AS
Amount (mg) of TV-oxide substance
= (W s /W 1 )x(A Th /A s )x2.32
Each amount (mg) of related substances other than quinone
and TV-oxide substances = ( W s / W T ) x (A Ti /A s ) x 2
W s : Amount [mg (potency)] of Rifampicin Reference
Standard
Wj\ Amount [mg (potency)] of sample
^4 S : Peak area of the standard solution
A T z- Peak area of quinone substance
A Jb : Peak area of TV-oxide substance
A Tl : Each peak area of related substances other than
quinone and TV-oxide substances
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 2.1 g of sodium perchlorate, 6.5 g
of citric acid monohydrate and 2.3 g of potassium dihydro-
gen phosphate in 1100 mL of water, and add 900 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
rifampicin is about 12 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of rifampicin.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add a mixture of acetonitrile and methanol
(1:1) to make exactly 20 mL. Confirm that the peak area of
rifampicin obtained with 20 /xh of this solution is equivalent
to 3.5 to 6.5% of that with 20 /uL of the standard solution.
System performance: When the procedure is run with 20
1066 Ringer's Solution / Official Monographs
JP XV
fiL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of rifampicin is not less than 2500 and not
more than 4.0, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
rifampicin is not more than 2.0%.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Assay Open the capsules of not less than 20 Rifampicin
Capsules, take out the content, weigh accurately the mass of
the content, and powder. Weigh accurately a portion of the
powder, equivalent to about 75 mg (potency) of Rifampicin,
dissolve in a mixture of acetonitrile and methanol (1:1) to
make exactly 50 mL. Pipet 10 mL of this solution, and add
acetonitrile to make exactly 50 mL. Pipet 5 mL of this solu-
tion prepared by dissolving 2.1 g of citric acid monohydrate,
27.6 g of disodium hydrogen phosphate dodecahydrate and
3.1 g of potassium dihydrogen phosphate in 1000 mL of a
mixture of water and acetonitrile (3:1) to make exactly 50
mL, and use this solution as the sample solution. Separately,
weigh accurately about 30 mg (potency) of Rifampicin Refer-
ence Standard, dissolve in 20 mL of a mixture of acetonitrile
and methanol (1:1), and add acetonitrile to make exactly 100
mL. Pipet 5 mL of this solution, add the solution prepared
by dissolving 2.1 g of citric acid monohydrate, 27.6 g of diso-
dium hydrogen phosphate dodecahydrate and 3.1 g of potas-
sium dihydrogen phosphate in 1000 mL of the mixture of
water and acetonitrile (3:1) to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
exactly 50 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of rifampicin.
Amount [mg (potency)] of rifampicin (C43H5 8 N40 12 )
= W s x (Aj/As) x (5/2)
W s : Amount [mg (potency)] of Rifampicin Reference
Standard
Operating conditions-
Proceed as directed in the operating conditions in the As-
say under Rifampicin.
System suitability —
System performance: Dissolve 30 mg (potency) of Rifam-
picin Reference Standard in 20 mL of the mixture of acetoni-
trile and methanol (1:1), and add acetonitrile to make 100
mL. To 5 mL of this solution add 2 mL of a solution of butyl
parahydroxybenzoate in the mixture of acetonitrile and
methanol (1:1) (1 in 5000), then add the solution prepared by
dissolving 2.1 g of citric acid monohydrate, 27.6 g of disodi-
um hydrogen phosphate dodecahydrate and 3.1 g of potassi-
um dihydrogen phosphate in 1000 mL of a mixture of water
and acetonitrile (3:1) to make exactly 50 mL. When the
procedure is run with 50 /uL of this solution under the above
operating conditions, butyl parahydroxybenzoate and rifam-
picin are eluted in this order with the resolution between these
peaks being not less than 1.5.
System repeatability: When the test is repeated 5 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
rifampicin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Ringer's Solution
Ringer's Solution is an aqueous solution for injec-
tion.
It contains not less than 0.53 w/v% and not more
than 0.58 w/v% of chlorine [as (CI: 35.45)], and not
less than 0.030 w/v% and not more than 0.036 w/v%
of calcium chloride hydrate (CaCl 2 .2H 2 0: 147.01).
Method of preparation
Sodium Chloride
Potassium Chloride
Calcium Chloride Hydrate
Water for Injection
0.3 g
0.33 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
No preservative may be added.
Description Ringer's Solution is a clear and colorless liquid.
It has a slightly saline taste.
Identification (1) Evaporate 10 mL of Ringer's Solution
to 5 mL: the solution responds to the Qualitative Tests <1.09>
for potassium salt and calcium salt.
(2) Ringer's Solution responds to the Qualitative Tests
<1.09> for sodium salt and chloride.
pH <2.54> 5.0-7.5
Purity (1) Heavy metals <1.07> — Evaporate 100 mL of
Ringer's Solution to about 40 mL on a water bath. Add 2 mL
of dilute acetic acid and water to make 50 mL, and perform
the test using this solution as the test solution. Control solu-
tion: to 3.0 mL of Standard Lead Solution add 2 mL of dilute
acetic acid and water to make 50 mL (not more than 0.3
ppm).
(2) Arsenic <1.11>— Perform the test with 20 mL of Rin-
ger's Solution as the test solution (not more than 0.1 ppm).
Bacterial endotoxins <4.01> Less than 0.50EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay (1) Chlorine — To 20 mL of Ringer's Solution, ac-
curately measured, add 30 mL of water. Titrate <2.50> with
0.1 mol/L silver nitrate VS while shaking vigorously (indica-
tor: 3 drops of sodium fluorescein TS).
Each mL of 0.1 mol/L silver nitrate VS
= 3.545 mg of CI
(2) Calcium chloride Hydrate — To 50 mL of Ringer's So-
lution, exactly measured, add 2 mL of 8 mol/L potassium
hydroxide TS and 0.05 g of NN indicator, and titrate <2.50>
immediately with 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS, until the color of the solu-
tion changes from red-purple to blue.
Each mL of 0.01 mol/L disodium dihydrogen
JPXV
Official Monographs / Ritodrine Hydrochloride 1067
ethylenediamine tetraacetate VS
= 1.470 mg of CaCl 2 .2H 2
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous infusions may be used.
Ritodrine Hydrochloride
'J r- K U >WI£ik
H OH
I 'HCI
and enantiomer
C 17 H 21 NCvHCl: 323.81
(l/?5',25'i?)-l-(4-Hydroxyphenyl)-2-
{[2-(4-hydroxyphenyl)ethyl]amino)propan-l-ol
monohydrochloride
[23239-51-2]
Ritodrine Hydrochloride, when dried, contains not
less than 98.0% and not more than 102.0% of
C 17 H 21 N0 3 .HC1.
Description Ritodrine Hydrochloride occurs as a white
crystalline powder.
It is freely soluble in water, in methanol and in ethanol
(99.5).
It dissolves in 0.01 mol/L hydrochloric acid TS.
A solution of Ritodrine Hydrochloride (1 in 10) shows no
optical rotation.
Melting point: about 196°C (with decomposition).
It is gradually colored to a light yellow by light.
Identification (1) Determine the absorption spectrum of a
solution of Ritodrine Hydrochloride (1 in 20,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Ritodrine Hydrochloride Reference
Standard prepared in the same manner as the sample solu-
tion: both spectra exhibit similar intensities of absorption at
the same wavelengths.
(2) Determine the infrared absorption spectrum of Rito-
drine Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Ritodrine Hydrochloride Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) A solution of Ritodrine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Ritodrine Hydrochloride in 50 mL of water is between 4.5
and 5.5.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 1.0 g of Ritodrine Hydrochloride in 10
mL of water is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Ritodrine
Hydrochloride according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Related substances — Dissolve 20 mg of Ritodrine
Hydrochloride in 20 mL of the mobile phase, and use this so-
lution as the sample solution. Pipet 1 mL of the sample solu-
tion, add the mobile phase to make exactly 200 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak of ritodrine threo-isomer, having the relative retention
time of about 1.2 with respect to ritodrine, is not larger than
4/5 times the peak area of ritodrine from the standard solu-
tion, the area of the peak other than ritodrine and ritodrine
threo-isomer is not larger than 3/10 times the peak area of
ritodrine from the standard solution, and the total area of the
peaks other than ritodrine and ritodrine threo-isomer is not
larger than 4 times the peak area of ritodrine from the stan-
dard solution.
Operating conditions —
Column, column temperature, and mobile phase: Proceed
as directed in the operating conditions in the Assay.
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Flow rate: Adjust the flow rate so that the retention time of
ritodrine is about 10 minutes.
Time span of measurement: About 3 times as long as the
retention time of ritodrine beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add exactly 50 mL of the mobile phase. Con-
firm that the peak area of ritodrine obtained with 10 fiL of
this solution is equivalent to 7 to 13% of that with 10 jxL of
the standard solution.
System performance: To about 20 mg of ritodrine
hydrochloride add 50 mL of the mobile phase and 5.6 mL of
sulfuric acid, and add the mobile phase to make 100 mL.
Heat a portion of this solution at about 85 °C for about 2
hours, and allow to cool. Pipet 10 mL of this solution, and
add exactly 10 mL of 2 mol/L sodium hydroxide TS. When
the procedure is run with 10 fiL of this solution under the
above operating conditions, ritodrine and the threo-isomer
are eluted in this order with the resolution between these
peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ritodrine is not more than 2.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C, 2
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 30 mg each of Ritodrine
Hydrochloride and Ritodrine Hydrochloride Reference Stan-
dard, previously dried, and dissolve in methanol to make ex-
actly 50 mL. Pipet 25 mL of these solutions, add exactly 5
mL of the internal standard solution, then add water to make
50 mL, and use these solutions as the sample solution and
standard solution. Perform the test with 10 /uL of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the ratios, Q T and Q s , of the peak area
of ritodrine to that of the internal standard.
Amount (mg) of C 17 H 21 N0 3 .HC1
1068 Ritodrine Hydrochloride Tablets / Official Monographs
JP XV
= W s x(Q T /Qs)
W s : Amount (mg) of Ritodrine Hydrochloride Reference
Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in methanol (3 in 5000)
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.6 g of diammonium hydrogen
phosphate and 1.1 g of sodium 1-heptanesulfonate in 700 mL
of water, and add 300 mL of methanol. Adjust to pH 3.0
with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
ritodrine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, ritodrine and the internal standard are eluted in this
order with the resolution between these peaks being not less
then 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of ritodrine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ritodrine Hydrochloride Tablets
U r- K U >WI£ik&
Ritodrine Hydrochloride Tablets contain not less
than 93.0% and not more than 107. 0%> of the labeled
amount of ritodrine hydrochloride (C 17 H2iN0 3 .HCl:
323.81).
Method of preparation Prepare as directed under Tablets,
with Ritodrine Hydrochloride.
Identification To 10 mL of the filtrate obtained in the Assay
add 0.01 mol/L hydrochloric acid TS to make 100 mL. De-
termine the absorption spectrum of this solution as directed
under Ultraviolet-visible Spectrophotometry <2.24>: it ex-
hibits a maximum between 272 nm and 276 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Ritodrine Hydrochloride Tablets add 9 mL
of 0.01 mol/L hydrochloric acid TS, shake until the tablet is
completely disintegrated, add 0.01 mol/L hydrochloric acid
TS to make exactly 10 mL, and filter through a membrane
filter having pore size of 0.45 /xm. Pipet 3 mL of the filtrate,
add exactly 1 mL of the internal standard solution, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Ritodrine Hydrochloride Reference
Standard, previously dried at 105 °C for 2 hours, and dissolve
in 0.01 mol/L hydrochloric acid TS to make exactly 50 mL.
Pipet 3 mL of this solution, add exactly 1 mL of the internal
standard solution, and use this solution as the standard solu-
tion. Perform the test with 10 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the ratios, g T and Q s , of the peak area of rito-
drine to that of the internal standard.
Amount (mg) of ritodrine hydrochloride (C 17 H 2 iN03.HCl)
= W s x(Q T /Q s )x(l/5)
W s : Amount (mg) of Ritodrine Hydrochloride Reference
Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in methanol (3 in 10,000)
Operating conditions —
Proceed as directed in the Assay.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, ritodrine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of ritodrine to that of the internal standard is not
more than 1.0%.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Ritodrine Hydrochloride
Tablets at 50 revolutions per minute according to the Paddle
method using 900 mL of water as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 15
minutes after starting the test, and filter through a membrane
filter with pore size of not more than 0. 45 /xm. Discard the
first 10 mL of the filtrate, pipet the subsequent FmL, add
water to make exactly V mL so that each mL contains about
5.6 //g of ritodrine hydrochloride (C 17 H2iN0 3 .HCl) accord-
ing to the labeled amount, and use this solution as the sample
solution. Separately, weigh accurately about 28 mg of Rito-
drine Hydrochloride Reference Standard, previously dried at
105 °C for 2 hours, and dissolve in water to make exactly 100
mL. Pipet 2 mL of this solution, add water to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 80 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of ritodrine. The
dissolution rate in 15 minutes is not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
ritodrine hydrochloride (C 17 H 21 N0 3 .HC1)
= W s x (A T /A S ) x (V'/V) X (1/C) x 18
W s : Amount (mg) of Ritodrine Hydrochloride Reference
Standard
C: Labeled amount (mg) of ritodrine hydrochloride
(C 17 H 21 N0 3 .HC1) in 1 tablet
JPXV
Official Monographs / Rokitamycin 1069
Operating conditions —
Proceed as directed in the Assay.
System suitability —
System performance: When the procedure is run with 80
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of ritodrine are not less than 3000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
80 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ritodrine is not more than 1.5%.
Assay To 20 Ritodrine Hydrochloride Tablets add 150 mL
of 0.01 mol/L hydrochloric acid TS, shake for 20 minutes,
and add 0.01 mol/L hydrochloric acid TS to make exactly
200 mL. Filter through a glass filter (G4), and discard the first
20 mL of the filtrate. Pipet 30 mL of the subsequent filtrate,
add exactly 5 mL of the internal standard solution and 0.01
mol/L hydrochloric acid TS to make 50 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 25 mg of Ritodrine Hydrochloride Reference Stan-
dard, previously dried at 105°C for 2 hours, and dissolve in
0.01 mol/L hydrochloric acid TS to make exactly 50 mL.
Pipet 30 mL of this solution, add exactly 5 mL of the internal
standard solution and 0.01 mol/L hydrochloric acid TS to
make 50 mL, and use this solution as the standard solution.
Perform the test with 10 /xh each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, g T and Q s , of the peak area of ritodrine to that of
the internal standard.
Amount (mg) of ritodrine hydrochloride (C 17 H 2 iN0 3 .HCl)
= ^ s x(g T /g s )x4
W s : Amount (mg) of Ritodrine Hydrochloride Reference
Standard
Internal standard solution — A solution of methyl para-
hydroxybenzoate in methanol (3 in 5000)
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 6.6 g of diammonium hydrogen
phosphate and 1.1 g of sodium 1-heptanesulfonate in 700 mL
of water, and add 300 mL of methanol. Adjust to pH 3.0
with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
ritodrine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, ritodrine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of ritodrine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Rokitamycin
n+^v-f
HjC
-JU
C 42 H 69 N0 15 : 827.99
(3i?,4S,5S,6fl,8i?,9i?,10£',12£',15i?)-5-[4-O-Butanoyl-
2,6-dideoxy-3-C-methyl-3-0-propanoyl-a-L-n7j>o-
hexopyranosyl-(l- > 4)-3,6-dideoxy-3-dimethylamino-/?-D-
glucopyranosyloxy]-6-formylmethyl-3,9-dihydroxy-4-
methoxy-8-methyihexadeca- 10,1 2-dien- 1 5-olide
[74014-51-0]
Rokitamycin is a derivative of leucomycin A 5 , which
is a macrolide antibiotic produced by the growth of the
mutants of Streptomyces kitasatoensis.
Rokitamycin contains not less than 900 Lig (potency)
and not more than 1050 Lig (potency) per mg, calculat-
ed on the anhydrous basis. The potency of Rokitamy-
cin is expressed as mass (potency) of rokitamycin
(C 42 H 69 N0 15 ).
Description Rokitamycin occurs as a white to yellowish
white powder.
It is very soluble in methanol and in chloroform, freely
soluble in ethanol (99.5) and in acetonitrile, and practically
insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Rokitamycin in methanol (1 in 50,000), as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Rokitamycin Reference Standard
prepared in the same manner as the sample solution: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Rokitamycin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Rokitamycin Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
1070 Roxatidine Acetate Hydrochloride / Official Monographs
JP XV
(3) Determine the spectrum of a solution of Rokitamycin
in deuterated chloroform for nuclear magnetic resonance
spectroscopy (1 in 20), using tetramethylsilane for nuclear
magnetic resonance spectroscopy as an internal reference
compound, as directed under Nuclear Magnetic Resonance
Spectroscopy <2.21> ('H): it exhibits single signals A, B, C
and D at around 8 1.4 ppm, at around <5 2.5 ppm, at around 5
3.5 ppm and at around 5 9.8 ppm, respectively. The ratio of
integrated intensity of these signals, A:B:C:D, is about
3:6:3:1.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Rokitamycin according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Related substances — Dissolve 50 mg of Rokitamycin
in 50 mL of acetonitrile, and use this solution as the sample
solution. Pipet 3 mL of the sample solution, add acetonitrile
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 5 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the peak areas of 3"-0-propionylleucomycin A 7
having the relative retention time of about 0.72, 3"-0-
propionylisoleucomycin A 5 having the relative retention time
of about 0.86 and 3"-0-propionylleucomycin A t having the
relative retention time of about 1.36 with respect to
rokitamycin obtained with the sample solution are not larger
than the peak area of rokitamycin with the standard solution,
the area of the peak other than rokitamycin, 3"-0-
propionylleucomycin A 7 , 3"-0-propionylisoleucomycin A 5
and 3"-0-propionylleucomycin Ai obtained with the sample
solution is not larger than 23/100 times the peak area of
rokitamycin with the standard solution, and the total area of
the peaks other than rokitamycin obtained with the sample
solution is not larger than 3 times the peak area of rokitamy-
cin with the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
55°C.
Mobile phase: A mixture of methanol, diluted 0.5 mol/L
ammonium acetate TS (2 in 5) and acetonitrile (124:63:13).
Flow rate: Adjust the flow rate so that the retention time of
rokitamycin is about 11 minutes.
Time span of measurement: About 2.5 times as long as the
retention time of rokitamycin beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 1 mL of the sam-
ple solution add acetonitrile to make exactly 10 mL. Confirm
that the peak area of rokitamycin obtained with 5 /uL of this
solution is equivalent to 7 to 13% of that with 5 /xL of the
standard solution.
System performance: When the procedure is run with 5 fiL
of the sample solution under the above operating conditions,
the number of theoretical plates and the symmetry factor of
the peak of rokitamycin are not less than 3000 and not more
than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
rokitamycin is not more than 2.0%.
Water <2.48> Not more than 3.0% (0.2 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Micrococcus luteus ATCC 9341
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base lay-
er. Adjust the pH <2.54> of the medium so that it will be 7.8
to 8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Rokitamycin Reference Standard equivalent to about 40 mg
(potency), dissolve in 50 mL of methanol, add 0.1 mol/L
phosphate buffer solution, pH 4.5 to make exactly 100 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution at 5 C C or below and use within 10
days. Take exactly a suitable amount of the standard stock
solution before use, add 0.1 mol/L phosphate buffer solu-
tion, pH 8.0 containing 0.01% of polysorbate 80 to make so-
lutions so that each mL contains 2//g (potency) and 0.5 /xg
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Rokitamycin equivalent to about 40 mg (potency), dissolve in
50 mL of methanol, and add 0.1 mol/L phosphate buffer so-
lution, pH 4.5 to make exactly 100 mL. Take exactly a suita-
ble amount of the solution, add 0.1 mol/L phosphate buffer
solution, pH 8.0 containing 0.01% of polysorbate 80 to make
solutions so that each mL contains 2 fig (potency) and 0.5 fig
(potency), and use these solutions as the high concentration
sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Roxatidine Acetate Hydrochloride
P +H7-5 1 i> >Br^i7.xJU£it±£
HCI
C 19 H 28 N 2 4 -HC1: 384.90
(3- {3-[(Piperidin-
l-yl)methyl]phenoxy}propylcarbamoyl)methyl
acetate monohydrochloride [93793-83-0]
Roxatidine Acetate Hydrochloride, when dried, con-
JP XV Official Monographs / Roxatidine Acetate Hydrochloride Extended-release Capsules 1071
tains not less than 99.0% and not more than 101.0% of
C 19 H 28 N 2 4 .HC1.
Description Roxatidine Acetate Hydrochloride occurs as
white, crystals or crystalline powder.
It is very soluble in water, freely soluble in acetic acid
(100), and sparingly soluble in ethanol (99.5).
Identification (1) Determine the absorption spectrum of a
solution of Roxatidine Acetate Hydrochloride in ethanol
(99.5) (1 in 10,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum or the spectrum of a solution of Roxati-
dine Acetate Hydrochloride Reference Standard prepared in
the same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Rox-
atidine Acetate Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Roxatidine Acetate Hydrochloride
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Roxatidine Acetate Hydrochloride (1 in
50) responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Roxatidine Acetate Hydrochlo-
ride in 20 mL of water: the pH of this solution is between 4.0
and 6.0.
Melting point <2.60> 147 - 151°C (after drying).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Roxatidine Acetate Hydrochloride in 10 mL of water: the so-
lution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Roxati-
dine Acetate Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 50 mg of Roxatidine
Acetate Hydrochloride in 10 mL of ethanol (99.5), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add ethanol (99.5) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak other than roxatidine acetate is not larger than 1/5 times
the peak area of roxatidine acetate obtained from the stan-
dard solution, and the total area of the peaks other than rox-
atidine acetate is not larger than 1/2 times the peak area of
roxatidine acetate from the standard solution.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4 mm in inside diameter
and 25 cm in length, packed with cyanopropylsilanized silica
gel for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of hexane, ethanol (99.5),
triethylamine and acetic acid (100) (384:16:2:1).
Flow rate: Adjust the flow rate so that the retention time of
roxatidine acetate is about 10 minutes.
Time span of measurement: About 1.5 times as long as the
retention time of roxatidine acetate beginning after the sol-
vent peak.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add ethanol (99.5) to make exactly 10 mL, and
use this solution as the solution for system suitability test.
Pipet 1 mL of the solution for system suitability test, and add
ethanol (99.5) to make exactly 10 mL. Confirm that the peak
area of roxatidine acetate obtained with 10 //L of this solu-
tion is equivalent to 7 to 13% of that with 10 /xL of the solu-
tion for system suitability test.
System performance: Dissolve 50 mg of Roxatidine
Acetate Hydrochloride and 10 mg of benzoic acid in 25 mL
of ethanol (99.5). When the procedure is run with 10 /uL of
this solution under the above operating conditions, benzoic
acid and roxatidine acetate are eluted in this order with the
resolution between these peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
roxatidine acetate is not more than 1.0%.
Loss on drying <2.41> Not more than 0.3% (1 g, in vacuum,
phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.3 g of Roxatidine Acetate
Hydrochloride, previously dried, dissolve in 5 mL of acetic
acid (100), add 50 mL of acetic anhydride, and titrate <2.50>
with 0.1 mol/L perchloric acid VS (potentiometric titration).
Perform a blank determination in the same manner, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 38.49 mg of C 19 H 28 N 2 4 .HC1
Containers and storage Containers — Tight containers.
Roxatidine Acetate Hydrochloride
Extended-release Capsules
Roxatidine Acetate Hydrochloride Extended-release
Capsules contains not less than 93.0% and not more
than 107.0% of the labeled amount of roxatidine
acetate hydrochloride (C 19 H 28 N 2 4 .HC1: 384.90).
Method of preparation Prepare as directed under Capsules,
with Roxatidine Acetate Hydrochloride.
Identification To 1 mL of the filtrate obtained in the Assay
add ethanol (99.5) to make 20 mL, and determine the absorp-
tion spectrum as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits maxima between 275 nm
and 278 nm, and between 282 nm and 285 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Take out the contents of 1 capsule of Roxatidine Acetate
Hydrochloride Extended-release Capsules, add exactly FmL
of ethanol (99.5) so that each mL contains about 2.5 mg of
1072 Roxatidine Acetate Hydrochloride Extended-release Capsules / Official Monographs JP XV
roxatidine acetate hydrochloride (Q9H28N2O4.HCI) accord-
ing to the labeled amount, disperse the particles with the aid
of ultrasonic wave, and filter through a membrane filter with
pore size of not more than 1.0 11m. To exactly 8 mL of the
filtrate add exactly 2 mL of the internal standard solution,
mix, and use this solution as the sample solution. Proceed as
directed in the Assay.
Amount (mg) of roxatidine acetate hydrochloride
(C 19 H 28 N 2 4 .HC1) = W s x (Qt/Qs) x (K/20)
W s : Amount (mg) of Roxatidine Acetate Hydrochloride
Reference Standard
Internal standard solution — A solution of benzoic acid in
ethanol (99.5) (1 in 500).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 capsule of Roxatidine Acetate
Hydrochloride Extended-release Capsules at 50 revolutions
per minute according to the Paddle method, using the sinker,
using 900 mL of water as the dissolution medium. Withdraw
exactly 20 mL each of the dissolution medium 45 minutes, 90
minutes and 8 hours after starting the test for a 37.5-mg cap-
sule and 60 minutes, 90 minutes and 8 hours after starting the
test for a 75-mg capsule. Supply exactly 20 mL of water,
warmed at 37±0.5°C, immediately after withdrawing of the
dissolution medium every time. Filter the dissolution media
withdrawn through a membrane filter with pore size of not
more than 0.45 ^m. Discard the first 10 mL of the filtrate,
pipet FmL of the subsequent filtrate, add water to make ex-
actly V mL so that each mL contains about 42 fig of roxati-
dine acetate hydrochloride (Q9H28N2O4.HQ) according to
the labeled amount, and use these solutions as the sample so-
lutions. Separately, weigh accurately about 21 mg of Roxati-
dine Acetate Hydrochloride Reference Standard, previously
dried in a desiccator (in vacuum, phosphorus (V) oxide) for 4
hours, and dissolve in water to make exactly 50 mL. Pipet 2
mL of this solution, add water to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with exactly 100 /uL each of the sample solutions and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A TM and A s , of roxatidine acetate of each so-
lution. The dissolution rates for a 37.5-mg capsule in 45
minutes, in 90 minutes and in 8 hours are 10 - 40%, 35-65
%, and not less than 70%, respectively, and for a 75-mg cap-
sule in 60 minutes, in 90 minutes and in 8 hours are 20 - 50%,
35 - 65%, and not less than 70%, respectively.
Dissolution rate (%) with respect to the labeled amount of
roxatidine acetate hydrochloride (C19H28N2O4.HCI) on the
Mth dissolution medium withdrawing (n = 1,2,3)
n-\i
W s x
^S ; = ,
1 T(Q v
1
A s X 45
V 1
x — X-X180
W s : Amount (mg) of Roxatidine Acetate Hydrochloride
Reference Standard
C: Labeled amount (mg) of roxatidine acetate hydrochlo-
ride (Q9H28N2O4.HQ) in 1 capsule
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [im in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile, triethyla-
mine and acetic acid (100) (340:60:2:1).
Flow rate: Adjust the flow rate so that the retention time of
roxatidine acetate is about 5 minutes.
System suitability —
System performance: When the procedure is run with 100
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of roxatidine acetate are not less than 3000
and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
100 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
roxatidine acetate is not more than 1.0%.
Assay Take out the contents of not less than 20 Roxatidine
Acetate Hydrochloride Extended-release Capsules, weigh ac-
curately the mass of the contents, and powder. Weigh ac-
curately a portion of the powder, equivalent to about 75 mg
of roxatidine acetate hydrochloride (Q9H28N2O4.HCI), add
exactly 30 mL of ethanol (99.5), shake, and filter through a
membrane filter with pore size of not more than 1,0 fim. To
exactly 8 mL of the filtrate add exactly 2 mL of the internal
standard solution, mix, and use this solution as the sample
solution. Separately, weigh accurately about 50 mg of Rox-
atidine Acetate Hydrochloride Reference Standard, previous-
ly dried in a desiccator (in vacuum, phosphorus (V) oxide) for
4 hours, and dissolve in ethanol (99.5) to make exactly 20
mL. To exactly 8 mL of this solution add exactly 2 mL of the
internal standard solution, mix, and use this solution as the
standard solution. Perform the test with 10 11L each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Q T and Q s , of the peak area
of roxatidine acetate to that of the internal standard.
Amount (mg) of roxatidine acetate hydrochloride
(Q9H28N2O4.HCI)
= ^ s x(e T /es)x(3/2)
W s : Amount (mg) of Roxatidine Acetate Hydrochloride
Reference Standard
Internal standard solution — A solution of benzoic acid in
ethanol (99.5) (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with cyanopropyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of hexane, ethanol (99.5),
triethylamine and acetic acid (100) (384:16:2:1).
Flow rate: Adjust the flow rate so that the retention time of
roxatidine acetate is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
JPXV
Official Monographs / Roxithromycin 1073
/uh of the standard solution under the above operating condi-
tions, the internal standard and roxatidine acetate are elute in
this order with the resolution between these peaks being not
less than 10.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of roxatidine acetate to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Roxithromycin
C 41 H 76 N 2 15 : 837.05
(2R,3S,4S,5R,6R,8R,10RMR,12S,13R)-
5-(3,4,6-Trideoxy-3-dimethylamino-/?-D-.r>'/o-
hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-0-methyl-
a-L-n7jo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-
(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-
hexamethylpentadecan-13-olide [80214-83-1]
Roxithromycin is a derivative of erythromycin.
It contains not less than 970 fig (potency) per mg,
calculated on the anhydrous basis. The potency of
Roxithromycin is expressed as mass (potency) of rox-
ithromycin (C 41 H7 6 N20 15 ).
Description Roxithromycin occurs as a white crystalline
powder.
It is freely soluble in ethanol (95) and in acetone, soluble in
methanol, sparingly soluble in acetonitrile, and practically in-
soluble in water.
Identification Determine the infrared absorption spectrum
of Roxithromycin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Roxithromycin Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
Optical rotation <2.49> [a]g": - 93 - - 96° (0.5 g calculated
on the anhydrous basis, acetone, 50 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Roxithromycin according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Related substances — Dissolve 40 mg of Roxithromy-
cin in the mobile phase A to make 10 mL, and use this solu-
tion as the sample solution. Separately, dissolve 20 mg of
Roxithromycin Reference Standard in the mobile phase A to
make exactly 10 mL. Pipet 1 mL of this solution, add the mo-
bile phase A to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 20 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas by the auto-
matic integration method: the area of a peak having the rela-
tive retention time of about 1.05 to the retention time of rox-
ithromycin from the sample solution is not larger than 2
times of the peak area of roxithromycin from the standard
solution. The areas of other than the peak of roxithromycin
and the peak having the relative retention time of about 1.05
to the retention time of roxithromycin are not larger than the
peak area of roxithromycin from the standard solution, and
the total area of the peaks other than roxithromycin from the
sample solution is not larger than 6 times of the peak area of
roxithromycin from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 205 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: To 200 mL of a solution of ammonium
dihydrogenphosphate (17 in 100) add 510 mL of water, and
adjust to pH 5.3 with 2 mol/L sodium hydroxide TS. To this
solution add 315 mL of acetonitrile.
Mobile phase B: A mixture of acetonitrole and water (7:3).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following ta-
ble.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-38
38-39
39-80
100
100^90
90
0^10
10
Flow rate: Adjust the flow rate so that the retention time of
roxithromycin is about 21 minutes.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase A to make exactly 10 mL.
Confirm that the peak area of roxithromycin obtained from
20 [iL of this solution is equivalent to 15 to 25% of that of
roxithromycin obtained from 20 fiL of the standard solution.
System performance: Dissolve 5 mg each of Roxithromy-
cin Reference Standard and 7V-demethylroxithromycin in the
mobile phase A to make 100 mL. When the procedure is run
with 20 fiL of this solution under the above operating condi-
tions, 7V-demethylroxithromycin and roxithromycin are elut-
ed in this order with the resolution between these peaks being
not less than 6.
System repeatability: When the test is repeated 5 times with
20 fiL of the standard solution under the above operating
1074 Freeze-dried Live Attenuated Rubella Vaccine / Official Monographs
JP XV
conditions, the relative standard deviation of the peak areas
of roxithromycin is not more than 2.0%.
Water <2.48> Not more than 3.0% (0.3 g, volumetric titra-
tion, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Roxithromycin and
Roxithromycin Reference Standard, equivalent to about 20
mg (potency), and dissolve separately in the mobile phase to
make exactly 10 mL, and use these solutions as the sample so-
lution and standard solution. Perform the test with exactly 20
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and calculate the peak areas, A T
and A s , of roxithromycin.
Amount \p.g (potency)] of C 4I H 76 N20 15
= H'sXG4tA4s)x1000
W s : Amount [mg (potency)] of Roxithromycin Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 205 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 200 mL of a solution of ammonium di-
hydrogenphosphate (17 in 100) add 510 mL of water, and ad-
just to pH 5.3 with 2 mol/L sodium hydroxide TS. To this
solution add 315 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
roxithromycin is about 11 minutes.
System suitability —
System performance: Dissolve 5 mg each of Roxithromy-
cin Reference Standard and Af-demethylroxithromycin in the
mobile phase to make 100 mL. When the procedure is run
with 20 iuL of this solution under the above operating condi-
tions, /V-demethylroxithromycin and roxithromycin are elut-
ed in this order with the resolution between these peaks being
not less than 6 and the symmetry factor of the peak of rox-
ithromycin is not more than 1.5.
System repeatability: When, the test is repeated 6 times
with 20 /xL of the standard solution under the above operat-
ing conditions, the relative standard deviation of the peak
areas of roxithromycin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Freeze-dried Live Attenuated
Rubella Vaccine
Freeze-dried Live Attenuated Rubella Vaccine is a
preparation for injection which is dissolved before use.
It contains live attenuated rubella virus.
It conforms to the requirements of Freeze-dried Live
Attenuated Rubella Vaccine in the Minimum Require-
ments for Biological Products.
Description Freeze-dried Live Attenuated Rubella Vaccine
becomes a colorless, yellowish or reddish clear liquid on addi-
tion of solvent.
Saccharated Pepsin
#*f^->>
Saccharated Pepsin is a mixture of pepsin obtained
from the gastric mucosa of hog or cattle and Lactose
Hydrate, and it is an enzyme drug having a proteolytic
activity.
Saccharated Pepsin contains not less than 3800 units
and not more than 6000 units per g.
Description Saccharated Pepsin occurs as a white powder.
It has a characteristic odor, and has a slightly sweet taste.
It dissolves in water to give a slightly turbid liquid, and
does not dissolve in ethanol (95) and in diethyl ether.
It is slightly hygroscopic.
Purity (1) Rancidity — Saccharated Pepsin has no un-
pleasant or rancid odor.
(2) Acidity — Dissolve 0.5 g of Saccharated Pepsin in 50
mL of water, and add 0.50 mL of 0.1 mol/L sodium
hydroxide VS and 2 drops of phenolphthalein TS: the solu-
tion is red in color.
Loss on drying <2.41> Not more than 1.0% (1 g, 80°C,
4 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay (i) Substrate solution — Use the substrate solution 1
described in (2) Assay for protein digestive activity under the
Digestion Test <4.03> after adjusting the pH to 2.0.
(ii) Sample solution — Weigh accurately an amount of
Saccharated Pepsin equivalent to about 1250 units, dissolve
in ice-cold 0.01 mol/L hydrochloric acid TS to make exactly
50 ml.
(iii) Standard solution — Weigh accurately a suitable
amount of Saccharated Pepsin Reference Standard, and dis-
solve in ice-cold 0.01 mol/L hydrochloric acid TS to make a
solution containing about 25 units per ml.
(iv) Procedure — Proceed as directed in (2) Assay for pro-
tein digestive activity under Digestion Test <4.03>, and deter-
mine the absorbances, A T and A TB , of the sample solution,
using trichloroacetic acid TS A as the precipitation reagent.
Separately, determine the absorbances, A s and ^4 SB , of the
standard solution in the same manner as the sample solution.
Units in 1 g of Saccharated Pepsin
= U S X {(^t-^tb)/(^s-^sb)} *(\/W)
U s ; Units per ml of the standard solution
W: Amount (g) of Saccharated Pepsin per ml of the sam-
ple solution
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
JPXV
Official Monographs / Saccharin 1075
Saccharin
■■ff
V
C 7 H 5 N0 3 S: 183.18
1 ,2-Benzo[rf]isothiazol-3(2//)-one 1 , 1 -dioxide
[81-07-2]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Saccharin contains not less than 99.0% and not
more than 101.0% of C 7 H 5 N0 3 S, calculated on the
dried basis.
♦Description Saccharin occurs as colorless or white crystals
or a white crystalline powder. It has a very sweet taste.
It is sparingly soluble in ethanol (95), and slightly soluble
in water.
It dissolves in sodium hydroxide TS.»
♦Identification Determine the infrared absorption spec-
trum of Saccharin as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.*
Melting point <2.60> 226 - 230°C
Purity *(1) Clarity and color of solution — Dissolve 1.0 g
of Saccharin in 30 mL of hot water or in 50 mL of ethanol
(95): the solution is clear and colorless in each case.»
*(2) Heavy metals <1.07> — Proceed with 2.0 g of Saccha-
rin according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).»
(3) Benzoate and salicylate — To 10 mL of a saturated so-
lution of Saccharin in hot water add 3 drops of iron (III)
chloride TS: no precipitate is formed, and no red-purple to
purple color develops.
*(4) o-Toluene sulfonamide — Dissolve 10 g of Saccharin
in 70 mL of sodium hydroxide TS, and extract with three
30-mL portions of ethyl acetate. Combine all the ethyl
acetate extracts, wash with 30 mL of a solution of sodium
chloride (1 in 4), dehydrate with 5 g of anhydrous sodium sul-
fate, then evaporate the solvent. To the residue add exactly 5
mL of the internal standard solution to dissolve, and use this
solution as the sample solution. Separately, dissolve 0.10 g of
o-toluene sulfonamide in ethyl acetate to make exactly 100
mL. Pipet 1 mL of this solution, evaporate to dryness on a
water bath, dissolve the residue in exactly 5 mL of the inter-
nal standard solution, and use this solution as the standard
solution. Perform the test with 1 /xL each of the sample solu-
tion and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, Q T and gs, of the peak height of o-
toluene sulfonamide to that of the internal standard: g T is
not larger than g s .
Internal standard solution — A solution of caffeine in ethyl
acetate (1 in 500).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 1 m
in length, packed with siliceous earth for gas chro-
matography coated 3% with diethylene glycol succinate poly-
ester for gas chromatography (180- 250 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
200°C.
Temperature of injection port: A constant temperature of
about 225 °C.
Temperature of detector: A constant temperature of about
250°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
caffeine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 1 /uL
of the standard solution under the above operating condi-
tions, the internal standard and o-toluene sulfonamide are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 6 times with
1 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak height of o-toluene sulfonamide to that of the internal
standard is not more than 2.0%. »
(5) Readily carbonizable substances <1.15> — Perform the
test with 0.20 g of Saccharin, by warming at 48 to 50°C for 10
minutes: the color of the solution is not more intense than the
matching fluid A.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C, 2
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Saccharin, dissolve
in 40 mL of ethanol (95), add 40 mL of water, and titrate
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina-
tion in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 18.32 mg of C 7 H 5 N0 3 S
Containers and storage Containers — Well-closed contain-
ers.
1076 Saccharin Sodium Hydrate / Official Monographs
JP XV
Saccharin Sodium Hydrate
Saccharin Sodium
C 7 H 4 NNa0 3 S.2H 2 0: 241.20
2-Sodio-l,2-benzo[rf]isothiazol-3(2//)-one 1,1-dioxide
dihydrate [6155-57-3]
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Saccharin Sodium Hydrate, contains not less than
99.0% and not more than 101.0% of saccharin sodium
(C 6 H 4 NNa0 3 S: 205.17), calculated on the anhydrous
basis.
♦Description Saccharin Sodium Hydrate occurs as color-
less crystals or a white, crystalline powder. It has an intensely
sweet taste, even in 10,000 dilutions.
It is freely soluble in water and in methanol, and sparingly
soluble in ethanol (95) and in acetic acid (100).
It effloresces slowly and loses about half the amount of
water of crystallization in air.»
Identification *(1) Determine the infrared absorption
spectrum of Saccharin Sodium Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.*
(2) A solution of Saccharin Sodium Hydrate (1 in 10)
responds to the Qualitative Tests <1.09> for sodium salt.
Purity *(1) Clarity and color of solution — Dissolve 1.0 g
of Saccharin Sodium Hydrate in 1.5 mL of water or in 50 mL
of ethanol (95): the solution is clear and colorless. ♦
(2) Acidity or alkalinity — Dissolve 1.0 g of Saccharin So-
dium Hydrate in 10 mL of water, and add 1 drop of
phenolphthalein TS: the solution is colorless. Add 1 drop of
0.1 mol/L sodium hydroxide VS to the solution: the color
changes to red.
*(3) Heavy metals <1.07> — Dissolve 2.0 g of Saccharin
Sodium Hydrate in 40 mL of water, add 0.7 mL of dilute
hydrochloric acid, dilute with water to make 50 mL, and rub
the inner wall of the vessel with a glass rod until crystalliza-
tion begins. Allow the solution to stand for 1 hour after the
beginning of crystallization, and then filter through dry filter
paper. Reject the first 10 mL of the filtrate, and take 25 mL
of the subsequent filtrate. Add 2 mL of dilute acetic acid and
water to make 50 mL, and perform the test, using this solu-
tion as the test solution. To 1.0 mL of Standard Lead Solu-
tion add 2 mL of dilute acetic acid and water to make 50 mL,
and use this solution as the control solution (not more than
10ppm).»
(4) Benzoate and salicylate — Dissolve 0.5 g of Saccharin
Sodium Hydrate in 10 mL of water, add 5 drops of acetic
acid (31) and 3 drops of iron (III) chloride TS: no turbidity is
produced, and no red-purple to purple color develops.
*(5) o-Toluene sulfonamide — Dissolve 10 g of Saccharin
Sodium Hydrate in 50 mL of water, and extract with three
30-mL portions of ethyl acetate. Combine all the ethyl
acetate extracts, wash with 30 mL of a solution of sodium
chloride (1 in 4), dehydrate with 5 g of anhydrous sodium sul-
fate, and evaporate ethyl acetate. To the residue add exactly 5
mL of the internal standard solution to dissolve, and use this
solution as the sample solution. Separately, dissolve 0.10 g of
o-toluene sulfonamide in ethyl acetate to make exactly 100
mL. Pipet 1 mL of this solution, evaporate on a water bath to
dryness, dissolve the residue in exactly 5 mL of the internal
standard solution, and use this solution as the standard solu-
tion. Perform the test with 1 fiL each of the sample solution
and standard solution as directed under Gas Chro-
matography <2.02> according to the following conditions,
and calculate the ratios, Qj and Qs, of the peak height of o-
toluene sulfonamide to that of the internal standard: Q T is
not more than Q s .
Internal standard solution — A solution of caffeine in ethyl
acetate (1 in 500).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A column 3 mm in inside diameter and 1 m in
length, packed with siliceous earth for gas chromatography
(180 to 250 fim in diameter), coated with diethyleneglycol
succinate polyester for gas chromatography at the ratio of 3
%.
Column temperature: A constant temperature of about
200°C.
Injection port temperature: A constant temperature of
about 225 °C.
Detector temperature: A constant temperature of about
250°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
caffeine is about 6 minutes.
System suitability —
System performance: When the procedure is run with 1 /uL
of the standard solution under the above operating condi-
tions, the internal standard and o-toluene sulfonamide are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 6 times with
1 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak height of o-toluene sulfonamide to that of the internal
standard is not more than 2.0%.*
(6) Readily carbonizable substances <1.15> — Perform the
test with 0.20 g of Saccharin Sodium Hydrate. Allow the so-
lution to stand between 48°C and 50°C for 10 minutes: the
solution has no more color than Matching Fluid A.
Water <2.48> Not more than 15.0% (0.1 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately about 0.15 g of Saccharin Sodium
Hydrate, dissolve in 50 mL of acetic acid (100), heat slightly
if necessary, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
JPXV
Official Monographs / Salazosulfapyridine 1077
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 20.52 mg of C 7 H 4 NNa0 3 S
♦Containers and storage Containers — Well-closed contain-
ers. ♦
Salazosulfapyridine
Sulfasalazine
tT7'/Xil,7 7 tf' J i?>
o o
N
H
CO E H
OH
C 18 H 14 N 4 05S: 398.39
2-Hydroxy-5-[4-(pyridin-2-ylsulfamoyl)phenylazo]benzoic
acid [599-79-1]
Salazosulfapyridine, when dried, contains not less
than 96.0% of C 18 H 14 N40 5 S.
Description Salazosulfapyridine occurs as a yellow to yel-
low-brown, fine powder. It is odorless and tasteless.
It is sparingly soluble in pyridine, slightly soluble in
ethanol (95), practically insoluble in water, in chloroform
and in diethyl ether.
It dissolves in sodium hydroxide TS.
Melting point: 240 - 249°C (with decomposition).
Identification (1) Dissolve 0.1 g of Salazosulfapyridine in
20 mL of dilute sodium hydroxide TS: a red-brown color de-
velops. This color gradually fades upon gradual addition of
0.5 g of sodium hydrosulfite with shaking. Use this solution
in the following tests (2) to (4).
(2) To 1 mL of the solution obtained in (1) add 40 mL of
water, neutralize with 0.1 mol/L hydrochloric acid TS, and
add water to make 50 mL. To 5 mL of this solution add 2 to 3
drops of dilute iron (III) chloride TS: a red color develops
and changes to purple, then fades when dilute hydrochloric
acid is added dropwise.
(3) The solution obtained in (1) responds to the Qualita-
tive Tests <1.09> for primary aromatic amines.
(4) To 1 mL of the solution obtained in (1) add 1 mL of
pyridine and 2 drops of copper (II) sulfate TS, and shake.
Add 3 mL of water and 5 mL of chloroform, shake, and al-
low to stand: a green color develops in the chloroform layer.
(5) Determine the absorption spectrum of a solution of
Salazosulfapyridine in dilute sodium hydroxide TS (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths.
Purity (1) Chloride <1.03>— Dissolve 2.0 g of Salazosul-
fapyridine in 12 mL of sodium hydroxide TS and 36 mL of
water, add 2 mL of nitric acid, shake, and filter. To 25 mL of
the filtrate add 6 mL of dilute nitric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 0.40 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.014%).
(2) Sulfate <1.14> — Dissolve 2.0 g of Salazosulfapyridine
in 12 mL of sodium hydroxide TS and 36 mL of water, add 2
mL of hydrochloric acid, shake, and filter. To 25 mL of the
filtrate add 1 mL of dilute hydrochloric acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution with 1.0 mL of
0.005 mol/L sulfuric acid VS (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of
Salazosulfapyridine according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Take 1.0 g of Salazosulfapyridine in
a decomposition flask, add 20 mL of nitric acid, and heat
gently until it becomes fluid. After cooling, add 5 mL of sul-
furic acid, and heat until white fumes are evolved. Add, if
necessary, 5 mL of nitric acid after cooling, and heat again.
Repeat this operation until the solution becomes colorless to
slightly yellow. After cooling, add 15 mL of a saturated solu-
tion of ammonium oxalate monohydrate, and heat until
white fumes are evolved again. After cooling, add water to
make 25 mL. Perform the test with 5 mL of this solution as
the test solution: the color of the test solution is not deeper
than that of the following standard stain.
Standard stain: Proceed in the same manner without
Salazosulfapyridine, transfer 5 mL of the obtained solution
to a generator bottle, add exactly 2 mL of Standard Arsenic
Solution, and proceed in the same manner as the test with the
test solution (not more than 10 ppm).
(5) Related substances — Dissolve 0.20 g of Salazosul-
fapyridine in 20 mL of pyridine, and use this solution as the
sample solution. Pipet 1 mL of this solution, add pyridine to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 10 /uh each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography.
Develop the plate with diluted methanol (9 in 10) to a dis-
tance of about 10 cm, and air-dry the plate. Examine the
plate under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
(6) Salicylic acid — To 0.10 g of Salazosulfapyridine add
15 mL of diethyl ether, and shake vigorously. Add 5 mL of
dilute hydrochloric acid, shake vigorously for 3 minutes, col-
lect the diethyl ether layer, and filter. To the water layer add
15 mL of diethyl ether, shake vigorously for 3 minutes, col-
lect the diethyl ether layer, filter, and combine the filtrates.
Wash the residue on the filter paper with a small quantity of
diethyl ether, and combine the washings and the filtrate.
Evaporate the diethyl ether with the aid of air-stream at room
temperature. To the residue add dilute ammonium iron (III)
sulfate TS, shake, and filter, if necessary. Wash the residue
on the filter paper with a small quantity of dilute ammonium
iron (III) sulfate TS, combine the washings and the filtrate,
add dilute ammonium iron (III) sulfate TS to make exactly 20
mL, and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of salicylic acid for assay,
previously dried in a desiccator (silica gel) for 3 hours, dis-
solve in dilute ammonium iron (III) sulfate TS to make exact-
ly 400 mL, and use this solution as the standard solution. De-
1078 Salbutamol Sulfate / Official Monographs
JP XV
termine the absorbances, A T and A s , at 535 nm of the sample
solution and the standard solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: salicylic acid content
is not more than 0.5%.
Content (%) of salicylic acid (C 7 H 6 3 )
= W s x(A T /A s )x 0.05
W s : Amount (mg) of salicylic acid for assay
Loss on drying <2.41> Not more than 2.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 20 mg of Salazosulfapyri-
dine, previously dried, and perform the test as directed in the
procedure of determination for sulfur under the Oxygen
Flask Combustion Method <1.06>, using 10 mL of diluted
hydrogen peroxide (30) (1 in 40) as an absorbing liquid.
Each mL of 0.005 mol/L barium perchlorate VS
= 1.992 mg of C 18 H 14 N 4 05S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Salbutamol Sulfate
H OH
• HjSOj
and enanttomer
(C 13 H 21 N0 3 ) 2 .H 2 S0 4 : 576.70
( 1 RS)-2-{\ , 1 -Dimethylethyl)amino- 1 -(4-hydroxy-3-
hydroxymethylphenyl)ethanol hemisulfate [51022-70-9]
Salbutamol Sulfate, when dried, contains not less
than 98.0% of (C 13 H 21 N0 3 ) 2 .H 2 S04.
Description Salbutamol Sulfate occurs as a white powder.
It is freely soluble in water, slightly soluble in ethanol (95),
and in acetic acid (100) and practically insoluble in diethyl
ether.
A solution of Salbutamol Sulfate (1 in 20) shows no optical
rotation.
Identification (1) Determine the absorption spectrum of a
solution of Salbutamol Sulfate in 0.1 mol/L hydrochloric
acid TS (1 in 12,500) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Sal-
butamol Sulfate, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Salbutamol Sulfate (1 in 20) responds to
the Qualitative Tests <1.09> for sulfate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Salbutamol Sulfate in 20 mL of water: the solution is clear
and colorless.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Sal-
butamol Sulfate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 20 mg of Salbutamol
Sulfate in 10 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethyl acetate, 2-propanol, water and ammonia so-
lution (28) (25:15:8:2) to a distance of about 15 cm, and air-
dry the plate. Leave the plate in a well-closed vessel saturated
with diethylamine vapor for 5 minutes, and spray evenly 4-
nitrobenzenediazonium chloride TS: the spots other than the
principal spot from the sample solution are not more intense
than the spot from the standard solution in color.
(4) Boron— Take 50 mg of Salbutamol Sulfate and 5.0
mL of the boron standard solution, and transfer to a plati-
num crucible. Add 5 mL of potassium carbonate-sodium car-
bonate TS, evaporate on a water bath to dryness, and dry at
120°C for 1 hour. Ignite the residue immediately. After cool-
ing, add 0.5 mL of water and 3 mL of curcumin TS to the
residue, warm gently in a water bath for 5 minutes. After
cooling, add 3 mL of acetic acid-sulfuric acid TS, mix, and
allow to stand for 30 minutes. Add ethanol (95) to make ex-
actly 100 mL, and filter. Discard the first 10 mL of the
filtrate, and use the subsequent filtrate as the sample solution
and standard solution. Perform the test with these solutions
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using ethanol (95) as the blank: the absorbance of the
sample solution at 555 nm is not larger than that of the stan-
dard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 100°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.9 g of Salbutamol Sulfate,
previously dried, and dissolve in 50 mL of acetic acid (100) by
warming. After cooling, titrate <2.50> with 0.1 mol/L per-
chloric acid VS until the color of the solution changes from
purple through blue to blue-green (indicator: 3 drops of crys-
tal violet TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 57.67 mg of (C 13 H 21 N0 3 ) 2 .H 2 S0 4
Containers and storage Containers — Tight containers.
Salicylated Alum Powder
^■U^JU-S a >>/<>*
Salicylated Alum Powder contains not less than
2.7% and not more than 3.3% of salicylic acid
(C 7 H 6 3 : 138.12).
JPXV
Official Monographs / Salicylic Acid 1079
Method of preparation
Salicylic Acid, finely powdered 30 g
Dried Aluminum Potassium Sulfate,
very finely powdered 640 g
Talc, very finely powdered a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Salicylated Alum Powder occurs as a white
powder.
Identification (1) The colored solution obtained in the As-
say has a red-purple color and exhibits an absorption maxi-
mum <2.24> between 520 nm and 535 nm (salicylic acid).
(2) Shake 0.3 g of Salicylated Alum Powder with 5 mL of
methanol, filter, and use the filtrate as the sample solution.
Separately, dissolve 0.01 g of salicylic acid in 5 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 [iL each of the sample so-
lution and standard solution on the plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of chloroform, acetone and acetic
acid (100) (45:5:1) to a distance of about 10 cm, and air-dry
the plate. Examine the plate under ultraviolet light (main
wavelength: 254 nm): the spot from the sample solution and
that from the standard solution show the same Ri value.
Spray evenly iron (III) chloride TS upon the plate: the spot
from the standard solution and the corresponding spot from
the sample solution reveal a purple color.
Assay Weigh accurately about 0.33 g of Salicylated Alum
Powder, add 80 mL of ethanol (95), and shake vigorously.
Dilute with ethanol (95) to make exactly 100 mL, filter, and
discard the first 10 mL of the filtrate. Use the subsequent
filtrate as the sample solution. Dissolve about 0.1 g of salicyl-
ic acid for assay, previously dried in a desiccator (silica gel)
for 3 hours and accurately weighed, in sufficient ethanol (95)
to make exactly 100 mL. Pipet 10 mL of this solution, dilute
with ethanol (95) to make exactly 100 mL, and use the solu-
tion as the standard solution. Pipet 10 mL each of the sample
solution and standard solution into stoppered test tubes
respectively, to each add exactly 5 mL of a solution of iron
(III) nitrate enneahydrate (1 in 200), and dilute with
hydrochloric acid-potassium chloride buffer solution, pH
2.0, to make exactly 25 mL. Determine the absorbances, A T
and A s , of both solutions at 530 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using a solution pre-
pared in the same manner with ethanol (95), instead of the
sample solution, as the blank.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= W s x(A T /A s )x(l/l0)
W s : Amount (mg) of salicylic acid for assay
Containers and storage Containers — Well-closed contain-
ers.
Salicylic Acid
COjH
OH
C 7 H 6 3 : 138.12
2-Hydroxybenzoic acid
[69-72-7]
Salicylic Acid, when dried, contains not less than
99.5% of C 7 H 6 3 .
Description Salicylic Acid occurs as white crystals or crys-
talline powder. It is odorless and has a slightly acid, followed
by an acrid taste.
It is freely soluble in ethanol (95), in acetone and in diethyl
ether, soluble in hot water, and slightly soluble in water.
Identification A solution of Salicylic Acid (1 in 500)
responds to the Qualitative Tests <1.09> (1) and (3) for salicy-
late.
Melting point <2.60> 158-161°C
Purity (1) Chloride <1.03>— Dissolve 5.0 g of Salicylic
Acid in 90 mL of water by heating, cool, dilute with water to
100 mL, and filter. Discard the first 20 mL of the filtrate, take
subsequent 30 mL of the filtrate, add 6 mL of dilute nitric
acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 0.35 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.008%).
(2) Sulfate <1.14>— To 30 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL, and perform the test using this solution as the test so-
lution. Prepare the control solution with 0.35 mL of 0.005
mol/L sulfuric acid VS (not more than 0.011%).
(3) Heavy metals <1.07> — Dissolve 2.0 g of Salicylic Acid
in 25 mL of acetone, add 4 mL of sodium hydroxide TS, 2
mL of dilute acetic acid and water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: to 2.0 mL of Standard Lead
Solution add 25 mL of acetone, 2 mL of dilute acetic acid
and water to make 50 mL (not more than 10 ppm).
(4) Readily carbonizable substances <1.15> — Perform the
test with 0.5 g of Salicylic Acid: the solution has no more
color than Matching Fluid C.
Loss on drying <2.41> Not more than 0.5% (2 g, silica gel, 3
hours).
Residue on ignition <2.44> Not more than 0.05% (1 g).
Assay Weigh accurately about 0.5 g of Salicylic Acid,
previously dried, dissolve in 25 mL of neutralized ethanol,
and titrate <2.50> with 0.1 mol/L sodium hydroxide VS (indi-
cator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 13.81 mg of C 7 H 6 3
Containers and storage Containers — Well-closed contain-
ers.
1080 Salicylic Acid Adhesive Plaster / Official Monographs
JP XV
Salicylic Acid Adhesive Plaster
Method of preparation
Adhesive Plaster consists of a mixture of the below in-
gredients with carefully selected rubber, resins, zinc oxide
and other substances. It has adhesive properties. It spreads
evenly on a fabric.
Salicylic Acid, finely powdered
Adhesive plaster base
500 g
a sufficient quantity
To make 1000 g
Description The surface of Salicylic Acid Adhesive Plaster
is whitish in color and adheres well to the skin.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Salicylic Acid Spirit
Salicylic Acid Spirit contains not less than 2.7 w/v%
and not more than 3.3 w/v% of salicylic acid (C 7 H 6 3 :
138.12).
Method of preparation
Salicylic Acid
Glycerin
Ethanol
30 g
50 mL
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients.
Description Salicylic Acid Spirit is a clear, colorless liquid.
Specific gravity df : about 0.86
Identification The solution obtained in the Assay has a red-
purple color. Determine the absorption spectrum of the solu-
tion as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits a maximum between 520 nm and 535 nm
(salicylic acid).
Alcohol number <7.07> Not less than 8.8 (Method 2).
Assay Measure exactly 10 mL of Salicylic Acid Spirit, add
10 mL of ethanol (95) and water to make exactly 100 mL.
Pipet 3 mL of this solution, and dilute with hydrochloric
acid-potassium chloride buffer solution, pH 2.0, to make ex-
actly 100 mL. Use this solution as the sample solution. Dis-
solve about 0.3 g of salicylic acid for assay, previously dried
in a desiccator (silica gel) for 3 hours and accurately weighed,
in 10 mL of alcohol and water to make exactly 100 mL. Pipet
3 mL of this solution, dilute with hydrochloric acid-potassi-
um chloride buffer solution, pH 2.0, to make exactly 100 mL,
and use this solution as the standard solution. Pipet 10 mL
each of the sample solution and standard solution, to each
add 5 mL of a solution of iron (III) nitrate enneahydrate (1 in
200), dilute with hydrochloric acid-potassium chloride buffer
solution, pH 2.0, to exactly 25 mL. Determine the absor-
bances <2.24>, A T and A s , of both solutions at 530 nm, using
a blank solution prepared in the same manner with water in-
stead of the sample solution.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= W s x(A T /A s )
W s : Amount (mg) of salicylic acid for assay
Containers and storage Containers — Tight containers.
Compound Salicylic Acid Spirit
Compound Salicylic Acid Spirit contains not less
than 1.8 w/v% and not more than 2.2 w/v% of salicyl-
ic acid (C 7 H 6 3 : 138.12), and not less than 0.43 w/v%
and not more than 0.53 w/v% of phenol (C 6 H 6 0:
94.11).
Method of preparation
Salicylic Acid
Liquefied Phenol
Glycerin
Ethanol
Water or Purified Water
20 g
5mL
40 mL
800 mL
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients.
Description Compound Salicylic Acid Spirit is a
colorless to light red liquid.
Specific gravity
clear,
df : about 0.88
Identification (1) To 1 mL of Compound Salicylic Acid
Spirit add hydrochloric acid-potassium chloride buffer solu-
tion, pH 2.0, to make 200 mL, and to 5 mL of this solution
add 5 mL of a solution of iron (III) nitrate enneahydrate (1 in
200): a red-purple color is produced (salicylic acid).
(2) To 1 mL of Compound Salicylic Acid Spirit add 20
mL of water and 5 mL of dilute hydrochloric acid, and ex-
tract with 20 mL of diethyl ether. Wash the diethyl ether ex-
tract with two 5-mL portions of sodium hydrogen carbonate
TS, and extract with 10 mL of dilute sodium hydroxide TS.
Shake 1 mL of the extract with 1 mL of sodium nitrite TS and
1 mL of dilute hydrochloric acid, allow to stand for 10
minutes, and add 3 mL of sodium hydroxide TS: a yellow
color is produced (phenol).
(3) To 0.5 mL of Compound Salicylic Acid Spirit add 5
mL of dilute hydrochloric acid, extract with 5 mL of chlo-
roform, and use the extract as the sample solution (1). To 2
mL of Compound Salicylic Acid Spirit add 5 mL of dilute
hydrochloric acid, extract with 5 mL of chrloroform, wash
the extract with two 5-mL portions of sodium hydrogen car-
bonate TS, and use the chloroform extract as the sample so-
lution (2). Separately, dissolve 0.01 g each of salicylic acid
and phenol in 5 mL each of chloroform, and use both solu-
tions as the standard solutions (1) and (2). Perform the test
with these solutions as directed under Thin-layer Chro-
JP XV
Official Monographs / Santonin 1081
matography <2.03>. Spot 5 fiL each of the sample solutions
and standard solutions on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, acetone and acetic acid (100)
(45:5:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots from the sample solution (1) and standard solution
(1) show the same Rf value, and the spots from the sample so-
lution (2) and standard solution (2) show the same Rf value.
Spray evenly iron (III) chloride TS upon the plate: the spot
from the standard solution (1) and the corresponding spot
from the sample solution (1) reveal a purple color.
Alcohol number <7.07> Not less than 7.5 (Method 2).
Assay Measure accurately 2 mL of Compound Salicylic
Acid Spirit, add exactly 5 mL of the internal standard solu-
tion and diluted methanol (1 in 2) to make 100 mL, and use
this solution as the sample solution. Weigh accurately about
0.2 g of salicylic acid for assay, previously dried in a desicca-
tor (silica gel) for 3 hours, and about 50 mg of phenol for as-
say, dissolve in diluted methanol (1 in 2) to make exactly 100
mL . Pipet 20 mL of this solution, add exactly 5 mL of the in-
ternal standard solution and diluted methanol (1 in 2) to
make 100 mL, and use this solution as the standard solution.
Perform the test with 15 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g Ta and Q Tb , of the peak area of salicylic acid and
phenol to that of the internal standard in the sample solution,
and the ratios, gsa and Q sb , of the peak area of salicylic acid
and phenol to that of the internal standard in the standard so-
lution.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= ^saX(Q Ta /Gsa)x(l/5)
Amount (mg) of phenol (C 6 H 6 0)
= W A Sb><(GTa/eTb)X(l/5)
W Sa : Amount (mg) of salicylic acid for assay
W sb : Amount (mg) of phenol for assay
Internal standard solution — A solution of theophylline in
methanol (1 in 1250).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 270 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 25 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography, 5 /xm in parti-
cle diameter.
Column temperature: Room temperature.
Mobile phase: A mixture of 0.1 mol/L phosphate buffer
solution, pH 7.0, and methanol (3:1).
Flow rate: Adjust the flow rate so that the retention time of
salicylic acid is about 6 minutes.
Selection of column: Dissolve 0.2 g of benzoic acid, 0.2 g
of salicylic acid and 0.05 g of theophylline in 1 00 mL of dilut-
ed methanol (1 in 2). To 10 mL of this solution add 90 mL of
diluted methanol (1 in 2). Proceed with 10 /xh of this solution
under the above operating conditions. Use a column giving e-
lution of benzoic acid, salicylic acid and theophylline in this
order, and clearly dividing each peak.
Containers and storage Containers — Tight containers.
Santonin
Ci 5 H 18 3 : 246.30
(3S,3aS,5aS,9bS)-3,5a,9-Trimethyl-3a,5,5a,9b-
tetrahydronaphtho[l,2-6]furan-2,8(3i7,4i7)-dione
[481-06-1]
Santonin, when dried, contains not less than 98.5%
and not more than 101.0% of C 15 H 18 3 .
Description Santonin occurs as colorless crystals, or a
white, crystalline powder.
It is freely soluble in chloroform, sparingly soluble in
ethanol (95), and practically insoluble in water.
It becomes yellow by light.
Identification (1) Determine the absorption spectrum of a
solution of Cortisone Acetate in ethanol (95) (3 in 250,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Corti-
sone Acetate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™: -170- -175° (0.2 g, chlo-
roform, 10 mL, 100 mm).
Melting point <2.60> 172 - 175°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Santonin according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm)
(2) Alkaloids— Boil 0.5 g of Santonin with 20 mL of
diluted sulfuric acid (1 in 100), cool, and filter. Dilute 10 mL
of the filtrate with water to 30 mL, add 3 drops of iodine TS,
and allow to stand for 3 hours: no turbidity is produced.
(3) Artemisin — Dissolve 1.0 g of powdered Santonin in 2
mL of chloroform by slight warming: the solution is clear
and colorless, or any yellow color produced is not darker
than Matching Fluid A.
(4) Phenols— Boil 0.20 g of Santonin with 10 mL of
water, cool, and filter. To the filtrate add bromine TS until
the color of the solution becomes yellow: no turbidity is pro-
duced.
(5) Acid-coloring substances — Moisten 10 mg of Santo-
nin with nitric acid: no color develops immediately. Moisten
Santonin with sulfuric acid, previously cooled to 0°C: no
color is produced immediately.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
1082 Scopolamine Butylbromide / Official Monographs
JP XV
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.25 g of Santonin, previ-
ously dried, dissolve in 10 mL of ethanol (95) by warming,
add exactly 20 mL of 0.1 mol/L sodium hydroxide VS, and
heat on a water bath under a reflux condenser for 5 minutes.
Cool quickly, and titrate <2.50> the excess sodium hydroxide
with 0.05 mol/L hydrochloric acid VS (indicator: 3 drops of
phenolphthalein TS). Perform a blank determination.
Each mL of 0.1 mol/L sodium hydroxide VS
= 24.63 mg of C 15 H, 8 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Scopolamine Butylbromide
7>;L-x=i7fc 7$ >fkik%Q
,N-CH 3
Br
/
\
H
C 2 iH 3 oBrN0 4 : 440.37
(lS,2S,4.R,5i^7s)-9-Butyl-7-[(2S)-3-riydroxy-2-
phenylpropanoyloxy]-9-methyl-3-oxa-9-
azoniatricyclo[3.3.1.0 24 ]nonane bromide [149-64-4]
Scopolamine Butylbromide, when dried, contains
not less than 98.5% of C 21 H 30 BrNO 4 .
Description Scopolamine Butylbromide occurs as white
crystals or crystalline powder.
It is very soluble in water, freely soluble in acetic acid
(100), soluble in ethanol (95), sparingly soluble in methanol,
slightly soluble in acetic anhydride, and practically insoluble
in diethyl ether.
Melting point: about 140°C (with decomposition).
Identification (1) To 1 mg of Scopolamine Butylbromide
add 3 to 4 drops of fuming nitric acid, and evaporate on a
water bath to dryness. After cooling, dissolve the residue in 1
mL of A^TV-dimethylformamide, and add 6 drops of
tetraethylammonium hydroxide TS: a red-purple color de-
velops.
(2) Determine the absorption spectrum of a solution of
Scopolamine Butylbromide (1 in 1000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Scopolamine Butylbromide, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Scopolamine Butylbromide (1 in 20)
responds to the Qualitative Tests <1.09> for bromide.
Optical rotation <2.49> [ a ]g>:
18.0- -20.0° (after
drying, 1 g, water, 10 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Scopolamine Butylbromide in
10 mL of water: the pH of this solution is between 5.5 and
6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Scopolamine Butylbromide in 10 mL of water: the solution is
clear, and has no more color than the following control solu-
tion.
Control solution: To 0.5 mL of Matching Fluid F add
diluted hydrochloric acid (1 in 40) to make 20 mL.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Scopola-
mine Butylbromide according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 0.10 g of Scopolamine
Butylbromide in the mobile phase to make exactly 10 mL,
and use this solution as the sample solution. Separately, dis-
solve 10 mg of scopolamine hydrobromide in the mobile
phase to make exactly 100 mL. Pipet 10 mL of this solution,
add the mobile phase to make exactly 50 mL, and use this so-
lution as the standard solution (1). Pipet 5 mL of the stan-
dard solution (1), add the mobile phase to make exactly 10
mL, and use this solution as the standard solution (2). Per-
form the test with exactly 20 /uL each of the sample solution
and standard solutions (1) and (2) as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of these solutions by the au-
tomatic integration method: the peak area of scopolamine
from the sample solution is not larger than that from the
standard solution (2), and each area of the peaks other than
the peak appearing in the first elution and the peak of
scopolamine and butylscopolamine from the sample solution
are not larger than the peak area from the standard solution
(1).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octylsilanized silica
gel for liquid chromatography (10,wm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 2 g of sodium lauryl sulfate in 370
mL of water and 680 mL of methanol, and adjust the pH to
3.6 with diluted phosphoric acid (1 in 10).
Flow rate: Adjust the flow rate so that the retention time of
butylscopolamine is about 7 minutes.
Time span of measurement: About twice as long as the
retention time of butylscopolamine.
System suitability —
System performance: Dissolve 5 mg each of Scopolamine
Butylbromide and scopolamine hydrobromide in 50 mL of
the mobile phase. When the procedure is run with 20 /uL of
this solution under the above operating conditions, scopola-
mine and butylscopolamine are eluted in this order with the
resolution between these peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution (2) under the above operating
conditions, the relative standard deviation of the peak area of
scopolamine is not more than 2.0%.
Loss on drying <2.41> Not more than 1.0% (1 g, 105 C C,
JPXV
Official Monographs / Serrapeptase 1083
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Scopolamine Butyl-
bromide, previously dried, dissolve in 40 mL of acetic acid
(100) and 30 mL of acetic anhydride, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 44.04 mg of C 21 H 30 BrNO 4
Containers and storage Containers — Tight containers.
Scopolamine Hydrobromide
Hydrate
H
H ,■ — OH
\ _Ol X a
•HBr-3H 2
C 17 H 2 iN0 4 .HBr.3H 2 0: 438.31
(lS',2S,4i?,5fl,7.y)-9-Methyl-3-oxa-
9-azatricyclo-[3 .3 . 1 .0 2,4 ]non-7-yl (25)-3-hydroxy-
2-phenylpropanoate monohydrobromide trihydrate
[6533-68-2]
Scopolamine Hydrobromide Hydrate, when dried,
contains not less than 98.5% of scopolamine
hydrobromide (Ci 7 H 21 N0 4 .HBr: 384.26).
Description Scopolamine Hydrobromide Hydrate occurs as
colorless or white crystals, or white granules or powder. It is
odorless.
It is freely soluble in water, sparingly soluble in ethanol
(95) and in acetic acid (100), and practically insoluble in
diethyl ether.
Identification (1) To 1 mg of Scopolamine Hydrobromide
Hydrate add 3 to 4 drops of fuming nitric acid, evaporate on
a water bath to dryness, and cool. Dissolve the residue in 1
mL of Af,iV-dimethylformamide, and add 6 drops of
tetraethylammonium hydroxide TS: a red-purple color is
produced.
(2) A solution of Scopolamine Hydrobromide Hydrate (1
in 20) responds to the Qualitative Tests <1.09> for bromide.
Optical rotation <2.49> [a]™: -24.0- -26.0° (after
drying, 0.5 g, water, 10 mL, 100 mm).
Melting point <2.60> 195 - 15
heat the bath to 180°C).
°C (after drying; previously
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Scopolamine Hydrobromide Hydrate in 10 mL of water: the
solution is clear and colorless.
(2) Acidity — Dissolve 0.50 g of Scopolamine
Hydrobromide Hydrate in 15 mL of water, and add 0.50 mL
of 0.02 mol/L sodium hydroxide and 1 drop of methyl red-
methylene blue TS: a green color develops.
(3) Apoatropine — Dissolve 0.20 g of Scopolamine
Hydrobromide Hydrate in 20 mL of water, add 0.60 mL of
0.002 mol/L potassium permanganate VS, and allow to
stand for 5 minutes: the red color in the solution does not dis-
appear.
(4) Related substances — Dissolve 0.15 g of Scopolamine
Hydrobromide Hydrate in 3 mL of water, and use this solu-
tion as the sample solution.
(i) To 1 mL of the sample solution add 2 to 3 drops of
ammonia TS: no turbidity is produced.
(ii) To 1 mL of the sample solution add 2 to 3 drops of
potassium hydroxide TS: a transient white turbidity might be
produced, and disappears clearly in a little while.
Loss on drying <2.41> Not more than 13.0% [1.5 g; first dry
in a desiccator (silica gel) for 24 hours, then dry at 105 °C for
3 hours].
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Scopolamine
Hydrobromide Hydrate, previously dried in 10 mL of acetic
acid (100) by warming. After cooling, add 40 mL of acetic
anhydride, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 38.43 mg of C 17 H 21 N0 4 .HBr
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Serrapeptase
[95077-02-4]
Serrapeptase is the enzyme preparation having pro-
teolytic activity, produced by the growth of Serratia
species.
Usually, it is diluted with Lactose Hydrate.
It contains not less than 2000 serrapeptase Units and
not more than 2600 serrapeptase Units per mg.
It is hygroscopic.
Description Serrapeptase occurs as a grayish white to light
brown powder, having a slight characteristic odor.
Identification Dissolve 0.4 g of Serrapeptase in 100 mL of
acetic acid-sodium acetate buffer solution, pH 5.0, transfer
exactly 1 mL each of this solution into three tubes, and refer
to them as A, B and C. To tube A add exactly 1 mL of water,
to tubes B and C add exactly 1 mL of 0.04 mol/L disodium
dihydrogen ethylenediamine tetraacetate TS, mix gently, and
allow them to stand in a water bath at 4 ± 1 °C for about 1
hour. Then, to the tube B add exactly 2 mL of 0.04 mol/L
zinc chloride TS, to the tubes A and C add exactly 2 mL of
water, mix gently, and allow them to stand in a water bath at
4± 1°C for about 1 hour. Pipet 1 mL each of these solutions,
add borate-hydrochloric acid buffer solution, pH 9.0 to the
solutions A and B to make exactly 200 mL, to the solution C
to make exactly 50 mL, and use these solutions as the sample
solutions. Proceed with these sample solutions as directed in
1084 Sesame Oil / Official Monographs
JP XV
the Assay: the activities of the solutions A and B are almost
the same, and the activity of the solution C is not more than
5% of that of the solution A.
Activity of solutions A, B or C = (A T /A S ) x (1/20) x£)x 176
A s : Absorbance of the standard solution
A T : Absorbance of the sample solution
20: Reaction time (minute)
D: Dilution rate (200 for solution A and B, 50 for solution
C)
176: Conversion factor (Total volume of enzyme reaction
solution/volume of filtrate taken x amount of tyrosine in 2
mL of tyrosine standard solution)
Purity (1) Heavy metals <1.07> — Put 1.0 g of Serrapep-
tase in a porcelain crucible, add 2 drops each of sulfuric acid
and nitric acid, and incinerate by ignition. After cooling, to
the residue add 2 mL of hydrochloric acid, evaporate to dry-
ness on a water bath, add 10 mL of a solution of hydroxyla-
mine hydrochloride (3 in 100) and 2 mL of dilute acetic acid,
and heat on a water bath for 5 minutes. After cooling, filter if
necessary, wash the filter paper with 10 mL of water, put the
filtrate and washing in a Nessler tube, add water to make 50
mL, and use this solution as the test solution. Prepare the
control solution as follows: Evaporate to dryness 2 drops
each of sulfuric acid and nitric acid on a sand bath, add 2 mL
of hydrochloric acid to the residue, evaporate to dryness on a
water bath, add 2.0 mL of Standard Lead Solution, 10 mL of
a solution of hydroxylamine hydrochloride (3 in 100) and 2
mL of dilute acetic acid, and heat on a water bath for
5 minutes. Proceed in the same manner as directed for the
preparation of the test solution, and add water to make 50
mL (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 0.40 g
of Serrapeptase according to Method 3, excepting addition of
5 mL of a solution of magnesium nitrate hexahydrate in
ethanol (95) (3 in 10) instead of a solution of magnesium ni-
trate hexahydrate in ethanol (95) (1 in 50), evaporating to
dryness on a water bath, then incinerating with a small flame,
and perform the test (not more than 5 ppm).
Loss on drying <2.41> Not more than 7.0% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 1.5% (1 g).
Assay (i) Sample solution: Dissolve exactly 0.100 g of Ser-
rapeptase in a solution of ammonium sulfate (1 in 20) to
make exactly 100 mL. Pipet 1 mL of this solution, add
borate-hydrochloric acid buffer solution, pH 9.0 to make
exactly 200 mL, and use this solution as the sample solution.
(ii) Tyrosine standard solution: Dissolve exactly 0.160 g
of Tyrosine Reference Standard, previously dried at 105 °C
for 3 hours, in 0.2 mol/L hydrochloric acid TS to make
exactly 1000 mL. Pipet 10 mL of this solution, and add
0.2 mol/L hydrochloric acid TS to make exactly 100 mL.
Prepare before use.
(iii) Substrate solution: Previously determine the loss on
drying <2.41> (60°C, reduced pressure not exceeding 0.67
kPa, 3 hours) of milk casein, previously dried. To exactly
1.20 g of the milk casein, calculated based on the loss on
drying, add 160 mL of a solution of sodium borate (19 in
1000), and heat in a water bath to dissolve. After cooling, ad-
just the pH to exactly 9.0 with 1 mol/L hydrochloric acid TS,
and add borate-hydrochloric acid buffer solution, pH 9.0 to
make exactly 200 mL. Use after warming to 37±0.5°C. Pre-
pare before use.
(iv) Precipitation reagent: Trichloroacetic acid TS for
serrapeptase. Use after warming to 37±0.5°C.
(v) Procedure: Pipet 1 mL of the sample solution, put in
a glass-stoppered tube (15 x 130 mm), allow to stand at 37 ±
0.5°C for 5 minutes, add exactly 5 mL of the substrate
solution, and mix well immediately. Allow to stand at 37 ±
0.5°C for exactly 20 minutes, add exactly 5 mL of trichloroa-
cetic acid TS for serrapeptase, mix, allow to stand at 37 ±
0.5°C for 30 minutes, and filter through a dried filter paper.
Pipet 2 mL of the filtrate, add exactly 5 mL of a solution of
anhydrous sodium carbonate (3 in 50), mix, add exactly 1 mL
of diluted Folin's TS (1 in 3), mix well, and allow to stand at
37±0.5°C for 30 minutes. Determine the absorbance of this
solution at 660 nm, 4,, as directed under Ultraviolet-visible
Spectrophotometry <2.24> using water as the blank. Separate-
ly, pipet 1 mL of the sample solution, add exactly 5 mL of
trichloroacetic acid TS for serrapeptase, mix, add exactly 5
mL of the substrate solution, allow to stand at 37±0.5°C for
30 minutes, and proceed in the same manner as directed
above to determine the absorbance A 2 . Separately, pipet 2
mL of the tyrosine standard solution, add exactly 5 mL of a
solution of anhydrous sodium carbonate (3 in 50), mix, add
exactly 1 mL of diluted Folin's TS (1 in 3), mix well, and pro-
ceed in the same manner as directed above to determine the
absorbance A 3 . Separately, pipet 2 mL of 0.2 mol/L
hydrochloric acid TS, and proceed in the same manner as
directed above to determine the absorbance A 4 .
Serrapeptase Unit per mg of Serrapeptase
= {(A i -A 2 )/(A 3 -A A )} x (1/20) x 200 x 176
20: Reaction time (minute)
200: Dilution rate
176: Conversion factor (Total volume of enzyme reaction
solution/volume of filtrate taken x amount of tyro-
sine in 2 mL of tyrosine standard solution)
One serrapeptase Unit corresponds to the amount of
serrapeptase which produces 5 n% of tyrosine per minute
from 5 mL of the substrate solution under the above condi-
tions.
Containers and storage Containers — Tight containers.
Sesame Oil
Oleum Sesami
=fv>ft
Sesame Oil is the fixed oil obtained from the seeds of
Sesamum indicum Linne (Pedaliaceae).
Description Sesame Oil is a clear, pale yellow oil. It is odor-
less or has a faint, characteristic odor, and has a bland taste.
It is miscible with diethyl ether and with petroleum ether.
It is slightly soluble in ethanol (95).
It congeals between 0°C and — 5°C.
Congealing point of the fatty acids: 20 - 25 °C
Identification To 1 mL of Sesame Oil add 0.1 g of sucrose
and 10 mL of hydrochloric acid, and shake for 30 seconds:
the acid layer becomes light red and changes to red on stand-
JPXV
Official Monographs / White Shellac 1085
ing.
Specific gravity <7.73> d*jj: 0.914 - 0.921
Acid value <1.13> Not more than 0.2.
Saponification value <7.73> 187-194
Unsaponifiable matters <1.13> Not more than 2.0%.
Iodine value <7.73> 103-118
Containers and storage Containers — Tight containers.
Purified Shellac
Purified Shellac is a resin-like substance obtained
from a purified secretion of Laccifer lacca Kerr (Cocci-
dae).
Description Purified Shellac occurs as light yellow-brown to
brown, lustrous, hard, brittle scutella. It has no odor or has a
faint, characteristic odor.
It is freely soluble in ethanol (95) and in ethanol (99.5), and
practically insoluble in water and in diethyl ether.
It dissolves in sodium hydroxide TS.
Acid value 60- 80 <1.13> Weigh accurately about 1 g of
Purified Shellac, add 40 mL of neutralized ethanol, and dis-
solve by warming. After cooling, titrate <2.50> with 0.1 mol/
L potassium hydroxide VS (potentiometric titration).
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Purified Shellac according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Arsenic <7.77> — Prepare the test solution with 0.40 g
of Purified Shellac according to Method 3, and perform the
test. Add 10 mL of a solution of magnesium nitrate hexahy-
drate in ethanol (95) (1 in 50), then add 1.5 mL of hydrogen
peroxide (30), and fire to burn (not more than 5 ppm).
(3) Ethanol-insoluble substances — Dissolve about 5 g of
Purified Shellac, accurately weighed, in 50 mL of ethanol (95)
on a water bath while shaking. Pour the ethanol solution into
a tared extraction thimble, previously dried at 105°C for 2
hours, in a Soxhlet extractor, and extract with ethanol (95)
for 3 hours: the mass of the residue is not more than 2.0%.
Use a cylindrical weighing bottle for taring the extraction
thimble.
(4) Rosin — Dissolve 2.0 g of Purified Shellac in 10 mL of
ethanol (99.5) with thorough shaking, add gradually 50 mL
of petroleum ether while shaking, and filter, if necessary.
Wash the solution with two 50-mL portions of water, filter
the upper layer, and evaporate the filtrate on a water bath to
dryness. Dissolve the residue in 2 mL of a mixture of carbon
tetrachloride and phenol (2:1), transfer the solution to a
depression of a spot plate, and fill the neighboring depression
with a mixture of carbon tetrachloride and bromine (4:1).
Immediately cover both depressions with a watch glass, and
allow to stand: the solution of the residue exhibits no purple
or blue color within 1 minute.
(5) Wax— Dissolve 10.0 g of Purified Shellac in 150 mL
of a solution of sodium carbonate decahydrate (9 in 200) with
shaking on a water bath, and continue the heating for 2
hours. After cooling, collect the floating wax by filtration,
wash the wax and the filter paper with water, transfer to a
beaker, and dry at 65°C until the water is almost evaporated.
Transfer the wax together with the filter paper to an extrac-
tion thimble in a Soxhlet extractor. Dissolve the wax remain-
ing in the beaker with a suitable quantity of chloroform by
warming. Pour the solution into the thimble, and extract with
chloroform for 2 hours. Evaporate the chloroform solution
to dryness, ad dry the residue at 105 C C for 3 hours: the mass
of the residue is not more than 20 mg.
Loss on drying Not more than 2.0%. Weigh accurately
about 1 g of medium powder of Purified Shellac, and dry at
40°C for 4 hours, then for 15 hours in a desiccator (calcium
chloride for drying).
Total ash <5.07> Not more than 1.0% (1 g).
Containers and storage Containers — Well-closed contain-
ers.
White Shellac
White Shellac is a resin-like substance obtained from
a bleached secretion of Laccifer lacca Kerr (Coccidae).
Description White Shellac occurs as yellowish white to light
yellow, hard, brittle granules. It is odorless or has a faint,
characteristic odor.
It is sparingly soluble in ethanol (95), very slightly soluble
in petroleum ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Acid value <1.13> 65-90 Weigh accurately about 0.5 g of
White Shellac, add 50 mL of neutralized ethanol as a solvent,
and dissolve by warming. After cooling, perform the test as
directed in the Acid value under Purified Shellac.
Purity (1) Chloride <7.ft?>— Shake and dissolve 0.40 g of
White Shellac in 5 mL of ethanol (95) while warming, add 40
mL of water, and cool. Add 12 mL of dilute nitric acid and
water to make 100 mL, and filter. Perform the test using 50
mL of the filtrate as the test solution. Prepare the control so-
lution as follows: to 0.80 mL of 0.01 mol/L hydrochloric
acid VS add 2.5 mL of ethanol (95), 6 mL of dilute nitric acid
and water to make 50 mL (not more than 0.140%).
(2) Sulfate <1.14>— Shake and dissolve 0.40 g of White
Shellac in 5 mL of ethanol (95) by warming, add 40 mL of
water, and cool. Add 2 mL of dilute hydrochloric acid and
water to make 100 mL, and filter. Perform the test using 50
mL of the filtrate as the test solution. Prepare the control so-
lution as follows: to 0.45 mL of 0.005 mol/L sulfuric acid VS
add 2.5 mL of ethanol (95), 1 mL of dilute hydrochloric acid
and water to make 50 mL (not more than 0.110%).
(3) Heavy metals <1.07>— Proceed with 2.0 g of White
Shellac according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm.)
(4) Arsenic <7.77> — Prepare the test solution with 0.40 g
of White Shellac according to Method 3, and perform the
test. Add 10 mL of a solution of magnesium nitrate hexahy-
1086 Siccanin / Official Monographs
JP XV
drate in ethanol (95) (1 in 50), then add 1.5 mL of hydrogen
peroxide (30), and fire to burn (not more than 5 ppm).
(5) Ethanol-insoluble substances — Dissolve about 5 g of
White Shellac, accurately weighed, in 50 mL of ethanol (95)
on a water bath while shaking. Pour the ethanol solution into
a tared extraction thimble, previously dried at 105°C for 2
hours, in a Soxhlet extractor, and extract with ethanol (95)
for 3 hours: the mass of the residue is not more than 2.0%.
Use a cylindrical weighing bottle for taring the extraction
thimble.
(6) Rosin — Dissolve 2.0 g of White Shellac in 10 mL of
ethanol (99.5) with thorough shaking, add gradually 50 mL
of petroleum ether while shaking, and filter, if necessary.
Wash the solution with two 50-mL portions of water, filter
the upper layer, and evaporate the filtrate on a water bath to
dryness. Dissolve the residue in 2 mL of a mixture of carbon
tetrachloride and phenol (2:1), transfer the solution to a
depression of a spot plate, and fill the neighboring depression
with a mixture of carbon tetrachloride and bromine (4:1).
Immediately cover both depressions with a watch glass, and
allow to stand: the solution of the residue exhibits no purple
or blue color within 1 minute.
(7) Wax— Dissolve 10.0 g of White Shellac in 150 mL of
a solution of sodium carbonate decahydrate (9 in 200) with
shaking on a water bath, and continue the heating for 2
hours. After cooling, collect the floating wax by filtration,
wash the wax and the filter paper with water, transfer to a
beaker, and dry at 65 °C until the water is almost evaporated.
Transfer the wax together with the filter paper to an extrac-
tion thimble in a Soxhlet extractor. Dissolve the wax remain-
ing in the beaker with a suitable quantity of chloroform by
warming. Pour the solution into the thimble, and extract with
chloroform for 2 hours. Evaporate the chloroform solution
to dryness, and dry the residue at 105 °C for 3 hours: the mass
of the residue is not more than 20 mg.
Loss on drying Not more than 6.0%. Weigh accurately
about 1 g of medium powder of White Shellac, and dry at 40°
C for 4 hours, then for 15 hours in a desiccator (calcium chlo-
ride for drying).
Total ash <J.07> Not more than 1.0% (1 g).
Containers and storage Containers — Well-closed contain-
ers.
Storage — In a cold place.
Siccanin
C 22 H 30 O 3 : 342.47
(4aS,6a5,llbi?,13a5',13b5')-4,4,6a,9-Tetramethyl-
l,2,3,4,4a,5,6,6a,llb,13b-decahydro-13//-
benzo[a]furo[2,3,4-m«]xanthen-ll-ol [22733-60-4]
Siccanin is a substance having antifungal activity
produced by the growth of Helminthosporium siccans.
It contains not less than 980 ,ug (potency) and not
more than 1010 fig (potency) per mg, calculated on the
dried basis. The potency of Siccanin is expressed as
mass (potency) of siccanin (C22H30O3).
Description Siccanin occurs as white to light yellow, crys-
tals or crystalline powder.
It is freely soluble in acetone, soluble in methanol and in
ethanol (99.5), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Siccanin in ethanol (99.5) (1 in 10,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Siccanin Reference Standard ob-
tained in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Sicca-
nin as directed in the potassium bromide disk method under
the Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Siccanin Reference Standard: both spectra exhibit similar in-
tensities of absorption at the same wave numbers.
Optical rotation <2.49> [a] 2 D : - 165 - - 175° (0.1 g, ethanol
(99.5), 10 mL, 100 mm).
Melting point <2.60> 138 - 142°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Siccanin according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(2) Related substances — Dissolve 0.20 g of Siccanin in 10
mL of acetone, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add acetone to make ex-
actly 200 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 fiL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of cyclohexane and acetone (5:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly 4-chlorobenzenedia-
zonium TS on the plate: the number of the spots other than
the principal spot obtained from the sample solution is not
more than three, and they are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, reduced
pressure not exceeding 0.67 kPa, 80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately an amount of Siccanin and Sicca-
nin Reference Standard, equivalent to about 50 mg (poten-
cy), dissolve each in the internal standard solution to make
exactly 50 mL, and use these solutions as the sample solution
and the standard solution. Perform the test with 10 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the ratios, Qj and Q s , of the
peak area of siccanin to that of the internal standard.
Amount [fig (potency)] of C22H 30 O 3
= W s x (Qj/Qs) x 1000
W s : Amount [mg (potency)] of Siccanin Reference Stand-
JPXV
Official Monographs / Light Anhydrous Silicic Acid 1087
ard
Internal standard solution — A solution of 1,4-diphenylben-
zene in methanol (1 in 30,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of methanol and phosphate
buffer solution, pH 5.9 (19:6).
Flow rate: Adjust the flow rate so that the retention time of
siccanin is about 17 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, siccanin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of siccanin to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Light Anhydrous Silicic Acid
Light Anhydrous Silicic Acid, calculated on the in-
cinerated basis, contains not less than 98.0% of silicon
dioxide (Si0 2 : 60.08).
Description Light Anhydrous Silicic Acid occurs as a white
to bluish white, light, fine power. It is odorless and tasteless,
and smooth to the touch.
It is practically insoluble in water, in ethanol (95), and in
diethyl ether.
It dissolves in hydrofluoric acid, in hot potassium
hydroxide TS and in hot sodium hydroxide TS, and does not
dissolve in dilute hydrochloric acid.
Identification (1) Dissolve 0.1 g of Light Anhydrous Silic-
ic Acid in 20 mL of sodium hydroxide TS by boiling, and add
12 mL of ammonium chloride TS: a white, gelatinous
precipitate is produced. The precipitate does not dissolve in
dilute hydrochloric acid.
(2) To the precipitate obtained in (1) add 10 mL of a solu-
tion of methylene blue trihydrate (1 in 10,000), and wash with
water: the precipitate has a blue color.
(3) Prepare a bead by fusing ammonium sodium
hydrogenphosphate tetrahydrate on a platinum loop. Bring
the hot, transparent bead into contact with Light Anhydrous
Silicic Acid, and fuse again: an insoluble matter is perceptible
in the bead. The resulting bead, upon cooling, becomes
opaque and acquires a reticulated appearance.
Purity (1) Chloride <1.03>— Dissolve 0.5 g of Light Anhy-
drous Silicic Acid in 20 mL of sodium hydroxide TS by boil-
ing, cool, filter if necessary, and wash with 10 mL of water.
Combine the filtrate and washings, add 18 mL of dilute nitric
acid, shake, and add water to make 50 mL. Perform the test
using this solution as the test solution. To 0.15 mL of 0.01
mol/L hydrochloric acid VS add 20 mL of sodium hydroxide
TS, 18 mL of dilute nitric acid and water to make 50 mL, and
use this solution as the control solution (not more than
0.011%).
(2) Heavy metals <1.07>— Dissolve 0.5 g of Light Anhy-
drous Silicic Acid in 20 mL of sodium hydroxide TS by boil-
ing, cool, add 15 mL of acetic acid (31), shake, filter if neces-
sary, wash with 10 mL of water, combine the filtrate and
washings, and add water to make 50 mL. Perform the test us-
ing this solution as the test solution. Add acetic acid (31) to
20 mL of sodium hydroxide TS and 1 drop of
phenolphthalein TS until the color of this solution disap-
pears, add 2.0 mL of Standard Lead Solution, 2 mL of dilute
acetic acid and water to make 50 mL, and use this solution as
the control solution (not more than 40 ppm).
(3) Aluminum — Dissolve 0.5 g of Light Anhydrous Silic-
ic Acid in 40 mL of sodium hydroxide TS by boiling, cool,
add sodium hydroxide TS to make 50 mL, and filter. Meas-
ure 10 mL of the filtrate, add 17 mL of acetic acid (31),
shake, add 2 mL of aluminon TS and water to make 50 mL,
and allow to stand for 30 minutes: the color of this solution is
not deeper than that of the following control solution.
Control solution: Dissolve 0.176 g of aluminum potassium
sulfate 12-water in water, and add water to make 1000 mL.
To 15.5 mL of this solution add 10 mL of sodium hydroxide
TS, 17 mL of acetic acid (31), 2 mL of aluminon TS and
water to make 50 mL.
(4) Iron <1.10> — To 40 mg of Light Anhydrous Silicic
Acid add 10 mL of dilute hydrochloric acid, and heat for 10
minutes in a water bath while shaking. After cooling, add 0.5
g of L-tartaric acid to dissolve by shaking. Prepare the test so-
lution with this solution according to Method 2, and perform
the test according to Method B. Prepare the control solution
with 2.0 mL of Standard Iron Solution (not more than 500
ppm).
(5) Calcium — Dissolve 1.0 g of Light Anhydrous Silicic
Acid in 30 mL of sodium hydroxide TS by boiling, cool, add
20 mL of water, 1 drop of phenolphthalein TS and dilute
nitric acid until the color of this solution disappears, immedi-
ately add 5 mL of dilute acetic acid, shake, add water to
make 100 mL, and obtain a clear liquid by centrifugation or
filtration. To 25 mL of this liquid add 1 mL of oxalic acid TS
and ethanol (95) to make 50 mL, immediately shake, and al-
low to stand for 10 minutes: the turbidity of this solution is
not deeper than that of the following control solution.
Control solution: Dissolve 0.250 g of calcium carbonate,
previously dried at 180°C for 4 hours, in 3 mL of dilute
hydrochloric acid, and add water to make 100 mL. To 4 mL
of this solution add 5 mL of dilute acetic acid and water to
make 100 mL. To 25 mL of this solution add 1 mL of oxalic
acid TS and ethanol (95) to make 50 mL, and shake.
(6) Arsenic <1.11> — Dissolve 0.40 g of Light Anhydrous
Silicic Acid in 10 mL of sodium hydroxide TS by boiling in a
porcelain crucible, cool, add 5 mL of water and 5 mL of di-
lute hydrochloric acid, shake, and perform the test with this
solution as the test solution (not more than 5 ppm).
Loss on drying <2.41> Not more than 7.0% (1 g, 105 C C,
1088 Silver Nitrate / Official Monographs
JP XV
4 hours).
Loss on ignition <2.43> Not more than 12.0% (1 g, 850 -
900°C, constant mass).
Volume test Weigh 5.0 g of Light Anhydrous Silicic Acid,
transfer gradually to a 200-mL measuring cylinder, and allow
to stand: the volume is not less than 70 mL.
Assay Weigh accurately about 1 g of Light Anhydrous Si-
licic Acid, add 20 mL of hydrochloric acid, and evaporate to
dryness on a sand bath. Moisten the residue with hydrochlor-
ic acid, evaporate to dryness, and heat between 110°C and
120°C for 2 hours. Cool, add 5 mL of dilute hydrochloric
acid, and heat. Allow to cool to room temperature, add 20 to
25 mL of hot water, filter rapidly, and wash the residue with
warm water until the last washing becomes negative to the
Qualitative Tests <1.09> (2) for chloride. Transfer the residue
together with the filter paper to a platinum crucible, ignite to
ash, and continue the ignition for 30 minutes. Cool, weigh
the crucible, and designate the mass as a(g). Moisten the
residue in the crucible with water, add 6 mL of hydrofluoric
acid and 3 drops of sulfuric acid, and evaporate to dryness.
Heat strongly for 5 minutes, cool, weigh the crucible, and
designate the mass as b (g).
Content (g) of silicon dioxide (Si0 2 ) = a — b
Containers and storage Containers — Tight containers.
Silver Nitrate
mmm
AgN0 3 : 169.87
Silver Nitrate, when dried, contains not less than
99.8% of AgN0 3 .
Description Silver Nitrate occurs as lustrous, colorless or
white crystals.
It is very soluble in water, soluble in ethanol (95), and prac-
tically insoluble in diethyl ether.
It gradually turns grayish black by light.
Identification A solution of Silver Nitrate (1 in 50) responds
to the Qualitative Tests <1.09> for silver salt and for nitrate.
Purity (1) Clarity and color of solution, and acidity or
alkalinity — Dissolve 1.0 g of Silver Nitrate in 10 mL of fresh-
ly boiled and cooled water: the solution is clear and colorless.
It is neutral.
(2) Bismuth, copper and lead — To 5 mL of a solution of
Silver Nitrate (1 in 10) add 3 mL of ammonia TS: the solution
is clear and colorless.
Loss on drying <2.41> Not more than 0.20% (2 g, silica gel,
light resistant, 4 hours).
Assay Weigh accurately about 0.7 g of Silver Nitrate, previ-
ously powdered and dried, dissolve in 50 mL of water, add 2
mL of nitric acid, and titrate <2.50> with 0.1 mol/L ammoni-
um thiocyanate VS (indicator: 2 mL of ammonium iron (III)
sulfate TS).
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 16.99 mg of AgN0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Silver Nitrate Ophthalmic Solution
Silver Nitrate Ophthalmic Solution is an aqueous eye
lotion.
It contains not less than 0.95 w/v% and not more
than 1.05 w/v%> of silver nitrate (AgN0 3 : 169.87).
Method of preparation
Silver Nitrate
Purified Water
10g
a sufficient quantity
To make 1000 mL
Prepare as directed under Ophthalmic Solution, with the
above ingredients.
Description Silver Nitrate Ophthalmic Solution is a clear,
colorless liquid.
Identification Silver Nitrate Ophthalmic Solution responds
to the Qualitative Tests <1.09> for silver salt and for nitrate.
Assay Measure accurately 20 mL of Silver Nitrate
Ophthalmic Solution, add 30 mL of water and 2 mL of nitric
acid, and titrate <2.50> with 0.1 mol/L ammonium thioc-
yanate VS (indicator: 2 mL of ammonium iron (III) sulfate
TS).
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 16.99 mg of AgN0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Silver Protein
Silver Protein is a compound of silver and proteins.
It contains not less than 7.5% and not more than
8.5% of silver (Ag: 107.87).
Description Silver Protein occurs as a light yellow-brown to
brown powder. It is odorless. It (1 g) dissolves slowly in 2 mL
of water. It is practically insoluble in ethanol (95), in diethyl
ether and in chloroform.
The pH of a solution of Silver Protein (1 in 10) is between
7.0 and 8.5.
It is slightly hygroscopic.
It is affected by light.
Identification (1) To 10 mL of a solution of Silver Protein
(1 in 100) add 2 mL of dilute hydrochloric acid, shake fre-
quently for 5 minutes, and filter. To the filtrate add 5 mL of a
solution of sodium hydroxide (1 in 10), and add 2 mL of
diluted copper (II) sulfate TS (2 in 25): a purple color de-
velops.
JPXV
Official Monographs / Simple Syrup 1089
(2) To 5 mL of a solution of Silver Protein (1 in 100) add
dropwise iron (III) chloride TS: the color of the solution
fades and a precipitate is gradually formed.
(3) Incinerate 0.2 g of Silver Protein by strong heating,
dissolve the residue in 1 mL of nitric acid by warming, and
add 10 mL of water: this solution responds to the Qualitative
Tests <1.09> (1) for silver salt.
Purity Silver salt — Dissolve 0.10 g of Silver Protein in 10
mL of water, and filter. To the filtrate add 1 mL of potassium
chromate TS: no turbidity is produced.
Assay Transfer about 1 g of Silver Protein, accurately
weighed, to a 100-mL decomposition flask, add 10 mL of sul-
furic acid, cover the flask with a funnel, and boil for 5
minutes. Cool, add dropwise 3 mL of nitric acid with cau-
tion, and heat for 30 minutes without boiling. Cool, add 1
mL of nitric acid, boil, and, if necessary, repeat this opera-
tion until the solution becomes colorless. After cooling,
transfer the solution to a 250-mL conical flask with 100 mL
of water, and titrate <2.50> with 0.1 mol/L ammonium thioc-
yanate VS (indicator: 3 mL of ammonium iron (III) sulfate
TS).
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 10.79 mg of Ag
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Silver Protein Solution
"/nx'f >IS;«
Silver Protein Solution contains not less than 0.22
w/v% and not more than 0.26 w/v% of silver (Ag:
107.87).
Method of preparation
Silver Protein
Glycerin
Mentha Water
30 g
100 mL
a sufficient quantity
To make 1000 mL
Dissolve and mix the above ingredients.
Description Silver Protein Solution is a clear, brown liquid,
having the odor of mentha oil.
Identification (1) To 1 mL of Silver Protein Solution add
10 mL of ethanol (95), mix, and add 2 mL of sodium
hydroxide TS. Add immediately 1 mL of a solution of copper
(II) chloride dihydrate in ethanol (95) (1 in 10), shake, and
filter: the filtrate is blue in color (glycerin).
(2) To 3 mL of Silver Protein Solution add water to make
10 mL, add 2 mL of dilute hydrochloric acid, shake frequent-
ly for 5 minutes, and filter. Add 5 mL of a solution of sodium
hydroxide (1 in 10) to the filtrate, and add 2 mL of diluted
copper (II) sulfate TS (2 in 25): a purple color develops (silver
protein).
(3) To 5 mL of the sample solution obtained in (2) add
iron (III) chloride TS dropwise: a brown precipitate is formed
(silver protein).
(4) Place 3 mL of Silver Protein Solution in a crucible,
heat cautiously, and evaporate almost to dryness. Then in-
cinerate gradually by strong heating, dissolve the residue in 1
mL of nitric acid by warming, and add 10 mL of water: the
solution responds to the Qualitative Tests <1.09> (1) for silver
salt.
Assay Pipet 25 mL of Silver Protein Solution into a 250-mL
Kjeldahl flask, and heat cautiously until a white gas of glyce-
rin is evolved. After cooling, add 25 mL of sulfuric acid,
cover the flask with a funnel, and heat gently for 5 minutes.
After cooling, drop gradually 5 mL of nitric acid, heat with
occasional shaking in a water bath for 45 minutes, and cool.
Add 2 mL of nitric acid, boil gently, and repeat this opera-
tion until the solution becomes colorless upon cooling.
Transfer cautiously the cooled content in the flask into a
500-mL conical flask with 250 mL of water. Boil gently for 5
minutes, cool, and titrate <2.50> with 0.1 mol/L ammonium
thiocyanate VS (indicator: 3 mL of ammonium iron (III) sul-
fate TS).
Each mL of 0.1 mol/L ammonium thiocyanate VS
= 10.79 mg of Ag
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Simple Ointment
Method of preparation
Yellow Beeswax
Fixed oil
330 g
a sufficient quantity
To make 1000 g
Prepare as directed under Ointments, with the above in-
gredients.
Description Simple Ointment is yellow in color. It has a
slight, characteristic odor.
Containers and storage Containers — Tight containers.
Simple Syrup
Simple Syrup is an aqueous solution of Sucrose.
Method of preparation
Sucrose
Purified Water
850 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Syrups, with the above materi-
als.
Description Simple Syrup is a clear, colorless to pale yel-
low, viscous liquid.
It is odorless and has a sweet taste.
Identification (1) Evaporate Simple Syrup on a water bath
1090 Sisomicin Sulfate / Official Monographs
JP XV
to dryness. 1 g of the residue so obtained, when ignited, melts
to swell, and decomposes, emitting an odor of caramel, to
bulky charcoal.
(2) To 0. 1 g of the residue obtained in (1) add 2 mL of di-
lute sulfuric acid, boil, add 4 mL of sodium hydroxide TS
and 3 mL of Fehling's TS, and heat to boiling: a red to dark
red precipitate is produced.
Specific gravity <2.56> df : 1.310 - 1.325
Purity (1) Artificial sweetening agents — To 100 mL of
Simple Syrup add 100 mL of water, shake, acidify a 50-mL
portion of the solution with dilute sulfuric acid, and make
another 50-mL portion alkaline with sodium hydroxide TS.
To each portion add 100 mL of diethyl ether, shake, separate
the diethyl ether layer, and evaporate the combined diethyl
ether extract on a water bath to dryness: the residue has no
sweet taste.
(2) Salicylic acid — To the residue obtained in (1) add 2 to
3 drops of dilute iron (III) chloride TS: no purple color de-
velops.
Containers and storage Containers — Tight containers.
Sisomicin Sulfate
•> 7 T <<*>>«*!
H-,N
•2^-HzSO,
C 19 H3 7 N 5 7 .2i/2H2S04: 692.72
3-Deoxy-4-C-methyl-3-methylamino-/?-L-
arabinopyranosyl-(l->6)-[2,6-diamino-2,3,4,6-tetradeoxy-
a-D-g(ycero-hex-4-enopyranosyl-(l- > 4)]-2-
deoxy-D-streptamine hemipentasulfate [53179-09-2]
Sisomicin Sulfate is the sulfate of an aminoglycoside
substance having antibacterial activity produced by the
growth of Micromonospora inyoensis.
It contains not less than 590 fig (potency) and not
more than 700 fig (potency) per mg, calculated on the
dried basis. The potency of Sisomicin Sulfate is ex-
pressed as mass (potency) of sisomicin (Q9H37N5O7:
447.53).
Description Sisomicin Sulfate occurs as a white to light yel-
lowish white powder.
It is very soluble in water, and practically insoluble in
ethanol (95).
It is hygroscopic.
Identification (1) Dissolve 50 mg of Sisomicin Sulfate in 5
mL of water, and add 0.3 mL of bromine TS: the solution is
immediately decolorized.
(2) Dissolve 15 mg each of Sisomicin Sulfate and Sisomi-
cin Sulfate Reference Standard in 5 mL of water, and use
these solutions as the sample solution and the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 fiL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of methanol, chloroform, ammonia water (28) and ace-
tone (2:2:1:1) to a distance of about 15 cm, and air-dry the
plate. Spray evenly 0.2% ninhydrin-water satutated 1-
butabol TS on the plate, and heat at 100°C for 5 minutes: the
principal spots from the sample solution and standard solu-
tion exhibit a red-purple to red-brown color and show the
same Ri value.
(3) A solution of Sisomicin Sulfate (1 in 100) responds to
the Qualitative Tests <1.09> (1) for sulfate.
Optical rotation <2.49> [a]™: + 100 - +110° (0.25 g calcu-
lated on the dried basis, water, 25 mL, 100 mm).
pH <2.54> Dissolve 0.5 g of Sisomicin Sulfate in 5 mL of
water: the pH of the solution is between 3.5 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Sisomicin Sulfate in 5 mL of water: the solution is clear and
colorless to light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Sisomi-
cin Sulfate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Related substances — Dissolve 50 mg of Sisomicin Sul-
fate, calculated on the dried basis, in water to make 10 mL,
and use this solution as the sample solution. Pipet 0.5 mL, 1
mL and 1.5 mL of the sample solution, add water to each to
make exactly 50 mL, and use these solutions as the standard
solution (1), the standard solution (2) and the standard solu-
tion (3), respectively. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solutions (1), (2)
and (3) on a plate of silica gel for thin-layer chromatography.
Develop the plate with a mixture of methanol, chloroform,
ammonia water (28) and acetone (2:2:1:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly 0.2% nin-
hydrin-water saturated 1-butabol TS on the plate, and heat at
100°C for 5 minutes. The spots corresponding to Ri about
0.35 and Ri about 0.30 are not more intense than that of the
spot from the standard solution (3), and the spot of gallamine
corresponding to Ri about 0.25 is not more intense than the
spot from the standard solution (1). The total amount of the
related substances is not more than 6%.
Loss on drying <2.41> Not more than 15.0% (0.15 g, in
vacuum not exceeding 0.67 kPa, 110°C, 3 hours). Sampling
should be carried out in a manner to avoid moisture absorp-
tion.
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus epidermidis ATCC
12228
(ii) Culture medium — Use the medium ii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer. Adjust the pH of the medium so that it will be 7.8 to
JPXV
Official Monographs / Sodium Acetate Hydrate 1091
8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Sisomicin Sulfate Reference Standard equivalent to about 25
mg (potency), add 0.1 mol/L phosphate buffer solution, pH
8.0 to make exactly 25 mL, and use this solution as the stan-
dard stock solution. Keep the standard stock solution at 5°C
or below and use within 7 days. Take exactly a suitable
amount of the standard stock solution before use, add 0.1
mol/L phosphate buffer solution, pH 8.0 to make solutions
so that each mL contains 1 /ug (potency) and 0.25 /xg (poten-
cy), and use these solutions as the high concentration stan-
dard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Sisomicin sulfate equivalent to about 25 mg (potency), add
0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly
25 mL. Take exactly a suitable amount of the solution, add
0.1 mol/L phosphate buffer solution, pH 8.0 to make solu-
tions so that each mL contains 1 /ug (potency) and 0.25 /xg
(potency), and use these solutions as the high concentration
sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, not exceeding -20°C, under
nitrogen or argon atmosphere.
Freeze-dried Smallpox Vaccine
^ig-e^^f:
Freeze-dried Smallpox Vaccine is a preparation for
injection which is dissolved before use. It contains live
vaccinia virus.
It conforms to the requirements of Freeze-dried
Smallpox Vaccine in the Minimum Requirements for
Biological Products.
Description Freeze-dried Smallpox Vaccine becomes a
white to gray, turbid liquid on addition of solvent.
Freeze-dried Smallpox Vaccine
Prepared in Cell Culture
Freeze-dried Smallpox Vaccine Prepared in Cell Cul-
ture is a preparation for injection which is dissolved be-
fore use. It contains live vaccinia virus.
It conforms to the requirements of Freeze-dried
Smallpox Vaccine Prepared in Cell Culture in the Mini-
mum Requirements for Biological Products.
Description Freeze-dried Smallpox Vaccine Prepared in
Cell Culture becomes a reddish clear liquid on addition of
solvent.
Sodium Acetate Hydrate
mmi- r- u ^A7Kftti
HjC-COjNa .ahfeO
C 2 H 3 Na0 2 .3H 2 0: 136.08
Monosodium acetate trihydrate
[6131-90-4]
Sodium Acetate Hydrate, when dried, contains not
less than 99.5% of sodium acetate (C 2 H 3 Na0 2 : 82.03).
Description Sodium Acetate Hydrate occurs as colorless
crystals or a white, crystalline powder. It is odorless or has a
slight, acetous odor. It has a cool, saline and slightly bitter
taste.
It is very soluble in water, freely soluble in acetic acid
(100), soluble in ethanol (95), and practically insoluble in
diethyl ether.
It is efflorescent in warm, dry air.
Identification A solution of Sodium Acetate Hydrate (1 in
10) responds to the Qualitative Tests <1.09> for acetate and
for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 2.0 g of
Sodium Acetate Hydrate in 20 mL of water: the solution is
clear and colorless.
(2) Acidity or alkalinity — Dissolve 1.0 g of Sodium
Acetate Hydrate in 20 mL of freshly boiled and cooled water,
and add 3 drops of phenolphthalein TS: a red color develops.
When cooled to 10°C, or 1.0 mL of 0.01 mol/L hydrochloric
acid VS is added after cooling to 10°C, the red color disap-
pears.
(3) Chloride <1.03>— Perform the test with 1 .0 g of Sodi-
um Acetate Hydrate. Prepare the control solution with 0.30
mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011
%).
(4) Sulfate <1.14>— Perform the test with 1 .0 g of Sodium
Acetate Hydrate. Prepare the control solution with 0.35 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.017%).
(5) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Acetate Hydrate according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(6) Calcium and magnesium — Dissolve 4.0 g of Sodium
Acetate Hydrate in 25 mL of water, add 6 g of ammonium
chloride, 20 mL of ammonia solution (28) and 0.25 mL of a
solution of sodium hydrogensulfite (1 in 10), and titrate <2.50
> with 0.01 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS until the blue color changes to grayish blue
(indicator: 0.1 g of methylthymol blue-potassium nitrate in-
dicator): the amount of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS consumed is not more than
0.5 mL.
(7) Arsenic </.//> — Prepare the test solution with 1.0 g
of Sodium Acetate Hydrate, according to Method 1, and per-
form the test (not more than 2 ppm).
(8) Potassium permanganate-reducing substance — Dis-
solve 1.0 g of Sodium Acetate Hydrate in 100 mL of water,
add 5 mL of dilute sulfuric acid, boil, add 0.50 mL of 0.002
mol/L potassium permanganate VS, and further boil for 5
1092 Sodium Aurothiomalate / Official Monographs
JP XV
minutes: the red color of the solution does not disappear.
Loss on drying <2.41> 39.0 - 40.5% (1 g, first at 80°C for 2
hours, and then at 130°C for 2 hours).
Assay Weigh accurately about 0.2 g of Sodium Acetate Hy-
drate, previously dried, dissolve in 50 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from yellow to green (indicator:
1 mL of /?-naphtholbenzein TS). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 8.203 mg of C 2 H 3 Na0 2
Containers and storage Containers — Tight containers.
Sodium Aurothiomalate
H S-Au
and enantiomer
Mixture of C 4 H 3 AuNa 2 4 S: 390.08 and
C 4 H 4 AuNa0 4 S: 368.09
Monogold monosodium monohydrogen (2RS)-2-
sulfidobutane-1 ,4-dioate
Monogold disodium (2i?5)-2-sulfidobutane-l,4-dioate
[12244-57-4, Sodium Aurothiomalate]
Sodium Aurothiomalate contains not less than
49.0% and not more than 52.5%> of gold (Au: 196.97),
calculated on the anhydrous basis and corrected by the
amount of ethanol.
Description Sodium Aurothiomalate occurs as white to
light yellow, powder or granules.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
It is hygroscopic.
It changes in color by light to greenish pale yellow.
Identification (1) To 2 mL of a solution of Sodium
Aurothiomalate (1 in 10) add 1 mL of a solution of calcium
nitrate tetrahydrate (1 in 10): a white precipitate is produced,
and it dissolves in dilute nitric acid and reappears on the addi-
tion of ammonium acetate TS.
(2) To 2 mL of a solution of Sodium Aurothiomalate (1
in 10) add 3 mL of silver nitrate TS: a yellow precipitate is
produced, and it dissolves in an excess of ammonia TS.
(3) Place 2 mL of a solution of Sodium Aurothiomalate
(1 in 10) in a porcelain crucible, add 1 mL of ammonia TS
and 1 mL of hydrogen peroxide (30), evaporate to dryness,
and ignite. Add 20 mL of water to the residue, and filter: the
residue on the filter paper occurs as a yellow or dark yellow,
powder or granules.
(4) The filtrate obtained in (3) responds to the Qualitative
Tests <1.09> for sodium salt.
(5) The filtrate obtained in (3) responds to the Qualitative
Tests <1.09> for sulfate.
pH <2.54> Dissolve 1.0 g of Sodium Aurothiomalate in 10
mL of water: the pH of this solution is between 5.8 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Aurothiomalate in 10 mL of water: the solution is
clear and light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Sodium
Aurothiomalate according to Method 2, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 30 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sodium Aurothiomalate according to Method 3, and per-
form the test (not more than 2 ppm).
(4) Ethanol — Weigh accurately about 0.2 g of Sodium
Aurothiomalate, add exactly 3 mL of the internal standard
solution and 2 mL of water to dissolve, and use this solution
as the sample solution. Separately, pipet 3 mL of ethanol
(99.5), and add water to make exactly 1000 mL. Pipet 2 mL
of this solution, add exactly 3 mL of the internal standard so-
lution, and use this solution as the standard solution.
Perform the test with 2/uL each of the sample solution and
standard solution as directed under Gas Chromatography
<2.02> according to the following conditions, and determine
the ratios of the peak area of ethanol to that of the internal
standard, g T and Q s : the amount of ethanol is not more than
3.0%.
Amount (mg) of ethanol = (Qt/Qs) x 6 x 0.793
0.793: Density (g/mL) of ethanol (99.5) at 20°C
Internal standard solution — A solution of 2-propanol (1 in
500).
Operating conditions —
Detector: Hydrogen flame-ionization detector.
Column: A column 3 mm in inside diameter and 3 m in
length, packed with porous styrene-divinylbenzene
copolymer for gas chromatography (particle diameter: 150 -
180 jum) (average pore size: 0.0085 /urn; 300 - 400 m 2 /g).
Column temperature: A constant temperature of about
180°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 7 minutes.
System suitability —
System performance: When the procedure is run with 2 /iL
of the standard solution under the above operating condi-
tions, ethanol and the internal standard are eluted in this ord-
er with the resolution between these peaks being not less than
4.
System repeatability: When the test is repeated 6 times with
2 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of ethanol to that of the internal standard is not
more than 2.0%.
Water <2.48> Not more than 5.0% (0.1 g, coulometric titra-
tion). Use a water vaporizer (heating temperature: 105°C;
heating time: 30 minutes).
Assay Weigh accurately about 25 mg of Sodium
Aurothiomalate, and dissolve in 2 mL of aqua regia by heat-
ing. After cooling, add water to make exactly 100 mL. Pipet
2 mL of the solution, add water to make exactly 25 mL, and
use this solution as the sample solution. Separately, pipet 5
mL, 10 mL and 15 mL of Standard Gold Solution for atomic
absorption spectrophotometry, add water to make exactly 25
mL, and use these solutions as the standard solutions (1), (2)
and (3), respectively. Perform the test with the sample solu-
JPXV
Official Monographs / Sodium Bicarbonate 1093
tion and standard solutions (1), (2) and (3) as directed under
Atomic Absorption Spectrophotometry <2.23> under the fol-
lowing conditions. Determine the amount of gold in the sam-
ple solution using the calibration curve obtained from the ab-
sorbances of the standard solutions.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: Gold hollow-cathode lamp
Wavelength: 242.8 nm
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Sodium Benzoate
^MMM-)- r- U OA
a
C0 2 Na
C 7 H 5 Na0 2 : 144.10
Monosodium benzoate [532-32-1]
Sodium Benzoate, when dried, contains not less than
99.0% of C 7 H 5 Na0 2 .
Description Sodium Benzoate occurs as white granules,
crystals or crystalline powder. It is odorless, and has a sweet
and saline taste.
It is freely soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
Identification A solution of Sodium Benzoate (1 in 100)
responds to the Qualitative Tests <1.09> for benzoate and the
Qualitative Tests <1.09> (1) and (2) for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Benzoate in 5 mL of water: the solution is clear and
colorless.
(2) Acidity or alkalinity — Dissolve 2.0 g of Sodium Ben-
zoate in 20 mL of freshly boiled and cooled water, and add 2
drops of phenolphthalein TS and 0.20 mL of 0.05 mol/L sul-
furic acid VS: the solution remains colorless. To this solution
add 0.40 mL of 0.1 mol/L sodium hydroxide VS: a red color
develops.
(3) Sulfate <1.14>— Dissolve 0.40 g of Sodium Benzoate
in 40 mL of water, add slowly 3.5 mL of dilute hydrochloric
acid with thorough stirring, allow to stand for 5 minutes, and
filter. Discard the first 5 mL of the filtrate, take the subse-
quent 20 mL of the filtrate, add water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 0.40 mL of 0.005 mol/L sul-
furic acid VS (not more than 0.120%).
(4) Heavy metals <1.07> — Dissolve 2.0 g of Sodium Ben-
zoate in 44 mL of water, add gradually 6 mL of dilute
hydrochloric acid with thorough stirring, and filter. Discard
the first 5 mL of the filtrate, take the subsequent 25 mL of the
filtrate, neutralize with ammonia TS, add 2 mL of dilute acet-
ic acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 2.0 mL of Standard Lead Solution add 2 mL of
dilute acetic acid and water to make 50 mL (not more than 20
ppm).
(5) Arsenic <1.11> — Mix well 1.0 g of Sodium Benzoate
with 0.40 g of calcium hydroxide, ignite, dissolve the residue
in 10 mL of dilute hydrochloric acid, and perform the test us-
ing this solution as the test solution (not more than 2 ppm).
(6) Chlorinated compounds — Dissolve 1.0 g of Sodium
Benzoate in 10 mL of water, add 10 mL of dilute sulfuric
acid, and extract with two 20-mL portions of diethyl ether.
Combine the diethyl ether extracts, and evaporate the diethyl
ether on a water bath. Place 0.5 g of the residue and 0.7 g of
calcium carbonate in a crucible, mix with a small amount of
water, and dry. Ignite it at about 600°C, dissolve in 20 mL of
dilute nitric acid, and filter. Wash the residue with 15 mL of
water, combine the filtrate and the washing, add water to
make 50 mL, and add 0.5 mL of silver nitrate TS: this solu-
tion has no more turbidity than the following control solu-
tion
Control solution: Dissolve 0.7 g of calcium carbonate in 20
mL of dilute nitric acid, and filter. Wash the residue with 15
mL of water, combine the filtrate and the washings, add 1.2
mL of 0.01 mol/L Hydrochloric acid VS and water to make
50 mL, and add 0.5 mL of silver nitrate TS.
(7) Phthalic acid — To 0.10 g of Sodium Benzoate add 1
mL of water and 1 mL of resorcinol-sulfuric acid TS, and
heat the mixture in an oil bath heated at a temperature be-
tween 120°C and 125°C to evaporate the water, then heat the
residue for further 90 minutes, cool, and dissolve in 5 mL of
water. To 1 mL of the solution add 10 mL of a solution of so-
dium hydroxide (43 in 500), shake, then examine under light
at a wavelength between 470 nm and 490 nm: the green
fluorescence of the solution is not more intense than that of
the following control solution.
Control solution: Dissolve 61 mg of potassium hydrogen
phthalate in water to make exactly 1000 mL. Pipet exactly 1
mL of the solution, add 1 mL of resorcinol-sulfuric acid TS,
and proceed as directed above.
Loss on drying <2.41> Not more than 1.5% (2 g, 110°C,
4 hours).
Assay Weigh accurately about 1.5 g of Sodium Benzoate,
previously dried, and transfer to a 300-mL glass-stoppered
flask. Dissolve in 25 mL of water, add 75 mL of diethyl ether
and 10 drops of bromophenol blue TS, and titrate <2.50> with
0.5 mol/L hydrochloric acid VS, while mixing the aqueous
and diethyl ether layers by vigorous shaking, until a persis-
tent, light green color is produced in the aqueous layer.
Each mL of 0.5 mol/L hydrochloric acid VS
= 72.05 mg of C 7 H 5 Na0 2
Containers and storage Containers — Well-closed contain-
ers.
Sodium Bicarbonate
NaHCO,: 84.01
Sodium Bicarbonate contains not less than 99.0% of
NaHC0 3 .
Description Sodium Bicarbonate occurs as white crystals or
1094 Sodium Bicarbonate Injection / Official Monographs
JP XV
crystalline powder. It is odorless, and has a characteristic, sa-
line taste.
It is soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
It slowly decomposes in moist air.
Identification A solution of Sodium Bicarbonate (1 in 30)
responds to the Qualitative Tests <1.09> for sodium salt and
for bicarbonate.
pH <2.54> Dissolve 1.0 g of Sodium Bicarbonate in 20 mL
of water: the pH of this solution is between 7.9 and 8.4.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Bicarbonate in 20 mL of water: the solution is clear
and colorless.
(2) Chloride <1.03>— To 0.40 g of Sodium Bicarbonate
add 4 mL of dilute nitric acid, heat to boil, cool, and add 6
mL of dilute nitric acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 0.45 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.040%).
(3) Carbonate — Dissolve 1.0 g of Sodium Bicarbonate in
20 mL of freshly boiled and cooled water with very gentle
swirling at a temperature not exceeding 15°C. Add 2.0 mL of
0.1 mol/L hydrochloric acid VS and 2 drops of
phenolphthalein TS: no red color develops immediately.
(4) Ammonium — Heat 1.0 g of Sodium Bicarbonate: the
gas evolved does not change moistened red litmus paper to
blue.
(5) Heavy metals <1.07> — Dissolve 4.0 g of Sodium Bi-
carbonate in 5 mL of water and 4.5 mL of hydrochloric acid,
and evaporate on a water bath to dryness. Dissolve the
residue in 2 mL of dilute acetic acid, 35 mL of water and 1
drop of ammonium TS, dilute with water to 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: evaporate 4.5 mL of
hydrochloric acid to dryness, and add 2 mL of dilute acetic
acid, 2.0 mL of Standard Lead Solution and water to make
50 mL (not more than 5 ppm).
(6) Arsenic <1.11> — Dissolve 1.0 g of Sodium Bicar-
bonate in 3 mL of water and 2 mL of hydrochloric acid, and
perform the test using this solution as the test solution (not
more than 2 ppm).
Assay Weigh accurately about 2 g of Sodium Bicarbonate,
dissolve in 25 mL of water, and titrate with 0.5 mol/L sulfur-
ic acid VS. When the color of the solution changes from blue
to yellow-green, boil with caution, cool, and continue the
titration <2.50> until a greenish yellow color develops (indica-
tor: 2 drops of bromocresol green TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 84.01 mg of NaHC0 3
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Sodium Bicarbonate Injection
It contains not less than 95% and not more than
105% of the labeled amount of sodium hydrogen car-
bonate (NaHC0 3 : 84.01).
Method of preparation Prepare as directed under Injec-
tions, with Sodium Bicarbonate.
Description
less liquid.
Sodium Bicarbonate Injection is a clear, color-
j*sg7kiti- h- u *Aa*i*
Sodium Bicarbonate Injection is an aqueous solution
for injection.
Identification To a volume of Sodium Bicarbonate Injec-
tion, equivalent to 1 g of Sodium Bicarbonate according to
the labeled amount, add water to make 30 mL: the solution
responds to the Qualitative Tests <1.09> for sodium salt and
for bicarbonate.
pH <2.54> 7.0-8.5
Bacterial endotoxins <4.01> Less than 5.0 EU/mEq.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Sodium Bicarbonate In-
jection, equivalent to about 2 g of sodium hydrogen car-
bonate (NaHC0 3 ), titrate with 0.5 mol/L sulfuric acid VS,
and proceed as directed in the Assay under Sodium Bicar-
bonate.
Each mL of 0.5 mol/L sulfuric acid VS
= 84.01 mg of NaHCC-3
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Sodium Bisulfite
Sodium Hydrogen Sulfite
wmwk&Ks-t- r u^a
NaHS0 3 : 104.06
Sodium Bisulfite is a mixture of sodium hydrogen-
sulfite and sodium pyrosulfite.
It contains not less than 64.0% and not more than
67.4% of sulfur dioxide (S0 2 : 64.06).
Description Sodium Bisulfite occurs as white granules or
powder, having the odor of sulfur dioxide.
Sodium Bisulfite is freely soluble in water, and practically
insoluble in ethanol (95) and in diethyl ether.
A solution of Sodium Bisulfite (1 in 20) is acid.
Sodium Bisulfite is slowly affected by air or by light.
Identification A solution of Sodium Bisulfite (1 in 20)
responds to the Qualitative Tests <1.09> for sodium salt and
for bisulfite.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Bisulfite in 10 mL of water: the solution is clear and
colorless.
(2) Thiosulfate — Dissolve 1.0 g of Sodium Bisulfite in 15
mL of water, add slowly 5 mL of dilute hydrochloric acid,
shake, and allow to stand for 5 minutes: no turbidity is
produced.
(3) Heavy metals <1.07> — Dissolve 1 .0 g of Sodium Bisul-
fite in 10 mL of water, add 5 mL of hydrochloric acid, and
JPXV
Official Monographs / Sodium Bromide 1095
evaporate on a water bath to dryness. To the residue add 2
mL of dilute acetic acid and water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution as follows: evaporate 5 mL of
hydrochloric acid on a water bath to dryness, and add 2 mL
of dilute acetic acid and 2.0 mL of Standard Lead Solution,
and dilute with water to make 50 mL (not more than 20
ppm).
(4) Iron <1.10> — Prepare the test solution with 1.0 g of
Sodium Bisulfite according to Method 1, and perform the
test according to Method A. Prepare the control solution
with 2.0 mL of Standard Iron Solution (not more than 20
ppm).
(5) Arsenic <1.11> — Dissolve 0.5 g of Sodium Bisulfite in
10 mL of water. Add 1 mL of sulfuric acid, heat on a sand
bath until white fumes are evolved, add water to make 5 mL,
and perform the test with this solution as the test solution
(not more than 4 ppm).
Assay Weigh accurately about 0.15 g of Sodium Bisulfite,
and transfer immediately into an iodine flask containing ex-
actly 50 mL of 0.05 mol/L iodine VS, stopper, shake, and al-
low to stand for 5 minutes in a dark place. Add 1 mL of
hydrochloric acid, and titrate <2.50> the excess iodine with
0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch
TS). Perform a blank determination.
Each mL of 0.05 mol/L iodine VS = 3.203 mg of S0 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant, preferably well-filled, and not
exceeding 30°C.
Sodium Borate
Na 2 B 4 O 7 .10H 2 O: 381.37
Sodium Borate contains not less than 99.0% and not
more than 103.0% of Na 2 B 4 O 7 .10H 2 O.
Description Sodium Borate occurs as colorless or white
crystals or a white, crystalline powder. It is odorless, and has
a slightly characteristic, saline taste.
It is freely soluble in glycerin, soluble in water, and practi-
cally insoluble in ethanol (95), in ethanol (99.5) and in diethyl
ether.
When placed in dry air, Sodium Borate effloresces and is
coated with a white powder.
Identification A solution of Sodium Borate (1 in 20)
responds to the Qualitative Tests <1.09> for sodium salt and
for borate.
pH <2.54> Dissolve 1.0 g of Sodium Borate in 20 mL of
water: the pH of this solution is between 9.1 and 9.6.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Borate in 20 mL of water by warming slightly: the so-
lution is clear and colorless.
(2) Carbonate or bicarbonate — Dissolve 1.0 g of pow-
dered Sodium Borate in 20 mL of freshly boiled and cooled
water, and add 3 mL of dilute hydrochloric acid: the solution
does not effervesce.
(3) Heavy metals <1.07> — Dissolve 1.5 g of Sodium
Borate in 25 mL of water and 7 mL of 1 mol/L hydrochloric
acid TS, add 1 drop of phenolphthalein TS, and add ammo-
nia TS until a pale red color develops. Then add dilute acetic
acid until the solution becomes colorless again, add 2 mL of
dilute acetic acid, and add water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: to 3.0 mL of Standard Lead Solution
add 2 mL of dilute acetic acid and water to make 50 mL (not
more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Sodium Borate according to Method 1, and perform the
test (not more than 5 ppm).
Assay Weigh accurately about 2 g of Sodium Borate, dis-
solve in 50 mL of water, and titrate <2.50> with 0.5 mol/L
hydrochloric acid VS (indicator: 3 drops of methyl red TS).
Each mL of 0.5 mol/L hydrochloric acid VS
= 95.34 mg of Na 2 B 4 O 7 .10H 2 O
Containers and storage Containers — Tight containers.
Sodium Bromide
NaBr: 102.89
Sodium Bromide, when dried,
99.0% of NaBr.
contains not less than
Description Sodium Bromide occurs as colorless or white
crystals or crystalline powder. It is odorless.
It is freely soluble in water, and soluble in ethanol (95).
It is hygroscopic, but not deliquescent.
Identification A solution of Sodium Bromide (1 in 10)
responds to the Qualitative Tests <1.09> for sodium salt and
for bromide.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Bromide in 3 mL of water: the solution is clear and
colorless.
(2) Alkalinity — Dissolve 1.0 g of Sodium Bromide in 10
mL of water, add 0.10 mL of 0.005 mol/L sulfuric acid VS
and 1 drop of phenolphthalein TS, heat to boil, and cool: the
solution is colorless.
(3) Chloride — Make a calculation from the result ob-
tained in the Assay. Not more than 97.9 mL of 0.1 mol/L sil-
ver nitrate VS is consumed for 1 g of Sodium Bromide.
(4) Sulfate <1.14>— Perform the test with 2.0 g of Sodium
Bromide. Prepare the control solution with 1.0 mL of 0.005
mol/L sulfuric acid VS (not more than 0.024%).
(5) Iodide — Dissolve 0.5 g of Sodium Bromide in 10 mL
of water, add 2 to 3 drops of iron (III) chloride TS and 1 mL
of chloroform, and shake: no red-purple color develops in
the chloroform layer.
(6) Bromate — Dissolve 1.0 g of Sodium Bromide in 10
mL of freshly boiled and cooled water, and add 2 drops of
potassium iodide TS, 1 mL of starch TS and 3 drops of dilute
sulfuric acid. Shake the mixture gently, and allow to stand
for 5 minutes: no blue color develops.
1096 Sodium Carbonate Hydrate / Official Monographs
JP XV
(7) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Bromide according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(8) Barium — Dissolve 0.5 g of Sodium Bromide in 10 mL
of water, add 0.5 mL of dilute hydrochloric acid and 1 mL of
potassium sulfate TS, and allow to stand for 10 minutes: no
turbidity is produced.
(9) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sodium Bromide according to Method 1, and perform the
test (not more than 2 ppm).
Loss on drying <2.41> Not more than 5.0% (1 g, 110°C,
4 hours).
Assay Weigh accurately about 0.4 g of Sodium Bromide,
previously dried, and dissolve in 50 mL of water. Add 10 mL
of dilute nitric acid and 50 mL of 0.1 mol/L silver nitrate VS,
exactly measured, and titrate <2.50> the excess silver nitrate
with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL
of ammonium iron (III) sulfate TS). Perform a blank deter-
mination.
Each mL of 0.1 mol/L silver nitrate VS
= 10.29 mg of NaBr
Containers and storage Containers — Tight containers.
Sodium Carbonate Hydrate
BURT r- U ^A7jan$9
Na 2 CO 3 .10H 2 O: 286.14
Sodium Carbonate Hydrate contains not less than
99.0% and not more than 103.0% of Na 2 CO 3 .10H 2 O.
Description Sodium Carbonate Hydrate occurs as colorless
or white crystals.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
A solution of Sodium Carbonate Hydrate (1 in 10) is alka-
line.
It is efflorescent in air.
It liquefies in its water of crystallization at 34°C, and
becomes anhydrous at above 100°C.
Identification A solution of Sodium Carbonate Hydrate (1
in 20) responds to the Qualitative Tests <1.09> for sodium salt
and for carbonate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Carbonate Hydrate in 5 mL of water: the solution is
clear and colorless.
(2) Chloride <1.03> — Dissolve 0.5 g of Sodium Carbonate
Hydrate in 10 mL of water, add 7 mL of dilute nitric acid, di-
lute with water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.071%).
(3) Heavy metals <1.07> — Dissolve 2.0 g of Sodium Car-
bonate Hydrate in 10 mL of water, add 8 mL of dilute
hydrochloric acid, and evaporate to dryness on a water bath.
Dissolve the residue in 35 mL of water and 2 mL of dilute a-
cetic acid, dilute with water to make 50 mL, and perform the
test using this solution as the test solution. Prepare the con-
trol solution as follows: evaporate 8 mL of dilute hydrochlor-
ic acid on a water bath to dryness, add 2 mL of dilute acetic
acid and 2.0 mL of Standard Lead Solution, and dilute with
water to make 50 mL (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.65 of
Sodium Carbonate Hydrate according to Method 1, and per-
form the test (not more than 3.1 ppm).
Loss on drying <2.41> 61.0 ~ 63.0% (1 g, 105°C, 4 hours).
Assay Dissolve about 3 g of Sodium Carbonate Hydrate,
weighed accurately, in 25 mL of water, and titrate with 0.5
mol/L sulfuric acid VS until the color of the solution changes
from blue to yellow-green. Boil cautiously, cool, and further
titrate <2.50> until a greenish yellow color appears (indicator:
2 drops of bromocresol green TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 143.1 mg of Na 2 CO 3 .10H 2 O
Containers and storage Containers — Tight containers.
Dried Sodium Carbonate
Na 2 C0 3 : 105.99
Dried Sodium Carbonate, when dried, contains not
less than 99.0%, of Na 2 C0 3 .
Description Dried Sodium Carbonate occurs as white crys-
tals or crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
A solution of Dried Sodium Carbonate (1 in 10) is alkaline.
It is hygroscopic.
Identification A solution of Dried Sodium Carbonate (1 in
20) responds to the Qualitative Tests <1.09> for sodium salt
and for carbonate.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Dried Sodium Carbonate in 10 mL of water: the solution is
clear and colorless.
(2) Chloride <7.0?>— Dissolve 0.5 g of Dried Sodium
Carbonate in 1 mL of water ,addl2mLof dilute nitric acid ,
dilute with water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.071%).
(3) Heavy metals <1.07> — Dissolve 1 .0 g of Dried Sodium
Carbonate in 10 mL of water, add 7.5 mL of dilute
hydrochloric acid, and evaporate on a water bath to dryness.
Dissolve the residue in 35 mL of water and 2 mL of dilute
acetic acid, dilute with water to make 50 mL, and perform
the test using this solution as the test solution. Prepare the
control solution as follows: evaporate 7.5 mL of dilute
hydrochloric acid on a water bath to dryness, add 2 mL of di-
lute acetic acid and 2.0 mL of Standard Lead Solution, and
dilute with water to make 50 mL (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.65 g
JPXV
Official Monographs / Sodium Chloride 1097
of Dried Sodium Carbonate according to Method 1, and per-
form the test (not more than 3.1 ppm).
Loss on drying <2.41> Not more than 2.0% (2 g, 106°C,
4 hours).
Assay Dissolve about 1.2 g of Dried Sodium Carbonate,
weighed accurately, in 25 mL of water, and titrate with 0.5
mol/L sulfuric acid VS until the color of the solution changes
from blue to yellow-green. Then boil cautiously, cool, and
further titrate <2.50> until a greenish yellow color develops
(indicator: 2 drops of bromocresol green TS).
Each mL of 0.5 mol/L sulfuric acid VS
= 52.99 mg of Na 2 C0 3
Containers and storage Containers — Tight containers.
Sodium Chloride
Met r- U OA
NaCl: 58.44
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦).
Sodium Chloride contains not less than 99.0% and
not more than 100.5% of NaCl, calculated on the dried
basis.
♦Description Sodium Chloride occurs as colorless or white,
crystals or crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5). »
Identification (1) A solution of Sodium Chloride (1 in 20)
responds to the Qualitative Tests <1.09> for sodium salt.
(2) A solution of Sodium Chloride (1 in 20) responds to
the Qualitative Tests <1.09> for chloride.
*Purity (1) Clarity and color of solution — Dissolve 1.0 g
of Sodium Chloride in 5 mL of water: the solution is clear
and colorless. ♦
(2) Acidity or alkalinity — Dissolve 20.0 g of Sodium
Chloride in 100.0 mL of freshly boiled and cooled water, and
use this solution as the sample solution. To 20 mL of the sam-
ple solution add 0.1 mL of bromothymol blue TS and 0.5 mL
of 0.01 mol/L hydrochloric acid VS: the color of the solution
is yellow. Separately, to 20 mL of the sample solution add 0.1
mL of bromothymol blue TS and 0.5 mL of 0.01 mol/L sodi-
um hydroxide VS: the color of the solution is blue.
(3) Sulfates — To 7.5 mL of the sample solution obtained
in (2) add water to make 30 mL, and use this solution as the
sample solution. Separately, dissolve 0.181 g of potassium
sulfate in diluted ethanol (99.5) (3 in 10) to make exactly 500
mL. Pipet 5 mL of this solution, and add diluted ethanol
(99.5) (3 in 10) to make exactly 100 mL. To 4.5 mL of this so-
lution add 3 mL of a solution of barium chloride dihydrate (1
in 4), shake, and allow to stand for 1 minutes. To 2.5 mL of
this solution add 15 mL of the sample solution and 0.5 mL of
acetic acid (31), and allow to stand for 5 minutes: any turbidi-
ty produced does not more than that produced in the follow-
ing control solution.
Control solution: Dissolve 0.181 g of potassium sulfate in
water to make exactly 500 mL. Pipet 5 mL of this solution,
add water to make exactly 100 mL, and proceed in the same
manner as directed above using this solution instead of the
sample solution.
(4) Phosphates — To 2.0 mL of the sample solution ob-
tained in (2) add 5 mL of 2 mol/L sulfuric acid TS and water
to make 100.0 mL, then add 4 mL of ammonium molybdate-
sulfuric acid TS and 0.1 mL of tin (II) chloride-hydrochloric
acid TS, and allow to stand for 10 minutes: the color of the
solution is not darker than the following control solution.
Control solution: To 1.0 mL of Standard Phosphoric Acid
Solution add 12.5 mL of 2 mol/L sulfuric acid TS and water
to make exactly 250 mL. To 100 mL of this solution add 4
mL of ammonium molybdate-sulfuric acid TS and 0.1 mL of
tin (II) chloride-hydrochloric acid TS, and allow to stand for
10 minutes.
(5) Bromides — To 0.50 mL of the sample solution ob-
tained in (2) add 4.0 mL of water, 2.0 mL of dilute phenol
red TS and 1.0 mL of a solution of sodium toluenesulfon-
chloramide trihydrate (1 in 10,000), and mix immediately.
After allowing to stand for 2 minutes, add 0.15 mL of 0.1
mol/L sodium thiosulfate VS, mix, add water to make ex-
actly 10 mL, and use this solution as the sample solution.
Separately, to 5.0 mL of a solution of potassium bromide (3
in 1 ,000,000) add 2.0 mL of dilute phenol red TS and 1 .0 mL
of a solution of sodium toluenesulfonchloramide trihydrate
(1 in 10,000), and mix immediately. Proceed in the same
manner as for the preparation of the sample solution, and use
the solution so obtained as the standard solution. Perform
the test with the sample solution and standard solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>
using water as the control: the absorbance at 590 nm of the
sample solution is not more than that of the standard solu-
tion.
(6) Iodides — Wet 5 g of Sodium Chloride with drop-wise-
ly added 0.15 mL of a freshly prepared mixture of starch TS,
0.5 mol/L sulfuric acid TS and sodium nitrite TS
(1000:40:3), allow to stand for 5 minutes, and examine under
daylight: a blue color does not appear.
(7) Ferrocyanides — Dissolve 2.0 g of Sodium Chloride in
6 mL of water, and add 0.5 mL of a mixture of a solution of
iron (II) sulfate heptahydrate (1 in 100) and a solution of am-
monium iron (III) sulfate dodecahydrate in diluted sulfuric
acid (1 in 400) (1 in 100) (19:1): a blue color does not develop
within 10 minutes.
*(8) Heavy metals <1.07> — Proceed with 5.0 g of Sodium
Chloride according to Method 1, and perform the test. Pre-
pare the control solution with 1.5 mL of Standard Lead Solu-
tion (not more than 3 ppm).»
(9) Iron — To 10 mL of the sample solution obtained in
(2) add 2 mL of a solution of citric acid monohydrate (1 in 5)
and 0.1 mL of mercapto acetic acid, alkalize with ammonia
TS, add water to make 20 mL, and allow to stand for 5
minutes: the solution has not more color than the following
control solution.
Control solution: Pipet 1 mL of Standard Iron Solution,
and add water to make exactly 25 mL. To 10 mL of this solu-
tion add 2 mL of a solution of citric acid monohydrate (1 in
5) and 0.1 mL of mercapto acetic acid, and proceed in the
same manner as directed for the sample solution.
(10) Barium — To 5.0 mL of the sample solution obtained
1098 10% Sodium Chloride Injection / Official Monographs
JP XV
in (2) add 5.0 mL of water and 2.0 mL of dilute sulfuric acid,
and allow to stand for 2 hours: the solution has not more tur-
bidity than the following control solution.
Control solution: To 5.0 mL of the sample solution ob-
tained in (2) add 7.0 mL of water, and allow to stand for 2
hours.
(11) Magnesium and alkaline-earth materials — To 200
mL of water add 0.1 g of hydroxylammonium chloride, 10
mL of ammonium chloride buffer solution, pH 10, 1 mL of
0.1 mol/L zinc sulfate VS and 0.2 g of eriochrome black T-
sodium chloride indicator, and warm to 40 C C. Add 0.01
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
dropwise until the red-purple color of the solution changes to
blue-purple. To this solution add a solution prepared by dis-
solving 10.0 g of Sodium Chloride in 100 mL of water, and
add 2.5 mL of 0.01 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS: the color of the solution is a blue-pur-
ple.
*(12) Arsenic <1.11> — Prepare the test solution with 1.0
g of Sodium Chloride according to Method 1, and perform
the test (not more than 2 ppm).»
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Assay Weigh accurately about 50 mg of Sodium Chloride,
dissolve in 50 mL of water, and titrate <2.50> with 0.1 mol/L
silver nitrate VS (potentiometric titration).
Each mL of 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
♦Containers and storage Containers — Tight containers. ♦
10% Sodium Chloride Injection
io% i&it-t h U^Aaff*
10% Sodium Chloride Injection is an aqueous solu-
tion for injection.
It contains not less than 9.5 w/v% and not more
than 10.5 w/v%> of sodium chloride (NaCl: 58.44).
Method of preparation
Sodium Chloride 100 g
Distilled Water for Injection a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
Description 10% Sodium Chloride Injection is a clear,
colorless liquid. It has a saline taste.
It is neutral.
Identification 10% Sodium Chloride Injection responds to
the Qualitative Tests <1.09> for sodium salt and for chloride.
Bactetial endotoxins <4.01> Less than 3.6 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Pipet 10 mL of 10% Sodium Chloride Injection, and
add water to make exactly 100 mL. Pipet 20 mL of this solu-
tion, add 30 mL of water, and titrate <2.50>, with vigorous
shaking, with 0.1 mol/L silver nitrate VS (indicator: 3 drops
of fluorescein sodium TS).
Each mL 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Isotonic Sodium Chloride Solution
0.9% Sodium Chloride Injection
Isotonic Salt Solution
Isotonic Sodium Chloride Injection
Isotonic Sodium Chloride Solution is an aqueous so-
lution for injection.
It contains not less than 0.85 w/v% and not more
than 0.95 w/v% of sodium chloride (NaCl: 58.44).
Method of preparation
Sodium Chloride
Water for Injection
a sufficient quantity
To make
1000 mL
Prepare as directed under Injections, with the above in-
gredients.
No preservative is added.
Description Isotonic Sodium Chloride Solution is a clear,
colorless liquid. It has a slightly saline taste.
Identification Isotonic Sodium Chloride Solution responds
to the Qualitative Tests <1.09> for sodium salt and for chlo-
ride.
pH <2.54> 4.5-8.0
Purity (1) Heavy metals <1.07> — Concentrate 100 mL of
Isotonic Sodium Chloride Solution to about 40 mL on a
water bath, and add 2 mL of dilute acetic acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution and 2 mL of dilute acetic acid, and add
water to make 50 mL (not more than 0.3 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 20 mL
of Isotonic Sodium Chloride Solution, and perform the test
(not more than 0.1 ppm).
Bacterial endotoxins <4.01> Less than 0.50 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly 20 mL of Isotonic Sodium Chloride
Solution, add 30 mL of water, and titrate <2.50> with 0.1
mol/L silver nitrate VS with vigorous shaking (indicator: 3
drops of fluorescein sodium TS).
Each mL of 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
JPXV
Official Monographs / Sodium Citrate Injection for Transfusion 1099
Sodium Chromate ( 51 Cr) Injection
^PASth^A ( 51 Cr) aW*
Sodium Chromate ( 51 Cr) Injection is an aqueous so-
lution for injection containing chromium-51 ( 51 Cr) in
the form of sodium chromate.
It conforms to the requirements of Sodium Chro-
mate ( 51 Cr) Injection in the Minimum Requirements
for Radiopharmaceuticals.
Test for Extractable Volume of Parentaral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Sodium Chromate ( 51 Cr) Injection is a clear,
light yellow liquid. It is odorless or has an odor of the preser-
vatives.
Sodium Citrate Hydrate
HO COjNa
NaOjC^X^-COjNa '2H 2
C 6 H 5 Na 3 7 .2H 2 0: 294.10
Trisodium 2-hydroxypropane-l,2,3-tricarboxylate dihydrate
[6132-04-3]
Sodium Citrate Hydrate, when dried, contains not
less than 99.0% and not more than 101.0% of sodium
citrate (C 6 H 5 Na 3 7 : 258.07).
Description Sodium Citrate Hydrate occurs as colorless
crystals, or a white, crystalline powder. It is odorless, and has
a cooling, saline taste.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
Identification A solution of Sodium Citrate Hydrate (1 in
20) responds to the Qualitative Tests <1.09> for citrate and for
sodium salt.
pH <2.54> Dissolve 1.0 g of Sodium Citrate Hydrate in 20
mL of water: the pH of this solution is between 7.5 and 8.5.
Purity (1) Clarity and color of solution — A solution of
1.0 g of Sodium Citrate Hydrate in 10 mL of water is clear
and colorless.
(2) Chloride <1.03>— Take 0.6 g of Sodium Citrate Hy-
drate, and perform the test. Prepare the control solution with
0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.015%).
(3) Sulfate <1.14>— To 0.5 g of Sodium Citrate Hydrate
add water to make 40 mL, then add 3.0 mL of dilute
hydrochloric acid and water to make 50 mL, and perform the
test. Prepare the control solution with 0.50 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 2.5 g of Sodium
Citrate Hydrate according to Method 2, and perform the test.
Prepare the control solution with 2.5 mL of Standard Lead
Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sodium Citrate Hydrate according to Method 1, and per-
form the test (not more than 2 ppm).
(6) Tartrate — To a solution of 1.0 g of Sodium Citrate
Hydrate in 2 mL of water add 1 mL of potassium acetate TS
and 1 mL of acetic acid (31): no crystalline precipitate is
formed after the sides of the tube have been rubbed with a g-
lass rod.
(7) Oxalate — Dissolve 1 .0 g of Sodium Citrate Hydrate in
a mixture of 1 mL of water and 3 mL of dilute hydrochloric
acid, add 4 mL of ethanol (95) and 0.2 mL of calcium chlo-
ride TS, and allow to stand for 1 hour: the solution is clear.
(8) Readily carbonizable substances <1.15> — Take 0.5 g
of Sodium Citrate Hydrate, and perform the test by heating
at 90°C for 1 hour: the solution has no more color than
Matching Fluid K.
Loss on drying <2.4I> 10.0 - 13.0% (1 g, 180°C, 2 hours).
Assay Weigh accurately about 0.2 g of Sodium Citrate Hy-
drate, previously dried, add 30 mL of acetic acid for nona-
queous titration, warm to dissolve, and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 8.602 mg of C 6 H 5 Na 3 7
Containers and storage Containers — Tight containers.
Sodium Citrate Injection for
Transfusion
?i>igi-h u^Aaw*
Sodium Citrate Injection for Transfusion is an aque-
ous solution for injection.
It contains not less than 9.5 w/v% and not more
than 10.5 w / v % of sodium citrate hydrate
(C 6 H 5 Na 3 7 .2H 2 0: 294.10).
Method of preparation
Sodium Citrate Hydrate
Water for Injection
100 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
No preservatives may be added.
Description Sodium Citrate Injection for Transfusion is a
clear, colorless liquid.
Identification Sodium Citrate Injection for Transfusion
responds to the Qualitative Tests <1.09> for sodium salt and
for citrate.
pH <2.54> 7.0-8.5
Bacterial endotoxins <4.01> Less than 5.6 EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Pipet 5 mL of Sodium Citrate Injection for Transfu-
1 100 Sodium Cromoglicate / Official Monographs
JP XV
sion, and add water to make exactly 25 mL. Evaporate 10 mL
of this solution, exactly measured, on a water bath to dry-
ness, dry the residue at 180°C for 2 hours, and dissolve in 30
mL of acetic acid (100) by warming. Cool, titrate <2.50> with
0.1 mol/L perchloric acid VS (indicator: 3 drops of crystal
violet TS). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 9.803 mg of C 6 H 5 Na 3 7 .2H 2
Containers and storage Containers — Hermetic containers.
Diagnostic Sodium Citrate Solution
W)fffl^l>»-^r»U^A»
Diagnostic Sodium Citrate Solution contains not less
than 3.3 w/v% and not more than 4.3 w/v% of sodi-
um citrate hydrate (C 6 H5Na 3 7 .2H 2 0: 294.10).
The requirements as described for aqueous injec-
tions under Injections are applicable.
Method of preparation
Sodium Citrate Hydrate
Water for Injection
38 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
gredients.
No preservative may be added.
Description Diagnostic Sodium Citrate Solution is a clear,
colorless liquid.
Identification Diagnostic Sodium Citrate Solution responds
to the Qualitative Tests <1.09> for sodium salt and for citrate.
pH <2.54> 7.0 - 8.5
Assay Pipet 5 mL of Diagnostic Sodium Citrate Solution,
evaporate on a water bath to dryness, dry the residue at
180°C for 2 hours, and dissolve in 30 mL of acetic acid (100)
by warming. Cool, and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (indicator: 3 drops of crystal violet TS). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 9.803 mg of C 6 H 5 Na 3 7 .2H 2
Containers and storage Containers — Hermetic containers.
Sodium Cromoglicate
1
.O. ,CO ? Na
OO 5 °
C 2 3H 14 Na 2 On: 512.33
Disodium 5,5' -(2-hydroxytrimethylenedioxy)bis(4-oxo-4//-
1 -benzopyran-2-carboxylate) [15826-3 7-6]
Sodium Cromoglicate contains not less than 98.0%
of C23H 14 Na 2 11 , calculated on the dried basis.
Description Sodium Cromoglicate occurs as a white, crys-
talline powder. It is odorless and tasteless at first, and later
develops a slightly bitter taste.
It is freely soluble in water, sparingly soluble in propylene
glycol, very slightly soluble in ethanol (95), and practically in-
soluble in 2-propanol and in diethyl ether.
It is hygroscopic.
It gradually acquires a yellow color by light.
Identification (1) Dissolve 0.1 g of Sodium Cromoglicate
in 2 mL of water, add 2 mL of sodium hydroxide TS, and
boil for 1 minute: a yellow color is produced. After cooling,
add 0.5 mL of concentrated diazobenzene sulfonic acid TS: a
dark red color is produced.
(2) Determine the absorption spectrum of a solution of
Sodium Cromoglicate in phosphate buffer solution, pH 7.4 (1
in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Sodium Cromoglicate responds to the Qualitative
Tests <1.09> for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 0.50 g
of Sodium Cromoglicate in 10 mL of water: the solution is
clear and colorless to pale yellow.
(2) Acidity or alkalinity — Dissolve 2.0 g of Sodium
Cromoglicate in 40 mL of freshly boiled and cooled water,
add 6 drops of bromothymol blue TS, and use this solution
as the sample solution. To 20 mL of the sample solution add
0.25 mL of 0.1 mol/L sodium hydroxide VS: a blue color is
produced. To another 20 mL of the sample solution add 0.25
mL of 0.1 mol/L hydrochloric acid VS: a yellow color is
produced.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Sodium
Cromoglicate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Oxalate — Dissolve 0.25 g of Sodium Cromoglicate in
water to make exactly 50 mL, and use this solution as the
sample solution. Separately, dissolve 0.049 g of oxalic acid
dihydrate, exactly weighed, in water to make exactly 100 mL.
Pipet 5 mL of this solution, add water to make exactly 100
mL, and use this solution as the standard solution. Pipet 20
mL each of the sample solution and standard solution, add
exactly 5 mL of iron salicylate TS to each solution, and add
water to make 50 mL. Determine the absorbances of these so-
lutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> using water as the blank: the absorbance of the
sample solution at 480 nm is not smaller than that of the stan-
dard solution.
(5) Related substances — Dissolve 0.20 g of Sodium
Cromoglicate in 10 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 10 mL, pipet 1 mL of this solution,
add water to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL each of the sample solution and standard solution on a
JPXV
Official Monographs / Sodium Hydroxide 1101
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
methanol, chloroform and acetic acid (100) (9:9:2) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): spots other than
the principal spot from the sample solution is not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 10.0% (1 g, in vacu-
um, 105°C, 4 hours).
Assay Weigh accurately about 0.18 g of Sodium Cromogli-
cate, and dissolve in a mixture of 25 mL of propylene glycol
and 5 mL of 2-propanol by warming. After cooling, add 30
mL of 1,4-dioxane, and titrate <2.50> with 0.1 mol/L per-
chloric acid-dioxane VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid-dioxane VS
= 25.62 mg of C 2 3H 14 Na 2 O u
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Sodium Fusidate
7->v>Shr-r- U7A
H^C^ jCHj
CH :
H 3 CH
C 3 iH 47 Na0 6 : 538.69
Monosodium (17Z)-ewM6a-acetoxy-3/?,ll/?-dihydroxy-
40, 80, 1 4a-trimethyl- 1 8-nor-5/?, 1 Oa-cholesta-1 7(20),24-
dien-21-oate [751-94-0]
Sodium Fusidate is the sodium salt of a substance
having antibacterial activity produced by the growth of
Fusidium coccineum.
It contains not less than 935 fig (potency) and not
more than 969 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Sodium Fusidate is
expressed as mass (potency) of fusidic acid (C 3 iH 48 6 :
516.71).
Description Sodium Fusidate occurs as white, crystals of
crystalline powder.
It is freely soluble in water, in methanol and in ethanol
(99.5).
Identification (1) Determine the infrared absorption spec-
tra of Sodium Fusidate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(2) Sodium Fusidate responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Purity Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Fusidate according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
Water <2.48> Not more than 2.0% (1 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Staphylococcus aureus ATCC 6538 P
(ii) Culture medium — Use the medium ii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer.
(iii) Standard solutions — Weigh accurately an amount of
Diethanolamine Fusidate Reference Standard, equivalent to
about 20 mg (potency), dissolve in 2 mL of ethanol (95), add
water to make exactly 20 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
a temperature not exceeding 5°C and use within 7 days. Take
exactly a suitable amount of the standard stock solution be-
fore use, add phosphate buffer solution, pH 6.0 to make solu-
tions so that each mL contains 4 fig (potency) and 1 fig
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Sodium Fusidate, equivalent to about 20 mg (potency), and
dissolve in water to make exactly 20 mL. Take exactly a suita-
ble amount of this solution, add phosphate buffer solution,
pH 6.0 to make solutions so that each mL contains 4 fig
(potency) and 1 fig (potency), and use these solutions as the
high concentration sample solution and low concentration
sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and at a temperature 2 to 8°C.
Sodium Hydroxide
7)Mit-f r- U 7 A
NaOH: 40.00
Sodium Hydroxide contains not less than 95.0% of
NaOH.
Description Sodium Hydroxide occurs as white fused mass-
es, in small pellets, in flakes, in sticks, and in other forms. It
is hard and brittle, and shows a crystalline fracture.
It is freely soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
It rapidly absorbs carbon dioxide in air.
It deliquesces in moist air.
Identification (1) A solution of Sodium Hydroxide (1 in
500) is alkaline.
(2) A solution of Sodium Hydroxide (1 in 25) responds to
the Qualitative Tests <1.09> for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
1 102 Sodium Iodide / Official Monographs
JP XV
Sodium Hydroxide in 20 mL of water: the solution is clear
and colorless.
(2) Chloride < 1.03 > —Dissolve 2.0 g of Sodium
Hydroxide in water, and add water to make 100 mL. To 25
mL of the solution add 10 mL of dilute nitric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution with 0.7 mL of
0.01 mol/L hydrochloric acid VS (not more than 0.050%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Sodium
Hydroxide in 5 mL of water, add 1 1 mL of dilute hydrochlor-
ic acid, and evaporate on a water bath to dryness. Dissolve
the residue in 35 mL of water, add 2 mL of dilute acetic acid
and 1 drop of ammonia TS, add water to make 50 mL, and
perform the test using this solution as the test solution.
Evaporate 11 mL of dilute hydrochloric acid on a water bath
to dryness, dissolve the residue in 2 mL of dilute acetic acid
and 3.0 mL of Standard Lead Solution, add water to make 50
mL, and use this solution as the control solution (not more
than 30 ppm).
(4) Potassium — Dissolve 0.10 g of Sodium Hydroxide in
water and dilute with water to make 40 mL. Add 1.0 mL of
dilute acetic acid to 4.0 mL of this solution, and shake. Add
5.0 mL of a solution of sodium tetraphenylboron (1 in 30),
shake immediately, and allow to stand for 10 minutes: the so-
lution has no more turbidity than the following control solu-
tion.
Control solution: Dissolve 9.5 mg of potassium chloride in
water, and dilute with water to make 1000 mL. Add 1.0 mL
of dilute acetic acid to 4.0 mL of this solution, shake, and
proceed as directed above.
(5) Sodium carbonate — The amount of sodium car-
bonate (Na 2 C0 3 : 105.99) is not more than 2.0%, when calcu-
lated by the following equation using B (mL) which is ob-
tained in the Assay.
Amount (mg) of sodium carbonate = 105.99x5
(6) Mercury — Dissolve 2.0 g of Sodium Hydroxide in 1
mL of a solution of potassium permanganate (3 in 50) and 30
mL of water, neutralize gradually with purified hydrochloric
acid, and add 5 mL of diluted sulfuric acid (1 in 2). To this
solution add a solution of hydroxylammonium chloride (1 in
5) until the precipitate of manganese dioxide disappears, add
water to make exactly 100 mL, and use this solution as the
sample solution. Perform the tests according to the Atomic
Absorption Spectrophotometry <2.23> (Cold vapor type) with
the sample solution. Place the sample solution in the sample
bottle of an atomic absorption spectrophotometer, add 10
mL of tin (II) chloride-sulfuric acid TS, connect the bottle
immediately to the atomic absorption spectrophotometer,
and circulate air. Read the absorbance A T of the sample solu-
tion when the indication of the recorder rises rapidly and
becomes constant at the wavelength of 253.7 nm. On the
other hand, to 2.0 mL of Standard Mercury Solution add 1
mL of a solution of potassium permanganate (3 in 50), 30 mL
of water and a volume of purified hydrochloric acid equal to
that used in the preparation of the sample solution, and read
the absorbance A s of the solution obtained by the same
procedure as used for the sample solution: A T is smaller than
A s .
Assay Weigh accurately about 1.5 g of Sodium Hydroxide,
and dissolve in 40 mL of freshly boiled and cooled water.
Cool the solution to 15°C, add 2 drops of phenolphthalein
TS, and titrate with 0.5 mol/L sulfuric acid VS until the red
color of the solution disappears. Record the amount, A
(mL), of 0.5 mol/L sulfuric acid VS consumed. Then add 2
drops of methyl orange TS to the solution, and further titrate
<2.50> with 0.5 mol/L sulfuric acid VS until the solution
shows a persistent light red color. Record the amount, B
(mL), of 0.5 mol/L sulfuric acid VS consumed. Calculate the
amount of NaOH from the difference, A (mL) — B (mL).
Each mL of 0.5 mol/L sulfuric acid VS
= 40.00 mg of NaOH
Containers and storage Containers — Tight containers.
Sodium Iodide
Nal: 149.89
Sodium Iodide, when dried, contains not less than
99.0% of Nal.
Description Sodium Iodide occurs as colorless crystals or a
white, crystalline powder. It is odorless.
Sodium Iodide is very soluble in water, and freely soluble
in glycerin and in ethanol (95).
It deliquesces in moist air.
Identification A solution of Sodium Iodide (1 in 20)
responds to the Qualitative Tests <1.09> for sodium salt and
for iodide.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Iodide in 2 mL of water: the solution is clear and
colorless.
(2) Alkalinity — Dissolve 1 .0 g of Sodium Iodide in 10 mL
of freshly boiled and cooled water, and add 1.0 mL of 0.005
mol/L sulfuric acid VS and 1 drop of phenolphthalein TS: no
color is produced.
(3) Chloride, bromide and thiosulfate — Dissolve 0.20 g
of Sodium Iodide in 5 mL of ammonia TS, add 15.0 mL of
0.1 mol/L silver nitrate VS, shake for a few minutes, and
filter. To 10 mL of the filtrate add 15 mL of dilute nitric acid:
no brown color appears. The solution has no more turbidity
than the following control solution.
Control solution: To 0.30 mL of 0.01 mol/L hydrochloric
acid VS add 2.5 mL of ammonia TS, 7.5 mL of 0.1 mol/L
silver nitrate VS and 15 mL of dilute nitric acid.
(4) Nitrate, nitrite and ammonium — Place 1.0 g of Sodi-
um Iodide in a 40-mL test tube, and add 5 mL of water, 5 mL
of sodium hydroxide TS and 0.2 g of aluminum wire. Insert a
pledget of absorbent cotton in the mouth of the test tube, and
place a piece of moistened red litmus paper on the cotton.
Heat the test tube on a water bath for 15 minutes: the evolved
gas does not turn moistened red litmus paper to blue.
(5) Cyanide — Dissolve 0.5 g of Sodium Iodide in 10 mL
of water. To 5 mL of this solution add 1 drop of iron (II) sul-
fate TS and 2 mL of sodium hydroxide TS, warm, and add 4
mL of hydrochloric acid: no green color develops.
(6) Iodate — Dissolve 0.5 g of Sodium Iodide in 10 mL of
freshly boiled and cooled water, and add 2 drops of dilute
sulfuric acid and 1 drop of starch TS: no blue color develops
JPXV
Official Monographs / Sodium Iotalamate Injection 1103
immediately.
(7) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Iodide according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than lOppm).
(8) Barium — Dissolve 0.5 g of Sodium Iodide in 10 mL of
water, add 1 mL of dilute sulfuric acid, and allow to stand
for 5 minutes: no turbidity is produced.
(9) Potassium — Dissolve 1.0 g of Sodium Iodide in
water, and add water to make 100 mL. To 4.0 mL of this
solution add 1.0 mL of dilute acetic acid, shake, add 5.0 mL
of a solution of sodium tetraphenylboron (1 in 30), immedi-
ately shake, and allow to stand for 10 minutes: the solution
has no more turbidity than the following control solution.
Control solution: Dissolve 9.5 mg of potassium chloride in
water, and add water to make 1000 mL. To 4.0 mL of this so-
lution add 1.0 mL of dilute acetic acid, shake, and then pro-
ceed as directed above.
(10) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Sodium Iodide according to Method 1, and perform the
test (not more than 5 ppm).
Loss on drying <2.41>
2 hours).
Not more than 5.0% (2 g, 120°C,
Assay Weigh accurately about 0.4 g of Sodium Iodide,
previously dried, in an iodine flask, dissolve in 10 mL of
water, add 35 mL of hydrochloric acid and 5 mL of chlo-
roform, and titrate <2.50> with 0.05 mol/L potassium iodate
VS while shaking vigorously until the red-purple color of the
chloroform layer disappears. The end point is attained when
the red-purple color does not reappear in the chloroform lay-
er within 5 minutes after the layer has been decolorized.
Each mL of 0.05 mol/L potassium iodate VS
= 14.99 mg of Nal
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Sodium Iodide ( 131 I) Solution
Sodium Iodide ( 131 I) Solution contains iodine-131
( 131 I) in the form of sodium iodide.
It conforms to the requirements of Sodium Iodide
( 131 I) Solution in the Minimum Requirements for
Radiopharmaceuticals .
Description Sodium Iodide ( 131 I) Solution is a clear, color-
less liquid. It is odorless, or has an odor due to the preserva-
tives or stabilizers.
Sodium Iodohippurate ( 131 I)
Injection
BWkt-fJl&T r- U ^A ( 131 I) aW*
Sodium Iodohippurate ( 131 I) Injection is an aqueous
solution for injection containing iodine-131 ( 131 I) in the
form of sodium o-iodohippurate.
It conforms to the requirements of Sodium Iodohip-
purate ( 131 I) Injection in the Minimum Requirements
for Radiopharmaceuticals.
Test for Extractable Volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Sodium Iodohippurate ( 131 I) Injection is a
clear, colorless liquid. It is odorless or has an odor of the
preservatives or stabilizers.
Sodium Iodide ( 123 I) Capsules
Sodium Iotalamate Injection
Sodium Iodide ( 123 I) Capsules contain iodine-123 in
the form of sodium iodide.
It conforms to the requirements of Sodium Iodide
( 123 I) Capsules in the Minimum Requirements for
Radiopharmaceuticals .
Sodium Iodide ( 131 I) Capsules
B^iti-\- U^7A( 131 i)^)^irJL.
Sodium Iodide ( 131 I) Capsules are prepared by dis-
pensing Sodium Iodide ( 131 I) Solution into capsules and
drying it.
Sodium Iodide ( 131 I) Capsules conform to the re-
quirements of Sodium Iodide ( 131 I) Capsules in the
Minimum Requirements for Radiopharmaceuticals.
Sodium Iotalamate Injection is an aqueous solution
for injection.
It contains not less than 95% and not more than
105% of the labeled amount of iotalamic acid
(C u H 9 l3N 2 4 : 613.91).
Method of preparation
(1)
Iotalamic Acid
Sodium Hydroxide
Water for Injection
(2)
Iotalamic Acid
Sodium Hydroxide
Water for Injection
645 g
42 g
a sufficient quantity
To make 1000 mL
772.5 g
50.5 g
a sufficient quantity
To make 1000 mL
Prepare as directed under Injections, with the above in-
1 104 Sodium Lauryl Sulfate / Official Monographs
JP XV
gredients (1) or (2).
Description Sodium Iotalamate Injection is a clear, color-
less or pale yellow, slightly viscous liquid.
It is gradually colored by light.
Identification (1) To a volume of Sodium Iotalamate In-
jection, equivalent to 1 g of Iotalamic Acid according to the
labeled amount, add 25 mL of water, and add 2.5 mL of
dilute hydrochloric acid with thorough stirring: a white
precipitate is produced. Filter the precipitate by suction
through a glass filter (G4), wash the precipitate with two
10-mL portions of water, and dry at 105°C for 1 hour. Pro-
ceed with the precipitate as directed in the Identification (2)
under Iotalamic Acid.
(2) Sodium Iotalamate Injection responds to the Qualita-
tive Tests <1.09> (1) for sodium salt.
pH <2.54> 6.5 - 7.7
Purity (1) Primary aromatic amines — To a volume of So-
dium Iotalamate Injection, equivalent to 0.20 g of Iotalamic
Acid according to the labeled amount, add 15 mL of water,
shake, add 4 mL of a solution of sodium nitrite (1 in 100) un-
der ice-cooling, and proceed as directed in the Purity (2) un-
der Iotalamic Acid: the absorbance is not more than 0.17.
(2) Iodine and iodide — To a volume of Sodium Iotala-
mate Injection, equivalent to 1.5 g of Iotalamic Acid accord-
ing to the labeled amount, add 20 mL of water and 5 mL of
dilute sulfuric acid, shake well, and filter the precipitate by
suction through a glass filter (G4). To the filtrate add 5 mL of
toluene, and shake vigorously: the toluene layer is colorless.
Then add 2 mL of a solution of sodium nitrite (1 in 100), and
shake vigorously: the toluene layer has no more color than
the following control solution.
Control solution: Dissolve 0.25 g of potassium iodide in
water to make 1000 mL. To 2.0 mL of this solution add 20
mL of water, 5 mL of dilute sulfuric acid, 5 mL of toluene
and 2 mL of a solution of sodium nitrite (1 in 100), and shake
vigorously.
Bacterial endotoxins <4.01> Less than 3.4EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Pipet a volume of Sodium Iotalamate Injection,
equivalent to about 4 g of iotalamic acid (CHH9LN204), add
water to make exactly 200 mL. Pipet 2 mL of this solution,
add water to make exactly 200 mL. To exactly 5 mL of this
solution add exactly 5 mL of the internal standard solution,
add the mobile phase to make 100 mL, and use this solution
as the sample solution. Separately, weigh accurately about
0.4 g of iotalamic acid for assay, previously dried at 105°C
for 4 hours, dissolve in 100 mL of water and 1 mL of sodium
hydroxide TS, and add water to make exactly 200 mL. Pipet
5 mL of this solution, add water to make exactly 50 mL. To
exactly 5 mL of this solution add exactly 5 mL of the internal
standard solution, add the mobile phase to make 100 mL,
and use this solution as the standard solution. Perform the
test with 10 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of iotalamic acid to that of the
internal standard.
Amount (mg) of iotalamic acid (C11H9I3N2O4)
= W s x(Q T /Q s )
W s : Amount (mg) of iotalamic acid for assay
Internal standard solution — A solution of L-tryptophan in
the mobile phase (3 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: To 3.9 g of phosphoric acid and 2.8 mL of
triethylamine add water to make 2000 mL. To this solution
add 100 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
iotalamic acid is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, iotalamic acid and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 5.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of iotalamic acid to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant.
Sodium Lauryl Sulfate
Sodium Lauryl Sulfate is a mixture of sodium alkyl
sulfate consisting chiefly of sodium lauryl sulfate
(C 12 H 2 5Na0 4 S: 288.38).
Description Sodium Lauryl Sulfate occurs as white to light
yellow crystals or powder. It has a slightly characteristic
odor.
It is sparingly soluble in methanol and in ethanol (95).
A solution of Sodium Lauryl Sulfate (1 in 10) is a clear or
an opalescent solution, which foams on agitation.
Identification (1) To 0.2 g of the residue obtained in Total
alcohol content add 4 mL of bromine-cyclohexane TS with
vigorous shaking, add 0.3 g of ./V-bromosuccinimide, and
heat in a water bath at 80°C for 5 minutes: a red color de-
velops.
(2) A solution of Sodium Lauryl Sulfate (1 in 10)
responds to the Qualitative Tests <1.09> (1) for sodium salt.
(3) To a solution of Sodium Lauryl Sulfate (1 in 10) add
dilute hydrochloric acid to make acid, boil gently, and cool:
the solution responds to the Qualitative Tests <1.09> for sul-
fate.
Purity (1) Alkalinity — Dissolve 1.0 g of Sodium Lauryl
Sulfate in 100 mL of water, add 2 drops of phenol red TS and
JPXV
Official Monographs / Sodium Picosulfate Hydrate 1105
0.60 mL of 0.1 mol/L hydrochloric acid VS: the solution
remains yellow.
(2) Sodium chloride — Dissolve about 5 g of Sodium
Lauryl Sulfate, accurately weighed, in 50 mL of water, neu-
tralize the solution with dilute nitric acid, if necessary, add
exactly 5 mL of 0.1 mol/L sodium chloride TS, and titrate
<2.50> with 0.1 mol/L silver nitrate VS (indicator: 2 drops of
fluorescein sodium TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
The combined content of sodium chloride (NaCl: 58.44)
and sodium sulfate (Na 2 S0 4 : 142.04) obtained in the next
paragraph (3) is not more than 8.0%.
(3) Sodium sulfate — Dissolve about 1 g of Sodium
Lauryl Sulfate, accurately weighed, in 10 mL of water, add
100 mL of ethanol (95), and heat at a temperature just below
the boiling point for 2 hours. Filter through a glass filter (G4)
while hot, and wash with 100 mL of boiling ethanol (95). Dis-
solve the precipitate by washing with 150 mL of water, col-
lecting the washings in a beaker. Add 10 mL of hydrochloric
acid, heat to boiling, add 25 mL of barium chloride TS, and
allow to stand overnight. Collect the precipitate, and wash
with water until the last washing shows no opalescence with
silver nitrate TS. Dry the precipitate, ignite to a constant
mass between 500°C and 600°C by raising the temperature
gradually, and weigh as barium sulfate (BaS0 4 : 233.39).
Amount (mg) of sodium sulfate (Na 2 S0 4 )
= amount (mg) of barium sulfate (BaS0 4 ) x 0.6086
(4) Unsulfated alcohols — Dissolve about 10 g of Sodium
Lauryl Sulfate, accurately weighed, in 100 mL of water, add
100 mL of ethanol (95), and transfer to a separator. Extract
the solution with three 50-mL portions of petroleum benzin.
If an emulsion forms, sodium chloride may be added to pro-
mote separation of the two layers. Combine the petroleum
benzin extracts and wash with three 50-mL portions of water.
Evaporate the petroleum benzin on a water bath, and dry the
residue at 105°C for 30 minutes. The mass of the dried
residue is not more than 4.0% of the mass of the Sodium
Lauryl Sulfate taken.
Water <2.48> Not more than 5.0% (0.5 g, direct titration).
Total alcohol content Dissolve about 5 g of Sodium Lauryl
Sulfate, accurately weighed, in 150 mL of water and 50 mL
of hydrochloric acid, and boil under a reflux condenser for 4
hours. Cool, extract with two 75-mL portions of diethyl
ether, and evaporate the combined diethyl ether extracts on a
water bath. Dry the residue at 105°C for 30 minutes. The
mass of the residue is not less than 59.0%.
Containers and storage Containers — Well-closed contain-
ers.
Sodium Pertechnetate ( 99 ™Tc)
Injection
'17-
vittAWtf- r- y ""j a ( 99m Tc) aw*
ous solution for injection containing technetium- - -/??
( 99m Tc) in the form of sodium pertechnetate.
It conforms to the requirements of Sodium Per-
technetate ( 99m Tc) Injection in the Minimum Require-
ments for Radiopharmaceuticals.
Test for Extractable Volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Sodium Pertechnetate ( 99m Tc) Injection is a
clear, colorless liquid.
Sodium Picosulfate Hydrate
tf=l7JU7?-- r-7-r- y^ATkJfPftl
•HiO
C l8 H l3 NNa 2 8 S 2 .H 2 0: 499.42
Disodium 4,4'-(pyridin-2-ylmethylene)bis(phenyl sulfate)
monohydrate [10040-45-6, anhydride]
Sodium Picosulfate Hydrate contains not less than
98.5%> of sodium picosulfate (C 18 H 13 NNa 2 8 S 2 :
481.41), calculated on the anhydrous basis.
Description Sodium Picosulfate Hydrate occurs as a white,
crystalline powder. It is odorless and tasteless.
It is very soluble in water, soluble in methanol, slightly
soluble in ethanol (99.5), and practically insoluble in diethyl
ether.
It is gradually colored by light.
The pH of a solution of Sodium Picosulfate Hydrate (1 in
20) is between 7.4 and 9.4.
Identification (1) Mix 5 mg of Sodium Picosulfate Hy-
drate with 0.01 g of l-chloro-2,4-dinitrobenzene, and melt by
gentle heating for 5 to 6 seconds. After cooling, add 4 mL of
potassium hydroxide-ethanol TS: an orange-red color de-
velops.
(2) To 0.2 g of Sodium Picosulfate Hydrate add 5 mL of
dilute hydrochloric acid, boil for 5 minutes, cool, and add 1
mL of barium chloride TS: a white precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Sodium Picosulfate Hydrate (1 in 25,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(4) Determine the infrared absorption spectrum of Sodi-
um Picosulfate Hydrate, previously dried at 105° C in vacu-
um for 4 hours, as directed in the potassium bromide disk
method under the Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(5) A solution of Sodium Picosulfate Hydrate (1 in 10)
responds to the Qualitative Tests <1.09> for sodium salt.
Sodium Pertechnetate ( 99m Tc) Injection is an aque- Absorbance <2.24> E\* (263 nm): 120 - 130 (calculated on
1 106 Sodium Polystyrene Sulfonate / Official Monographs
JP XV
the anhydrous basis, 4 mg, water, 100 mL).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Picosulfate Hydrate in 10 mL of water: the solution
is clear and colorless to pale yellow.
(2) Chloride <1.03>— Perform the test with 0.5 g of Sodi-
um Picosulfate Hydrate. Prepare the control solution with
0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.028%).
(3) Sulfate <1.14>— Perform the test with 0.40 g of Sodi-
um Picosulfate Hydrate. Prepare the control solution with
0.35 mL of 0.005 mol/L sulfuric acid VS (not more than
0.042%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Picosulfate Hydrate according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Sodium Picosulfate Hydrate according to Method 3, and
perform the test (not more than 1 ppm).
(6) Related substances — Dissolve 0.25 g of Sodium
Picosulfate Hydrate in 5 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add methanol to make exactly 500 mL, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 fXL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of 1-butanol, water and acetic acid (100) (74:20:19) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Water <2.48> 3.0 - 4.5% (0.5 g, direct titration).
Assay Weigh accurately about 0.4 g of Sodium Picosulfate
Hydrate, dissolve in 50 mL of methanol, add 7 mL of acetic
acid (100), and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 48.14 mg of C 18 H 13 NNa 2 8 S 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Sodium Polystyrene Sulfonate
Sodium Polystyrene Sulfonate is a cation exchange
resin prepared as the sodium form of the sulfonated
styrene divinylbenzene copolymer.
It contains not less than 9.4% and not more than
11.0% of sodium (Na: 22.99), calculated on the anhy-
drous basis.
Each g of Sodium Polystyrene Sulfonate, calculated
on the anhydrous basis, exchanges with not less than
O.llOg and not more than 0.135 g of potassium (K:
39.10).
Description Sodium Polystyrene Sulfonate occurs as a yel-
low-brown powder. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95), in ace-
tone and in diethyl ether.
Identification (1) Determine the infrared absorption spec-
trum of Sodium Polystyrene Sulfonate as directed in the
potassium bromide disk method under the Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(2) To 1 g of Sodium Polystyrene Sulfonate add 10 mL of
dilute hydrochloric acid, stir, and filter. Add ammonia TS to
the filtrate to neutralize: the solution responds to the Qualita-
tive Tests <1.09> for sodium salt.
Purity (1) Ammonium — Place 1.0 g of Sodium Polysty-
rene Sulfonate in a flask, add 5 mL of sodium hydroxide TS,
cover the flask with a watch glass having a moistened strip of
red litmus paper on the underside, and boil for 15 minutes:
the gas evolved does not change the red litmus paper to blue.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Polystyrene Sulfonate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 2.0 g
of Sodium Polystyrene Sulfonate according to Method 3, and
perform the test (not more than 1 ppm).
(4) Styrene — To 10.0 g of Sodium Polystyrene Sulfonate
add 10 mL of acetone, shake for 30 minutes, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 10 mg of styrene in acetone to make exactly 100 mL.
Pipet 1 mL of this solution, add acetone to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with exactly 20 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine peak areas, A T and A s , of styrene in each solu-
tion: A T is not larger than A s .
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
styrene is about 8 minutes.
System suitability —
System performance: Dissolve 20 mg each of styrene and
butyl parahydroxybenzoate in 100 mL of acetone. To 5 mL
of this solution add acetone to make 100 mL. When the
procedure is run with 20 /xh of this solution under the above
operating conditions, butyl parahydroxybenzoate and sty-
rene are eluted in this order with the resolution between these
peaks being not less than 5.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
styrene is not more than 2.0%.
Water <2.48> Not more than 10.0% (0.2 g, direct titration).
JPXV
Official Monographs / Sodium Prasterone Sulfate Hydrate 1107
Assay (1) Sodium — Weigh accurately about 1 g of Sodi-
um Polystyrene Sulfonate, calculated on the anhydrous ba-
sis, in a glass-stoppered flask, add exactly 50 mL of 3 mol/L
hydrochloric acid TS, shake for 60 minutes, and filter. Dis-
card the first 20 mL of the filtarte, pipet the subsequent 5 mL
of the filtrate, and add water to make exactly 100 mL. Pipet
20 mL of this solution, add water to make exactly 1000 mL,
and use this solution as the sample solution. Separately, pipet
a suitable quantity of Standard Sodium Stock Solution, di-
lute exactly with water so that each ml of the solution con-
tains 1 to 3 n% of sodium (Na: 22.99), and use these solutions
as the standard solution. Perform the test with the sample so-
lution and the standard solution according to the Atomic Ab-
sorption Spectrophotometry <2.23> under the following con-
ditions, and determine the amount of sodium in the sample
solution using the calibration curve obtained from the stan-
dard solutions.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A sodium hollow-cathode lamp
Wavelength: 589.0 nm
(2) Potassium exchange capacity — Weigh accurately
about 1.5 g of Sodium Polystyrene Sulfonate, calculated on
the anhydrous basis, in a glass-stoppered flask, add exactly
100 mL of Standard Potassium Stock Solution, shake for 15
minutes, and filter. Discard the first 20 mL of the filtrate,
pipet the subsequent 10 mL of the filtrate, and add water to
make exactly 100 mL. Pipet 10 mL of this solution, add
water to make exactly 1000 mL, and use this solution as the
sample solution. Separately, pipet a suitable quantity of
Standard Potassium Stock Solution, dilute with water so that
each mL of the solution contains 1 to 5 //g of potassium (K:
39.10), and use the solution as the standard solution. Per-
form the test with these solutions as directed under Atomic
Absorption Spectrophotometry <2.23>, and determine the
amount Y (mg) of potassium in 1000 mL of the sample solu-
tion using the calibration curve obtained from the standard
solution. The quantity of potassium absorbed on each g of
Sodium Polystyrene Sulfonate, calculated on the anhydrous
basis, is calculated from the following equation: it is between
0.110 g and 0.135 g.
Quantity (mg) of potassium (K) absorbed on 1 g of Sodium
Polystyrene Sulfonate, calculated on the anhydrous basis
= (X - \00Y)/W
X: Amount (mg) of potassium in 100 mL of the Standard
Potassium Stock Solution before exchange.
W: Mass (g) of Sodium Polystyrene Sulfonate taken, cal-
culated on the anhydrous basis.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A potassium hollow-cathode lamp
Wavelength: 766.5 nm
Containers and storage Containers — Tight containers.
Sodium Prasterone Sulfate Hydrate
H 3 C -°
HsC
NaOaS-0
«2H 2
Ci 9 H 27 Na0 5 S.2H 2 0: 426.50
Monosodium 17-oxoandrost-5-en-3/?-yl sulfate dihydrate
[1099-87-2, anhydride]
Sodium Prasterone Sulfate Hydrate contains not less
than 98.0% of sodium prasterone sulfate (C 19 H 2 7Na0 5
S: 390.47), calculated on the dried basis.
Description Sodium Prasterone Sulfate Hydrate occurs as
white crystals or crystalline powder. It is odorless.
It is soluble in methanol, sparingly soluble in water and in
ethanol (95), and practically insoluble in acetone and in
diethyl ether.
The pH of a solution of Sodium Prasterone Sulfate Hy-
drate (1 in 200) is between 4.5 and 6.5.
Melting point: about 160°C (with decomposition, after
drying).
Identification (1) Dissolve 0.01 g of Sodium Prasterone
Sulfate Hydrate in 4 mL of ethanol (95), add 2 mL of 1,3-
dinitrobenzene TS and 2 mL of a solution of sodium
hydroxide (1 in 8): a red-purple color develops, and gradually
changes to brown.
(2) To 10 mL of a solution of Sodium Prasterone Sulfate
Hydrate (1 in 200) add 0.5 mL of bromine TS: the color of
bromine TS immediately disappears.
(3) Determine the infrared absorption spectrum of Sodi-
um Prasterone Sulfate Hydrate as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(4) A solution of Sodium Prasterone Sulfate Hydrate (1
in 200) responds to the Qualitative Tests <1.09> for sodium
salt.
Optical rotation <2.49> [a]™: + 10.7 - +12.1° (0.73 g, cal-
culated on the dried basis, methanol, 20 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.25 g
of Sodium Prasterone Sulfate Hydrate in 50 mL of water: the
solution is clear and colorless.
(2) Chloride <1.03>— Dissolve 1.0 g of Sodium Praster-
one Sulfate Hydrate in 20 mL of acetone and 20 mL of water,
and add 6 mL of dilute nitric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution as follows: to 0.30 mL of 0.01 mol/
L hydrochloric acid VS add 20 mL of acetone, 6 mL of dilute
nitric acid and water to make 50 mL (not more than 0.011%).
(3) Sulfate <1.14>— To 1.2 g of Sodium Prasterone Sul-
fate Hydrate add 20 mL of water, shake vigorously for 5
minutes, and filter. To 10 mL of the filtrate add 20 mL of ace-
1 108 Sodium Pyrosulfite / Official Monographs
JP XV
tone, 1 mL of dilute hydrochloric acid and water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution as follows: to 0.40 mL of 0.005
mol/L sulfuric acid VS add 20 mL of acetone, 1 mL of dilute
hydrochloric acid and water to make 50 mL (not more than
0.032%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Sodium
Prasterone Sulfate Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(5) Related substances — Dissolve 0.10 g of Sodium
Prasterone Sulfate Hydrate in 10 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add methanol to make exactly 200 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol and water (75:22:3) to a distance of
about 10 cm, and air-dry the plate. Spray evenly a mixture of
sulfuric acid and ethanol (95) (1:1) on the plate, and heat at
80°C for 5 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> 8.0 - 9.0% (0.5 g, in vacuum, phos-
phorus (V) oxide, 60°C, 3 hours).
Assay Weigh accurately about 0.25 g of Sodium Prasterone
Sulfate Hydrate, dissolve in 30 mL of water. Apply this solu-
tion to a chromatographic column 10 mm in inside diameter,
previously prepared by pouring 5 mL of strongly acidic ion-
exchange resin (H type) for column chromatography, and
elute at the rate of 4 mL per minute. Wash the chromato-
graphic column with 100 mL of water, combine the washings
with above effluent solution, and titrate <2.50> with 0.05
mol/L sodium hydroxide VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.05 mol/L sodium hydroxide VS
= 19.52 mg of C 19 H 27 Na0 5 S
Containers and storage Containers — Tight containers.
Sodium Pyrosulfite
Sodium Metabisulfite
Na 2 S 2 5 : 190.11
Sodium Pyrosulfite contains not less than 95.0% of
Na 2 S 2 O s .
Description Sodium Pyrosulfite occurs as white crystals or
crystalline powder. It has the odor of sulfur dioxide.
It is freely soluble in water, very slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
A solution of Sodium Pyrosulfite (1 in 20) is acid.
It is hygroscopic.
It decomposes slowly on exposure to air.
Identification A solution of Sodium Pyrosulfite (1 in 20)
responds to the Qualitative Tests <1.09> for sodium salt and
for bisulfite.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Pyrosulfite in 10 mL of water: the solution is clear
and colorless.
(2) Thiosulfate — Dissolve 1.0 g of Sodium Pyrosulfite in
15 mL of water, add slowly 5 mL of dilute hydrochloric acid,
shake, and allow to stand for 5 minutes: no turbidity is
produced.
(3) Heavy metals <1.07> — Dissolve 1.0 g of Sodium
Pyrosulfite in 10 mL of water, and evaporate with 5 mL of
hydrochloric acid on a water bath to dryness. Dissolve the
residue in 10 mL of water, add 1 drop of phenolphthalein TS,
and add ammonia TS until the solution becomes slightly red.
Add 2 mL of dilute acetic acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution as follows: evaporate 5 mL of
hydrochloric acid on a water bath to dryness, and to the
residue add 2 mL of dilute acetic acid, 2.0 mL of Standard
Lead Solution and water to make 50 mL (not more than 20
ppm).
(4) Iron <1J0> — Prepare the test solution with 1.0 g of
Sodium Pyrosulfite according to Method 1, and perform the
test according to Method A. Prepare the control solution
with 2.0 mL of Standard Iron Solution (not more than 20
ppm).
(5) Arsenic <1.11> — Dissolve 0.5 g of Sodium Pyrosulfite
in 10 mL of water, heat with 1 mL of sulfuric acid on a sand
bath until white fumes are evolved, and add water to make 5
mL. Perform the test with this solution as the test solution
(not more than 4 ppm).
Assay Weigh accurately about 0.15 g of Sodium Pyrosul-
fite, and transfer to an iodine flask containing an exactly
measured 50 mL of 0.05 mol/L iodine VS. Stopper tightly,
shake well, and allow to stand for 5 minutes in a dark place.
Add 1 mL of hydrochloric acid VS, and titrate <2.50> the ex-
cess of iodine with 0.1 mol/L sodium thiosulfate VS (indica-
tor: 1 mL of starch TS). Perform a blank determination.
Each mL of 0.05 mol/L iodine VS = 4.753 mg of Na 2 S 2 5
Containers and storage Containers — Tight containers.
Storage — Light-resistant, preferably well-filled, and not
exceeding 30°C.
Sodium Salicylate
C02Na
OH
C 7 H 5 Na0 3 : 160.10
Monosodium 2-hydroxybenzoate [54-21-7]
Sodium Salicylate, when dried, contains not less
than 99.5% of C 7 H 5 Na0 3 .
Description Sodium Salicylate occurs as white, crystals or
JPXV
Official Monographs / Sodium Thiosulfate Hydrate 1109
crystalline powder.
It is very soluble in water, freely soluble in acetic acid
(100), and soluble in ethanol (95).
It is gradually colored by light.
Identification (1) Determine the infrared absorption spec-
trum of Sodium Salicylate, previously dried, as directed in
the potassium bromide disk method under the Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(2) A solution of Sodium Salicylate (1 in 20) responds to
the Qualitative Tests <1.09> for sodium salt.
pH <2.54> The pH of a solution of 2.0 g of Sodium Salicy-
late in 20 mL of water is between 6.0 and 8.0.
Purity (1) Clarity of solution — Dissolve 1.0 g of Sodium
Salicylate in 10 mL of water: the solution is clear, and its ab-
sorbance at 420 nm determined as directed under Ultraviolet-
visible Spectrophotometry <2.24> is not more than 0.02.
(2) Chloride <1.03>— Dissolve 0.5 g of Sodium Salicylate
in 15 mL of water, add 6 mL of dilute nitric acid and ethanol
(95) to make 50 mL, and perform the test using this solution
as the test solution. Prepare the control solution with 0.30
mL of 0.01 mol/L hydrochloric acid VS, 28 mL of ethanol
(95), 6 mL of dilute nitric acid and water to make 50 mL (not
more than 0.021%).
(3) Sulfate — Dissolve 0.25 g of Sodium Salicylate in 5 mL
of water, and add 0.5 mL of barium chloride TS: the solution
shows no change.
(4) Sulfite and thiosulfate — Dissolve 1 .0 g of Sodium
Salicylate in 20 mL of water, add 1 mL of hydrochloric acid,
and filter. Add 0.15 mL of 0.05 mol/L iodine VS to the
filtrate: a yellow color develops.
(5) Heavy metals <1.07> — Proceed with 1.0 g of Sodium
Salicylate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(6) Arsenic <1.11> — To 1.0 g of Sodium Salicylate in a
decomposition flask add 5 mL of nitric acid and 2 mL of sul-
furic acid, and heat carefully until white fumes are evolved.
After cooling, add 2 mL of nitric acid, and heat. After cool-
ing, add several 2-mL portions of hydrogen peroxide (30),
and heat until the solution is colorless to pale yellow. Repeat
the procedure of adding nitric acid and hydrogen peroxide
(30) and heating, if necessary. After cooling, add 2 mL of a
saturated solution of ammonium oxalate monohydrate, and
heat until white fumes are evolved. After cooling, add water
to make 5 mL, and perform the test with this solution (not
more than 2 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Assay Weigh accurately about 0.3 g of Sodium Salicylate,
previously dried, dissolve in 50 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.01 mg of C 7 H 5 Na0 3
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dried Sodium Sulfite
Na 2 S0 3 : 126.04
Dried Sodium Sulfite contains not less than 97.0% of
Na 2 S0 3 .
Description Dried Sodium Sulfite is white crystals or pow-
der. It is odorless.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
The pH of a solution of Dried Sodium Sulfite (1 in 10) is
about 10.
It gradually changes in moist air.
Identification An aqueous solution of Dried Sodium Sulfite
(1 in 20) responds to the Qualitative Tests <1.09> for sodium
salt and sulfite.
Purity (1) Thiosulfate — Dissolve 1.0 g of Dried Sodium
Sulfite in 15 mL of water, add gradually 5 mL of hydrochlor-
ic acid, shake, and allow to stand for 5 minutes: no turbidity
is produced.
(2) Heavy metals <1.07> — Dissolve 1 .0 g of Dried Sodium
Sulfite in 5 mL of water, add 2 mL of hydrochloric acid grad-
ually, and evaporate the mixture on a water bath to dryness.
Add 3 mL of boiling water and 1 mL of hydrochloric acid to
the residue, and again evaporate to dryness on a water bath.
Dissolve the residue in 2 mL of dilute acetic acid and water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution as follows:
evaporate 3 mL of hydrochloric acid to dryness, and add 2
mL of dilute acetic acid, 2.0 mL of Standard Lead Solution
and water to make 50 mL (not more than 20 ppm).
(3) Arsenic <1.11> — Dissolve 0.5 g of Dried Sodium Sul-
fite in 5 mL of water, add 1 mL of sulfuric acid, and
evaporate on a sand bath until white fumes are evolved. Add
water to make 5 mL, take this solution as the sample solu-
tion, and perform the test (not more than 4 ppm).
Assay Weigh accurately about 0.2 g of Dried Sodium Sul-
fite, transfer immediately to an iodine flask containing exact-
ly 50 mL of 0.05 mol/L iodine VS, stopper, shake, and allow
to stand for 5 minutes in a dark place. Add 1 mL of
hydrochloric acid, and titrate <2.50> the excess iodine with
0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch
TS). Perform a blank determination.
Each mL of 0.05 mol/L iodine VS = 6.302 mg of Na 2 S0 3
Containers and storage Containers — Tight containers.
Sodium Thiosulfate Hydrate
Na 2 S 2 3 .5H 2 0: 248.18
Sodium Thiosulfate Hydrate, when dried, contains
1110 Sodium Thiosulfate Injection / Official Monographs
JP XV
not less than 99.0% and not more than 101.0% of sodi-
um thiosulfate (Na 2 S 2 3 : 158.11).
Description Sodium Thiosulfate Hydrate occurs as color-
less, crystals or crystalline powder. It is odorless.
It is very soluble in water, and practically insoluble in
ethanol (99.5).
It effloresces in dry air, and is deliquescent in moist air.
Identification (1) A solution of Sodium Thiosulfate Hy-
drate (1 in 10) responds to the Qualitative Tests <1.09> for
thiosulfate.
(2) A solution of Sodium Thiosulfate Hydrate (1 in 10)
responds to the Qualitative Tests <1.09> for sodium salt.
pH <2.54> Dissolve 1.0 g of Sodium Thiosulfate Hydrate in
10 mL of water: the pH of the solution is between 6.0 and
8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Thiosulfate Hydrate in 10 mL of water: the solution
is clear and colorless.
(2) Heavy metals <1.07> — Dissolve 1.0 g of Sodium
Thiosulfate Hydrate in 10 mL of water, add slowly 5 mL of
dilute hydrochloric acid, and evaporate on a water bath to
dryness. Add 15 mL of water to the residue, boil gently for 2
minutes, and filter. Heat the filtrate to boil, and add bromine
TS to the hot filtrate to produce a clear solution and provide a
slight excess of bromine. Boil the solution to expel the bro-
mine. Cool, add 1 drop of phenolphthalein TS, and add
dropwise sodium hydroxide TS until a slight red color is
produced. Add 2 mL of dilute acetic acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution as follows: to 2.0 mL of
Standard Lead Solution add 2 mL of dilute acetic acid and
water to make 50 mL (not more than 20 ppm).
(3) Calcium — Dissolve 1.0 g of Sodium Thiosulfate in 10
mL of water, add 2 mL of ammonium oxalate TS, and allow
to stand for 4 minutes: no turbidity is produced.
(4) Arsenic </.//>— To 0.40 g of Sodium Thiosulfate add
3 mL of nitric acid and 5 mL of water, evaporate on a water
bath to dryness, and perform the test with the residue. Pre-
pare the test solution according to Method 2, and perform
the test (not more than 5 ppm).
Loss on drying <2.41> 32.0 - 37.0% (1 g, in vacuum, 40 -
45°C, 16 hours).
Assay Weigh accurately about 0.4 g of Sodium Thiosulfate,
previously dried, dissolve in 30 mL of water, and titrate
<2.50> with 0.05 mol/L iodine VS (indicator: 1 mL of starch
TS).
Each mL of 0.05 mol/L iodine VS
= 15.81 mg of Na 2 S 2 3
Containers and storage Containers — Tight containers.
Sodium Thiosulfate Injection
ttm&T r- U -*A&*f«
Sodium Thiosulfate Injection is an aqueous solution
for injection.
It contains not less than 95% and not more than 105
% of the labeled amount of sodium thiosulfate hydrate
(Na 2 S 2 3 .5H 2 0: 248.18).
Method of preparation Prepare as directed under Injec-
tions, with Sodium Thiosulfate Hydrate.
Description Sodium Thiosulfate Injection is a clear, color-
less liquid.
Identification Sodium Thiosulfate Injection responds to the
Qualitative Tests <1.09> for sodium salt and for thiosulfate.
Extractable volume <6.05> It meets the requirement.
Pyrogen <4.04> Perform the test with Sodium Thiosulfate
Injection stored in a container in a volume exceeding 10 mL:
it meets the requirements.
Assay Measure exactly a volume of Sodium Thiosulfate In-
jection, equivalent to about 0.5 g of sodium thiosulfate hy-
drate (Na 2 S 2 03.5H 2 0), add water to make 30 mL, and titrate
<2.50> with 0.05 mol/L iodine VS (indicator: 1 mL of starch
TS).
Each mL of 0.05 mol/L iodine VS
= 24.82 mg of Na 2 S 2 3 .5H 2
Containers and storage Containers — Hermetic containers.
Sodium Valproate
H)\,-fum-tY U-5A
COjNa
C 8 H 15 Na0 2 : 166.19
Monosodium 2-propylpentanoate [1069-66-5]
Sodium Valproate, when dried, contains not less
than 98.5% of QH 15 Na0 2 .
Description Sodium Valproate occurs as a white, crystalline
powder. It has a characteristic odor and a slightly bitter taste.
It is very soluble in water, freely soluble in formic acid, in
ethanol (95), in ethanol (99.5) and in acetic acid (100), and
practically insoluble in chloroform and in diethyl ether.
It is hygroscopic.
Identification (1) To 1 mL of a solution of Sodium Val-
proate in ethanol (99.5) (1 in 200) add 4 mL of hydroxyla-
mine perchlorate-dehydrated ethanol TS and 1 mL of N,N'-
dicyclohexylcarbodiimide-dehydrated ethanol TS, shake
well, and allow to stand in lukewarm water for 20 minutes.
After cooling, add 1 mL of iron (III) perchlorate-dehydrated
ethanol TS, and shake: a purple color develops.
(2) To 5 mL of a solution of Sodium Valproate (1 in 20)
add 1 mL of a solution of cobalt (II) nitrate hexahydrate (1 in
20), and warm on a water bath: a purple precipitate is
formed.
(3) Dissolve 0.5 g of Sodium Valproate in 5 mL of water,
add 5 mL of chloroform and 1 mL of 2 mol/L hydrochloric
acid TS, and shake vigorously for 1 minute. After allowing to
stand, separate the chloroform layer, dehydrate the chlo-
roform with anhydrous sodium sulfate, then filter, and
evaporate the filtrate to dryness. Determine the infrared ab-
sorption spectrum of the residue as directed in the liquid film
JPXV
Official Monographs / Sorbitan Sesquioleate 1111
method under the Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(4) A solution of Sodium Valproate (1 in 10) responds to
the Qualitative Tests <1.09> for sodium salt.
pH <2.54> Dissolve 1.0 g of Sodium Valproate in 20 mL of
water: the pH of this solution is between 7.0 and 8.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sodium Valproate in 10 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Dissolve 0.5 g of Sodium Valproate
in 25 mL of ethanol (95), and add 6 mL of dilute nitric acid
and water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution as fol-
lows: to 0.70 mL of 0.01 mol/L hydrochloric acid VS add 25
mL of ethanol (95), 6 mL of dilute nitric acid and water to
make 50 mL (not more than 0.050%).
(3) Sulfate <1.14>— Dissolve 0.5 g of Sodium Valproate
in 25 mL of ethanol (95), and add 1 mL of dilute hydrochlor-
ic acid and water to make 50 mL. Perform the test using this
solution as the test solution. Prepare the control solution as
follows: to 0.50 mL of 0.005 mol/L sulfuric acid VS add 25
mL of ethanol (95), 1 mL of dilute hydrochloric acid and
water to make 50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Dissolve 2.0 g of Sodium Val-
proate in 44 mL of water, shake with 6 mL of dilute
hydrochloric acid, allow to stand for 5 minutes, and filter.
Discard the first 5 mL of the filtrate, neutralize the subse-
quent 25 mL with ammonia TS, and add 2 mL of dilute acetic
acid and water to make 50 mL. Perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 2.0 mL of Standard Lead Solution add 2 mL of di-
lute acetic acid and water to make 50 mL (not more than 20
ppm).
(5) Arsenic </.//> — Dissolve 2.0 g of Sodium Valproate
in 10 mL of water, shake with 10 mL of dilute hydrochloric
acid, allow to stand for 5 minutes, and filter. Discard the first
5 mL of the filtrate, and perform the test with the subsequent
10 mL (not more than 2 ppm).
(6) Related substances — Dissolve 0.10 g of Sodium Val-
proate in 10 mL of a mixture of formic acid and chloroform
(1:1), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add a mixture of formic acid and
chloroform (1:1) to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 2
/uL each of the sample solution and the standard solution as
directed under Gas Chromatography <2.02> according to the
following conditions. Determine each peak area of both solu-
tions by the automatic integration method: the total area of
all peaks other than the area of the valproic acid from the
sample solution is not larger than the peak area of the val-
proic acid from the standard solution.
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with siliceous earth for gas chro-
matography (150 to 180 //m in particle diameter) coated with
diethylene glycol adipate ester for gas chromatography and
phosphoric acid at the ratios of 5% and 1%, respectively.
Column temperature: A constant temperature of about
145°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
valproic acid is between 6 and 10 minutes.
Selection of column: Mix 1 mL of the sample solution and
4 mL of a solution of ^-valerianic acid in a mixture of formic
acid and chloroform (1:1) (1 in 1000). Proceed with 2//L of
this solution under the above operating conditions, and cal-
culate the resolution. Use a column giving elution of n-
valerianic acid and valproic acid in this order with the resolu-
tion between these peaks being not less than 3.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of valproic acid obtained from 2 /uL of
the standard solution is between 4 mm and 10 mm.
Time span of measurement: About twice as long as the
retention time of valproic acid, beginning after the solvent
peak.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 0.2 g of Sodium Valproate,
previously dried, dissolve in 80 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 16.62 mg of C 8 H 15 Na0 2
Containers and storage Containers — Tight containers.
Sorbitan Sesquioleate
7 )l £9 >-k X+Tt" U -OW.3L Xf )l
Sorbitan Sesquioleate is a mixture of monoester and
diester of sorbitol anhydride, partially esterified with
oleic acid.
Description Sorbitan Sesquioleate is a pale yellow to light
yellow-brown, viscous oily liquid. It has a faint, characteris-
tic odor and a slightly bitter taste.
It is freely soluble in diethyl ether, slightly soluble in
ethanol (95), and very slightly soluble in methanol.
It is dispersed as fine oily drops in water.
Identification (1) To 0.5 g of Sorbitan Sesquioleate add 5
mL of ethanol (95) and 5 mL of dilute sulfuric acid, and heat
on a water bath for 30 minutes. Cool, shake with 5 mL of
petroleum ether, and allow to stand, and separate the upper
layer and the lower layer. Shake 2 mL of the lower layer with
2 mL of freshly prepared catechol solution (1 in 10), then
with 5 mL of sulfuric acid: a red to red-brown color develops.
(2) Heat the upper layer obtained in (1) on a water bath,
and evaporate petroleum ether. To the residue add 2 mL of
diluted nitric acid (1 in 2), and then add 0.5 g of potassium ni-
trite between 30°C and 35°C with stirring: the solution de-
velops an opalescence, and, when cooled, crystals are
formed.
Specific gravity <7.75> d\\: 0.960 - 1.020
Saponification value <1.13> 150 - 168
Purity (1) Acidity — To 2.0 g of Sorbitan Sesquioleate add
1112 D-Sorbitol / Official Monographs
JP XV
50 mL of neutralized ethanol, and heat on a water bath near-
ly to boiling with stirring once or twice. Cool, add 4.3 mL of
O.lmol/L sodium hydroxide VS and 5 drops of
phenolphthalein TS: a red color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Sorbitan
Sesquioleate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sorbitan Sesquioleate according to Method 2, and per-
form the test (not more than 2 ppm).
Water <2.48> Not more than 3.0% (1 g, direct titration, stir
for 30 minutes).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Containers and storage Containers — Tight containers.
D-Sorbitol
D-VJUfcfh-JU
HO H H OH
HO H H OH
C 6 H 14 6 : 182.17
D-Glucitol [50-70-4]
D-Sorbitol, when dried, contains not less than 97.0%
of C 6 H 14 6 .
Description D-Sorbitol occurs as white granules, powder, or
crystalline masses. It is odorless, and has a sweet taste with a
cold sensation.
It is very soluble in water, sparingly soluble in ethanol (95),
and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) To 1 mL of a solution of D-Sorbitol (7 in
10) add 2 mL of iron (II) sulfate TS and 1 mL of a solution of
sodium hydroxide (1 in 5): a blue-green color develops, but
no turbidity is produced.
(2) Shake thoroughly 1 mL of a solution of D-Sorbitol (1
in 20) with 1 mL of a freshly prepared solution of catechol (1
in 10), add rapidly 2 mL of sulfuric acid, and shake: a reddish
purple to red-purple color immediately develops.
(3) Boil 0.5 g of D-Sorbitol with 10 mL of acetic anhy-
dride and 1 mL of pyridine under a reflux condenser for 10
minutes, cool, shake with 25 mL of water, and allow to stand
in a cold place. Transfer the solution to a separator, extract
with 30 mL of chloroform, and evaporate the extract on a
water bath. Add 80 mL of water to the oily residue, heat for
10 minutes on a water bath, then filter the hot mixture. After
cooling, collect the produced precipitate through a glass filter
(G3), wash with water, recrystallize once from ethanol (95),
and dry in a desiccator (in vacuum, silica gel) for 4 hours: the
precipitate melts between 97°C and 101 °C.
Purity (1) Clarity and color of solution, and acidity or
alkalinity — Dissolve 5 g of D-Sorbitol in 20 mL of water by
warming with shaking: the solution is clear, colorless, and
neutral.
(2) Chloride <1.03>— Perform the test with 2.0 g of D-
Sorbitol. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.005%).
(3) Sulfate <I.14>— Perform the test with 4.0 g of D-Sor-
bitol. Prepare the control solution with 0.50 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.006%).
(4) Heavy metals <1.07> — Proceed with 5.0 g of D-Sor-
bitol according to Method 1, and perform the test. Prepare
the control solution with 2.5 mL of Standard Lead Solution
(not more than 5 ppm).
(5) Nickel — Dissolve 0.5 g of D-Sorbitol in 5 mL of
water, add 3 drops of dimethylglyoxime TS and 3 drops of
ammonia TS, and allow to stand for 5 minutes: no red color
develops.
(6) Arsenic <1.11> — Prepare the test solution with 1.5 g
of D-Sorbitol according to Method 1, and perform the test
(not more than 1.3 ppm).
(7) Glucose— Dissolve 20.0 g of D-Sorbitol in 25 mL of
water, and boil gently with 40 mL of Fehling's TS for 3
minutes. After cooling, filter the supernatant liquid cautious-
ly through a glass filter (G4), leaving the precipitate in the
flask as much as possible, wash the precipitate with hot water
until the last washings no longer show an alkali reaction, and
filter the washings through the glass filter. Dissolve the
precipitate in the flask in 20 mL of iron (III) sulfate TS, filter
through the glass filter, and wash with water. Combine the
filtrate and the washings, heat at 80°C, and titrate <2.50> with
0.02 mol/L potassium permanganate VS: not more than 6.3
mL of volume for titration consumed or consumption is re-
quired.
(8) Sugars — Dissolve 20.0 g of D-Sorbitol in 25 mL of
water, and heat with 8 mL of dilute hydrochloric acid under a
reflux condenser in a water bath for 3 hours. After cooling,
add 2 drops of methyl orange TS, followed by sodium
hydroxide TS until an orange color develops, and add water
to make 100 mL. Boil gently 10 mL of this solution with 10
mL of water and 40 mL of Fehling's TS for 3 minutes and
proceed as directed in (7).
Loss on drying <2.41> Not more than 2.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 80°C, 3 hours).
Residue on ignition <2.44> Not more than 0.02% (5 g).
Assay Weigh accurately about 0.2 g of D-Sorbitol, previ-
ously dried, dissolve in water and add water to make exactly
100 mL. Pipet 10 mL of the solution into an iodine flask, add
exactly 50 mL of potassium periodate TS, and heat for 15
minutes in a water bath. Cool, add 2.5 g of potassium iodide,
immediately stopper tightly, and shake well. Allow to stand
for 5 minutes in a dark place, and titrate <2.50> with 0. 1 mol/
L sodium thiosulfate VS (indicator: 3 mL of starch TS). Per-
form a blank determination.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.822 mg of C 6 H 14 6
Containers and storage Containers — Tight containers.
D-Sorbitol Solution
D-y;u£h-ju>ft
D-Sorbitol Solution contains not less than 97%
JP XV
Official Monographs / Soybean Oil 1113
and not more than 103% of the labeled amount of
D-sorbitol (C 6 Hi 4 6 : 182.17).
Description D-Sorbitol Solution is a clear, colorless liquid.
It is odorless, and has a sweet taste.
It is miscible with water, with ethanol (95), with glycerin
and with propylene glycol.
It sometimes separates crystalline masses.
Identification (1) To a volume of D-Sorbitol Solution,
equivalent to 0.7 g of D-Sorbitol according to the labeled
amount, add 2 mL of iron (II) sulfate TS and 1 mL of a solu-
tion of sodium hydroxide (1 in 5): a blue-green color de-
velops, but no turbidity is produced.
(2) To a volume of D-Sorbitol Solution, equivalent to 1 g
of D-Sorbitol according to the labeled amount, add water to
make 20 mL. To 1 mL of this solution add 1 mL of a freshly
prepared solution of catechol (1 in 10), mix well, then add
rapidly 2 mL of sulfuric acid, and mix: a reddish purple to
red-purple color immediately develops.
Purity (1) Acidity or alkalinity — D-Sorbitol Solution is
neutral.
(2) Chloride <1.03> — Proceed with a volume of D-Sor-
bitol Solution, equivalent to 2.0 g of D-Sorbitol according to
the labeled amount, and perform the test. Prepare the control
solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS
(not more than 0.005%).
(3) Sulfate <1.14>— To a volume of D-Sorbitol Solution,
equivalent to 4.0 g of D-Sorbitol according to the labeled
amount, and perform the test. Prepare the control solution
with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than
0.006%).
(4) Heavy metals <1.07> — Proceed with a volume of D-
Sorbitol Solution, equivalent to 5.0 g of D-Sorbitol according
to the labeled amount, and according to Method 1, perform
the test. Prepare the control solution with 2.5 mL of Stan-
dard Lead Solution (not more than 5 ppm).
(5) Nickel — Take a volume of D-Sorbitol Solution,
equivalent to 0.5 g of D-Sorbitol according to the labeled
amount, add 3 drops of dimethylglyoxime TS and 3 drops of
ammonia TS, and allow to stand for 5 minutes: no red color
develops.
(6) Arsenic <1.11> — Take a volume of D-Sorbitol Solu-
tion, equivalent to 1.5 g of D-Sorbitol according to the la-
beled amount, dilute with water or concentrate to 5 mL on a
water bath, if necessary, cool, and perform the test using this
solution as the test solution (not more than 1.3 ppm).
(7) Glucose — Take a volume of D-Sorbitol Solution,
equivalent to 20.0 g of D-Sorbitol according to the labeled
amount, dilute with water or concentrate to 40 mL on a water
bath, if necessary, add 40 mL of Fehling's TS, and boil gently
for 3 minutes. After cooling, filter the supernatant liquid cau-
tiously through a glass filter (G4), leaving the precipitate in
the flask as much as possible, wash the precipitate with hot
water until the last washings no longer show alkalinity, and
filter the washings through the glass filter. Dissolve the
precipitate in the flask in 20 mL of iron (III) sulfate TS, filter
through the glass filter, and wash the filter with water. Com-
bine the filtrate and the washings, heat at 80°C, and titrate
<2.50> with 0.02 mol/L potassium permanganate VS: not
more than 6.3 mL of 0.02 mol/L potassium permanganate
VS is required.
(8) Sugars — Take a volume of D-Sorbitol Solution,
equivalent to 20.0 g of D-Sorbitol according to the labeled
amount, dilute with water or concentrate to 40 mL of a water
bath, if necessary, add 8 mL of dilute hydrochloric acid, and
heat under a reflux condenser in a water bath for 3 hours. Af-
ter cooling, add 2 drops of methyl orange TS, followed by so-
dium hydroxide TS until an orange color develops, and add
water to make 100 mL. Boil gently 10 mL of this solution
with 10 mL of water and 40 mL of Fehling's TS for 3 minutes
and proceed as directed in (7).
Residue on ignition <2.44> Measure exactly a volume of D-
Sorbitol Solution, equivalent to 5 g of D-Sorbitol according
to the labeled amount, add 3 to 4 drops of sulfuric acid, and
heat gently to evaporate. Ignite to burn, cool, and perform
the test with the residue: not more than 1 mg.
Assay Measure exactly a volume of D-Sorbitol Solution,
equivalent to about 5 g of D-sorbitol (C 6 H I4 6 ) according to
the labeled amount, and add water to make exactly 250 mL.
Pipet 10 mL of this solution, add water to make exactly 100
mL. Pipet exactly 10 mL of the solution into an iodine flask,
add exactly 50 mL of potassium periodate TS, and heat for
15 minutes in a water bath. Cool, add 2.5 g of potassium
iodide, immediately stopper tightly, and shake well. Allow to
stand for 5 minutes in a dark place, and titrate <2.50> with 0.1
mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS).
Perform a blank determination.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.822mgof C 6 H 14 6
Containers and storage Containers — Tight containers.
Soybean Oil
Oleum Sojae
$M X>ft
Soybean Oil is the fixed oil obtained from the seeds
of Glycine max merrill (Leguminosae).
Description Soybean Oil is a clear, pale yellow oil. It is
odorless or has a slight odor, and has a bland taste.
It is miscible with diethyl ether and with petroleum ether.
It is slightly soluble in ethanol (95), and practically insolu-
ble in water.
It congeals between - 10°C and - 17°C.
Congealing point of the fatty acids: 22 - 27°C
Specific gravity <1.13> d\\: 0.916 - 0.922
Acid value <1.13> Not more than 0.2.
Saponification value <7.75> 188 - 195
Unsaponifiable matter <1.13> Not more than 1.0%.
Iodine value <7.75> 126 - 140
Containers and storage Containers — Tight containers.
1114 Spectinomycin Hydrochloride Hydrate / Official Monographs
JP XV
Spectinomycin Hydrochloride
Hydrate
•EHCI-SHjO
C 14 H 24 N 2 7 .2HC1.5H 2 0: 495.35
(2fl,4ai?,5ai?,6S,7S,8fl,9S,9afl,10aS)-
4a , 7 , 9-Trihy droxy-2-methyl-6 , 8 -
bis(methylamino)-2,3,4a,5a,6,7,8,9,9a,10a-decahydro-
4//-pyrano[2,3-6][l,4]benzodioxin-4-one dihydrochloride
pentahydrate [22189-32-8]
Spectinomycin Hydrochloride Hydrate is the
hydrochloride of a substance having antibacterial ac-
tivity produced by the growth of Streptomyces spec-
tabilis.
It contains not less than 603 fig (potency) and not
more than 713 fig (potency) per mg. The potency of
Spectinomycin Hydrochloride Hydrate is expressed as
mass (potency) of spectinomycin (Q4H24N2O7:
332.35).
Description Spectinomycin Hydrochloride Hydrate occurs
as a white to light yellowish white crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (95).
Identification (1) To 5 mL of a solution of Spectinomycin
Hydrochloride Hydrate (1 in 100) add gently anthrone TS: a
blue to blue-green color is produced at the zone of contact.
(2) Determine the infrared absorption spectra of Spec-
tinomycin Hydrochloride Hydrate and Spectinomycin
Hydrochloride Reference Standard as directed in the paste
method under the Infrared Spectrophotometry <2.25>, and
compare these spectra: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) To 3 mL of a solution of Spectinomycin Hydrochlo-
ride Hydrate (1 in 150) add 1 drop of silver nitrate TS: a white
turbidity is produced.
Optical rotation <2.49> [a]g>: + 15 - +21° (2.1 g calculated
on the anhydrous basis, water, 25 mL, 200 mm).
pH <2.54> Dissolve 0.10 g of Spectinomycin Hydrochloride
Hydrate in 10 mL of water: the pH of the solution is between
4.0 and 5.6.
Water <2.48> Not less than 16.0% and not more than
20.0% (0.3 g, volumetric titration, direct titration).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism— Klebsiella pneumoniae ATCC 10031
(ii) Culture medium — Use the medium i in 3) under (1)
Agar media for seed and base layer. Adjust the pH of the
medium so that it will be 7.8 to 8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Spectinomycin Hydrochloride Reference Standard, equiva-
lent to about 20 mg (potency), dissolve in 0.1 mol/L phos-
phate buffer solution, pH 8.0 to make exactly 20 mL, and use
this solution as the standard stock solution. Keep the stan-
dard stock solution at a temperature not exceeding 5°C and
use within 10 days. Take exactly a suitable amount of the
standard stock solution before use, add 0.1 mol/L phosphate
buffer solution, pH 8.0 to make solutions so that each mL
contains 200 fig (potency) and 50,Mg (potency), and use these
solutions as the high concentration standard solution and low
concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Spectinomycin Hydrochloride Hydrate, equivalent to about
20 mg (potency), and dissolve in 0.1 mol/L phosphate buffer
solution, pH 8.0 to make exactly 20 mL. Take exactly a suita-
ble amount of this solution, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
200 fig (potency) and 50 fig (potency), and use these solutions
as the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Spiramycin Acetate
H 3 C.
H 3 C"
CH 3
») —
/\
^ /
\ /I
H 3 C
H -
OHC
H
CH 3
1 'f\
-)—
H
H
RO
-CH 3
HaC
Spiramycin II Acetate . R= J.
(Spiramycin I Acetate} ' ^CHn
O a
Spiramycin III Acetate : Ft= — r XH 3
(Spiramycin II Acetate (Spiramycin I Acetate))
(3R,4R,5S,6R,8R,9R,10E,12E,15R)-3-
Acetoxy-5-[4-0-acetyl-2,6-dideoxy-3-C-methyl-a-
L-n7j>o-hexopyranosyl-(l->4)-3,6-dideoxy-3-
dimethylamino-/?-D-glucopyranosyloxy]-9-(2, 3,4,6-
tetradeoxy-4-dimethylamino-/6-D-e/"v?/!ro-
hexopyranosyloxy)-6-formylmethyl-9-hydroxy-4-methoxy-
8-methylhexadeca-10,12-dien-15-olide
(Spiramycin III Acetate)
(3R,4R,SS,6R,8R,9R,l0E,l2E,l5R)-5-
[4-0-Acetyl-2,6-dideoxy-3-C-methyl-a-L-n7j>o-
hexopyranosyl-(l->4)-3,6-dideoxy-3-dimethylamino-/3-
D-glucopyranosyloxy]-9-(2,3,4,6-tetradeoxy-4-
dimethylamino-/?-D-ery?/!ro-hexopyranosyloxy)-6-
formylmethyl-9-hydroxy-4-methoxy-8-methyl-3-
JP XV
Official Monographs / Spiramycin Acetate 1115
propionyloxyhexadeca- 1 0, 1 2-dien- 1 5-olide
[74014-51-0, Spiramycin Acetate]
Spiramycin Acetate is a derivative of a mixture of
macrolide substances having antibacterial activity pro-
duced by the growth of Streptomyces ambofaciens.
It contains not less than 900 /ug (potency) and not
more than 1450 fig (potency) per mg, calculated on the
dried basis. The potency of Spiramycin Acetate is
expressed as mass (potency) of spiramycin acetate II
(C4 7 H7 8 N 2 16 : 927.13). One mg (potency) of Spiramy-
cin Acetate is equivalent to 0.7225 mg of spiramycin
acetate II (C 47 H 78 N 2 16 ).
Description Spiramycin Acetate occurs as a white to light
yellowish white powder.
It is very soluble in acetonitrile and in methanol, freely
soluble in ethanol (99.5), and practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Spiramycin Acetate in methanol (1 in 50,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Spiramycin Acetate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Content ratio of the active principle Dissolve 25 mg of
Spiramycin Acetate in 25 mL of the mobile phase, and use
this solution as the sample solution. Perform the test with 5
fiL of the sample solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the areas, A n , A m , A lv , A y , A vl and A yu , of
the peaks of spiramycin acetate II, spiramycin acetate III,
spiramycin acetate IV, spiramycin acetate V, spiramycin
acetate VI and spiramycin acetate VII, respectively, by the
automatic integration method, and calculate the ratios of the
amounts of A u , A w and the total of A m and A v to the total
amount of all these peaks: the amount of A n is 30 - 45%, A ly
is 30 - 45%, and the total of A m and A Y is not more than
25%. The relative retention times of spiramycin acetate III,
spiramycin acetate IV, spiramycin acetate V, spiramycin
acetate VI and spiramycin acetate VII with respect to
spiramycin acetate II are 1.3, 1.7, 2.3, 0.85 and 1.4, respec-
tively.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 231 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (3 fim in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of acetonitrile, 0.02 mol/L potas-
sium dihydrogen phosphate TS and a solution of dipotassium
hydrogen phosphate (87 in 25,000) (26:7:7).
Flow rate: Adjust the flow rate so that the retention time of
acetylspiramycin II is about 10 minutes.
System suitability —
System performance: Dissolve 25 mg of Spiramycin
Acetate II Reference Standard in the mobile phase to make
100 mL. When the procedure is run with 5 fiL of this solution
under the above operating conditions, the number of theoret-
ical plates and the symmetry factor of the peak of spiramycin
acetate II are not less than 14,500 and not more than 2.0, re-
spectively.
System repeatability: When the test is repeated 6 times with
5 fiL of the sample solution under the above operating condi-
tions, the relative standard deviation of the peak area of
spiramycin acetate II is not more than 2.0%.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Spiramycin Acetate according to Method 2, and perform the
test. Prepare the control solution with 1.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 2.0 g
of Spiramycin Acetate according to Method 3, and perform
the test (not more than 1 ppm).
Loss on drying <2.41> Not more than 3.0% (1 g, in vacuum,
phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.5% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount
of Spiramycin Acetate II Reference Standard, equivalent to
about 50 mg (potency), dissolve in 20 mL of methanol, add
0.1 mol/L phosphate buffer solution for antibiotics, pH 8.0
to make exactly 50 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at not
exceeding 5°C, and use within 3 days. Take exactly a suitable
amount of the standard stock solution before use, add
0.1 mol/L phosphate buffer solution for antibiotics, pH 8.0
to make solutions so that each mL contains 80 //g (potency)
and 20,Mg (potency), and use these solutions as the high
concentration standard solution and low concentration stan-
dard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Spiramycin Acetate, equivalent to about 50 mg (potency),
dissolve in 20 mL of methanol, and add 0. 1 mol/L phosphate
buffer solution for antibiotics, pH 8.0 to make exactly
50 mL. Take exactly a suitable amount of this solution, add
0.1 mol/L phosphate buffer solution for antibiotics, pH 8.0
to make solutions so that each mL contains 80 fig (potency)
and 20 ^g (potency), and use these solutions as the high
concentration sample solution and low concentration sample
solution, respectively.
Containers and storage Containers — Tight containers.
1116 Spironolactone / Official Monographs
JP XV
Spironolactone
xtfn/^? V>
C24H32O4S: 416.57
7«-Acetylsulf anyl-3-oxo- 1 7«-pregn-4-ene-2 1 , 1 7/?-
carbolactone [52-01-7]
Spironolactone, when dried, contains not less than
97.0% and not more than 103.0% of C 2 4H 32 4 S.
Description Spironolactone occurs as a white to light yel-
low-brown, fine powder.
It is freely soluble in chloroform, soluble in ethanol (95),
slightly soluble in methanol, and practically insoluble in
water.
Melting point: 198 - 207°C (Insert the capillary tube into a
bath at about 125 °C, and continue the heating so that the
temperature rises at a rate of about 10°C per minute in the
range between 140°C and 185°C, and when the temperature
is near the expected melting range, reduce the heating so that
the temperature rises at a rate of about 3°C per minute.)
Identification (1) Determine the absorption spectrum of a
solution of Spironolactone in methanol (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Spironolactone Reference Stan-
dard prepared in the same manner as the sample solution:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Spironolactone, previously dried, as directed in the potassi-
um bromide disk method under the Infrared Spectrophoto-
metry <2.25>, and compare the spectrum with the Reference
Spectrum or the spectrum of Spironolactone Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers. If any difference appears between
the spectra, dissolve Spironolactone and Spironolactone
Reference Standard in methanol, respectively, then evaporate
methanol to dryness, and repeat the test on the residues.
Optical rotation <2.49> [<*]£,: - 33
0.25 g, chloroform, 25 mL, 200 mm).
■37° (after drying,
Purity (1) Mercapto compounds — Shake 2.0 g of
Spironolactone with 20 mL of water, and filter. To 10 mL of
the filtrate add 1 mL of starch TS and 0.05 mL of 0.01 mol/L
iodine VS, and mix: a blue color develops.
(2) Related substances — Dissolve 0.20 g of Spironolac-
tone in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL of this solution, add ethanol (95)
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 [iL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with n-
butyl acetate to a distance of about 15 cm, and air-dry the
plate. Spray evenly a solution of sulfuric acid in methanol (1
in 10) on the plate, and heat the plate at 105 °C for 10
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41>
hours).
Not more than 0.5% (1 g, 105°C, 2
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Spironolac-
tone and Spironolactone Reference Standard, previously
dried at 105 °C for 2 hours, dissolve in methanol to make ex-
actly 250 mL. Pipet 5 mL each of these solutions, add
methanol to make exactly 100 mL, and use these solutions as
the sample solution and standard solution, respectively. Per-
form the test with these solutions as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and determine the ab-
sorbances, A T and A s , of the sample solution and the stan-
dard solution at 238 nm.
Amount (mg) of C24H32O4S
= W s x(A T /A s )
W s : amount (mg) of Spironolactone Reference Standard
Containers and storage Containers — Tight containers.
Stearic Acid
Xx7U>
Stearic Acid is solid fatty acids obtained from fats,
and it consists chiefly of stearic acid (C 18 H 36 2 : 284.48)
and palmitic acid (C 16 H 32 2 : 256.42).
Description Stearic Acid occurs as white, unctuous or crys-
talline masses or powder. It has a faint, fatty odor.
It is freely soluble in diethyl ether, soluble in ethanol (95),
and practically insoluble in water.
Melting point: 56-72°C.
Acid value <I.I3> 194-210
Iodine value <1.13> Not more than 4.0.
Purity (1) Mineral acid — Melt 5 g of Stearic Acid by
warming, shake with 5 mL of boiling water for 2 minutes,
filter after cooling, and add 1 drop of methyl orange TS to
the filtrate: no red color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Stearic
Acid according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Fat and paraffin — Boil 1 .0 g of Stearic Acid with 0.5 g
of anhydrous sodium carbonate and 30 mL of water: the so-
lution, while hot, is clear or not more turbid than the follow-
ing control solution.
Control solution: To 0.70 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 30
mL, and add 1 mL of silver nitrate TS.
Residue on ignition <2.44> Not more than 0.1% (1 g).
JPXV
Official Monographs / Streptomycin Sulfate 1117
Containers and storage Containers — Well-closed contain-
ers.
Stearyl Alcohol
Xx7UJU7JU=l-JU
Streptomycin Sulfate
Stearyl Alcohol is a mixture of solid alcohols, and
consists chiefly of stearyl alcohol (C 18 H 38 0: 270.49).
Description Stearyl Alcohol occurs as a white, unctuous
matter. It has a faint, characteristic odor. It is tasteless.
It is freely soluble in ethanol (95), in ethanol (99.5), in
diethyl ether, and practically insoluble in water.
Melting point <1.13> 56 - 62°C Prepare the sample accord-
ing to Method 2, then attach tightly a capillary tube to the
bottom of the thermometer by means of a rubber band or by
any suitable means, and make the bottom of the capillary
tube equal in position to the lower end of the thermometer.
Insert this thermometer into a test tube 17 mm in inside di-
ameter and about 170 mm in height, fasten the thermometer
with cork stopper so that the lower end of the thermometer is
about 25 mm distant from the bottom of the test tube. Sus-
pend the test tube in a beaker containing water, and heat the
beaker with constant stirring until the temperature rises to
5°C below the expected melting point. Then regulate the rate
of increase to 1°C per minute. The temperature at which the
sample is transparent and no turbidity is produced is taken as
the melting point.
Acid value <1.13> Not more than 1.0.
Ester value <1.13> Not more than 3.0.
Hydroxyl value <I.I3> 200 - 220
Iodine value <1.13> Not more than 2.0.
Purity (1) Clarity of solution — Dissolve 3.0 g of Stearyl
Alcohol in 25 mL of ethanol (99.5) by warming: the solution
is clear.
(2) Alkalinity — To the solution obtained in (1) add 2
drops of phenolphthalein TS: no red color develops.
Residue on ignition <2.44> Not more than 0.05% (2 g).
Containers and storage Containers — Well-closed contain-
ers.
OH H
CnO39N7Oi2.il/2H2SO4: 728.69
2-Deoxy-2-methylamino-a-L-glucopyranosyl-(l -» 2)-
5-deoxy-3-C-formyl-a-L-lyxofuranosyl-(l->4)-7V,./V'-
diamidino-D-streptamine sesquisulfate [3810-74-0]
Streptomycin Sulfate is the sulfate of an
aminoglycoside substance having antibacterial activity
produced by the growth of Streptomyces griseus.
It contains not less than 740 fig (potency) and not
more than 820 fig (potency) per mg, calculated on the
dried basis. The potency of Streptomycin Sulfate is
expressed as mass (potency) of streptomycin
(C 21 H 39 N 7 12 : 581.57).
Description Streptomycin Sulfate occurs as a white to light
yellowish white powder.
It is freely soluble in water, and very slightly soluble in
ethanol (95).
Identification (1) Dissolve 50 mg of Streptomycin Sulfate
in 5 mL of water, add 1 mL of ninhydrin TS and 0.5 mL of
pyridine, and heat for 10 minutes: a purple color is devel-
oped.
(2) Dissolve 10 mg each of Streptomycin Sulfate and
Streptomycin Sulfate Reference Standard in 10 mL of water,
and use these solutions as the sample solution and the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 10 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a solution of potassium dihydrogen phosphate (7 in 100)
to a distance of about 12 cm, and air-dry the plate. Spray
evenly a mixture of a solution of 1,3-dihydroxynaphthalene
in ethanol (95) (1 in 500) and diluted sulfuric acid (1 in 5)
(1:1) on the plate, and heat at about 150°C for about 5
minutes: the principal spots from the sample solution and the
standard solution show the same in color tone and Rf value.
(3) A solution of Streptomycin Sulfate (1 in 5) responds
to the Qualitative Tests <1.09> for sulfate.
Optical rotation <2.49> [a]™: - 79 - - 88° (0.5 g calculated
on the dried basis, water, 50 mL, 100 mm).
1118 Sucralfate Hydrate / Official Monographs
JP XV
pH <2.54> The pH of a solution obtained by dissolving 2.0 g
of Streptomycin Sulfate in 10 mL of water is between 4.5 and
7.0.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 1.0 g of Streptomycin Sulfate in 5 mL of
water is clear, and colorless or pale yellow.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Strep-
tomycin Sulfate according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Streptomycin Sulfate according to Method 3 and perform
the test (not more than 1 ppm).
(4) Related substances — Dissolve exactly 0.20 g of Strep-
tomycin Sulfate in a mixture of methanol and sulfuric acid
(97:3) to make 5 mL, and heat under a reflux condenser for 1
hour. After cooling, wash the inside of the condenser with a
suitable amount of a mixture of methanol and sulfuric acid
(97:3), add a mixture of methanol and sulfuric acid (97:3) to
make exactly 20 mL, and use this solution as the sample solu-
tion. Separately, dissolve exactly 36 mg of D( + )-mannose in
a mixture of methanol and sulfuric acid (97:3) to make 5 mL,
and heat under a reflux condenser for 1 hour. After cooling,
wash the inside of the condenser with a suitable amount of a
mixture of methanol and sulfuric acid (97:3), and add a mix-
ture of methanol and sulfuric acid (97:3) to make exactly 50
mL. Pipet 5 mL of this solution, add a mixture of methanol
and sulfuric acid (97:3) to make exactly 50 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography, develop with a mixture of toluene, methanol
and acetic acid (100) (2:1:1) to a distance of 13 to 15 cm, and
air-dry the plate. Spray evenly a mixture of a solution of 1,3-
dihydroxynaphthalene in ethanol (95) (1 in 500) and diluted
sulfuric acid (1 in 5) (1:1) on the plate, and heat at 110°C for
5 minutes: the spot from the sample solution corresponding
to the spot from the standard solution is not more intense
than the spot from the standard solution.
Loss on drying <2.4I> Not more than 5.0% (0.5 g, reduced
pressure not exceeding 0.67 kPa, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er, having pH 7.8 - 8.0 after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Streptomycin Sulfate Reference Standard, previously dried,
equivalent to about 20 mg (potency), dissolve in diluted phos-
phate buffer solution, pH 6.0 (1 in 2) to make exactly 50 mL,
and use this solution as the standard stock solution. Keep the
standard stock solution between 5°C and 15°C, and use wi-
thin 30 days. Take exactly a suitable amount of the standard
stock solution before use, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
8 n% (potency) and 2//g (potency), and use these solutions as
the high concentration standard solution and low concentra-
tion standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Streptomycin Sulfate, equivalent to about 20 mg (potency),
dissolve in water to make exactly 50 mL. Take exactly a suita-
ble amount of this solution, add 0.1 mol/L phosphate buffer
solution, pH 8.0 to make solutions so that each mL contains
8 //g (potency) and 2 n% (potency), and use these solutions as
the high concentration sample solution and low concentra-
tion sample solution, respectively.
Containers and storage Containers — Tight containers.
Sucralfate Hydrate
Aluminum Sucrose Sulfate Ester
• xAI(OH) 3 • >HjO
FfcSOsAlfOH^
C 12 H 30 Al 8 O 51 S 8 .xAl(OH) 3 .j'H 2 O
[54182-58-0]
Sucralfate Hydrate contains not less than 17.0% and
not more than 21.0% of aluminum (Al: 26.98) and not
less than 34.0% and not more than 43.0% of sucrose
octasulfate ester (Cnt^C^Sg: 982.80), calculated on
the dried basis.
Description Sucralfate Hydrate occurs as a white powder.
It is odorless and tasteless.
It is practically insoluble in water, in hot water, in ethanol
(95) and in diethyl ether.
It dissolves in dilute hydrochloric acid and in sulfuric acid-
sodium hydroxide TS.
Identification (1) To 0.05 g of Sucralfate Hydrate in a
small test tube add 0.05 g of fresh pieces of sodium, and melt
by careful heating. Immerse the test tube immediately in 100
mL of water, break the test tube, shake well, and filter. To 5
mL of the filtrate add 1 drop of sodium pentacyanonitrosyl-
ferrate (III) TS: a red-purple color develops.
(2) Dissolve 40 mg of Sucralfate Hydrate in 2 mL of di-
lute sulfuric acid, and add gently 2 mL of anthrone TS to
make 2 layers: a blue color develops at the zone of contact,
and gradually changes to blue-green.
(3) Dissolve 0.5 g of Sucralfate Hydrate in 10 mL of di-
lute hydrochloric acid: the solution responds to the Qualita-
tive Tests <1.09> for aluminum.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sucralfate Hydrate in 10 mL of dilute sulfuric acid: the solu-
tion is clear and colorless.
(2) Chloride <1.03>— Dissolve 0.5 g of Sucralfate Hy-
drate in 30 mL of dilute nitric acid, and heat gently to boil-
ing. After cooling, add water to make 100 mL, and to 10 mL
of this solution add 3 mL of dilute nitric acid and water to
make 50 mL. Perform the test using this solution as the test
solution. Prepare the control solution with 0.70 mL of 0.01
JPXV
Official Monographs / Sucralfate Hydrate 1119
mol/L hydrochloric acid VS (not more than 0.50%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Sucralfate
Hydrate in 20 mL of a solution of sodium chloride (1 in 5)
and 1 mL of dilute hydrochloric acid, and to this solution
add 2 mL of dilute acetic acid and water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution as follows: evaporate 1 mL of dilute
hydrochloric acid on a water bath to dryness, and add 20 mL
of a solution of sodium chloride (1 in 5), 2 mL of dilute acetic
acid, 2.0 mL of Standard Lead Solution and water to make
50 mL (not more than 20 ppm).
(4) Arsenic <1.11> — Dissolve 1.0 g of Sucralfate Hydrate
in 5 mL of dilute hydrochloric acid, use this solution as the
test solution, and perform the test (not more than 2 ppm).
(5) Free aluminum — To 3.0 g of Sucralfate Hydrate add
50 mL of water, heat in a water bath for 5 minutes, cool, and
filter. Wash the residue with four 5-mL portions of water,
combine the filtrate with the washings, add 2 mL of dilute
hydrochloric acid, and heat in a water bath for 30 minutes.
After cooling, neutralize the solution with sodium hydroxide
TS, add water to make exactly 100 mL, and use this solution
as the sample solution. Pipet 50 mL of the sample solution,
add exactly 25 mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS and 20 mL of acetic acid-
ammonium acetate buffer solution, pH 4.5, and boil for 5
minutes. After cooling, add 50 mL of ethanol (95), and ti-
trate <2.50> the excess disodium dihydrogen ethylenediamine
tetraacetate with 0.05 mol/L zinc acetate VS until the color
of the solution changes from green-purple through purple to
red (indicator: 3 mL of dithizone TS). Perform a blank deter-
mination (not more than 0.2%).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 1.349 mg of Al
(6) Related substances — Proceed with 50 /xL of the sam-
ple solution obtained in the Assay (2) Sucrose octasulfate es-
ter as directed in the Assay (2) Sucrose octasulfate ester, and
perform the test as directed under Liquid Chromatography
<2.01>. Determine the peak area of sucrose octasulfate ester
from the sample solution and that of a related substance with
the relative retention time about 0.7 to the peak of sucrose
octasulfate ester by the automatic integration method, and
calculate the ratio of the peak area of the related substance to
that of sucrose octasulfate ester: it is not more than 0.1.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of sucrose octasulfate ester from 50 /xL
of the standard solution obtained in the Assay (2) Sucrose oc-
tasulfate ester composes 60% to 100% of the full scale.
Loss on drying <2.41> Not more than 14.0% (1 g, 105°C,
3 hours).
Acid-consuming capacity Weigh accurately about 0.25 g of
Sucralfate Hydrate, previously dried, place in a 200-mL
glass-stoppered conical flask, add exactly 100 mL of 0.1 mol/
L hydrochloric acid VS, stopper the flask tightly, and shake
at 37 ± 2°C for exactly 1 hour (150 shakings per minute, am-
plitude: 20 mm). After cooling in water for 5 minutes, pipet
10 mL of the supernatant liquid, and titrate <2.50> the excess
acid with 0.1 mol/L sodium hydroxide VS until the pH
becomes 3.5. Perform a blank determination. The amount of
0.1 mol/L hydrochloric acid VS consumed per g of Sucral-
fate is not less than 130 mL.
Assay (1) Aluminum — Weigh accurately about 1 g of
Sucralfate Hydrate, dissolve in 10 mL of dilute hydrochloric
acid by warming on a water bath, cool, and add water to
make exactly 250 mL. Pipet 25 mL of this solution, add ex-
actly 25 mL of 0.05 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS and 20 mL of acetic acid-ammonium
acetate buffer solution, pH 4.5, and boil for 5 minutes. After
cooling, add 50 mL of ethanol (95), and titrate <2.50> the ex-
cess disodium dihydrogen ethylenediamine tetraacetate with
0.05 mol/L zinc acetate VS until the color of the solution
changes from green-purple through purple to red (indicator:
3 mL of dithizone TS). Perform a blank determination.
Each mL of 0.05 mol/L disodium dihydrogen ethylenedia-
mine tetraacetate VS
= 1.349 mg of Al
(2) Sucrose octasulfate ester — Weigh accurately about
0.55 g of Sucralfate Hydrate, add exactly 10 mL of sulfuric
acid-sodium hydroxide TS, shake vigorously, and dissolve
with ultrasonic wave at below 30°C for 5 minutes. To this so-
lution add 0.1 mol/L sodium hydroxide VS to make exactly
25 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.25 g of Potassium Su-
crose Octasulfate Reference Standard, add the mobile phase
to make exactly 25 mL, and use this solution as the standard
solution. Prepare rapidly the sample solution and the stan-
dard solution, and perform the test immediately. Pipet 50 /xL
each of the sample solution and standard solution, and per-
form the test as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine the
peak areas, A T and A s , of sucrose octasulfate ester from each
solution.
Amount (mg) of sucrose octasulfate ester (C 12 H 2 2035S 8 )
= W s x(Aj/A s )x 0.7633
W s : Amount (mg) of Potassium Sucrose Octasulfate
Reference Standard, calculated on the anhydrous basis
Operating conditions —
Detector: A differential refractometer.
Column: A stainless steel column about 4 mm in inside di-
ameter and about 30 cm in length, packed with aminopropyl-
silanized silica gel for liquid chromatography (about 8 /xm in
particle diameter).
Column temperature: Room temperature.
Mobile phase: Dissolve a suitable amount (26 to 132 g) of
ammonium sulfate in 1000 mL of water, and adjust with
phosphoric acid to a pH of 3.5. Allow a solution of Potassi-
um Sucrose Octasulfate Reference Standard in dilute
hydrochloric acid (1 in 100) to stand at 60°C for 10 minutes,
cool, and perform the test immediately. Adjust the amount
of ammonium sulfate in the mobile phase so that the peak of
a related substance with the relative retention time being
about 0.7 to that of sucrose octasulfate ester almost returns
to the base line, and the peak of sucrose octasulfate ester
elutes most rapidly.
Flow rate: Adjust the flow rate so that the retention time of
sucrose octasulfate ester is between 6 and 11 minutes.
Selection of column: Allow a solution of Potassium Su-
crose Octasulfate Reference Standard in dilute hydrochloric
acid (1 in 100) to stand at 60°C for 10 minutes, cool, and pro-
ceed immediately with 50 /xL of this solution under the above
operating conditions. Use a column with a resolution being
1120 Sucrose / Official Monographs
JP XV
not less than 1.5 between sucrose octasulfate ester and a relat-
ed substance with the relative retention time being about 0.7
to sucrose octasulfate ester.
System repeatability: Repeat the test 6 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of sucrose octasul-
fate ester is not more than 2.0%.
Containers and storage Containers — Tight containers.
Sucrose
OH H
C 12 H 22 O n : 342.30
/?-D-Fructofuranosyl a-D-glucopyranoside [57-50-1]
Sucrose contains no additives.
For Sucrose used for preparation of the large volume
infusions, the label states the purpose.
Description Sucrose is a white crystalline powder, or lus-
trous colorless or white crystals.
It is very soluble in water, and slightly soluble in ethanol
(95).
Identification (1) To 10 mg each of Sucrose and white soft
sugar add diluted methanol (3 in 5) to make 20 mL each, and
use these solutions as the sample solution and the standard
solution (a), respectively. Separately, to 10 mg each of glu-
cose, lactose hydrate, fructose and white soft sugar add
methanol (3 in 5) to make 20 mL, and use this solution as the
standard solution (b). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2//L
each of the sample solution and standard solutions (a) and (b)
on a plate of silica gel for thin-layer chromatography, and
dry the plate completely. Develop the plate with a mixture of
1,2-dichloroethane, acetic acid (100), methanol and water
(10:5:3:2) to a distance of about 15 cm, and dry the plate with
a hot air. And immediately repeat the development with
replaced developing mixture, and dry the plate in the same
way. Spray evenly a solution of 0.5 g of thymol in 100 mL of
a mixture of ethanol (95) and sulfuric acid (19:1), heat at
130°C for 10 minutes: the principal spot from the sample so-
lution is the same with the principal spot from the standard
solution (a) in the Ri, color and size, and four spots from the
standard solution (b) are apparently distinguishable.
(2) Dissolve 50.0 g of Sucrose in recently boiled and
cooled water to make 100 mL, and use this solution as the
sample solution. To 1 mL of the sample solution add water to
make 100 mL, then to 5 mL of this solution add 0.15 mL of
freshly prepared copper (II) sulfate TS and 2 mL of freshly
prepared 2 mol/L sodium hydroxide TS: the solution is clear
and blue, and not changes on boiling. Then to this solution
add 4 mL of dilute hydrochloric acid, boil, and add 4 mL of 2
mol/L sodium hydroxide TS: orange precipitates are immedi-
ately produced.
Optical rotation <2.49> [a]™: +66.3 - +67.0° (26 g, water,
100 mL, 100 mm).
Purity (1) Clarity and color of solution — The sample so-
lution obtained in the Identification (2) is clear, and has no
more color than the following control solution.
Control solution: To exactly 2.4 mL of iron (III) chloride
colorimetric stock solution and exactly 0.6 mL of cobalt (II)
chloride colorimetric stock solution add 7.0 mL of diluted
hydrochloric acid (7 in 250). To 5.0 mL of this solution add
95.0 mL of diluted hydrochloric acid (7 in 250).
(2) Acidity or alkalinity — To 10 mL of the sample solu-
tion obtained in the Identification (2) add 0.3 mL of
phenolphthalein TS: the solution is colorless, and develops a
red color on addition of 0.3 mL of 0.01 mol/L sodium
hydroxide VS.
(3) Sulfite — Dissolve 5.0 g of Sucrose in 40 mL of water,
add 2.0 mL of dilute sodium hydroxide TS and water to make
exactly 50 mL, and use this solution as the sample solution.
Separately, dissolve 0.076 g of sodium disulfite in water to
make exactly 50 mL, then pipet 5 mL of this solution, add
water to make exactly 100 mL. Pipet 3 mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Immediately, pipet 10 mL each of the
sample solution and the standard solution, add 1.0 mL of 3
mol/L hydrochloric acid, 2.0 mL of decolorized fuchsin TS
and 2.0 mL of formaldehyde solution TS, and allow to stand
for 30 minutes. Determine the absorbance at 583 nm of these
solutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> using the control solution obtained by proceed-
ing with 10.0 mL of water in the same manner as above: the
absorbance of the sample solution is not larger than that of
the standard solution (not more than 15 ppm as S0 2 ). When
the standard solution does not show a red-purple to blue-pur-
ple color, result of the test is invalid.
(4) Lead — Put exactly 50 mg of Sucrose in a poly-
tetrafuruoroethylene decomposition-vessel, add 0.5 mL of
nitric acid to dissolve, seal up the vessel, and heat at 150°C
for 5 hours. After cooling, add water to make exactly 5 mL,
and use this solution as the sample solution. Perform the test
with more than 3 parts of the sample solution as directed in
the standard addition method under Atomic Absorption
Spectrophotometry <2.23> (electrothermal type) according to
the following conditions. The standard solution is prepared
by adding water to a suitable volume of Standard Lead Solu-
tion exactly volumed, and perform a blank determination
with a solution prepared by adding water to 10.0 mL of nitric
acid to make exactly 100 mL, and make any necessary correc-
tion (not more than 0.5 ppm).
Operating conditions —
Lamp: A hollow cathode lamp
Wavelengh: 283.3 mm
Temperature for drying: 110°C
Temperature for incineration: 600°C
Temperature for atomization: 2100°C
(5) Invert sugar — Transfer 5 mL of the sample solution
obtained in the Identification (2) to a test-tube about 150 mm
long and about 16 mm in diameter, add 5 mL of water, 1.0
mL of 1 mol/L sodium hydroxide VS and 1.0 mL of methy-
JP XV
Official Monographs / White Soft Sugar 1121
lene blue TS, mix, and place in a water bath. After exactly 2
minutes, take the tube out of the bath, and examine the solu-
tion immediately: the blue color does not disappear com-
pletely (0.04%). Ignore any blue color at the air and solution
interface.
Conductivity
(i) Potassium chloride conductivity calibration standard
solution — Dissolve powdered potassium chloride, previously
dried at 500 - 600°C for 4 hours, in newly distillated water
having less conductivity than 2//S-cm _1 to get three kinds of
the standard solution containing 0.7455 g, 0.0746 g and
0.0149 g of potassium chloride in 1000.0 g, respectively. The
conductivities of these solutions at 20°C are shown in the fol-
lowing table.
Standard solution
Conductivity
Resistivity
(g/1000.0 g)
CMS-cm" 1 )
(Q ■ cm)
0.7455
1330
752
0.0746
133.0
7519
0.0149
26.6
37594
Xo (fiS-cm l ) = JG
Determine the corrected conductivity, Xc> of the sample
solution by the following expression: not more than
35^8-cm- 1 .
Xc (^S-cm- 1 ) = ^T-0.35^
Loss on drying <2.41> Not more than 0.1% (2 g, 105°C,
3 hours).
Dextrins For Sucrose used to prepare large volume aqueous
infusions, to 2 mL of the sample solution obtained in the
Identification (2) add 8 mL of water, 0.05 mL of dilute
hydrochloric acid and 0.05 mL of iodine TS: the solution
remains yellow.
Bacterial endotoxins <4.01> Less than 0.25 EU/mg, for Su-
crose exclusively to be used to prepare Injections for in-
travenous infusion of larger volume.
Containers and storage Containers — Well-closed contain-
ers.
(ii) Apparatus — Use an appropriate conductivity meter.
The conductivity is determined to measure the electrical
resistance of the column of liquid between the electrodes of
the immersed measuring device (conductivity cell). The ap-
paratus is supplied with alternative current to avoid the
effects of electrode polarization. It is usually equipped with a
temperature compensation device. The conductivity cell con-
tains of two parallel platinum electrodes coated with plati-
num black, and both electrodes are generally protected by a
glass tube which allows good exchange between the solution
and the electrodes. Use a cell giving the cell constant of 0.01
to 1 cm -1 .
(iii) Procedure — Use the suitable potassium chloride con-
ductivity calibration standard solution to the measurement.
After washing the well with water, rinse 2 to 3 times with the
calibration standard solution, fill up the cell with the calibra-
tion standard solution, and determine the conductivity of the
calibration standard solution kept at 20±0.1°C.
Repeat the determination, and measure the conductivity of
the calibration standard solution, G Xo (/xS), after a stable
reading of ± 3% is obtained. The cell constant, /, is calculat-
ed by the following:
t _ Xkci
G Xo
J: Cell constant (cm -1 )
^ KC1 : Conductivity constant of the potassium chloride con-
ductivity calibration standard solution (//S-cm -1 )
(20°C)
G Xi> : Conductivity measured (//S)
Dissolve 31.3 g of Sucrose in newly distillated water to
make exactly 100 mL, and use this solution as the sample so-
lution. After washing well the cell with water, rinse the cell
with the sample solution 2 to 3 times , fill up with the sample
solution, and determine the conductivity of the sample solu-
tion, G T (/uS), kept at 20 ± 0.1 °C, while stirring. Determine
the conductivity of the water used for preparation of the sam-
ple solution, G (jiS), in the same manner as above, and cal-
culate the conductivity, Xt (/US-cm -1 ) and Xo (^S-cm -1 ), by
the following expressions:
X T (^S-cm-») = /G T
White Soft Sugar
C 12 H 22 0„: 342.30
/?-D-Fructofuranosyl a-D-glucopyranoside [57-50-1]
Description White Soft Sugar is colorless or white crystals
or crystalline powder. It is odorless and has a sweet taste.
It is very soluble in water, very slightly soluble in ethanol
(95), and practically insoluble in diethyl ether.
A solution of White Soft Sugar (1 in 10) is neutral.
Identification (1) When 1 g of White Soft Sugar is ignited,
it melts and swells, and decomposes, emitting an odor of
caramel, to bulky charcoal.
(2) To 0. 1 g of White Soft Sugar add 2 mL of dilute sul-
furic acid, boil, add 4 mL of sodium hydroxide TS and 3 mL
of Fehling's TS, and heat to boiling: a red to dark red
precipitate is produced.
Optical rotation <2.49> [a]™: +65.0- +67.0° (after
drying, 13 g, water, 50 mL, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 100 g
of White Soft Sugar in 100 mL of water, take 50 mL of this
solution in a Nessler tube, and view transversely the Nessler
tube against a white background: the solution is colorless or
only slightly yellow and has no blue color. Fill the solution in
the Nessler tube, stopper, and allow to stand for 2 days: no
precipitate is produced.
(2) Chloride <1.03>— To 10.0 g of White Soft Sugar add
water to make 100 mL, and use this solution as the sample so-
1 122 Sulbactam Sodium / Official Monographs
JP XV
lution. To 20 mL of the sample solution add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more
than 0.005%).
(3) Sulfate <1.14> — To 40 mL of the sample solution ob-
tained in (2) add 1 mL of dilute hydrochloric acid and water
to make 50 mL. Perform the test using this solution as the
test solution. Propare the control solution with 0.50 mL of
0.005 mol/L sulfuric acid VS (not more than 0.006%).
(4) Calcium — To 10 mL of the sample solution obtained
in (2) add 1 mL of ammonium oxalate TS: this solution
shows immediately no change.
(5) Heavy metals <1.07>— Proceed with 5.0 g of White
Soft Sugar according to Method 1, and perform the test. Pre-
pare the control solution with 2.5 mL of Standard Lead Solu-
tion (not more than 5 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of White Soft Sugar according to Method 1, and perform the
test (not more than 2 ppm).
(7) Invert sugar — Dissolve 5.0 g of White Soft Sugar in
water to make 100 mL, filter if necessary, and use this solu-
tion as the sample solution. Separately place 100 mL of alka-
line copper (II) sulfate solution in a 300-mL beaker, cover the
beaker with a watch glass, and boil. Immediately add 50.0
mL of the sample solution, boil the mixture exactly for 5
minutes, add at once 50 mL of freshly boiled and cooled
water, dip it in a water bath of a temperature below 10°C for
5 minutes, and collect the precipitate in a tared glass filter
(G4). Wash the residue on the filter with water until the last
washing is neutral, then wash with 10 mL of ethanol (95), add
10 mL of diethyl ether, and dry at 105 °C for 30 minutes: the
mass of the residual precipitate is not more than 0.120 g.
Loss on drying <2.41> Not more than 1.30% (15 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (2 g).
Containers and storage Containers — Well-closed contain-
ers.
Sulbactam Sodium
TJL-M^A^r- U^7A
,C0 2 Na
CH :!
C 8 H 10 NNaO 5 S: 255.22
Monosodium (2S,5R )-3, 3-dimethyl-7-oxo-4-thia-
l-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide
[69388-84-7]
Sulbactam Sodium contains not less than 875 fig
(potency) per mg, calculated on the anhydrous basis.
The potency of Sulbactam Sodium is expressed as mass
(potency) of sulbactam (C 8 H„N0 5 S: 233.24).
Description Sulbactam Sodium occurs as a white to yellow-
ish white crystalline powder.
It is freely soluble in water, sparingly soluble in methanol,
very slightly soluble in ethanol (99.5), and practically insolu-
ble in acetonitrile.
Identification (1) Determine the infrared absorption spec-
trum of Sulbactam Sodium as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) Sulbactam Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [a]^: +219- +233° (1 g, water,
100 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Sulbactam Sodium in 20 mL of
water: the pH of the solution is between 5.2 and 7.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sulbactam Sodium in 20 mL of water: the solution is clear,
and colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Sulbac-
tam Sodium according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sulbactam Sodium as directed in Method 3, and perform
the test (not more than 2 ppm).
(4) Sulbactam penicillamine — Weigh accurately about
0.2 g of Sulbactam Sodium, dissolve in the mobile phase to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 40 mg of sulbactam
sodium for sulbactam penicillamine, dissolve in 2 mL of
water, add 0.5 mL of sodium hydroxide TS, allow to stand
for 10 minutes at a room temperature, and add 0.5 mL of 1
mol/L hydrochloric acid TS, then add the mobile phase to
make exactly 100 mL. Pipet 5 mL of this solution, add the
mobile phase to make exactly 50 mL, and use this solution as
the standard solution. Perform the test with exactly 10 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of sulbactam penicillamine by the automatic integration
method: the amount of sulbactam penicillamine is not more
than 1.0%.
Amount (%) of sulbactam penicillamine
= (W s /W T )x(A 1 /A s )x 5
W s : Amount (mg) of sulbactam sodium for sulbactam
penicillamine
Wj\ Amount (mg) of the sample
Operating conditions —
Column, column temperature, mobile phase, and flow
rate: Proceed as directed in the operating conditions in the
Assay.
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
System suitability —
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of sulbactam penicillamine is not more than 2.0%.
JPXV
Official Monographs / Sulbenicillin Sodium 1123
Water <2.48> Not more than 1.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Sulbactam Sodium
and Sulbactam Reference Standard, equivalent to about 0.1 g
(potency), dissolve each in a suitable amount of the mobile
phase, add exactly 10 mL of the internal standard solution,
then add the mobile phase to make 100 mL, and use these so-
lutions as the sample solution and standard solution. Per-
form the test with 10 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, Q T and Q s , of the peak area of sulbactam to that
of the internal standard.
Amount [/<g (potency)] of sulbactam (C 8 H u N0 5 S)
= W s x(Q T /Q s )x 1000
fV s : Amount [mg (potency)] of Sulbactam Reference
Standard
Internal standard solution — A solution of ethyl parahydrox-
ybenzoate in the mobile phase (7 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: To 750 mL of 0.005 mol/L tetrabutylam-
monium hydroxide TS add 250 mL of acetonitrile for liquid
chromatography.
Flow rate: Adjust the flow rate so that the retention time of
sulbactam is about 6 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, sulbactam and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of sulbactam is not more than 1.0%.
Containers and storage Containers — Tight containers.
Sulbenicillin Sodium
XM-yU >i-Y U^A
1 COjNa
- S ^CH 3
N+
C 16 H 16 N 2 Na 2 7 S 2 : 458.42
Disodium (2S,5.R,6.R)-3,3-dimethyl-7-oxo-6-[(2.R)-2-phenyl-
2-sulfonatoacetylamino]-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate [28002-18-8]
Sulbenicillin Sodium contains not less than 900 tig
(potency) and not more than 970 tig (potency) per mg,
calculated on the anhydrous basis. The potency of Sul-
benicillin Sodium is expressed as mass (potency) of sul-
benicillin (C 16 H 18 N 2 7 S 2 : 414.45).
Description Sulbenicillin Sodium occurs as white to light
yellowish white powder.
It is very soluble in water, freely soluble in methanol, and
slightly soluble in ethanol (99.5).
It is hygroscopic.
Identification (1) Determine the infrared absorption spec-
trum of Sulbenicillin Sodium as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Sulbenicillin Sodium Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(2) Sulbenicillin Sodium responds to the Qualitative Tests
<1.09> (1) for sodium salt.
Optical rotation <2.49> [a]g>: + 167 - + 182° (1 g calculated
on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.20
g of Sulbenicillin Sodium in 10 mL of water is between 4.5
and 7.0.
Purity (1) Clarity and color of solution — Dissolve 2.5 g of
Sulbenicillin Sodium in 5 mL of water: the solution is clear
and colorless to pale yellow.
(2) Heavy metals <1. 07>— Proceed with 1.0 g of Sul-
benicillin Sodium according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sulbenicillin Sodium according to Method 1, and perform
the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Sulbenicillin
Sodium in 15 mL of the mobile phase, and use this solution
as the sample solution. Perform the test with 10 /xL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, determine each
peak area by the automatic integration method, and calculate
the amount of these peaks by the area percentage method: the
amount of the each peak other than the two peaks of sul-
benicillin is not more than 2.0%, and the total amount of the
peaks other than the two peaks of sulbenicillin is not more
than 5.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 10 g of potassium dihydrogen
phosphate in 750 mL of water, adjust the pH to 6.0 ± 0.1
with sodium hydroxide TS, and add water to make 1000 mL.
To 940 mL of this solution add 60 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
1 124 Sulfadiazine Silver / Official Monographs
JP XV
the lately eluted peak of sulbenicillin is about 18 minutes.
Time span of measurement: About 1.5 times as long as the
retention time of the lately eluted peak of sulbenicillin begin-
ning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the sample solution, add the mobile phase to make exactly
100 mL, and use this solution as the solution for system
suitability test. Pipet 1 mL of the solution for system suitabil-
ity test, and add the mobile phase to make exactly 10 mL.
Confirm that the total area of the two peaks of sulbenicillin
obtained from 10,mL of this solutionis equivalent to 7 to 13%
of that from 10 /xL of the solution for system suitability test.
System performance: When the procedure is run with 10
/uL of the sample solution under the above operating condi-
tions, the resolution between the two peaks of sulbenicillin is
not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the total areas of the two peaks of sulbenicillin is not more
than 5.0%.
Water <2.48> Not more than 6.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er. Adjust the pH of the medium so that it will be 6.4 to 6.6
after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Sulbenicillin Sodium Reference Standard, equivalent to
about 50 mg (potency), dissolve in phosphate buffer solution,
pH 6.0 to make exactly 50 mL, and use this solution as the
standard stock solution. Keep the standard stock solution in
a freezer, and use within 4 days. Take exactly a suitable
amount of the standard stock solution before use, add phos-
phate buffer solution, pH 6.0 to make solutions so that each
mL contains 40 /xg (potency) and 10 fig (potency), and use
these solutions as the high concentration standard solution
and low concentration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Sulbenicillin Sodium, equivalent to about 50 mg (potency),
and dissolve in phosphate buffer solution, pH 6.0 to make ex-
actly 50 mL. Take exactly a suitable amount of this solution,
add phosphate buffer solution, pH 6.0 to make solutions so
that each mL contains 40 /xg (potency) and 10 /ug (potency),
and use these solutions as the high concentration sample solu-
tion and low concentration sample solution, respectively.
Containers and storage Containers — Hermetic containers.
Sulfadiazine Silver
X^/y/y^
XT
0..0
HT^*
A
Aa
C 10 H 9 AgN 4 O 2 S: 357.14
Monosilver 4-amino-A L (pyrimidin-2-yl)-
benzenesulfonamidate [22199-08-2]
Sulfadiazine Silver, when dried, contains not less
than 99.0% and not more than 102.0%, of
C 10 H 9 AgN 4 O 2 S.
Description Sulfadiazine Silver occurs as a white to pale
yellow, crystalline powder. It is odorless.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
It dissolves in ammonia TS.
It is gradually colored by light.
Melting point: about 275°C (with decomposition).
Identification Determine the infrared absorption spectrum
of Sulfadiazine Silver, previously dried, as directed in the
paste method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of previously dried Sulfadiazine Silver Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Purity (1) Nitrate— To 250 mL of water add 1 .0 g of Sul-
fadiazine Silver, shake well for 50 minutes, filter, and use this
filtrate as the sample solution. Separately, weigh accurately
0.25 g of potassium nitrate, and dissolve in water to make ex-
actly 2000 mL. Pipet 5 mL of this solution and add water to
make exactly 200 mL, and use this solution as the standard
solution. Pipet 2.0 mL each of the sample solution and the
standard solution, and add 5 mL of a solution of cromotrop-
ic acid in sulfuric acid (1 in 10,000) and sulfuric acid to make
exactly 10 mL. Determine the absorbances, A T and A s , of the
sample solution and standard solution at 408 nm as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using a
solution, prepared with exactly 2.0 mL of water in the same
manner, as the blank: A T is not larger than A s (not more than
0.05%).
(2) Related substances — Dissolve 50 mg of Sulfadiazine
Silver in 5 mL of a mixture of ethanol (95) and ammonia so-
lution (28) (3:2), and use this solution as the sample solution.
Pipet 2 mL of the sample solution, and add a mixture of
ethanol (95) and ammonia solution (28) (3:2) to make exactly
20 mL. Pipet 2 mL of this solution, add a mixture of ethanol
(95) and ammonia solution (28) (3:2) to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, methanol and ammonia solu-
tion (28) (10:5:2) to a distance of about 15 cm, and air-dry the
JP XV
Official Monographs / Sulfamethizole 1125
plate. Examine under ultraviolet light (main wavelength: 254
nm): the spots other than the principal spot and spot of the
starting point from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
phosphorus (V) oxide, 80%, 4 hours).
Residue on ignition <2.44> 41.0 - 45.0% (1 g).
Silver content Weigh accurately about 50 mg of Sulfadia-
zine Silver, previously dried, dissolve in 2 mL of nitric acid,
and add water to make exactly 100 mL. Pipet 1 mL of this so-
lution, add water to make exactly 100 mL, and use this solu-
tion as the sample solution. Measure accurately a suitable
quantity of Standard Silver Solution for Atomic Absorption
Spectrophotometry <2.23>, dilute with water to make a solu-
tion containing 1.0 to 2.0 ng of silver (Ag:107.87) per ml, and
use this solution as the standard solution. Perform the test
with the sample solution and the standard solution as direct-
ed under Atomic Absorption Spectrophotometry <2.23> ac-
cording to the following conditions, and calculate the silver
content of the sample solution from the calibration curve ob-
tained from the absorbance of the standard solution: it con-
tains not less than 28.7% and not more than 30.8% of silver.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A silver hollow cathode lamp
Wavelength: 328.1 nm
Assay Weigh accurately about 0.1 g each of Sulfadiazine
Silver and Sulfadiazine Silver Reference Standard, each
previously dried, and add ammonia TS to make exactly 100
mL, respectively. Pipet 1 mL each of these solutions, add
water to make exactly 100 mL, and use these solutions as the
sample solution and the standard solution, respectively. De-
termine the absorbances, A T and A s , of the sample solution
and the standard solution at 255 nm, as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using a solu-
tion, prepared with exactly 1 mL of ammonia TS and a
sufficient water to make exactly 100 mL, as the blank.
Amount (mg) of C 10 H 9 AgN 4 O 2 S
= W s x(A T /A s )
W s : Amount (mg) of Sulfadiazine Silver
Reference Standard
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Sulfamethizole
XJU7 7 *3-V-)\,
o o s-"i
H N
h,N
C 9 H 10 N4O 2 S 2 : 270.33
4-Amino-A^-(5-methyl-l,3,4-thiadiazol-2-yl)-
benzenesulfonamide [144-82-1]
Sulfamethizole, when dried, contains not less than
99.0% of C 9 H 10 N 4 O 2 S 2 .
Description Sulfamethizole occurs as white to yellowish
white crystals or crystalline powder. It is odorless.
It is slightly soluble in ethanol (95), and in acetic acid (100)
and practical insoluble in water and in diethyl ether.
It dissolves in dilute hydrochloric acid and in sodium
hydroxide TS.
It is gradually colored by light.
Identification Determine the infrared absorption spectrum
of Sulfamethizole, previously dried, as directed in the potas-
sium bromide disk method under Infrared Spectrophotomet-
ry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Melting point <2.60> 208 - 211°C
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Sulfamethizole in 3 mL of sodium hydroxide TS and 20 mL
of water: the solution is clear and colorless.
(2) Acidity— To 1.0 g of Sulfamethizole add 50 mL of
water, warm at 70°C for 5 minutes, allow to stand for 1 hour
in an ice bath, and filter. To 25 mL of the filtrate add 2 drops
of methyl red TS and 0.60 mL of 0.1 mol/L sodium
hydroxide VS: a yellow color develops.
(3) Heavy metals <1. 07>— Proceed with 1.0 g of Sul-
famethizole according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sulfamethizole according to Method 3, and perform the
test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Sulfamethi-
zole in acetone to make 10 mL, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add ace-
tone to make exactly 50 mL, then pipet 5 mL of this solution,
add acetone to make exactly 20 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 5
/uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate and acetic acid (100) (20:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Sulfamethizole,
previously dried, dissolve in 5 mL of hydrochloric acid and
50 mL of water, add 10 mL of a solution of potassium
bromide (3 in 10), cool below 15°C, and titrate <2.50> with
0.1 mol/L sodium nitrite VS according to the potentiometric
titration method or the amperometric titration method.
Each mL of 0.1 mol/L sodium nitrite VS
= 27.03 mg of C 9 H 10 N4O 2 S 2
Containers and storage Containers — Well-closed contain-
1126 Sulfamethoxazole / Official Monographs
JP XV
Storage — Light-resistant.
Sulfamethoxazole
Sulfisomezole
7,)V~Jt * h^-tf-V-JU
o o
jCn
doHnNjOjS: 253.28
4-Amino-A r -(5-methylisoxazol-3-yl)benzenesulfonamide
[723-46-6]
Sulfamethoxazole, when dried,
than 99.0% of QqHuNjOjS.
contains not less
Description Sulfamethoxazole occurs as white crystals or
crystalline powder. It is odorless, and has a slightly bitter
taste.
It is very soluble in A^Af-dimethylformamide, sparingly
soluble in ethanol (95), slightly soluble in diethyl ether, and
very slightly soluble in water.
It dissolves in sodium hydroxide TS.
It is gradually colored by light.
Identification Determine the infrared absorption spectrum
of Sulfamethoxazole, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Melting point <2.60> 169 - 172°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sulfamethoxazole in 5 mL of sodium hydroxide TS, and add
20 mL of water: the solution is clear and colorless.
(2) Acidity — To 1.0 g of Sulfamethoxazole add 50 mL of
water, heat at 70°C for 5 minutes, allow to stand in ice water
for 1 hour, and filter. To 25 mL of the filtrate add 2 drops of
methyl red TS and 0.60 mL of 0.1 mol/L sodium hydroxide
VS: a yellow color develops.
(3) Heavy metals <1.07>— Proceed with 1.0 g of Sul-
famethoxazole according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(4) Arsenic </.//> — Prepare the test solution with 1.0 g
of Sulfamethoxazole according to Method 3, and perform
the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.20 g of Sulfamethox-
azole in 10 mL of a solution of ammonia solution (28) in
methanol (1 in 50), and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add a solution of
ammonia solution (28) in methanol (1 in 50) to make exactly
10 mL. Pipet 1 mL of this solution, add a solution of ammo-
nia solution (28) in methanol (1 in 50) to make exactly 20 mL,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 ,uL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, acetonitrile and diluted am-
monia solution (28) (7 in 100) (10:8:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41>
4 hours).
Not more than 0.5% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Sulfamethoxazole,
previously dried, dissolve in 30 mL of A^TV-dimethylfor-
mamide, add 10 mL of water, and titrate <2.50> with 0.1 mol
/L sodium hydroxide VS until a light blue color is produced
(indicator: 0.5 mL of thymolphthalein TS). Separately, per-
form a blank determination in the same manner with a mix-
ture of 30 mL of A^N-dimethylformamide and 26 mL of
water, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 25.33 mg of doHnNaOaS
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Sulfamonomethoxine Hydrate
X)\,7t=E/ / r-+v>7Kfttl
„CH 3
°w
N
H
J
■ HjP
CiiH 12 N 4 3 S.H 2 0: 298.32
4-Amino-A f -(6-methoxypyrimidin-4-yl)benzenesulfonamide
monohydrate [1220-83-3, anhydride]
Sulfamonomethoxine Hydrate, when dried, contains
not less than 99.0% of sulfamonomethoxine (C 11 H 12 N 4
3 S: 280.31).
Description Sulfamonomethoxine Hydrate occurs as white
to pale yellow crystals, granules or crystalline powder. It is
odorless.
It is soluble in acetone, slightly soluble in ethanol (95), very
slightly soluble in diethyl ether, and practically insoluble in
water.
It dissolves in dilute hydrochloric acid and in sodium
hydroxide TS.
It is gradually colored by light.
Identification Determine the infrared absorption spectrum
of Sulfamonomethoxine Hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
JPXV
Official Monographs / Sulfinpyrazone 1127
Melting point <2.60> 204 - 206 °C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sulfamonomethoxine Hydrate in 5 mL of sodium hydroxide
TS and 20 mL of water: the solution is clear and colorless to
pale yellow. Dissolve 0.5 g of Sulfamonomethoxine Hydrate
in 5 mL of sodium hydroxide TS, and heat: no turbidity is
produced. After cooling, add 5 mL of acetone: the solution is
clear.
(2) Heavy metals <1.07>— Proceed with 1.0 g of Sul-
famonomethoxine Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Sulfamonomethoxine Hydrate according to Method 3,
and perform the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.02 g of Sul-
famonomethoxine Hydrate in ethanol (95) to make 10 mL,
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add ethanol (95) to make exactly 200
mL, and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 5 /xL each of the sample solu-
tion and standard solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol and ammonia solution (28)
(4:1) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 254 nm): the
spots other than the principal spot from the sample solution
are not more intense than the spot from the standard solu-
tion.
Loss on drying <2.41> 4.5 - 6.5% (1 g, 105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.10% (1 g).
Assay Weigh accurately about 0.5 g of Sul-
famonomethoxine Hydrate, previously dried, dissolve in 5
mL of hydrochloric acid and 50 mL of water, add 10 mL of a
solution of potassium bromide (3 in 10), cool below 15°C,
and titrate <2.50> with 0.1 mol/L sodium nitrite VS (poten-
tiometric titration or amperometric titration).
Each mL of 0.1 mol/L sodium nitrite VS
= 28.03 mg of C„H 12 N 4 03S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Sulfinpyrazone
7JU7 -f >b°v'/>
C 23 H 2 „N 2 3 S: 404.48
l,2-Diphenyl-4-[2-(phenylsulfinyl)ethyl]pyrazolidine-3,5-
dione [57-96-5]
Sulfinpyrazone, when dried, contains not less than
98.5% of C 23 H 2 oN 2 3 S.
Description Sulfinpyrazone occurs as a white to pale yel-
lowish white powder. It is odorless, and has a bitter taste.
It is freely soluble in acetic acid (100) and in acetone, solu-
ble in ethanol (95), slightly soluble in diethyl ether, and prac-
tically insoluble in water.
It dissolves in sodium hydroxide TS.
Melting point: about 138°C (with decomposition).
Identification (1) Dissolve 2 mg of Sulfinpyrazone in 1 mL
of acetic acid (100), add 1 mL of palladium (II) chloride TS
and 2 mL of chloroform, and shake: a yellow color develops
in the chloroform layer.
(2) Determine the absorption spectrum of a solution of
Sulfinpyrazone in 0.01 mol/L sodium hydroxide TS (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Sulfinpyrazone
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Sulfin-
pyrazone, previously dried, as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Sulfinpyrazone in 10 mL of acetone: the solution is clear and
colorless. Dissolve 0.5 g of Sulfinpyrazone in 10 mL of sodi-
um hydroxide TS: the solution is clear and colorless.
(2) Heavy metals <1. 07>— Proceed with 2.0 g of Sulfin-
pyrazone according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sulfinpyrazone according to Method 3, and perform the
test (not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Sulfinpyra-
zone in 5 mL of acetone, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add acetone to
make exactly 100 mL, and use this solution as the standard
solution (1). Pipet 1 mL of the sample solution, add acetone
to make exactly 200 mL, and use this solution as the standard
solution (2). Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot rapidly 5 /uL
each of the sample solution and standard solutions (1) and (2)
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography under a stream of nitrogen. Develop the
plate with a mixture of chloroform and acetic acid (100) (4:1)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the most
intense spot other than the principal spot from the sample so-
lution is not more intense than the spot from the standard so-
lution (1), and the spots other than the principal and above
spots from the sample solution are not more intense than the
spot from the standard solution (2).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
1 128 Sulfinpyrazone Tablets / Official Monographs
JP XV
Assay Weigh accurately about 0.5 g of Sulfinpyrazone,
previously dried, dissolve in 40 mL of acetone, add 40 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 40.45 mg of C 23 H 20 N 2 O 3 S
Containers and storage Containers — Well-closed contain-
ers.
Sulfinpyrazone Tablets
Sulfisoxazole
Sulfafurazole
*l
CH,
CH,
CuHoNjOjS: 267.30
4-Amino-iV-(3,4-dimethylisoxazol-5-yi)benzene-
sulfonamide [127-69-5]
Sulfinpyrazone Tablets contain not less than 93%
and not more than 107% of the labeled amount of sul-
finpyrazone (C 23 H 20 N 2 O 3 S: 404.48).
Sulfisoxazole, when dried,
99.0% of C 11 H 1 3N 3 3 S.
contains not less than
Method of preparation
with Sulfinpyrazone.
Prepare as directed under Tablets,
Identification (1) Weigh a portion of powdered Sulfin-
pyrazone Tablets, equivalent to 2 mg of Sulfinpyrazone ac-
cording to the labeled amount, add 1 mL of acetic acid (100),
and shake. To this solution add 1 mL of palladium (II) chlo-
ride TS and 2 mL of chloroform, and shake: a yellow color
develops in the chloroform layer.
(2) Determine the absorption spectrum of the sample so-
lution obtained in the Assay as directed under Ultraviolet-
visible Spectrophotometry <2.24>: it exhibits a maximum be-
tween 257 nm and 261 nm.
Dissolution Being specified separately.
Assay Weigh accurately, and powder not less than 20 Sul-
finpyrazone Tablets. Weigh accurately a portion of the pow-
der, equivalent to about 50 mg of sulfinpyrazone
(C23H20N2O3S), add 25 mL of methanol, and shake for 15
minutes. To this solution add 50 mL of sodium hydroxide
TS, shake, cool, and add water to make exactly 200 mL. Af-
ter centrifuging, pipet 4 mL of the supernatant liquid, add
water to make exactly 100 mL, and use this solution as the
sample solution. Separately, weigh accurately about 0.05 g of
Sulfinpyrazone Reference Standard, previously dried at
105 °C for 2 hours, dissolve in 25 mL of methanol, add 50 mL
of sodium hydroxide TS, and cool. To this solution add
water to make exactly 200 mL, pipet 4mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Determine the absorbances, A T and
A s , of these solutions at 260 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>.
Amount (mg) of sulfinpyrazone (C23H20N2O3S)
= W s x(A T /A s )
W s : Amount (mg) of Sulfinpyrazone
Reference Standard
Containers and storage Containers — Well-closed contain-
ers.
Description Sulfisoxazole occurs as white crystals or crys-
talline powder. It is odorless, and has a slightly bitter taste.
It is freely soluble in pyridine and in M-butylamine, soluble
in methanol, sparingly soluble in ethanol (95), slightly soluble
in acetic acid (100), and very slightly soluble in water and in
diethyl ether.
It dissolves in dilute hydrochloric acid, in sodium
hydroxide TS and in ammonia TS.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Sulfisoxazole in 1 mL
of dilute hydrochloric acid and 4 mL of water: the solution
responds to the Qualitative Tests <1.09> for primary aromatic
amines.
(2) Dissolve 0.02 g of Sulfisoxazole in 5 mL of water and
1 mL of n-butylamine, add 2 to 3 drops of copper (II) sulfate
TS, and shake well. Add 5 mL of chloroform, shake, and al-
low to stand: a blue-green color develops in the chloroform
layer.
(3) Dissolve 0.01 g of Sulfisoxazole in 1 mL of pyridine,
add 2 drops of copper (II) sulfate TS, and shake. Add 3 mL
of water and 5 mL of chloroform, shake, and allow to stand:
a light yellow-brown color develops in the chloroform layer.
(4) To 0.5 g of Sulfisoxazole add 2 mL of acetic acid
(100), dissolve by heating under a reflux condenser, add 1 mL
of acetic anhydride, and boil for 10 minutes. Add 10 mL of
water, cool, and alkalize with about 7 mL of a solution of so-
dium hydroxide (3 in 10). Filter, if necessary, immediately
acidify by adding acetic acid (100) dropwise, collect the
produced precipitate, recrystallize from methanol, and dry at
105 C C for 1 hour: the crystals melt <2.60> between 208°C and
210°C.
Melting point <2.60> 192 - 196°C (with decomposition).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Sulfisoxazole in 5 mL of sodium hydroxide TS and 20 mL of
water: the solution is clear and colorless to pale yellow.
(2) Acidity— To 1.0 g of Sulfisoxazole add 50 mL of
water, warm at 70°C for 5 minutes, allow to stand in an ice
bath for 1 hour, and filter. To 25 mL of the filtrate add 2
drops of methyl red TS and 0.20 mL of 0.1 mol/L sodium
hydroxide VS: a yellow color develops.
(3) Heavy metals <1.07> — Proceed with 1.0 g of Sulfisox-
azole according to Method 2, and perform the test. Prepare
JPXV
Official Monographs / Sulfobromophthalein Sodium 1129
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
Loss on drying <2.41> Not more than 0.5% (2 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Sulfisoxazole, previ-
ously dried, dissolve in 50 mL of methanol by warming, cool
and titrate <2.50> with 0.2 mol/L sodium hydroxide VS (indi-
cator: 3 drops of phenolphthalein TS). Perform a blank de-
termination using a mixture of 50 mL of methanol and 18 mL
of water, and make any necessary correction.
Each mL of 0.2 mol/L sodium hydroxide VS
= 53.46 mg of CnHuNjOaS
Containers and storage Containers — Well-closed contain-
ers.
Sulfobromophthalein Sodium
xiifcifn^y? i>-\ >i- V U ^7A
SO,Na
Na0 3 S
C 20 H 8 Br 4 Na 2 O 10 S 2 : 838.00
Disodium 5,5'-(4,5,6,7-tetrabromo-3-oxo-l,3-dihydro-
isobenzofuran-l,l-diyl)bis(2-hydroxybenzenesulfonate)
[71-67-0]
Sulfobromophthalein Sodium, when dried, contains
not less than 96.0% and not more than 104.0% of
C2oH 8 Br 4 Na 2 10 S 2 .
Description Sulfobromophthalein Sodium occurs as a
white, crystalline powder. It is odorless.
It is soluble in water, and practically insoluble in ethanol
(95) and in diethyl ether.
It is hygroscopic.
Identification (1) Dissolve 0.02 g of Sulfobromo-
phthalein Sodium in 10 mL of water, and add 1 mL of sodi-
um carbonate TS: a blue-purple color is produced. Add 1 mL
of dilute hydrochloric acid to the solution: the color of the
solution disappears.
(2) Transfer 0.2 g of Sulfobromophthalein Sodium to a
porcelain crucible, mix well with 0.5 g of anhydrous sodium
carbonate, and ignite until the mixture is charred. After cool-
ing, add 15 mL of hot water to the residue, heat for 5 minutes
on a water bath, filter, and render the filtrate slightly acid
with hydrochloric acid: the solution responds to the Qualita-
tive Tests <1.09> for bromide, and the Qualitative Tests
<1.09> (1) and (2) for sulfate.
(3) Sulfobromophthalein Sodium responds to the
Qualitative Tests <1.09> (1) for sodium salt.
pH <2.54> The pH of a solution of 1.0 g of Sul-
fobromophthalein Sodium in 20 mL of water is between 4.0
and 5.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Sulfobromophthalein Sodium in 10 mL of water: the solu-
tion is clear and colorless to pale yellow.
(2) Chloride <1.03>— Perform the test with 2.0 g of Sul-
fobromophthalein Sodium. Prepare the control solution with
0.10 mL of 0.01 mol/L hydrochloric acid VS (not more than
0.002%).
(3) Sulfate — To 10 mL of a solution of Sul-
fobromophthalein Sodium (1 in 500) add 5 drops of dilute
hydrochloric acid, heat to boil, and add 1 mL of hot barium
chloride TS: the solution is clear when observed 1 minute af-
ter the addition of the barium chloride TS.
(4) Calcium — Weigh accurately about 5 g of Sul-
fobromophthalein Sodium, transfer to a porcelain dish, heat
gently to char, and heat strongly between 700°C and 750°C
until the residue is incinerated. After cooling, add 10 mL of
dilute hydrochloric acid, and heat for 5 minutes on a water
bath. Transfer the contents to a flask with 50 mL of water,
and add 5 mL of 8 mol/L potassium hydroxide TS and 0.1 g
of NN indicator. Titrate <2. 50> with 0.01 mol/L disodium di-
hydrogen ethylenediamine tetraacetate VS until the red-pur-
ple color of the solution changes to blue.
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 0.4008 mg of Ca
The content of calcium (Ca: 40.08) is not more than
0.05%.
(5) Heavy metals <1. 07>— Proceed with 1.0 g of Sul-
fobromophthalein Sodium according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(6) Arsenic <1.11> — Transfer 0.65 g of Sulfobromo-
phthalein Sodium to a crucible, add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 50), fire
to burn, then heat gently until the residue is incinerated. If
any carbon remains, moisten the residue with a small amount
of nitric acid, and incinerate again by ignition. After cooling,
add 10 mL of dilute sulfuric acid, and heat until white fumes
are evolved. After cooling, add 5 mL of water to the residue,
and perform the test with this solution as the test solution
(not more than 3.1 ppm).
Loss on drying <2.41> Not more than 5.0% (0.5 g, 105°C,
3 hours).
Residue on ignition <2.44>
700-750°C).
14 - 19% (after drying, 0.5 g,
Assay Dissolve about 0.1 g of Sulfobromophthalein Sodi-
um, previously dried and accurately weighed, in water to
make exactly 500 mL. Pipet 5 mL of this solution, and add a
solution of anhydrous sodium carbonate (1 in 100) to make
exactly 200 mL. Perform the test with this solution as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>. De-
termine the absorbance A of this solution at the wavelength
of maximum absorption at about 580 nm, using water as the
blank.
Amount (mg) of C2oH8Br 4 Na20 10 S2
= (,4/881) X 200,000
Containers and storage Containers — Tight containers.
1130 Sulfobromophthalein Sodium Injection / Official Monographs
JP XV
Storage — Light-resistant.
Sulfobromophthalein Sodium
Injection
Sulfobromophthalein Sodium Injection is an aque-
ous solution for Injection. It contains not less than 94
% and not more than 106% of the labeled amount of
sulfobromophthalein sodium (C 2 oH 8 Br 4 Na20 10 S2:
838.00).
Method of preparation Prepare as directed under Injec-
tions, with Sulfobromophthalein Sodium.
Description Sulfobromophthalein Sodium Injection is a
clear and colorless or pale yellow liquid.
pH: 5.0-6.0
Identification (1) Measure a volume of Sulfobromo-
phthalein Sodium Injection, equivalent to 0.02 g of Sul-
fobromophthalein Sodium according to the labeled amount,
and proceed as directed in the Identification (1) under Sul-
fobromophthalein Sodium.
(2) Measure a volume of Sulfobromophthalein Sodium
Injection, equivalent to 0.1 g of Sulfobromophthalein Sodi-
um according to the labeled amount, add 0.5 g of anhydrous
sodium carbonate, and evaporate on a water bath to dryness.
Ignite the residue until it is charred. Proceed as directed in the
Identification (2) under Sulfobromophthalein Sodium.
Extractble volume <6.05> It meets the repequirement.
Pyrogen <4.04> Add isotonic sodium chloride solution to
Sulfobromophthalein Sodium Injection to make a 0.5 w/v%
solution of Sulfobromophthalein Sodium according to the la-
beled amount. Inject into each of the rabbits 5 mL of this so-
lution per kg of body mass: it meets the requirements.
Assay Measure exactly a volume of Sulfobromophthalein
Sodium Injection, equivalent to about 0.1 g of sul-
fobromophthalein sodium (C2oH 8 Br 4 Na20 10 S2), add water to
make exactly 500 mL, and proceed as directed in the Assay
under Sulfobromophthalein Sodium.
Amount (mg) of sulfobromophthalein sodium
(C 20 H 8 Br 4 Na 2 O 10 S2)
= (.4/881) x 200,000
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Sulfur
S: 32.07
Sulfur, when dried, contains not less than 99.5%
of S.
Description Sulfur occurs as a light yellow to yellow pow-
der. It is odorless and tasteless.
It is freely soluble in carbon disulfide, and practically in-
soluble in water, in ethanol (95) and in diethyl ether.
Identification (1) Ignite Sulfur: it burns with a blue flame
and gives a pungent odor of sulfur dioxide.
(2) Dissolve 5 mg of Sulfur in 5 mL of sodium hydroxide
TS by heating in a water bath, cool, and add 1 drop of sodi-
um pentacyanonitrosylferrate (III) TS: a blue-purple color
develops.
(3) Boil 1 mg of sulfur with 2 mL of pyridine and 0.2 mL
of sodium hydrogen carbonate TS: a blue color develops.
Purity (1) Clarity of solution — Dissolve 1.0 g of Sulfur in
a mixture of 20 mL of a solution of sodium hydroxide (1 in 6)
and 2 mL of ethanol (95) by boiling: the solution is clear. Dis-
solve 2.0 g of Sulfur in 10 mL of carbon disulfide: the solu-
tion is almost clear or slightly opalescent.
(2) Acidity or alkalinity— Shake 2.0 g of Sulfur with 50
mL of freshly boiled and cooled water, and add 2 drops of
phenolphthalein TS: no red color develops. Further add 1.0
mL of 0.1 mol/L sodium hydroxide VS: a red color develops.
(3) Arsenic </.//> — Prepare the test solution with 0.20 g
of Sulfur according to Method 3, and perform the test (not
more than 10 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
not more than 0.67 kPa, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.4 g of Sulfur, previously
dried, dissolve in 20 mL of potassium hydroxide-ethanol TS
and 10 mL of water by boiling, cool, and add water to make
exactly 100 mL. Transfer exactly 25 mL of the solution to a
400-mL beaker, add 50-mL of hydrogen peroxide TS, and
heat on a water bath for 1 hour. Acidify the solution with di-
lute hydrochloric acid, add 200 mL of water, heat to boil,
add hot barium chloride TS dropwise until no more
precipitate is formed, and heat on a water bath for 1 hour.
Collect the precipitate, and wash with water until the last
washing shows no opalescence with silver nitrate TS. Dry the
precipitate, heat strongly to constant mass, and weigh as
barium sulfate (BaS0 4 : 233.39). Perform a blank determina-
tion, and make any necessary correction.
Amount (mg) of S
= amount (mg) of barium sulfate (BaS0 4 ) x 0.13739
Containers and storage Containers — Well-closed contain-
ers.
Sulfur and Camphor Lotion
Method of preparation
Sulfur
60 g
tf-Camphor or tf/-Camphor
5g
Hydroxypropylcellulose
4g
Calcium Hydroxide
lg
Ethanol
4mL
Water or Purified Water
a sufficient quantity
To make 1000 mL
JPXV
Official Monographs / Sulpiride 1131
Dissolve Hydroxypropylcellulose in 200 mL of Water or
Purified Water. Add this solution in small portions to the
triturate of Sulfur with the Ethanol solution of (/-Camphor
or (//-Camphor, and triturate again the mixture. Separately,
dissolve Calcium Hydroxide in 500 mL of Water or Purified
Water, stopper tightly, shake, and allow to stand. Add 300
mL of this supernatant liquid to the above mixture, then add
Water or Purified Water to make 1000 mL, and shake
thoroughly.
Description Sulfur and Camphor Lotion is a light yellow
suspension.
A part of the components separates out on standing.
Identification (1) To 5 mL of well shaken Sulfur and
Camphor Lotion add 25 mL of water, and centrifuge [use
this supernatant liquid for test (3)]. To 0.02 g of the
precipitate add 2 mL of pyridine and 0.2 mL of sodium
hydrogen carbonate TS, and boil: a blue color develops (sul-
fur).
(2) To 10 mL of well shaken Sulfur and Comphor Lotion
add 5 mL of diethyl ether, and mix. Separate the diethyl ether
layer, and filter through a pledget of cotton. Wash the cotton
with a small portion of diethyl ether, combine the washings
with the filtrate, and distil cautiously on a water bath to re-
move the diethyl ether. Dissolve the residue in 1 mL of
methanol, add 1 mL of 2,4-dinitrophenylhydrazine TS, and
heat for about 2 minutes on a water bath. Cool, dilute with
water to make about 5mL, and allow to stand. Filter the
produced precipitate through a glass filter (G4), and wash the
residue on the filter with water until the last washing is color-
less. Dissolve the residue in 10 mL of ethanol (95), add 5 mL
of sodium hydroxide TS, and allow to stand for 2 minutes: a
red color develops ((/-camphor or (//-camphor).
(3) The supernatant liquid obtained in (1) responds to the
Qualitative Tests <1.09> (2) and (3) for calcium salt.
Containers and storage Containers — Tight containers.
Sulfur, Salicylic Acid and
Thianthol Ointment
^7t-^-^U5 1 ;ng-5 1 7> \--iim
Method of preparation
Sulfur
100 g
Salicylic Acid, finely powdered
30 g
Thianthol
100 mL
Zinc Oxide, very finely powdered
100 g
Simple Ointment or a suitable
ointment base
a sufficient quantity
To make
1000 g
Prepare as directed under Ointments, with above in-
gredients.
Description Sulfur, Salicylic Acid and Thianthol Ointment
is light yellow in color.
Identification (1) Stir well 0.5 g of Sulfur, Salicylic Acid
and Thianthol Ointment with 10 mL of water while heating,
cool, and filter. To 1 mL of the filtrate add 5 mL of iron (III)
nitrate TS: a purple color is produced (salicylic acid).
(2) Shake 1 g of Sulfur, Salicylic Acid and Thianthol
Ointment with 20 mL of diethyl ether, remove the super-
natant liquid and floating materials. Wash the residue with 10
mL of diethyl ether, and remove the diethyl ether by suction.
To the residue add 2 mL of pyridine and 0.2 mL of sodium
hydrogen carbonate TS, and boil: a light blue to blue color is
produced (sulfur).
(3) To 1 g of Sulfur, Salicylic Acid and Thianthol Oint-
ment add 15 mL of ethanol (95), stir well while warming on a
water bath, cool, and filter. Use the filtrate as the sample so-
lution. Dissolve 0.01 g each of salicylic acid and thianthol in 5
mL of ethanol (95), and use these solutions as the standard
solution (1) and standard solution (2). Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solutions (1) and (2) on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, acetone and acetic acid (100)
(45:5:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
the spots of each component obtained from the sample solu-
tion and standard solutions (1) and (2) show the same Rf
value. Spray iron (III) chloride TS upon the plate evenly: the
spot from the standard solution (1) and that from the cor-
responding sample solution reveal a purple color.
Containers and storage Containers — Tight containers.
Sulpiride
xjutru K
°,,°
¥
o
i
r
C 15 H 23 N 3 04S: 341.43
A r -(l-Ethylpyrolidin-2-ylmethyl)-2-methoxy-5-
sulfamoylbenzamide [15676-16-1]
Sulpiride, when dried, contains not less than 98.5%
and not more than 101.0% of Q5H23N3O4S.
Description Sulpiride is a white, crystalline powder.
It is freely soluble in acetic acid (100) and in dilute acetic
acid, sparingly soluble in methanol, slightly soluble in
ethanol (99.5), and practically insoluble in water.
It is soluble in 0.05 mol/L sulfuric acid TS.
A solution of Sulpiride in methanol (1 in 100) shows no op-
tical rotation.
Melting point: about 178°C (with decomposition).
Identification (1) Dissolve 0.1 g of Sulpiride in 0.05
mol/L sulfuric acid TS to make 100 mL. Dilute 5 mL of the
solution with water to make 100 mL. Determine the absorp-
tion spectrum of the solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, using water as the blank,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Sulpi-
1132 Sulpiride Capsules / Official Monographs
JP XV
ride as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
Purity (1) Clarity of solution — Dissolve 2.0 g of Sulpiride
in 7 mL of dilute acetic acid, and add water to make 20 mL:
the solution is clear. Perform the test with the solution as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using water as the blank: the absorbance at a wavelength of
450 nm does not exceed 0.020.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Sulpiride
as directed under Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Related substances — Dissolve 50 mg of Sulpiride in 10
mL of methanol, and use this solution as the sample solution.
Dilute 1 mL of the sample solution, accurately measured,
with methanol to make exactly 100 mL. Dilute 2 mL of this
solution, accurately measured, with methanol to make ex-
actly 10 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 20 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of 1-butanol, water and acetic acid
(100) (4:2:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): the number of the spots other than the principal spot
from the sample solution is not more than 2, and they have
no more color than the spot from the standard solution.
When the plate is exposed to iodine vapor for 30 minutes, the
number of the spots other than the principal spot from the
sample solution is not more than 2, and they have no more
color than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Dissolve about 0.4 g of Sulpiride, previously dried
and accurately weighed, in 80 mL of acetic acid (100), and ti-
trate <2.50> with 0.1 mol/L perchloric acid VS (indicator: 2
drops of crystal violet TS) until the color of the solution
changes from violet through blue to bluish green. Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.14 mg of C 15 H 2 3N 3 04S
Containers and storage Containers — Well-closed contain-
ers.
Sulpiride Capsules
Sulpiride Capsules contain not less than 95.0% and
not more than 105.0% of the labeled amount of sulpi-
ride (C 15 H 2 3N 3 04S: 341.43).
Method of preparation Prepare as directed under Capsules,
with Sulpiride.
Identification Determine the absorption spectrum of the
sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using water as
the blank: it exhibits a maximum between 289 nm and 293
nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 capsule of Sulpiride Capsules add 30 mL of 0.05
mol/L sulfuric acid TS, shake for 30 minutes, add 0.05
mol/L sulfuric acid TS to make exactly FmL so that each
mL of the solution contains about 1 mg of sulpiride
(C 15 H 2 3N 3 04S), and filter the solution. Discard the first 20
mL of the filtrate, pipet the subsequent 5 mL of the filtrate,
add water to make exactly 100 mL, and use this solution as
the sample solution. Proceed as directed in the Assay.
Amount (mg) of sulpiride (C15H23N3O4S)
= W s x(A T /A s )x(V/50)
W s : Amount (mg) of sulpiride for assay
Dissolution Being specified separately.
Assay Cut the capsule of not less than 20 Sulpiride Cap-
sules, weigh accurately the mass of the contents, and powder.
Weigh accurately a portion of the powder, equivalent to
about 0.1 g of sulpiride (C^H^NjC^S), add 70 mL of 0.05
mol/L sulfuric acid TS, shake for 30 minutes, and add 0.05
mol/L sulfuric acid TS to make exactly 100 mL, and filter.
Discard the first 20 mL of the filtrate, pipet the subsequent 5
mL of the filtrate, add water to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 50 mg of sulpiride for assay, previously dried
at 105°C for 3 hours, and dissolve in 0.05 m/L sulfuric acid
TS to make exactly 50 mL. Pipet 5 mL of this solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Determine the absorbances, A T and ^4 S , of
the sample solution and standard solution at 291 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using water as the blank.
Amount (mg) of sulpiride (C 15 H M N 3 C>4S) = W s x (A T /A S ) x 2
W s : Amount (mg) of sulpiride for assay
Containers and storage Containers — Tight containers.
Sulpiride Tablets
XJUtfU Ktt
Sulpiride Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of sulpi-
ride (C 15 H 2 3N30 4 S: 341.43).
Method of preparation Prepare as directed under Tablets,
with Sulpiride.
Identification Determine the absorption spectrum of the
sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using water as
the blank: it exhibits a maximum between 289 nm and 293
nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
JPXV
Official Monographs / Sulpyrine Hydrate 1133
Content uniformity test.
To 1 tablet of Sulpiride Tablets add 30 mL of 0.05 mol/L
sulfuric acid TS, shake for 30 minutes, add 0.05 mol/L sul-
furic acid TS to make exactly V mL so that each mL of the
solution contains about 1 mg of sulpiride (Q5H23N3O4S), and
filter the solution. Discard the first 20 mL of the filtrate, pipet
the subsequent 5 mL of the filtrate, add water to make exactly
100 mL, and use this solution as the sample solution. Proceed
as directed in the Assay.
Amount (mg) of sulpiride (Q5H23N3O4S)
= W s x(A T /A s )x(V/50)
W s : Amount (mg) of sulpiride for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Sulpiride Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of 2nd fluid for dissolution test as the dissolution
medium. Withdraw 20 mL or more of the dissolution medi-
um 30 minutes after starting the test for a 50-mg tablet and 45
minutes after for a 100-mg or a 200-mg tablet, and filter
through a membrane filter with pore size of not more than 0.5
/um. Discard the first 10 mL of the filtrate, pipet the subse-
quent KmL, add 2nd fluid for dissolution test to make ex-
actly V mL so that each mL contains about 56 n% of sulpi-
ride (C 15 H23N 3 4 S) according to the labeled amount, and use
this solution as the sample solution. Separately, weigh ac-
curately about 28 mg of sulpiride for assay, previously dried
at 105°C for 3 hours, and dissolve in 2nd fluid for dissolution
test to make exactly 100 mL. Pipet 5 mL of this solution, add
2nd fluid for dissolution test to make exactly 25 mL, and use
this solution as the standard solution. Perform the test with
the sample solution and the standard solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>, and deter-
mine the absorbances, A T and/4 s , at 291 nm. The dissolution
rate of a 50-mg tablet in 30 minutes is not less than 80%, that
of a 100-mg tablet in 45 minutes is not less than 75%, and
that of a 200-mg tablet in 45 minutes is not less than 70%.
Dissolution rate (%) with respect to the labeled amount of
sulpiride (C^H^C^S)
= W s x (Aj/A s ) x (V'/V)x (I/O x 180
W s : Amount (mg) of sulpiride for assay
C: Labeled amount (mg) of sulpiride (Q5H23N3O4S) in 1
tablet
Assay Weigh accurately, and powder not less than 20 Sulpi-
ride Tablets. Weigh accurately a portion of the powder,
equivalent to about 0.1 g of sulpiride (C 15 H 2 3N30 4 S), add 70
mL of 0.05 mol/L sulfuric acid TS, shake for 30 minutes,
and add 0.05 mol/L sulfuric acid TS to make exactly 100 mL,
and filter. Discard the first 20 mL of the filtrate, pipet the sub-
sequent 5 mL of the filtrate, add water to make exactly 100
mL, and use this solution as the sample solution. Separately,
weigh accurately about 50 mg of sulpiride for assay, previ-
ously dried at 105 °C for 3 hours, and dissolve in 0.05 mL sul-
furic acid TS to make exactly 50 mL. Pipet 5 mL of this solu-
tion, add water to make exactly 100 mL, and use this solution
as the standard solution. Determine the absorbances, A T and
A s , of the sample solution and standard solution at 291 nm as
directed under Ultraviolet-visible Spectrophotometry<2.24>,
using water as the blank.
Amount (mg) of sulpiride (Q5H23N3O4S) = W s x (A T /A S ) x 2
W s : Amount (mg) of sulpiride for assay
Containers and storage Containers — Tight containers.
Sulpyrine Hydrate
1
N
N
• H 3 Q
Na0 3 S
K
-N O
CH,
C 1 3H 16 N 3 Na04S.H 2 0: 351.35
Monosodium [(1 ,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
l//-pyrazol-4-yl)(methyl)amino]methanesulfonate
monohydrate [5907-38-0]
Sulpyrine Hydrate contains not less than 98.5% of
sulpyrine (C 13 H 16 N 3 Na0 4 S: 333.34), calculated on the
dried basis.
Description Sulpyrine Hydrate occurs as white to light yel-
low crystals or crystalline powder. It is odorless, and has a
bitter taste.
It is very soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
It is colored by light.
Identification (1) Add 2 drops of dilute sulfuric acid and 1
mL of chlorinated lime TS to 3 mL of a solution of Sulpyrine
Hydrate (1 in 15): a deep blue color develops at first, but the
color immediately turns red, then gradually changes to yel-
low.
(2) Boil 5 mL of a solution of Sulpyrine Hydrate (1 in 25)
with 3 mL of dilute hydrochloric acid: the odor of sulfur di-
oxide is perceptible at first, and on further boiling, the odor
of formaldehyde is perceptible.
(3) A solution of Sulpyrine Hydrate (1 in 10) responds to
the Qualitative Tests <1.09> for sodium salt.
Purity (1) Clarity of solution, and acidity or
alkalinity — Dissolve 1.0 g of Sulpyrine Hydrate in 10 mL of
water: the solution is clear and neutral.
(2) Sulfate <1.14>— Dissolve 0.20 g of Sulpyrine Hydrate
in 0.05 mol/L hydrochloric acid VS to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution with 0.50 mL of 0.005 mol/L sulfuric
acid VS and 0.05 mol/L hydrochloric acid VS to make 50 mL
(not more than 0.120%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Sulpyrine
Hydrate according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(4) Merbuline — Transfer 0.10 g of Sulpyrine Hydrate
with 2 mL of water and 1 mL of dilute sulfuric acid into a
flask, cover with a funnel, and boil gently for 15 minutes.
Cool, add 2 mL of a solution of sodium acetate trihydrate (1
in 2) and water to make 5 mL, shake this solution with 5 mL
of benzaldehyde-saturated solution, and allow to stand for 5
minutes: the solution is clear.
1134 Sulpyrine Injection / Official Monographs
JP XV
(5) Chloroform-soluble substances — Mix, by frequent
shaking, 1.0 g of Sulpyrine Hydrate and 10 mL of chlo-
roform for 30 minutes. Collect the precipitate, wash with two
5-mL portions of chloroform, combine the washings with the
filtrate, and evaporate on a water bath to dryness. Dry the
residue at 105 °C for 4 hours: the mass of the residue is not
more than 5.0 mg.
Loss on drying <2.41> Not more than 6.0% (1 g, 105°C,
4 hours).
Assay Weigh accurately about 0.25 g of Sulpyrine Hydrate,
dissolve in 100 mL of diluted hydrochloric acid (1 in 20),
previously cooled below 10°C. Titrate <2.50> immediately
with 0.05 mol/L iodine VS while keeping the temperature be-
tween 5°C and 10°C, until the color of the solution remains
blue upon shaking vigorously for 1 minute after the addition
of 0.05 mol/L iodine VS (indicator: 1 mL of starch TS).
Each mL of 0.05 mol/L iodine VS
= 16.67 mg of C 13 H I6 N 3 Na0 4 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Sulpyrine Injection
x;ufc?u>a*t*
Sulpyrine Injection is an aqueous solution for injec-
tion. It contains not less than 95% and not more than
105% of the labeled amount of sulpyrine hydrate
(C 13 H 16 N3Na0 4 S.H 2 0: 351.35).
Method of preparation Prepare as directed under Injec-
tions, with Sulpyrine Hydrate.
Description Sulpyrine Injection is a clear, colorless or pale
yellow liquid.
pH: 5.0-8.5
Identification (1) To a volume of Sulpyrine Injection,
equivalent to 0.2 g of Sulpyrine Hydrate according to the la-
beled amount, add water to make 3 mL, then add 2 drops of
dilute sulfuric acid and 1 mL of chlorinated lime TS: a deep
blue color develops at first, and the color immediately turns
red and gradually changes to yellow.
(2) To a volume of Sulpyrine Injection, equivalent to 0.2
g of Sulpyrine according to the labeled amount, add water to
make 5 mL, and boil this solution with 3 mL of dilute
hydrochloric acid: the odor of sulfur dioxide is perceptible at
first, and on further boiling the odor of formaldehyde is per-
ceptible.
Assay Pipet 2 mL of Sulpyrine Injection, dilute with water
to exactly 100 mL. Measure exactly a volume (FmL) of this
solution, equivalent to about 0.05 g of sulpyrine hydrate
(C 13 H 16 N 3 Na0 4 S.H 2 0), and add water to make exactly 100
mL. Pipet 5 mL of this solution, add water to exactly 100
mL, and use this solution as the sample solution. Weigh ac-
curately about 0.05 g of sulpyrine for assay (previously deter-
mine the loss on drying <2.41> in the same manner as Sulpy-
rine Hydrate), and dissolve in water to make exactly 100 mL.
Pipet 5 mL of this solution, add water to exactly 100 mL, and
use this solution as the standard solution. Pipet 2 mL each of
the sample solution and standard solution into separate
25-mL volumetric flasks, add 5 mL of ethanol (95), 2 mL of a
solution of 4-dimethylaminocinnamaldehyde in ethanol (95)
(1 in 250) and 2 mL of acetic acid (100) to each of these solu-
tions, shake well, allow to stand for 15 minutes, and add
water to exactly 25 mL. Perform the test with these solutions
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, using a solution prepared with 2 mL of water in the
same manner as the blank. Determine the absorbances, A T
and A s , of the subsequent solutions of the sample solution
and the standard solution at 510 nm.
Amount (mg) of sulpyrine hydrate (C 13 H I6 N 3 Na0 4 S.H 2 0)
in 1 mL of Sulpyrine Injection
= H's x (-4t/-4s)x(50/I / ) x L0540
W s : Amount (mg) of sulpyrine for assay, calculated
on the dried basis
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant, and under nitrogen atmosphere.
Sultamicillin Tosilate Hydrate
o
h * n . H u Vf v CHi °V n X
SO3H
•2H;0
C 2 5H 3 „N 4 9 S 2 .C 7 H 8 3 S.2H 2 0: 802.89
(2S,5i?)-(3,3-Dimethyl-4,4,7-trioxo-4-thia-l-azabicy-
clo[3.2.0]hept-2-ylcarbonyloxy)methyl (2S,5R,6R)-6-[(2R)-
2-amino-2-phenylacetylamino]-3,3-dimethyl-
7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate
monotosylate dihydrate [83105-70-8, anhydride]
Sultamicillin Tosilate Hydrate contains not less than
698 fig (potency) per mg, calculated on the anhydrous
basis and corrected by the amount of residual solvent.
The potency of Sultamicillin Tosilate is expressed as
mass (potency) of sultamicillin (C 2 5H3 N4O 9 S2:
594.66).
Description Sultamicillin Tosilate Hydrate occurs as a
white to yellowish white crystalline powder.
It is freely soluble in acetonitrile, in methanol and in
ethanol (99.5), and very slightly soluble in water.
Identification Determine the infrared absorption spectrum
of Sultamicillin Tosilate Hydrate as directed in the paste
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Sultamicillin Tosilate Reference Standard: both spec-
tra exhibit similar intensities of absorption at the same wave
numbers.
Optical rotation <2.49> [a]™: + 173 -+ 187° (0.5 g calcu-
lated on the anhydrous bases, a mixture of water and acetoni-
JPXV
Official Monographs / Sultamicillin Tosilate Hydrate 1135
trile (3:2), 25 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Sultamicillin Tosilate Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sultamicillin Tosilate Hydrate, according to Method 3,
and perform the test (not more than 2 ppm).
(3) Ampicillin — Perform the procedure rapidly. Weigh
accurately about 20 mg of Sultamicillin Tosilate Hydrate,
dissolve in the mobile phase to make exactly 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 20 mg (potency) of Ampicillin Reference Stan-
dard, dissolve in the mobile phase to make exactly 100 mL.
Pipet 6 mL of this solution, add the mobile phase to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 25 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the area of the peak of ampicillin by the auto-
matic integration method: the peak area from the sample so-
lution is not more than that from the standard solution.
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Assay.
Mobile phase: Dissolve 3.12 g of sodium dihydrogen
phosphate dihydrate in about 750 mL of water, adjust to pH
3.0 with diluted phosphoric acid (1 in 10), and add water to
make
1000 mL. To 80 mL of acetonitrile for liquid chromato-
graphy add this solution to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
ampicillin is about 14 minutes.
System suitability —
System performance: Dissolve 12 mg of Ampicillin Refer-
ence Standard, 4 mg of Sulbactam Reference Standard and 4
mg of /?-toluenesulfonic acid monohydrate in 1000 mL of the
mobile phase. When the procedure is run with 25 [xL of this
solution under the above operating conditions, sulbactam, p-
toluenesulfonic acid and ampicillin are eluted in this order
with the resolution between these peaks being not less than
2.0.
System repeatability: When the test is repeated 6 times with
25 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of ampicillin is not more than 2.0%.
(4) Sulbactam — Perform the procedure rapidly. Weigh
accurately about 20 mg of Sultamicillin Tosilate Hydrate,
dissolve in the mobile phase to make exactly 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately an amount of Sulbactam Reference Standard, e-
quivalent to about 20 mg (potency), dissolve in the mobile
phase to make exactly 100 mL. Pipet 2 mL of this solution,
add the mobile phase to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 25 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the area of the
peak of sulbactam by the automatic integration method: the
peak area from the sample solution is not more than that
from the standard solution.
Operating conditions —
Proceed as directed in the operating conditions in the Puri-
ty (3).
System suitability —
Proceed as directed in the system suitability in the Purity
(3).
(5) Penicilloic acids — Weigh accurately about 25 mg of
Sultamicillin Tosilate Hydrate, dissolve in 1 mL of acetoni-
trile, and add 25 mL of 0.02 mol/L phosphate buffer solu-
tion, pH 3.0, in a 100-mL flask with stopper. Add exactly 5
mL of 0.005 mol/L iodine VS, and allow to stand the stop-
pered flask for 5 minutes. Titrate <2.50> with 0.005 mol/L so-
dium thiosulfate VS (indicator: 1.0 mL of starch TS). Per-
form a blank determination in the same manner, and make
any necessary correction. Calculate the amount of penicilloic
acid (C25H34N4O11S2: 630.70) by using the following equa-
tion: it is not more than 3.0%.
Each mL of 0.005 mol/L sodium thiosulfate VS
= 0.2585 mg of C25H34N4OHS2
(6) Residual solvent <2.46> — Weigh accurately about 0.1
g of Sultamicillin Tosilate Hydrate, dissolve in 2 mL of
methanol, add water to make exactly 20 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 1 g of ethyl acetate, and mix with water to make exact-
ly 200 mL. Pipet 2 mL of this solution, add 10 mL of
methanol and water to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with exactly
5 /xL each of the sample solution and standard solution as
directed under Gas Chromatography <2.02> according to the
following conditions, and determine the peak areas, A T and
j4 s , of ethyl acetate of these solutions. Calculate the amount
of ethyl acetate by the following equation: not more than 2.0
%.
Amount (%) of ethyl acetate
= (W s /W J )x(A J /A s )x (1/5)
W s : Amount (mg) of ethyl acetate
W T : Amount (mg) of the sample
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A column 3 mm in inside diameter and 1 m in
length, packed with porous stylene-divinylbenzene
copolymer for gas chromatography (0.0085 /xm in average
pore size and 300 - 400 m 2 /g in specific surface area) (150 to
180//m in particle diameter).
Column temperature: A constant temperature of about
155°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethyl acetate is about 6 minutes.
System suitability —
System performance: When the procedure is run with 5 /uL
of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of ethyl acetate are not less than 500 steps and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
5 fXL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
ethyl acetate is not more than 5%.
Water <2.48> 4.0 - 6.0% (0.5 g, volumetric titration, direct
titration).
1136 Sultiame / Official Monographs
JP XV
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Perform the procedure rapidly. Weigh accurately an
amount of Sultamicillin Tosilate Hydrate and Sultamicillin
Tosilate Reference Standard, equivalent to about 50 mg
(potency), dissolve each in the mobile phase to make exactly
50 mL. Pipet 5 mL each of these solutions, add exactly 5 mL
of the internal standard solution, add the mobile phase to
make 25 mL, and use these solutions as the sample solution
and standard solution, respectively. Perform the test with 10
/uL each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the ratios, g T and Q s , of the peak area of sul-
tamicillin to that of the internal standard of each solution.
Amount \p.g (potency)] of sultamicillin (C25H30N4O9S2)
= ^sX(Gt/Gs)x1000
fV s : Amount [mg (potency)] of Sultamicillin Tosilate
Reference Standard
Internal standard solution — A solution of isopropyl-4-
aminobenzoate in the mobile phase (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 215 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Dissolve 3.12 g of sodium dihydrogen-
phosphate in about 750 mL of water, adjust to pH 3.0 with
diluted phosphoric acid (1 in 10), and add water to make 1000
mL. To 400 mL of acetonitrile for liquid chromatography
add this solution to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
sultamicillin is about 4 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, p-toluenesulfonic acid, sultamicillin and the internal
standard are eluted in this order with the resolution between
these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sultamicillin is not more than 2.0%.
Containers and storage Containers — Tight containers.
Sultiame
gxr
NH 2
C 10 H 14 N 2 O 4 S 2 : 290.36
4-(3,4,5,6-Tetrahydro-2//-l,2-thiazin-
2-yl)benzenesulfonamide S.S-dioxide [61-56-3]
Sultiame, when dried, contains not less than 98.5%
of C 10 H 14 N 2 O 4 S 2 .
Description Sultiame occurs as white crystals or crystalline
powder. It is odorless, and has a slightly bitter taste.
It is very soluble in A^Af-dimethylformamide, freely solu-
ble in «-butylamine, slightly soluble in methanol and in
ethanol (95), very slightly soluble in water, and practically in-
soluble in diethyl ether.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 0.02 g of Sultiame in 5 mL of
water and 1 mL of M-butylamine, add 2 to 3 drops of copper
(II) sulfate TS, and shake well. To this solution add 5 mL of
chloroform, shake, and allow to stand: a green color de-
velops in the chloroform layer.
(2) Mix 0.1 g of Sultiame with 0.5 g of sodium carbonate
decahydrate, and melt carefully: the gas evolved changes
moistened red litmus paper to blue. After cooling, crush the
fused substance with a glass rod, stir with 10 mL of water,
and filter. To 4 mL of the filtrate add 2 drops of hydrogen
peroxide (30), 5 mL of diluted hydrochloric acid (1 in 5) and 2
to 3 drops of barium chloride TS: a white precipitate is
formed.
(3) Determine the absorption spectrum of a solution of
Sultiame in methanol (1 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
Melting point <2.60> 185 - 188 C C
Purity (1) Chloride <1.03>— Dissolve 1.0 g of Sultiame in
20 mL of sodium hydroxide TS by warming, cool, and add 2
mL of acetic acid (100) and water to make 100 mL. After
shaking, filter, and discard the first 10 mL of the filtrate. To
the subsequent 40 mL add 6 mL of dilute nitric acid and
water to make 50 mL, and perform the test using this solution
as the test solution. Prepare the control solution as follows:
to 0.25 mL of 0.01 mol/L hydrochloric acid VS add 8 mL of
sodium hydroxide TS, 0.8 mL of acetic acid (100), 6 mL of
dilute nitric acid and water to make 50 mL (not more than
0.022%).
(2) Sulfate <1.14>— Dissolve 1.0 g of Sultiame in 20 mL
of sodium hydroxide TS by warming, cool, and add 8 mL of
dilute hydrochloric acid and water to make 100 mL. After
shaking, filter, and discard the first 10 mL of the filtrate. To
the subsequent 40 mL add 1 mL of dilute hydrochloric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 0.40 mL of 0.005 mol/L sulfuric acid VS add 8 mL
of sodium hydroxide TS, 4.2 mL of dilute hydrochloric acid
and water to make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 2.0 g of Sultiame
according to Method 2, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Sultiame according to Method 3, and perform the test (not
more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Sultiame in
methanol to make exactly 20 mL, and use this solution as the
sample solution. Separately, dissolve 10 mg of sulfanilamide
JPXV
Official Monographs / Suxamethonium Chloride for Injection 1137
in methanol to make exactly 100 mL. Pipet 10 mL of this so-
lution, add methanol to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 20 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol and ammonia solution (28) (30:8:1)
to a distance of about 12 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Sultiame, previously
dried, dissolve in 70 mL of ./V,7V-dimethylformamide, and ti-
trate <2.50> with 0.2mol/L tetramethylammonium
hydroxide VS (potentiometric titration). Perform a blank de-
termination, and make any necessary correction.
Each mL of 0.2 mol/L tetramethylammonium
hydroxide VS
= 58.07 mg of C 10 H 14 N 2 O4S 2
Containers and storage Containers — Well-closed contain-
ers.
Suxamethonium Chloride Hydrate
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Suxamethonium Chloride Hydrate in 10 mL of water: the so-
lution is clear and colorless.
(2) Related substances — Dissolve 0.25 g of Suxamethoni-
um Chloride Hydrate in 5 mL of water, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add water to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot 1
/iL each of the sample solution and standard solution on a
plate of cellulose for thin-layer chromatography. Develop the
plate with a mixture of a solution of ammonium acetate (1 in
100), acetone, n-butanol and formic acid (20:20:20:1) to a
distance of about 10 cm, and dry the plate at 105°C for 15
minutes. Spray evenly platinic chloride-potassium iodide TS
on the plate, and allow to stand for 15 minutes: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Water <2.48> 8.0 - 10.0% (0.4 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Suxamethonium
Chloride Hydrate, dissolve in 80 mL of a mixture of acetic
anhydride and acetic acid (100) (7:3), and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration).
Each mL of 0.1 mol/L perchloric acid VS
= 18.07 mg of C 14 H 30 Cl 2 N 2 O 4
Containers and storage Containers — Tight containers.
Suxamethonium Chloride
for Injection
SCI • 2H^>
%Mmx*v* v-oj^xtm
C 14 H 30 Cl 2 N 2 O 4 .2H 2 O: 397.34
2,2'-Succinyldioxybis(Af,A r ,Af-trimethylethylaminium)
dichloride dihydrate [6101-15-1]
Suxamethonium Chloride Hydrate contains not less
than 98.0% of suxamethonium chloride (C 14 H 3 oCl 2 N2
4 : 361.31), calculated on the anhydrous basis.
Description Suxamethonium Chloride Hydrate occurs as a
white, crystalline powder.
It is freely soluble in water, in methanol and in acetic acid
(100), slightly soluble in ethanol (95), very slightly soluble in
acetic anhydride, and practically insoluble in diethyl ether.
Identification (1) Determine the infrared absorption spec-
trum of Suxamethonium Chloride Hydrate as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(2) A solution of Suxamethonium Chloride Hydrate (1 in
20) responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> The pH of a solution of Suxamethonium Chlo-
ride Hydrate (1 in 100) is between 4.0 and 5.0.
Melting point <2.60> 159 - 164°C (hydrate form).
Suxamethonium Chloride for Injection is a prepara-
tion for injection which is dissolved before use. It con-
tains not less than 93% and not more than 107% of the
labeled amount of suxamethonium chloride
(C 14 H3oCl 2 N 2 4 : 361.31).
The concentration of Suxamethonium Chloride for
Injection should be stated as the amount of sux-
amethonium chloride (C 14 H 30 Cl2N 2 O 4 ).
Method of preparation Prepare as directed under Injec-
tions, with Suxamethonium Chloride Hydrate.
Description Suxamethonium Chloride for Injection occurs
as a white, crystalline powder or mass.
Identification Take an amount of Suxamethonium Chloride
for Injection, equivalent to 0.05 g of Suxamethonium Chlo-
ride Hydrate according to the labeled amount, dissolve in
water to make 10 mL, and use this solution as the sample so-
lution. Separately, dissolve 0.05 g of suxamethonium chlo-
ride for thin-layer chromatography in 10 mL of water, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 1 /xL each of the sample solution
and standard solution on a plate of cellulose for thin-layer
chromatography. Develop the plate with a mixture of a solu-
1138 Suxamethonium Chloride Injection / Official Monographs
JP XV
tion of ammonium acetate (1 in 100), acetone, 1-butanol and
formic acid (20:20:20:1) to a distance of about 10 cm, and
dry the plate at 105°C for 15 minutes. Spray evenly platinic
chloride-potassium iodide TS on the plate: the spots obtained
from the sample solution and standard solution are blue-pur-
ple in color and have similar Ri.
pH <2.54> The pH of a solution of Suxamethonium Chlo-
ride for Injection (1 in 100) is between 4.0 and 5.0.
Purity Related substances — Take an amount of Sux-
amethonium Chloride for Injection, equivalent to 0.25 g of
Suxamethonium Chloride Hydrate according to the labeled
amount, and proceed as directed in the Purity (2) under Sux-
amethonium Chloride Hydrate.
Assay Weigh accurately the contents of not less than 10
preparations of Suxamethonium Chloride for Injection.
Weigh accurately about 0.5 g of the contents, and proceed as
directed in the Assay under Suxamethonium Chloride Hy-
drate.
Each mL of 0.1 mol/L perchloric acid VS
= 18.07 mg of C 14 H 30 Cl 2 N 2 O 4
Containers and storage Containers — Hermetic containers.
Suxamethonium Chloride Injection
x*y/ [■ -O Ai&ikmiftyfc
Suxamethonium Chloride Injection is an aqueous
solution for injection. It contains not less than 93%
and not more than 107% of the labeled amount of sux-
amethonium chloride (Q4H30CI2N2O4: 361.31).
The concentration of Suxamethonium Chloride In-
jection should be stated as the amount of suxamethoni-
um chloride (Q4H30CI2N2O4).
Method of preparation Prepare as directed under Injec-
tions, with Suxamethonium Chloride Hydrate.
Description Suxamethonium Chloride Injection is a clear,
colorless liquid.
Identification Take a volume of Suxamethonium Chloride
Injection, equivalent to 0.05 g of Suxamethonium Chloride
Hydrate according to the labeled amount, add water to make
10 mL, and use this solution as the sample solution.
Separately, dissolve 0.05 g of suxamethonium chloride for
thin-later chromatography in 10 mL of water, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 1 fiL each of the sample solution and standard
solution on a plate of cellulose for thin-layer chro-
matography. Develop the plate with a mixture of a solution
of ammonium acetate (1 in 100), acetone, 1-butanol and for-
mic acid (20:20:20:1) to a distance of about 10 cm, and dry
the plate at 105 °C for 15 minutes. Spray evenly platinic chlo-
ride-potassium iodide TS on the plate: the spots obtained
from the sample solution and the standard solution are blue-
purple in color and have similar Ri.
pH <2.54> 3.0-5.0
Purity Hydrolysis products — Perform the preliminary neu-
tralization with 0.1 mol/L sodium hydroxide VS in the As-
say: not more than 0.7 mL of 0.1 mol/L sodium hydroxide
VS is required for each 200 mg of Suxamethonium Chloride
(C I4 H 30 Cl 2 N 2 O 4 ) taken.
Extractable volume <6.05> It meets the requiremeut.
Assay Transfer to a separator an accurately measured
volume of Suxamethonium Chloride Injection, equivalent to
about 0.2 g of suxamethonium chloride (C I4 H3oCl 2 N 2 4 ),
add 30 mL of freshly boiled and cooled water, and wash the
solution with five 20-mL portions of diethyl ether. Combine
the diethyl ether washings, and extract the combined diethyl
ether layer with two 10-mL portions of freshly boiled and
cooled water. Wash the combined water extracts with two
10-mL portions of diethyl ether. Combine the solution and
the water extracts, add 2 drops of bromothymol blue TS, and
neutralize with 0.1 mol/L sodium hydroxide VS. Add ac-
curately measured 25 mL of 0.1 mol/L sodium hydroxide
VS, and boil for 40 minutes under a reflux condenser, and
cool. Titrate <2.50> the excess sodium hydroxide with 0.1 mol
/L hydrochloric acid VS. Transfer 50 mL of the freshly
boiled and cooled water to a flask, add 2 drops of
bromothymol blue TS, neutralize the solution with 0.1 mol/
L sodium hydroxide VS, and perform a blank determination.
Each mL of 0.1 mol/L sodium hydroxide VS
= 18.07 mg of C 14 H 30 Cl 2 N 2 O 4
Containers and storage Containers — Hermetic containers.
Storage — Not exceeding 5°C, and avoid freezing.
Expiration date 12 months after preparation.
Talampicillin Hydrochloride
Ampicillinphthalidyl Hydrochloride
o
HCI
C 24 H 23 N 3 6 S.HC1: 517.98
3-Oxo- 1 ,3-dihydroisobenzofuran- 1 -yl (2S,5R ,6R )-6-[(2R )-2-
amino-2-phenylacetylamino]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate
monohydrochloride [47747-56-8]
Talampicillin Hydrochloride is the hydrochloride of
ampicillin phthalidyl ester.
It contains not less than 600 fig (potency) and not
more than 700 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Talampicillin
Hydrochloride is expressed as mass (potency) of am-
picillin (C 16 H 19 N 3 4 S: 349.40).
Description Talampicillin Hydrochloride occurs as a white
to light yellowish white powder.
It is very soluble in methanol, and freely soluble in water
JPXV
Official Monographs / Talc 1139
and in ethanol (99.5).
Identification (1) To 1 mL of a solution of Talampicillin
Hydrochloride (1 in 30) add 1 mL of sodium hydroxide TS,
mix, allow to stand for 5 minutes, and add 2 mL of dilute sul-
furic acid and 2 to 3 drops of 2,4-dinitrophenylhydrazine TS:
an orange-yellow precipitate is formed.
(2) Determine the infrared absorption spectrum of
Talampicillin Hydrochloride as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Talampicillin Hydrochloride Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
(3) To 10 mL of a solution of Talampicillin Hydrochlo-
ride (1 in 300) add 1 mL of dilute nitric acid, and add silver
nitrate TS: a white precipitate is formed.
Optical rotation <2.49> [ a ]g>: +151 - +171° (0.2 g calcu-
lated on the anhydrous basis, ethanol (99.5), 20 mL, 100
mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Talampicillin Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Talampicillin Hydrochloride according to Method 4, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Talampicillin
Hydrochloride in ethanol (99.5) to make exactly 10 mL, and
use this solution as the sample solution. Pipet 1 mL, 2 mL
and 3 mL of the sample solution, add ethanol (99.5) to each
to make exactly 100 mL, and use these solutions as the stan-
dard solution (1), the standard solution (2) and the standard
solution (3), respectively. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10,mL each of the sample solution and standard solu-
tions (1), (2) and (3) on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of tetra-
hydrofuran, ethyl acetate, water and ethanol (95) (4:4:2:1) to
a distance of about 13 cm, and air-dry the plate. Spray evenly
a solution of ninhydrin in ethanol (99.5) (1 in 500) on the
plate, and heat at 110°C for 5 minutes: the spots other than
the principal spot from the sample solution is not more in-
tense than the spot from the standard solution (3), and the
total of the amount of each spot other than the principal spot
from the sample solution, which is calculated by the compari-
son with the spots obtained from the standard solutions (1),
(2) and (3), is not more than 5%.
(4) 2-Formylbenzoic acid — Dissolve 50 mg of Talampicil-
lin Hydrochloride in ethanol (99.5) to make exactly 10 mL,
and use this solution as the sample solution. Separately, dis-
solve 10 mg of 2-formylbenzoic acid in ethanol (99.5) to
make exactly 100 mL. Pipet 5 mL of this solution, add
ethanol (99.5) to make exactly 10 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography, develop the
plate with a mixture of chloroform and acetic acid (100) (4:1)
to a distance of about 13 cm, and air-dry the plate. Spray
evenly a solution of 2,4-dinitrophenylhydrazine in diluted
sulfuric acid (6 in 25) (1 in 500): the spot of 2-formylbenzoic
acid obtained from the sample solution is not more intense
than that obtained from the standard solution.
Water <2.48> Not more than 3.0% (0.5 g, volumetric titra-
tion, direct titration).
Assay Weigh accurately an amount of Talampicillin
Hydrochloride and Talampicillin Hydrochloride Reference
Standard, equivalent to about 20 mg (potency), dissolve in
water to make exactly 20 mL each, and use these solutions as
the sample solution and the standard solution. The standard
solution should be prepared before use. Pipet 2 mL each of
the sample solution and the standard solution in separate
100-mL glass-stoppered flasks, add 2.0 mL of sodium
hydroxide TS, and allow them to stand for exactly 15
minutes. Add 2.0 mL of diluted hydrochloric acid (1 in 10)
and exactly 10 mL of 0.005 mol/L iodine VS, allow them to
stand for exactly 15 minutes, and titrate <2.50> with 0.01
mol/L sodium thiosulfate VS until the color of the solution is
disappeared. If necessary, add 0.2 to 0.5 mL of starch TS.
Separately, pipet 2 mL each of the sample solution and the
standard solution in separate 100-mL glass-stoppered flasks,
add exactly 10 mL of 0.005 mol/L iodine VS, titrate <2.50>
with 0.01 mol/L sodium thiosulfate VS until the color of the
solution is disappeared, and make any necessary correction.
For this titration, add 0.2 to 0.5 mL of starch TS, if necessa-
ry. Calculate the amount (mL) of 0.005 mol/L iodine VS, K T
and V s , consumed by the sample solution and the standard
solution, respectively.
Amount [fig (potency)] of ampicillin (C 16 H 19 N 3 04S)
= ^ s x(I/ T /Fs)xl000
W s : Amount [mg (potency)] of Talampicillin Hydrochlo-
ride Reference Standard
Containers and storage Containers — Tight containers.
Talc
Talc is a native, hydrous magnesium silicate, some-
times containing a small portion of aluminum silicate.
Description Talc occurs as a white to grayish white, fine,
crystalline powder. It is odorless and tasteless.
It is unctuous, and adheres readily to the skin.
It is practically insoluble in water, in ethanol (95) and in
diethyl ether.
Identification (1) Mix 0.2 g of Talc with 0.9 g of anhy-
drous sodium carbonate and 1.3 g of potassium carbonate,
and heat the mixture in a platinum or nickel crucible until fu-
sion is complete. Cool, and transfer the fused mixture to a
beaker with the aid of 50 mL of hot water. Add hydrochloric
acid until it ceases to cause effervescence, add 10 mL of
hydrochloric acid, and evaporate the mixture on a water bath
to dryness. Cool, add 20 mL of water, boil, and filter. Add 10
mL of a solution of methylene blue trihydrate (1 in 10,000) to
the residue, and wash with water: the precipitate is blue in
color.
(2) Dissolve 2 g of ammonium chloride and 5 mL of am-
monia TS in the filtrate obtained in (1), filter if necessary, and
1 140 Tamsulosin Hydrochloride / Official Monographs
JP XV
add disodium hydrogenphosphate TS: a white, crystalline
precipitate is produced.
Purity (1) Acid-soluble substances — Weigh accurately
about 1 g of Talc, heat with 20 mL of dilute hydrochloric
acid at 50°C for 15 minutes with stirring. Cool, add water to
make exactly 50 mL, and filter. Centrifuge, if necessary, until
the filtrate becomes clear. To 25 mL of this filtrate add 1 mL
of dilute sulfuric acid, evaporate to dryness, and ignite to
constant mass at 800 ± 25 °C: the amount of the residue is
not more than 2.0%.
(2) Acid or alkali, and water-soluble substances — To 10.0
g of Talc, add 50 mL of water, weigh, and boil for 30
minutes, supplying water lost by evaporation. Cool, add
water to restore the original mass, and filter. Centrifuge, if
necessary, until the filtrate becomes clear: the filtrate is neu-
tral. Evaporate 20 mL of the filtrate to dryness, and dry the
residue at 105°C for 1 hour: the mass of the residue is not
more than 4.0 mg.
(3) Water-soluble iron — Make 10 mL of the filtrate ob-
tained in (2) weakly acidic with hydrochloric acid, and add
dropwise potassium hexacyanoferrate (II) TS: the liquid does
not acquire a blue color.
(4) Arsenic <1.11>— To 0.5 g of Talc add 5 mL of dilute
sulfuric acid, and heat gently to boiling with shaking. Cool
immediately, filter, and wash the residue with 5 mL of dilute
sulfuric acid, then with 10 mL of water. Combine the filtrate
and the washings, evaporate to 5 mL on a water bath, and
perform the test with this solution as the test solution (not
more than 4 ppm).
Loss on ignition <2.43> Not more than 5.0% (1 g, 450 -
550°C, 3 hours).
Containers and storage Containers — Well-closed contain-
ers.
Tamsulosin Hydrochloride
^AXny^lS
C 20 H 28 N 2 O 5 S.HCl: 444.97
5- {(2/?)-2-[2-(2-Ethoxyphenoxy)ethylamino]propyl} -
2-methoxybenzenesulfonamide monohydrochloride
[106463-1 7-6]
Tamsulosin Hydrochloride, when dried, contains
not less than 98.5% and not more than 101.0% of
C 20 H 28 N 2 O 5 S.HCl.
Description Tamsulosin Hydrochloride occurs as white
crystals.
It is freely soluble in formic acid, sparingly soluble in
water, slightly soluble in acetic acid (100), and very slightly
soluble in ethanol (99.5).
Melting point: about 230°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Tamsulosin Hydrochloride (3 in 160,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of Tam-
sulosin Hydrochloride as directed in the potassium chloride
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
(3) To 5 mL of an ice cooled solution of Tamsulosin
Hydrochloride (3 in 400) add 3 mL of dilute nitric acid, shake
well, allow to stand at room temperature for 30 minutes, and
filter: the filtrate responds to the Qualitative Tests <1.09> for
chloride.
Optical rotation <2.49> [a]™: -17.5 --20.5° (after
drying, 0.15 g, water, warming, after cooling, 20 mL, 100
mm).
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tamsulosin Hydrochloride according to Method 4, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances —
(i) Dissolve 50 mg of Tamsulosin Hydrochloride in 10
mL of the mobile phase, and use this solution as the sample
solution. Pipet 2 mL of the sample solution, and add the mo-
bile phase to make exactly 50 mL. Pipet 2.5 mL of this solu-
tion, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
10 ^L each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of the peak other
than tamsulosin obtained from the sample solution is not
larger than the peak area of tamsulosin from the standard so-
lution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 225 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 4.4 mL of perchloric acid and 1.5 g
of sodium hydroxide in 950 mL of water, adjust the pH to 2.0
with sodium hydroxide TS, and add water to make 1000 mL.
To 700 mL of this solution add 300 mL of acetonitrile for liq-
uid chromatography.
Flow rate: Adjust the flow rate so that the retention time of
tamsulosin is about 6 minutes.
Time span of measurement: Until tamsulosin is eluted, be-
ginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase to make ex-
actly 50 mL. Confirm that the peak area of tamsulosin ob-
tained from 10 /xL of this solution is equivalent to 1.4 to
2.6% of that from 10 /uL of the standard solution.
System performance: Dissolve 5 mg of Tamsulosin
Hydrochloride and 10 mg of propyl parahydroxybenzoate in
20 mL of the mobile phase. To 2 mL of this solution add the
JPXV
Official Monographs / Tartaric Acid 1141
mobile phase to make 20 mL. When the procedure is run with
10 /xL of this solution under the above operating conditions,
tamsulosin and propyl parahydroxybenzoate are eluted in
this order with the resoluton between these peaks being not
less than 12.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tamsulosin is not more than 4.0%.
(ii) Perform the test with 10 /uL each of the sample solu-
tion and standard solution which are obtained in above (i) as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine each peak area by
the automatic integration method: the area of the peak other
than tamsulosin obtained from the sample solution is not
larger than the peak area of tamsulosin from the standard so-
lution.
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity (2) (i).
Mobile phase: Dissolve 4.4 mL of perchloric acid and 1 .5 g
of sodium hydroxide in 950 mL of water, adjust the pH to 2.0
with sodium hydroxide TS, and add water to make 1000 mL.
To this solution add 1000 mL of acetonitrile for liquid chro-
matography.
Flow rate: Adjust the flow rate so that the retention time of
tamsulosin is about 2.5 minutes.
Time span of measurement: About 5 times as long as the
retention time of tamsulosin, beginning after the peak of
tamsulosin.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add the mobile phase used in the
Purity (2) (i) to make exactly 50 mL. Confirm that the peak
area of tamsulosin obtained from 10 /xh of this solution is
equivalent to 1 .4 to 2.6% of that from 10 /uL of the standard
solution.
System performance: Proceed as directed in the system
suitability in the Purity (2) (i).
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tamsulosin is not more than 4.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Tamsulosin
Hydrochloride, previously dried, dissolve in 5 mL of formic
acid, add 75 mL of a mixture of acetic acid (100) and acetic
anhydride (3:2), and immediately titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 44.50 mg of C 20 H 28 N 2 O 5 S.HCl
Containers and storage Containers — Well-closed contain-
ers.
Tannic Acid
?>->m
Tannic Acid is the tannin usually obtained from nut-
galls or rhusgalls.
Description Tannic Acid occurs as a yellowish white to light
brown, amorphous powder, glistening leaflets, or spongy
masses. It is odorless or has a faint, characteristic odor, and
has a strongly astringent taste.
It is very soluble in water and in ethanol (95), and practi-
cally insoluble in diethyl ether.
Identification (1) To 5 mL of a solution of Tannic Acid (1
in 400) add 2 drops of iron (III) chloride TS: a blue-black
color develops. Allow the solution to stand: a blue-black
precipitate is produced.
(2) To 5 mL of a solution of Tannic Acid (1 in 20) add 1
drop each of albumin TS, gelatin TS, or 1 mL of starch TS: a
precipitate is produced in each solution.
Purity (1) Gum, dextrin and sucrose — Dissolve 3.0 g of
Tannic Acid in 15 mL of boiling water: the solution is clear or
slightly turbid. Cool, and filter the solution. To 5 mL of the
filtrate add 5 mL of ethanol (95): no turbidity is produced.
Add further 3 mL of diethyl ether to this solution: no turbidi-
ty is produced.
(2) Resinous substances — To 5 mL of the filtrate ob-
tained in (1) add 10 mL of water: no turbidity is produced.
Loss on drying <2.41> Not more than 12.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 1.0% (0.5 g).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tartaric Acid
>mw.
H OH
HO ? C
„CO;H
H OH
C 4 H 6 6 : 150.09
(2i?,3/?)-2,3-Dihydroxybutanedioic acid [87-69-4]
Tartaric Acid, when dried, contains not less than
99.7% of C 4 H 6 6 .
Description Tartaric Acid occurs as colorless crystals or a
white, crystalline powder. It is odorless, and has a strong acid
taste.
It is very soluble in water, freely soluble in ethanol (95),
and slightly soluble in diethyl ether.
A solution of Tartaric Acid (1 in 10) is dextrorotatory.
Identification (1) Ignite Tartaric Acid gradually: it
decomposes and an odor of burning sugar is perceptible.
(2) A solution of Tartaric Acid (1 in 10) changes blue lit-
1 142 Taurine / Official Monographs
JP XV
mus paper to red, and responds to the Qualitative Tests
<1.09> for tartrate.
Purity (1) Sulfate <1.14>— Perform the test with 0.5 g of
Tartaric Acid. Prepare the control solution with 0.50 mL of
0.005 mol/L sulfuric acid VS (not more than 0.048%).
(2) Oxalate — Dissolve 1.0 g of Tartaric Acid in 10 mL of
water, and add 2 mL of calcium chloride TS: no turbidity is
produced.
(3) Heavy metals <1.07> — Proceed with 2.0 g of Tartaric
Acid according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(4) Calcium — Neutralize a solution of 1.0 g of Tartaric
Acid in 10 mL of water with ammonia TS, and add 1 mL of
ammonium oxalate TS: no turbidity is produced.
(5) Arsenic </.//> — Prepare the test solution with 2.0 g
of Tartaric Acid according to Method 1 , and perform the test
(not more than 1 ppm).
Loss on drying <2.41>
3 hours).
Not more than 0.5% (3 g, silica gel,
Residue on ignition <2.44> Not more than 0.05% (1 g).
Assay Weigh accurately about 1 .5 g of Tartaric Acid, previ-
ously dried, dissolve in 40 mL of water, and titrate <2.50>
with 1 mol/L sodium hydroxide VS (indicator: 2 drops of
phenolphthalein TS).
Each mL of 1 mol/L sodium hydroxide VS
= 75.04 mg of C 4 H 6 6
Containers and storage Containers — Well-closed contain-
ers.
Taurine
^^,S0 3 H
H 2 N
C 2 H 7 N0 3 S: 125.15
2-Aminoethanesulfonic acid
[107-35-7]
Taurine, when dried, contains not less than 99.0%
and not more than 101.0% of C 2 H 7 N0 3 S.
Description Taurine occurs as colorless or white crystals, or
a white crystalline powder.
It is soluble in water, and practically insoluble in ethanol
(99.5).
The pH of a solution prepared by dissolving 1.0 g of Tau-
rine in 20 mL of freshly boiled and cooled water is between
4.1 and 5.6.
Identification Determine the infrared absorption spectrum
of Taurine as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Purity (1) Clarity and color of solution — A solution ob-
tained by dissolving 1.0 g of Taurine in 20 mL of water is
clear and colorless.
(2) Chloride</.03>— Perform the test with 1.0 g of Tau-
rine. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.011%).
(3) Sulfated. 74>— Perform the test with 2.0 g of Tau-
rine. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.010%).
(4) Ammonium</.ft2> — Perform the test with 0.25 g of
Taurine. Prepare the control solution with 5.0 mL of Stan-
dard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 2.0 g of Taurine
according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 10 ppm).
(6) Iron<7.70> — Prepare the test solution with 2.0 g of
Taurine according to Method 1, and perform the test accord-
ing to Method A. Prepare the control solution with 2.0 mL of
Standard Iron Solution (not more than 10 ppm).
(7) Related substances — Dissolve 1.0 g of Taurine in 50
mL of water, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add water to make ex-
actly 50 mL. Pipet 1 mL of this solution, add water to make
exactly 10 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /iL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of water, ethanol (99.5), 1-butanol and acetic acid (100)
(150:150:100:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly ninhydrin-butanol TS on the plate, and
heat at 105°C for 5 minutes: the spot other than the principle
spot with the sample solution is not more than one spot, and
it is not more intense than the spot with the standard solu-
tion.
Loss on drying <2.41> Not more than 0.20% (1 g, 105 °C, 2
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Taurine, previously
dried, dissolve in 50 mL of water, add 5 mL of formaldehyde
solution, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (potentiometric titration). Perform a blank determination
in the same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 12.52 mg of C 2 H 7 N0 3 S
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Teceleukin (Genetical Recombination) 1143
Teceleukin (Genetical
Recombination)
Ttn^>(IB»i)
Met-Ala-Pro-ThrSer-Sei-Ser-TTirLys-Lys-Thr Gin Leu Gin Lou- Glu His Leu Leu Leu-
Asp Leu Gin Met- lie Lau-Asn-Gly-lle-Asn ■Asn-Tyr-Lys-Asn-Pii>-Ly3-Leu-Tlir-Aig-Mel-
Leu-Thr - Phe -Lys Phe -Tyr - Met ■ Pro Lys - Lys - Ala-Th r- G lu - Leu-Lys-His- Leu - Gin- Cys-Lou -
\
Glu-GkrGru-Leu LysProLau Glu Gru Val-Lau-AsnLeu-Ala-Gtn-Ser-Lys-Asn Pha His-
Lsu - Arg- Pro - Arg- Asp - Leu- 1 le - Ser - Asn ■■ I le Asji ■ Val I la - Val - Leu - Glu - Leu-Lys - Gly -Ser-
Glu-Thr-ThfPha Mai Cys-Glu-Tyr-Ala-Asp-Glu-Thr-AlaThr lle-Val-GTu-Phe-Leu-Asni-
Arg-Trp-llB-Thir-Phe-Cys-Gln-Ser-lle-lla SarThr-Lau Thr
C 698 H 1127 N 179 O 2 4 S 8 : 15547.01
[136279-32-8]
The desired product of Teceleukin (Genetical
Recombination) is a protein consisting of 134 amino
acid residues manufactured by E. coli through expres-
sion of human interleukin-2 cDNA. It is a solution and
possesses a T-lymphocyte activating effect.
It contains potency between 7.7 x 10 6 and 1.54 x 10 7
units/mL, and not less than 7.7 x 10 6 units permg of
protein.
Description Teceleukin (Genetical Recombination) occurs
as a clear and colorless liquid.
Identification (1) Measure accurately an appropriate
amount of Teceleukin (Genetical Recombination), add ac-
curately to a concentration of 200 units per mL of culture
medium for assay of teceleukin, and use this solution as the
sample stock solution. Dilute reference anti-interleukin-2 an-
tibody for teceleukin with culture medium for assay of
teceleukin to a concentration of approximately 200 neutral
units/mL and use this solution as the interleukin-2 neutral
antibody solution. Accurately add an equivalent volume of
the interleukin-2 neutral antibody solution to the sample
stock solution, shake, and then leave for 1 hour in a 37 °C in-
cubator in air containing 5% carbon dioxide. This solution is
the sample solution. Prepare a standard solution by accurate-
ly adding an equivalent volume of culture medium for assay
of teceleukin to the sample stock solution, mixing, and then
processing in the same way. Process the sample and standard
solutions according to the assay method, determine their
respective dilution coefficients, D N and D T , and then deter-
mine the neutralization rate, which should be at least 90%,
using the following formula.
Neutralization rate (%) = {(D T -D K )/D T } x 100
However, please note if the mean values of the absorbance of
the maximum uptake control solution and absorbance of the
minimum uptake control solution do not fit the standard
curve, the neutralization coefficient is to be determined within
the following range.
Neutralization coefficient (%) > {(D T - 2)/D T } x 100
(2) Place a volume of Teceleukin (Genetical Recombina-
tion) corresponding to approximately 50 /xg of protein into 2
test tubes for hydrolysis, evaporate to dryness under vacuum,
and use one as the sample (1). To the other, add 50,«L of a
mixture of formic acid and hydrogen peroxide (30) (9:1) that
has been left at room temperature for one hour, cool for 4
hours in ice, add 0.5 mL of water, and then evaporate to dry-
ness under vacuum to give the sample (2). To 1.3 mL of
methanesulfonic acid add 3.7 mL of water, mix well, add and
dissolve 10 mg of 3-(2-aminoethyl)indole, to make a 4 mol/L
methanesulfonic acid solution. Dissolve 39.2 g of trisodium
citrate dihydrate, 33 mL of hydrochloric acid, 40 mL of thio-
diglycol, and 4 mL of lauromacrogol solution (1 in 4) in 700
mL of water, adjust the pH to 2.2, add water to 1000 mL,
add 100 //L of capric acid, and mix to make a sodium citrate
solution for dilution. Add 50 /xL of freshly prepared 4 mol/L
methanesulfonic acid to the sample (1) and sample (2), cool
to -70°C, and then deaerate under vacuum. Heat to 115°C
±2°C for 24 hours after sealing these test tubes under
reduced pressure. After cooling, unseal, add 50 /xL of 4
mol/L sodium hydroxide TS followed by 0.4 mL of sodium
citrate solution for dilution to make the sample solution (1)
and sample solution (2). Separately, accurately measure 0.25
mmol amounts of L-aspartic acid, L-threonine, L-serine, L-
glutamic acid, L-proline, glycine, L-alanine, L-valine, l-
methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylala-
nine, L-lysine hydrochloride, ammonium chloride, L-histi-
dine hydrochloride monohydrate, and L-arginine hydrochlo-
ride as well as 0.125 mmol of L-cysteine and then dissolve in
0.1 mol/L hydrochloric acid TS to make exactly 100 mL.
This is the amino acid standard stock solution. Accurately
measure 1 mL of this solution, and add sodium citrate solu-
tion for dilution to make exactly 25 mL. This is solution A.
Accurately weigh approximately 20 mg of L-tryptophan and
dissolve in water to make exactly 1000 mL. This is solution B.
Accurately measure 10 mL of both solution A and solution
B, combine together, and add sodium citrate solution for di-
lution to make exactly 50 mL. This is the amino acid standard
solution. Separately, accurately weigh approximately 17 mg
of L-cysteic acid and dissolve in sodium citrate solution for
dilution to make exactly 50 mL. Accurately measure 1 mL of
this solution and add sodium citrate solution for dilution to
make exactly 100 mL. This is the cysteic acid standard solu-
tion. Accurately measure 0.25 mL of the sample solution (1),
the sample solution (2), amino acid standard solution, and
the cysteic acid standard solution. When the test is conducted
by Liquid Chromatography <2.01> under the following condi-
tions, peaks for the 18 amino acids are observed in the chro-
matogram obtained from the sample solution (1). Also,
measure the peak area of each amino acid in the sample solu-
tion (1) and the amino acid standard solution, and taking the
molar number of alanine in the sample solution (1) as 5.0, de-
termine the concentrations of aspartic acid, glutamic acid,
proline, glycine, methionine, leucine, tyrosine, phenylala-
nine, lysine, histidine, tryptophan, and arginine and then cal-
culate the molar ratio for each amino acid. Also, measure the
cysteic acid peak areas of the sample solution (2) and the cys-
teic acid standard solution, determine the concentration of
cysteine, and, taking the molar number of alanine in the sam-
ple solution (2) as 5.0, calculate the molar ratio of cysteine.
When determining the molar ratios of the respective amino
acids, aspartic acid is 11.4 to 12.6, glutamic acid 17.1 to 18.9,
proline 4.5 to 5.5, glycine 1.8 to 2.2, cysteine 2.7 to 3.3,
methionine 4.5 to 5.5, leucine 20.9 to 23.1, tyrosine 2.7 to
3.3, phenylalanine 5.4 to 6.6, lysine 10.5 to 11.6, histidine 2.7
to 3.3, tryptophan 0.7 to 1.2, and arginine 3.6 to 4.4.
1144 Teceleukin (Genetical Recombination) / Official Monographs
JP XV
Operating conditions —
Detector: Visible absorption photometer [wavelengths: 440
nm (proline) and 570 nm (amino acids other than proline)]
Column: A stainless steel column with an inside diameter
of 4 mm and length of 25 cm packed with a strongly acidic
ion exchange resin for liquid chromatography consisting of
polystyrene to which sulphonate group binds.
Column temperature: A constant temperature of about
50°C when the sample is injected. After a certain time, in-
crease the temperature to a constant temperature of about
62°C
Reaction temperature: A constant temperature of about
98°C.
Time for color formation: Approximately 2 minutes
Mobile phase: After preparing mobile phases A, B, and C
according to the following table, add 0.1 mL of capric acid to
each.
Mobile phase
A
Mobile phase
B
Mobile phase
C
Citric acid
monohydrate
18.70 g
10.50 g
7.10g
Trisodium citrate
dihydrate
7.74g
14.71 g
26.67 g
Sodium chloride
7.07 g
2.92 g
54.35 g
Ethanol (99.5)
60 mL
—
—
Benzyl alcohol
—
—
10 mL
Thiodiglycol
5mL
5mL
—
Lauromacrogol
solution (1 in 4)
4mL
4mL
4mL
Water
Appropriate
Appropriate
Appropriate
amount
amount
amount
pH
3.2
4.3
4.7
Total volume
1000 mL
1000 mL
1000 mL
Changing mobile phases and column temperature: When
operating under the above conditions using 0.25 mL of ami-
no acid standard solution, the amino acids will elute in the
following order; aspartic acid, threonine, serine, glutamic
acid, proline, glycine, alanine, cystine, valine, methionine,
isoleucine, leucine, tyrosine, phenylalanine, lysine, ammo-
nia, histidine, tryptophan, and arginine. Switchover to mo-
bile phase A, mobile phase B, and mobile phase C, in se-
quence so that the resolution between the peaks of cystine
and valine is 2.0 or more and that between ammonia and
histidine is 1.5 or more. Also, increase the temperature after
a constant length of time so that the resolution between the
peaks of glutamic acid and proline is at least 2.0.
Reaction reagents: Dissolve 408 g of lithium acetate dihy-
drate in water, and add 100 mL of acetic acid (100) and water
to make 1000 mL. To this solution add 1200 mL of dimethyl-
sulfoxide and 800 mL of 2-methoxyethanol. This is solution
(I). Separately, mix together 600 mL of dimethylsulf oxide
and 400 mL of 2-methoxyethanol and then add 80 g of nin-
hydrin and 0.15 g of sodium borohydride. This is solution
(II). After gassing 3000 mL of the solution (I) for 20 minutes
with nitrogen, rapidly add 1000 mL of the solution (II) and
then mix by gassing for 10 minutes with nitrogen.
Mobile phase flow rate: About 0.275 mL every minute
Reaction reagent flow rate: About 0.3 mL every minute
System suitability —
System performance: When 0.25 mL of the amino acid
standard solution is run under the above conditions, the reso-
lution between the peaks of threonine and serine is at least
1.5.
(3) Dissolve 0.242 g of 2-amino-2-hydroxymethyl-l,3-
propanediol, 5.0 g of sodium lauryl sulfate, and 74 mg of dis-
odium dihydrogen ethylenediamine tetraacetate dihydrate in
60 mL of water. After adjusting the pH to 8.0 using 1 mol/L
hydrochloric acid TS, add water to make 100 mL. This is the
molecular weight determination buffer solution. Accurately
measure 20 fiL of Teceleukin (Genetical Recombination), add
exactly 20 fiL of the molecular weight determination buffer
solution and 2ftL of 2-mercaptoethanol, and then heat for 5
minutes on a 90 to 100°C water bath without allowing any
water evaporation from the mixture. After cooling, add ex-
actly 1 fiL of bromophenol blue solution (1 in 2000) and then
shake. This is the sample solution. Separately, accurately
measure 5 fiL of molecular weight marker for teceleukin, and
add exactly 50 fiL of water, 55 fiL of the molecular weight de-
termination buffer solution, and 5 fiL of 2-mercaptoethanol,
and then heat for 5 minutes on a 90 to 100°C water bath
without allowing any water evaporation from the mixture.
After cooling, add exactly 1 fiL of bromophenol blue solu-
tion (1 in 2000), and shake well. This is the molecular weight
standard solution. When conducting a test using SDS-
polyacrylamide gel electrophoresis with 1 fiL each of the sam-
ple solution and the molecular weight standard solution, the
molecular weight of the main band is between the range of
14,000 and 16,000.
Operating conditions —
Equipment: Horizontal electrophoresis vessel equipped
with a cooling unit, a device that accumulates load voltage
over time, and a direct current power source device that con-
trols the amperage, voltage, wattage.
Spotting of solutions: Solutions are spotted on concentrat-
ing gel of polyacrylamide gel sheets.
Electrophoresis conditions
Polyacrylamide gel sheet: Polyester sheet to which a
polyacrylamide gel (width, about 43 mm, length, about 50
mm, and thickness, about 0.5 mm) is closely adhered. The
polyacrylamide gel consists of a concentrating gel with a
gel support concentration of 7.5% and a 3% degree of
crosslinking and a separating gel with corresponding values
of 20% and 2%. The gel contains pH 6.5 Tris-acetate
buffer.
Buffer solution for electrode: Prepared by dissolving
35.83 g of tricine, 24.23 g of 2-amino-2-hydroxymethyl-
1,3-propanediol, and 5.5 g of sodium lauryl sulfate in
water to make 1000 mL.
Cooling temperature of gel support plate: 15°C
Running conditions
Pre-electrophoresis and electrophoresis: The voltage,
amperage, and wattage should not exceed 250 V, 10 mA,
and 3 W, respectively. The amperage and wattage should
be proportional to the number of polyacrylamide sheets.
Immediately after adding sample: The voltage, am-
perage, and wattage should not exceed 250 V, 1 mA, and 3
W, respectively. The amperage and wattage should be
proportional to the number of polyacrylamide sheets.
Electrophoresis time
Before adding sample: Until value of load voltage in-
tegrated with respect to time reaches 60 V-h.
Immediately after adding sample: Until value of load
voltage integrated with respect to time reaches 1 V-h.
Main electrophoresis: Until value of load voltage in-
tegrated with respect to time reaches 140 V-h.
Fixation and staining
JPXV
Official Monographs / Teceleukin (Genetical Recombination) 1145
Dissolve 25 g of anhydrous sodium carbonate anhydride
and 0.8 mL of formaldehyde solution in water to make
1000 mL. This is the developing solution. After immersing
the polyacrylamide gel sheet in a mixture of ethanol (99.5),
water and acetic acid (100) (5:4:1) for 2 minutes, immerse
for 2 minutes in a mixture of water, ethanol (99.5) and a-
cetic acid (100) (17:2:1). Change the mixture, immerse for
another 4 minutes, immerse in water for 2 minutes to rinse
the polyacrylamide gel sheet, and change the water to im-
merse for 2 minutes. This procedure is carried out with
heating to 50°C. Next, while heating at 40°C, immerse for
10 to 15 minutes in diluted silver nitrate TS (1 in 7), warm
to 30°C, and gently rinse the polyacrylamide gel sheet with
water. While warming at 30°C, immerse the poly-
acrylamide gel sheet in freshly prepared developing solu-
tion. After obtaining adequate color formation, immerse
the polyacrylamide gel sheet in diluted acetic acid (100) (1
in 20) to terminate the color formation.
Estimation of molecular weight
Plot graphs for each band obtained from the molecular
weight standard solution, distance from the border of the
concentrating gel and separating gel, and the logarithm of
the molecular weight of proteins in each band. Determine
the molecular mass by reading the corresponding position
of the major band obtained from the sample solution on
the graph.
(4) The isoelectric point determined from the electropho-
resis position is 7.4 to 7.9 when 3 fiL of Teceleukin (Genetical
Recombination) and 8 fiL of isoelectric marker for teceleukin
are tested by the polyacrylamide gel isoelectric method.
Operating conditions —
Equipment: Horizontal electrophoretic vessel with cooling
unit and direct current power source that can perform con-
stant wattage control.
Preparation of polyacrylamide gel: Dissolve 1 .62 g of poly-
acrylamide and 50 mg of 7V,iV'-methylenebisacrylamide in
water to make 25 mL. Accurately measure 7.5 mL of this so-
lution, 2 mL of a 10 mL solution prepared by adding water to
5 g of glycerin, and 0.64 mL of a pH 3 to pH 10 amphoteric
electrolyte solution, and degas under reduced pressure while
stirring thoroughly. Next, accurately measure 74 fiL of fresh-
ly prepared ammonium peroxodisulfate solution (1 in 50), 3
fiL of A f ,A r ,A f ',./V'-tetramethylethylenediamine, and 50 ^L of
freshly prepared riboflavin sodium phosphate solution (1 in
1000), stir well, immediately pour on a gel preparation plate
(10 cm wide, 1 1 cm long, and 0.8 mm thick), and then expose
to a fluorescent light source for 60 minutes to gelate.
Spotting
Add Teceleukin (Genetical Recombination) or isoelectric
marker for teceleukin 30 minutes after starting electropho-
resis to wells in gel plates to which plastic tape (3.5 mm
wide, 3.5 mm long, 0.4 mm thick) has been applied in ad-
vance and that have undergone gelation.
Electrophoresis conditions
Cathode solution: Sodium hydroxide TS
Anode solution: DL-aspartic acid solution (133 in 25,000)
Cooling temperature of gel support plate: 2±1°C
Running conditions: After starting the electrophoresis, a
constant wattage of 10 W for 20 minutes and 20 W there-
after. However, the voltage should be 3000 V or less.
Running time: 120 to 140 minutes while blowing Nitrogen
into the electrophoresis vessel.
Fixation and washing
Dissolve 28.75 g of trichloroacetic acid and 8.65 g of 5-
sulfosalicylic acid dihydrate in 75 mL of methanol and 175
mL of water. Immerse the gel in this solution for 60
minutes to fix the protein to the gel. After fixation, im-
merse for 10 minutes in a mixture of water, ethanol (99.5)
and acetic acid (100) (67:25:8).
Staining and decolorization
Dissolve 0.11 g of Coomassie brilliant blue G-250 in 25
mL of ethanol (99.5), and add 8 mL of acetic acid (100)
and water to make 100 mL. This is the staining solution.
Immerse the gel for 10 minutes while heating at 60°C in
freshly filtered staining solution. After staining, decolorize
by immersing in a mixture of water, ethanol (99.5) and
acetic acid (100) (67:25:8).
Determination of isoelectric point
Plot the protein isoelectric points and the distance from
the cathode of each band obtained from the isoelectric
markers for teceleukin. Determine the isoelectric point
from the corresponding position of the major bands ob-
tained from the sample solution.
pH <2.54> 2.7 to 3.5
Purity (1) Host cell-derived protein — Take an appropriate
amount of Teceleukin (Genetical Recombination) and add an
exact amount of diluted acetic acid (100) (1 in 350) to make a
solution containing between 0.68 and 0.72 mg of protein in
one mL. This is the sample stock solution. Dissolve 1.52 g of
2-amino-2-hydroxymethyl-l ,3-propanediol hydrochloride
and 10.94 g of 2-amino-2-hydroxymethyl-l,3-propanediol in
water to make 200 mL. Dissolve 0.5 g of bovine serum albu-
min in 25 mL of this solution. This is 2 w/v% bovine serum
albumin-Tris-hydrochloride buffer solution. Accurately
measure 0.5 mL of the sample stock solution, add exactly 30
fiL of sodium carbonate TS, stir, and immediately add ex-
actly 0.47 mL of the 2 w/v% bovine serum albumin-Tris-
hydrochloride buffer solution to make the sample solution.
Accurately measure 10 mL of dilute acetic acid (100) (1 in
350), add 0.6 mL of sodium carbonate TS, and then add 2
w/v% bovine serum albumin-Tris-hydrochloride buffer solu-
tion to make exactly 20 mL. This is the dilution solution.
Add the E. coli protein stock solution to this dilution solution
to make a solution containing 0.015 fig of E. coli protein in
one mL. This is standard solution (1). Accurately dilute this
solution serially two-fold with the dilution solution to make
standard solutions (2) to (8) having different concentrations
of E. coli protein. Dissolve 0.5 g of bovine serum albumin in
100 mL of 0.01 mol/L phosphate buffer-sodium chloride TS,
pH 7.4. This is the wash solution. Accurately measure 0.1 mL
of the sample solution, standard solutions (1) to (8), and dilu-
tion solution as a blank standard solution and place each in 3
wells in solid phase plates (place dilution solution in 6 wells),
cover with plastic wrap, mix by shaking in a horizontal direc-
tion, and then leave standing at a constant temperature of
about 25 °C for 5 to 16 hours. Next, remove the solution from
each well by aspiration, add 0.25 mL of the the wash solu-
tion, mix again by shaking in a horizontal direction, and then
remove by aspiration. Repeat this procedure 2 more times by
adding 0.25 mL of the wash solution to each well. Freshly di-
lute peroxidase marker antibody stock solution with 1 w/v%
bovine serum albumin-phosphate buffer-sodium chloride TS,
add exactly 0.1 mL to each well, cover with plastic wrap, mix
by shaking in a horizontal direction, and then leave standing
at a constant temperature of about 25°C for 16 to 24 hours.
1146 Teceleukin (Genetical Recombination) / Official Monographs
JP XV
Next, remove the solution in the wells by aspiration, add 0.25
mL of the wash solution, mix by shaking in a horizontal
direction, and then remove the solution by aspiration. Using
0.25 mL of the wash solution, repeat this procedure 2 more
times for each well. To each well, accurately add 0.1 mL of
teceleukin chromophore solution, stir gently, and then shield
from light and leave standing for 30 minutes at a constant
temperature of about 25 °C. Add exactly 0.1 mL of diluted
sulfuric acid (3 in 50) to each well and then mix by gently
shaking horizontally. Perform the test with these solutions as
directed under Ultraviolet-visible spectrophotometry <2.24>,
and measure the absorbances A T2 and A S2 at 450 nm and A Tl
and A sl at 510 nm. Prepare a standard curve by plotting the
values obtained from each standard solution (A S2 - A sl ) on a
graph having the concentration of E. coli protein (ng/mL) in
logarithmic scale on the horizontal axis and the absorbance
values on the vertical axis. Match the values obtained from
the sample solution (A T2 - A Tl ) to the standard curve, deter-
mine the concentration A of E. coli protein in the sample so-
lution, and take the mean. The amount of E. coli protein is
not more than 5 ng when the amount of E. coli protein per
mg of protein is determined using the following formula.
Amount (ng) of E. coli-derived protein per mg protein
= A/C
C: Protein concentration (mg/mL) in sample solution
The test is valid if the E. coli protein concentration is 0.3
ng/mL or less when the concentration is obtained by fitting
the absorbance value at detection limit, calculated from the
following formula using absorvance value of the dilution so-
lution, to the standard curve.
Absorbance at detection limit
= X+3.3x
£(Xi-JQ 2
/(6-1)
X\: Individual absorbance values obtained from the dilu-
tion solution
X: The mean of absorbance values obtained from the dilu-
tion solution
6: The number of wells in the microplate containing dilu-
tion solution
(2) Tetracycline hydrochloride — Serially subculture
through 2 passages at 35 to 37°C the test bacteria Micrococ-
cus luteus A T CC9341 in a slant culture of test bacteria inocu-
lation media for teceleukin and then dilute this 100-fold by
adding sterilized purified water. This is the test bacteria solu-
tion. Store the test bacteria solution at 5°C or less and use the
solution within 5 days. Dilute the test bacteria solution serial-
ly by adding sterilized purified water, add an appropriate
amount to 100 mL of normal agar medium for teceleukin,
conduct a preliminary test, and determine the amount of
tetracycline hydrochloride that shows an inhibition zone cor-
responding to standard solution containing 0.5 ^g (potency)
in 1 mL. Add this amount to 100 mL of normal agar medium
for teceleukin dissolved and then cooled to 45 to 50°C and
mix. Pipet 25 mL of this solution into square Petri dishes
(135x95 mm) and spread horizontally to solidify. Prepare
plates for testing by making an appropriate number of wells
in this agar medium. The volume of the test bacteria solution
to which 100 mL of normal agar medium for teceleukin has
been added is 0.25 to 1.0 mL. Accurately measure an ap-
propriate amount of Tetracycline Hydrochloride Reference
Standard and dilute accurately with water to make a clear so-
lution with a concentration of 1 mg (potency)/mL. Accurate-
ly measure an appropriate amount of this solution and dilute
precisely with water to make standard solutions with concen-
trations of 4, 2, 1 and 0.5 //g (potency)/mL. Separately, di-
lute Teceleukin (Genetical Recombination) with diluted acet-
ic acid (100) (3 in 1000) if needed, or alternatively concentrate
under reduced pressure, to make a sample solution with a
protein concentration of 0.8 to 1.2 mg/mL. Accurately meas-
ure 25 /uL of the sample solution and each standard solution,
and add each to the wells in the same test plate. Repeat the
same procedure for at least 3 more test plates. Leave the test
plates at room temperature for 30 to 60 minutes and then in-
cubate for 16 to 18 hours at 35 to 37°C. Measure the inhibi-
tions zones to a diameter of 0.25 mm. Determine the mean
among the test plates for each of the solutions.
Prepare a standard curve by plotting a graph with the con-
centration of each standard solution in logarithmic scale on
the horizontal axis and the diameter of the inhibition zone on
the vertical axis. Match the diameter of the inhibition zone of
teceleukin from the standard curve and determine A, the con-
centration of tetracycline hydrochloride. When the amount
of tetracycline hydrochloride per mg of protein is determined
by the following formula, the amount is not more than 0.7
fig. However, if an inhibition zone is not seen, or is seen but
the diameter is smaller than 0.5 fig/mL on the standard
curve, A is taken as being 0.5//g/mL or less.
Amount [fig (potency)] of tetracycline hydrochloride
(C 22 H 24 N 2 8 .HC1) per mg of protein =A/P
P: The protein concentration (mg/mL) of the sample solu-
tion.
(3) Desmethionyl form — Add water to an appropriate
amount of teceleukin to make a sample solution with a pro-
tein concentration of about 0.17 mg/mL. Perform the test
with 1.2 mL of this solution as directd under Liquid Chro-
matography <2.01> under the following conditions. Deter-
mine using automatic integration the peak area, A 2 , of
teceleukin and the peak area of the desmethionyl form with a
relative retention time of about 0.8 relative to teceleukin, Ay.
The content of the desmethionyl form is not more than 1 .0%
when determined using the following formula.
Amount (%) of desmethionyl form = {A X /(A X +A 2 )} x 100
Operating conditions —
Detector: Ultraviolet absorption photometer (wavelength:
280 nm)
Columns: Two stainless steel columns with inside di-
ameters of 7.5 mm and lengths of 7.5 cm connected in se-
quence and packed with 10 ftm synthetic polymer bound to
diethylaminoethyl base for liquid chromatography.
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Mix 0.658 g of diethanolamine in 400 mL
of water, adjust the pH to 9.0 by adding 1 mol/L hydrochlor-
ic acid TS, and then add water to make 500 mL.
Mobile phase B: Add 300 mL of water to 2.6 mL of a pH 6
to 9 amphoteric electrolyte solution and 0.5 mL of a pH 8 to
10.5 amphoteric electrolyte solution, adjust to pH 7 with
diluted hydrochloric acid (9 in 100), and then add water to
make 400 mL.
JPXV
Official Monographs / Teceleukin (Genetical Recombination) 1147
Switching mobile phases and sample injection: Inject the
sample solution while running the mobile phase A. Repeated-
ly inject 10 times a sample solution volume of 0.11 mL fol-
lowed by a single injection of 100 ,mL. After injecting the en-
tire volume and running mobile phase A for 60 minutes,
switch to mobile phase B. After measuring the sample solu-
tion and after running 1 mol/L sodium chloride TS for 10
minutes for postreatment and cleaning of the columns, inject
100 fiL of sodium hydroxide TS while running the mobile
phase A and then 55 minutes later start injection of the next
sample solution.
Flow: Adjust the flow of the mobile phase B so that the
retention time for teceleukin is 45 to 65 minutes. Measure the
retention time from the point at which the mobile phase is
switched to the mobile phase B.
System suitability —
System performance: Dissolve in water a mixture of two
kinds of equine heart-derived myoglobin whose isoelectric
points are 6.76 and 7.16 to make a concentration of approxi-
mately 0.5 mg/mL. Mix together 50 iiL of this solution, 50
luL of Teceleukin (Genetical Recombination), and 1.47 mL of
water. When 1.2 mL of this solution is run under the above
conditions, myoglobin and teceleukin are eluted in this order,
and their respective peaks are completely separated.
(4) Dimer — Prepare a sample solution by adding 20 iiL
of 0.2% sodium laurylsulfate TS to 20 /uL of Teceleukin (Ge-
netical Recombination). Perform the test as directed under
Liquid Chromatography <2.01> using 20 fiL of this solution
under the following conditions. Determine using automated
integration the teceleukin peak area, A 2 , and the peak area,
A lt of the dimer with a relative retention time of 0.8 to 0.9 in
relation to teceleukin. The amount of the dimer is not more
than 1.0% by the following formula.
Amount (%) of dimer = \A X /(A X +A 2 )\ x 100
Operating conditions —
Detector: Ultraviolet absorption photometer (wavelength:
220 nm)
Column: A stainless steel column with an inside diameter
of 7.5 mm and 60 cm in length, packed with gycol etherifized
silica gel for liquid chromatography (particle diameter: 10
/urn)
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in 0.1
mol/L sodium phosphate buffer, pH 7.0, to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
teceleukin is between 30 and 40 minutes.
System suitability —
System performance: Add 20 /uL of 0.2% sodium lauryl
sulfate TS to 20 /uL of a solution consisting of 5 mg of car-
bonic anhydrase and 5 mg of a-lactoalbumin dissolved in
100 mL of water. When 20 iiL of this solution is tested under
the above conditions, carbonic anhydrase and a-lactoalbu-
min are eluted in this order with the resolution between these
peaks being not less than 1.5.
System repeatability: Measure exactly 1.0 mL of the sam-
ple solution, add the mobile phase to exactly 20 mL. To ex-
actly 1 mL of this solution add the mobile phase to make ex-
actly 10 mL. When the test is repeated 3 times with 20 jiL of
this solution under the above conditions, the relative stan-
dard deviation of the teceleukin peak area is not more than
7%.
(5) Other related proteins — Perform the test on 5 iuL of
Teceleukin (Genetical Recombination) as directed under Liq-
uid Chromatography <2.01> under the following conditions,
and measure the area of each peak using automatic integra-
tion. When the amounts are determined by the area percent
method, the total amount of peaks other than the teceleukin
and solvent peaks is not more than 1.0%.
Operating conditions —
Detector: Ultraviolet absorption photometer (wavelength:
220 nm)
Column: A stainless steel column with an inside diameter
of 4.6 mm and 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter)
Column temperature: A constant temperature of about
30°C.
Mobile phase A: A solution of trifluoroacetic acid in a mix-
ture of water and acetonitrile (19:1) (1 in 1000)
Mobile phase B: A solution of trifluoroacetic acid in
acetonitrile (7 in 10,000)
Mobile phase flow: Control the concentration gradient by
changing the mobile phase A and mobile phase B as shown in
the table below.
Time after injection Mobile phase Mobile phase
of sample (min) A (vol%) B (vol%)
0-12
12-25
25-45
45-50
60^50 40^50
50 50
50^0 50^100
100
Flow rate: l.OmL/min
Time span of measurement: a range that is approximately
1.2-fold the retention time of teceleukin.
System suitability —
System performance: Add 3.8 //L of water and 16.6 /XL of
polysorbet 80 solution (1 in 100) to 83.6 //L of Teceleukin
(Genetical Recombination) and let stand for at least one
hour. When 5 ,«L of this solution is tested by running under
the above conditions, there is complete separation between
the teceleukin peak and the peak with a relative retention
time of about 0.98 in relation to the teceleukin peak.
(6) Acetic acid — Measure exactly 0.25 mL of Teceleukin
(Genetical Recombination) and add exactly 0.25 mL of the
internal standard solution to make the sample solution.
Separately, measure exactly 3 mL of acetic acid (100) and add
water to make exactly 100 mL. Take exactly 10 mL of this so-
lution and add water to make exactly 100 mL. Measure ex-
actly 2 mL of this solution and add exactly 2 mL of the inter-
nal standard solution to make the standard solution. Perform
the test with 1 /uL each of the sample solution and the stan-
dard solution by Gas Chromatography <2.02> under the fol-
lowing conditions. Determine the ratios of the peak area of
acetic acid to that of the internal standard, Q T and Q s , and
the amount of acetic acid (C 2 H 4 2 ) in 1 mL of Teceleukin
(Genetical Recombination) determined by the following for-
mula is between 2.85 and 3.15 mg.
Amount (mg) of acetic acid (C 2 H 4 2 ) in 1 mL of Teceleukin
(Genetical Recombination)
= (2t/2s)x 1.5x1.049x2
1 148 Teceleukin for Injection (Genetical Recombination) / Official Monographs
JP XV
1.5: Concentration (wL/mL) of acetic acid (100) in the
standard solution
1.049: Density (mg/^L) of acetic acid (100) at 25°C
2: Dilution coefficient
Internal standard solution — Diluted propionic acid (1 in 500)
Operating conditions —
Detector: Hydrogen flame ionization detector
Column: A glass column with an inside diameter of 1.2
mm and 40 m in length, whose inside is covered with
chemically-bound polyethylene glycol for gas chro-
matography 1.0 /xm in thickness.
Column temperature: A constant temperature of about 1 10
°C.
Carrier gas: Helium
Flow rate: Adjust the flow rate so that the retention time of
acetic acid is about 8 minutes.
System suitability —
System performance: When 1 fiL of the standard solution
is run under the above conditions, acetic acid and the internal
standard are eluted in this order with the resolution between
these peaks being not less than 3.0.
System repeatability: When the test is repeatedly run 6
times under the above conditions using 1 fiL of standard so-
lution, the relative standard deviation of the ratio of the acet-
ic acid peak area to the internal standard peak area is not
more than 5%.
Bacterial endotoxins <4.01> Less than 5EU per mg of pro-
tein
Specific activity Accurately measure an appropriate amount
of Teceleukin (Genetical Recombination), and add water ac-
curately so that 1 mL contains about 0.1 mg. This is the sam-
ple solution. Separately, measure precisely about 25 mg of
human serum albumin for assay, dissolve in water, and add
water to make 50 mL. Measure exactly an appropriate
amount of this solution, and accurately dilute with water to
make standard solutions with concentrations of 0.05, 0.10,
and 0.15 mg/mL. Accurately measure 1 mL each of the sam-
ple solution, the standard solutions, and water, add 2.5 mL
of alkaline copper solution, mix, leave for at least 10 minutes
to dissolve, add exactly 2.5 mL of water and 0.5 mL of dilut-
ed Folin reagent (1 in 2), immediately shake vigorously, and
then leave for 30 minutes at 37 °C. Perform the test on these
solutions, with water as a control, as directd under Ultrav-
iolet-visible Spectrophotometry <2.24>, and measure the ab-
sorbance at 750 nm. With the concentration of the standard
solution as the x-axis and the absorbance as the j'-axis, per-
form linear regression using their respective reciprocals, and
determine the protein content.
Determine the ratio of the potency determined by Assay
and the protein content.
Assay Accurately measure an appropriate amount of
Teceleukin (Genetical Recombination) and, depending on the
cell sensitivity, dilute precisely by adding culture medium for
assay of teceleukin to a constant concentration of 10 to 50
units/mL (estimated value). This is the sample solution.
Separately, dissolve Interleukin-2 Reference Substance in 1
mL of sterilized purified water, and, depending on the cell
sensitivity, dilute precisely by adding culture medium for as-
say of teceleukin to a constant concentration of 10 to 50
units/mL. This is the standard solution. Add exactly 50 fiL
of culture medium for assay of teceleukin to all but 8 wells in
a microtest plate. Add 50 fiL of the sample solution and the
standard solution to 2 wells each containing culture medium
for assay of teceleukin. From these 4 wells, remove exactly 50
fiL and add to 4 other wells containing culture medium for
assay of teceleukin. From these 4 wells, remove exactly 50 fiL
and add to 4 other wells containing culture medium for assay
of teceleukin and repeat this procedure to prepare 2 wells that
contain each of 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, and
1/256 dilutions of the sample and standard solutions. Add 50
fiL of the standard solution to each of the 8 empty wells to
make maximum uptake controls. Eight wells containing only
culture medium for assay of teceleukin serve as the minimum
uptake controls. After adding exactly 50 /xL of cell suspen-
sion solution for teceleukin to each well in a microtest plate,
leave for 15 to 17 hours in an incubator at 37°C filled with air
containing 5% carbon dioxide. After adding 25 fiL of MTT
TS to each of the wells in the plate, leave for 4 hours in an in-
cubator at 37°C filled with air containing 5% carbon dioxide.
Transfer the culture medium in all of the wells to empty wells
in another microtest plate. To each of the empty wells from
which the culture medium was removed, add 100 fiL of
hydrochloric acid-2-propanol TS, and then shake the plates
horizontally for 5 minutes to elute the pigment. After return-
ing the transferred culture medium to each original well, per-
form the test with the solution in each well, determine the
difference in absorption at wavelengths of 560 nm and 690
nm, and calculate the mean values of the identical respective
solutions in the two wells (dilution solutions of the sample so-
lution and standard solutions) as well as the 8 wells contain-
ing the maximum or minimum uptake controls. Prepare stan-
dard curves by plotting the values obtained from each dilu-
tion solution of the sample solution, with the dilution
coefficient of the sample solution on the microtest plates in
logarithmic scale on the horizontal axis and the absorbance
on the vertical axis. Determine the mean absorbance values
of the maximum and minimum uptake controls, find the
values on the standard curve, and then determine the dilution
coefficient, D T . Perform the same plot for the dilution solu-
tion of the standard solution, determine the dilution
coefficient, D s , and then determine the potency in 1 mL by
the following formula.
Teceleukin potency (units) in 1 mL of Teceleukin (Genetical
Recombination)
= S x (D T /D S ) x d
S: Concentration of standard solution (units/mL)
d: Dilution coefficient when sample solution prepared
Containers and storage Containers — Tight containers
Storage — Store at -70°C or below.
Teceleukin for Injection (Genetical
Recombination)
Teceleukin for Injection (Genetical Recombination)
is a preparation for injection which is dissolved before
use.
It contains not less than 70.0% and not more than
JP XV
Official Monographs / Tegafur 1149
150.0% of the labeled amount of teceleukin (genetical
recombination) (C 698 H 112 7N 17 9O204S 8 : 15547.01).
Method of preparation Prepare as directed under Injection,
with Teceleukin (Genetical Recombination).
Description Teceleukin for Injection (Genetical Recombi-
nation) occurs as a white, light mass or powder.
Identification (1) Dissolve the content of 1 vial of Teceleu-
kin for Injection (Genetical Recombination) in 1 mL of steri-
lized purified water, dilute exactly with culture medium for
assay of teceleukin to make the sample stock solution con-
taining about 200 units/mL (estimate). Proceed as directed in
the Identification (1) and (3) under Teceleukin (Genetical
Recombination).
(2) Dissolve 0.242 g of 2-amino-2-hydroxymethyl-l,3-
propanediol, 5.0 g of sodium lauryl sulfate and 74 mg of dis-
odium dihydrogen ethylenediamine tetraacetate dihydrate in
60 mL of water. Adjust to pH 8.0 with 1 mol/L hydrochloric
acid TS, add water to make 100 mL, and use this solution as
the buffer solution for molecular mass determination.
Separately, dissolve the content of 1 vial of Teceleukin for In-
jection (Genetical Recombination) in exactly 1 mL of water.
To exactly 100 liL of this solution add exactly 100 ^L of the
buffer solution for molecular mass determination and 10 iiL
of 2-mercaptoethanol, and heat on a water bath for 5 minutes
without allowing any water evaporation from the mixture.
After cooling, add exactly 1 iiL of bromophenol blue solu-
tion (1 in 2000), mix, and use this solution as the sample solu-
tion. Proceed as directed in Identification (3) under Teceleu-
kin (Genetical Recombination): a band appears in the range
of molecular mass between 14,000 and 16,000.
pH <2.54> Dissolve the content of one vial of Teceleukin for
Injection (Genetical Recombination) in 1 mL of water: the
pH of the solution is between 7.0 and 7.7.
Purity Clarity and color of solution — Dissolve the content
of one vial of Teceleukin for Injection (Genetical Recombi-
nation) in 1 mL of water: the solution is clear and colorless.
Loss on drying <2.41> Transfer the content of the vial of
Teceleukin for Injection (Genetical Recombination) to a
weighing bottle under the atmosphere not exceeding 10% rel-
ative humidity, and perform the test as directed in the Water
content determination described in the Minimum Require-
ments for Biological Products: not more than 3%.
Bacterial endotoxins <4.01> Less than 5 EU/350,000 units.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test. Calculate as \M— A I =0.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Dissolve the content of 1 vial of Teceleukin for Injec-
tion (Genetical Recombination) in exactly 1 mL of sterilized
purified water, dilute exactly with culture medium for assay
of teceleukin to make the sample solution containing a
definite concentration of 10 to 50 units/mL (estimate). Pro-
ceed as directed in the Assay under Teceleukin (Genetical
Recombination), and calculate the amount (unit) of teceleu-
kin in 1 vial by the following formula.
Amount (unit) of teceleukin in 1 vial = S x (D T /D S ) xrfxi
S: Concentration (unit/mL) of the standard solution
d: Rate of dilution to prepare the sample solution
/: Volume (mL) of the sample solution
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, not exceeding 10°C, avoiding
freezing.
Tegafur
n
NH
and enantsomer
C 8 H 9 FN 2 3 : 200.17
5-Fluoro-l-[(2i?S)-tetrahydrofuran-2-yl]uracil
[17902-23-7]
Tegafur, when dried, contains not less than 98.0%
of C 8 H 9 FN 2 3 .
Description Tegafur occurs as a white, crystalline powder.
It is soluble in methanol and in acetone, and sparingly
soluble in water and in ethanol (95).
It dissolves in dilute sodium hydroxide TS.
A solution of Tegafur in methanol (1 in 50) shows no opti-
cal rotation.
Identification (1) Prepare the test solution with 0.01 g of
Tegafur as directed under Oxygen Flask Combustion Method
<1.06>, using a mixture of 0.5 mL of 0.01 mol/L sodium
hydroxide TS and 20 mL of water as an absorbing liquid: the
test solution responds to the Qualitative Tests <1.09> (2) for
fluoride.
(2) Determine the absorption spectrum of a solution of
Tegafur in 0.01 mol/L sodium hydroxide TS (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(3) Determine the infrared absorption spectrum of
Tegafur, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> Dissolve 0.5 g of Tegafur in 50 mL of water: the
pH of this solution is between 4.2 and 5.2.
Melting point <2.60> 166 - 171 °C
Purity (1) Clarity and color of solution — Dissolve 0.2 g of
Tegafur in 10 mL of dilute sodium hydroxide TS: the solu-
tion is clear and colorless.
(2) Chloride <1.03>— Dissolve 0.8 g of Tegafur in 40 mL
1150 Teicoplanin / Official Monographs
JP XV
of water by warming, cool, filter if necessary, and add 6 mL
of dilute nitric acid and water to make 50 mL. Perform the
test using this solution as the test solution. Prepare the con-
trol solution with 0.25 mL of 0.01 mol/L hydrochloric acid
VS (not more than 0.011%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Tegafur in 40
mL of water by warming, cool, filter if necessary, and add 2
mL of dilute acetic acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution with 1.0 mL of Standard Lead Solution (not
more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution in a plati-
num crucible with 1.0 g of Tegafur according to Method 4,
incinerating by ignition between 750°C and 850°C, and per-
form the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.10 g of Tegafur in 10
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add methanol to make ex-
actly 200 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /uL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of chloroform and ethanol (95) (5:1)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.15 g of Tegafur, previous-
ly dried, place in an iodine bottle, dissolve in 75 mL of water,
and add exactly 25 mL of 1/60 mol/L potassium bromate
VS. Add rapidly 1.0 g of potassium bromide and 12 mL of
hydrochloric acid, stopper the bottle tightly at once, and al-
low to stand for 30 minutes with occasional shaking. To this
solution add 1 .6 g of potassium iodide, shake gently, allow to
stand for exactly 5 minutes, and titrate <2.50> the liberated io-
dine with 0.1 mol/L sodium thiosulfate VS (indicator: 2 mL
of starch TS). Perform a blank determination.
Each mL of Veo mol/L potassium bromate VS
= 10.01 mg of C 8 H 9 FN 2 3
Containers and storage Containers — Tight containers.
Teicoplanin
Teicoplanin Ay., : R :
Teicoplanin A? ? : Ft 3
Teicoplanin A23 : R a
Teicoplanin Am : R :
Teicoplanin A^ s : R^ =
Teicoplanin A 31 : R 3 = H
Teicoplanin A 2 .i
C 88 H 95 C1 2 N 9 3 3: 1877.64
(3S,15/?,18i?,34i?,355,38S,48i?,50ai?)-34-(2-Acetylamino-2-
deoxy-/?-D-glucopyranosyloxy)-15-amino-22,31-dichloro-
56-[2-(4Z)-dec-4-enoylamino-2-deoxy-/?-D-glucopyranosylox-
y]-6, 1 1 ,40,44-tetrahydroxy-42-(a-D-mannopyranosyloxy)-
2,16, 36, 50,51, 59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
li/,15i/,34i/-20,23:30,33-dietheno-3, 18:35,48-
bis(iminomethano)-4, 8: 10, 14:25,28:43 , 47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
ffj][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [91032-34-7]
Teicoplanin A 2 . 2
C 88 H 97 C1 2 N 9 33 : 1879.66
(3S,15/?,18i?,34i?,35S,38S,48i?,50ai?)-34-
(2-Acetylamino-2-deoxy-/?-D-glucopyranosyloxy)-
15-amino-22,31-dichloro-56-[2-deoxy-
2-(8-methylnonanoylamino)-/?-D-glucopyranosyloxy]-
6,11 ,40,44-tetrahydroxy-42-(a-D-mannopyranosyloxy)-
2,16, 36, 50,51, 59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
l//,15//,34//-20,23:30,33-dietheno-3, 18:35,48-
JPXV
Official Monographs / Teicoplanin 1151
bis(iminomethano)-4,8 : 10, 14:25 ,28 :43 ,47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
/w][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [91032-26-7]
Teicoplanin A 2 . 3
C 88 H 97 C1 2 N 9 033: 1879.66
(3S,15i?,18fl,34fl,35S,38S,48fl,50afl)-34-(2-Acetylamino-
2-deoxy-/?-D-glucopyranosyloxy)-15-amino-22,31-dichloro-
56-(2-decanoylamino-2-deoxy-/?-D-glucopyranosyloxy)-
6,11 ,40,44-tetrahydroxy-42-(a-D-mannopyranosyloxy)-
2,16,36,50,51,59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
lH,l5H,34H-20, 23:30, 33-dietheno-3, 18:35,48-
bis(iminomethano)-4,8:10,14:25,28:43,47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
m][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [91032-36-9]
Teicoplanin A 2 . 4
C 89 H 99 C1 2 N 9 33 : 1893.68
(35,15i?,18/?,34/?,35S,38S,48/?,50a/?)-34-
(2-Acetylamino-2-deoxy-/?-D-glucopyranosyloxy)-
15-amino-22,31-dichloro-56-[2-deoxy-
2-(8-methyldecanoylamino)-/?-D-glucopyranosyloxy]-
6,11 ,40,44-tetrahydroxy-42-(a-D-mannopyranosyloxy)-
2,16,36,50,51,59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
lH,l5H,34H-20, 23:30, 33-dietheno-3, 18:35,48-
bis(iminomethano)-4,8:10,14:25,28:43,47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
m][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [91032-37-0]
Teicoplanin A 2 . 5
C 89 H 99 C1 2 N 9 33 : 1893.68
(35,15i?,18/?,34/?,35S,38S,48/?,50a/?)-34-
(2-Acetylamino-2-deoxy-/?-D-glucopyranosyloxy)-
15-amino-22,31-dichloro-56-[2-deoxy-
2-(9-methyldecanoylamino)-/?-D-glucopyranosyloxy]-
6,11 ,40,44-tetrahydroxy-42-(a-D-mannopyranosyloxy)-
2,16,36,50,51,59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
lH,l5H,34H-20, 23:30, 33-dietheno-3, 18:35, 48-
bis(iminomethano)-4,8:10,14:25,28:43,47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
»i][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [91032-38-1]
Teicoplanin A 3 .j
C 72 H 68 C1 2 N 8 28 : 1564.25
(35,15i?,18/?,34/?,35S,38S,48/?,50a/?)-34-(2-Acetylamino-2-
deoxy-/?-D-glucopyranosyloxy)-15-amino-22,31-dichloro-
6,11 , 40,44, 56-pentahydroxy-42-(a-D-mannopyranosyloxy)-
2,16,36,50,51,59-hexaoxo-
2,3,16,17,18,19,35,36,37,38,48,49,50,50a-tetradecahydro-
\H,\5H,T>4H-20, 23:30, 33-dietheno-3, 18:35,48-
bis(iminomethano)-4,8:10,14:25,28:43,47-tetrametheno-
28//-[l,14,6,22]dioxadiazacyclooctacosino[4,5-
m][10,2,16]benzoxadiazacyclotetracosine-38-
carboxylic acid [93616-27-4]
[61036-62-2, Teicoplanin]
Teicoplanin is a mixture of glycopeptide substances
having antibacterial activity produced by the growth of
Actinoplanes teichomyceticus.
It contains not less than 900 /ug (potency) per mg,
calculated on the anhydrous, de-sodium chloride and
de-residual solvents basis. The potency of Teicoplanin
is expressed as mass (potency) of teicoplanin
(C72-89H68-99Cl2Ng.9O2g.33).
Description Teicoplanin occurs as a white to light yellowish
white powder.
It is freely soluble in water, sparingly soluble in N,N-
dimethylformamide, and practically insoluble in acetonitrile,
in methanol, in ethanol (95), in acetone, in acetic acid (100)
and in diethyl ether.
Identification (1) To 1 mL of a solution of Teicoplanin (1
in 100) add 2 mL of ninhydrin TS, and warm for 5 minutes: a
blue-purple color develops.
(2) To 1 mL of a solution of Teicoplanin (3 in 100) add
slowly 2 mL of anthrone TS, and shake gently: a dark brown
color develops.
(3) Determine the infrared absorption spectra of Tei-
coplanin and Teicoplanin Reference Standard as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
spectrum of Teicoplanin Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
pH <2.54> Dissolve 0.5 g of Teicoplanin in 10 mL of water:
the pH of the solution is between 6.3 and 7.7.
Content ratio of the active principle Dissolve about 20 mg
of Teicoplanin in water to make 10 mL, and use this solution
as the sample solution. Perform the test with 20 /uL of the
sample solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the sum of peak areas of teicoplanin A 2 group, 5 a , the sum of
peak areas of teicoplanin A 3 group, 5b, and the sum of peak
areas of other contents, S c from the sample solution by the
automatic integration method. Calculate the content ratio of
them by the formula given below: teicoplanin A 2 group, tei-
coplanin A 3 group, and the other are not less than 80.0%, not
more than 15.0% and not more than 5.0%, respectively.
The elution order of each content and the relative retention
time of each content to the retention time of teicoplanin A 2 . 2
are shown in the following table.
Name of content
Elution
order
Relative
retention time
teicoplanin A 3 group
^0.42
teicoplanin A 31
1
0.29
teicoplanin A 2 group
0.42<,^1.25
teicoplanin A 2I
2
0.91
teicoplanin A 22
3
1.00
teicoplanin A 23
4
1.04
teicoplanin A 24
5
1.17
teicoplanin A 25
6
1.20
others
1.25 <
1152 Teicoplanin / Official Monographs
JP XV
Content ratio (%) of teicoplanin A 2 group
= {S a /(S a + 0.83S b + S c )} xlOO
Content ratio (%) of teicoplanin A 3 group
= {0.83S b /(S a + 0.83S„ + S c )} x 100
Content ratio (%) of others
= {5 c /(S a + 0.83S b + 5 c )} xlOO
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: Dissolve 7.80 g of sodium dihydrogen
phosphate dihydrate in 1650 mL of water, add 300 mL of
acetonitrile, adjust pH to 6.0 with sodium hydroxide TS, and
add water to make 2000 mL.
Mobile phase B: Dissolve 7.80 g of sodium dihydrogen
phosphate dihydrate in 550 mL of water, add 1400 mL of
acetonitrile, adjust the pH to 6.0 with sodium hydroxide TS,
and add water to make 2000 mL.
Flowing of the mobile phase: Flow mobile phase A for 10
minutes before injection. After injection, control the
gradient by mixing the mobile A and B as directed in the fol-
lowing table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-32
100-
* 70
0-
+ 30
32-40
70-
* 50
30-
+ 50
40-42
50-
MOO
50-
+
Flow rate: 1.8 mL per minute.
Time span of measurement: About 1.7 times as long as the
retention time of teicoplanin A 2 .2, beginning after the solvent
peak.
System suitability —
Test for required detection: Confirm that peak height of
teicoplanin A 2 _ 2 obtained from the sample solution is equiva-
lent to 90% of the full scale.
System performance: When the procedure is run with 20
/uL of the sample solution under the above operating condi-
tions, the symmetry factor of the peak of teicoplanin A iA is
not more than 2.2.
System repeatability: When the test is repeated 3 times with
20 /xL of the sample solution under the above operating con-
ditions, the relative standard deviation of the peak area of
teicoplanin A 2 _ 2 is not more than 2.0%.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Sodium chloride — Weigh accurately about 0.5 g of
Teicoplanin, dissolve in 50 mL of water, titrate <2.50> with
0.1 mol/L silver nitrate VS (indicator: 1 mL of potassium
chromate TS), and calculate an amount of sodium chloride:
not more than 5.0%.
Each mL of 0.1 mol/L silver nitrate VS
= 5.844 mg of NaCl
(4) Arsenic <1.U> — Being specified separately.
(5) Residual solvents <2.46> — Weigh accurately about 0.1
g of Teicoplanin, dissolve in A^A^dimethylformamide to
make exactly 10 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 1 g each of methanol
and acetone, and add Af/V-dimethylformamide to make ex-
actly 100 mL. Pipet 1 mL of this solution, add A^/V-dimethyl-
formamide to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 4 /uL each
of the sample solution and standard solution as directed un-
der Gas Chromatography <2.02> according to the following
condition. Calculate the peak area of methanol, Ay, and the
peak area of acetone, A 2 , obtained from the sample solution,
and the peak area of methanol, A sl , and the peak area of ace-
tone, A S2 , obtained from the standard solution by the auto-
matic integration method, and calculate the amounts of
methanol and acetone by the following formula: not more
than 0.5% and not more than 1.0%, respectively.
Amount (%) of methanol
= W sl x(Ai/A sl )x 0.001 x(l/W T1 )x 100
W sl : Amount (g) of methanol
W Ti : Amount (g) of Teicoplanin
Amount (%) of acetone
= W S2 x (A 2 /A S2 ) x 0.001 x (i/W J2 ) x 100
Wsi'- Amount (g) of acetone
W J2 : Amount (g) of Teicoplanin
Operating conditions —
Detector: Hydrogen flame-ionization detector.
Column: A glass column 2 mm in inside diameter and 3 m
in length, packed with graphite carbon for gas chro-
matography, 150 to 180^m in particle diameter, coated with
0.1% of polyethylene glycol esterified.
Column temperature: Inject the sample at a constant tem-
perature of about 70°C, maintain the temperature for 4
minutes, then program to increase the temperature at the rate
of 8°C per minute to 210°C.
Detector temperature: A constant temperature of about
240°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention times
of methanol and acetone are about 2 minutes and 5 minutes,
respectively.
System suitability —
Test for required detection: Confirm that the peak height
of acetone obtained from 4 /uL of the standard solution is e-
quivalent to about the full scale.
System performance: When the procedure is run with 4 /iL
of the standard solution under the above operating condi-
tions, methanol and acetone are eluted in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 3 times with
4 /xL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
acetone is not more than 3%.
Water <2.48> Not more than 15.0% (0.2 g, volumetric titra-
tion, direct titration).
Bacterial endotoxins <4.01> Less than 0.75 EU/mg (poten-
cy).
(3) Heavy metals <1.07> — Being specified separately.
Blood pressure depressant Being specified separately.
JPXV
Official Monographs / Terbutaline Sulfate 1153
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount of
Teicoplanin Reference Standard equivalent to about 50 mg
(potency), dissolve in phosphate buffer solution, pH 6.0 to
make exactly 50 mL, and use this solution as the standard
stock solution. Keep the standard stock solution at not ex-
ceeding 5°C and use within 14 days. Take exactly a suitable
amount of this solution before use, add phosphate buffer so-
lution, pH 6.0 to make solutions so that each mL contains
160 /ug (potency) and 40 /ug (potency), and use these solutions
as the high concentration standard solution and low concen-
tration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Teicoplanin equivalent to about 50 mg (potency), dissolve in
phosphate buffer solution, pH 6.0 to make exactly 50 mL.
Take exactly a suitable amount of this solution, add phos-
phate buffer solution, pH 6.0 to make solutions so that each
mL contains 160 /xg (potency) and 40 fig (potency), and use
these solutions as the high concentration sample solution and
low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 5°C.
Terbutaline Sulfate
H OH
• H 2 S0 4
and enantiomer
(C 12 H 19 N0 3 )2.H 2 S0 4 : 548.65
5-[(li?S)-2-(l,l-Dimethylethylamino)-
l-hydroxyethyl]benzene-l,3-diol hemisulfate [23031-32-5]
Terbutaline Sulfate contains not less than 98.5% of
(C 12 H 1 9N03)2.H 2 S04, calculated on the anhydrous ba-
sis.
Description Terbutaline Sulfate is white to slightly
brownish white crystals or crystalline powder It is odorless or
has a faint odor of acetic acid.
It is freely soluble in water, and practically insoluble in
acetonitrile, in ethanol (95), in acetic acid (100), in chlo-
roform, and in diethyl ether.
It is gradually colored by light and by air.
Melting point: about 255 °C (with decomposition).
Identification (1) Dissolve 1 mg of Terbutaline Sulfate in
1 mL of water, and add 5 mL of Tris buffer solution, pH 9.5,
0.5 mL of 4-aminoantipyrine solution (1 in 50) and 2 drops of
potassium hexacyanoferrate (III) solution (2 in 25): a reddish
purple color is produced.
(2) Determine the absorption spectrum of a solution of
Terbutaline Sulfate in 0.01 mol/L hydrochloric acid TS (1 in
10,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wavelengths. This maximum can be biphasic.
(3) A solution of Terbutaline Sulfate (1 in 50) responds to
the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve Terbutaline Sulfate in 10 mL of water:
the pH of this solution is between 4.0 and 4.8.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Terbutaline Sulfate in 10 mL of water: the solution is clear
and colorless or slightly yellow.
(2) Chloride <7.0?>— Perform the test with 2.0 g of Ter-
butaline Sulfate. Prepare the control solution with 0.25 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.004%).
(3) Acetic acid — Dissolve 0.50 g of Terbutaline Sulfate in
a solution of phosphoric acid (59 in 1000) to make exactly 10
mL, and use this solution as the sample solution. Separately,
dissolve 1.50 g of acetic acid (100) in a solution of phosphoric
acid (59 in 1000) to make exactly 100 mL. Dilute 2 mL of this
solution, accurately measured, with a solution of phosphoric
acid (59 in 1000) to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 2
,uL each of the sample solution and standard solution as
directed under Gas Chromatography <2.02> according to the
following operating conditions. Measure the peak areas, A T
and A s , of acetic acid for the two solutions: A T is not larger
than A s .
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass column 3 mm in inside diameter and 1 m
in length, packed with 10% of macrogol 6000 on 180- to
250-^m terephthalic acid for gas chromatography.
Column temperature: A constant temperature at about
120°C.
Carrier gas: Nitrogen.
Flow rate: Adjust the flow rate so that the retention time of
acetic acid is about 5 minutes.
System suitability —
System performance: Mix 0.05 g each of acetic acid (100)
and propionic acid in 100 mL of diluted phosphoric acid (59
in 1000). When the procedure is run with 2 iuL of this solution
under the above conditions, acetic acid and propionic acid
are eluted in this order with the resolution between these
peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
2 juL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
acetic acid is not more than 3.0%.
(4) 3,5-Dihydroxy-a)-tert-butylaminoacetophenone sul-
fate — Dissolve 0.50 g of Terbutaline Sulfate in 0.01 mol/L
hydrochloric acid TS to make exactly 25 mL, and perform
the test as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: the absorbance at a wavelength of 330 nm does
not exceed 0.47.
(5) Heavy metals <1.07> — Proceed with 2.0 g of Terbuta-
line Sulfate as directed under Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(6) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Terbutaline Sulfate according to method 3, and perform
the test (not more than 2 ppm).
Water <2.48> Not more than 0.5% (1 g, direct titration).
1154 Testosterone Enanthate / Official Monographs
JP XV
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Terbutaline Sulfate,
dissolve in 50 mL of a mixture of acetonitrile and acetic acid
(100) (1:1) by stirring and warming. Allow to cool, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration, substituting a saturated solution of potassium chlo-
ride in methanol for the internal fluid).
Each mL of 0.1 mol/L perchloric acid VS
= 54.87 mg of (C 12 H 19 N0 3 ) 2 .H 2 S0 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Testosterone Enanthate
exactly 100 mL. Measure exactly 10 mL of this solution, and
dilute with ethanol (95) to make exactly 100 mL. Measure ex-
actly 10 mL of this solution, and dilute with ethanol (95) to
make exactly 100 mL. Perform the test as directed under
Ultraviolet-visible Spectrophotometry <2.24> with this solu-
tion. Read the absorbance A of this solution at the
wavelength of maximum absorption at about 241 nm.
Amount (mg) of C 26 H 40 O 3
= (.4/426) x 100,000
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 30°C.
Testosterone Enanthate Injection
7X r-XxP>H-> HiXxJL-
CH S
C 26 H 40 O 3 : 400.59
3-Oxoandrost-4-en-17/?-yl heptanoate [315-37-7]
Testosterone Enanthate, when dried, contains not
less than 95.0% and not more than 105.0% of
C26H40O3.
Description Testosterone Enanthate occurs as white to pale
yellow crystals, crystalline powder or a pale yellow-brown,
viscous liquid. It is odorless or has a slight, characteristic
odor.
It is very soluble in ethanol (95), in 1,4-dioxane and in
diethyl ether, and practically insoluble in water.
Melting point: about 36°C
Identification Heat 25 mg of Testosterone Enanthate with 2
mL of a solution of potassium hydroxide in methanol (1 in
100) under a reflux condenser on a water bath for 1 hour,
cool, and add 10 mL of water. Collect the produced
precipitate by suction, wash with water until the last washing
is neutral, and dry the precipitate in a desiccator (in vacuum,
phosphorus (V) oxide) for 4 hours: the precipitate melts be-
tween 151°C and 157°C.
Optical rotation <2.49> [a]™: + 77 - + 88° (after drying, 0.1
g, 1,4-dioxane, 10 mL, 100 mm).
Purity Acidity — Dissolve 0.5 g of Testosterone Enanthate
in 10 mL of ethanol (95) which has previously been rendered
neutral to bromothymol blue TS, and add 2 drops of
bromothymol blue TS and 0.50 mL of 0.01 mol/L sodium
hydroxide VS: the color of the solution is light blue.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately about 0.1 g of Testosterone Enan-
thate, previously dried, and dissolve in ethanol (95) to make
Testosterone Enanthate Injection is an oily solution
for injection.
It contains not less than 90% and not more than 110
% of the labeled amount of testosterone enanthate (C 26
H 40 O 3 : 400.59).
Method of preparation Prepare as directed under Injec-
tions, with Testosterone Enanthate.
Description Testosterone Enanthate Injection is a clear,
colorless or pale yellow oily liquid.
Identification Measure a volume of Testosterone Enanthate
Injection, equivalent to 0.05 g of Testosterone Enanthate ac-
cording to the labeled amount, add 8 mL of petroleum ether,
and extract with three 10-mL portions of diluted acetic acid
(31) (7 in 10). Combine the extracts, wash with 10 mL of
petroleum ether, add 0.5 mL of diluted sulfuric acid (7 in 10)
to 0.1 mL of the extract, and heat on a water bath for 5
minutes. Cool, and add 0.5 mL of iron (III) chloride-acetic
acid TS: the color of the solution is blue.
Extractable volume <6.05> It meets the requirement.
Assay Measure accurately a volume of Testosterone Enan-
thate Injection, equivalent to about 25 mg of testosterone
enanthate (C 26 H 4 o0 3 ), and dissolve in chloroform to make ex-
actly 25 mL. Pipet 3 mL of this solution, add chloroform to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 25 mg of Testoster-
one Propionate Reference Standard, proceed in the same
manner as for the sample solution, and use this solution as
the standard solution. Pipet 5 mL each of the sample solution
and standard solution, add exactly 10 mL of isoniazid TS,
add methanol to make exactly 20 mL, and allow to stand for
45 minutes. Determine the absorbances, A T and A s , of these
solutions at 380 nm, respectively, as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using a solution ob-
tained by proceeding with 5 mL of chloroform as the blank.
Amount (mg) of testosterone enanthate (C 26 H 40 O 3 )
= W s x(A T /As)x 1.1629
W s : Amount (mg) of Testosterone Propionate
Reference Standard
Containers and storage Containers-
Storage — Light-resistant.
■Hermetic containers.
JPXV
Official Monographs / Testosterone Propionate Injection 1155
Testosterone Propionate
o
H,C
A^ CH *
C 22 H 32 3 : 344.49
3-Oxoandrost-4-en-17/?-yl propanoate [57-85-2]
Testosterone Propionate, when dried, contains not
less than 97.0% and not more than 103.0% of
C22H32O3.
Description Testosterone Propionate occurs as white to
pale yellow crystals or crystalline powder.
It is freely soluble in methanol and in ethanol (95), and
practically insoluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Testosterone Propionate in ethanol (95) (1 in
100,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Testosterone
Propionate Reference Standard prepared in the same manner
as the sample solution: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Testosterone Propionate, previously dried, as directed in the
potassium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum or the spectrum of Testosterone
Propionate Reference Standard: both spectra exhibit similar
intensities of absorption at the same wave numbers.
Optical rotation <2.49> [a]™: + 83
g, ethanol (95), 10 mL, 100 mm).
Melting point <2.60> 1 18 - 123°C
+ 90° (after drying, 0.1
Purity Related substances — Dissolve 40 mg of Testosterone
Propionate in 2 mL of ethanol (95), and use this solution as
the sample solution. Pipet 1 mL of this solution, add ethanol
(95) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and diethylamine (19:1) to a distance of about 15 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spot other than the principal spot
from the sample solution is not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (0.5 g).
Assay Weigh accurately each about 10 mg of Testosterone
Propionate and Testosterone Propionate Reference
Standard, previously dried, and dissolve in methanol to make
exactly 100 mL. To exactly 5 mL of these solutions add ex-
actly 5 mL of the internal standard solution and methanol to
make 20 mL, and use these solutions as the sample solution
and standard solution. Perform the test with 5 [iL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the ratios, Qt and Q s , of the peak area
of testosterone propionate to that of the internal standard.
Amount (mg) of C22H32O3
= W s x (Q T /Q S )
W s : Amount (mg) of Testosterone Propionate Reference
Standard
Internal standard solution — A solution of Progesterone in
methanol (9 in 100,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 241 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [tm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of acetonitrile and water (7:3).
Flow rate: Adjust the flow rate so that the retention time of
testosterone propionate is about 10 minutes.
System suitability —
System performance: When the procedure is run with 5 [iL
of the standard solution under the above operating condi-
tions, the internal standard and testosterone propionate are
eluted in this order with the resolution between these peaks
being not less than 9.
System repeatability: When the test is repeated 6 times with
5 [iL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of testosterone propionate to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Testosterone Propionate Injection
T7 hXxP>"/Ptf7|->^i7.TjUait>S
Testosterone Propionate Injection is an oily solution
for injection.
It contains not less than 92.5% and not more than
107.5% of the labeled amount of testosterone
propionate (C22H32O3: 344.49).
Method of preparation Prepare as directed under Injec-
tions, with Testosterone Propionate.
Description Testosterone Propionate Injection is a clear,
colorless or pale yellow oily liquid.
Identification Dissolve the residue obtained as directed in
the procedure in the Assay in exactly 20 mL of methanol, and
1156 Freeze-dried Tetanus Antitoxin, Equine / Official Monographs
JP XV
use this solution as the sample solution. Separately, dissolve 1
mg of Testosterone Propionate Reference Standard in 10 mL
of methanol, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of chloroform and diethylamine
(19:1) to a distance of about 15 cm, and air-dry the plate. Ex-
amine under ultraviolet light (main wavelength: 254 nm): the
Rf values of the principal spot with the sample solution and
of the spot with the standard solution are not different each
other.
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay (i) Chromatographic tube A glass tube about 1
cm in inside diameter and about 18 cm in length, with a glass
filter (G3) at the lower end.
(ii) Chromatographic column To about 2 g of silica gel
for liquid chromatography add 5 mL of dichloromethane,
and mix gently. Transfer and wash into the chromatographic
tube with the aid of dichloromethane, allow to elute the
dichloromethane through the column, and put a filter paper
on the upper end of the silica gel.
(iii) Standard solution Weigh accurately about 10 mg
of Testosterone Propionate Reference Standard, previously
dried at 105 °C for 4 hours, and dissolve in methanol to make
exactly 100 mL. Pipet 5 mL of this solution, add exactly 5
mL of the internal standard solution and methanol to make
exactly 20 mL.
(iv) Sample stock solution To exactly a volume of
Testosterone Propionate Injection, equivalent to about
20 mg of testosterone propionate (C22H32O3), add
dichloromethane to make exactly 20 mL.
(v) Procedure Transfer exactly 2 mL of the sample
stock solution into the chromatographic column, and elute to
the upper surface of the silica gel. Wash the inner surface of
the chromatographic tube with 15 mL of dichloromethane,
elute to the upper surface of the silica gel, and discard the
effluent. Elute 15 mL of a mixture of dichloromethane and
methanol (39:1), discard the first 5 mL of the effluent, and
collect the subsequent effluent. Wash the lower part of the
column with a few amount of dichloromethane, combine the
washings and the effluent, and evaporate the solvent under
reduced pressure. Dissolve the residue so obtained with
methanol to make exactly 20 mL. Pipet 5 mL of this solution,
add exactly 5 mL of the internal standard solution and
methanol to make exactly 20 mL, and use this solution as the
sample solution. Perform the test with 5 /uL each of the sam-
ple solution and standard solution as directed in the Assay
under Testosterone Propionate.
Amount (mg) of testosterone propionate (C22H32O3)
= W s x(Qt/Q s )x2
W s : Amount (mg) of Testosterone Propionate Reference
Standard
Internal standard solution — A solution of Progesterone in
methanol (9 in 100,000).
Containers and storage Containers — Hermetic containers.
Freeze-dried Tetanus Antitoxin,
Equine
IB*****-****
Freeze-dried Tetanus Antitoxin, Equine, is a prepa-
ration for injection which is dissolved before use.
It contains tetanus antitoxin in immunoglobulin of
horse origin.
It conforms to the requirements of Freeze-dried
Tetanus Antitoxin, Equine, in the Minimum Require-
ments for Biological Products.
Description Freeze-dried Tetanus Antitoxin, Equine,
becomes a clear, colorless to light yellow-brown liquid or
slightly white-turbid liquid on addition of solvent.
Adsorbed Tetanus Toxoid
JfcPHMWl 1**7 4 K
Adsorbed Tetanus Toxoid is a liquid for injection
containing tetanus toxoid prepared by treating tetanus
toxin with formaldehyde by a method involving no
appreciable loss of the immunogenicity and rendered
insoluble by the addition of aluminum salt.
It conforms to the requirements of Adsorbed
Tetanus Toxoid in the Minimum Requirements for
Biological Products.
Description Adsorbed Tetanus Toxoid becomes a uniform
white-turbid liquid on shaking.
Tetracaine Hydrochloride
Tr7*-f >m&k
HjC
C 15 H 24 N 2 2 .HC1: 300.82
2-(Dimethylamino)ethyl 4-(butylamino)benzoate
monohydrochloride [136-47-0]
Tetracaine Hydrochloried, when dried, contains not
less than 98.5% of C 15 H 25 N 2 2 .HC1.
Description Tetracaine Hydrochloride occurs as white crys-
tals or crystalline powder. It is odorless, and has a slightly
bitter taste followed by a sense of numbness on the tongue.
It is very soluble in formic acid, freely soluble in water,
soluble in ethanol (95), sparingly soluble in ethanol (99.5),
slightly soluble in acetic anhydride, and practically insoluble
JPXV
Official Monographs / Tetracycline Hydrochloride 1157
in diethyl ether.
A solution of Tetracaine Hydrochloride (1 in 10) is neutral.
Melting point: about 148°C
Identification (1) Dissolve 0.5 g of Tetracaine Hydrochlo-
ride in 50 mL of water, add 5 mL of ammonia TS, shake,
and allow to stand in a cold place. Collect the precipitate,
wash with water until the washings is neutral, and dry in a
desiccator (silica gel) for 24 hours: it melts <2.60> between 42
°C and 44°C.
(2) Dissolve 0.1 g of Tetracaine Hydrochloride in 8 mL
of water, and add 3 mL of ammonium thiocyanate TS: a
crystalline precipitate is produced. Collect the precipitate,
recrystallize from water, and dry at 80°C for 2 hours: it
melts <2.60> between 130°C and 132°C.
(3) Determine the absorption spectrum of a solution of
Tetracaine Hydrochloride in ethanol (99.5) (1 in 200,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) A solution of Tetracaine Hydrochloride (1 in 10)
responds to the Qualitative Tests <1.09> for chloride.
Purity Heavy metals <1.07> — Proceed with 1.0 g of Tetra-
caine Hydrochloride according to Method 1, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tetracaine
Hydrochloride, previously dried, dissolve in 2 mL of formic
acid, add 80 mL of acetic anhydride, allow to stand at 30°C
on a water bath for 15 minutes, cool, and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 30.08 mg of C 15 H 24 N 2 2 .HC1
Containers and storage Containers — Tight containers.
Tetracycline Hydrochloride
xr-TiM^y^i&S
NH,
■ HCI
HO CH;
C 22 H 24 N 2 8 .HC1: 480.90
(4S,4aS,5aS,6S,12aS)-4-Dimethylamino-
3,6,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-
l,4,4a,5,5a,6,ll,12a-octahydrotetracene-2-
carboxamide monohydrochloride [64-75-5]
Tetracycline Hydrochloride is the hydrochloride of a
tetracycline substance having antibacterial activity pro-
duced by the growth of Streptomyces aureofaciens.
It contains not less than 950 fig (potency) and not
more than 1010 fig (potency) per mg, calculated on the
dried basis. The potency of Tetracycline Hydrochlo-
ride is expressed as mass (potency) of tetracycline
hydrochloride (C 22 H 24 N 2 8 .HC1).
Description Tetracycline Hydrochloride occurs as a yellow
to pale brownish yellow crystalline powder.
It is freely soluble in water, and sparingly soluble in
ethanol (95).
Identification (1) Determine the absorption spectrum of a
solution of Tetracycline Hydrochloride (1 in 62,500) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Tetracycline Hydrochloride
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Tetracycline Hydrochloride as directed in the potassium chlo-
ride disk method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum or
the spectrum of Tetracycline Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Tetracycline Hydrochloride (1 in 100)
responds to the Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1.0 g of Tetracycline Hydrochloride in
100 mL of water: the pH of the solution is between 1.8 and
2.8.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tetracycline Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 1 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Tetracycline Hydrochloride according to Method 4, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 25 mg of Tetracycline
Hydrochloride in 50 mL of 0.01 mol/L hydrochloric acid TS,
and use this solution as the sample solution. Pipet 3 mL of
the sample solution, add 0.01 mol/L hydrochloric acid TS to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /uL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the areas of each peak by the automatic
integration method: the peak areas other than tetracycline
from the sample solution is not more than the peak area of
tetracycline from the standard solution, and the total of the
peak areas other than tetracycline from the sample solution is
not more than 3 times of the peak area of tetracycline from
the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 7 times as long as the
retention time of tetracycline beginning after the solvent
peak.
System suitability —
Test for required detection: Pipet 3 mL of the standard so-
1158 Thallium ( 201 T1) Chloride Injection / Official Monographs
JP XV
lution, add 0.1 mol/L hydrochloric acid TS to make exactly
100 mL, and confirm that the peak area of tetracycline ob-
tained from 20 /xL of this solution is equivalent to 1 to 5% of
that of tetracycline obtained from 20 /uL of the standard solu-
tion.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of tetracycline is not more than 1.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.3% (1.0 g).
Assay Weigh accurately an amount of Tetracycline
Hydrochloride and Tetracycline Hydrochloride Reference
Standard, equivalent to about 25 mg (potency), and dissolve
each in 0.1 mol/L hydrochloric acid TS to make exactly 50
mL, and use these solutions as the sample solution and stan-
dard solution, respectively. Perform the test with exactly 20
fiL each of these solutions as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and calculate the peak area, A T and A s , of tetracycline of
each solution.
Amount [fig (potency)] of C22H24N2CVHCI
= ^ s x(y4 T M s )xl000
W s : Amount [mg (potency)] of Tetracycline
Hydrochloride Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with stylene-divinylben-
zene copolymer for liquid chromatography (0.01 /um in pore
diameter).
Column temperature: A constant temperature of about
60°C.
Mobile phase: Dissolve 3.5 g of dipotassium hydrogen-
phosphate, 2.0 g of tetrabutylammonium hydrogensulfate
and 0.4 g of disodium dihydrogen ethylenediamine tetraa-
cetate dihydrate in 300 mL of water, adjust to pH 9.0 with so-
dium hydroxide TS, add 90.0 g of Z-butanol, and add water
to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
tetracycline is about 5 minutes.
System suitability —
System performance: Dissolve 0.05 g of Tetracycline
Hydrochloride Reference Standard in water to make 25 mL.
Heat 5 mL of this solution on a water bath for 60 minutes,
then add water to make 25 mL. When the procedure is run
with 20 fiL of this solution under the above operating condi-
tions, the retention time of 4-epitetracycline is about 3
minutes, and 4-epitetracycline and tetracycline are eluted in
this order with the resolution between these peaks being not
less than 2.5.
System repeatability: When, the test is repeated 6 times
with 20 /xL of the standard solution under the above operat-
ing conditions, the relative standard deviation of the peak
areas of tetracycline is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thallium ( 201 T1) Chloride Injection
^{t^U^ApiTDait*
Thallium ( 201 T1) Chloride Injection is an aqueous
solution for injection
It contains thallium-201 ( 201 T1) in the form of thal-
lous chloride.
It conforms to the requirements of Thallium ( 201 T1)
Chloride Injection in the Minimum Requirements for
Radiopharmaceuticals .
Test for Extractable Volume of Parenteral Prepara-
tions and Insoluble Particulate Matter Test for Injec-
tions are not applied to this injection.
Description Thallium ( 201 T1) Chloride Injection is a clear,
colorless liquid.
Theophylline
CH 3
C 7 H 8 N 4 2 : 180.16
1 ,3-Dimethyl-li7-purine-2,6(3/f,7i/)-dione [58-55-9]
Theophylline, when dried, contains not less than
99.0% of C 7 H 8 N 4 2 .
Description Theophylline occurs as white crystals or crys-
talline powder.
It is soluble in /V.A'-dimethylformamide, and slightly solu-
ble in water and in ethanol (99.5).
It dissolves in 0.1 mol/L hydrochloric acid TS.
Identification (1) Determine the absorption spectrum of a
solution of Theophylline in 0.1 mol/L hydrochloric acid TS
(1 in 200,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of The-
ophylline, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 271 - 275 °C
Purity (1) Acidity — To 0.5 g of Theophylline add 75 mL
of water, 2.0 mL of 0.01 mol/L sodium hydroxide VS and 1
drop of methyl red TS: a yellow color develops.
(2) Heavy metals <1.07> — Proceed with 1.0 g of The-
ophylline according to Method 4, and perform the test. Pre-
JP XV
Official Monographs / Thiamazole 1159
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(3) Arsenic </.//> — Prepare the test solution with 1.0 g
of Theophylline according to Method 3, and perform the test
(not more than 2 ppm).
(4) Related substances — Dissolve 0.10 g of Theophylline
in 3 mL of Af,7V-dimethylformamide, add 10 mL of
methanol, and use this solution as the sample solution. Pipet
1 mL of the sample solution, add methanol to make exactly
200 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. De-
velop the plate with a mixture of acetone, chloroform,
methanol, 1-butanol and ammonia solution (28) (3:3:2:2:1)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Theophylline,
previously dried, and dissolve in 100 mL of water, add ex-
actly 20 mL of 0.1 mol/L silver nitrate VS, shake the mix-
ture, and titrate <2.50> with 0.1 mol/L sodium hydroxide VS
(potentiometric titration). Perform a blank determination,
and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 18.02 mg of C 7 H 8 N 4 2
Containers and storage Containers — Well-closed contain-
ers.
Thiamazole
f77V'-Jl
CH 3
C 4 H 6 N 2 S: 114.17
l-Methyl-l//-imidazole-2-thiol
SH
[60-56-0]
Thiamazole, when dried, contains not less than
98.0% of C 4 H 6 N 2 S.
Description Thiamazole occurs as white to pale yellowish
white crystals or crystalline powder. It has a faint, charac-
teristic odor, and has a bitter taste.
It is freely soluble in water and in ethanol (95), and slightly
soluble in diethyl ether.
The pH of the solution (1 in 50) is between 5.0 and 7.0.
Identification (1) Dissolve 5 mg of Thiamazole in 1 mL of
water, shake with 1 mL of sodium hydroxide TS, and add 3
drops of sodium pentacyanonitrosylferrate (III) TS: a yellow
color develops, and it gradually changes to yellow-green to
green. To this solution add 1 mL of acetic acid (31): it
changes to blue.
(2) To 2 mL of a solution of Thiamazole (1 in 200) add 1
mL of sodium carbonate TS and 1 mL of diluted Folin's TS
(1 in 5): a deep blue color develops.
Melting point <2.60> 144 - 147°C
Purity (1) Selenium — Proceed with 0.10 g of Thiamazole
as directed under Oxygen Flask Combustion Method <1.06>,
using 25 mL of diluted nitric acid (1 in 30) as the absorbing
liquid, and prepare the test solution. Apply a small amount
of water to the upper part of apparatus A, pull out C careful-
ly, and transfer the test solution to a beaker. Wash C, B and
the inner side of A with 25 mL of water, and combine the
washings with the test solution. Boil gently for 10 minutes,
cool to room temperature, add water to make exactly 50 mL,
and use this solution as the sample solution. Separately,
weigh exactly 40 mg of selenium, dissolve in 100 mL of dilut-
ed nitric acid (1 in 2), heat to dissolve on a water bath if
necessary, and add water to make exactly 1000 mL. Pipet 5
mL of this solution, and add water to make exactly 200 mL.
To 2 mL of this solution, exactly measured, add diluted nitric
acid (1 in 60) to make exactly 50 mL, and use this solution as
the standard solution. Pipet 40 mL each of the sample solu-
tion and standard solution into separate beakers, and adjust
each solution with ammonia solution (28) to a pH of 1.8 to
2.2. To each solution add 0.2 g of hydroxylammonium chlo-
ride, shake gently to dissolve, then add 5 mL of 2,3-di-
aminonaphthalene TS, shake, and allow to stand for 100
minutes. Transfer these solutions to corresponding separa-
tors, rinse the beakers with 10 mL of water, combine the rins-
ings in the respective separators, shake well with 5.0 mL of
cyclohexane for 2 minutes, and extract. Centrifuge the cyclo-
hexane extracts to remove any water remaining in these solu-
tions. Perform the test with these solutions as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using a solu-
tion prepared with 40 mL of diluted nitric acid (1 in 60) in the
same manner as the blank. The absorbance of of the sample
solution at the wavelength of maximum absorbance at about
378 nm does not exceed the absorbance of the standard solu-
tion.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Thiama-
zole according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Thiamazole according to Method 1, and perform the test
(not more than 2 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.25 g of Thiamazole, previ-
ously dried, dissolve in 75 mL of water, add 15 mL of 0.1
mol/L sodium hydroxide VS from a burette, and add 30 mL
of 0.1 mol/L silver nitrate VS with stirring. Add 1 mL of
bromothymol blue TS, and titrate <2.50> with 0.1 mol/L so-
dium hydroxide VS, until a persistent blue-green color is
produced. Determine the total volume of 0.1 mol/L sodium
hydroxide VS consumed.
Each mL of 0.1 mol/L sodium hydroxide VS
= 11.42 mg of C 4 H 6 N 2 S
Containers and storage Containers — Well-closed contain-
1160 Thiamazole Tablets / Official Monographs
JP XV
Storage — Light-resistant.
Thiamazole Tablets
Thiamazole Tablets contain not less than 94% and
not more than 106% of the labeled amount of
thiamazole (C 4 H 6 N 2 S: 114.17).
Method of preparation
with Thiamazole.
Prepare as directed under Tablets,
Identification (1) To a quantity of powdered Thiamazole
Tablets, equivalent to 0.05 g of Thiamazole according to the
labeled amount, add 20 mL of hot ethanol (95), shake for 15
minutes, filter, and evaporate the filtrate on a water bath to
dryness. Dissolve the residue in 10 mL of water, filter if
necessary, and use this solution as the sample solution. To 1
mL of the sample solution add 1 mL of sodium hydroxide
TS, shake, and add 3 drops of sodium pentacyanonitrosylfer-
rate (III) TS: a yellow color develops, and it gradually
changes to yellow-green to green. To this solution add 1 mL
of acetic acid (31): it changes to blue.
(2) With 2 mL of the sample solution obtained in (1),
proceed as directed in the Identification (2) under Thiama-
zole.
Assay Weigh accurately and powder not less than 20
Thiamazole Tablets. Weigh accurately a quantity of the pow-
der, equivalent to about 0.15 g of thiamazole (C 4 H 6 N 2 S), add
80 mL of water, shake for 15 minutes, add water to make ex-
actly 100 mL, and centrifuge. Filter, discard the first 20 mL
of the filtrate, pipet 50 mL of the subsequent filtrate, add 1
mL of bromothymol blue TS, and if a blue color develops,
neutralize with 0.1 mol/L hydrochloric acid VS until the
color of the solution changes to green. To this solution add
4.5 mL of 0.1 mol/L sodium hydroxide VS from a burette,
add 15 mL of 0.1 mol/L silver nitrate VS while stirring, and
titrate <2.50> with 0.1 mol/L sodium hydroxide VS. Continue
the titration until a persistent blue-green color is produced,
and determine the total volume of 0.1 mol/L sodium
hydroxide VS consumed.
Each mL of 0.1 mol/L sodium hydroxide VS
= 11.42 mg of C 4 H 6 N 2 S
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Thiamine Chloride Hydrochloride
Vitamin Bi Hydrochloride
^7 5 >mk®m®&
CI -HOI
C 12 H I7 ClN 4 OS.HCl: 337.27
3-(4-Amino-2-methylpyrimidin-5-ylmethyi)-5-(2-
hydroxyethyl)-4-methylthiazolium chloride
monohydrochloride [67-03-8]
Thiamine Chloride Hydrochloride contains not less
than 98.5% of C 12 H 17 ClN 4 OS.HCl, calculated on the
anhydrous basis.
Description Thiamine Chloride Hydrochloride occurs as
white crystals or crystalline powder. It is odorless or has a
slight, characteristic odor.
It is freely soluble in water, sparingly soluble in methanol,
slightly soluble in ethanol (95), and practically insoluble in
diethyl ether.
Melting point: about 245°C (with decomposition).
Identification (1) To 5 mL of a solution of Thiamine
Chloride Hydrochloride (1 in 500) add 2.5 mL of sodium
hydroxide TS and 0.5 mL of potassium hexacyanoferrate
(III) TS. Then add 5 mL of 2-methyl-l-propanol, shake the
mixture vigorously for 2 minutes, allow to stand, and exa-
mine under ultraviolet light (main wavelength: 365 nm): the
2-methyl-l-propanol layer shows a blue-purple fluorescence.
This fluorescence disappears when the mixture is acidified,
but reappears when it is again made alkaline.
(2) Determine the absorption spectrum of a solution of
Thiamine Chloride Hydrochloride (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum or the
spectrum of a solution of Thiamine Chloride Hydrochloride
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Thia-
mine Chloride Hydrochloride, previously dried at 105°C for
2 hours, as directed in the potassium bromide disk method
under the Infrared Spectrophotometry <2.25>, and compare
the spectrum with the Reference Spectrum, or the spectrum
of Thiamine Chloride Hydrochloride Reference Standard
previously driedat 105°C for 2 hours: both spectra exhibit
similar intensities of absorption at the same wave numbers.
In case when some differences are found between the spectra,
repeat the test with residues obtained by dissolving these sub-
stances in water, evaporating to dryness, and drying at 105°C
for 2 hours.
(4) A solution of Thiamine Chloride Hydrochloride (1 in
500) responds to the Qualitative Tests <1.09> for chloride.
pH <2.54> Dissolve 1.0 g of Thiamine Chloride Hydrochlo-
ride in 100 mL of water: the pH of this solution is between
2.7 and 3.4.
JPXV
Official Monographs / Thiamine Chloride Hydrochloride Injection 1161
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Thiamine Chloride Hydrochloride in 10 mL of water: the so-
lution is clear, and has no more color than the following con-
trol solution.
Control solution: To 1.5 mL of l/60mol/L potassium
dichromate VS add water to make 1000 mL.
(2) Sulfate <1.14>— Weigh 1.5 g of Thiamine Chloride
Hydrochloride, and perform the test. Prepare the control so-
lution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not
more than 0.011%).
(3) Nitrate — Dissolve 0.5 g of Thiamine Chloride
Hydrochloride in 25 mL of water. Add 2 mL of sulfuric acid
to 2 mL of this solution, shake, cool, and superimpose iron
(II) sulfate TS: no dark brown ring is produced at the junc-
tion of the two layers.
(4) Heavy metals <1.07> — Proceed with 1 .0 g of Thiamine
Chloride Hydrochloride according to Method 1, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(5) Related substances — Dissolve 0.10 g of Thiamine
Chloride Hydrochloride in 100 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 100
mL, and use this solution, as the standard solution. Perform
the test with exactly 10 /uL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the area of each peak by the automatic integration method:
the total area of the peaks other than thiamine is not larger
than the peak area of thiamine from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of thiamine.
System suitability —
Test for required detectability: To exactly 5 mL of the stan-
dard solution add water to make exactly 50 mL. Confirm that
the peak area of thiamine obtained from 10 ^L of this solu-
tion is equivalent to 7 to 13% of that of thiamine obtained
from 10 fiL of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
10 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
thiamine is not more than 1.0%.
Water <2.48> Not more than 5.0% (30 mg, coulometric
titration).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.1 g each of Thiamine
Chloride Hydrochloride and Thiamine Chloride Hydrochlo-
ride Reference Standard (separately determine the water
<2.48> in the same manner as Thiamine Chloride Hydrochlo-
ride), and dissolve them in the mobile phase to make exactly
50 mL. To 10 mL each of the solutions, accurately measured,
add exactly 5 mL each of the internal standard solution, add
the mobile phase to make 50 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and calculate the ratios, Q T and
Qs, of the peak area of thiamine to that of the internal stan-
dard.
Amount (mg) of C 12 H 17 ClN 4 OS.HCl
= W s x (Q T /Q S )
W s : Amount (mg) of Thiamine Chloride Hydrochloride
Reference Standard, calculated on the anhydrous
basis
Internal standard solution — A solution methyl benzoate in
methanol (1 in 50).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.1 g of sodium 1-octanesulfonate
in 1000 mL of diluted acetic acid (100) (1 in 100). To 600 mL
of this solution add 400 mL of a mixture of methanol and
acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
thiamine is about 12 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, thiamine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 6.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of thiamine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiamine Chloride Hydrochloride
Injection
Vitamin Bi Hydrochloride Injection
Thiamine Chloride Hydrochloride Injection is an
aqueous solution for injection.
It contains not less than 95% and not more than
115% of the labeled amount of thiamine Chloride
hydrochloride (C 12 H 17 ClN 4 OS.HCl: 337.27).
Method of preparation Prepare as directed under Injec-
tions, with Thiamine Chloride Hydrochloride.
Description Thiamine Chloride Hydrochloride Injection is
a clear, colorless liquid.
pH: 2.5-4.5
Identification To a volume of Thiamine Chloride
1162 Thiamine Chloride Hydrochloride Powder / Official Monographs
JP XV
Hydrochloride Injection, equivalent to 0.05 g of Thiamine
Chloride Hydrochloride according to the labeled amount,
add water to make 25 mL. Proceed with 5 mL of this solution
as directed in the Identification (1) under Thiamine Chloride
Hydrochloride.
Extractable volume <6.05> It meets the requirement.
Assay Dilute with 0.001 mol/L hydrochloric acid TS if
necessary, then measure exactly a volume of Thiamine Chlo-
ride Hydrochloride Injection, equivalent to about 20 mg of
thiamine chloride hydrochloride (C I2 H I7 ClN 4 OS.HCl), and
add 20 mL of methanol and 0.001 mol/L hydrochloric acid
TS to make 100 mL. To 25 mL of this solution, exactly meas-
ured, add exactly 5 mL of the internal standard solution, add
0.001 mol/L hydrochloric acid TS to make 50 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 0.1 g of Thiamine Chloride Hydrochloride
Reference Standard (separately determine the water <2.48> in
the same manner as Thiamine Chloride Hydrochloride), and
dissolve in 0.001 mol/L hydrochloric acid TS to make exactly
50 mL. To 10 mL of this solution, exactly measured, add 20
mL of methanol and 0.001 mol/L hydrochloric acid TS to
make exactly 100 mL. To 25 mL of this solution, exactly
measured, add exactly 5 mL of the internal standard solu-
tion, add 0.001 mol/L hydrochloric acid TS to make 50 mL,
and use this solution as the standard solution. Proceed as
directed in the Assay under Thiamine Chloride Hydrochlo-
ride.
Amount (mg) of thiamine chloride hydrochloride
(C 12 H 17 C1N 4 0S.HC1)
= ^sX(e T /es)x(l/5)
W s : Amount (mg) of Thiamine Chloride Hydrochloride
Reference Standard, calculated on the anhydrous
basis
Internal standard solution — A solution of methyl benzoate in
methanol (1 in 200).
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Thiamine Chloride Hydrochloride
Powder
Vitamin B 1 Hydrochloride Powder
Thiamine Chloride Hydrochloride Powder contains
not less than 95% and not more than 115% of the la-
beled amount of thiamine chloride hydrochloride
(C 12 H 17 ClN 4 OS.HCl: 337.27).
Method of preparation Prepare as directed under Powders,
with Thiamine Chloride Hydrochloride.
Identification To a portion of Thiamine Chloride
Hydrochloride Powder, equivalent to 0.02 g of Thiamine
Chloride Hydrochloride according to the labeled amount,
add 50 mL of water and 10 mL of dilute acetic acid, shake,
and filter. Proceed with 5 mL of the filtrate as directed in the
Identification (1) under Thiamine Chloride Hydrochloride.
Purity Rancidity — Thiamine Chloride Hydrochloride Pow-
der has no unpleasant or rancid odor. It is tasteless.
Assay Weigh accurately a quantity of Thiamine Chloride
Hydrochloride Powder, equivalent to about 20 mg of thia-
mine chloride hydrochloride (C 12 H 17 ClN 4 OS.HCl), add 60
mL of 0.01 mol/L hydrochloric acid TS, and heat on a water
bath for 30 minutes. Shake vigorously for 10 minutes, cool,
add methanol to make exactly 100 mL, and centrifuge. Pipet
25 mL of the supernatant, add exactly 5 mL of the internal
standard solution, add water to make 50 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 0.1 g of Thiamine Chloride Hydrochloride Reference
Standard (separately determine the water <2.48> in the same
manner as Thiamine Chloride Hydrochloride), and dissolve
in 0.01 mol/L hydrochloric acid TS to make exactly 50 mL.
To 10 mL of this solution, exactly measured, add 50 mL of
0.01 mol/L hydrochloric acid TS, and add methanol to make
exactly 100 mL. To 25 mL of this solution, exactly measured,
add exactly 5 mL of the internal standard solution, add water
to make 50 mL, and use this solution as the standard solu-
tion. Proceed as directed in the Assay under Thiamine Chlo-
ride Hydrochloride.
Amount (mg) of thiamine chloride hydrochloride
(C I2 H 17 ClN 4 OS.HCl)
= W s x(Q T /Q s )x(l/5)
W s : Amount (mg) of Thiamine Chloride Hydrochloride
Reference Standard, calculated on the anhydrous ba-
sis
Internal standard solution — A solution of methyl benzoate in
methanol (1 in 200).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiamine Nitrate
Vitamin Bi Nitrate
^7 5 >mvtn
NO,
C 12 H 17 N 5 4 S: 327.36
3-(4-Amino-2-methylpyrimidin-5-ylmethyl)-5-(2-
hydroxyethyl)-4-methylthiazolium nitrate [532-43-4]
Thiamine Nitrate, when dried, contains not less than
98.0 % and not more than 102.0 % of
C 12 H 17 N 5 4 S.
Description Thiamine Nitrate occurs as white crystals or
crystalline powder. It is odorless or a slight, characteristic
odor.
It is sparingly soluble in water, and very slightly soluble in
ethanol (95), and practically insoluble in diethyl ether.
Melting point: about 193°C (with decomposition).
Identification (1) Take 2-mL portions of a solution of
Thiamine Nitrate (1 in 500), and add 2 to 3 drops of iodine
JP XV
Official Monographs / Thiamylal Sodium 1163
TS: a red-brown precipitate or turbidity is produced. Upon
further addition of 1 mL of 2,4,6-trinitrophenol TS, a yellow
precipitate or turbidity is produced.
(2) To 1 mL of a solution of Thiamine Nitrate (1 in 500)
add 1 mL of lead (II) acetate TS and 1 mL of a solution of so-
dium hydroxide (1 in 10), and warm: the color of the solution
changes through yellow to brown, and on standing, a black-
brown precipitate is produced.
(3) To 5 mL of a solution of Thiamine Nitrate (1 in 500)
add 2.5 mL of sodium ;hydroxide TS and 0.5 mL of potassi-
um hexacyanoferrate (III) TS. Then add 5 mL of 2-methyl-l-
propanol, shake the mixture vigorously for 2 minutes, allow
to stand, and examine under ultraviolet light (main
wavelength: 365 nm): the 2-methyl-l-propanol layer shows a
blue-purple fluorescence. This fluorescence disappears when
the mixture is acidified, but reappears when it is again made
alkaline.
(4) A solution of Thiamine Nitrate (1 in 50) responds to
the Qualitative Tests <1.09> (1) and (2) for nitrate.
pH <2.54> Dissolve 1.0 g of Thiamine Nitrate in 100 mL of
water: the pH of this solution is between 6.5 and 8.0.
Purity (1) Chloride <1.03>— Perform the test with 0.20 g
of Thiamine Nitrate. Prepare the control solution with 0.30
mL of 0.01 mol/L hydrochloric acid VS (not more than
0.053%).
(2) Sulfate <1.14> — Dissolve 1.5 g of Thiamine Nitrate in
30 mL of water and 2 mL of dilute hydrochloric acid, and
add water to make 50 mL. Perform the test using this solu-
tion as the test solution. Prepare the control solution with
0.35 mL of 0.005 mol/L sulfuric acid VS and 2 mL of dilute
hydrochloric acid, and add water to make 50 mL (not more
than 0.011%).
(3) Heavy metals <1.07> — Dissolve 1.0 g of Thiamine Ni-
trate in 30 mL of water by warming, cool, and add 12 mL of
6 mol/L acetic acid TS and water to make 50 mL. Perform
the test with this solution as the test solution. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
Loss on drying <2.41> Not more than 1.0% (0.5 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.1 g each of Thiamine Ni-
trate, previously dried, and Thiamine Chloride Hydrochlo-
ride Reference Standard (separately determine the water
<2.48> in the same manner as Thiamine Chloride Hydrochlo-
ride), and dissolve them in the mobile phase to make exactly
50 mL. To 10 mL each of the solutions, accurately measured,
add exactly 5 mL each of the internal standard solution, add
the mobile phase to make 50 mL, and use these solutions as
the sample solution and standard solution. Perform the test
with 10 /xL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions and calculate the ratios, Qt and
Q s , of the peak area of thiamine to that of the internal stan-
dard.
Amount (mg) of C 12 H 17 N 5 4 S = W s X (Q T /Q S ) x 0.9706
W s : Amount (mg) of Thiamine Chloride Hydrochloride
Reference Standard, calculated on the anhydrous ba-
sis
Internal standard solution — A solution of methyl benzoate in
methanol (1 in 50).
Operating conditions —
Detector: An ultraviolet spectrophotometer (wavelength:
254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: Dissolve 1.1 g of sodium 1-octanesulfonate
in 1000 mL of diluted acetic acid (100) (1 in 100). To 600 mL
of this solution add 400 mL of a mixture of methanol and
acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
thiamine is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, thiamine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 6.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of thiamine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiamylal Sodium
SNri
and enantiomer
C 12 H 17 N 2 Na0 2 S: 276.33
Monosodium 5-allyl-5-[(li?S)-l-methylbutyl]-4,6-
dioxo- 1,4, 5 ,6-tetrahydropyrimidine-2-thiolate [33 7-4 7-3 ]
Thiamylal Sodium contains not less than 97.5% and
not more than 101.0% of C 12 H 17 N2Na0 2 S, calculated
on the dried basis.
Description Thiamylal Sodium occurs as light yellow crys-
tals or powder.
It is very soluble in water, and freely soluble in ethanol
(95).
The pH of a solution of Thiamylal Sodium (1 in 10) is
between 10.0 and 11.0.
It is hygroscopic.
It is gradually decomposed by light.
Its solution in ethanol (95) (1 in 10) shows no optical
rotation.
Identification (1) Determine the absorption spectrum of a
solution of Thiamylal Sodium in ethanol (95) (7 in 1,000,000)
1164 Thiamylal Sodium for Injection / Official Monographs
JP XV
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Thiamylal Sodium, previously dried, as directed in the potas-
sium bromide disk method under Infrared
Spectrophotometry <2.25>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wave numbers.
(3) A solution of Thiamylal Sodium (1 in 10) responds to
Qualitative Tests <1.09> for sodium salt.
Purity (1) Clarity and color of solution — To 1 .0 g of
Thiamylal Sodium in a 11- to 13-mL glass-stoppered test tube
add 10 mL of freshly boiled and cooled water, stopper tight-
ly, allow to stand, and dissolve by occasional gentle shaking:
the solution is clear and light yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Thiamylal Sodium according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Thiamylal So-
dium in 10 mL of ethanol (95), and use this solution as the
sample solution. Pipet 1 mL and 3 mL of the sample solu-
tion, add ethanol (95) to make exactly 200 mL, and use these
solutions as the standard solution (1) and the standard solu-
tion (2). Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solutions (1) and (2) on a
plate of silica gel for thin-layer chromatography, develop
with a mixture of toluene, methanol and ethyl acetate
(40:7:3) to a distance of about 12 cm, and air-dry the plate.
Allow the plate to stand in iodine vapor for a night: the spot
appeared around Rf 0.1 obtained with the sample solution is
not more intense than the spot with the standard solution (2),
and the spot other than the principal spot, the spot at origin
and the spot mentioned above obtained with the sample solu-
tion is not more intense than the spot with the standard solu-
tion (1).
Loss on drying <2.41> Not more than 2.0% (1 g, 105°C,
1 hour).
Assay Weigh accurately about 0.25 g of Thiamylal Sodium,
dissolve in 50 mL of methanol and 5 mL of dilute
hydrochloric acid, and add methanol to make exactly 100
mL. Pipet 10 mL of this solution, and add methanol to make
exactly 100 mL. Pipet 5 mL of this solution, add exactly 10
mL of the internal standard solution and the mobile phase to
make 200 mL, and use this solution as the sample solution.
Separately, weigh accurately about 23 mg of Thiamylal
Reference Standard, previously dried at 105°C for 1 hour,
dissolve in 50 mL of methanol and 0.5 mL of dilute
hydrochloric acid, and add methanol to make exactly 100
mL. Pipet 5 mL of this solution, add exactly 10 mL of the in-
ternal standard solution and the mobile phase to make 200
mL, and use this solution as the standard solution. Perform
the test with 20 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qt and Q s , of the peak area of thiamylal to that of the inter-
nal standard.
Amount (mg) of C 12 H 17 N 2 Na0 2 S
= W s x (Q T /Q S ) x 10 x 1 .0864
W s : Amount (mg) of Thiamylal Reference Standard
Internal standard solution — A solution of phenyl benzoate in
methanol (3 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 289 nm).
Column: A stainless steel column about 4 mm in inside
diameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and 0.05 mol/L
acetic acid-sodium acetate buffer solution, pH 4.6 (13:7).
Flow rate: Adjust the flow rate so that the retention time of
thiamylal is about 6 minutes.
System suitability —
System performance: When the procedure is run with
20 [iL of the standard solution under the above operating
conditions, thiamylal and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 12.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of thiamylal to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiamylal Sodium for Injection
Thiamylal Sodium for Injection is a preparation for
injection which is dissolved before use.
It contains not less than 93.0% and not more than
107.0% of the labeled amount of thiamylal sodium
(C 12 H 17 N 2 Na0 2 S: 276.33).
Method of preparation Prepare as directed under Injec-
tions, with 100 parts of Thiamylal Sodium and 7 parts of
Dried Sodium Carbonate in mass.
Description Thiamylal Sodium for Injection occurs as light
yellow crystals, powder or masses.
It is hygroscopic.
It is gradually decomposed by light.
Identification (1) To 1.0 g of Thiamylal Sodium for
Injection add 20 mL of ethanol (95), shake vigorously, and
filter. Dissolve the precipitate so obtained in 1 mL of water,
and add 1 mL of barium chloride TS: a white precipitate is
produced. Centrifuge this solution, take off the supernatant
liquid, and to the precipitate add dilute hydrochloric acid
dropwise: the precipitate dissolves with effervescence.
(2) To 50 mg of Thiamylal Sodium for Injection add
100 mL of ethanol (95), shake vigorously, and filter. To 3 mL
of the filtrate add ethanol (95) to make 200 mL. Determine
the absorption spectrum of this solution as directed under
JPXV
Official Monographs / Compound Thianthol and Salicylic Acid Solution 1165
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits max-
ima between 236 nm and 240 nm, and between 287 nm and
291 nm.
pH <2.54> The pH of a solution obtained by dissolving 1 .0 g
of Thiamylal Sodium for Injection in 40 mL of water is be-
tween 10.5 and 11.5.
Purity Related substances — To 0. 10 g of Thiamylal Sodium
for Injection add 10 mL of ethanol (95), shake vigorously,
filter, and use the filtrate as the sample solution. Proceed as
diected in the Purity (3) under Thiamylal Sodium.
Bacterial endotoxins <4.01> Less than l.OEU/mg.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Open carefully 10 containers of Thiamylal Sodium
for Injection, dissolve the contents with water, wash out the
inside of each container with water, combine them, and add
water to make exactly KmL so that each mL contains about 5
mg of thiamylal sodium (C 12 H 17 N 2 Na02S). Pipet 5 mL of this
solution, and add 0.5 mL of dilute hydrochloric acid and
methanol to make exactly 100 mL. Pipet 5 mL of this solu-
tion, add exactly 10 mL of the internal standard solution and
the mobile phase to make 200 mL, and use this solution as the
sample solution. Proceed the test with the sample solution as
directed in the Assay under Thiamylal Sodium.
Amount (mg) of thiamylal sodium (Ci 2 H 17 N 2 Na02S) in 1
container
= W s x (Qj/Qs) x (K/50) x 1 .0864
W s : Amount (mg) of Thiamylal Reference Standard
Internal standard solution — A solution of phenyl benzoate in
methanol (3 in 500).
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Thianthol
^7> I — JU
Thianthol consists of dimethylthianthrene and ditol-
uene disulfide.
It contains not less than 23.5% and not more than
26.5% of sulfur (S: 32.07).
Description Thianthol is a yellowish, viscous liquid. It has a
faint, agreeable odor.
It is freely soluble in diethyl ether, slightly soluble in
ethanol (95), and practically insoluble in water.
It, when cold, may separate crystals, which melt on warm-
ing.
Specific gravity df Q : 1.19-1.23
Identification To 0. 1 g of Thianthol add cautiously 5 mL of
sulfuric acid: a blue-purple color develops. Add 5 to 6 drops
of nitric acid to the solution: the color of the solution changes
to yellow-red with evolution of gas.
Purity (1) Acidity or alkalinity — Shake 10 g of Thianthol
with 20 mL of water, allow to stand, and separate the water
layer. The solution is neutral.
(2) Sulfate — To 10 mL of the water layer obtained in (1)
add 2 to 3 drops of barium chloride TS: no opalescence is
produced.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 10 mg of Thianthol, and
proceed as directed in the sulfur determination of Oxygen
Flask Combustion Method <1.06>, using a mixture of 5 mL
of diluted sodium hydroxide TS (1 in 10) and 1.0 mL of
hydrogen peroxide TS as an absorbing liquid.
Containers and storage Containers — Tight containers.
Compound Thianthol and
Salicylic Acid Solution
W5^-t> h-ju-y-u^ua*
Compound Thianthol and Salicylic Acid Solution
contains not less than 1.8 w/v% and not more than 2.2
w/v% of salicylic acid (C 7 H 6 3 : 138.12), and not less
than 1.8 w/v% and not more than 2.2 w/v% of phenol
(C 6 H 6 Q: 94.11).
Method of preparation
Thianthol
Salicylic Acid
Phenol
Olive Oil
Ether
Petroleum Benzin
200 mL
20 g
20 g
50 mL
100 mL
a sufficient quantity
To make 1000 mL
Dissolve Salicylic Acid and Phenol in Ether, add Thian-
thol, Olive Oil and Petroleum Benzin to this solution, mix
and dissolve to make 1000 mL.
Description Compound Thianthol and Salicylic Acid Solu-
tion is a light yellow liquid, having a characteristic odor.
Identification (1) Place 1 mL of Compound Thianthol
and Salicylic Acid Solution to a porcelain dish, and evaporate
on a water bath to dryness. To the residue add cautiously 5
mL of sulfuric acid: the color of the solution changes to yel-
low-red with evolution of gas (thianthol).
(2) Shake 10 mL of Compound Thianthol and Salicylic
Acid Solution with 10 mL of sodium hydrogen carbonate TS,
and separate the water layer. To 0.5 mL of the water layer
add hydrochloric acid-potassium chloride buffer solution, pH
2.0, to make 50 mL, and to 5 mL of this solution add 5 mL of
a solution of iron (III) nitrate enneahydrate (1 in 200): a red-
purple color is produced (salicylic acid).
(3) Wash the upper phase obtained in (2) with 10 mL of
sodium hydrogen carbonate TS, and extract with 10 mL of
1166 Thiopental Sodium / Official Monographs
JP XV
dilute sodium hydroxide TS. Shake 1 mL of the extract with 1
mL of sodium nitrate TS and 1 mL of dilute hydrochloric
acid, and add 3 mL of sodium hydroxide TS: a yellow color is
produced (phenol).
(4) To 1 mL of Compound Thianthol and Salicylic Acid
Solution add 10 mL of ethanol (95), mix, and use this solu-
tion as the sample solution. Dissolve 0.01 g each of salicylic
acid, phenol and thianthol in 5 mL each of ethanol (95), and
use each solution as standard solutions (1), (2) and (3). Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 [iL each of the sample so-
lution and standard solutions on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of chloroform, acetone and acetic
acid (100) (45:5:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): three spots obtained from the sample solution and
the corresponding spots of standard solutions (1), (2) and (3)
show the same Ri value. Spray evenly iron (III) chloride TS
on the plate: the spot from standard solution (1) and the cor-
responding spot from the sample solution reveal a purple
color.
Assay Measure exactly 2 mL of Compound Thianthol and
Salicylic Acid Solution, add exactly 10 mL of the internal
standard solution, then add 70 mL of diluted methanol (1 in
2), mix well, and add diluted methanol (1 in 2) to make 100
mL. Filter, discard the first 10 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Weigh accurately
about 0.2 g of salicylic acid for assay, previously dried in a
desiccator (silica gel) for 3 hours, and about 0.2 g of phenol
for assay, dissolve in diluted methanol (1 in 2) to make exact-
ly 50 mL. Pipet 10 mL of this solution, add exactly 10 mL of
the internal standard solution and diluted methanol (1 in 2) to
make 100 mL, and use this solution as the standard solution.
With 5 fiL each of the sample solution and standard solution,
perform the test as directed under Liquid Chromatography
<2.01> according to the following conditions, and calculate
the ratios, g Ta and Q Tb , of the peak area of salicylic acid and
phenol to that of the internal standard in the sample solution,
and the ratios, gsa and Q sb , of the peak area of salicylic acid
and phenol to that of the internal standard in the standard so-
lution.
Amount (mg) of salicylic acid (C 7 H 6 3 )
= ^s a x(e Ta /e S a)x(i/5)
Amount (mg) of phenol (C 6 H 6 0)
= ^sbX(G T b/esb)x(l/5)
W Sll : Amount (mg) of salicylic acid for assay
W sb : Amount (mg) of phenol for assay
Internal standard solution — A solution of theophylline in
methanol (1 in 1000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 270 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 25 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 jum in parti-
cle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of 0.1 mol/L phosphate buffer
solution, pH 7.0, and methanol (3:1).
Flow rate: Adjust the flow rate so that the retention time of
salicylic acid is about 6 minutes.
Selection of column: Dissolve 0.2 g of benzoic acid, 0.2 g
of salicylic acid and 0.05 g of theophylline in 100 mL of dilut-
ed methanol (1 in 2). To 10 mL of this solution add 90 mL of
diluted methanol (1 in 2). Proceed with 10 /jL of this solution
under the above operating conditions. Use a column giving
elution of benzoic acid, salicylic acid and theophylline in this
order, and clearly dividing each peak.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding 25 °C.
Thiopental Sodium
S\s
and enantiomer
C„H l7 N 2 Na0 2 S: 264.32
Monosodium 5-ethyl-5-[(li?S')-l-methylbutyl]-4,6-
dioxo- 1,4, 5 ,6-tetrahydropyrimidine-2-thiolate [71-73-8]
Thiopental Sodium, when dried, contains not less
than 97.0% of C„H 17 N 2 Na0 2 S.
Description Thiopental Sodium occurs as a light yellow
powder. It has a faint, characteristic odor.
It is very soluble in water, freely soluble in ethanol (95),
and practically insoluble in diethyl ether.
A solution of Thiopental Sodium (1 in 10) is alkaline.
It is hygroscopic.
Its solution gradually decomposes on standing.
Identification (1) Dissolve 0.2 g of Thiopental Sodium in
5 mL of sodium hydroxide TS, and add 2 mL of lead (II)
acetate TS: a white precipitate, which dissolves upon heating,
is produced. Boil the solution thus obtained: a black
precipitate forms gradually, and the precipitate responds to
the Qualitative Tests <1.09> for sulfide.
(2) Dissolve 0.5 g of Thiopental Sodium in 15 mL of
water, add 10 mL of dilute hydrochloric acid to produce
white precipitate, and extract with four 25-mL portions of
chloroform. Combine the chloroform extracts, evaporate on
a water bath, and dry at 105°C for 2 hours: the residue melts
<2.60> between 157°C and 162°C.
(3) A solution of Thiopental Sodium (1 in 10) responds to
the Qualitative Tests <1.09> (1) and (2) for sodium salt.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Thiopental Sodium in 10 mL of freshly boiled and cooled
water: the solution is clear and light yellow.
(2) Heavy metals <1.07> — Dissolve 2.0 g of Thiopental
Sodium in 76 mL of water, add 4 mL of dilute hydrochloric
acid, shake, and filter through a glass filter (G4). To 40 mL
of the filtrate add 2 mL of ammonium acetate TS, dilute with
water to 50 mL, and perform the test using this solution as
the test solution. Prepare a control solution as follows: to 2.0
mL of Standard Lead Solution add 2 mL of dilute acetic acid,
2 mL of ammonium acetate TS and water to make 50 mL
JPXV
Official Monographs / Thiopental Sodium for Injection 1167
(not more than 20 ppm).
(3) Neutral and basic substances — Weigh accurately
about 1 g of Thiopental Sodium, dissolve in 10 mL of water
and 5 mL of sodium hydroxide TS, and shake vigorously
with 40 mL of chloroform. Separate the chloroform layer,
wash with two 5-mL portions of water, filter, and evaporate
the filtrate on a water bath to dryness. Dry the residue at
105 °C for 1 hour: the amount of the residue is not more than
0.50%.
(4) Related substances — Dissolve 50 mg of Thiopental
Sodium in 50 mL of the mobile phase, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add the mobile phase to make exactly 200 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 20 fiL each of the sample solution and the standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Measure each peak area
of each solution by the automatic integration method: the
total area of peaks other than those of thiopental in the sam-
ple solution is not larger than the peak area of thiopental in
the standard solution.
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel (5 fim in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1 g of potassium dihydrogen phos-
phate in 1000 mL of water, and adjust the pH to 3.0 with
phosphoric acid. To 700 mL of this solution add 300 mL of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
thiopental is about 15 minutes.
Time span of measurement: About 1.5 times as long as the
retention time of thiopental.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 10 mL.
Confirm that the peak area of thiopental obtained from 20
/uL of this solution is equivalent to 15 to 25% of that of
thiopental obtained from 20 fiL of the standard solution.
System performance: Dissolve 5 mg each of isopropyl
parahydroxybenzoate and propyl parahydroxybenzoate in 50
mL of acetonitrile, and add water to make 100 mL. When the
procedure is run with 20 fiL of this solution under the above
operating conditions, isopropyl parahydroxybenzoate and
propyl parahydroxybenzoate are eluted in this order with the
resolution between these peaks being not less than 1.9.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
thiopental is not more than 2.0%.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
80°C, 4 hours).
Assay Weigh accurately about 0.5 g of Thiopental Sodium,
previously dried, transfer to a separator, dissolve in 20 mL of
water, add 5 mL of ethanol (95) and 10 mL of dilute
hydrochloric acid, and extract with 50 mL of chloroform,
then with three 25-mL portions of chloroform. Combine the
chloroform extracts, wash with two 5-mL portions of water,
and extract the washings with two 10-mL portions of chlo-
roform. Filter the combined chloroform extracts into a coni-
cal flask, and wash the filter paper with three 5-mL portions
of chloroform. Combine the filtrate and the washings, and
add 10 mL of ethanol (95). Titrate <2.50> with O.lmol/L
potassium hydroxide-ethanol VS until the color of the solu-
tion changes from yellow through light blue to purple (indica-
tor: 2 mL of alizarin yellow GG-thymolphthalein TS). Per-
form a blank determination with a mixture of 160 mL of
chloroform and 30 mL of ethanol (95), and make any neces-
sary correction.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 26.43 mg of C„H 17 N 2 Na0 2 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiopental Sodium for Injection
>±%tmi L l-<<>9-)l1- r- U ?A
Thiopental Sodium for Injection is a preparation for
injection which is dissolved before use.
It contains not less than 93% and not more than
107% of the labeled amount of thiopental sodium
(C„H 17 N 2 Na0 2 S: 264.32).
Method of preparation Prepare as directed under Injec-
tions, with 100 parts of Thiopental Sodium and 6 parts of
Dried Sodium Carbonate in mass.
Description Thiopental Sodium for Injection is a light yel-
low powder or mass, and has a slight, characteristic odor.
It is very soluble in water, and practically insoluble in de-
hydrated diethyl ether.
It is hygroscopic.
Identification (1) Dissolve 0.1 g of Thiopental Sodium for
Injection in 10 mL of water, and add 0.5 mL of barium chlo-
ride TS: a white precipitate is formed. Collect the precipitate,
and add dilute hydrochloric acid drop wise: the precipitate
dissolves with effervescence.
(2) Proceed as directed in the Identification under
Thiopental Sodium.
pH <2.54> Dissolve 1 g of Thiopental Sodium for Injection
in 40 mL of water: the pH of this solution is between 10.2 and
11.2.
Purity Proceed as directed in the Purity under Thiopental
Sodium.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
80°C, 4 hours).
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Take 10 samples of Thiopental Sodium for Injection,
and open each container carefully. Dissolve each content
with water, wash each container with water, combine the
washings with the former solution, and add water to make
exactly 1000 mL. Pipet 10 mL of this solution, and add water
to make exactly 100 mL. Measure exactly a volume (V mL)
of this solution, equivalent to about 15 mg of thiopental sodi-
1168 Thioridazine Hydrochloride / Official Monographs
JP XV
um (C 11 H 1 7N 2 Na02S), and add water to make exactly 1000
mL. Pipet 10 mL of this solution, add 15 mL of diluted dilute
sodium hydroxide TS (1 in 100), add water to make exactly 30
mL, and use this solution as the sample solution. Separately,
weigh accurately about 46 mg of thiopental for assay, previ-
ously dried at 105°C for 3 hours, dissolve in 50 mL of dilute
sodium hydroxide TS, and add water to make exactly 200
mL. Pipet 2 mL of this solution, add water to make exactly
100 mL, and use this solution as the standard solution. Per-
form the test with the sample solution and standard solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and determine the absorbances, A T and A s , at 304
nm.
Amount (mg) of thiopental sodium (CnH 17 N 2 Na0 2 S)
in each sample of Thiopental Sodium for Injection
= W s x(A T /A s )x(300/V)x 1.0907
W s : Amount (mg) of thiopental sodium for assay
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant.
Thioridazine Hydrochloride
^/fU #>*>£&£
and enantiomer
C 21 H 26 N 2 S 2 .HC1: 407.04
10- {2-[(2/?5')-l-Methylpiperidin-2-yl]ethyl} -2-
methylsulf anyl- 1 0//-phenothiazine monohydrochloride
[130-61-0]
Thioridazine Hydrochloride, when dried,
not less than 99.0% of C 21 H 26 N 2 S 2 .HC1.
contains
Description Thioridazine Hydrochloride occurs as a white
to pale yellow, crystalline powder. It is odorless, and has a
bitter taste.
It is freely soluble in water, in methanol, in ethanol (95)
and in acetic acid (100), sparingly soluble in acetic anhydride,
and practically insoluble in diethyl ether.
The pH of a solution of Thioridazine Hydrochloride (1 in
100) is between 4.2 and 5.2.
It is gradually colored by light.
Identification (1) Dissolve 0.01 g of Thioridazine
Hydrochloride in 2 mL of sulfuric acid: a deep blue color de-
velops.
(2) Dissolve 0.01 g of Thioridazine Hydrochloride in 2
mL of water, and add 1 drop of cerium (IV) tetraammonium
sulfate TS: a blue color develops, and the color disappears on
the addition of excess of the reagent.
(3) Determine the infrared absorption spectrum of
Thioridazine Hydrochloride, previously dried, as directed in
the potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) To 5 mL of a solution of Thioridazine Hydrochloride
(1 in 100) add 2 mL of ammonia TS, and heat on a water bath
for 5 minutes. After cooling, filter, and acidify the filtrate
with dilute nitric acid: the solution responds to the Qualita-
tive Tests <1.09> (2) for chloride.
Melting point <2.60> 159 - 164 C C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Thioridazine Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Thioridazine Hydrochloride, according to Method 3, and
perform the test (not more than 2 ppm).
(3) Related substances — Conduct this procedure under
the protection from the sunlight. Dissolve 0. 10 g of Thiorida-
zine Hydrochloride in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, and add methanol to make exactly 20 mL. Pipet 2mL
of this solution, add methanol to make exactly 10 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, 2-propanol and ammonia so-
lution (28) (74:25:1) to a distance of about 10 cm, and air-dry
the plate. Examine the plate under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Thioridazine
Hydrochloride, previously dried, dissolve in 80 mL of a mix-
ture of acetic anhydride and acetic acid (100) (1:1), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 40.70 mg of C 21 H 26 N 2 S 2 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Thiotepa
V7
K
I
... P..
^N II N— 7
V s \f
C 6 H 12 N 3 PS: 189.22
Tris(aziridin-l-yl)phosphine sulfide [52-24-4]
Thiotepa, when dried, contains not less than 98.0%
JPXV
Official Monographs / L-Threonine 1169
of QH 12 N 3 PS.
Description Thiotepa occurs as colorless or white crystals,
or white, crystalline powder. It is odorless.
It is freely soluble in water, in ethanol (95) and in diethyl
ether.
A solution of Thiotepa (1 in 10) is neutral.
Identification (1) To 5 mL of a solution of Thiotepa (1 in
100) add 1 mL of hexaammonium heptamolybdate TS, and
allow to stand: a dark blue color develops slowly when the so-
lution is cold, or quickly when warm.
(2) To 5 mL of a solution of Thiotepa (1 in 100) add 1 mL
of nitric acid: this solution responds to the Qualitative Tests
<1.09> (2) for phosphate.
(3) Dissolve 0.1 g of Thiotepa in a mixture of 1 mL of
lead (II) acetate TS and 10 mL of sodium hydroxide TS, and
boil: the gas evolved changes moistened red litmus paper to
blue, and the solution shows a grayish red color.
Melting point <2.60> 52 - 57°C
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Thiotepa in 20 mL of water: the solution is clear and color-
less.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Thiotepa
in a platinum crucible according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(3) Arsenic </.//> — Dissolve 0.20 g of Thiotepa in 5 mL
of water, and add 1 mL of nitric acid and 1 mL of sulfuric
acid. Take this solution, prepare the test solution according
to Method 2, and perform the test (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.20% (1 g, in vacu-
um, silica gel, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g, plati-
num crucible).
Assay Weigh accurately about 0.1 g of Thiotepa, previous-
ly dried, dissolve in 50 mL of a solution of potassium thioc-
yanate (3 in 20), add 25 mL of 0.05 mol/L sulfuric acid VS,
exactly measured, and allow to stand for 20 minutes with oc-
casional shaking. Titrate <2.50> the excess sulfuric acid with
0.1 mol/L sodium hydroxide VS until the color of the solu-
tion changes from red to light yellow (indicator: 3 drops of
methyl red TS). Perform a blank determination.
Each mL of 0.05 mol/L sulfuric acid VS
= 6.307 mg of C 6 H 12 N 3 PS
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
L-Threonine
L-hU7|-->
H 3 C
H OH
CQjH
H NHj
C 4 H 9 N0 3 : 119.12
(2S,3.R)-2-Amino-3-hydroxybutanoic acid [72-19-5]
L-Threonine, when dried, contains not less than
98.5% of C 4 H 9 N0 3 .
Description L-Threonine occurs as white crystals or crystal-
line powder. It is odorless or has a slight, characteristic odor,
and has a slightly sweet taste.
It is freely soluble in formic acid, soluble in water, and
practically insoluble in ethanol (95).
Identification Determine the infrared absorption spectrum
of L-Threonine, previously dried, as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]™: -26.0- -29.0° (after
drying, 1.5 g, water, 25 mL, 100 mm).
pH <2.54> Dissolve 0.20 g of L-Threonine in 20 mL of
water: the pH of this solution is between 5.2 and 6.2.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
L-Threonine in 20 mL of water: the solution is clear and
colorless.
(2) Chloride <7.0?>— Perform the test with 0.5 g of L-
Threonine. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <I.I4>— Perform the test with 0.6 g of l-
Threonine. Prepare the control solution with 0.35 mL of
0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Threonine. Prepare the control solution with 5.0 mL of
Standard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of L-Threo-
nine according to Method 1, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(6) Arsenic <1.11> — Dissolve 1.0 g of L-Threonine in 5
mL of dilute hydrochloric acid, and perform the test with this
solution as the test solution (not more than 2 ppm).
(7) Related substances — Dissolve 0.30 g of L-Threonine
in 50 mL of water, and use this solution as the sample solu-
tion. Pipet 1 mL of this solution, and add water to make ex-
actly 50 mL. Pipet 5 mL of this solution, add water to make
exactly 20 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /iL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of 1-butanol, water and acetic acid (100) (3:1:1) to a distance
of about 10 cm, and dry the plate at 80°C for 30 minutes.
Spray evenly the plate with a solution of ninhydrin in acetone
(1 in 50), and heat the plate at 80°C for 5 minutes: the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.20% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0. 12 g of L-Threonine, previ-
ously dried, dissolve in 3 mL of formic acid, add 50 mL of
acetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
1170 Thrombin / Official Monographs
JP XV
= 11.91 mg of C 4 H 9 N0 3
Containers and storage Containers — Tight containers.
Thrombin
h n>tr>
Thrombin is prepared from prothrombin obtained
from blood of man or bull, through interaction with
added thromboplastin in the presence of calcium ions,
sterilized and lyophilized.
It contains not less than 80% and not more than
150% of the labeled Units of thrombin.
Each mg contains not less than 10 Units of throm-
bin.
Description Thrombin is a white to light yellow, amor-
phous substance.
Thrombin (500 Units) dissolves in 1.0 mL of isotonic sodi-
um chloride solution clearly or with slight turbidity within 1
minute.
Loss on drying <2.41> Not more than 3% (50 mg, in vacu-
um, phosphorus (V) oxide, 4 hours).
Sterility <4.06> It meets the requirement.
Assay (i) Fibrinogen solution — Weigh accurately about
30 mg of fibrinogen, and dissolve in 3 mL of isotonic sodium
chloride solution. Allow the solution to clot sufficiently with
frequent shaking after the addition of about 3 Units of
thrombin. Wash the precipitated clot thoroughly until the
washings yield no turbidity on addition of silver nitrate TS,
weigh the clot after drying at 105°C for 3 hours, and calcu-
late the percentage of the clot in the fibrinogen. Dissolve the
fibrinogen in isotonic sodium chloride solution so that the
clot should be 0.20%, adjust the pH of the solution between
7.0 and 7.4 by addition of 0.05 mol/L dibasic sodium phos-
hate TS (or if necesary, use 0.5 mol/L disodium hydrogen-
phosphate TS), and dilute with isotonic sodium chloride solu-
tion to make a 0.10% solution.
(ii) Procedure — Dissolve Thrombin Reference Standard
in isotonic sodium chloride solution, and prepare four kinds
of standard solutions which contain 4.0, 5.0, 6.2, and 7.5
Units in 1 mL. Transfer accurately 0.10 mL each of the stan-
dard solutions maintained at a given degree ± 1°C between
20°C and 30°C to a small test tube, 10 mm in inside diameter,
100 mm in length, blow out 0.90 mL of the fibrinogen solu-
tion at the same temperature into the test tube from a pipet,
start a stop watch simultaneously, shake the tube constantly,
and determine the time for the first appearance of clot. Cal-
culate the average values of five determinations for the four
kinds of standard solutions, respectively. If the deviation be-
tween the maximum and the minimum values of five determi-
nations is more than 10% of the average value, reject the
whole run, and try the experiment again. The concentration
of the standard solution may be changed appropriately wi-
thin the range between 14 and 60 seconds of the clotting time.
The determination proceeds at the same temperature
described above. Next, weigh accurately the whole contents
of a single container of Thrombin, dissolve it in isotonic sodi-
um chloride solution to provide a solution which is presumed
to contain about 5 Units in each mL, treat 0.10 mL of the so-
lution with the same reagents in the same manner five times,
determine the clotting times, and calculate the average value.
Plot the average values of the clotting times of the four kinds
of the standard solutions on a logarithmic graph, using Units
as the abscissa and clotting times as the ordinate, and draw a
calibration line which best fits the four plotted points. Using
this line, read the Units U from the average value of the clot-
ting times of the sample solution.
Units of a single container of Thrombin = U X 10 x V
V: The number of mL of the volume in which the contents
of a single container of Thrombin has been dissolved.
Calculate the units for 1 mg of the contents.
Containers and storage Containers — Hermetic containers.
Storage — Not exceeding 10°C.
Expiration date Use within 36 months after the date of
manufacture.
Thymol
3-=E-)l
CH 6
C 10 H 14 O: 150.22
5-Methyl-2-(l-methylethyl)phenol [89-83-8]
Thymol contains not less than 98.0% of C 10 H 14 O.
Description Thymol occurs as colorless crystals or white,
crystalline masses. It has an aromatic odor, and has a burn-
ing taste.
It is very soluble in acetic acid (100), freely soluble in
ethanol (95) and in diethyl ether, and slightly soluble in
water.
It sinks in water, but when warmed, it melts and rises to the
surface of water.
Identification (1) To 1 mL of a solution of Thymol in
acetic acid (100) (1 in 300) add 6 drops of sulfuric acid and 1
drop of nitric acid: a blue-green color develops by reflected
light and a red-purple color develops by transmitted light.
(2) Dissolve 1 g of Thymol in 5 mL of a solution of sodi-
um hydroxide (1 in 10) by heating in a water bath, and con-
tinue heating for several minutes: a light yellow-red color
slowly develops. Allow this solution to stand at room temper-
ature: the color changes to dark yellow-brown. Shake this so-
lution with 2 to 3 drops of chloroform: a purple color grad-
ually develops.
(3) Triturate Thymol with an equal mass of camphor or
menthol: the mixture liquefies.
Melting point <2.60> 49 - 51°C
Purity (1) Non-volatile residue — Volatilize 2.0 g of
Thymol by heating on a water bath, and dry the residue at
105 °C for 2 hours: the mass is not more than 1.0 mg.
(2) Other phenols — Shake vigorously 1 .0 g of Thymol
with 20 mL of warm water for 1 minute, and filter. To 5 mL
JPXV
Official Monographs / Tiaramide Hydrochloride 1171
of the filtrate add 1 drop of iron (III) chloride TS: a green
color may develop, but no blue to purple color develops.
Assay Weigh accurately about 0.5 g of Thymol, dissolve in
10 mL of sodium hydroxide TS, and add water to make ex-
actly 100 mL. Measure exactly 10 mL of the solution into an
iodine flask, add 50 mL of water and 20 mL of dilute sulfuric
acid, and cool in ice water for 30 minutes. Add exactly 20 mL
of 0.05 mol/L bromine VS, stopper tightly immediately, al-
low to stand for 30 minutes in ice water with occasional shak-
ing in a dark place, add 14 mL of potassium iodide TS and 5
mL of chloroform, stopper tightly, shake vigorously, and ti-
trate <2.50> the liberated iodine with 0.1 mol/L sodium
thiosulfate VS (indicator: 3 mL of starch TS). Stopper tight-
ly, shake vigorously near the end point, and continue the
titration until the blue color in the chloroform layer disap-
pears. Perform a blank determination.
Each mL of 0.05 mol/L bromine VS
= 3.755 mg of C 10 H 14 O
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Dried Thyroid
Dried Thyroid is the fresh thyroid gland, previously
deprived of connective tissue and fat, minced, dried
rapidly at a temperature not above 50°C, and pow-
dered, or diluted with suitable diluents. It is obtained
from domesticated animals that are used for food by
man.
It contains not less than 0.30% and not more than
0.35% of iodine (I: 126.90) in the form of organic com-
pounds peculiar to the thyroid gland.
Description Dried Thyroid occurs as a light yellow to
grayish brown powder. It has a slight, characteristic, meat-
like odor.
Identification Mount Dried Thyroid in diluted formalde-
hyde solution (1 in 10), stain in hematoxylin TS for 10 to 30
minutes, wash with water, soak in a mixture of 1 mL of
hydrochloric acid and 99 mL of diluted ethanol (7 in 10) for 5
to 10 seconds, and again wash with water for about 1 hour.
Stain in a solution of eosin Y (1 in 100) for 1 to 5 minutes,
wash with water, dehydrate, and soak successively in diluted
ethanol (7 in 10) for 5 to 10 seconds, in diluted ethanol (4 in
5) for 5 to 10 seconds, in diluted ethanol (9 in 10) for 1 to 2
minutes, in ethanol (95) for 1 to 5 minutes then in ethanol
(99.5) for 1 to 5 minutes. Interpenetrate in xylene, seal with
balsam, and examine under a microscope: epithelial nuclei
forming follicles peculiar to the thyroid gland are observed.
Purity (1) Inorganic iodides — Mix 1.0 g of Dried Thyroid
with 10 mL of a saturated solution of zinc sulfate heptahy-
drate, shake for 5 minutes, and filter. To 5 mL of the filtrate
add 0.5 mL of starch TS, 4 drops of sodium nitrite TS and 4
drops of dilute sulfuric acid with thorough shaking: no blue
color is produced.
(2) Fat — Extract 1.0 g of Dried Thyroid with diethyl
ether for 2 hours using a Soxhlet extractor. Evaporate the
diethyl ether extract, and dry the residue at 105°C to constant
mass: the mass of the residue is not more than 0.030 g.
Loss on drying <2.41> Not more than 6.0% (1 g, 105°C,
constant mass).
Total ash <J.07> Not more than 5.0% (0.5 g).
Assay Transfer about 1 g of Dried Thyroid, accurately
weighed, to a crucible, add 7 g of potassium carbonate, mix
carefully, and gently tap the crucible on the table to compact
the mixture. Overlay with 10 g of potassium carbonate, and
compact again thoroughly by tapping. Place the crucible in a
muffle furnace preheated to a temperature between 600°C
and 700°C, and ignite the mixture for 25 minutes. Cool, add
20 mL of water, heat gently to boiling, and filter into a flask.
To the residue add 20 mL of water, boil, and filter into the
same flask. Rinse the crucible and the char on the funnel with
boiling water until the filtrate measures 200 mL. Add slowly 7
mL of freshly prepared bromine TS, 40 mL of diluted phos-
phoric acid (1 in 2), and boil until starch iodide paper is no
longer colored blue by the evolved gas. Wash down inside of
the flask with water, and continue boiling for 5 minutes. Dur-
ing the boiling add water from time to time to maintain a
volume at not less than 200 mL. Cool, add 5 mL of a solution
of phenol (1 in 20), again rinse inside of the flask with water,
and allow to stand for 5 minutes. Add 2 mL of diluted phos-
phoric acid (1 in 2) and 5 mL of potassium iodide TS, and ti-
trate <2.50> immediately the liberated iodine with 0.01 mol/L
sodium thiosulfate VS (indicator: 3 mL of starch TS). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.01 mol/L sodium thiosulfate VS
= 0.2115 mg of I
Containers and storage Containers — Tight containers.
Tiaramide Hydrochloride
C 15 H 18 C1N 3 3 S.HC1: 392.30
5-Chloro-3-{2-[4-(2-hydroxyethyl)piperazin-l-yl]-
2-oxoethyl} -1 ,3-benzothiazol-2(3//)-one monohydrochloride
[35941-71-0]
Tiaramide Hydrochloride, when dried, contains not
less than 98.5% of C 15 H 18 C1N 3 3 S.HC1.
Description Tiaramide Hydrochloride occurs as a white,
crystalline powder. It is odorless.
It is freely soluble in water, slightly soluble in ethanol (95)
and in acetic acid (100), and practically insoluble in acetic an-
hydride and in diethyl ether.
The pH of a solution of Tiaramide Hydrochloride (1 in 20)
is between 3.0 and 4.5.
Melting point: about 265°C (with decomposition).
Identification (1) Dissolve 5 mg of Tiaramide Hydrochlo-
1 172 Tiaramide Hydrochloride Tablets / Official Monographs
JP XV
ride in 5 mL of 0.1 mol/L hydrochloric acid TS, and add 3
drops of Dragendorff's TS: an orange precipitate is formed.
(2) Determine the infrared absorption spectrum of
Tiaramide Hydrochloride, previously dried, as directed in the
potassium chloride disk method under the Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Tiaramide Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
Tiaramide Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Tiaramide Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Tiaramide Hydrochloride according to Method 1, and per-
form the test. In the procedure, add 20 mL of diluted
hydrochloric acid (1 in 2) (not more than 2 ppm).
(4) Related substances — Dissolve 0.20 g of Tiaramide
Hydrochloride in 10 mL of diluted ethanol (99.5) (7 in 10),
and use this solution as the sample solution. Pipet 1 mL of
the sample solution, and add diluted ethanol (99.5) (7 in 10)
to make exactly 100 mL. Pipet 2 mL of this solution, add
diluted ethanol (99.5) (7 in 10) to make exactly 10 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /XL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. After air-drying,
immediately develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (4:2:1) to a distance of about 10
cm, air-dry the plate, and then dry at 100°C for 30 minutes.
After cooling, examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
and the spot of the starting point from the sample solution
are not more intense than the spot from the standard solu-
tion. Allow the plate to stand in iodine vapor for 30 minutes:
the spots other than the principal spot and the spot of the
starting point from the sample solution are not more intense
than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tiaramide
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3) by warm-
ing, cool, and titrate <2.50> with 0.1 mol/L perchloric acid
VS until the color of the solution changes from red through
purple to blue-purple (indicator: 3 drops of neutral red TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L perchloric acid VS
= 39.23 mg of C 15 H 18 C1N 3 03S.HC1
Containers and storage Containers — Well-closed contain-
ers.
Tiaramide Hydrochloride Tablets
Tiaramide Hydrochloride Tablets contain not less
than 95.0% and not more than 105.0% of the labeled
amount of tiaramide (Ci 5 H 18 ClN30 3 S: 355.84).
Method of preparation Prepare as directed under Tablets,
with Tiaramide Hydrochloride.
Identification (1) Determine the absorption spectrum of
the sample solution obtained in the Assay as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits
maxima between 285 nm and 289 nm, and between 292 nm
and 296 nm.
(2) To a quantity of powdered Tiaramide Hydrochloride
Tablets, equivalent to 0.1 g of tiaramide according to the
labeled amount, add 10 mL of diluted ethanol (7 in 10), shake
well, filter, and use the filtrate as the sample solution.
Separately, dissolve 0.1 1 g of tiaramide hydrochloride for as-
say in 10 mL of diluted ethanol (7 in 10), and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, de-
velop with a mixture of 1-butamol, water and acetic acid
(100) (4:2:1) to a distance of about 10 cm, and dry the plate at
100°C for 30 minutes. Spray evenly Dragendorff's TS for
spraying followed by diluted nitric acid (1 in 50) on the plate:
the principal spot obtained with the sample solution and the
spot with the standard solution are yellow-red in color and
have the same Rf value.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Tiaramide Hydrochloride Tablets add a
volume of 0.1 mol/L hydrochloric acid TS, equivalent to 3/5
volume of KmL which makes a solution so that each mL
contains about 1 mg of tiaramide (C 15 H 18 C1N 3 03S) according
to the labeled amount, shake for 60 minutes, then add 0.1
mol/L hydrochloric acid TS to make exactly KmL, and
filter. Discard the first 20 mL of the filtrate, pipet 5 mL of the
subsequent filtrate, add water to make exactly 100 mL, and
use this solution as the sample solution. Separately, weigh ac-
curately about 55 mg of tiaramide hydrochloride for assay,
previously dried at 105°C for 3 hours, and dissolve in 0.1
mol/L hydrochloric acid TS to make exactly 50 mL. Pipet 5
mL of this solution, add water to make exactly 100 mL, and
use this solution as the standard solution. Determine the ab-
sorbances, A T and A s , of the sample solution and standard
solution at 294 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of tiaramide (C 15 H 18 C1N 3 03S)
= W s x (A T /A S ) x (K/50) x 0.907
W s : Amount (mg) of tiaramide hydrochloride for assay
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Tiaramide Hydrochloride
JPXV
Official Monographs / Ticlopidine Hydrochloride 1173
Tablets at 50 revolutions per minute according to the Paddle
method, using 900 mL of water as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 15
minutes after starting the test for a 50-mg tablet or 30
minutes after starting the test for a 100-mg tablet, and filter
through a membrane filter with pore size of not more than 0.5
fim. Discard the first 10 mL of the filtrate, pipet FmL of the
subsequent filtrate, add water to make exactly V mL so that
each mL contains about 56 n% of tiaramide (C 15 H 18 C1N 3 3 S)
according to the labeled amount, and use this solution as the
sample solution. Separately, weigh accurately about 15 mg of
tiaramide hydrochloride for assay, previously dried at 105°C
for 3 hours, and dissolve in water to make exactly 50 mL.
Pipet 5 mL of this solution, add water to make exactly 25
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , at 294 nm of the sample solution
and standard solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>. The dissolution rates for a 50-mg
tablet in 15 minutes and a 100-mg tablet in 30 minutes are not
less than 80%, respectively.
Dissolution rate (%) with respect to the labeled amount of
tiaramide (C 15 H 18 C1N 3 3 S)
= W s x (Aj/A s ) x (V/ V) x (I/O x 360 x 0.907
W s : Amount (mg) of tiaramide hydrochloride for assay
C: Labeled amount (mg) of tiaramide (C 15 H I8 C1N 3 3 S) in
1 tablet
Assay Weigh accurately the mass of more than 20
Tiaramide Hydrochloride Tablets, and powder. Weigh ac-
curately an amount of the powder, equivalent to about 0.1 g
of tiaramide (C 15 H 18 C1N 3 3 S), add 60 mL of O.lmol/L
hydrochloric acid TS, shake for 30 minutes, add 0.1 mol/L
hydrochloric acid TS to make exactly 100 mL, and filter. Dis-
card the first 20 mL of the filtrate, pipet 5 mL of the subse-
quent filtrate, add water to make exactly 100 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 0.11 g of tiaramide hydrochloride for assay, previously
dried at 105°C for 3 hours, and dissolve in 0.1 mol/L
hydrochloric acid TS to make exactly 100 mL. Pipet 5 mL of
this solution, add water to make exactly 100 mL, and use this
solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 294 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>.
Amount (mg) of tiaramide (C 15 H 18 C1N 3 3 S)
= W s x (A T /A S ) x 0.907
W s : Amount (mg) of tiaramide hydrochloride for assay
Containers and storage Containers — Tight containers.
Ticlopidine Hydrochloride
^ntfv>>i£|gi£
•HC"
C M H 14 C1NS.HC1: 300.25
5-(2-Chlorobenzyl)-4,5 ,6,7-
tetrahydrothieno[3,2-c]pyridine monohydrochloride
[53885-35-1]
Ticlopidine Hydrochloride contains not less than
99.0% of C 14 H 14 C1NS.HC1, calculated on the anhy-
drous basis.
Description Ticlopidine Hydrochloride occurs as a white to
pale yellowish white crystalline powder.
It is freely soluble in acetic acid (100), soluble in water and
in methanol, sparingly soluble in ethanol (95), and practically
insoluble in diethyl ether.
Identification (1) Determine the infrared absorption spec-
trum of Ticlopidine Hydrochloride as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) A solution of Ticlopidine Hydrochloride (1 in 20)
responds to the Qualitative Tests <1.09> (2) for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Ticlopidine Hydrochloride according to Method 3, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Ticlopidine Hydrochloride according to Method 4, and
perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.5 g of Ticlopidine
Hydrochloride in 20 mL of a solution of hydrochloric acid in
methanol (1 in 20,000), and use this solution as the sample so-
lution. To exactly 5 mL of the sample solution add a solution
of hydrochloric acid in methanol (1 in 20,000) to make exact-
ly 200 mL, and use this solution as the standard solution (1).
Separately, pipet 1 mL of the sample solution, add a solution
of hydrochloric acid in methanol (1 in 20,000) to make exact-
ly 50 mL, and use this solution as the standard solution (2).
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution (1) on a plate of silica gel for
thin-layer chromatography (Plate 1), and spot 10 /uL each of
the sample solution and standard solution (2) on another
plate of silica gel for thin-layer chromatography (Plate 2).
Develop the plates with an upper layer of a mixture of water,
1-butanol and acetic acid (100) (5:4:1) to a distance of about
15 cm, and air-dry the plates. Spray evenly a solution of nin-
hydrin in acetone (1 in 50) on Plate 1, and heat at 100°C for
20 minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution (1). Allow Plate 2 to stand in an iodine
vapor for 30 minutes: the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution (2).
(4) Formaldehyde — Dissolve 0.80 g of Ticlopidine
Hydrochloride in 19.0 mL of water, add 1 .0 mL of 4 mol/L
sodium hydroxide TS, shake well, centrifuge, and filter the
supernatant liquid. To 5.0 mL of the filtrate add 5.0 mL of
acetylacetone TS, mix, and warm at 40°C for 40 minutes: the
solution has no more color than the following control solu-
tion.
Control solution: Weigh accurately 0.54 g of formalde-
hyde solution, and add water to make exactly 1000 mL. To
exactly 10 mL of this solution add water to make exactly 1000
1 174 Timepidium Bromide Hydrate / Official Monographs
JP XV
mL. Prepare before use. To 8.0 mL of this solution add water
to make 20.0 mL, and filter. To 5.0 mL of the filtrate add 5.0
mL of acetylacetone TS, and proceed in the same manner.
Water <2.48> Not more than 1.0% (0.3 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Ticlopidine
Hydrochloride, dissolve in 20 mL of acetic acid (100), add 40
mL of acetic anhydride, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 30.03 mg of C 14 H 14 C1NS.HC1
Containers and storage Containers — Well-closed contain-
ers.
Timepidium Bromide Hydrate
5 1 / tr^Ajuk***]*
H 3 C,
s 7 Br • HjO
and enantiomer
C 17 H 22 BrNOS 2 .H 2 0: 418.41
(5 /?5')-3-(Dithien-2-ylmethylene)-5-methoxy- 1,1-
dimethylpiperidinium bromide monohydrate
[35035-05-3, anhydride]
Timepidium Bromide Hydrate contains not less than
98.5% of timepidium bromide (C 17 H 2 2BrNOS 2 :
400.40), calculated on the anhydrous basis.
Description Timepidium Bromide Hydrate occurs as white
crystals or crystalline powder.
It is very soluble in methanol and in acetic acid (100), freely
soluble in ethanol (99.5), sparingly soluble in water and in
acetic anhydride, and practically insoluble in diethyl ether.
The pH of a solution of Timepidium Bromide Hydrate in
freshly boiled and cooled water (1 in 100) is between 5.3 and
6.3.
A solution of Timepidium Bromide Hydrate in methanol
(1 in 20) shows no optical rotation.
Identification (1) To 1 mL of a solution of Timepidium
Bromide Hydrate (1 in 100) add 1 mL of ninhydrin-sulfuric
acid TS: a red purple color develops.
(2) Determine the absorption spectrum of a solution of
Timepidium Bromide Hydrate (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of
Timepidium Bromide Hydrate as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhitit similar intensities of absorption at
the same wave numbers.
(4) A solution of Timepidium Bromide Hydrate (1 in 100)
responds to the Qualitative Tests <1.09> (1) for Bromide.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Timepidium Bromide Hydrate in 10 mL of water: the solu-
tion is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Timepidium Bromide Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Timepidium
Bromide Hydrate in 10 mL of methanol, and use this solu-
tion as the sample solution. Pipet 1 mL of the sample solu-
tion, and add methanol to make exactly 100 mL. Pipet 1 mL
of this solution, add methanol to make exactly 10 mL, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of chloroform, methanol, water, acetic acid
(100) and ethyl acetate (5:4:1:1:1) to a distance of about 13
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Water <2.48> 3.5 - 5.0% (0.4 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.6 g of Timepidium
Bromide Hydrate, dissolve in 60 mL of a mixture of acetic
anhydride and acetic acid (100) (2:1), and titrate <2.50> with
0.1 mol/L perchloric acid VS (potentiometric titration). Per-
form a blank determination, and make any necessary correc-
tion.
Each mL of 0.1 mol/L perchloric acid VS
= 40.04 mg of C 17 H 22 BrNOS 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Timolol Maleate
H OH
C0 2 H
CO ? H
C^H^C^S-C^Cv 432.49
(2S)- 1 -[(1,1 -Dimethylethyl)amino]-3-(4-morpholin-4-yl-
1 ,2,5-thiadiazol-3-yloxy)propan-2-ol monomaleate
[26921-17-5]
Timolol Maleate, when dried, contains not less than
98.0 % and not more than 101.0 % of
(^ 1 3 rl 24^4030.(^41^1404.
Description Timolol Maleate occurs as a white to pale yel-
JPXV
Official Monographs / Imidazole 1175
lowish white crystalline powder.
It is freely soluble in acetic acid (100), and soluble in water
and in ethanol (99.5).
It dissolves in 0.1 mol/L hydrochloric acid TS.
Melting point: about 197°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Timolol Maleate in 0.1 mol/L hydrochloric acid
TS (3 in 100,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Timolol Maleate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) To 5 mL of a solution of Timolol Maleate (1 in 500)
add 1 drop of potassium permanganate TS: the red color of
the TS disappears immediately.
Optical rotation <2.49> [a]^ : -5.7 - -6.2° (after drying,
1.25 g, 1 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of Timolol Maleate in 20 mL of water is between 3.8 and
4.3.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Timolol Maleate in 20 mL of water: the solution is clear, and
its absorbance at 440 nm, determined as directed under
Ultraviolet-visible Spectrophotometry <2.24>, is not more
than 0.05.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Timolol
Maleate according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(3) Related substances — Dissolve 30 mg of Timolol Male-
ate in 20 mL of the mobile phase, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add the
mobile phase to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 25 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
timolol and maleic acid is not larger than 1/5 times the peak
area of timolol from the standard solution, and the total area
of the peaks other than the peak of timolol and maleic acid is
not larger than 1/2 times the peak area of timolol from the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with phenylsilanized sili-
ca gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 1.9 g of sodium 1-hexanesulfonate
in 1800 mL of water, add 6.0 mL of triethylamine and 8.0
mL of formic acid, adjust to pH 3.0 with formic acid, and
add water to make 2000 mL. To 1400 mL of this solution add
500 mL of methanol and 100 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
timolol is about 18 minutes.
Time span of measurement: About 2 times as long as the
retention time of timolol beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mobile phase to make 10 mL. Confirm
that the peak area of timolol obtained from 25 fiL of this so-
lution is equivalent to 7 to 13% of that from 25 /xL of the
standard solution.
System performance: When the procedure is run with 25
/xL of the sample solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of timolol are not less than 1500 and not more
than 2.5, respectively.
System repeatability: When the test is repeated 6 times with
25 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
timolol is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
100°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Timolol Maleate,
previously dried, dissolve in 90 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
metric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 43.25 mg of C^H^N^S^LLA,
Containers and storage Containers — Tight containers.
Tinidazole
^--¥ s f-)V
C 8 H 13 N 3 04S: 247.27
l-[2-(Ethylsulfonyl)ethyl]-2-methyl-5-nitro-l//-imidazole
[19387-91-8]
Tinidazole, when dried, contains not less than
98.5% and not more than 101.0% of C 8 H 13 N 3 4 S.
Description Tinidazole occurs as a light yellow, crystalline
powder.
It is soluble in acetic anhydride and in acetone, sparingly
soluble in methanol, slightly soluble in ethanol (99.5), and
very slightly soluble in water.
Identification (1) Determine the absorption spectrum of a
solution of Tinidazole in methanol (1 in 50,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
1176 Tipepidine Hibenzate / Official Monographs
JP XV
Tinidazole as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Melting point <2.60> 125 - 129°C
Purity (1) Sulfate <1.14>— To 2.0 g of Tinidazole add 100
mL of water, boil for 5 minutes, cool, add water to make 100
mL, and filter. Take 25 mL of the filtrate, and add 1 mL of
dilute hydrochloric acid and water to make 50 mL. Use this
solution as the test solution, and perform the test. Prepare
the control solution with 0.45 mL of 0.005 mol/L sulfuric
acid VS (not more than 0.043%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Tinida-
zole according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Tinidazole according to Method 3, and perform the test
(not more than 1 ppm).
(4) Related substances — Dissolve 50 mg of Tinidazole in
2 mL of acetone, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add acetone to make ex-
actly 200 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate and diethyla-
mine (19:1) to a distance of about 10 cm, air-dry the plate,
heat at 100°C for 5 minute, and cool. Examine under ultrav-
iolet light (main wavelength: 254 nm): the spots other than
the principal spot from the sample solution are not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.35 g of Tinidazole, previ-
ously dried, dissolve in 50 mL of acetic anhydride, and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 24.73 mg of QH13N3O4S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tipepidine Hibenzate
<3 L <\±i}>£'<>Xffl&
COiW o
C 15 H 17 NS 2 .C 14 H 10 O 4 : 517.
3-(Dithien-2-ylmethylene)- 1 -methylpiperidine mono [2-(4-
hydroxybenzoyl)benzoate] [31139-87-4]
Tipepidine Hibenzate, when dried, contains not less
than 98.5% of C 15 H 17 NS 2 .C 14 H 1() 04.
Description Tipepidine Hibenzate occurs as a white to light
yellow, crystalline powder. It is odorless and tasteless.
It is freely soluble in acetic acid (100), slightly soluble in
methanol and in ethanol (95), very slightly soluble in water,
and practically insoluble in diethyl ether.
Identification (1) Dissolve 0.01 g of Tipepidine Hibenzate
in 5 mL of sulfuric acid: an orange-red color develops.
(2) Dissolve 0.3 g of Tipepidine Hibenzate in 10 mL of
sodium hydroxide TS and 5 mL of water, and extract with
two 20-mL portions of chloroform. Wash the chloroform ex-
tracts with 10 mL of water, and filter the chloroform layer.
Evaporate the filtrate on a water bath to dryness, and dis-
solve the residue in 0.5 mL of 1 mol/L hydrochloric acid TS
and 5 mL of water. To 2 mL of this solution add 5 mL of
Reinecke salt TS: a light red precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Tipepidine Hibenzate in ethanol (99.5) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum;
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(4) Determine the infrared absorption spectrum of
Tipepidine Hibenzate, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Melting point <2.60> 189 - 193 °C
Purity (1) Clarity of solution — Dissolve 1.0 g of Tipepi-
dine Hibenzate in 10 mL of acetic acid (100): the solution is
clear. Perform the test with this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: its absorbance
at 400 nm is not more than 0.16.
(2) Heavy metals <1.07> — Proceed with 2.0 g of Tipepi-
dine Hibenzate according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Tipepidine Hibenzate according to Method 3, and perform
the test (not more than 2 ppm).
(4) Related substances — (i) Dissolve 10 mg of Tipep-
idine Hibenzate in 20 mL of the mobile phase, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 20 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine each peak
area of both solutions by the automatic integration method:
the total area of all peaks other than the area of the hibenzic
acid and tipepidine from the sample solution is not larger
than the peak area of the tipepidine from the standard solu-
tion.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
JPXV
Official Monographs / Tipepidine Hibenzate Tablets 1177
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of a solution of ammonium
acetate (1 in 100) and tetrahydrofuran (32:13).
Flow rate: Adjust the flow rate so that the retention time of
tipepidine is about 12 minutes.
Time span of measurement: As long as the retention time
of tipepidine beginning after the solvent peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of tipepidine obtained from 20 /xL
of this solutionis equivalent to 7 to 13% of that of tipepidine
obtained from 20 /xL of the standard solution.
System performance: Dissolve 10 mg of Tipepidine Hiben-
zate and 3 mg of propyl parahydroxybenzoate in 100 mL of
the mobile phase. When the procedure is run with 20 /xh of
this solution under the above operating conditions, hibenzic
acid, tipepidine and propyl parahydroxybenzoate are eluted
in this order with the resolution between the peaks of tipepi-
dine and propyl parahydroxybenzoate being not less than 3.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tipepidine is not more than 1.5%.
(ii) Dissolve 10 mg of Tipepidine Hibenzate in 20 mL of
the mobile phase, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add the mobile phase
to make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 20 /xL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine each peak area by the automatic integra-
tion method: the total area of all peaks other than the area of
the hibenzic acid and tipepidine from the sample solution is
not larger than 1 /2 times the peak area of the tipepidine from
the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of methanol and a solution of
ammonium acetate (1 in 500) (13:7).
Flow rate: Adjust the flow rate so that the retention time of
tipepidine is about 10 minutes.
Time span of measurement: As long as the retention time
of tipepidine beginning after the solvent peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of tipepidine obtained from 20 /xL
of this solutionis equivalent to 7 to 13% of that of tipepidine
obtained from 20 fiL of the standard solution.
System performance: Dissolve 12 mg of Tipepidine Hiben-
zate and 4 mg of xanthene in 50 mL of the mobile phase.
When the procedure is run with 10 /xL of this solution under
the above operating conditions, hibenzic acid, tipepidine and
xanthene are eluted in this order with the resolution between
the peaks of tipepidine and xanthene being not less than 3.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tipepidine is not more than 3.0%.
Loss on drying <2.4I> Not more than 0.5% (1 g, 60°C, in
vacuum, phosphorus (V) oxide, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 1 g of Tipepidine Hibenzate,
previously dried, dissolve in 40 mL of acetic acid (100), and
titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from purple through blue to
green (indicator: 3 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each ml of 0.1 mol/L perchloric acid VS
= 51.77 mg of C 15 H 17 NS 2 .C 14 H 10 O4
Containers and storage Containers — Well-closed contain-
ers.
Storagle — Light-resistant.
Tipepidine Hibenzate Tablets
^<&t>£.'<>Xffl&&
Tipepidine Hibenzate Tablets contain not less than
95% and not more than 105% of the labeled amount of
tipepidine hibenzate (C15H17NS2.C14H10O4: 517.66).
Method of preparation Prepare as directed under Tablets,
with Tipepidine Hibenzate.
Identification (1) To a quantity of powdered Tipepidine
Hibenzate Tablets, equivalent to 44 mg of Tipepidine Hiben-
zate according to the labeled amount, add 5 mL of water,
shake for 1 minute, add 10 mL of sodium hydroxide TS, and
extract with two 20-mL portions of chloroform. Combine the
extracts, wash with 10 mL of water, and filter the chloroform
layer. Evaporate the filtrate on a water bath to dryness, dis-
solve the residue in 0.2 mL of 1 mol/L hydrochloric acid TS
and 2 mL of water, and add 5 mL of Reinecke salt TS: a light
red precipitate is formed.
(2) To a quantity of powdered Tipepidine Hibenzate
Tablets, equivalent to 1 1 mg of Tipepidine Hibenzate accord-
ing to the labeled amount, add 30 mL of ethanol (99.5), and
warm for 10 minutes with occasional shaking. After cooling,
add ethanol (99.5) to make 50 mL, and filter. To 1 mL of the
filtrate add ethanol (99.5) to make 20 mL, and determine the
absorption spectrum of this solution as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 282 nm and 286 nm.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Tipepidine Hibenzate
Tablets at 50 revolutions per minute according to the Paddle
method, using 900 mL of water as the dissolution medium.
1 178 Titanium Oxide / Official Monographs
JP XV
Use the dissolved solution 30 minutes after starting the test as
the sample solution. Separately, weigh accurately about 0.11
g of tipepidine hibenzate for assay, previously dried in a
desiccator (in vacuum, phosphorus (V) oxide, 60°C) for 3
hours, and dissolve in 80 mL of diluted ethanol (99.5) (3 in 4)
by warming occasionally. After cooling, add diluted ethanol
(99.5) (3 in 4) to make exactly 100 mL, then pipet 20 mL of
this solution, add water to make exactly 900 mL, and use this
solution as the standard solution. Determine the absor-
bances, A Tl and ^4 S i, at 286 nm, and A T2 and A S2 , at 360 nm
of the sample solution and standard solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>. The disso-
lution rate of Tipepidine Hibenzate Tablets in 30 minutes is
not less than 80%.
Dissolution rate (%) with respect to the labeled amount
of tipepidine hibenzate (Ci5H 17 NS2.C 14 H 10 O 4 )
= W s x {{A T - A J2 /A S - A S2 )} X (20/Q
W s : Amount (mg) of tipepidine hibenzate for assay.
C: Labeled amount (mg) of tipepidine hibenzate
(C 15 H 17 NS 2 .C M H 10 O4) in 1 tablet.
Assay Weigh accurately and powder not less than 20
Tipepidine Hibenzate Tablets. Weigh accurately a portion of
the powder, equivalent to about 22 mg of tipepidine hiben-
zate (C 15 H 17 NS2.C 14 H 10 O4), add 10 mL of diluted acetic acid
(100) (1 in 2) and 30 mL of methanol, and warm for 10
minutes with occasional shaking. After cooling, add diluted
methanol (1 in 2) to make exactly 50 mL, and filter. Discard
the first 10 mL of the filtrate, pipet the subsequent 5 mL, add
exactly 5 mL of the internal standard solution, then add
diluted methanol (1 in 2) to make 25 mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 22 mg of tipepidine hibenzate for assay, previously
dried in a desiccator (in vacuum, phosphorus (V) oxide,
60°C) for 3 hours, dissolve in 10 mL of diluted acetic acid
(100) (1 in 2) and 30 mL of methanol, and add diluted
methanol (1 in 2) to make exactly 50 mL. Pipet 5 mL of this
solution, add exactly 5 mL of the internal standard solution,
then add diluted methanol (1 in 2) to make exactly 25 mL,
and use this solution as the standard solution. Perform the
test with 20 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of tipepidine to that of the inter-
nal standard, respectively.
Amount (mg) of tipepidine hibenzate
(C 15 H 17 NS 2 .C 14 H 10 O 4 )
= W s x(Q T /Q s )
W s : Amount (mg) of tipepidine hibenzate for assay
Internal standard solution — A solution of dibucaine
hydrochloride in methanol (1 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of a solution of sodium lauryl
sulfate in diluted phosphoric acid (1 in 1000) (1 in 500),
acetonitrile and 2-propanol (3:2:1).
Flow rate: Adjust the flow rate so that the retention time of
tipepidine is about 7 minutes.
System suitability —
System performance: When the procedure is run with 20
/xL of the standard solution under the above operating condi-
tions, tipepidine and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of tipepidine to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Titanium Oxide
Ti0 2 : 79.87
Titanium Oxide, when dried, contains not less than
98.5% of Ti0 2 .
Description Titanium Oxide occurs as a white powder. It is
odorless and tasteless.
It is practically insoluble in water, in ethanol (99.5) and in
diethyl ether.
It dissolves in hot sulfuric acid and in hydrofluoric acid,
and does not dissolve in hydrochloric acid, in nitric acid and
in dilute sulfuric acid.
When fused by heating with potassium hydrogen sulfate,
with potassium hydroxide, or with potassium carbonate, it
changes to soluble salts.
Shake 1 g of Titanium Oxide with 10 mL of water: the mix-
ture is neutral.
Identification Heat 0.5 g of Titanium Oxide with 5 mL of
sulfuric acid until white fumes are evolved, cool, add cauti-
ously water to make 100 mL, and filter. To 5 mL of the
filtrate add 2 to 3 drops of hydrogen peroxide TS: a yellow-
red color develops.
Purity (1) Lead — Place 1 .0 g of Titanium Oxide in a plati-
num crucible, add 10.0 g of potassium hydrogen sulfate, heat
gently with caution at the beginning, then raise the tempera-
ture gradually, and heat strongly with occasional shaking un-
til the contents fuse to yield a clear liquid. Cool, add 30 mL
of a solution of diammonium hydrogen citrate (9 in 20) and
50 mL of water, dissolve by heating on a water bath, cool,
add water to make 100 mL, and use this solution as the sam-
ple stock solution. Take 25 mL of the solution to a separator,
add 10 mL of a solution of ammonium sulfate (2 in 5) and 5
drops of thymol blue TS, neutralize with ammonia TS, and
add 2.5 mL of ammonia TS. To this solution add exactly 20
mL of a solution of dithizone in w-butyl acetate (1 in 500),
shake for 10 minutes, and use this w-butyl acetate solution as
the sample solution. Separately, place 6.0 mL of Standard
Lead Solution in a platinum crucible, proceed as directed in
JPXV
Official Monographs / Tizanidine Hydrochloride 1179
the sample solution, and use this solution as the standard so-
lution. Determine the absorbances of the sample solution and
standard solution as directed under Atomic Absorption Spec-
trophotometry <2.23> according to the following conditions:
the absorbance of the sample solution is smaller than that of
the standard solution (not more than 60 ppm).
Gas: Combustible gas — Acetylene gas or hydrogen gas
Supporting gas — Air
Lamp: Lead hollow-cathode lamp
Wavelength: 283.3 nm
(2) Arsenic <1.11> — Perform the test with 20 mL of the
sample stock solution obtained in (1) as the test solution: the
stain is not deeper than the following standard stain.
Standard stain: Proceed in the same manner without
Titanium Oxide, transfer 20 mL of the obtained solution to a
generator bottle, add 2.0 mL of Standard Arsenic Solution,
and proceed in the same manner as the test with the test solu-
tion (not more than 10 ppm).
(3) Water-soluble substances — Shake thoroughly 4.0 g of
Titanium Oxide with 50 mL of water, and allow to stand
overnight. Shake thoroughly with 2 mL of ammonium chlo-
ride TS, add further 2 mL of ammonium chloride TS if neces-
sary, and allow titanium oxide to settle. Add water to make
200 mL, shake thoroughly, and filter through double filter
paper. Discard the first 10 mL of the filtrate, evaporate 100
mL of the clear filtrate on a water bath, and heat strongly at
650°C to constant mass: the mass of the residue is not more
than 5.0 mg.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Assay Weigh accurately about 0.2 g of Titanium Oxide,
previously dried, transfer to a crucible, and add 3 g of potas-
sium disulfate. Cover, and heat gently at first, gradually raise
the temperature, and then heat the fused contents for 30
minutes. Continue heating for 30 minutes at a higher temper-
ature to make the fused mixture a deep yellow-red, almost
clear liquid. Cool, transfer the contents of the crucible to a
250-mL beaker, wash the crucible with a mixture of 75 mL of
water and 2.5 mL of sulfuric acid into the beaker, and heat
on a water bath until the solution becomes almost clear. Dis-
solve 2 g of L-tartaric acid in the solution, add 2 to 3 drops of
bromothymol blue TS, neutralize with ammonia TS, and
acidify with 1 to 2 mL of diluted sulfuric acid (1 in 2). Pass
hydrogen sulfide sufficiently through the solution, add 30 mL
of ammonia TS, again saturate the solution with hydrogen
sulfide, allow to stand for 10 minutes, and filter. Wash the
precipitate on the filter paper with ten 25-mL portions of a
mixture of ammonium L-tartrate solution (1 in 100) and am-
monium sulfide TS (9:1). When the precipitate is filtered and
washed, prevent iron (II) sulfide from oxidation by filling the
solution on the filter paper. Combine the filtrate and the
washings, add 40 mL of diluted sulfuric acid (1 in 2), and boil
to expel hydrogen sulfide. Cool, and dilute with water to
make 400 mL. Add gradually 40 mL of cupferron TS to the
solution with stirring, and allow to stand. After sedimenta-
tion of a yellow precipitate, add again cupferron TS until a
white precipitate is produced. Filter by slight suction using
quantitative filter paper, wash with twenty portions of diluted
hydrochloric acid (1 in 10), and remove water by stronger
suction at the last washing. Dry the precipitate together with
the filter paper at 70°C, transfer to a tared crucible, and heat
very gently at first, and raise the temperature gradually after
smoke stops evolving. Heat strongly between 900°C and
950°C to constant mass, cool, and weigh as titanium oxide
(Ti0 2 ).
Containers and storage Containers — Well-closed contain-
Tizanidine Hydrochloride
^Hf=>5 >±£Sgi£
N
^
■ HCI
N
- M
HN.
C 9 H 8 C1N 5 S.HC1: 290.17
5-Chloro-7V-(4,5-dihydro-l//-imidazol-2-yl)-
2, 1 ,3-benzothiadiazole-4-amine monohydrochloride
[64461-82-1]
Tizanidine Hydrochloride, when dried, contains
not less than 99.0% and not more than 101.0% of
C 9 H 8 C1N 5 S.HC1.
Description Tizanidine Hydrochloride occurs as a white to
light yellowish white crystalline powder.
It is soluble in water, slightly soluble in ethanol (99.5), and
practically insoluble in acetic anhydride and in acetic acid
(100).
Melting point: about 290°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Tizanidine Hydrochloride in diluted 1 mol/L am-
monia TS (1 in 10) (1 in 125,000) as directed under Ultra-
violet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Tizanidine Hydrochloride as directed in the potassium chlo-
ride disk method under Infrared Spectrophotometry <2.25>,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) A solution of Tizanidine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tizanidine Hydrochloride according to Method 3, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 60 mg of Tizanidine
Hydrochloride in 10 mL of a mixture of water and acetoni-
trile (17:3), and use this solution as the sample solution. Pipet
1 mL of the sample solution, add the mixture of water and
acetonitrile (17:3) to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/xL of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than
tizanidine is not larger than 1/5 times the peak area of tizani-
dine with the standard solution.
1180 Tobramycin / Official Monographs
JP XV
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm for about 3 minutes after sample injec-
tion and 318 nm subsequently).
Column: A stainless steel column 4.6 mm in inside
diameter and 12.5 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /xm in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of water and formic acid
(200:1), adjusted to pH 8.5 with ammonia water (28).
Mobile phase B: A mixture of acetonitrile and the mobile
phase A (4:1).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
ers.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0- 10
81 -> 68
19- 32
10- 13
68
32
13-26
68 -> 10
32- 90
26-28
10
90
Flow rate: Adjust the flow rate so that the retention time of
tizanidine is about 7 minute.
Time span of measurement: About 4 times as long as the
retention time of tizanidine beginning after the solvent peak.
System suitability —
Test for required detectability: Measure exactly 2 mL of
the standard solution, and add the mixture of water and
acetonitrile (17:3) to make exactly 10 mL. Confirm that the
peak area of tizanidine obtained with 10 ,aL of this solution is
equivalent to 14 to 26% of that with 10 /uL of the standard so-
lution.
System performance: Dissolve 2 mg each of Tizanidine
Hydrochloride and p-toluenesulfonic acid monohydrate in
100 mL of the mixture of water and acetonitrile (17:3). When
the procedure is run with 10 juL of this solution under the
above operating conditions, p-toluenesulfonic acid and
tizanidine are eluted in this order with the resolution between
these peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tizanidine is not more than 2.0%.
Loss on drying <2.41>
hours).
Not more than 0.2% (1 g, 105°C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Tizanidine
Hydrochloride, previously dried, dissolve in 60 mL of a
mixture of acetic anhydride and acetic acid (100) (7:3) with
the aid of warming. After cooling, titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 29.02 mg of C 9 H 8 C1N 5 S.HC1
Tobramycin
h7"7V'f'»
C 18 H 37 N 5 9 : 467.51
3-Amino-3-deoxy-a-D-glucopyranosyl-(l — 6)-
[2,6-diamino-2,3,6-trideoxy-a-D-n7j>o-hexopyranosyl-
(1 -4)]-2-deoxy-D-streptamine [32986-56-4]
Tobramycin is an aminoglycoside substance having
antibacterial activity produced by the growth of Strep-
tomyces tenebrarius.
It contains not less than 900 Lig (potency) and not
more than 1060 Lig (potency) per mg, calculated on the
anhydrous basis. The potency of Tobramycin is ex-
pressed as mass (potency) of tobramycin (Qgt^NsOg).
Description Tobramycin occurs as a white to pale yellowish
white powder.
It is very soluble in water, freely soluble in formamide,
slightly soluble in methanol, and very slightly soluble in
ethanol (95).
It is hygroscopic.
Identification (1) Determine the spectrum of a solution of
Tobramycin in heavy water for nuclear magnetic resonance
spectroscopy (1 in 125) as directed under Nuclear Magnetic
Resonance Spectroscopy <2.21> ('H), using sodium 3-
trimethylsilylpropanesulfonate for nuclear magnetic
resonance spectroscopy as an internal reference compound: it
exhibits a double signal A at around <55.1 ppm, a multiple
signal B between <52.6 ppm and <54.0 ppm, and a multiple sig-
nal C between <5 1 .0 ppm and <52.1 ppm. The ratio of the in-
tegrated intensity of these signals, A:B:C, is about 1:8:2.
(2) Dissolve 10 mg each of Tobramycin and Tobramycin
Reference Standard in 1 mL of water, and use these solutions
as the sample solution and the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 4,mL of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ammo-
nia TS, 1-butanol and methanol (5:5:2) to a distance of about
10 cm, and air-dry the plate. Spray evenly ninhydrin TS on
the plate, and heat at 100°C for 5 minutes: the Rf values of
the principal spots obtained from the sample solution and the
standard solution are the same.
Containers and storage Containers— Well-closed contain- Optical rotation <2.49> [ a ]^°: +138- +148° (1 g calculated
JPXV
Official Monographs / Tocopherol 1181
on the anhydrous basis, water, 25 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.10
g of Tobramycin in 10 mL of water is between 9.5 and 11.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Tobramycin in 10 mL of water: the solution is clear and
colorless to pale yellow.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Tobramycin according to Method 2, and perform the test.
Prepare the control solution with 3.0 mL of Standard Lead
Solution (not more than 30 ppm).
(3) Related substances — Dissolve 80 mg of Tobramycin
in 10 mL of diluted ammonia solution (28) (1 in 250), and use
this solution as the sample solution. Pipet 1 mL of the sample
solution, add diluted ammonia solution (28) (1 in 250) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /xL of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ammonia solution (28), ethanol (95) and 2-butanone
(1:1:1) to a distance of about 10 cm, air-dry the plate, then
further dry at 110°C for 10 minutes. Immediately spray even-
ly a mixture of water and sodium hypochlorite TS (4:1) on
the plate, air-dry the plate, then spray potassium iodide-
starch TS on the plate: the spot other than the principal spot
from the sample solution is not more intense than the spot
from the standard solution.
Water <2.48> Not more than 11.0% (0.1 g, volumetric titra-
tion, direct titration). Use a mixture of formamide for water
determination and methanol for water determination (3:1) in-
stead of methanol for water determination.
Residue on ignition <2.44> Not more than 1.0% (0.5 g).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism — Bacillus subtilis ATCC 6633
(ii) Culture medium — Use the medium i in 1) under (1)
Agar media for seed and base layer.
(iii) Standard solutions — Weigh accurately an amount of
Tobramycin Reference Standard, equivalent to about 25 mg
(potency), dissolve in 0.1 mol/L phosphate buffer solution,
pH 8.0 to make exactly 25 mL, and use this solution as the
standard stock solution. Keep the standard stock solution be-
tween 5°C and 15°C, and use within 30 days. Take exactly a
suitable amount of the standard stock solution before use,
add 0.1 mol/L phosphate buffer solution, pH 8.0 to make so-
lutions so that each mL contains 8 /xg (potency) and 2,ug
(potency), and use these solutions as the high concentration
standard solution and low concentration standard solution,
respectively.
(iv) Sample solutions — Weigh accurately an amount of
Tobramycin, equivalent to about 25 mg (potency), and dis-
solve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make
exactly 25 mL. Take exactly a suitable amount of this solu-
tion, add 0.1 mol/L phosphate buffer solution, pH 8.0 to
make solutions so that each mL contains 8 fig (potency) and 2
H% (potency), and use these solutions as the high concentra-
tion sample solution and low concentration sample solution,
respectively.
Containers and storage Containers — Tight containers.
Tocopherol
Vitamin E
rf/-a-Tocopherol
h=l7xP-JU
Crl a
C 29 H 50 O 2 : 430.71
2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-
6-ol [10191-41-0]
Tocopherol contains not less than 96.0% and not
more than 102.0% of C 29 H 50 O 2 .
Description Tocopherol is a clear, yellow to red-brown, vis-
cous liquid. It is odorless.
It is miscible with ethanol (99.5), with acetone, with chlo-
roform, with diethyl ether and with vegetable oils.
It is freely soluble in ethanol (95), and practically insoluble
in water.
It is optically inactive.
It is oxidized by air and light, and acquires a dark red
color.
Identification (1) Dissolve 0.01 g of Tocopherol in 10 mL
of ethanol (99.5), add 2 mL of nitric acid, and heat at 75°C
for 15 minutes: a red to orange color develops.
(2) Determine the infrared absorption spectrum of
Tocopherol as directed in the liquid film method under In-
frared Spectrophotometry <2.25>, and compare the spectrum
with the Reference Spectrum or the spectrum of Tocopherol
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
Absorbance <2.24> E\ 0/ ° m (292 nm): 71.0-76.0 (10 mg,
ethanol (99.5), 200 mL).
Refractive index <2.45> n™: 1.503 - 1.507
Specific gravity <2.56> df : 0.947 - 0.955
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Tocopherol in 10 mL of ethanol (99.5): the solution is
clear and has no more color than Matching Fluid C.
(2) Heavy metals <1.07> — Proceed with 1.0 g of
Tocopherol according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
Assay Dissolve about 50 mg each of Tocopherol and To-
copherol Reference Standard, accurately weighed, in ethanol
(99.5) to make exactly 50 mL, and use these solutions as the
sample solution and standard solution. Perform the test with
exactly 20 /xL each of these solutions as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak heights, H T and H s , of
tocopherol in the sample solution and standard solution.
Amount (mg) of C29H50O2 = W s x (H T /H S )
1182 Tocopherol Acetate / Official Monographs
JP XV
W s : Amount (mg) of Tocopherol Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 292 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xL in particle
diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of methanol and water (49:1).
Flow rate: Adjust the flow rate so that the retention time of
tocopherol is about 10 minutes.
System suitability —
System performance: Dissolve 0.05 g each of Tocopherol
and tocopherol acetate in 50 mL of ethanol (99.5). When the
procedure is run with 20 fiL of this solution under the above
operating conditions, tocopherol and tocopherol acetate are
eluted in this order with the resolution between these peaks
being not less than 2.6.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak height-
s of tocopherol is not more than 0.8%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and well-filled, or under nitro-
gen atmosphere.
Tocopherol Acetate
Vitamin E Acetate
e?/-a-Tocopherol Acetate
hH7iP-Jl,iii7 l fJI,
C 31 H 52 3 : 472.74
2, 5 ,7 , 8-Tetramethyl-2-(4,8 , 1 2-trimethyltridecyl)chroman-
6-yl acetate [7695-91-2]
Tocopherol Acetate contains not less than 96.0%
and not more than 102.0% of C 31 H520 3 .
Description Tocopherol Acetate is a clear, colorless or yel-
low, viscous and odorless liquid.
It is miscible with ethanol (99.5), with acetone, with chlo-
roform, with diethyl ether, with hexane and with fixed oils.
It is freely soluble in ethanol (95), and practically insoluble
in water.
It is optically inactive.
It is affected by air and light.
Identification (1) Dissolve 0.05 g of Tocopherol Acetate
in 10 mL of ethanol (99.5), add 2 mL of nitric acid, and heat
at 75°C for 15 minutes: a red to orange color is produced.
(2) Determine the infrared absorption spectrum of
Tocopherol Acetate as directed in the liquid film method un-
der Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum or the spectrum of
Tocopherol Acetate Reference Standard: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Absorbance <2.24> E\ v ° m (284 nm): 41.0-45.0 (10 mg,
ethanol (99.5), 100 mL).
Refractive index <2.45> n™: 1.494 - 1.499
Specific gravity <2.56> df : 0.952 - 0.966
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Tocopherol Acetate in 10 mL of ethanol (99.5): the solu-
tion is clear, and has no more color than the following con-
trol solution.
Control solution: To 0.5 mL of Ferric Chloride Colorimet-
ric Stock Solution add 0.5 mol/L hydrochloric acid TS to
make 100 mL.
(2) Heavy metals <1.07> — Carbonize 1.0 g of Tocopherol
Acetate by gentle heating. Cool, add 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10), and
ignite the ethanol to burn. Cool, add 1 mL of sulfuric acid,
proceed according to Method 4, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (20 ppm).
(3) a-Tocopherol — Dissolve 0.10 g of Tocopherol
Acetate in exactly 10 mL of hexane, and use this solution as
the sample solution. Separately, dissolve 50 mg of
Tocopherol Reference Standard in hexane to make exactly
100 mL. Pipet 1 mL of this solution, add hexane to make ex-
actly 10 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of toluene and acetic acid (100) (19:1) to a distance of about
10 cm, and air-dry the plate. Spray evenly a solution of iron
(III) chloride hexahydrate in ethanol (99.5) (1 in 500) on the
plate, then spray evenly a solution of a,a'-dipyridyl in
ethanol (99.5) (1 in 200) on the same plate, and allow to stand
for 2 to 3 minutes: the spot from the sample solution cor-
responding to that from the standard solution is not larger
than and not more intense than the spot from the standard
solution.
Assay Dissolve 50 mg each of Tocopherol Acetate and
Tocopherol Acetate Reference Standard, accurately weighed,
in ethanol (99.5) to make exactly 50 mL, and use these solu-
tions as the sample solution and standard solution. Perform
the test with exactly 20 /xh each of these solutions as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak heights, H T and
H s , of tocopherol acetate in the sample solution and the stan-
dard solution, respectively.
Amount (mg) of C 31 H 52 3 = W s x (H T /H S )
W s : Amount (mg) of Tocopherol Acetate Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 284 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
JPXV
Official Monographs / Tocopherol Calcium Succinate 1183
silica gel (5 jum in particle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of methanol and water (49:1).
Flow rate: Adjust the flow rate so that the retention time of
tocopherol acetate is about 12 minutes.
System suitability —
System performance: Dissolve 0.05 g each of Tocopherol
Acetate and tocopherol in 50 mL of ethanol (99.5). When the
procedure is run with 20 fiL of this solution under the above
operating conditions, tocopherol and tocopherol acetate are
eluted in this order with the resolution between these peaks
being not less than 2.6.
System repeatability: When the test is repeated 5 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak height-
s of tocopherol acetate is not more than 0.8%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tocopherol Calcium Succinate
Vitamin E Calcium Succinate
---\A
Q;C
C 66 H 106 CaO 10 : 1099.62
Monocalcium bis {3-[2, 5,7, 8-tetramethyl-2-(4, 8,12-
trimethyltridecyl)chroman-6-yloxycarbonyl]propanoate}
[14638-18-7]
Tocopherol Calcium Succinate, when dried, con-
tains not less than 96.0% and not more than 102.0% of
C66Hi06CaO 10 .
Description Tocopherol Calcium Succinate occurs as a
white to yellowish white powder. It is odorless.
It is freely soluble in chloroform and in carbon tetrachlo-
ride, and practically insoluble in water, in ethanol (95) and in
acetone.
Shake 1 g of Tocopherol Calcium Succinate with 7 mL of
acetic acid (100): it dissolves, and produces a turbidity after
being allowed to stand for a while.
It dissolves in acetic acid (100).
It is optically inactive.
Identification (1) Dissolve 0.05 g of Tocopherol Calcium
Succinate in 1 mL of glacial aetic acid, add 9 mL of ethanol
(99.5), and mix. To this solution add 2 mL of fuming nitric
acid, and heat at 75°C for 15 minutes: a red to orange color
develops.
(2) Dissolve 0.08 g of Tocopherol Calcium Succinate,
previously dried, in 0.2 mL of carbon tetrachloride. Deter-
mine the infrared absorption spectrum of the solution as
directed in the liquid film method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) Dissolve 5 g of Tocopherol Calcium Succinate in 30
mL of chloroform, add 10 mL of hydrochloric acid, shake
for 10 minutes, then draw off the water layer, and neutralize
with ammonia TS: the solution responds to the Qualitative
Tests <1.09> for calcium salt.
Absorbance <2.24> E 1 ^ (286 nm): 36.0 - 40.0 (10 mg, chlo-
roform, 100 mL).
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Tocopherol Calcium Succinate in 10 mL of chloroform:
the solution is clear, and has no more color than the follow-
ing control solution.
Control solution: To 0.5 mL of Ferric Chloride Colorimet-
ric Stock Solution add 0.5 mol/L hydrochloric acid TS to
make 100 mL.
(2) Alkalinity — To 0.20 g of Tocopherol Calcium Suc-
cinate add 10 mL of diethyl ether, 2 mL of water, 1 drop of
phenolphthalein TS and 0.10 mL of 0.1 mol/L hydrochloric
acid VS, and shake: no red color develops in the water layer.
(3) Chloride <1.03>— Dissolve 0.10 g of Tocopherol Cal-
cium Succinate in 4 mL of acetic acid (100), add 20 mL of
water and 50 mL of diethyl ether, shake thoroughly, and col-
lect the water layer. To the diethyl ether layer add 10 mL of
water, shake, and collect the water layer. Combine the water
layers, add 6 mL of dilute nitric acid and water to make 50
mL, and perform the test using this solution as the test solu-
tion. Prepare the control solution in the same manner using
0.60 mL of 0.01 mol/L hydrochloric acid VS in place of
Tocopherol Calcium Succinate (not more than 0.212%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of
Tocopherol Calcium Succinate according to Method 4, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Tocopherol Calcium Succinate according to Method 3,
and perform the test (not more than 2 ppm).
(6) a-Tocopherol — Dissolve 0.10 g of Tocopherol Calci-
um Succinate in exactly 10 mL of chloroform, and use this
solution as the sample solution. Separately, dissolve 50 mg of
Tocopherol Reference Standard in chloroform to make ex-
actly 100 mL. Pipet 1 mL of this solution, add chloroform to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /iL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of toluene and acetic acid (100) (19:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly a solution of
iron (III) chloride hexahydrate in ethanol (99.5) (1 in 500) on
the plate, then spray evenly a solution of a-a'-dipyridyl in
ethanol (99.5) (1 in 200) on the same plate, and allow to stand
for 2 to 3 minutes: the spots from the sample solution cor-
responding to the spots from the standard solution is not
larger than and not more intense than the spots from the
standard solution.
Loss on drying <2.41> Not more than 2.0% (1 g, in vacuum,
phosphorus (V) oxide, 24 hours).
Assay Weigh accurately about 50 mg each of Tocopherol
Calcium Succinate and Tocopherol Succinate Reference
Standard, previously dried, dissolve in a mixture of ethanol
1184 Tocopherol Nicotinate / Official Monographs
JP XV
(99.5) and diluted acetic acid (100) (1 in 5) (9:1) to make ex-
actly 50 mL, and use these solutions as the sample solution
and the standard solution. Pipet exactly 20 /uL each of the
sample solution and standard solution, and perform the test
as directed under Liquid Chromatography <2.01> according
to the following operating conditions. Determine the peak
heights, H T and H s , of tocopherol succinate in these solu-
tions, respectively.
Amount (mg) C 66 H 10 6CaO 10
= W s x(H T /H s )x 1.0358
W s : Amount (mg) of Tocopherol Succinate Reference
Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 284 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 fiL in particle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of methanol, water and acetic
acid (100) (97:2:1).
Flow rate: Adjust the flow rate so that the retention time of
tocopherol succinate is about 8 minutes.
Selection of column: Dissolve 0.05 g each of tocopherol
succinate and tocopherol in 50 mL of a mixture of ethanol
(99.5) and diluted acetic acid (100) (1 in 5) (9:1). Proceed with
20 /xL of this solution under the above operating conditions,
and calculate the resolution. Use a column giving elution of
tocopherol succinate and tocopherol in this order with the
resolution between these peaks being not less than 2.0.
System repeatability: Repeat the test five times with the
standard solution under the above operating conditions: the
relative standard deviation of the peak height of tocopherol
succinate is not more than 0.8%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tocopherol Nicotinate
Vitamin E Nicotinate
rf/-a-Tocopherol Nicotinate
CH :!
Xd
CH 3
C 3 5H 5 3N0 3 : 535.80
2, 5 ,7 , 8-Tetramethyl-2-(4,8 , 1 2-trimethyltridecyl)chroman-
6-yl nicotinate [51898-34-1]
Tocopherol Nicotinate contains not less than 96.0%
of nicotinic acid cW-a-tocopherol (C35H53NO3).
Description Tocopherol Nicotinate occurs as a yellow to
orange-yellow liquid or solid.
It is freely soluble in ethanol (99.5), and practically insolu-
ble in water.
A solution of Tocopherol Nicotinate in ethanol (99.5)
(1 in 10) shows no optical rotation.
It is affected by light.
Identification (1) Determine the absorption spectrum of a
solution of Tocopherol Nicotinate in ethanol (99.5) (1 in
20,000) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare the spectrum with the Reference
Spectrum or the spectrum of a solution of Tocopherol
Nicotinate Reference Standard prepared in the same manner
as the sample solution: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(2) Determine the infrared spectrum of Tocopherol
Nicotinate, if necessary melt by warming, as directed in the
liquid film method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of Tocopherol Nicotinate Reference
Standard: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tocopherol Nicotinate according to Method 4, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Tocopherol Nicotinate according to Method 4, and per-
form the test (not more than 2 ppm).
(3) Related substances — Dissolve 0.05 g of Tocopherol
Nicotinate in 50 mL of ethanol (99.5), and use this solution as
the sample solution. Pipet 7 mL of this solution, add ethanol
(99.5) to make exactly 200 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /xh each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine each peak area of both solutions
by the automatic integration method: the total area of the
peaks other than tocopherol nicotinate from the sample solu-
tion is not larger than the peak area of tocopherol nicotinate
from the standard solution, and the area of a peak which has
a retention time 0.8 to 0.9 times that of tocopherol nicotinate
from the sample solution is not larger than 4/7 of the peak
area of tocopherol nicotinate from the standard solution.
Operating conditions —
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Assay.
Mobile phase: A mixture of methanol and water (19:1).
Flow rate: Adjust the flow rate so that the retention time of
tocopherol nicotinate is about 20 minutes.
Time span of measurement: About 1.5 times as long as the
retention time of tocopherol nicotinate beginning after the
solvent peak.
System suitability —
Test for required detection: To exactly 1 mL of the sample
solution add ethanol (99.5) to make exactly 100 mL, and use
this solution as the test solution for system suitability. Pipet 1
mL of the test solution for system suitability, add ethanol
(99.5) to make exactly 10 mL. Confirm that the peak area of
tocopherol nicotinate obtained from 10,mL of this solution is
equivalent to 7 to 13% of that of tocopherol nicotinate ob-
tained from 10 /uL of the test solution for system suitability.
System performance: Dissolve 0.05 g of Tocopherol
Nicotinate and 0.25 g of tocopherol in 100 mL of ethanol
JPXV
Official Monographs / Todralazine Hydrochloride Hydrate 1185
(99.5). When the procedure is run with 10 jxL of this solution
under the above operating conditions, tocopherol and
tocopherol nicotinate are eluted in this order with the resolu-
tion between these peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of tocopherol nicotinate is not more than 2.0%.
Assay Weigh accurately about 50 mg each of Tocopherol
Nicotinate and Tocopherol Nicotinate Reference Standard,
dissolve each in ethanol (99.5) to make exactly 50 mL, and
use these solutions as the sample solution and standard solu-
tion, respectively. Perform the test with exactly 5 /xL each of
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, and determine peak areas, A T and A s , of
tocopherol nicotinate of these solutions.
Amount (mg) of C35H53NO3
= W s x(A T /A s )
W s : Amount (mg) of Tocopherol Nicotinate
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 264 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: Methanol
Flow rate: Adjust the flow rate so that the retention time of
tocopherol nicotinate is about 10 minutes.
System suitability —
System performance: Dissolve 0.05 g of Tocopherol
Nicotinate and 0.25 g of tocopherol in 100 mL of ethanol
(99.5). When the procedure is run with 5 fiL of this solution
under the above operating conditions, tocopherol and
tocopherol nicotinate are eluted in this order with the resolu-
tion between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
5 nL of the standard solution under the above operating con-
ditions: the relative standard deviation of the peak areas of
tocopherol nicotinate is not more than 0.8%
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Todralazine Hydrochloride Hydrate
Ecarazine Hydrochloride
CnH 12 N 4 2 .HCl.H 2 0: 286.71
Ethyl 2-(phthalazin- 1 -yl)hydrazinecarboxylate
monohydrochloride monohydrate [3778-76-5, anhydride]
Todralazine Hydrochloride Hydrate contains not
less than 98.5% of todralazine hydrochloride
(C„H 12 N 4 2 .HC1: 268.70), calculated on the anhy-
drous basis.
Description Todralazine Hydrochloride Hydrate occurs as
white crystals or crystalline powder. It has a slight, charac-
teristic odor, and has a bitter taste.
It is very soluble in formic acid, freely soluble in methanol,
soluble in water, sparingly soluble in ethanol (95), and practi-
cally insoluble in diethyl ether.
The pH of a solution of Todralazine Hydrochloride Hy-
drate (1 in 200) is between 3.0 and 4.0.
Identification (1) To 2 mL of a solution of Todralazine
Hydrochloride Hydrate (1 in 200) add 5 mL of silver nitrate-
ammonia TS: the solution becomes turbid, and a black
precipitate is formed.
(2) Determine the absorption spectrum of a solution of
Todralazine Hydrochloride Hydrate in 0.1 mol/L
hydrochloric acid TS (3 in 100,000) as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Todralazine Hydrochloride Hydrate as directed in the potas-
sium chloride disk method under the Infrared Spectrophoto-
metry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(4) A solution of Todralazine Hydrochloride Hydrate (1
in 50) responds to the Qualitative Tests <1.09> (1) for chlo-
ride.
Purity (1) Clarity and color of solution — Dissolve 0.30 g
of Todralazine Hydrochloride Hydrate in 10 mL of water:
the solution is clear and colorless to pale yellow.
(2) Sulfate <1.14>— Proceed the test with 2.0 g of
Todralazine Hydrochloride Hydrate. Prepare the control so-
lution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not
more than 0.012%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Todrala-
zine Hydrochloride Hydrate according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Todralazine Hydrochloride Hydrate according to Method
1, and perform the test (not more than 2 ppm).
(5) Related substances — Dissolve 50 mg of Todralazine
Hydrochloride Hydrate in 100 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
sample solution, add the mobile phase to make exactly 200
mL, and use this solution as the standard solution. Perform
the test with exactly 10 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine each
peak area of both solutions by the automatic integration
method: the total area of the peaks other than the peak of
todralazine from the sample solution is not larger than the
peak area of todralazine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
1186 Tofisopam / Official Monographs
JP XV
(wavelength: 240 nm).
Column: A stainless steel column 3.9 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (10 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1.10 g of sodium 1-heptane sul-
fonate in 1000 mL of diluted methanol (2 in 5). Adjust the
pH of the solution to between 3.0 and 3.5 with acetic acid
(100).
Flow rate: Adjust the flow rate so that the retention time of
todralazine is about 8 minutes.
Time span of measurement: About twice as long as the
retention time of todralazine beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 5 mL of the stan-
dard solution add the mobile phase to make exactly 25 mL.
Confirm that the peak area of todralazine obtained from 10
/uL of this solution is equivalent to 15 to 25% of that of
todralazine obtained from 10,mL of the standard solution.
System performance: Dissolve 5 mg each of Todralazine
Hydrochloride Hydrate and potassium biphthalate in 100 mL
of the mobile phase. When the procedure is run with 10 fiL of
this solution under the above operating conditions, phthalic
acid and todralazine are eluted in this order with the resolu-
tion between these peaks being not less than 8.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of todralazine is not more than 2.0%.
Water <2.48> 6.0 - 7.5% (0.5 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Todralazine
Hydrochloride, dissolve in 5 mL of formic acid, add 70 mL
of acetic anhydride, and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 26.87 mg of C„H 12 N 4 2 .HC1
Containers and storage Containers — Tight containers.
Tofisopam
r-7-f 7A°A
and enantiomGr
C 22 H 26 N 2 4 : 382.45
(5RS)-l -(3 ,4-Dimethoxyphenyl)-5-ethyl-7 , 8-dimethoxy-
4-methyl-5//-2,3-benzodiazepine [22345-47-7]
Tofisopam, when dried, contains not less than
98.0% of C 22 H 26 N 2 4 .
Description Tofisopam occurs as a pale yellowish white,
crystalline powder.
It is freely soluble in acetic acid (100), soluble in acetone,
sparingly soluble in ethanol (95), slightly soluble in diethyl
ether, and practically insoluble in water.
A solution of Tofisopam in ethanol (95) (1 in 100) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Tofisopam in ethanol (95) (1 in 100,000) as direct-
ed under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Tofisopam, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Melting point <2.60> 155 - 159°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tofisopam according to Method 2, and perform the test. Pre-
pare the control solution with 1.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Tofisopam according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.05 g of Tofisopam in
10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add acetone to make
exactly 25 mL, pipet 1 mL of this solution, add acetone to
make exactly 20 mL, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 //L each of the
sample solution and standard solution on a plate of silica gel
with fluorescent indicator for thin-layer chromatography.
Develop the plate with a mixture of ethyl acetate, acetone,
methanol and formic acid (24:12:2:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the principal
spot from the sample solution are not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Tofisopam, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 38.25 mg of C 22 H 26 N 2 4
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Tolazamide 1187
Tolazamide
h7+f* 5 K
w xr
H H
C 14 H 21 N 3 3 S: 311.40
7V-(Azepan- 1 -ylcarbamoyl)-4-methylbenzenesulf onamide
[1156-19-0]
Tolazamide, when dried, contains not less than
97.5% and not more than 102.0% of C 14 H 21 N30 3 S.
Description Tolazamide occurs as a white to pale yellow,
crystalline powder. It is odorless.
It is freely soluble in chloroform, soluble in acetone, slight-
ly soluble in ethanol (95) and in w-butylamine, and practically
insoluble in water and in diethyl ether.
Melting point: about 168°C (with decomposition).
Identification (1) Dissolve 0.02 g of Tolazamide in 5 mL
of water and 1 mL of M-butylamine, add 2 to 3 drops of cop-
per (II) sulfate TS, and shake well. Shake well this solution
with 5 mL of chloroform, and allow to stand: a green color
develops in the chloroform layer.
(2) Determine the absorption spectrum of a solution of
Tolazamide in ethanol (95) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Tolazamide Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of
Tolazamide, previously dried, as directed in the potassium
bromide disk method under the Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Tolazamide Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Tolazamide according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Tolazamide according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.20 g of Tolazamide in
acetone to make exactly 10 mL, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add ace-
tone to make exactly 200 mL, and use this solution as the
standard solution (1). Separately, dissolve 20 mg of p-tol-
uenesulf onamide in acetone to make exactly 200 mL, and use
this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /uL each of the sample solution
and standard solutions (1) and (2) on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of chloroform, methanol, cyclohexane and diluted ammonia
solution (28) (10 in 11) (200:100:60:23) to a distance of about
12 cm, and air-dry the plate. Heat the plate at 110°C for 10
minutes, and immediately expose to chlorine for 2 minutes.
Expose the plate to cold wind until a very pale blue color de-
velops when 1 drop of potassium iodide-starch TS is placed
on a site below the starting line on the plate. Spray evenly
potassium iodide-starch TS on the plate: the spot from the
sample solution corresponding to the spot from the standard
solution (2) is not more intense than the spot from the stan-
dard solution (2), and the spots other than the principal and
above spots from the sample solution are not more intense
than the spot from the standard solution (1).
(4) yV-Aminohexamethyleneimine — To 0.50 g of
Tolazamide add 2.0 mL of acetone, stopper the flask tightly,
shake vigorously for 15 minutes. Add 8.0 mL of disodium
hydrogenphosphate-citric acid buffer solution, pH 5.4,
shake, allow to stand for 15 minutes, and filter. To the
filtrate add 1.0 mL of trisodium ferrous pentacyanoamine
TS, and shake: the color developing within 30 minutes is not
deeper than that of the following control solution.
Control solution: Dissolve 0.125 g of iV-aminohex-
amethyleneimine in acetone to make exactly 100 mL. Pipet 1
mL of this solution, and add acetone to make exactly 100
mL. To 2.0 mL of this solution add 8.0 mL of disodium
hydrogenphosphate-citric acid buffer solution, pH 5.4,
shake, and proceed in the same manner.
Loss on drying <2.41> Not more 0.5% (1 g, in vacuum,
60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 30 mg each of Tolazamide
and Tolazamide Reference Standard, previously dried, dis-
solve each in 10 mL of the internal standard solution, and use
these solutions as the sample solution and standard solution,
respectively. Perform the test with 10 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, g T and Q s , of the peak area of
tolazamide to that of the internal standard, respectively.
Amount (mg) of Q4H21N3O3S
= ^ s x(e T /es)
W s : Amount (mg) of Tolazamide Reference Standard
Internal standard solution — A solution of tolbutamide in
ethanol-free chloroform (3 in 2000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 30 cm in length, packed with silica gel for liquid
chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of hexane, water-saturated
hexane, tetrahydrofuran, ethanol (95) and acetic acid (100)
(475:475:20:15:9).
Flow rate: Adjust the flow rate so that the retention time of
tolazamide is about 12 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the internal standard and tolazamide are eluted in this
1188 Tolbutamide / Official Monographs
JP XV
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak area of tolazamide to that of the internal standard is not
more than 1.0%.
Containers and storage Containers — Well-closed contain-
ers.
Tolbutamide
HnC
o o o
X X ,
N N
H H
CHj
C 12 H 18 N 2 3 S: 270.35
A r -(Butylcarbamoyl)-4-methylbenzenesulfonamide
[64-77-7]
Tolbutamide, when dried, contains not less than
99.0% of C 12 H 18 N 2 3 S.
Description Tolbutamide occurs as white crystals or crystal-
line powder. It is odorless or has a slight, characteristic odor.
It is tasteless.
It is soluble in ethanol (95), slightly soluble in diethyl ether,
and practically insoluble in water.
Identification (1) Boil 0.2 g of Tolbutamide with 8 mL of
diluted sulfuric acid (1 in 3) under a reflux condenser for 30
minutes. Cool the solution in ice water, collect the precipitat-
ed crystals, recrystallize from water, and dry at 105°C for 3
hours: the crystals melt <2.60> between 135°C and 139°C.
(2) Render the filtrate obtained in (1) alkaline with about
20 mL of a solution of sodium hydroxide (1 in 5), and heat:
an ammonia-like odor is perceptible.
Melting point <2.60> 126 - 132°C
Purity (1) Acidity — Warm 3.0 g of Tolbutamide with 150
mL of water at 70°C for 5 minutes, allow to stand for 1 hour
in ice water, and filter. To 25 mL of the filtrate add 2 drops
of methyl red TS and 0.20 mL of 0.1 mol/L sodium
hydroxide VS: a yellow color develops.
(2) Chloride <1.03>— To 40 mL of the filtrate obtained in
(1) add 6 mL of dilute nitric acid and water to make 50 mL.
Perform the test using this solution as the test solution. Pre-
pare the control solution with 0.25 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.011%).
(3) Sulfate <1.14>— To 40 mL of the filtrate obtained in
(1) add 1 mL of dilute hydrochloric acid and water to make
50 mL. Perform the test using this solution as the test solu-
tion. Prepare the control solution with 0.35 mL of 0.005 mol
/L sulfuric acid VS (not more than 0.021%).
(4) Heavy metals <1.07> — Proceed with 2.0 g of Tol-
butamide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tolbutamide, previ-
ously dried, and dissolve in 30 mL of neutralized ethanol.
Add 20 mL of water, and titrate <2.50> with 0.1 mol/L sodi-
um hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 27.04 mg of C 12 H 18 N 2 3 S
Containers and storage Containers — Well-closed contain-
ers.
Tolbutamide Tablets
Tolbutamide Tablets contain not less than 95% and
not more than 105% of the labeled amount of tol-
butamide (C 12 H 18 N 2 3 S: 270.35).
Method of preparation
with Tolbutamide.
Prepare as directed under Tablets,
Identification Shake a quantity of powdered Tolbutamide
Tablets, equivalent to 0.5 g of Tolbutamide according to the
labeled amount, with 50 mL of chloroform, filter, and
evaporate the filtrate to dryness. Proceed with the residue as
directed in the Identification under Tolbutamide.
Dissolution <6.10> Perform the test according to the fol-
lowing method: it meets the requirement.
Take 1 tablet of Tolbutamide Tablets at 100 revolutions
per minute according to the using 900 mL of phosphate
buffer solution, pH 7.4, as the dissolution medium. Take 20
mL or more of the dissolved solution 30 minutes after the
start of the test, and filter through a membrane filter (less
than 0.8 jxm. in pore size). Discard the first 10 mL of the
filtrate, pipet the subsequent FmL, add water to make exact-
ly K'mL of a solution containing about 10 /xg of tol-
butamide (C 12 H 18 N 2 3 S) per mL according to the labeled
amount, and use this solution as the sample solution.
Separately, weigh accurately about 50 mg of Tolbutamide
Reference Standard, previously dried at 105°C for 3 hours,
dissolve in 10 mL of methanol, and add phosphate buffer so-
lution, pH 7.4, to make exactly 100 mL. Pipet 2 mL of this
solution, add water to make exactly 100 mL, and use this so-
lution as standard solution. Determine the absorbances, A T
and A s , of the sample solution and the standard solution at
226 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, respectively. The dissolution rate of Tol-
butamide Tablets after 30 minutes should be not less than
80%.
Dissolution rate (%) to labeled amount
of tolbutamide (C 12 H, 8 N 2 3 S)
= W s x (A T /A S ) x (V'/V) x (90/Q x (1/5)
W s : Amount (mg) of Tolbutamide Reference Standard
C: Labeled amount (mg) of tolbutamide (C I2 H 18 N 2 3 S) per
tablet
Assay Weigh accurately and powder not less than 20 Tol-
butamide Tablets. Weigh accurately a portion of the powder,
JPXV
Official Monographs / Tolnaftate Solution 1189
equivalent to about 0.5 g of tolbutamide (C 12 H 18 N 2 03S), dis-
solve in 50 mL of neutralized ethanol, add 25 mL of water,
and titrate <2.50> with 0.1 mol/L sodium hydroxide VS (indi-
cator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 27.04 mg of C 12 H 18 N 2 3 S
Containers and storage Containers — Well-closed contain-
ers.
Tolnaftate
HkJ-7*- h
C 19 H 17 NOS: 307.41
O-Naphthalen-2-yl jV-methyl-./V-(3-
methylphenyl)thiocarbamate [2398-96-1 ]
Tolnaftate, when dried,
98.0% of C 19 H 17 NOS.
contains not less than
Description Tolnaftate occurs as a white powder. It is odor-
less.
It is freely soluble in chloroform, sparingly soluble in
diethyl ether, slightly soluble in methanol and in ethanol (95),
and practically insoluble in water.
Identification (1) To 0.2 g of Tolnaftate add 20 mL of
potassium hydroxide-ethanol TS and 5 mL of water, and
heat under a reflux condenser for 3 hours. After cooling, to
10 mL of this solution add 2 mL of acetic acid (100), and
shake with 1 mL of lead (II) acetate TS: a black precipitate is
formed.
(2) Determine the absorption spectrum of a solution of
Tolnaftate in methanol (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Tolnaftate Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Tol-
naftate, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Tolnaftate Refer-
ence Standard: both spectra exhibit similar intensities of ab-
sorption at the same wave numbers.
Melting point <2.60> 111 - 114°C (after drying).
Purity (1) Heavy metals <1.07> — Carbonize 1.0 g of Tol-
naftate by gentle heating. After cooling, add 5 mL of nitric
acid and 1 mL of sulfuric acid, and heat until white fumes are
evolved. After cooling, add 2 mL of nitric acid, and heat un-
til white fumes are evolved. After cooling, add 2 mL of nitric
acid and 0.5 mL of perchloric acid, and heat gradually until
white fumes are evolved. Repeat this procedure twice, and
heat until white fumes are no longer evolved. Incinerate the
residue by igniting between 500°C and 600°C for 1 hour.
Proceed according to Method 2, and perform the test with 50
mL of the test solution so obtained. Prepare the control solu-
tion as follows: to 11 mL of nitric acid add 1 mL of sulfuric
acid, 1 mL of perchloric acid and 2 mL of hydrochloric acid,
proceed in the same manner as the test solution, and add 2.0
mL of Standard Lead Solution and water to make 50 mL (not
more than 20 ppm).
(2) Related substances — Dissolve 0.50 g of Tolnaftate in
10 mL of chloroform, and use this solution as the sample so-
lution. Pipet 2 mL of the sample solution, and add chlo-
roform to make exactly 100 mL. Pipet 5 mL of this solution,
add chloroform to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-lay-
er chromatography. Develop the plate with toluene to a dis-
tance of about 10 cm, and air-dry the plate. Allow the plate
to stand in iodine vapor for 5 minutes, and examine under
ultraviolet light (wavelength: 254 nm): the spots other than
the principal spot from the sample solution are not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum
at a pressure not exceeding 0.67 kPa, 65°C, 3 hours).
Residue on ignition <2.44> Weigh accurately about 2.0 g of
Tolnaftate, and carbonize by gradual heating. Moisten the
substance with 1 mL of sulfuric acid, heat gradually until
white fumes are no longer evolved, and ignite between 450°C
and 550°C for about 2 hours to constant mass: the residue is
not more than 0.1%.
Assay Weigh accurately about 50 mg of Tolnaftate and
Tolnaftate Reference Standard, previously dried, dissolve
each in 200 mL of methanol by warming in a water bath,
cool, and add methanol to make exactly 250 mL. Pipet 5 mL
each of the solutions, to each add methanol to make exactly
100 mL, and use these solutions as the sample solution and
standard solution, respectively. Determine the absorbances,
A-y and A s , of the sample solution and standard solution at
257 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>.
Amount (mg) of C 19 H 17 NOS
= W s x(Aj/A s )
W s : Amount (mg) of Tolnaftate Reference Standard
Containers and storage Containers — Tight containers.
Tolnaftate Solution
r-;L-1-7?-r-«
Tolnaftate Solution contains not less than 90% and
not more than 110% of the labeled amount of tol-
naftate (C 19 H 17 NOS: 307.41).
Method of preparation Prepare as directed under Liquids
and Solutions, with Tolnaftate.
Identification (1) Spot 1 drop of Tolnaftate Solution on
filter paper. Spray hydrogen hexachloroplatinate (IV)-potas-
1190 Tolperisone Hydrochloride / Official Monographs
JP XV
sium iodide TS on the paper: a light yellow color develops in
the spot.
(2) To a volume of Tolnaftate Solution, equivalent to
0.02 g of Tolnaftate according to the labeled amount, add
chloroform to make 10 mL, and use this solution as the sam-
ple solution. Separately, dissolve 0.02 g of Tolnaftate Refer-
ence Standard in 10 mL of chloroform, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /uL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-lay-
er chromatography. Develop the plate with toluene to a dis-
tance of about 12 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 254 nm): the spot from
the sample solution and that from the standard solution show
the same Rf value.
Assay Pipet a volume of Tolnaftate Solution, equivalent to
about 20 mg of tolnaftate (C 19 H 17 NOS), add exactly 4 mL of
the internal standard solution, then add chloroform to make
50 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.4 g of Tolnaftate Refer-
ence Standard, previously dried in vacuum at a pressure not
exceeding 0.67 kPa at 65 °C for 3 hours, and dissolve in chlo-
roform to make exactly 100 mL. Pipet 5 mL of this solution,
add exactly 4 mL of the internal standard solution, then add
chloroform to make 50 mL, and use this solution as the stan-
dard solution. Perform the test with 10 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and calculate the ratios, Qt and Q s , of the peak area of
tolnaftate to that of the internal standard, respectively.
Amount (mg) of tolnaftate (C 19 H 17 NOS)
= W s x(Q T /Q s )x (1/20)
fV s : Amount (mg) of Tolnaftate Reference Standard
Internal standard solution — A solution of diphenyl phthalate
in chloroform (3 in 200).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 ^m in
particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of methanol and water (7:3).
Flow rate: Adjust the flow rate so that the retention time of
tolnaftate is about 14 minutes.
Selection of column: Proceed with 10 iiL of the standard
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of the internal
standard and tolnaftate in this order with the resolution be-
tween these peaks being not less than 5.
Containers and storage Containers — Tight containers.
Tolperisone Hydrochloride
o
-HCI
CH;l and enantiomer
C 16 H 23 N0.HC1: 281.82
(2i?5')-2-Methyl-l-(4-methylphenyl)-3-piperidin-l-
ylpropan-1-one monohydrochloride [3644-61-9]
Tolperisone Hydrochloride, when dried,
not less than 98.5% of C 16 H 23 NO.HCl.
contains
Description Tolperisone Hydrochloride occurs as a white,
crystalline powder. It has a slight, characteristic odor.
It is very soluble in acetic acid (100), freely soluble in water
and in ethanol (95), soluble in acetic anhydride, slightly solu-
ble in acetone, and practically insoluble in diethyl ether.
It is hygroscopic.
The pH of a solution of Tolperisone Hydrochloride (1 in
20) is between 4.5 and 5.5.
Melting point: 167- 174°C
Identification (1) Dissolve 0.2 g of Tolperisone
Hydrochloride in 2 mL of ethanol (95), add 2 mL of 1,3-
dinitrobenzene TS and 2 mL of sodium hydroxide TS, and
heat: a red color develops.
(2) To 5 mL of a solution of Tolperisone Hydrochloride
(1 in 20) add 2 to 3 drops of iodine TS: a red-brown
precipitate is produced.
(3) Dissolve 0.5 g of Tolperisone Hydrochloride in 5 mL
of water, add 2 mL of ammonia TS, and filter. Acidify 5 mL
of the filtrate with dilute nitric acid: the solution responds to
the Qualitative Tests <1.09> for chloride.
Absorbance <2.24> E l /° m (257 nm): 555 - 585 (after drying,
5 mg, ethanol (95), 500 mL).
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Tolperisone Hydrochloride in 10 mL of water: the solution is
clear and colorless.
(2) Sulfate <1.14>— Perform the test using 4.0 g of Tol-
perisone Hydrochloride. Prepare the control solution with
0.40 mL of 0.005 mol/L sulfuric acid VS (not more than
0.005%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Tolperi-
sone Hydrochloride according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(4) Piperidine hydrochloride — Dissolve 0.20 g of Tolperi-
sone Hydrochloride in water to make exactly 10 mL, and use
this solution as the sample solution. Separately, dissolve 20
mg of piperidine hydrochloride in water to make exactly 1000
mL, and use this solution as the standard solution. Transfer
5.0 mL each of the sample solution and standard solution to
different separators, add 0.1 mL each of a solution of copper
(II) sulfate pentahydrate (1 in 20), then add 0.1 mL each of
ammonia solution (28) and exactly 10 mL each of a mixture
of isooctane and carbon disulfide (3:1), and shake vigorously
for 30 minutes. Immediately after allowing to stand, separate
JPXV
Official Monographs / Tranexamic Acid 1191
the isooctane-carbon disulfide mixture layer, and dehydrate
with anhydrous sodium sulfate. Perform the test with these
solutions as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>: the absorbance of the sample solution at 438
nm is not more than that of the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tolperisone
Hydrochloride, previously dried, dissolve in 70 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.18 mg C 16 H 23 NO.HCl
Containers and storage Containers — Well-closed contain-
ers.
Tranexamic Acid
H ? N
,CQjH
C 8 H 15 N0 2 : 157.21
?ra«5-4-(Aminomethyl)cyclohexanecarboxylic acid
[1197-18-8]
Tranexamic Acid, when dried, contains not less than
98.0% and not more than 101.0% of QH 15 N0 2 .
Description Tranexamic Acid occurs as white crystals or
crystalline powder.
It is freely soluble in water, and practically insoluble in
ethanol (99.5).
Identification Determine the infrared absorption spectrum
of Tranexamic Acid as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Tranexamic Acid Reference Standard: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
pH <2.54> The pH of a solution prepared by dissolving 1.0
g of Tranexamic Acid in 20 mL of water is between 7.0 and
8.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Tranexamic Acid in 10 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Perform the test with 1.0 g of
Tranexamic Acid. Prepare the control solution with 0.40 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.014%).
(3) Heavy metals — Dissolve 2.0 g of Tranexamic Acid in
water to make 20 mL, and use this solution as the sample
stock solution. To 12 mL of the sample stock solution add
2 mL of hydrochloric acid-ammonium acetate buffer solu-
tion, pH 3.5, mix, add 1.2 mL of thioacetamide TS , mix
immediately, and use this solution as the sample solution.
Separately, proceed in the same manner as above with a
mixture of 1 mL of Standard Lead Solution, 2 mL of the
sample stock solution and 9 mL of water, and use the solu-
tion so obtained as the standard solution. Separately, pro-
ceed in the same manner with a mixture of 10 mL of water
and 2 mL of the sample stock solution, and use the solution
so obtained as the control solution. Conform that the color
of the standard solution is slightly darker than that of the
control solution. Compare the sample solution and the stan-
dard solution 2 minutes after they are prepared: the color of
the sample solution is not more intense than that of the stan-
dard solution (not more than 10 ppm).
(4) Arsenic <1.11> — Prepare the test solution by dissolv-
ing 1 .0 g of Tranexamic Acid in 10 mL of water, and perform
the test (not more than 2 ppm).
(5) Related substances — Dissolve 0.20 g of Tranexamic
Acid in water to make exactly 20 mL, and use this solution as
the sample solution. Pipet 5 mL of the sample solution, and
add water to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add water to make exactly 10 mL, and use this solution
as the standard solution. Perform the test with exactly 20 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area multiplied by relative
response factor 1.2 of the peak, having the relative retention
time of about 1 .5 with respect to tranexamic acid, is not more
than 2/5 of the peak area of tranexamic acid from the stan-
dard solution, and the area of the peak, having the relative
retention time of about 2.1 with respect to tranexamic acid, is
not more than 1/5 of the peak area of tranexamic acid from
the standard solution. The area of each peak other than
tranexamic acid and other than the peaks mentioned above is
not more than 1/5 of the peak area of tranexamic acid from
the standard solution. For this comparison, use the area of
the peaks, having the relative retention time of about 1.1 and
about 1.3, after multiplying by their relative response factors
0.005 and 0.006, respectively. The total area of the peaks
other than tranexamic acid is not more than the peak area of
tranexamic acid from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 3 times as long as the
retention time of tranexamic acid beginning after the solvent
peak.
System suitability-
Test for required detectability: To exactly 5 mL of the stan-
dard solution add water to make exactly 25 mL. Confirm that
the peak area of tranexamic acid obtained from 20 nL of this
solution is equivalent to 14 to 26% of that from 20 /uh of the
standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 7%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 C C,
1192 Tranexamic Acid Capsules / Official Monographs
JP XV
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Tranexamic
Acid and Tranexamic Acid Reference Standard, previously
dried, dissolve in water to make exactly 25 mL, and use these
solutions as the sample solution and standard solution. Per-
form the test with exactly 20 /xL each of the sample solution
and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of tranexamic acid.
Amount (mg) of C 8 H 15 N0 2 = W s x(A T /A s )
W s : Amount (mg) of Tranexamic Acid Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 6.0 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /urn in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 1 1 .0 g of sodium dihydrogen phos-
phate in 500 mL of water, and add 5 mL of triethylamine and
1.4 g of sodium lauryl sulfate. Adjust the pH to 2.5 with
phosphoric acid or diluted phosphoric acid (1 in 10), add
water to make 600 mL, and add 400 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
tranexamic acid is about 20 minutes.
System suitability —
System performance: To 5 mL of the standard solution
add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in
10,000) and water to make 50 mL. When the procedure is run
with 20 fiL of this solution under the above operating condi-
tions, tranexamic acid and 4-(aminomethyl)benzoic acid are
eluted in this order with the resolution between these peaks
being not less than 5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 0.6%.
Containers and storage Containers — Well-closed contain-
ers.
Tranexamic Acid Capsules
Tranexamic Acid Capsules contain not less than
95.0% and not more than 105.0% of the labeled
amount of tranexamic acid (C 8 H 15 N0 2 : 157.21).
Method of preparation Prepare as directed under Capsules,
with Tranexamic Acid.
Identification Take an amount of powdered contents of
Tranexamic Acid Capsules, equivalent to 0.5 g of Tranexam-
ic Acid according to the labeled amount, add 50 mL of water,
shake well, and filter. To 5 mL of the filtrate add 1 mL of nin-
hydrin TS, and heat for 3 minutes: a dark purple color de-
velops.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 capsule of Tranexamic Acid Cap-
sules at 50 revolutions per minute according to the Paddle
methed using a sinker and using 900 mL of water as the disso-
lution medium. Withdraw 20 mL or more of the dissolved so-
lution 15 minutes after starting the test, and filter through a
membrane filter with pore size of not more than 0.45 /xm.
Discard the first 10 mL of the filtrate, pipet the subsequent V
mL, add water to make exactly V mL so that each mL con-
tains about 0.28 mg of tranexamic acid (C 8 H 15 N0 2 ) accord-
ing to the labeled amount, and use this solution as the sample
solution. Separately, weigh accurately about 28 mg of
Tranexamic Acid Reference Standard, previously dried at
105 °C for 2 hours, dissolve in water to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas of tranexamic acid, A T and A s . The dissolu-
tion rate in 15 minutes is not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
tranexamic acid (C 8 H 15 N0 2 )
= W s x (A T /A S ) x (V'/V) x (1/C) x 900
W s : Amount (mg) of Tranexamic Acid Reference Stan-
dard
C: Labeled amount (mg) of tranexamic acid (C 8 H 15 N0 2 ) in
1 capsule
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 220 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 11.0 g of anhydrous sodium di-
hydrogen phosphate in 500 mL of water, and add 10 mL of
triethylamine and 1.4 g of sodium lauryl sulfate. Adjust the
pH to 2.5 with phosphoric acid, add water to make 600 mL,
and add 400 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
tranexamic acid is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of tranexamic acid are not less than 4000 and
not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 2.0%.
Assay Weigh accurately the mass of the contents of not less
than 20 Tranexamic Acid Capsules, and powder. Weigh ac-
curately an amount of the powder, equivalent to about 0.1 g
JPXV
Official Monographs / Tranexamic Acid Tablets 1193
of tranexamic acid (C 8 H 15 N02), add 30 mL of water, shake
well, and add water to make exactly 50 mL. Centrifuge, filter
the supernatant liquid through a membrane filter with pore
size of not more than 0.45 //m, discard the first 10 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 50 mg of Tranexamic
Acid Reference Standard, previously dried at 105°C for 2
hours, dissolve in water to make exactly 25 mL, and use this
solution as the standard solution. Perform the test with ex-
actly 30 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions, and determine the peak areas, A T
and A s , of tranexamic acid.
Amount (mg) of tranexamic acid (C 8 H 15 N0 2 )
= W s x(A T /A s )x2
W s : Amount (mg) of Tranexamic Acid Reference Standard
Operating conditions —
Detector, column, and mobile phase: Proceed as directed
in the operating conditions in the Assay under Tranexamic
Acid.
Column temperature: A constant temperature of about
35°C.
Flow rate: Adjust the flow rate so that the retention time of
tranexamic acid is about 16 minutes.
System suitability —
System performance: To 5 mL of the standard solution
add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in
10,000) and water to make 50 mL. When the procedure is run
with 30 /uL of this solution under the above operating condi-
tions, tranexamic acid and 4-(aminomethyl)benzoic acid are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
30 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 1.0%.
Containers and storage Containers — Tight containers.
Tranexamic Acid Injection
Tranexamic Acid Injection is an aqueous injection.
It contains not less than 95.0% and not more than
105.0% of the labeled amount of tranexamic acid
(C 8 H 15 N0 2 : 157.21).
Method of preparation Prepare
Injections, with Tranexamic Acid.
as directed under
Description Tranexamic Acid Injection is a clear and color-
less liquid.
Identification To a volume of Tranexamic Acid Injection,
equivalent to 50 mg of Tranexamic Acid according to the
labeled amount, add water to make 5 mL, add 1 mL of
ninhydrin TS, and heat: a dark purple color develops.
pH <2.54> 7.0 - 8.0
Bacterial endotoxins <4.01> Not more than 0.12 EU/mg.
Extractable volume <6.05> It meets the requirement.
Foreign insoluble matter <6.06> Perform the test according
to Method 1: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Take accurately a volume of Tranexamic Acid
Injection, equivalent to about 0.1 g of tranexamic acid
(C 8 H 15 N0 2 ), add water to make exactly 50 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 50 mg of Tranexamic Acid Reference Standard,
previously dried at 105°C for 2 hours, dissolve in water to
make exactly 25 mL, and use this solution as the standard so-
lution. Perform the test with exactly 30 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following
conditions, and determine the peak areas, A T and A s , of
tranexamic acid.
Amount (mg) of tranexamic acid (C 8 H 15 N0 2 )
= W s x(A 1 /A s )
W s : Amount (mg) of Tranexamic Acid Reference Standard
Operating conditions —
Detector, column, and mobile phase: Proceed as directed
in the operating conditions in the Assay under Tranexamic
Acid.
Column temperature: A constant temperature of about
35°C.
Flow rate: Adjust the flow rate so that the retention time of
tranexamic acid is about 16 minutes.
System suitability —
System performance: To 5 mL of the standard solution
add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in
10,000) and water to make 50 mL. When the procedure is run
with 30 /xL of this solution under the above operating condi-
tions, tranexamic acid and 4-(aminomethyl)benzoic acid are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
30 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 1.0%.
Containers and storage Containers — Hermetic containers.
Tranexamic Acid Tablets
r7^fAIW
Tranexamic Acid Tablets contain not less than
95.0% and not more than 105.0%, of the labeled
amount of tranexamic acid (C 8 H 15 N0 2 : 157.21).
Method of preparation
with Tranexamic Acid.
Prepare as directed under Tablets,
Identification To an amount of powdered Tranexamic Acid
Tablets, equivalent to 0.5 g of Tranexamic Acid according to
the labeled amount, add 50 mL of water, shake well, and
filter. To 5 mL of the filtrate add 1 mL of ninhydrin TS, and
1194 Trapidil / Official Monographs
JP XV
heat for 3 minutes: a dark purple color develops.
Uniformity of dosage units <6.02> It meets the requirement
of the Mass variation test.
Dissolution Being specified separately.
Assay Weigh accurately the mass of not less than 20
Tranexamic Acid Tablets, and powder. Weigh accurately a
quantity of the powder, equivalent to about 5 g of tranexamic
acid (C 8 H 15 N0 2 ), add 150 mL of water, disintegrate the
tablets completely with the aid of ultrasonic waves, and add
water to make exactly 200 mL. Centrifuge, pipet 4 mL of the
supernatant liquid, and add water to make exactly 50 mL.
Filter through a membrane filter with pore size of not more
than 0.45 fim, discard the first 10 mL of the filtrate, and use
the subsequent filtrate as the sample solution. Separately,
weigh accurately about 50 mg of Tranexamic Acid Reference
Standard, previously dried at 105°C for 2 hours, dissolve in
water to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with exactly 30 /uL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
tranexamic acid.
Amount (mg) of tranexamic acid (C 8 H 15 N0 2 )
= Ws x G4tA4s)Xi00
W s : Amount (mg) of Tranexamic Acid Reference Standard
Operating conditions —
Detector, column, and mobile phase: Proceed as directed
in the operating conditions in the Assay under Tranexamic
Acid.
Column temperature: A constant temperature of about
35°C.
Flow rate: Adjust the flow rate so that the retention time of
tranexamic acid is about 16 minutes.
System suitability —
System performance: To 5 mL of the standard solution
add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in
10,000) and water to make 50 mL. When the procedure is run
with 30 fiL of this solution under the above operating condi-
tions, tranexamic acid and 4-(aminomethyl)benzoic acid are
eluted in this order with the resolution between these peaks
being not less than 3.
System repeatability: When the test is repeated 6 times with
30 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
tranexamic acid is not more than 1.0%.
Containers and storage Containers — Tight containers.
Trapidil
C 10 H 15 N 5 : 205.26
7-Diethylamino-5-methyl[l,2,4]triazolo[l,5-a]pyrimidine
[15421-84-8]
Trapidil, when dried, contains not less than 98.5%
of C 10 H 15 N 5 .
Description Trapidil occurs as a white to pale yellowish
white, crystalline powder.
It is very soluble in water and in methanol, freely soluble in
ethanol (95), in acetic anhydride and in acetic acid (100), and
sparingly soluble in diethyl ether.
The pH of a solution of Trapidil (1 in 100) is between 6.5
and 7.5.
Identification (1) To 5 mL of a solution of Trapidil (1 in
50) add 3 drops of Dragendorff's TS: an orange color de-
velops.
(2) Determine the absorption spectrum of a solution of
Trapidil (1 in 125,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
Absorbance <2.24> E\°^ (307 nm): 860 - 892 (after drying,
20 mg, water, 2500 mL).
Melting point <2.60> 101 - 105°C
Purity (1) Clarity and color of solution — Dissolve 2.5 g of
Trapidil in 10 mL of water: the solution is clear and colorless
to pale yellow.
(2) Chloride <1.03>— Perform the test with 0.5 g of
Trapidil. Prepare the control solution with 0.25 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.018%).
(3) Ammonium — Place 0.05 g of Trapidil in a glass-stop-
pered conical flask, thoroughly moisten with 10 drops of so-
dium hydroxide TS, and stopper the flask. Allow it to stand
at 37°C for 15 minutes: the gas evolved does not change
moistened red litmus paper to blue.
(4) Heavy metals <1.07> — Dissolve 1.0 g of Trapidil in 40
mL of water, and add 1.5 mL of dilute hydrochloric acid, 2
mL of dilute acetic acid and water to make 50 mL. Perform
the test using this solution as the test solution. Prepare the
control solution as follows: to 1.0 mL of Standard Lead So-
lution add 2 mL of dilute acetic acid and water to make 50
mL (not more than lOppm).
(5) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Trapidil according to Method 1, and perform the test (not
more than 2 ppm).
(6) Related substances — Dissolve 0.10 g of Trapidil in 4
mL of methanol, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add methanol to
make exactly 20 mL. Pipet 1 mL of this solution, add
methanol to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
20 /uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of chloroform, ethanol (95) and acetic
acid (100) (85:13:2) to a distance of about 10 cm, and air-dry
the plate. Allow the plate to stand in iodine vapor for 60
minutes: the spots other than the principal spot from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, in vacuum,
silica gel, 60°C, 3 hours).
JPXV
Official Monographs / Triamcinolone 1195
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Trapidil, previously
dried, dissolve in 20 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 20.53 mg of C 10 H 15 N 5
Containers and storage Containers — Tight containers.
Trepibutone
r-Ut°7'r->
C 16 H 22 6 : 310.34
4-Oxo-4-(2,4,5-triethoxyphenyl)butanoic acid
[41826-92-0]
Trepibutone, when dried, contains not less than
98.5% of C 16 H 22 6 .
Description Trepibutone occurs as white to yellowish white
crystals or crystalline powder. It is odorless, and is tasteless
or has a slight, characteristic aftertaste.
It is soluble in acetone, sparingly soluble in ethanol (95),
slightly soluble in diethyl ether, and practically insoluble in
water.
It dissolves in sodium hydroxide TS.
Identification (1) Determine the absorption spectrum of a
solution of Trepibutone in diluted dilute sodium hydroxide
TS (1 in 10) (1 in 100,000) as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
tensities of absorption at the same wavelengths.
(2) Determine the spectrum of a solution of Trepibutone
in deuterated chloroform for the nuclear magnetic resonance
spectroscopy (1 in 10), using tetramethylsilane for the nuclear
magnetic resonance spectroscopy as an internal reference
compound, as directed under Nuclear Magnetic Resonance
Spectroscopy <2.21> ('H): it exhibits a sharp multiple signal A
at around <51.5 ppm, a triplet signal B at around d 2.7 ppm, a
triplet signal C at around 5 3.3 ppm, a multiple signal D at
around 5 4.2 ppm, a sharp single signal E at around 5 6.4
ppm, a sharp single signal F at around 5 7.4 ppm, and a sin-
gle signal G at around 5 10.5 ppm. The ratio of integrated in-
tensity of each signal, A:B:C:D:E:F:G, is about
9:2:2:6:1:1:1.
Melting point <2.60> 146 - 150°C
Purity (1) Chloride <1.03> — Dissolve 0.5 g of Trepibutone
in 30 mL of acetone, and add 6 mL of dilute nitric acid and
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.30 mL of 0.01 mol/L hydrochloric acid VS add 30 mL of
acetone, 6 mL of dilute nitric acid and water to make 50 mL
(not more than 0.021%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Trepibu-
tone according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(3) Related substances — Dissolve 0.10 g of Trepibutone
in 10 mL of acetone, and use this solution as the sample solu-
tion. Pipet 2 mL of the sample solution, add acetone to make
exactly 100 mL. To exactly 10 mL of this solution add ace-
tone to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/uL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
isopropylether, acetone, water and formic acid (100:30:3:3)
to a distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): the spots
other than the principal spot from the sample solution are not
more intense than the spot from the standard solution.
Loss on drying <2.41>
4 hours).
Not more than 0.5% (1 g, 105°C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Trepibutone, previ-
ously dried, dissolve in 50 mL of ethanol (95), add 50 mL of
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (indicator: 5 drops of phenolphthalein TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 31.03 mg of C I6 H 22 6
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Triamcinolone
h l J7Av/P>
C 21 H 27 F0 6 : 394.43
9-Fluoro- 1 1/6, 1 6a, 1 7 ,2 1 -tetrahydroxypregna- 1 ,4-diene-
3,20-dione [124-94-7]
Triamcinolone, when dried, contains not less than
97.0% and not more than 103.0% of C 21 H 27 F0 6 .
Description Triamcinolone occurs as a white, crystalline
powder. It is odorless.
It is freely soluble in 7V,iV-dimethylformamide, slightly
soluble in methanol, in ethanol (95) and in acetone, and prac-
tically insoluble in water, in 2-propanol and in diethyl ether.
Melting point: about 264°C (with decomposition).
Identification (1) Dissolve 1 mg of Triamcinolone in 6 mL
of ethanol (95), add 5 mL of 2,6-di-fert-butylcresol TS and 5
mL of sodium hydroxide TS, and heat on a water bath for 30
1196 Triamcinolone Acetonide / Official Monographs
JP XV
minutes under a reflux condenser: a red-purple color de-
velops.
(2) Add 5 mL of water and 1 mL of Fehling's TS to 0.01
g of Triamcinolone, and heat: a red precipitate is produced.
(3) Proceed with 0.01 g of Triamcinolone as directed un-
der Oxygen Flask Combustion Method <1.06>, using a mix-
ture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20
mL of water as the absorbing liquid. When combustion is
completed, shake vigorously so as to absorb the gas evolved:
the solution responds to the Qualitative Tests <1.09> for fluo-
ride.
(4) Determine the infrared absorption spectrum of
Triamcinolone, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum or the spectrum of previously dried Triamcinolone
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers. If any difference
appears between the spectra, dissolve 0.1 g each of Triam-
cinolone and Triamcinolone Reference Standard in 7 mL of a
mixture of 2-propanol and water (2:1), respectively, by
warming. Allow the solutions to cool in ice to effect crystals,
filter, then wash the formed crystals with two 10-mL portions
of water, and repeat the test on the dried crystals.
Optical rotation <2.49> [a] d°: +65- +71° (after drying,
0.1 g, Af,7V-dimethylformamide, 10 mL, 100 mm).
Purity Heavy metals <1.07> — Proceed with 0.5 g of Triam-
cinolone according to Method 2, and perform the test. Pre-
pare the control solution with 1.5 mL of Standard Lead Solu-
tion (not more than 30 ppm).
Loss on drying <2.41> Not more than 2.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.3% (0.5 g,
platinum crucible).
Assay Dissolve about 20 mg each of Triamcinolone and
Triamcinolone Reference Standard, previously dried and ac-
curately weighed, in a solution of L-ascorbic acid in methanol
(1 in 1000) to make exactly 50 mL. Pipet 5 mL each of these
solutions, add exactly 5 mL each of the internal standard so-
lution, add a solution of L-ascorbic acid in methanol (1 in
1000) to make 20 mL, and use these solutions as the sample
solution and standard solution. Perform the test with 10 iiL
each of these solutions as directed under Liquid Chromatog-
raphy <2.01> according to the following conditions, and cal-
culate the ratios, Q T and Q s , of the peak height of tri-
amcinolone to that of the internal standard, respectively.
Amount (mg) of C 21 H 27 F0 6 = W s x (g T /g s )
W s : Amount (mg) of Triamcinolone Reference Standard
Internal standard solution — Dissolve 15 mg of methyl para-
hydroxybenzoate in a solution of L-ascorbic acid in methanol
(1 in 1000) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.0 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel (5 iim in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:1).
Flow rate: Adjust the flow rate so that the retention time of
triamcinolone is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
fiL of the standard solution under the above operating condi-
tions, triamcinolone and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 2.0.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of triamcinolone to that of the internal standard
is not more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Triamcinolone Acetonide
r- U7AW n>7-tz r-= K
C 24 H 3 iF0 6 : 434.50
9-Fluoro- 1 1/?,21 -dihydroxy- 1 6a, 17-
(1 -methylethylidenedioxy)pregna-l ,4-diene-3 ,20-dione
[76-25-5]
Triamcinolone Acetonide, when dried, contains not
less than 97.0%> and not more than 103. 0% of
C24H31FCV
Description Triamcinolone Acetonide occurs as a white,
crystalline powder. It is odorless.
It is sparingly soluble in ethanol (99.5), in acetone, and in
1,4-dioxane, slightly soluble in methanol and in ethanol (95),
and practically insoluble in water and in diethyl ether.
Melting point: about 290°C (with decomposition).
Identification (1) Dissolve 2 mg of Triamcinolone
Acetonide in 40 mL of ethanol (95), add 5 mL of 2,6-di-terf-
butylcresol TS and 5 mL of sodium hydroxide TS, and heat
on a water bath under a reflux condenser for 20 minutes: a
green color develops.
(2) Add 5 mL of water and 1 mL of Fehling's TS to 0.01
g of Triamcinolone Acetonide, and heat: a red precipitate is
produced.
(3) Proceed with 0.01 g of Triamcinolone Acetonide as
directed under Oxygen Flask Combustion Method <1.06>, us-
ing a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS
and 20 mL of water as the absorbing liquid. When combus-
tion is completed, shake vigorously so as to absorb the gas
evolved: the solution responds to the Qualitative Tests <1.09>
for fluoride.
(4) Determine the absorption spectrum of a solution of
Triamcinolone Acetonide in ethanol (95) (1 in 100,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
JPXV
Official Monographs / Triamterene 1197
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Triamcinolone Acetonide
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(5) Determine the infrared absorption spectrum of
Triamcinolone Acetonide, previously dried, as directed in the
potassium bromide disk method under the Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of previously dried
Triamcinolone Acetonide Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers. If any difference appears between the spectra, dis-
solve 0.1 g each of Triamcinolone Acetonide and Triamcino-
lone Acetonide Reference Standard in 20 mL of ethanol (95),
respectively, then evaporate the ethanol to dryness, and
repeat the test on the dried residue.
Optical rotation <2.49> [a]™: + 100 - + 107° (after drying,
0.1 g, 1,4-dioxane, 10 mL, 100 mm).
Purity (1) Heavy metals <1.07> — Proceed with 0.5 g of
Triamcinolone Acetonide according to Method 2, and per-
form the test. Prepare the control solution with 1.5 mL of
Standard Lead Solution (not more than 30 ppm).
(2) Related substances — Dissolve 40 mg of Triamcino-
lone Acetonide in 4 mL of acetone, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
acetone to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
[iL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and methanol (93:7) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 2.0% (0.5 g, in vacu-
um, phosphorus (V) oxide, 60°C, 3 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g,
platinum crucible).
Assay Dissolve about 20 mg each of Triamcinolone
Acetonide and Triamcinolone Acetonide Reference Stan-
dard, previously dried and accurately weighed, in methanol
to make exactly 50 mL. Pipet 10 mL each of these solutions,
add exactly 10 mL each of the internal standard solution,
then add the mobile phase to make 50 mL, and use these solu-
tions as the sample solution and standard solution. Perform
the test with 20 /xL each of these solutions as directed under
Liquid Chromatography <2.01> according to the following
conditions, and calculate the ratios, g T and Q s , of the peak
height of triamcinolone acetonide to that of the internal stan-
dard, respectively.
Amount (mg) of C 24 H 3I F0 6
= W s x(Q 1 /Q s )
W s : Amount (mg) of Triamcinolone Acetonide Reference
Standard
Internal standard solution — A solution of prednisolone in
methanol (1 in 50,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 30 cm in length, packed with octadecylsilanized
silica gel (10 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of water and acetonitrile (3:1).
Flow rate: Adjust the flow rate so that the retention time of
triamcinolone acetonide is about 13 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the internal standard and triamcinolone acetonide are
eluted in this order with the resolution between these peaks
being not less than 6.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratios of the
peak height of triamcinolone acetonide to that of the internal
standard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Triamterene
h U7AxU>
C 12 H„N 7 : 253.26
6-Phenylpteridine-2,4,7-triamine
[396-01-0]
Triamterene, when dried, contains not less than
98.5% of C 12 H„N 7 .
Description Triamterene occurs as a yellow, crystalline
powder. It is odorless, and tasteless.
It is sparingly soluble in dimethylsulfoxide, very slightly
soluble in acetic acid (100), and practically insoluble in water,
in ethanol (95), and in diethyl ether.
It dissolves in nitric acid and in sulfuric acid, but does not
dissolve in dilute nitric acid, in dilute sulfuric acid and in di-
lute hydrochloric acid.
Identification (1) To 0.01 g of Triamterene add 10 mL of
water, heat, and filter after cooling: the filtrate shows a pur-
ple fluorescence. To 2 mL of the filtrate add 0.5 mL of
hydrochloric acid: the fluorescence disappears.
(2) The filtrate obtained in (1) responds to the Qualitative
Tests <1.09> for primary aromatic amines.
(3) Dissolve 0.01 g of Triamterene in 100 mL of acetic
acid (100), and to 10 mL of the solution add water to make
100 mL. Determine the absorption spectrum of the solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wavelengths.
1198 Trichlormethiazide / Official Monographs
JP XV
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Triamterene according to Method 2, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Triamterene according to Method 3, and perform the test
(not more than 2 ppm).
(3) Related substances — Dissolve 0.10 g of Triamterene
in 20 mL of dimethylsulf oxide. To 2 mL of this solution add
methanol to make 50 mL, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add methanol to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethyl acetate, ammonia solution (28) and
methanol (9:1:1) to a distance of about 10 cm, and air-dry the
plate. Examine the plate under ultraviolet light (main
wavelength: 365 nm): the spots other than the principal spot
from the sample solution are not more intense than the spot
from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105°C,
4 hours).
Residue on ignition <2.44> Not more than 0.10% (1 g).
Assay Weigh accurately about 0.15 g of Triamterene, previ-
ously dried, and dissolve in 100 mL of acetic acid (100) by
warming. Titrate <2.50> with 0.05 mol/L perchloric acid VS
(indicator: 2 drops of crystal violet TS). Perform a blank de-
termination, and make any necessary correction.
Each mL of 0.05 mol/L perchloric acid VS
= 12.663 mg of C 12 H„N 7
Containers and storage Containers — Well-closed contain-
ers.
Trichlormethiazide
h U^njuy-^TvH
HjN
5?
Nh
A
H H
"
and enantiomer
C 8 H 8 Cl3N 3 04S 2 : 380.66
(3flS)-6-Chloro-3-dichloromethyl-3,4-dihydro-2i/-l,2,4-
benzothiadiazine-7-sulfonamide 1,1 -dioxide [133-67-5]
Trichlormethiazide, when dried, contains not less
than 97.5% and not more than 102.0% of
C 8 H 8 C1 3 N 3 4 S 2 .
Description Trichlormethiazide occurs as a white powder.
It is freely soluble in Af.A^dimethylformamide and in
acetone, slightly soluble in acetonitrile and in ethanol (95),
and practically insoluble in water.
A solution of Trichlormethiazide in acetone (1 in 50) shows
no optical rotation.
Melting point: about 270°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Trichlormethiazide in ethanol (95) (3 in 250,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Trichlormethiazide
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Trichlormethiazide as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Trichlormethiazide Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Perform the test with Trichlormethiazide as directed
under Flame Coloration Test <1.04> (2): a green color ap-
pears.
Purity (1) Chloride <7.0?>— Dissolve 1.0 g of Trichlor-
methiazide in 30 mL of acetone, add 6 mL of dilute nitric
acid and water to make 50 mL, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 1.0 mL of 0.01 mol/L hydrochloric acid VS
add 30 mL of acetone, 6 mL of dilute nitiric acid and water to
make 50 mL (not more than 0.036%).
(2) Sulfate <1.14>— Dissolve 1.0 g of Trichlormethiazide
in 30 mL of acetone, add 1 mL of dilute hydrochloric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution as fol-
lows: to 1.0 mL of 0.005 mol/L sulfuric acid VS add 30 mL
of acetone, 1 mL of dilute hydrochloric acid and water to
make 50 mL (not more than 0.048%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Trichlor-
methiazide according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 0.6 g
of Trichlormethiazide according to Method 5, using 20 mL
of Af.Af-dimethylformamide, and perform the test (not more
than 3.3 ppm).
(5) Related substances — Dissolve 25 mg of Trichlor-
methiazide in 50 mL of acetonitrile, and use the solution as
the sample solution. Perform the test with 10 juL of the sam-
ple solution as directed under Liquid Chromatography <2.01>
according to the following conditions, determine each peak
area by the automatic integration method, and calculate the
amount of related substances by the area percentage method:
the amount of 4-amino-6-chlorobenzene-l,3-disulfonamide,
having the relative retention time of about 0.3 with respect to
trichlormethiazide, is not more than 2.0%, and the total
amount of the related substances is not more than 2.5%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with phenylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of diluted phosphoric acid
(1 in 1000) and acetonitrile (3:1).
JPXV
Official Monographs / Triclilormethiazide Tablets 1199
Mobile phase B: A mixture of acetonitrile and diluted
phosphoric acid (1 in 1000) (3:1).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0- 10
10-20
100
100^
0^ 100
Flow rate: 1.5 mL per minute
Time span of measurement: About 2.5 times as long as the
retention time of trichlormethiazide beginning after the sol-
vent peak.
System suitability —
Test for required detectability: To exactly 1 mL of the sam-
ple solution add acetonitrile to make exactly 50 mL, and use
this solution as the solution for system suitability test. Pipet
1 mL of the solution, and add acetonitrile to make exactly
20 mL. Confirm that the peak area of trichlormethiazide
obtained from 10 /xL of this solution is equivalent to 3.5 to
6.5% of that of trichlormethiazide obtained from 10 /uL of
the solution for system suitability test.
System performance: To 5 mL of the solution for system
suitability test add 5 mL of water, and warm in a water bath
at 60°C for 30 minutes. When the procedure is run with
10 /xL of this solution, after cooling, under the above operat-
ing conditions, 4-amino-6-chlorobenzene-l,3-disulfonamide
and trichlormethiazide are eluted in this order, the relative
retention time of 4-amino-6-chlorobenzene-l,3-disul-
fonamide with respect to trichlormethiazide is about 0.3, and
the number of theoretical plates and the symmetry factor of
the peak of trichlormethiazide are not less than 5000 and not
more than 1.2, respectively.
System repeatability: When the test is repeated 3 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of trichlormethiazide is not more than 2.0% .
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 25 mg of Trichlormethiazide
and Trichlormethiazide Reference Standard, previously
dried, and dissolve separately in exactly 20 mL of the internal
standard solution. To 1 mL of these solutions add acetoni-
trile to make 20 mL, and use these solutions as the sample so-
lution and the standard solution. Perform the test with 10 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the ratios, Q T and Q s , of
the peak area of trichlormethiazide to that of the internal
standard.
Amount (mg) of C 8 H 8 C1 3 N 3 4 S 2 = W s x(Q T /Q s )
W s : Amount (mg) of Trichlormethiazide Reference Stan-
dard
Internal standard solution — A solution of 3-nitrophenol in
acetonitrile (1 in 800).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with phenylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (3:1).
Flow rate: Adjust the flow rate so that the retention time of
trichlormethiazide is about 8 minutes.
System suitability —
System performance: When the procedure is run with
10 /uL of the standard solution under the above operating
conditions, the internal standard and trichlormethiazide are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of trichlormethiazide to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Well-closed contain-
Trichlormethiazide Tablets
Trichlormethiazide Tablets contain not less than
93.0% and not more than 107.0%, of the labeled
amount of trichlormethiazide (C 8 H 8 Cl3N 3 4 S2:
380.66).
Method of preparation Prepare as directed under Tablets,
with Trichlormethiazide.
Identification To an amount of pulverized Trichlor-
methiazide Tablets, equivalent to 4 mg of Trichlormethiazide
according to the labeled amount, add 10 mL of acetone,
shake vigorously for 5 minutes, centrifuge, and use the super-
natant liquid as the sample solution. Separately, dissolve 4
mg of Trichlormethiazide Reference Standard in 10 mL of
acetone, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, hexane and
methanol (10:4:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): the principal spots from the sample solution and the
standard solution show the same i?f value.
Purity Related substances — Pulverize a suitable amount of
Trichlormethiazide Tablets in an agate mortar. Take an
amount of the powder, equivalent to 10 mg of Trichlor-
methiazide according to the labeled amount, add 20 mL of
acetonitrile, shake vigorously for 15 minutes, centrifuge, and
use the supernatant liquid as the sample solution. Perform
the test with 10 /xL of the sample solution as directed under
1200 inch lor me thiazide Tablets / Official Monographs
JP XV
Liquid Chromatography <2.01> according to the following
conditions, determine each peak area by the automatic in-
tegration method, and calculate the amount of each related
substance by the area percentage method: the amount of 4-
amino-6-chlorobenzene-l,3-disulfoneamide, having the rela-
tive retention time of about 0.3 with respect to trichlor-
methiazide, is not more than 4.0%, and the total amount of
the peaks other than trichlormethiazide is not more than
5.0%.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with phenylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of diluted phosphoric acid
(1 in 1000) and acetonitrile (3:1).
Mobile phase B: A mixture of acetonitrile and diluted
phosphoric acid (1 in 1000) (3:1).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A (vol%)
Mobile phase
B (vol%)
0-10
10-20
100
100^
0^ 100
Flow rate: 1.5 mL/minute
Time span of measurement: About 2.5 times as long as the
retention time of trichlormethiazide beginning after the sol-
vent peak.
System suitability —
Test for required detectability: Dissolve 25 mg of Trichlor-
methiazide in 50 mL of acetonitrile. Pipet 1 mL of this solu-
tion, add acetonitrile to make exactly 50 mL, and use this so-
lution as the solution for system suitability test. Pipet 1 mL
of the solution for system suitability test, and add acetonitrile
to make exactly 20 mL. Confirm that the peak area of
trichlormethiazide obtained from 10,mL of this solution is
equivalent to 3.5 to 6.5% of that obtained from 10 /xL of the
solution for system suitability test.
System performance: To 5 mL of the solution for system
suitability test add 5 mL of water, and warm in a water bath
of 60°C for 30 minutes. When the procedure is run with 10
[iL of this solution, after cooling, under the above operating
conditions, 4-amino-6-chlorobenzene-l,3-disulfonamide and
trichlormethiazide are eluted in this order, the relative reten-
tion time of 4-amino-6-chlorobenzene-l,3-disulfonamide
with respect to trichlormethiazide is about 0.3, and the num-
ber of theoretical plates and the symmetry factor of the peak
of trichlormethiazide are not less than 5000 and not more
than 1.2, respectively.
System repeatability: When the test is repeated 3 times with
10 /xL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of trichlormethiazide is not more than 2.0% .
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Perform the test according to the following method: it
meets the requirements of the Content Uniformity Test. To
one tablet of Trichlormethiazide Tablets add 5 mL of diluted
phosphoric acid (1 in 50) to disintegrate. Add exactly an
amount of the internal standard solution, equivalent to 10
mL per 2 mg of trichlormethiazide (C 8 H 8 C1 3 N 3 4 S 2 ) accord-
ing to the labeled amount, add acetonitrile to make 25 mL,
shake vigorously for 15 minutes, and centrifuge. To an
amount of the supernatant liquid add the mobile phase to
make a solution so that it contains about 40 fig of trichlor-
methiazide (C 8 H 8 C1 3 N 3 4 S 2 ) in each mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 20 mg of Trichlormethiazide Reference Standard,
previously dried at 105 C C for 3 hours, and dissolve in
acetonitrile to make exactly 100 mL. Pipet 5 mL of this solu-
tion, add exactly 5 mL of the internal standard solution, add
10 mL of acetonitrile and 5 mL of diluted phosphoric acid (1
in 50), and use this solution as the standard solution. Per-
form the test with 20 [iL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the conditions described in the Assay,
and determine the ratios, Q T and Q s , of the peak area of
trichlormethiazide to that of the internal standard.
Amount (mg) of trichlormethiazide (C 8 H 8 3 N 3 4 S 2 )
= ^ s x(g T /g s )xCx(l/20)
W s : Amount (mg) of Trichlormethiazide Reference
Standard
C: Labeled amount (mg) of trichlormethiazide
(C 8 H 8 C1 3 N 3 4 S 2 ) per tablet
Internal standard solution — A solution of 3-nitrophenol in
acetonitrile (1 in 5000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Trichlormethiazide
Tablets at 50 revolutions per minute according to the Paddle
method, using 900 mL of water as the dissolution medium.
Withdraw 20 mL or more of the dissolution medium 15
minutes after starting the test, and filter through a membrane
filter with pore size of not more than 0.45 //m. Discard the
first 10 mL of the filtrate, pipet the subsequent KmL of the
filtrate, add diluted phosphoric acid (1 in 50) to make exactly
V mL so that each mL contains about 1 . 1 //g of trichlor-
methiazide (C 8 H 8 3 N 3 4 S 2 ) according to the labeled
amount, and use this solution as the sample solution.
Separately, weigh accurately about 22 mg of Trichlor-
methiazide Reference Standard, previously dried at 105°C
for 3 hours, and dissolve in acetonitrile to make exactly
200 mL. Pipet 2 mL of this solution, add diluted phosphoric
acid (1 in 50) to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with exactly 40 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, y4 Ta and
A Se , of trichlormethiazide obtained with the sample solution
and the standard solution, and the area, A Tb , of the peak,
having the relative retention time of about 0.3 with respect to
trichlormethiazide, obtained with the sample solution. The
dissolution rate in 15 minutes is not less than 75%.
Dissolution rate (%) with respect to the labeled amount of
JPXV
Official Monographs / Trichomycin 1201
trichlormethiazide (C 8 H 8 C1 3 N 3 04S2)
= W s x [(A^ + A^ x 0.95)A4 Sa ] x (V'/V) X (1/Q x (9/2)
W s : Amount (mg) of Trichlormethiazide Reference Stan-
dard
C: Labeled amount (mg) of trichlormethiazide
(C 8 H 8 C1 3 N 3 4 S 2 ) per tablet
Operating conditions —
Proceed as directed in the operating conditions in the
Assay.
System suitability —
System performance: Dissolve 25 mg of Trichlor-
methiazide in 50 mL of acetonitrile. To 1 mL of this solution
add acetonitrile to make 50 mL. To 5 mL of this solution add
5 mL of water, and heat at 60°C in a water bath for 30
minutes. After cooling, when the procedure is run with 10 fiL
of this solution under the above operating conditions, 4-
amino-6-chlorobenzene-l,3-disulfonamide and trichlor-
methiazide are eluted in this order, the relative retention time
of 4-amino-6-chlorobenzene-l,3-disulfonamide with respect
to trichlormethiazide is about 0.3, and the number of theoret-
ical plates and the symmetry factor of the peak of trichlor-
methiazide are not less than 5000 and not more than 1.2, re-
spectively.
System repeatability: When the test is repeated 6 times with
40 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
trichlormethiazide is not more than 2.0%.
Assay Weigh accurately the mass of not less than 20
Trichlormethiazide Tablets, and pulverize the tablets in an
agate mortar. Weigh accurately an amount of the powder,
equivalent to about 2 mg of trichlormethiazide
(C 8 H 8 Cl3N 3 4 S2) according to the labeled amount, add 5 mL
of diluted phosphoric acid (1 in 50) and exactly 10 mL of the
internal standard solution, add 10 mL of acetonitrile, shake
vigorously for 15 minutes, and centrifuge. To 2 mL of the su-
pernatant liquid add 2 mL of the mobile phase, and use this
solution as the sample solution. Separately, weigh accurately
about 40 mg of Trichlormethiazide Reference Standard,
previously dried at 105 °C for 3 hours, and dissolve in
acetonitrile to make exactly 200 mL. Pipet 5 mL of this solu-
tion, add exactly 5 mL of the internal standard solution, add
10 mL of acetonitrile and 5 mL of diluted phosphoric acid (1
in 50), and use this solution as the standard solution. Per-
form the test with 20 fiL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the ratios, Q T and Q s , of the peak area of trichlormethiazide
to that of the internal standard.
Amount (mg) of trichlormethiazide (C 8 H 8 C1 3 N 3 4 S 2 )
= ^ S x(Gt/Gs)x(1/20)
W s : Amount (mg) of Trichlormethiazide Reference
Standard
Internal standard solution — A solution of 3-nitrophenol in
acetonitrile (1 in 5000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 268 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with phenylsilanized
silica gel for liquid chromatography (5 ftm in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (3:1).
Flow rate: Adjust the flow rate so that the retention time of
trichlormethiazide is about 8 minutes.
System suitability —
System performance: When the procedure is run with
20 fiL of the standard solution under the above operating
conditions, the internal standard and trichlormethiazide are
eluted in this order with the resolution between these peaks
being not less than 2.0.
System repeatability: When the test is repeated 6 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of trichlormethiazide to that of the internal stan-
dard is not more than 1.0%.
Containers and storage Containers — Tight containers.
Trichomycin
h U=it-<
k" jH V l TrichMnycki A: R'^H. H*-OH
1 Trichomycin B. R 1 -OH. R^-H
Trichomycin A
33-(3-Amino-3,6-dideoxy-/?-D-mannopyranosyloxy)-17-
[6-(4-aminophenyl)-4-hydroxy-l-methyl-6-oxohexyl]-
l,3,5,9,ll,37-hexahydroxy-18-methyl-13,15-dioxo-16,39-
dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-
heptaene-36-carboxylic acid [12698-99-6]
Trichomycin B
33-(3-Amino-3,6-dideoxy-/?-D-mannopyranosyloxy)-17-
[6-(4-aminophenyl)-4-hydroxy-l-methyl-6-oxohexyl]-
l,3,5,7,9,37-hexahydroxy-18-methyl-13,15-dioxo-16,39-
dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-
heptaene-36-carboxylic acid [12699-00-2]
[1394-02-1, Trichomycin]
Trichomycin is a mixture of polyene macrolide sub-
stances having antifungal and antiprotozoal activities
produced by the growth of Streptomyces hachijoensis.
It contains not less than 7000 Units per mg, calculat-
ed on the dried basis. The potency of Trichomycin is
expressed as unit based on the amount of trichomycin.
One unit of Trichomycin is equivalent to 0.05 fig of
trichomycin.
Description Trichomycin occurs as a yellow to yellow-
brown powder.
It is practically insoluble in water, in ethanol (99.5) and in
tetrahydrofuran.
It dissolves in dilute sodium hydroxide TS.
It is hygroscopic.
Identification (1) To 2 mg of Trichomycin add 2 mL of
1202 Triclofos Sodium / Official Monographs
JP XV
sulfuric acid: a blue color appears, and the color is changed
to a blue-purple after allowing to stand.
(2) Dissolve 1 mg of Trichomycin in 50 mL of a solution
of sodium hydroxide (1 in 200). Determine the absorption
spectrum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 359
nm and 365 nm, between 378 nm and 384 nm, and between
400 nm and 406 nm.
Content ratio of the active principle Conduct this proce-
dure without exposure to daylight, using light-resistant ves-
sels. Dissolve about 10 mg of Trichomycin in 50 mL of a mix-
ture of tetrahydrofuran for liquid chromatography and water
(3:1), and use this solution as the sample solution. Perform
the test with 5 /uL of the sample solution as directed under
Liquid Chromatography <2.01> according to the following
conditions, determine the peak areas by the automatic in-
tegration method, and calculate the amount of trichomycin A
and trichomycin B by the area percentage method: the
amount of trichomycin A is between 20% and 40%, and that
of trichomycin B is between 15% and 25%. The relative
retention time of trichomycin B with respect to trichomycin
A is about 1.2.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 360 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen
phosphate and 1.7 g of sodium lauryl sulfate in a mixture of
600 mL of water and 400 mL of acetonitrile for liquid chro-
matography.
Flow rate: Adjust the flow rate so that the retention time of
trichomycin A is about 8 minutes.
Time span of measurement: About 4 times as long as the
retention time of trichomycin A.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the sample solution, add a mixture of tetrahydrofuran for
liquid chromatography and water (3:1) to make exactly 50
mL, and use this solution as the solution for system suitabil-
ity test. Pipet 5 mL of the solution for system suitability test,
and add a mixture of tetrahydrofuran for liquid chro-
matography and water (3:1) to make exactly 30 mL. Confirm
that the peak area of trichomycin A obtained from 5 /uL of
this solution is equivalent to 12 to 22% of that from 5 /uL of
the solution for system suitability test.
System performance: When the procedure is run with 5 /uL
of the solution for system suitability test under the above
operating conditions, trichomycin A and trichomycin B are
eluted in this order with the resolution between these peaks
being not less than 2.5.
System repeatability: When the test is repeated 6 times with
5 /uL of the solution for system suitability test under the
above operating conditions, the relative standard deviation
of the peak area of trichomycin A is not more than 2.0%.
Loss on drying <2.41> Not more than 5.0% (1 g, in vacuum,
60°C, 3 hours).
Assay Conduct this procedure without exposure to day-
light, using light-resistant vessels. Weigh accurately an
amount of Trichomycin and Trichomycin Reference Stan-
dard, equivalent to about 150,000 units, dissolve them
separately in a mixture of tetrahydrofuran for liquid chro-
matography and water (3:1) to make exactly 100 mL, and use
these solutions as the sample solution and the standard solu-
tion. Perform the test with exactly 20 //L each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of trichomy-
cin.
Amount (unit) of trichomycin = W s X (A T /A S )
W s : Amount (unit) of Trichomycin Reference Standard
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 360 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with silica gel for liquid
chromatography (10 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 15 g of ammonium acetate in 120
mL of water, and add 1 000 mL of acetonitrile for liquid chro-
matography and 700 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
trichomycin is about 6 minutes.
System suitability —
System performance: Dissolve 5 mg of Trichomycin and 1
mg of berberine chloride in 100 mL of a mixture of tetra-
hydrofuran for liquid chromatography and water (3:1).
When the procedure is run with 20 fiL of this solution under
the above operating conditions, berberine and trichomycin
are eluted in this order with the resolution between these
peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
trichomycin is not more than 2.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Triclofos Sodium
Monosodium Trichloroethyl Phosphate
r- U?n*Xl-r- U^A
a ci
Cr ^-^ P0 3 HNa
C 2 H 3 Cl3Na0 4 P: 251.37
Monosodium 2,2,2-trichloroethyl
monohydrogenphosphate [7246-20-0]
Triclofos Sodium, when dried, contains not less than
97.0 % and not more than 102.0 % of
C2H 3 Cl3Na0 4 P, and not less than 41.0% and not more
than 43.2% of chlorine (CI: 35.45).
Description Triclofos Sodium is a white, crystalline pow-
JPXV
Official Monographs / Triclofos Sodium Syrup 1203
der.
It is freely soluble in water, slightly soluble in ethanol (95),
and practically insoluble in diethyl ether.
It is hygroscopic.
Identification (1) Determine the infrared absorption spec-
trum of Triclofos Sodium as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(2) To 0.5 g of Triclofos Sodium add 10 mL of nitric
acid, evaporate on a water bath to dryness, and ignite further
over a flame. Dissolve the residue in 5 mL of water, and filter
it necessary: the filtrate responds to Qualitative Tests <1.09>
for sodium salt.
(3) To 0.1 g of Triclofos Sodium add 1 g of anhydrous
sodium carbonate, and heat for 10 minutes. After cooling,
dissolve the residue in 40 mL of water, filter if necessary, and
render the filtrate acidic with dilute nitric acid: the solution
responds to the Qualitative Tests <1.09> (2) for chloride. The
remainder of the filtrate responds to the Qualitative Tests
<1.09> (1) for chloride and to the Qualitative Tests <1.09> for
phosphate.
pH <2.54> Dissolve 1.0 g of Triclofos Sodium in 50 mL of
water: the pH of this solution is between 3.0 and 4.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Triclofos Sodium in 50 mL of water: the solution is clear and
colorless.
(2) Chloride <1.03>— Perform the test with 0.20 g of
Triclofos Sodium. Prepare the control solution with 1.0 mL
of 0.01 mol/L hydrochloric acid VS (not more than 0.178%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Triclofos
Sodium according to Method 1, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 20 ppm).
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Triclofos Sodium according to Method 1 , and perform the
test (not more than 2 ppm).
(5) Free phosphoric acid — Weigh accurately about 0.3 g
of Triclofos Sodium, previously dried, dissolve in water to
make exactly 100 mL, and use this solution as the sample so-
lution. Pipet 5 mL each of the sample solution and Standard
Phosphoric Acid Solution, add 2.5 mL of hexaammonium
heptamolybdate-sulfuric acid TS and 1 mL of l-amino-2-
naphthol-4-sulfonic acid TS, shake, add water to make exact-
ly 25 mL, and allow to stand at 20°C for 30 minutes. Perform
the test with these solutions, using a solution obtained in the
same manner with 5 mL of water as the blank, as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>. Determine
the absorbances, A T and A s , of each solution from the sam-
ple solution and Standard Phosphoric Acid Solution at 740
nm: the content of the free phosphoric acid is not more than
1.0%.
Content (%) of the free phosphoric acid (H3PO4)
= (A T /A S ) x (\/W) x 287.8
W: Amount (mg) of the sample taken.
Loss on drying <2.41> Not more than 5.0% (1 g, in vacuum,
100°C, 3 hours).
Assay (1) Triclofos sodium — Weigh accurately about 0.2
g of Triclofos Sodium, previously dried, place in a Kjeldhal
flask, add 2 mL of sulfuric acid and 2.5 mL of nitric acid,
and heat until brown gas are not evolved. After cooling, add
1 mL of nitric acid, heat until white fumes are produced, and
cool. Repeat this procedure until the solution becomes color-
less. Transfer this solution to a flask using 150 mL of water,
add 50 mL of molybdenum (III) oxide-citric acid TS, heat
gently to boil, add gradually 25 mL of quinoline TS with stir-
ring, and heat on a water bath for 5 minutes. After cooling,
filter the precipitate, and wash repeatedly with water until the
washing does not indicate acidity. Transfer the precipitate to
a flask using 100 mL of water, add exactly 50 mL of 0.5 mol/
L sodium hydroxide VS, dissolve, and titrate <2.50> with 0.5
mol/L hydrochloric acid VS until the color of the solution
changes from purple to yellow (indicator: 3 drops of
phenolphthalein-thymol blue TS). Perform a blank determi-
nation.
Each mL of 0.5 mol/L sodium hydrochloride VS
= 4.834 mg of C 2 H 3 Cl 3 Na0 4 P
(2) Chlorine — Weigh accurately about 10 mg of Triclofos
Sodium, previously dried, perform the test according to the
procedure of determination for chlorine as directed under
Oxygen Flask Combustion Method <1.06>, using 1 mL of 1
mol/L sodium hydroxide TS and 20 mL of water as the ab-
sorbing liquid.
Containers and storage Containers — Tight containers.
Triclofos Sodium Syrup
Monosodium Trichloroethyl Phosphate Syrup
Triclofos Sodium Syrup contains not less than 90%
and not more than 110% of the labeled amount of
triclofos sodium (CzHjCljNaC^P: 251.37).
Method of preparation Prepare as directed under Syrups,
with Triclofos Sodium.
Identification (1) Weigh a portion of Triclofos Sodium
Syrup, equivalent to 0.25 g of Triclofos Sodium according to
the labeled amount, add 40 mL of water, shake well, add 5
mL of diluted sulfuric acid (3 in 50), and extract with 25 mL
of 3-methyl-l-butanol. Take 5 mL of the extract, evaporate
on a water bath to dryness, and add 1 mL of diluted sulfuric
acid (1 in 2) and 1 mL of a solution of potassium perman-
ganate (1 in 20) to the residue. Heat in a water bath for 5
minutes, add 7 mL of water, and then add a solution of oxal-
ic acid dihydrate (1 in 20) until the color of the solution disap-
pears. To 1 mL of this solution add 1 mL of pyridine and 1
mL of a solution of sodium hydroxide (1 in 5), and heat in a
water bath, while shaking, for 1 minute: a light red color de-
velops in the pyridine layer.
(2) Take 10 mL of the extract obtained in (1), evaporate
on a water bath to dryness, add 1 g of anhydrous sodium car-
bonate to the residue, and heat for 10 minutes. After cooling,
dissolve the residue in 40 mL of water, filter if necessary, and
render the filtrate acidic with dilute nitric acid: the solution
responds to the Qualitative Tests <1.09> (2) for chloride. The
1204 Trihexyphenidyl Hydrochloride / Official Monographs
JP XV
remainder of the filtrate responds to the Qualitative Tests
<1.09> (1) for chloride and to the Qualitative Tests <1.09> for
phosphate.
pH <2.54> 6.0 - 6.5
Assay Weigh accurately a portion of Triclofos Sodium
Syrup, equivalent to 0.13 g of Triclofos Sodium according to
the labeled amount, add 15 mL of water, 1 mL of sodium
hydroxide TS and 15 mL of diethyl ether, shake for 1 minute,
and separate the water layer. Wash the diethyl ether layer
with 1 mL of water, and combine the washing with above
water layer. To this solution add 2.5 mL of diluted sulfuric
acid (3 in 50), and extract with four 10-mL portions of 3-
methyl-1-butanol. Combine the 3-methyl-l-butanol extracts,
and add 3-methyl-l-butanol to make exactly 50 mL. Measure
exactly 10 mL each of this solution, and dilute with potassi-
um hydroxide-ethanol TS. Place in a glass ampule, fire-seal,
mix, and heat at 120°C for 2 hours in an autoclave. After
cooling, transfer the contents to a flask, add 20 mL of diluted
nitric acid (63 in 500) and exactly 25 mL of 0.02 mol/L silver
nitrate VS, shake well, and titrate <2.50> the excess silver ni-
trate with 0.02 mol/L ammonium thiocyanate VS (indicator:
2 to 3 drops of ammonium iron (III) sulfate TS). Perform a
blank determination.
Each mL of 0.02 mol/L silver nitrate VS
= 1.676 mg of C 2 H 3 Cl 3 Na0 4 P
Containers and storage Containers — Tight containers.
Storage — In a cold place.
Trihexyphenidyl Hydrochloride
r- U's*v7x=vJU£®J£
■HCI
and enantiomer
C 20 H 31 NO.HC1: 337.93
(l/?5')-l-Cyclohexyl-l-phenyl-3-(piperidin-l-yl)propan-l-ol
monohydrochloride [52-49-3]
Trihexyphenidyl Hydrochloride, when dried,
tains not less than 98.5% of C 20 H 31 NO.HC1.
con-
Description Trihexyphenidyl Hydrochloride occurs as a
white, crystalline powder. It is odorless, and has a bitter
taste.
It is soluble in ethanol (95), sparingly soluble in acetic acid
(100), slightly soluble in water, very slightly soluble in acetic
anhydride, and practically insoluble in diethyl ether.
Melting point: about 250°C (with decomposition).
Identification (1) Dissolve 1 g of Trihexyphenidyl
Hydrochloride in 100 mL of water by warming, and cool.
Use this solution as the sample solution. To 5 mL of the sam-
ple solution add 1 mL of a solution of 2,4,6-trinitrophenol in
chloroform (1 in 50), and shake vigorously: a yellow
precipitate is formed.
(2) To 20 mL of the sample solution obtained in (1) add 2
mL of sodium hydroxide TS: a white precipitate is formed.
Collect the precipitate, wash with a small amount of water,
recrystallize from methanol, and dry in a desiccator (in vacu-
um, silica gel) for 2 hours: the crystals so obtained melt
<2.60> between 113°C and 117°C.
(3) The sample solution obtained in (1) responds to the
Qualitative Tests <1.09> (2) for chloride.
pH <2.54> Dissolve 1 .0 g of Trihexyphenidyl Hydrochloride
in 100 mL of water by warming, and cool: the pH of this so-
lution is between 5.0 and 6.0.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Trihexyphenidyl Hydrochloride in 100 mL of water by warm-
ing: the solution is clear and colorless.
(2) Heavy metals <1.07> — Dissolve 1.5 g of Trihex-
yphenidyl Hydrochloride in 60 mL of water by warming on a
water bath at 80°C, cool, and filter. To 40 mL of the filtrate
add 2 mL of dilute acetic acid and water to make 50 mL, and
perform the test using this solution as the test solution. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion, 2 mL of dilute acetic acid and water to make 50 mL (not
more than 20 ppm).
(3) Piperidylpropiophenone — Dissolve 0.10 g of Trihex-
yphenidyl Hydrochloride in 40 mL of water and 1 mL of 1
mol/L hydrochloric acid VS by warming, cool, and add
water to make 100 mL. Determine the absorbance of this so-
lution at 247 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: the absorbance is not more than 0.50.
Loss on drying <2.41>
3 hours).
Not more than 0.5% (1 g, 105 °C,
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Trihexyphenidyl
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (1:1), and titrate
<2.50> with 0.1 mol/L perchloric acid-dioxane VS (potentio-
metric titration). Perform a blank determination, and make
any necessary correction.
Each mL of 0.1 mol/L perchloric acid-dioxane VS
= 33.793 mg of C 20 H 31 NO.HC1
Containers and storage Containers — Tight containers.
Trihexyphenidyl Hydrochloride
Tablets
Trihexyphenidyl Hydrochloride Tablets contain not
less than 93% and not more than 107% of the labeled
amount of trihexyphenidyl hydrochloride
(C 20 H 31 NO.HC1: 337.93).
Method of preparation Prepare as directed under Tablets,
with Trihexyphenidyl Hydrochloride.
Identification (1) Weigh a quantity of powdered Trihex-
yphenidyl Hydrochloride Tablets, equivalent to 0.1 g of Tri-
hexyphenidyl Hydrochloride according to the labeled
amount, add 30 mL of chloroform, shake, and filter.
Evaporate the filtrate on a water bath to dryness. Dissolve
the residue in 10 mL of water by warming, cool, and use this
JPXV
Official Monographs / Trihexyphenidyl Hydrochloride Tablets 1205
solution as the sample solution. With 5 mL of the sample so-
lution, proceed as directed in the Identification (1) under Tri-
hexyphenidyl Hydrochloride.
(2) Shake a quantity of powdered Trihexyphenidyl
Hydrochloride Tablets, equivalent to 0.01 g of Trihex-
yphenidyl Hydrochloride according to the labeled amount,
with 5 mL of chloroform, filter, and use the filtrate as the
sample solution. Dissolve 0.02 g of Trihexyphenidyl
Hydrochloride Reference Standard in 10 mL of chloroform,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10,mL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform and methanol (9:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly hydrogen hexachloroplatinate
(IV)-potassium iodide TS on the plate: the spots from the
sample solution and the standard solution show a blue-purple
color and the same Ri value.
(3) The sample solution obtained in (1) responds to the
Qualitative Tests <1.09> (2) for chloride.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To one tablet of Trihexyphenidyl Hydrochloride Tablets
add 2 mL of dilute hydrochloric acid and 60 mL of water,
disintegrate by vigorous shaking for 10 minutes, and warm
on a water bath with occasional shaking for 10 minutes.
Cool, add 2 mL of methannol, and add water to make exactly
V mL of the solution contains about 20 ^g of trihexyphenidyl
hydrochloride (C 20 H 31 NO.HC1) per ml. Centrifuge, if neces-
sary, and use the supernatant liquid as the sample solution.
Separately, dissolve about 20 mg of Trihexyphenidyl
Hydrochloride Reference Standard (determine previously its
loss on drying <2.41> in the same manner as Trihexyphenidyl
Hydrochloride) in methanol to make exactly 20 mL. Pipet 2
mL of this solution, and add 2 mL of dilute hydrochloric acid
and water to make exactly 100 mL, and use this solution as
the standard solution. Pipet 10 mL each of the sample solu-
tion and the standard solution, transfer to glass-stoppered
centrifuge tubes, add exactly 10 mL of bromocresol purple-
dipotassium hydrogenphosphate-citric acid TS and 15 mL of
chloroform, stopper tightly, shake well, and centrifuge. Pipet
10 mL each of the chloroform layers, add chloroform to
make exactly 50 mL. Determine the absorbances, A T and A s ,
of the subsequent solutions of the sample solution and stan-
dard solution at 408 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24>, respectively.
Amount (mg) of trihexyphenidyl hydrochloride
(C 2 „H 31 N0.HC1)
= W s x(A T /A s )x(V/l000)
W s : Amount (mg) of Trihexyphenidyl Hydrochlochloride
Reference Standard, calculated on the dried basis
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Trihexyphenidyl
Hydrochloride Tablets at 50 revolutions per minute accord-
ing to the Paddle method, using 900 mL of 2nd fluid for dis-
solution test as the dissolution medium. Take 30 mL or more
of the dissolved solution 30 minutes after starting the test,
and filter through a membrane filter with pore size of not
more than 0.8 /urn. Discard the first 10 mL of the filtrate, and
use the subsequent as the sample solution. Separately, weigh
accurately about 10 mg of Trihexyphenidyl Hydrochloride
Reference Standard, previously dried at 105°C for 3 hours,
and dissolve in 2nd fluid for dissolution test to make exactly
100 mL. Pipet 2 mL of this solution, add 2nd fluid for disso-
lution test to make exactly 100 mL, and use this solution as
the standard solution. Pipet 20 mL each of the sample solu-
tion, the standard solution and 2nd fluid for dissolution test
add exactly 1 mL of diluted acetic acid (31) (1 in 10), and im-
mediately add 5 mL of bromocresol green-sodium hydroxide-
acetic acid-sodium acetate TS, and shake. Then, add exactly
10 mL each of dichloromethane, shake well, centrifuge, and
take the dichloromethane layer. Determine the absorbances,
Aj, A s and A B , of these dichloromethane layers at 415 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using dichloromethane as a blank. The dissolution rate of
Trihexyphenidyl Hydrochloride Tablets in 30 minutes should
be not less than 70%.
Dissolution rate (%) with respect to the labeled amount
of trihexyphenidyl hydrochloride (C 20 H 31 NO.HC1)
= W s x [(A T - A B )/(A S - A B )] x (1/C) x 18
W s : Amount (mg) of Trihexyphenidyl Hydrochloride
Reference Standard
C: Labeled amount (mg) of trihexyphenidyl hydrochloride
(C 20 H 31 NO.HC1) in 1 tablet
Assay Weigh accurately and powder not less than 20 Tri-
hexyphenidyl Hydrochloride Tablets. Weigh accurately a
portion of the powder, equivalent to about 5 mg of trihex-
yphenidyl hydrochloride (C 20 H 3I NO.HC1), dissolve in 2 mL
of dilute hydrochloric acid and 60 mL of water by warming
on a water bath for 10 minutes with occasional shaking. Af-
ter cooling, add 2 mL of methanol and water to make exactly
100 mL, and use this solution as the sample solution. Dis-
solve about 50 mg of Trihexyphenidyl Hydrochloride Refer-
ence Standard (determine previously its loss on drying <2.41>
in the same manner as Trihexyphenidyl Hydrochloride),
weighed accurately, in methanol, add methanol to make ex-
actly 20 mL. Pipet 2 mL of this solution, add 2 mL of dilute
hydrochloric acid and water to make exactly 100 mL, and use
this solution as the standard solution. Pipet 10 mL each of
the sample solution and the standard solution into glass-stop-
pered centrifuge tubes, add exactly 10 mL each of
bromocresol purple-dipotassium hydrogenphosphate-citric
acid TS and 15 mL each of chloroform, stopper tightly,
shake thoroughly, and centrifuge. Pipet 10 mL each of the
chloroform layers, and add chloroform to make exactly 50
mL. Determine the absorbances, A T and A s , of the subse-
quent solutions of the sample solution and standard solution
at 408 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, respectively.
Amount (mg) of trihexyphenidyl hydrochloride
(C 20 H 31 NO.HC1)
= W s x(,4 T A4 s )x(l/10)
W s : Amount (mg) of Trihexyphenidyl Hydrochloride
Reference Standard, calculated on the dried basis
Containers and storage Containers — Tight containers.
1206 Trimebutine Maleate / Official Monographs
JP XV
Trimebutine Maleate
C 22 H 29 N05.C4H 4 04: 503.54
(2/?5)-2-Dimethylamino-2-phenylbutyl 3,4,5-
trimethoxybenzoate monomaleate [34140-59-5]
Trimebutine Maleate, when dried, contains not less
than 98.5% and not more than 101.0% of trimebutine
maleate (CmH^NOs.C^CM.
Description Trimebutine Maleate occurs as white, crystals
or crystalline powder.
It is freely soluble in /V,iV-dimethylformamide and in acetic
acid (100), soluble in acetonitrile, and slightly soluble in
water and in ethanol (99.5).
It dissolves in 0.01 mol/L hydrochloric acid TS.
A solution of it in /V,7V-dimethylformamide (1 in 20) shows
no optical rotation.
Identification (1) Determine the absorption spectrum of a
solution of Trimebutine Maleate in 0.01 mol/L hydrochloric
acid TS (1 in 50,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>, and compare the spectrum with
the Reference Spectrum: both spectra exhibit similar intensi-
ties of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Trimebutine Maleate as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wave numbers.
Melting point <2.60> 131 - 135°C
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Trimebutine Maleate according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 2.0 g
of Trimebutine Maleate according to Method 3, and perform
the test (not more than 1 ppm).
(3) Related substances — Dissolve 0.10 g of Trimebutine
Maleate in 100 mL of a mixture of 0.01 mol/L hydrochloric
acid TS and acetonitrile (13:7), and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add a
mixture of 0.01 mol/L hydrochloric acid TS and acetonitrile
(13:7) to make exactly 250 mL, and use this solution as the
standard solution. Perform the test with exactly 20 /uL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area by the automat-
ic integration method: the area of the peak other than maleic
acid and trimebutine from the sample solution is not more
than 1/2 times the peak area of trimebutine from the stan-
dard solution, and the total area of the peaks other than
maleic acid and trimebutine is not more than the peak area of
trimebutine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: To 650 mL of diluted perchloric acid (17 in
20,000), previously adjusted the pH to 3.0 with a solution of
ammonium acetate (1 in 1000), add 1 g of sodium 1-pen-
tanesulfonate to dissolve. To 650 mL of this solution add 350
mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
trimebutine is about 9 minutes.
Time span of measurement: About 2 times as long as the
retention time of trimebutine beginning after the peak of
maleic acid.
System suitability —
Test for required detectability: Measure exactly 5 mL of
the standard solution, and add a mixture of 0.01 mol/L
hydrochloric acid TS and acetonitrile (13:7) to make exactly
20 mL. Confirm that the peak area of trimebutine obtained
from 20 /xL of this solution is equivalent to 20 to 30% of that
from 20 /xh of the standard solution.
System performance: Dissolve 40 mg of Trimebutine Male-
ate and 20 mg of imipramine hydrochloride in 100 mL of a
mixture of 0.01 mol/L hydrochloric acid TS and acetonitrile
(13:7). When the procedure is run with 20//L of this solution
under the above operating conditions, trimebutine and im-
ipramine are eluted in this order with the resolution between
these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
trimebutine is not more than 5%.
Loss on drying <2.41>
hours).
Not more than 0.5% (1 g, 105 °C, 3
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.8 g of Trimebutine Male-
ate, previously dried, dissolve in 70 mL of acetic acid (100),
and titrate <2.50> with 0.1 mol/L perchloric acid VS until the
color of the solution changes from purple through blue to
blue-green (indicator: 3 drops of crystal violet TS). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 50.35 mg of C 22 H 29 N05.C4H 4 04
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / 1 rimetazidine Hydrochloride Tablets 1207
Trimetazidine Hydrochloride
hj **>?>?>£&£
H 3 C
H 3 C^
C U H 22 N,0 3 .2HC1: 339.26
r
l-(2,3,4-Trimethoxybenzyl)piperazine dihydrochloride
[13171-25-0]
Trimetazidine Hydrochloride contains not less than
98.0 % and not more than 101.0 % of
C14H22N2O3.2HCI, calculated on the dried basis.
Description Trimetazidine Hydrochloride occurs as a
white, crystalline powder.
It is very soluble in water and in formic acid, sparingly
soluble in methanol, and slightly soluble in ethanol (99.5).
The pH of a solution of Trimetazidine Hydrochloride (1 in
20) is between 2.3 and 3.3.
Melting point: about 227°C (with decomposition).
Identification (1) Determine the absorption spectrum of a
solution of Trimetazidine Hydrochloride in 0.1 mol/L
hydrochloric acid TS (1 in 6250) as directed under Ultra-
violet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Trimetazidine Hydrochloride as directed in the potassium
chloride disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) A solution of Trimetazidine Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Trimetazidine Hydrochloride according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Related substances — Dissolve 0.5 g of Trimetazidine
Hydrochloride in 10 mL of water, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of
diethylamine and cyclohexane (1:1) to a distance of about 10
cm, air-dry the plate, and then dry at 110°C for 1 hour. After
cooling, examine under ultraviolet light (main wavelength:
254 nm): the spot other than the principal spot and the spot at
the starting point are not more intense than the spots from
the standard solution.
Water <2.48> Not more than 1.5% (2 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.12 g of Trimetazidine
Hydrochloride, dissolve in 5 mL of formic acid, add exactly
15 mL of 0.1 mol/L perchloric acid VS, and heat at 90-
100°C for 30 minutes. After cooling, add 45 mL of acetic
acid (100), and titrate <2.50> the excess perchloric acid with
0.1 mol/L sodium acetate VS (potentiometric titration). Per-
form a blank determination in the same manner.
Each mL of 0.1 mol/L perchloric acid VS
= 16.96 mg of C 14 H 22 N 2 3 .2HC1
Containers and storage Containers — Tight containers.
Trimetazidine Hydrochloride
Tablets
YV*9
vv x
imfejlmife
Trimetazidine Hydrochloride Tablets contain not
less than 94.0% and not more than 106.0% of the
labeled amount of trimetazidine hydrochloride
(C 14 H 22 N 2 3 .2HC1: 339.26).
Method of preparation Prepare as directed under Tablets,
with Trimetazidine Hydrochloride.
Identification Shake a quantity of powdered Trimetazidine
Hydrochloride Tablets, equivalent to 10 mg of Trimetazidine
Hydrochloride according to the labeled amount, with 10 mL
of a mixture of ethanol (95) and water (3:1), and filter.
Evaporate the filtrate on a water bath, add 2 mL of water to
the residue, and shake. To 1 mL of this solution add 1 mL of
/?-benzoquinone TS, boil gently for 2 to 3 minutes, and cool:
a red color develops.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Trimetazidine Hydrochloride Tablets add 15
mL of a mixture of 0.1 mol/L hydrochloric acid TS and
ethanol (99.5) (1:1), and treat with ultrasonic waves for 10
minutes. Shake the solution for 10 minutes, and add the mix-
ture of 0.1 mol/L hydrochloric acid TS and ethanol (99.5)
(1:1) to make exactly 20 mL. Centrifuge, pipet KmL of the
supernatant liquid, equivalent to about 0.75 mg of trimetazi-
dine hydrochloride (C 14 H 22 N 2 3 .2HC1), add exactly 2.5 mL
of the internal standard solution, add 0.1 mol/L hydrochlor-
ic acid TS to make 50 mL, and use this solution as the sample
solution. Separately, weigh accurately about 30 mg of
trimetazidine hydrochloride for assay, separately determined
the water content <2.48> in the same manner as Trimetazidine
Hydrochloride, and dissolve in the mixture of 0.1 mol/L
hydrochloric acid TS and ethanol (99.5) (1:1) to make exactly
200 mL. Pipet 5 mL of this solution, add exactly 5 mL of the
internal standard solution and 0.1 mol/L hydrochloric acid
TS to make 50 mL, and use this solution as the standard solu-
tion. Proceed as directed in the Assay.
Amount (mg) of trimetazidine hydrochloride
(C 14 H 22 N 2 3 .2HC1) =W s x (Q T /Q S ) x (1/2 V)
W s : Amount (mg) of trimetazidine hydrochloride for as-
say, calculated on the anhydrous basis
Internal standard solution — A solution of parahydroxyben-
1208 Trimethadione / Official Monographs
JP XV
zoic acid in the mixture of 0.1 mol/L hydrochloric acid TS
and ethanol (99.5) (1:1) (7 in 40,000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Trimetazidine Hydrochlo-
ride Tablets at 50 revolutions per minute according to the
Paddle method using 900 mL of water as the dissolution
medium. Withdraw not less than 20 mL of the dissolved solu-
tion 45 minutes after starting the test, and filter through a
membrane filter with a pore size of not more than 0.45 fim.
Discard the first 10 mL of the filtrate, pipet FmL of the sub-
sequent filtrate, and add water to make exactly V mL so that
each mL contains about 3.3 [ig of trimetazidine hydrochlo-
ride (Q4H22N2O3.2HCI) according to the labeled amount.
Pipet 3 mL of this solution, add exactly 3 mL of 0.1 mol/L
hydrochloric acid TS, and use this solution as the sample so-
lution. Separately, weigh accurately about 17 mg of
trimetazidine hydrochloride for assay, separately determined
the water content in the same manner as Trimetazidine
Hydrochloride, and dissolve in water to make exactly 100
mL. Pipet 5 mL of this solution, and add water to make ex-
actly 50 mL. Pipet 5 mL of this solution, and add water to
make exactly 25 mL. Pipet 3 mL of this solution, add exactly
3 mL of 0.1 mol/L hydrochloric acid TS, and use this solu-
tion as the standard solution. Perform the test with exactly 50
/nL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of trimetazidine. The dissolution rate in 45 minutes is
not less than 80%.
Dissolution rate (%) with respect to the labeled amount of
trimetazidine hydrochloride (Q4H22N2O3.2HO)
= W s x (Aj/As) x (V'/V)x (I/O x 18
W s : Amount (mg) of trimetazidine hydrochloride for as-
say, calculated on the anhydrous basis
C: Labeled amount (mg) of trimetazidine hydrochloride
(C14H22N2O3.2HCI) in 1 tablet
Operating conditions —
Proceed as directed in the Assay.
System suitability —
System performance: When the procedure is run with 50
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of trimetazidine are not less than 5000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
trimetazidine is not more than 1.5%.
Assay Weigh accurately not less than 20 tablets of
Trimetazidine Hydrochloride Tablets, and powder. Weigh
accurately a portion of the powder, equivalent to about 3 mg
of trimetazidine hydrochloride (C14H22N2O3.2HO), add
about 15 mL of a mixture of 0.1 mol/L hydrochloric acid TS
and ethanol (99.5) (1:1), and treat with ultrasonic waves for
10 minutes. Then shake for 10 minutes, add the mixture of
0.1 mol/L hydrochloric acid TS and ethanol (99.5) (1:1) to
make exactly 20 mL, and centrifuge. To exactly 5 mL of the
supernatant liquid add exactly 5 mL of the internal standard
solution and 0.1 mol/L hydrochloric acid TS to make 50 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 30 mg of trimetazidine hydrochloride
for assay, separately determined the water content <2.48> in
the same manner as Trimetazidine Hydrochloride, and dis-
solve in the mixture of 0.1 mol/L hydrochloric acid TS and
ethanol (99.5) (1:1) to make exactly 200 mL. To exactly 5 mL
of this solution add exactly 5 mL of the internal standard so-
lution and 0.1 mol/L hydrochloric acid TS to make 50 mL,
and use this solution as the standard solution. Perform the
test with 10 /xL each of the sample solution and standard so-
lution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the ratios,
Qt and Q s , of the peak area of trimetazidine to that of the in-
ternal standard.
Amount (mg) of trimetazidine hydrochloride
(C4H22N2O3.2HCI) = W s x (Q T /Q S ) x 1/10
W s : Amount (mg) of trimetazidine hydrochloride for as-
say, calculated on the anhydrous basis
Internal standard solution — A solution of parahydroxyben-
zoic acid in the mixture of 0.1 mol/L hydrochloric acid TS
and ethanol (99.5) (1:1) (7 in 40,000).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 230 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L potassium di-
hydrogen phosphate TS, pH 3.0 and methanol (17:3).
Flow rate: Adjust the flow rate so that the retention time of
trimetazidine is about 7 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, trimetazidine and the internal standard are eluted in
this order with the resolution between these peaks being not
less than 3.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of trimetazidine to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Trimethadione
h U/^vTt-:
H 3 C
O'
CHj
C 6 H 9 N0 3 : 143.14
3,5,5-Trimethyl-l ,3-oxazolidine-2,4-dione [127-48-0]
Trimethadione, when dried, contains not less than
98.0% of C 6 H 9 N0 3 .
JPXV
Official Monographs / Trimetoquinol Hydrochloride Hydrate 1209
Description Trimethadione occurs as white crystals or crys-
talline powder. It has a camphor-like odor.
It is very soluble in ethanol (95) and in chloroform, freely
soluble in diethyl ether, and soluble in water.
Identification (1) To 5 mL of a solution of Trimethadione
(1 in 50) add 2 mL of barium hydroxide TS: a precipitate is
formed immediately.
(2) Determine the infrared absorption spectrum of a solu-
tion of Trimethadione in chloroform (1 in 50) as directed in
the solution method under Infrared Spectrophotometry
<2.25>, using a 0.1-mm fixed sodium chloride cell, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
Meting point <2.60> 45 - 47 °C
Purity Heavy metals <1.07> — Proceed with 2.0 g of
Trimethadione according to Method 1, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
Loss on drying <2.41> Not more than 0.5% (1 g, silica gel,
6 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Trimethadione,
previously dried, in a glass-stoppered conical flask, dissolve
in 5 mL of ethanol (95), add exactly measured 50 mL of 0.1
mol/L sodium hydroxide VS, stopper, and allow to stand for
15 minutes with occasional shaking. Titrate <2.50> the excess
sodium hydroxide with 0.1 mol/L hydrochloric acid VS (in-
dicator: 4 drops of cresol red TS). Perform a blank determi-
nation.
Each mL of 0.1 mol/L sodium hydroxide VS
= 14.31 mg of C 6 H 9 N0 3
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
Trimethadione Tablets
tion (1) under Trimethadione.
(2) Determine the infrared absorption spectrum of a solu-
tion of the residue obtained in (1) in chloroform (1 in 50) in a
0.1-mm fixed sodium chloride cell, as directed in the solution
method under Infrared Spectrophotometry <2.25>: it exhibits
absorption at the wave numbers of about 2960 cm" 1 , 1814
cm" 1 , 1735 cm" 1 , 1445 cm" 1 , 1394 cm" 1 , 1290 cm" 1 , 1100
cm" 1 and 1055 cm" 1 .
Assay Weigh accurately and powder not less than 20
Trimethadione Tablets. Weigh accurately a portion of the
powder, equivalent to about 1 g of trimethadione
(C 6 H 9 N0 3 ), add 50 mL of ethanol (95), and boil gently for 15
minutes under a reflux condenser. Filter the warm ethanol
(95) solution into a 100-mL volumetric flask through a glass
filter (G4), and wash the residue with three 10-mL portions of
warm ethanol (95). Combine the washings with the filtrate in
the flask, cool, and add ethanol (95) to make exactly 100 mL.
Pipet 25 mL of the solution into a glass-stoppered conical
flask, add 25 mL of water and exactly 30 mL of 0.1 mol/L
sodium hydroxide VS, stopper, allow to stand for 15 minutes
with occasional shaking, and titrate <2.50> the excess sodium
hydroxide with 0.1 mol/L hydrochloric acid VS (indicator: 4
drops of cresol red TS). Perform a blank determination.
Each mL of 0.1 mol/L sodium hydroxide VS
= 14.31 mg of C 6 H 9 N0 3
Containers and storage Containers — Tight containers.
Storage — Not exceeding 30°C.
Trimetoquinol Hydrochloride
Hydrate
Tretoquinol Hydrochloride
r- U/ h^Z-A^K^**!*
l J^ Q Zl • HC " H '°
HJ >*$>*>»
-o
If
Trimethadione Tabelts contain not less than 94%
and not more than 106% of the labeled amount of
trimethadione (C 6 H 9 N0 3 : 143.14).
Method of preparation
with Trimethadione.
Prepare as directed under Tablets,
Identification (1) Weigh a portion of powdered
Trimethadione Tablets, equivalent to 1 g of Trimethadione
according to the labeled amount, add 10 mL of petroleum
benzin, and shake frequently for 15 minutes. Decant, remove
the petroleum benzin, add another 10 mL of petroleum ben-
zin, and repeat the extraction in the same manner. To the
residue add 25 mL of diethyl ether, allow to stand for 20
minutes with occasional shaking, filter, evaporate the filtrate
at room temperature, and dry the residue in a desiccator (sili-
ca gel) for 6 hours: the residue melts <2.60> between 44°C and
47°C. Proceed with this residue as directed in the Identifica-
C 19 H 23 N0 5 .HC1.H 2 0: 399.87
(IS)- 1 -(3 ,4 , 5-Trimethoxybenzyl)- 1,2,3 ,4-tetrahydro-
isoquinoline-6,7-diol monohydrochloride monohydrate
[18559-59-6, anhydride]
Trimetoquinol Hydrochloride Hydrate contains not
less than 98.5% and not more than 101.0% of trimeto-
quinol hydrochloride (C 19 H 23 N0 5 .HC1: 381.85), calcu-
lated on the anhydrous basis.
Description Trimetoquinol Hydrochloride Hydrate occurs
as white, crystals or crystalline powder.
It is freely soluble in methanol, and sparingly soluble in
water and in ethanol (99.5).
Melting point: about 151 °C (with decomposition, after
drying in vacuum, 105°C, 4 hours).
Identification (1) Determine the absorption spectrum of a
1210 Dental Triozinc Paste / Official Monographs
JP XV
solution of Trimetoquinol Hydrochloride Hydrate in 0.01
mol/L hydrochloric acid TS (1 in 20,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Trimetoquinol Hydrochloride Hydrate as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Trimetoquinol Hydrochloride Hydrate
(1 in 50) responds to the Qualitative Tests <1.09> (1) for chlo-
ride.
Optical rotation <2.49> [a]g>: - 16 - - 19° (0.25 g, calculat-
ed on the anhydrous basis, water, after warming and cooling,
25 mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Trimetoquinol Hydrochloride
Hydrate in 100 mL of water by warming, and cool: the pH of
this solution is between 4.5 and 5.5.
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Trimetoquinol Hydrochloride Hydrate in 10 mL of water
by warming: the solution is clear and colorless.
(2) Sulfate <1.14>— Perform the test with 0.5 g of
Trimetoquinol Hydrochloride Hydrate. Prepare the control
solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not
more than 0.038%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Trimeto-
quinol Hydrochloride Hydrate according to Method 2, and
perform the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(4) Related substances — Dissolve 50 mg of Trimeto-
quinol Hydrochloride Hydrate in 50 mL of the mobile phase,
and use this solution as the sample solution. Pipet 1 mL of
this solution, add the mobile phase to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 20 /uL each of the sample solution and stan-
dard solution, as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine each
peak area of both solutions by the automatic integration
method: the total area of the peaks other than that of
trimetoquinol from the sample solution is not larger than the
peak area of trimetoquinol from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 283 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 //m in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2 g of potassium dihydrogen phos-
phate and 2 g of sodium 1-pentane sulfonate in 1000 mL of
water. Adjust with phosphoric acid to a pH between 2.8 and
3.2, and filter through a membrane filter with pore size of 0.4
fim. Add 200 mL of acetonitrile to 800 mL of the filtrate.
Flow rate: Adjust the flow rate so that the retention time of
trimetoquinol is about 7 minutes.
Time span of measurement: About twice as long as the
retention time of trimetoquinol beginning after the solvent
peak.
System suitability —
Test for required detection: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of trimetoquinol obtained from
20,mL of this solution is equivalent to 7 to 13% of that of
trimetoquinol obtained from 20 /xL of the standard solution.
System performance: Dissolve 5 mg of Trimetoquinol
Hydrochloride Hydrate and 1 mg of procaine hydrochloride
in 50 mL of the mobile phase. When the procedure is run
with 20 /xL of this solution under the above operating condi-
tions, procaine and trimetoquinol are eluted in this order
with the resolution between these peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
trimetoquinol is not more than 2.0%.
Water <2.48> 3.5 - 5.5% (0.3 g, volumetric titration, direct
titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Trimetoquinol
Hydrochloride Hydrate, dissolve in 2 mL of 0.1 mol/L
hydrochloric acid VS and 70 mL of ethanol (99.5) with
thorough shaking, and titrate <2.50> with 0.1 mol/L potassi-
um hydroxide-ethanol VS (potentiometric titration). Calcu-
late the consumed volume of 0.1 mol/L potassium hydrox-
ide-ethanol VS between the first inflection point and of the
second inflection point.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
= 38.19 mg of Ci 9 H 23 N0 5 .HCl
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Dental Triozinc Paste
Sf4ffl I- U7t-^>^/\°x^
Dental Triozinc Paste consists of a powder contain-
ing Paraformaldehyde, Thymol, anhydrous zinc sul-
fate and Zinc Oxide, and a solution containing Cresol,
Potash Soap and Glycerin. Suitable amounts of the
two components are triturated before use.
Method of preparation
(1) The powder
Paraformaldehyde, finely powdered
Thymol, finely powdered
Zinc Sulfate Hydrate
Zinc Oxide
10 g
3g
9g
82 g
To make about 100 g
Heat Zinc Sulfate Hydrate at about 250°C to obtain anhy-
drous zinc sulfate, cool, and pulverize to a fine powder. Mix
homogeneously this powder with Thymol, Paraformalde-
hyde, and Zinc Oxide.
(2) The solution
JP XV
Official Monographs / L-Tryptophan 1211
Cresol
Potash Soap
Glycerin
40;
40;
20;
To make 100 g
Dissolve Potash Soap in a mixture of Cresol and Glycerin.
Description The powder occurs as a fine, white powder,
having a characteristic odor. The solution is a clear, yellow-
brown to red-brown, viscous liquid, having the odor of
cresol.
Containers and storage Containers — Tight containers.
Tropicamide
hntij]= K
and enantiomer
C 17 H 20 N 2 O 2 : 284.35
(2/?5 , )-A^-Ethyl-3-hydroxy-2-phenyl-AT-(pyridin-4-
ylmethyl)propanamide [1508-75-4]
Tropicamide, when dried, contains not less than
98.5% of C 17 H 20 N 2 O 2 .
Description Tropicamide occurs as a white, crystalline pow-
der. It is odorless, and has a bitter taste.
It is freely soluble in ethanol (95) and in chloroform, slight-
ly soluble in water and in diethyl ether, and practically insolu-
ble in petroleum ether.
It dissolves in dilute hydrochloric acid.
The pH of a solution of Tropicamide (1 in 500) is between
6.5 and 8.0.
Identification (1) To 5 mg of Tropicamide add 0.5 mL of
a solution of ammonium vanadate (V) in sulfuric acid, (1 in
200), and heat: a blue-purple color develops.
(2) Dissolve 5 mg of Tropicamide in 1 mL of ethanol (95)
and 1 mL of water, add 0.1 g of l-chloro-2,4-dinitrobenzene,
and heat on a water bath for 5 minutes. Cool, and add 2 to 3
drops of a solution of sodium hydroxide (1 in 10) and 3 mL
of ethanol (95): a red-purple color develops.
Absorbance <2.24> E l /° m (255 nm): 166 - 180 (after drying,
5 mg, 2 mol/L hydrochloric acid TS, 200 mL).
Melting point <2.60> 96
°C
Purity (1) Chloride <1.03> — Dissolve 1.0 g of Tropica-
mide in 30 mL of ethanol (95), add 6 mL of dilute nitric acid
and water to make 50 mL, and perform the test using this so-
lution as the test solution. Prepare the control solution with
0.45 mL of 0.01 mol/L hydrochloric acid VS, 30 mL of
ethanol (95), 6 mL of dilute nitric acid, and add water to
make 50 mL (not more than 0.016%).
(2) Heavy metals <1.07> — Dissolve 1.0 g of Tropicamide
in 30 mL of ethanol (95), add 2 mL of dilute acetic acid and
water to make 50 mL, and perform the test using this solution
as the test solution. Prepare the control solution with 2.0 mL
of Standard Lead Solution, 30 mL of ethanol (95), 2 mL of
dilute acetic acid and water to make 50 mL (not more than 20
ppm).
(3) JV-Ethyl-y-picolylamine — Dissolve 0.10 g of
Tropicamide in 5 mL of water by heating, add 1 mL of a so-
lution of acetaldehyde (1 in 20), and shake well. Add 1 to 2
drops of sodium pentacyanonitrosylferrate (III) TS and 1 to 2
drops of sodium hydrogen carbonate TS, and shake: no blue
color develops.
(4) Tropic acid — To 10 mg of Tropicamide add 5 mg of
sodium borate and 7 drops of 4-dimethylaminobenzaldehyde
TS, and heat in a water bath for 3 minutes. Cool in ice water,
and add 5 mL of acetic anhydride: no red-purple color de-
velops.
Loss on drying <2.41> Not more than 0.30% (1 g, in vacu-
um, silica gel, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tropicamide, previ-
ously dried, dissolve in 50 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (indicator: 3 drops
of crystal violet TS). Perform a blank determination, and
make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 28.44 mg of C, 7 H 20 N 2 O 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
L-Tryptophan
l- r- U ~f h "7 r >
C u H 12 N 2 2 : 204.23
(2 S)-2-Amino-3 -(indol-3 -yl)propanoic acid
[73-22-3]
L-Tryptophan, when dried,
98.5% of C„H 12 N 2 2 .
contains not less than
Description L-Tryptophan occurs as white to yellowish
white crystals or crystalline powder. It is odorless, and has a
slightly bitter taste.
It is freely soluble in formic acid, slightly soluble in water,
and very slightly soluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Tryptophan, previously dried, as directed in the potassi-
um bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [ a ]g>: -30.0- -33.0° Weigh ac-
curately about 0.25 g of L-Tryptophan, previously dried, and
dissolve in 20 mL of water by warming. After cooling, add
water to make exactly 25 mL, and determine the optical rota-
tion of the solution in a 100-mm cell.
1212 Tubocurarine Chloride Hydrochloride Hydrate / Official Monographs
JP XV
pH <2.54> Dissolve 1.0 g in 100 mL of water by warming,
and cool: the pH of this solution is between 5.4 and 6.4.
Purity (1) Clarity of solution — Dissolve 0.20 g of L-Tryp-
tophan in 10 mL of 2 mol/L hydrochloric acid TS: the solu-
tion is clear.
(2) Chloride <1.03> — Dissolve 0.5 g of L-Tryptophan in 6
mL of dilute nitric acid, and add water to make 50 mL. Per-
form the test using this solution as the test solution. Prepare
the control solution with 0.30 mL of 0.01 mol/L hydrochloric
acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Dissolve 0.6 g of L-Tryptophan in 40
mL of water and 1 mL of dilute hydrochloric acid, and add
water to make 50 mL. Perform the test using this solution as
the test solution. Prepare the control solution with 0.35 mL
of 0.005 mol/L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Tryptophan. Prepare the control solution with 5.0 mL of
Standard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of L-Tryp-
tophan according to Method 4, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 20 ppm).
(6) Arsenic <1.11> — Dissolve 1.0 g of L-Tryptophan in 3
mL of 1 mol/L hydrochloric acid TS and 2 mL of water by
heating, and perform the test with this solution as the test so-
lution (not more than 2 ppm).
(7) Related substances — Dissolve 0.30 g of L-Tryptophan
in 1 mL of 1 mol/L hydrochloric acid TS, add water to make
50 mL, and use this solution as the sample solution. Pipet 1
mL of the sample solution, and add water to make exactly 50
mL. Pipet 5 mL of this solution, add water to make exactly
20 mL, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 [iL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (3:1:1) to a distance of
about 10 cm, and dry the plate at 80°C for 30 minutes. Spray
evenly a solution of ninhydrin in acetone (1 in 50) on the
plate, and heat at 80°C for 5 minutes: the spots other than
the principal spot from the sample solution are not more in-
tense than the spot from the standard solution.
Loss on drying <2.41> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of L-Tryptophan,
previously dried, dissolve in 3 mL of formic acid, add 50 mL
of acetic acid (100), and titrate <2.50> with 0.1 mol/L per-
chloric acid VS (potentiometric titration). Perform a blank
determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 20.42 mg of C„H 12 N 2 2
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tubocurarine Chloride
Hydrochloride Hydrate
Tubocurarine Hydrochloride
CI -HCI'SHjG
C 37 H 41 C1N 2 6 .HC1.5H 2 0: 771.72
7',12'-Dihydroxy-6,6'-dimethoxy-2,2',2'-
trimethyltubocuraranium chloride monohydrochloride
pentahydrate [41354-45-4]
Tubocurarine Chloride Hydrochloride Hydrate con-
tains not less than 98.0% of tubocurarine chloride
hydrochloride (C 37 H 41 C1N 2 6 .HC1: 681.65), calculated
on the dried basis.
Description Tubocurarine Chloride Hydrochloride Hydrate
occurs as white crystals or crystalline powder. It is odorless.
It is sparingly soluble in water and in ethanol (95), slightly
soluble in acetic acid (100), and practically insoluble in
diethyl ether and in chloroform.
The pH of a solution of Tubocurarine Chloride
Hydrochloride Hydrate (1 in 100) is between 4.0 and 6.0.
Melting point: about 270°C (with decomposition).
Identification (1) To 20 mL of a solution of Tubocurarine
Chloride Hydrochloride Hydrate (1 in 2000) add 0.2 mL of
sulfuric acid and 2 mL of a solution of potassium iodate (1 in
100), shake, and heat on a water bath for 30 minutes: a yel-
low color is produced.
(2) To 1 mL of a solution of Tubocurarine Chloride
Hydrochloride Hydrate (1 in 100) add 1 mL of a solution of
Reinecke salt (1 in 25): a red precipitate is formed.
(3) Determine the absorption spectrum of a solution of
Tubocurarine Chloride Hydrochloride Hydrate (3 in 100,000)
as directed under Ultraviolet-visible Spectrophotometry
<2.24>, and compare the spectrum with the Reference Spec-
trum or the spectrum of a solution of Tubocurarine Chloride
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(4) A solution of Tubocurarine Chloride Hydrochloride
Hydrate (1 in 50) responds to Qualitative Tests <1.09> (2) for
chloride.
Optical rotation <2.49> [a]™: +210- +220° (0.1 g, calcu-
lated on the dried basis, water, 10 mL, after allowing to stand
for 3 hours, 100 mm).
Purity (1) Clarity and color of solution — Dissolve 0.10 g
of Tubocurarine Chloride Hydrochloride Hydrate in 10 mL
JPXV
Official Monographs / Tulobuterol Hydrochloride 1213
of ethanol (95): the solution is clear and colorless.
(2) Chloroform-soluble substances — Weigh accurately
about 0.2 g of Tubocurarine Chloride Hydrochloride Hy-
drate, calculated on the dried basis, add 200 mL of water and
1 mL of a saturated solution of sodium hydrogen carbonate,
and extract with three 20-mL portions of chloroform. Com-
bine the chloroform extracts, wash with 10 mL of water,
filter the chloroform solution through absorbent cotton into
a tared beaker, wash the absorbent cotton with two 5-mL
portions of chloroform, and combine the filtrate and the
washings. Evaporate the chloroform on a water bath, and
dry the residue at 105 °C for 1 hour: the mass of the residue is
not more than 2.0% of the mass of Tubocurarine Chloride
Hydrochloride Hydrate taken. Add 10 mL of water to the
residue: the residue does not dissolve. Add 1 mL of
hydrochloric acid, and stir: the residue dissolves.
Loss on drying <2.41> 9 - 12% (0.5 g, in vacuum, phospho-
rus (V) oxide, 105°C, 4 hours).
Residue on ignition <2.44> Not more than 0.2% (0.5 g).
Assay Weigh accurately about 0.5 g of Tubocurarine Chlo-
ride Hydrochloride Hydrate, add 20 mL of acetic acid (100),
and dissolve by warming on a water bath. After cooling, add
60 mL of acetic anhydride, and titrate <2.50> with 0.1 mol/L
perchloric acid VS (potentiometric titration). Perform a
blank determination, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 34.08 mg of C 37 H 41 C1N 2 6 .HC1
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Tubocurarine Chloride
Hydrochloride Injection
Tubocurarine Hydrochloride Injection
V#9=7 'J >i&ik%>}imi&Mm
rine Chloride Hydrochloride Hydrate.
(3) To a volume of Tubocurarine Chloride Hydrochlo-
ride Injection, equivalent to 3 mg of Tubocurarine Chloride
Hydrochloride Hydrate according to the labeled amount, add
water to make 100 mL, and determine the absorption spec-
trum of the solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
279 nm and 281 nm, and a minimum between 253 nm and
257 nm.
Optical rotation <2.49> a™'- + 0.35 - + 0.42° (200 mm), cal-
culated with reference to the value of solution containing 1
mg of Tubocurarine Chloride Hydrochloride Hydrate per
mL, according to the labeled amount of Tubocurarine Chlo-
ride Hydrochloride Injection.
pH <2.54> 3.0-6.0
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Tubocurarine Chloride
Hydrochloride Injection, equivalent to about 15 mg of
tubocurarine chloride hydrochloride hydrate (C37H 41 C1N 2 6
.HC1.5H 2 0), add water to make exactly 500 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 15 mg of Tubocurarine Chloride Hydrochloride Refer-
ence Standard (separately determine the loss on drying <2.41>
in the same manner as Tubocurarine Chloride Hydrochloride
Hydrate), dissolve in water to make exactly 500 mL, and use
this solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and the standard
solution at 280 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, respectively.
Amount (mg) of tubocurarine chloride hydrochloride
hydrate (C 37 H 41 C1N 2 6 .HC1.5H 2 0)
= W s x(A T /A s )X 1.1321
W s : Amount (mg) of Tubocurarine Chloride Hydrochlo-
ride Reference Standard, calculated on the dried basis
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, and under Nitrogen atmosphere.
Tubocurarine Chloride Hydrochloride Injection is
an aqueous solution for injection. It contains not less
than 93% and not more than 107% of the labeled
amount of tubocurarine chloride hydrochloride hy-
drate (C 37 H 4 iClN 2 6 .HC1.5H 2 0: 771.72).
Method of preparation Prepare as directed under Injec-
tions, with Tubocurarine Chloride Hydrochloride Hydrate.
Description Tubocurarine Chloride Hydrochloride Injec-
tion is a clear, colorless liquid.
Identification (1) To a volume of Tubocurarine Chloride
Hydrochloride Injection, equivalent to 0.01 g of Tubocura-
rine Chloride Hydrochloride Hydrate according to the la-
beled amount, add water to make 20 mL, and proceed as
directed in the Identification (1) under Tubocurarine Chlo-
ride Hydrochloride Hydrate.
(2) Proceed with a volume of Tubocurarine Chloride
Hydrochloride Injection, equivalent to 3 mg of Tubocurarine
Chloride Hydrochloride Hydrate according to the labeled
amount, as directed in the Identification (2) under Tubocura-
Tulobuterol Hydrochloride
H OH
»HCI
and enantiomer
C 12 H I8 C1N0.HC1: 264.19
(li?S>l-(2-Chlorophenyl)-2-(l,l-
dimethylethyl)aminoethanol monohydrochloride
[56776-01-3]
Tulobuterol Hydrochloride, when dried, contains
not less than 98.5% of C 12 H 18 ClNO.HCl.
Description Tulobuterol Hydrochloride occurs as white
crystals or crystalline powder.
It is very soluble in methanol, freely soluble in water, in
ethanol (95) and in acetic acid (100), sparingly soluble in acet-
ic anhydride, and very slightly soluble in diethyl ether.
1214 Turpentine Oil / Official Monographs
JP XV
A solution of Tulobuterol Hydrochloride (1 in 20) shows
no optical rotation.
Melting point: about 163°C
Identification (1) Determine the absorption spectrum of a
solution of Tulobuterol Hydrochloride (1 in 2500) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Tulobuterol Hydrochloride, previously dried, as directed in
the potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spctra exhibit similar intensities of
absorption at the same wave numbers.
(3) A solution of Tulobuterol Hydrochloride (1 in 20)
responds to the Qualitative Tests <1.09> for chloride.
Purity (1) Clarity and color of solution — Dissolve
1.0 g of Tulobuterol Hydrochloride in 10 mL of water: the
solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 2.0 g of
Tulobuterol Hydrochloride according to Method 1, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Related substances — Dissolve 0.30 g of Tulobuterol
Hydrochloride in 5 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Use a
plate previously developed with the upper-layer of a mixture
of ethyl acetate and ammonia solution (28) (200:9) to the top
of the plate and air-dried. Spot 5 /xL each of the sample solu-
tion and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. De-
velop the plate with the upper layer of a mixture of ethyl
acetate and ammonia solution (28) (200:9) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): the spots other than the
principal spot and the spot of starting point from the sample
solution are not more intense than the spot from the standard
solution.
Loss on drying <2.41> Not more than 0.5% (0.5 g, in vacu-
um, 60°C, 4 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Tulobuterol
Hydrochloride, previously dried, dissolve in 80 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 26.42 mg of C 12 H 18 C1N0.HC1
Containers and storage Containers — Tight containers.
Turpentine Oil
Oleum Terebinthinae
Turpentine Oil is the essential oil distilled with steam
from the wood or balsam of Pinus species (Pinaceae).
Description Turpentine Oil is a clear, colorless to pale yel-
low liquid. It has a characteristic odor and a pungent, bitter
taste.
Turpentine Oil (1 mL) is miscible with 5 mL of ethanol (95)
and this solution is neutral.
Refractive index <2.45> n™: 1.465 - 1.478
Specific gravity <7.75> d^j: 0.860 - 0.875
Purity (1) Foreign matter — Turpentine Oil has no offen-
sive odor. Shake 5 mL of Turpentine Oil with 5 mL of a solu-
tion of potassium hydroxide (1 in 6): the aqueous layer does
not show a yellow-brown to dark brown color.
(2) Hydrochloric acid-coloring substances — Shake 5 mL
of Turpentine Oil with 5 mL of hydrochloric acid, and allow
to stand for 5 minutes: the hydrochloric acid layer is light yel-
low and not brown in color.
(3) Mineral oil — Place 5 mL of Turpentine Oil in a Cassia
flask, cool to a temperature not exceeding 15°C, add drop-
wise 25 mL of fuming sulfuric acid while shaking, warm be-
tween 60°C and 65 °C for 10 minutes, and add sulfuric acid to
raise the lower level of the oily layer to the graduated portion
of the neck: not more than 0.1 mL of oil separates.
Distilling range <2.57> 150 - 170°C, not less than 90 vol%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ubidecarenone
3.tT7]l//>
C 59 H 90 O 4 : 863.34
(2£,6£,10£,14£,18£,22£',26£',30£',347i,38£>2-
(3,7,11,15,19,23,27,31,35,39-Decamethyltetraconta-
2,6,10,14,18,22,26,30,34,38-decaen-l-yl)-5,6-dimethoxy-3-
methyl-1 ,4-benzoquinone [303-98-0]
Ubidecarenone contains not less than 98.0% of
C59H90O4, calculated on the anhydrous basis.
Description Ubidecarenone occurs as a yellow to orange
crystalline powder.
It is odorless and has no taste.
It is soluble in diethyl ether, very slightly soluble in ethanol
(99.5), and practically insoluble in water.
JPXV
Official Monographs / Ulinastatin 1215
It is gradually decomposed and colored by light.
Melting point: about 48°C
Identification (1) Dissolve 0.05 g of Ubidecarenone in 1
mL of diethyl ether, and add 10 mL of ethanol (99.5). To 2
mL of this solution add 3 mL of ethanol (99.5) and 2 mL of
dimethyl malonate, then add dropwise 1 mL of a solution of
potassium hydroxide (1 in 5), and mix: a blue color appears.
(2) Determine the infrared absorption spectrum of
Ubidecarenone as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Ubidecarenone Reference Standard: both spectra ex-
hibit similar intensities of absorption at the same wave num-
bers.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Ubidecarenone according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.05 g of Ubidecare-
none in 50 mL of ethanol (99.5) by warming at about 50°C
for 2 minutes, and after cooling use this solution as the sam-
ple solution. To exactly 1 mL of the sample solution add
ethanol (99.5) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 5 /uL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine each peak area of both solu-
tions by the automatic integration method: the total area of
the peaks other than the peak of ubidecarenone from the
sample solution is not larger than the peak area of ubidecare-
none from the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase,
flow rate, and selection of column: Proceed as directed in the
operating conditions in the Assay.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of ubidecarenone obtained from 5 juL of
the standard solution is between 20 mm and 40 mm.
Time span of measurement: About 2 times of the retention
time of ubidecarenone beginning after the solvent peak.
Water <2.48> Not more than 0.20% (1 g, direct titration).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 50 mg each of Ubidecare-
none and Ubidecarenone Reference Standard (separately de-
termined the water content <2.48> in the same manner as
Ubidecarenone) dissolve each in 40 mL of ethanol (99.5) by
warming at about 50°C for 2 minutes, and after cooling add
ethanol (99.5) to make exactly 50 mL each, and use these so-
lutions as the sample solution and the standard solution. Per-
form the test with exact 5 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
peak areas, A T and A s , of ubidecarenone of these solution.
Amount (mg) of C 59 H 90 O 4
= W s x(A T /A s )
W s : Amount (mg) of Ubidecarenone Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 275 nm).
Column: A stainless steel column about 5 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: A constant temperature of about
35°C.
Mobile phase: A mixture of methanol and ethanol (99.5)
(13:7).
Flow rate: Adjust the flow rate so that the retention time of
ubidecarenone is about 10 minutes.
Selection of column: Dissolve 0.01 g each of Ubidecare-
none and ubiquinone-9 in 20 mL of ethanol (99.5) by warm-
ing at about 50°C for 2 minutes. After cooling, proceed with
5 /xL of this solution under the above operating conditions,
and calculate the resolution. Use a column giving elution of
ubiquinone-9 and ubidecarenone in this order with the reso-
lution between these peaks being not less than 4.
System repeatability: Repeat the test five times with the
standard solution under the above operating conditions: the
relative standard deviation of the peak areas of ubidecare-
none is not more than 0.8%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ulinastatin
Ulinastatin is a solution of a glycoprotein having
trypsin inhibiting activity, which is separated and puri-
fied from human urine.
It contains ulinastatin of not less than 45,000 Units
per mL and not less than 2500 Units per mg protein.
Description Ulinastatin occurs as a light brown to brown,
clear liquid.
Identification (1) Dilute a suitable amount of Ulinastatin
with water to make a solution containing 4000 Units of
ulinastatin per mL. To 1 mL of this solution add 1 mL of a
solution of phenol (1 in 20), then carefully add 5 mL of sul-
furic acid, and mix: an orange to red-orange color develops.
(2) Dilute a suitable quantity of Ulinastatin with water to
make a solution containing 2000 units per mL. Determine the
absorption spectrum of the solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(3) Dilute a suitable amount of Ulinastatin with pH 7.8
2,2',2"-nitrilotrisethanol buffer solution to make a solution
containing 500 Units of ulinastatin per mL, and use this solu-
tion as the sample solution. Use the same buffer solution as
the control solution. To 0.1 mL each of the sample solution
and the control solution add 1.6 mL of the buffer solution
and 0.2 mL of trypsin TS for test of ulinastatin, mix, and al-
low them to stand in a water bath at 25 °C for 1 minute. Then
add 1 mL of A r -a-benzoyl-L-arginine-4-nitroanilide TS, mix,
and allow them to stand at 25 C C for 2 minutes: the solution
obtained with the sample solution develops no color while
that obtained with the control solution develops a yellow
1216 Ulinastatin / Official Monographs
JP XV
color.
(4) To 1.5 g of Powdered Agar add 100 mL of pH 8.4
boric acid-sodium hydroxide buffer solution, dissolve by
warming in a water bath, then pour immediately into a Petri
dish placed horizontally so that the agar layer is about 2 mm
in thickness. After the agar becomes hard, bore two wells
about 2.5 mm in diameter with a separation of 6 mm from
each other. In one of the wells place 10,mL of a solution of
Ulinastatin containing 500 Units per mL in pH 8.4 boric acid-
sodium hydroxide buffer solution, and in the other well place
10 /xL of anti-ulinastatin rabbit serum, cover the dish to
avoid drying of the agar, and allow to stand for overnight at
a room temperature: a clear precipitin line appears between
the wells.
pH <2.54> 6.0 - 8.0
Specific activity When calculated from the results obtained
by the Assay and the following method, the specific activity is
not less than 2500 Units per 1 mg protein.
(i) Sample solution — To an exactly measured volume of
Ulinastatin, equivalent to about 10,000 Units according to
the labeled amount, add water to make exactly 20 mL.
(ii) Standard solutions — Weigh accurately about 10 mg of
bovine serum albumin for test of ulinastatin, and dissolve in
water to make exactly 20 mL. To a suitable volume of this so-
lution add water to make four solutions containing exactly
300, 200, 100 and 50 /ug of the albumin per mL, respectively.
(iii) Procedure — Pipet 0.5 mL each of the sample solution
and standard solutions, put them in glass test tubes about 18
mm in internal diameter and about 130 mm in length, add ex-
actly 5 mL of alkaline copper TS, mix, and allow the tubes to
stand in a water bath at 30°C for 10 minutes. Then add exact-
ly 0.5 mL of diluted Folin' s TS (1 in 2), mix, and warm in the
water bath for 20 minutes. Determine the absorbances of
these solutions at 750 nm as directed under Ultraviolet-visible
Spectrophotometry <2.24> using a solution obtained in the
same manner with 0.5 mL of water as the blank.
Plot the absorbances of the standard solutions on the verti-
cal axis and their protein concentrations on the horizontal
axis to prepare a calibration curve, and determine the protein
content of the sample solution from its absorbance by using
this curve. Then calculate the amount of protein per mL of
Ulinastatin.
Purity (1) Heavy metals <1.07> — Proceed with 10 mL of
Ulinastatin according to Method 2, and perform the test.
Prepare the control solution with 1.0 mL of Standard Lead
Solution (not more than 1 ppm).
(2) Related substances — To a suitable volume of
Ulinastatin add water to make a solution containing exactly
12,500 Units per mL, and use this solution as the sample
stock solution. To exactly 0.25 mL of the sample stock solu-
tion add exactly 0.2 mL of glycerin and exactly 0.05 mL of
0.05% bromophenol blue TS, mix, and use this solution as
the sample solution. Separately, to exactly 1 mL of the sam-
ple stock solution add water to make exactly 100 mL. To ex-
actly 0.25 mL of this solution add exactly 0.2 mL of glycerin
and exactly 0.05 mL of 0.05% bromophenol blue TS, mix,
and use this solution as the standard solution. Perform the
following test with the sample solution and the standard solu-
tion: the bands other than the principal band obtained from
the sample solution are not more intense than the band from
the standard solution in the electrophoretogram.
(i) Tris buffer solution for polyacrylamide gel elec-
trophoresis A Dissolve 18.2 g of 2-amino-2-hydroxymethyl-
1,3-propanediol in 80 mL of water, adjust to pH 8.8 with 6
mol/L hydrochloric acid TS, and add water to make 100 mL.
(ii) Tris buffer solution for polyacrylamide gel elec-
trophoresis B Dissolve 6.0 g of 2-amino-2-hydroxymethyl-
1,3-propanediol in 80 mL of water, adjust to pH 8.8 with 6
mol/L hydrochloric acid TS, and add water to make 100 mL.
(iii) Tris buffer solution for polyacrylamide gel elec-
trophoresis C Dissolve 3.0 g of 2-amino-2-hydroxymethyl-
1,3-propanediol and 14.4 g of glycine in water to make 1000
mL.
(iv) Acrylamide solution for polyacrylamide gel elec-
trophoresis Dissolve 30 g of acrylamide and 0.8 g of N,N'-
methylenebisacrylamide in water to make 100 mL.
(v) Gel for separation Mix gently 15 mL of tris buffer
solution for polyacrylamide gel electrophoresis A, 20 mL of
acrylamide solution for polyacrylamide gel electrophoresis,
24.5 mL of water, 0.022 mL of N.N.N'.N'-
tetramethylethylenediamine, 0.32 mL of 10% ammonium
peroxodisulfate TS and 0.3 mL of 1 mol/L sodium sulfite TS,
pour into a plate for slab gel preparation, then cover the gel
mixture with a layer of water, and allow to set for 1 hour.
(vi) Gel for concentration Remove the water layer on
the gel for separation, and pour a mixture of 2.5 mL of tris
buffer solution for polyacrylamide gel electrophoresis B, 2.66
mL of acrylamide solution for polyacrylamide gel elec-
trophoresis, 14.6 mL of water, 0.01 mL of N,N,N',N'-
tetramethylethylenediamine, 0.2 mL of 10% ammonium
peroxodisulfate TS and 0.04 mL of 1 mol/L sodium sulfite
TS on the gel. Then position a plastic sample well former so
that the height of the gel for concentration is about 15 mm,
and allow to set for 2 hours.
(vii) Procedure
Electrophoresis — Set the gel in an apparatus for slab gel
electrophoresis, and fill the upper and lower reservoirs with
tris buffer solution for polyacrylamide gel electrophoresis C.
Introduce carefully 10 /xL each of the sample solution and
standard solution into the wells using a different well for each
solution, and allow electrophoresis to proceed using the elec-
trode of the lower reservoir as the anode. Switch off the pow-
er supply when the bromophenol blue band has migrated to
about 10 mm from the bottom of the gel.
Staining — Dissolve 2.0 g of Coomassie brilliant blue R-250
in 400 mL of a mixture of methanol and 100 mL of acetic
acid (100), add water to make 1000 mL, and use this solution
as the staining solution. Stain the gel for 2 hours in the stain-
ing solution warmed to 40°C.
Decolorization — To 100 mL of methanol and 75 mL of
acetic acid (100) add water to make 1000 mL, and use this so-
lution as the rinsing solution. Immerse the gel removed from
the staining solution in the rinsing solution to decolorise.
(3) Kallidinogenase — Dilute a suitable volume of
Ulinastatin with water so that each mL of the solution con-
tains about 50,000 Units, and use this solution as the sample
solution. Take exactly 0.4 mL of the sample solution into a
test tube, add exactly 0.5 mL of pH 8.2 tris buffer solution,
mix, and allow the tube to stand in a water bath at 37 ±
0.2°C for 5 minutes. Add exactly 0.1 mL of substrate TS for
kallidinogenase assay (4), mix, allow the tube to stand in the
water bath of 37 ± 0.2°C for exactly 30 minutes, then add
exactly 0.1 mL of diluted acetic acid (100) (1 in 2), mix, and
use this solution as the test solution. Separately, take exactly
JP XV
Official Monographs / Urapidil 1217
0.4 mL of the sample solution in a test tube, add exactly 0.5
mL of pH 8.2 tris buffer solution, mix, and allow the tube to
stand in the water bath of 37 ± 0.2°C for 35 minutes. Then
add exactly 0.1 mL of diluted acetic acid (100) (1 in 2), mix,
add exactly 0.1 mL of substrate TS for kallidinogenase assay
(4), mix, and use this solution as the control solution. Deter-
mine the absorbances of the test solution and the control so-
lution at 405 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24> using water as the blank, and calculate
the difference between them: the difference is not more than
0.050.
Molecular mass Dilute a suitable volume of Ulinastatin
with the mobile phase so that each mL of the solution con-
tains about 6500 Units, and use this solution as the sample so-
lution. Separately, dissolve 1.0 mg each of y-globulin (mol.
mass: 160,000), bovine serum albumin for test of ulinastatin
(mol. mass: 67,000), and myoglobin (mol. mass: 17,000) in
about 1 mL of the mobile phase, and use this solution as the
molecular mass reference solution. Perform the test with 50
/uL each of the sample solution and molecular mass reference
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Prepare a calibration
curve by plotting the logarithm of molecular masses on the
vertical axis and the retention times (min) of the molecular
mass reference substances on the horizontal axis, and deter-
mine the molecular mass of the sample using the calibration
curve and the retention time obtained with the sample solu-
tion: the molecular mass is 67,000 ± 5000.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 280 nm).
Column: A stainless steel column about 7 mm in inside di-
ameter and about 60 cm in length, packed with porous silica
gel for liquid chromatography (10-12//m in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: Dissolve 16.33 g of potassium dihydrogen-
phosphate and 124.15 g of ethylene glycol in water to make
1000 mL. If necessary, adjust to pH 4.0 with phosphoric
acid.
Flow rate: Adjust the flow rate so that the retention time of
bovine serum albumin is about 36 minutes.
Selection of column: Proceed with 50 /xh of the molecular
mass reference solution according to the above operating
conditions, and calculate the resolution. Use a column from
which y-globulin, bovine serum albumin and myoglobin are
eluted in this order with the resolution between their peaks
being not less than 1.5, respectively.
Antigenicity Dilute a suitable volume of Ulinastatin with
isotonic sodium chloride solution so that each mL of the so-
lution contains 15,000 Units, and use this solution as the sam-
ple solution. Inject 0.10 mL of the sample solution on 3 occa-
sions at intervals of 2 days into the peritoneal cavity of each
of 4 well-nourished, healthy guinea pigs weighing 250 to 300
g. Inject 0.10 mL of horse serum into the peritoneal cavity of
each of 4 guinea pigs of another group as a control. Inject
0.20 mL of the sample solution intravenously into each of 2
guinea pigs of the first group 14 days after the first in-
traperitoneal injection and into each of the remaining 2
guinea pigs 21 days after the injection, and inject 0.20 mL of
horse serum intravenously in the same manner into each
guinea pig of the second group. Observe the signs of respira-
tory distress, collapse or death of the animals for 30 minutes
after each intravenous injection and 24 hours later: the
animals of the first group exhibit none of the signs mentioned
above, and all the animals of the second group exhibit sym-
ptoms of respiratory distress or collapse and not less than 3
animals are killed.
Toxicity Inject intravenously 0.50 mL of Ulinastatin into
each of five well-fed, healthy albino mice weighing 18 to 25 g:
no mouse dies within 48 hours after injection. If any mouse
dies within 48 hours, repeat the test using 5 albino mice
weighing 19 to 21 g: all the animals survive for 48 hours.
Assay Measure exactly a suitable volume of Ulinastatin, di-
lute with 2,2',2"-nitrilotrisethanol buffer solution, pH 7.8 so
that each mL of the solution contains about 150 Units ac-
cording to the labeled amount, and use this solution as the
sample solution. Separately, dilute a suitable volume of
Ulinastatin Reference Standard with 2, 2', 2"-
nitrilotrisethanol buffer solution, pH 7.8 so that each mL of
the solution contains exactly 300, 200, 100, 50 or Units, and
use these solutions as the standard solutions. 2,2', 2"-
Nitrilotrisethanol buffer solution, pH 7.8 and A'-a-benzoyl-
L-arginine-4-nitroanilide TS are warmed in a water bath of 25
± 1°C for use as described below. Take exactly 0.1 mL each
of the sample solution and the standard solutions in test
tubes, add exactly 1.6 mL of 2,2',2"-nitrilotrisethanol buffer
solution, pH 7.8 mix, and put the tubes in the water bath of
25 ± 1°C. One minute after addition of the buffer solution
add exactly 0.2 mL of ice-cooled trypsin TS for test of
ulinastatin, mix, and put the tubes again in the water bath.
One minute later add exactly 1 mL of TV-a-benzoyl-L-ar-
ginine-4-nitroanilide TS, mix, and then put the tubes in the
water bath. Exactly 2 minutes later add exactly 0.1 mL of
diluted acetic acid (100) (1 in 2) to stop the enzyme reaction,
and determine the absorbances of the solutions so obtained at
405 nm as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> using water as the blank. Prepare a calibration
curve using the absorbances obtained with the standard solu-
tions, and calculate ulinastatin Units in the sample solution
from its absorbance by using this curve.
Containers and storage Containers — Tight containers.
Storage — Preserve at -20°C or lower.
Urapidil
^tfvJU
C 20 H 29 N 5 O 3 : 387.48
6- {3-[4-(2-Methoxyphenyl)piperazin-l-yl]propylamino} -
1,3-dimethyluracil [34661-75-1]
Urapidil, when dried, contains not less than 98.0%
and not more than 101.0% of C20H29N5O3.
1218 Urea / Official Monographs
JP XV
Description Urapidil occurs as white to pale yellowish,
white, crystals or crystalline powder. It has a bitter taste.
It is freely soluble in acetic acid (100), sparingly soluble in
ethanol (95) and in acetone, and very slightly soluble in
water.
Identification (1) Determine the absorption spectrum of a
solution of Urapidil in ethanol (95) (1 in 100,000) as directed
under Ultraviolet-visible Spectrophotometry <2.24>, and
compare the spectrum with the Reference Spectrum: both
spectra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of Ura-
pidil as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare the spec-
trum with the Reference Spectrum: both spectra exhibit simi-
lar intensities of absorption at the same wave numbers.
Melting point <2. 60> 1 56 - 1 6 1 °C
Purity (1) Chloride <7.0?>— Dissolve 3.0 g of Urapidil in
40 mL of acetone and 6 mL of dilute nitric acid, add water to
make 50 mL, and perform the test using this solution as the
test solution. Prepare the control solution as follows. To 0.25
mL of 0.01 mol/L hydrochloric acid VS add 40 mL of ace-
tone, 6 mL of dilute nitric acid and water to make 50 mL (not
more than 0.003%).
(2) Heavy metals <1.07> — Proceed with 1.0 g of Urapidil
according to Method 4, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(3) Related substances — Dissolve 40 mg of Urapidil in 5
mL of ethanol (95), and use this solution as the sample solu-
tion. Pipet 1 mL of the sample solution, add ethanol (95) to
make exactly 200 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed un-
der Thin-layer Chromatography <2.03>. Spot 5 /uL each of
the sample solution and standard solution on a plate of silica
gel with fluorescent indicator for thin-layer chromatography,
develop with a mixture of ethyl acetate, ethanol (95) and am-
monia water (28) (22:13:1) to a distance of about 15 cm, and
air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): the spot other than the principal spot
appears not more than one and it is not more intense than the
spot from the standard solution.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 3
hours).
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 70 mg of Urapidil, previous-
ly dried, dissolve in 80 mL of acetic acid (100), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination in the same man-
ner, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 12.92 mg of C 20 H 29 N 5 O 3
Containers and storage Containers — Tight containers.
Urea
o
NH a
CH 4 N 2 0: 60.06
Urea [57-13-6]
Urea contains not less than 99.0% of CH 4 N 2 0.
Description Urea occurs as colorless to white crystals or
crystalline powder. It is odorless, and has a cooling, saline
taste.
It is very soluble in water, freely soluble in boiling ethanol
(95), soluble in ethanol (95), and very slightly soluble in
diethyl ether.
A solution of Urea (1 in 100) is neutral.
Identification (1) Heat 0.5 g of Urea: it liquefies and the
odor of ammonia is perceptible. Continue heating until the
liquid becomes turbid, then cool. Dissolve the resulting lump
in a mixture of 10 mL of water and 2 mL of sodium
hydroxide TS, and add 1 drop of copper (II) sulfate TS: a
reddish purple color develops.
(2) Dissolve 0.1 g of Urea in 1 mL of water, and add 1
mL of nitric acid: a white, crystalline precipitate is formed.
Melting point <2.60> 132.5 - 134. 5°C
Purity (1) Chloride <7.0?>— Perform the test with 2.0 g of
Urea. Prepare the control solution with 0.40 mL of 0.01 mol/
L hydrochloric acid VS (not more than 0.007%).
(2) Sulfate <1.14>— Perform the test with 2.0 g of Urea.
Prepare the control solution with 0.40 mL of 0.005 mol/L
sulfuric acid VS (not more than 0.010%).
(3) Heavy metals <1.07> — Proceed with 1.0 g of Urea ac-
cording to Method 1, and perform the test. Prepare the con-
trol solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(4) Ethanol-insoluble substances — Dissolve 5.0 g of Urea
in 50 mL of warm ethanol (95), filter through a tared glass
filter (G4), wash the residue with 20 mL of warm ethanol
(95), and dry at 105 °C for 1 hour: the mass of the residue is
not more than 2.0 mg.
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Urea, dissolve in
water, and make exactly 200 mL. Measure exactly 5 mL of
this solution into a Kjeldahl flask, and proceed as directed
under Nitrogen Determination <1.08>.
Each mL of 0.005 mol/L sulfuric acid VS
= 0.3003 mg of CH 4 N 2
Containers and storage Containers — Well-closed contain-
ers.
JPXV
Official Monographs / Urokinase 1219
Urokinase
Vaf-i—V.
[9010-53-1 ]
Urokinase is an enzyme, obtained from human u-
rine, that activates plasminogen, and has the molecular
mass of about 54,000.
It is a solution using a suitable buffer solution as the
solvent.
It contains not less than 60,000 Units per mL, and
not less than 120,000 Units per mg of protein.
Description Urokinase is a clear and colorless liquid.
The pH is between 5.5 and 7.5.
Identification (1) Dissolve 0.07 g of fibrinogen in 10 mL
of phosphate buffer solution, pH 7.4. To this solution add 1
mL of a solution of thrombin containing 10 Units per mL in
isotonic sodium chloride solution, mix, place in a Petri dish
about 90 mm in inside diameter, and keep horizontally until
the solution is coagulated. On the surface drop 10 /xL of a so-
lution of Urokinase containing 100 Units per mL in gelatin-
tris buffer solution, and stand for overnight: lysis circle is ap-
peared.
(2) Dissolve 1.0 g of Powdered Agar in 100 mL of boric
acid-sodium hydroxide buffer solution, pH 8.4, by warming,
and pour the solution into a Petri dish until the height come
to about 2 mm. After cooling, make two wells of 2.5 mm in
diameter with the space of 6 mm. To each well place separate-
ly 10 ,uL of a solution of Urokinase containing 30,000 Units
per mL in isotonic sodium chloride solution and 10 /xL of an-
ti-urokinase serum, and stand for overnight: a clear precipi-
tin line is appeared.
Purity (1) Heavy metals <1.07> — Proceed with 2.0 mL of
Urokinase according to Method 2, and perform the test. Pre-
pare the control solution with 2.0 mL of Standard Lead Solu-
tion (not more than 10 ppm).
(2) Blood group substances — Dilute Urokinase with iso-
tonic sodium chloride solution so that each mL of the solu-
tion contains 12,000 Units, and use this solution as the sam-
ple solution. To anti-A type antibody for blood typing add
isotonic sodium chloride solution to dilute each 32, 64, 128,
256, 512 and 1024 times, place separately 25 /uL each of these
solutions in six wells on the first and second lane of a V-
shaped 96-wells microplate. Next, add 25 /xL of the sample
solution into the six wells on the first lane and 25 fiL of iso-
tonic sodium chloride solution into the six wells of the second
lane, mix, and allow to stand for 30 min. To each well add 50
jxL of A-type erythrocyte suspension, mix, allow to stand for
2 hours, and compare the agglutination of erythrocyte in
both lanes: dilution factor of anti-A type antibody of the
wells which show the agglutination is equal in both lanes.
Perform the same test by using anti-B type antibody for
blood typing and B-type erythrocyte suspension.
Abnormal toxicity Dilute Urokinase with isotonic sodium
chloride solution so that each mL of the solution contains
12,000 Units, and use this solution as the sample solution. In-
ject 5.0 mL of the sample solution into the peritoneal cavity
of each of 2 or more of well-nourished, healthy guinea pigs
weighing about 350 g, and inject 0.5 mL of the sample solu-
tion into the peritoneal cavity of each of 2 or more of well-
nourished, healthy mice aged about 5 weeks. Observe the
conditions of the animals for more than 7 days: all the
animals exhibit no abnormalities.
High molecular mass urokinase Dilute Urokinase with
gelatin-phosphate buffer solution so that each mL of the solu-
tion contains 10,000 Units, and use this solution as the sam-
ple solution. Perform the test with 100 /xL of the sample solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the peak
areas of two peaks eluted closely at about 35 minutes having
smaller retention time, A^, and larger retention time, A b , by
the automatic integration method: the value, A J (A^+A^), is
not less than 0.85.
Operating conditions —
Apparatus: Use a pumping system for the mobile phase, a
sample injection port, a column, a pumping system for the
reaction reagent, a reaction coil, a reaction chamber, a spec-
trofluorometer and a recorder. Attach a 3-way tube to the
outlet for the mobile phase of the column, connect the pump-
ing system for the reaction reagent and the reaction coil, and
join outlet of the reaction coil to the spectrofluorometer.
Detector: Spectrofluorometer (excitation wavelength: 365
nm, fluorescence wavelength: 460 nm).
Column: A stainless steel column 7.5 mm in inside di-
ameter and 60 cm in length, packed with porous silica gel for
liquid chromatography (10 to 12 /xm in particle diameter).
Column temperature: A constant temperature of about
20°C.
Reaction coil: A stainless steel column 0.25 mm in inside
diameter and 150 cm in length.
Reaction coil temperature: 37 °C
Mobile phase: Gelatin-phosphate buffer solution.
Flow rate of mobile phase: 0.5 mL per minute.
Reaction reagent: 7-(Glutarylglycyl-L-arginylamino)-4-
methylcoumarin TS.
Flow rate of reaction reagent: 0.75 mL per minute.
Selection of column: Adjust the pH of Urokinase to 7.5
with sodium hydroxide TS, allow to stand at 37°C for over 24
hours, and add gelatin-phosphate buffer solution to make the
solution containing 20,000 Units per mL. Proceed with 100
/xL of this solution under the above operating conditions, and
calculate the resolution. Use a column giving elution of high
molecular mass urokinase (mol. wt.: 54,000) and low molecu-
lar mass urokinase (mol. wt.: 33,000) in this order with the
resolution between these peaks being not less than 1.0.
Assay (1) Urokinase — Pipet 1 mL of Urokinase, dilute
exactly with gelatin-tris buffer solution so that each mL of the
solution contains about 30 Units, and use this solution as the
sample solution. Add exactly 2 mL of gelatin-tris buffer solu-
tion to contents of one ampoule of High Molecular Mass
Urokinase Reference Standard to dissolve, pipet 1 mL of this
solution, dilute exactly with gelatin-tris buffer solution so
that each mL of the solution contains about 30 Units, and use
this solution as the standard solution. Place 1.0 mL of L-
pyroglutamylglycyl-L-arginine-/>-nitroaniline hydrochloride
TS in two silicon-coated test tubes about 10 mm in inside di-
ameter, warm them in a water bath at 35 ± 0.2°C for 5
minutes, add separately 0.50 mL each of the sample solution
and the standard solution, warm in a water bath at 35 ±
1220 Ursodeoxycholic Acid / Official Monographs
JP XV
0.2°C for exactly 30 minutes, then add 0.50 mL of diluted
acetic acid (100) (2 in 5). Determine the absorbances, A T and
A s , of these solutions at 405 nm as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>, using water as the
blank. Separately place 1 .0 mL of L-pyroglutamylglycyl-L-ar-
ginine-/?-nitroaniline hydrochloride TS in two test tubes, add
0.50 mL of diluted acetic acid (100) (2 in 5), and 0.50 mL
each of the sample solution and the standard solution. Deter-
mine the absorbances, A TO and A so , of these solutions at 405
nm as the same manner, using water as the blank.
Amount (Units) of Urokinase
= l(A T -A TO )/(A s -A so )} xaxb
a: Amount (Units) of urokinase in 1 mL of the standard
solution
b: Total volume (mL) of the sample solution
(2) Protein — Measure exactly a volume of Urokinase,
equivalent to about 15 mg of protein, and perform the test as
directed under Nitrogen Determination <1.08>.
Each mL of 0.005 mol/L sulfuric acid VS
= 0.8754 mg of protein
Containers and storage Containers — Tight containers.
Storage — Not exceeding — 20°C.
Ursodeoxycholic Acid
Ursodesoxycholic Acid
H H H
C 2 4H4 O 4 : 392.57
3a,7/?-Dihydroxy-5/?-cholan-24-oic acid
[128-13-2]
Ursodeoxycholic Acid, when dried, contains not less
than 98.5% of C 2 4H 40 O 4 .
Description Ursodeoxycholic Acid occurs as white crystals
or powder. It is odorless, and has a bitter taste.
It is freely soluble in ethanol (95), in ethanol (99.5) and in
acetic acid (100), slightly soluble in chloroform, very slightly
soluble in diethyl ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Identification Dissolve 0.01 g of Ursodeoxycholic Acid in 1
mL of sulfuric acid, add 1 drop of formaldehyde solution,
and allow to stand for 5 minutes. To the solution add 5 mL
of water: a blue-green suspended substance is produced.
Optical rotation <2.49> [a]^: +59.0 - +62.0° (after
drying, 1.0 g, ethanol (99.5), 25 mL, 100 mm).
Melting point <2.60> 200 - 204°C
Purity (1) Odor — To 2.0 g of Ursodeoxycholic Acid add
100 mL of water, and boil for 2 minutes: no odor is percepti-
ble.
(2) Chloride <1.03> — Dissolve 2.0 g of Ursodeoxycholic
Acid in 20 mL of acetic acid (100) with shaking, add water to
make 200 mL, shake thoroughly, and allow to stand for 10
minutes. Filter this solution, discard the first 10 mL of the
filtrate, and use the subsequent filtrate as the sample solu-
tion. To 40 mL of the sample solution add 6 mL of dilute
nitric acid and water to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS
add 4 mL of acetic acid (100), 6 mL of dilute nitric acid and
water to make 50 mL (not more than 0.022%).
(3) Sulfate <1.14> — To 40 mL of the sample solution ob-
tained in (2) add 1 mL of dilute hydrochloric acid and water
to make 50 mL, and perform the test using this solution as
the test solution. Prepare the control solution as follows: to
0.40 mL of 0.005 mol/L sulfuric acid VS add 4 mL of acetic
acid (100), 1 mL of dilute hydrochloric acid and water to
make 50 mL (not more than 0.048%).
(4) Heavy metals <1.07> — Proceed with 1.0 g of Ur-
sodeoxycholic Acid according to Method 2, and perform the
test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 20 ppm).
(5) Barium — To the solution obtained in (1) add 2 mL of
hydrochloric acid, boil for 2 minutes, cool, filter, and wash
with water until the last washing makes 100 mL. To 10 mL of
the solution add 1 mL of dilute sulfuric acid: no turbidity is
produced.
(6) Arsenic </.//> — Prepare the test solution with 1.0 g
of Ursodeoxycholic Acid according to Method 3, and per-
form the test (not more than 2 ppm).
(7) Related substances — Dissolve 50 mg of Ursodeoxy-
cholic Acid in a mixture of chloform and ethanol (95) (9:1) to
make exactly 25 mL, and use this solution as the sample solu-
tion. Separately, dissolve 75 mg of chenodeoxycholic acid for
thin-layer chromatography in a mixture of chloroform and
ethanol (95) (9:1) to make exactly 100 mL. To exactly 2 mL
of this solution add a mixture of chloroform and ethanol (95)
(9:1) to make exactly 50 mL, and use this solution as the stan-
dard solution (1). Dissolve 25 mg of lithocholic acid for thin-
layer chromatography in a mixture of chloroform and
ethanol (95) (9:1) to make exactly 50 mL. To exactly 1 mL of
this solution add a mixture of chloroform and ethanol (95)
(9:1) to make exactly 50 mL. To exactly 2 mL of this solution
add a mixture of chloroform and ethanol (95) (9:1) to make
exactly 10 mL, and use this solution as the standard solution
(2). Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xh each of the
sample solution and standard solutions (1) and (2) on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of chloroform, acetone and acetic acid (100)
(7:2:1) to a distance of about 10 cm, and air-dry the plate.
Dry the plate at 120°C for 30 minutes, spray evenly a solution
of phosphomolybdic acid M-hydrate in ethanol (95) (1 in 5)
immediately, and heat at 120°C for 2 to 3 minutes: the spot
from the sample solution, corresponding to that from the
standard solution (1), is not more intense than the spot from
the standard solution (1), and the spot other than the prin-
cipal spot and the above spots from the sample solution are
not more intense than the spot from the standard solution
(2).
Loss on drying <2.41> Not more than 1.0% (1 g, 105 °C,
2 hours).
JPXV
Official Monographs / Vancomycin Hydrochloride 1221
Residue on ignition <2.44> Not more than 0.2% (1 g).
Assay Weigh accurately about 0.5 g of Ursodeoxycholic
Acid, previously dried, and dissolve in 40 mL of neutralized
ethanol and 20 mL of water. Add 2 drops of phenolphthalein
TS, titrate with 0.1 mol/L sodium hydroxide VS, and titrate
<2.50> again after adding 100 mL of freshly boiled and cooled
water near the end point.
Each mL of 0.1 mol/L sodium hydroxide VS
= 39.26 mg of C 24 H 40 O 4
Containers and storage Containers — Well-closed contain-
ers.
L-Valine
CH :i
CO^H
l-M'U
H NHj
C 5 H„N0 2 : 117.15
(25')-2-Amino-3-methylbutanoic acid
[72-18-4]
l- Valine, when dried,
of C 5 H„N0 2 .
contains not less than 98.5%
Description L-Valine occurs as white crystals or crystalline
powder. It is odorless or has a faint characteristic odor, and
has a slightly sweet taste, which becomes bitter.
It is freely soluble in formic acid, soluble in water, and
practically insoluble in ethanol (95).
It dissolves in dilute hydrochloric acid.
Identification Determine the infrared absorption spectrum
of L-Valine, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]o- +26.5- +29.0° (after
drying, 2 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm).
pH <2.54> Dissolve 0.5 g of L-Valine in 20 mL of water: the
pH of this solution is between 5.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 0.5 g of
L-Valine in 20 mL of water: the solution is clear and color-
less.
(2) Chloride <1.03>— Perform the test with 0.5 g of L-Va-
line. Prepare the control solution with 0.30 mL of 0.01 mol/L
hydrochloric acid VS (not more than 0.021%).
(3) Sulfate <1.14>— Perform the test with 0.6 g of L-Va-
line. Prepare the control solution with 0.35 mL of 0.005 mol/
L sulfuric acid VS (not more than 0.028%).
(4) Ammonium <1.02> — Perform the test with 0.25 g of
L-Valine. Prepare the control solution with 5.0 mL of Stan-
dard Ammonium Solution (not more than 0.02%).
(5) Heavy metals <1.07> — Proceed with 1.0 g of L-Valine
according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 20 ppm).
(6) Arsenic <1.11> — Proceed with 1.0 g of L-Valine, pre-
pare the test solution according to Method 2, and perform
the test (not more than 2 ppm).
(7) Related substances — Dissolve 0. 10 g of L-Valine in 25
mL of water, and use this solution as the sample solution.
Pipet 1 mL of the sample solution, and add water to make ex-
actly 50 mL. Pipet 5 mL of this solution, add water to make
exactly 20 mL, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 /xL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of 1-butanol, water and acetic acid (100) (3:1:1) to a distance
of about 10 cm, and dry the plate at 80°C for 30 minutes.
Spray evenly a solution of ninhydrin in acetone (1 in 50) on
the plate, and heat at 80°C for 5 minutes: the spots other
than the principal spot from the sample solution are not more
intense than the spot from the standard solution.
Loss on drying <2.4I> Not more than 0.30% (1 g, 105°C,
3 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.12 g of L-Valine, previous-
ly dried, and dissolve in 3 mL of formic acid, add 50 mL of a-
cetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric
acid VS (potentiometric titration). Perform a blank determi-
nation, and make any necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 11.72 mg of C 5 H„N0 2
Containers and storage Containers — Tight containers.
Vancomycin Hydrochloride
y\*>^7-f
-ikmik
N
! H H
O H NH
•HCI
Y
o
C 66 H 75 C1 2 N 9 24 .HC1: 1485.71
(lS,2i?,18i?,19i?,225,25i?,28/?,40S)-50-[3-Amino-
2,3,6-trideoxy-3-C-methyl-a-L-/j'xo-hexopyranosyl-
(l->2)-/?-D-glucopyranosyloxy]-22-carbamoylmethyl-
5,15-dichloro-2,18,32,35,37-pentahydroxy-19-[(2i?)-
1222 Vancomycin Hydrochloride / Official Monographs
JP XV
4-methyl-2-(methylamino)pentanoylamino]-
20,23,26,42, 44-pentaoxo-7,13-dioxa-21, 24, 27,41, 43-
pentaazaoctacyclo[26.14.2.2 3 ' 6 .2 14 . 17 .l 8 ' 12 .l 29 .".0 l0 . 25 .0 34 ' 39
jpentaconta-
3, 5, 8, 10,12(50), 14,16,29,31, 33(49),34,36,38,45,47-
pentadecaene-40-carboxylic acid monohydrochloride
[1404-93-9]
Vancomycin Hydrochloride is the hydrochloride of a
glycopeptide substance having antibacterial activity
produced by the growth of Streptomyces orientalis.
It contains not less than 1000 fig (potency) and not
more than 1200 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Vancomycin
Hydrochloride is expressed as mass (potency) of van-
comycin (C 66 H 75 C1 2 N 9 24 : 1449.25).
Description Vancomycin Hydrochloride occurs as a white
powder.
It is freely soluble in water, soluble in formamide, slightly
soluble in methanol, very slightly soluble in ethanol (95), and
practically insoluble in acetonitrile.
It is hygroscopic.
Identification (1) Determine the absorption spectrum of a
solution of Vancomycin Hydrochloride (1 in 10,000) as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
and compare the spectrum with the Reference Spectrum or
the spectrum of a solution of Vancomycin Hydrochloride
Reference Standard prepared in the same manner as the sam-
ple solution: both spectra exhibit similar intensities of ab-
sorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Van-
comycin Hydrochloride as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>, and
compare the spectrum with the Reference Spectrum or the
spectrum of Vancomycin Hydrochloride Reference Stan-
dard: both spectra exhibit similar intensities of absorption at
the same wave numbers.
(3) Dissolve 20 mg of Vancomycin Hydrochloride in 10
mL of water, and add 1 drop of silver nitrate TS: a white tur-
bidity is produced.
Optical rotation <2.49> [«]£?: - 30 - - 40° (0.2 g calculated
on the anhydrous basis, water, 20 mL, 100 mm).
pH <2.54> The pH of a solution obtained by dissolving 0.25
g of Vancomycin Hydrochloride in 5 mL of water is between
2.5 and 4.5.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Vancomycin Hydrochloride according to Method 2, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Related substances — Dissolve 0.10 g of Vancomycin
Hydrochloride in 10 mL of the mobile phase A, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add the mobile phase A to make exactly 25 mL, and
use this solution as the standard solution. Perform the test
with exactly 20 fiL each of the sample solution and standard
solution as directed under the Liquid Chromatography <2.01>
according to the following conditions. If necessary, proceed
with 20 ftL of the mobile phase A in the same manner to com-
pensate for the base line. Determine each peak area by the au-
tomatic integration method: the area of each peak other than
vancomycin from the sample solution is not more than the
peak area of vancomycin from the standard solution, and the
total area of the peaks other than vancomycin is not more
than 3 times of the peak area of vancomycin from the stan-
dard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 280 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase A: A mixture of triethylamine buffer solu-
tion, pH 3.2, acetonitrile and tetrahydrofuran (92:7:1). Ad-
just the amount of acetonitrile so that the retention time of
vancomycin is 7.5 to 10.5 minutes.
Mobile phase B: A mixture of triethylamine buffer solu-
tion, pH 3.2, acetonitrile and tetrahydrofuran (70:29:1).
Flowing of the mobile phase: Control the gradient by mix-
ing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase Mobile phase
A (vol%) B (vol%)
0-
- 12
12-
-20
20-
-22
100
100^0
0^ 100
100
Flow rate: 1.5 mL per minute.
Time span of measurement: As long as about 2.5 times of
the retention time of vancomycin beginning after the solvent
peak.
System suitability —
Test for required detectability: Confirm that the peak area
of vancomycin obtained from 20 fiL of the standard solution
is equivalent to 3 to 5% of that from 20 fiL of the sample so-
lution.
System performance: Dissolve 5 mg of Vancomycin
Hydrochloride in 10 mL of water, heat at 65°C for 48 hours,
and cool to the ordinal temperature. When the procedure is
run with 20 ftL of this solution under the above operating
conditions, related substance 1, vancomycin and related sub-
stance 2 are eluted in this order, the resolution between the
peaks of the related substance 1 and vancomycin is not less
than 3, the number of theoretical plates of the peak of van-
comycin is not less than 1500, and the related substance 2 is
eluted between 15 and 18 minutes.
System repeatability: When the test is repeated 5 times with
20 fiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
vancomycin is not more than 2.0%.
Water <2.48> Not more than 5.0% (0.1 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for water
determination and methanol for water determination (3:1)).
Residue on ignition <2.44> Not more than 1.0% (1 g).
Assay Perform the test according to the Cylinder-plate
method as directed under the Microbial Assay for Antibiotics
<4.02> according to the following conditions,
(i) Test organism — Bacillus subtilis ATCC 6633
JPXV
Official Monographs / Vasopressin Injection 1223
(ii) Culture medium — Use the medium i in 1) Medium for
test organism [5] under (1) Agar media for seed and base lay-
er. Adjust the pH of the medium so that it will be 6.2 to 6.4
after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Vancomycin Hydrochloride Reference Standard, equivalent
to about 25 mg (potency), dissolve in water to make exactly
25 mL, and use this solution as the standard stock solution.
Keep the standard stock solution at 5°C or below, and use wi-
thin 7 days. Take exactly a suitable amount of the standard
stock solution before use, add 0.1 mol/L phosphate buffer
solution, pH 4.5 to make solutions so that each mL contains
100 /ug (potency) and 25 ug (potency), and use these solutions
as the high concentration standard solution and low concen-
tration standard solution, respectively.
(iv) Sample solutions — Weigh accurately an amount of
Vancomycin Hydrochloride, equivalent to about 25 mg
(potency), and dissolve in water to make exactly 25 mL. Take
exactly a suitable amount of this solution, add 0.1 mol/L
phosphate buffer solution, pH 4.5 to make solutions so that
each mL contains 100 /xg (potency) and 25 /xg (potency), and
use these solutions as the high concentration sample solution
and low concentration sample solution, respectively.
Containers and storage Containers — Tight containers.
Vancomycin Hydrochloride for
Injection
iiltffl A' > =l -V 4 v >i&Mi&
Vancomycin Hydrochloride for Injection is a prepa-
ration for injection which is dissolved before use.
It contains not less than 90.0% and not more than
115.0% of the labeled amount of vancomycin
(C 66 H 75 C1 2 N 9 24 : 1449.25).
Method of preparation Prepare as directed under Injec-
tions, with Vancomycin Hydrochloride.
Description Vancomycin Hydrochloride for Injection oc-
curs as white masses or a white powder.
Identification (1) Dissolve an amount of Vancomycin
Hydrochloride for Injection, equivalent to 5 mg (potency) of
Vancomycin Hydrochloride, in 50 mL of water, and deter-
mine the absorption spectrum of this solution as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 279 and 283 nm.
(2) Dissolve an amount of Vancomycin Hydrochloride
for Injection, equivalent to 20 mg (potency) of Vancomycin
Hydrochloride, in 10 mL of water, and add 1 drop of silver
nitrate TS: a white turbidity is produced.
pH <2.54> The pH of a solution prepared by dissolving an
amount of Vancomycin Hydrochloride for Injection, equiva-
lent to 0.5 g (potency) of Vancomycin Hydrochloride accord-
ing to the labeled amount, in 10 mL of water is between 2.5
and 4.5.
Purity (1) Clarity and color of solution — Dissolve an
amount of Vancomycin Hydrochloride for Injection, equiva-
lent to 0.5 g (potency) of Vancomycin Hydrochloride accord-
ing to the labeled amount, in 10 mL of water: the solution is
clear and colorless to pale yellow, and the absorbance of the
solution, determined at 465 nm as directed under Ultraviolet-
visible Spectrophotometry <2.24>, is not more than 0.05.
(2) Related substances — Dissolve an amount of Van-
comycin Hydrochloride for Injection, equivalent to 0.1 g
(potency) of Vancomycin Hydrochloride according to the la-
beled amount, in 10 mL of the mobile phase A, and use this
solution as the sample solution.
Proceed as directed in the Purity (2) under Vancomycin
Hydrochloride.
Water <2.48> Not more than 5.0% (0.1 g, volumetric titra-
tion, direct titration. Use a mixture of formamide for Karl
Fisher method and methanol for Karl Fisher method (3:1)).
Bacterial endotoxins <4.01> Less than 0.25 EU/mg (poten-
cy).
Uniformity of dosage unit <6.02> It meets the requirement
of the Mass Variation Test.
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions.
(i) Test organism, culture medium, and standard
solutions — Proceed as directed in the Assay under Vancomy-
cin Hydrochloride.
(ii) Sample solutions — Weigh accurately the contents of
not less than 10 Vancomycin Hydrochloride for Injection.
Weigh accurately an amount of the content, equivalent to
about 25 mg (potency) of Vancomycin Hydrochloride ac-
cording to the labeled amount, and dissolve in water to make
exactly 25 mL. Take exactly a suitable amount of this solu-
tion, add 0.1 mol/L phosphate buffer solution, pH 4.5 to
make solutions so that each mL contains 100^g (potency)
and 25 /ug (potency), and use these solutions as the high con-
centration sample solution and low concentration sample so-
lution, respectively.
Containers and storage Containers — Hermetic containers.
Vasopressin Injection
/N*V7 p U->>>±l^>g
Vasopressin Injection is an aqueous solution for in-
jection.
It contains synthetic vasopressin or the pressor prin-
ciple, vasopressin, obtained from the posterior lobe of
the pituitary of healthy catties and pigs, from which
the majority of the oxytocic principle, oxytocin, has
been removed.
It contains not less than 85% and not more than
120% of the labeled vasopressin Units.
Method of preparation Prepare as directed under Injec-
1224 Vasopressin Injection / Official Monographs
JP XV
tions, with vasopressin prepared by synthesis or obtained
from the posterior lobe of the pituitary.
Description Vasopressin Injection is a clear and colorless
liquid. It is odorless or has a slight, characteristic odor.
pH: 3.0-4.0
Purity Oxytocic principle — When tested by the following
procedure, Vasopressin Injection contains not more than 0.6
oxytocin Units for each determined 10 vasopressin Units.
(i) Standard stock solution: Dissolve 200 Units of Oxyto-
cin Reference Standard, according to the labeled Units, in ex-
actly 10 mL of diluted acetic acid (100) (1 in 400). Pipet 1 mL
of this solution, and add diluted acetic acid (100) (1 in 400) to
make exactly 10 mL. Store in a cold place, avoiding freezing.
Use within 6 months from the date of preparation.
(ii) Standard solution: Dilute the standard stock solution
with isotonic sodium chloride solution so that each mL of the
solution contains 0.020 oxytocin Units.
(iii) Sample solution: Assume oxytocin Units as equiva-
lent to 6/100 of the determined vasopressin Units. Dilute
Vasopressin Injection with isotonic sodium chloride solution
so that each mL of the resulting solution is expected to con-
tain 0.020 oxytocin Unit.
(iv) Apparatus: Use the apparatus for the uterus contrac-
tion test, equipped with a thermostatic bath. Maintain a tem-
perature of the bath at 37°C to 38°C with a variation of not
more than 0. 1 °C during the course of the test. Use a 1 00-mL
Magnus' chamber for suspending the uterus.
(v) Test animal: Use healthy, virgin and metestrus guinea
pigs weighing between 175 g and 350 g. They have been bred
under conditions where they have been completely isolated
from the sight and smell of males since the time of weaning.
(vi) Procedure: Immerse the Magnus' chamber in the
bath maintained at a constant temperature, add Locke-Rin-
ger's solution to the chamber, and introduce oxygen into the
solution at a moderate rate. Sacrifice a guinea pig by means
of a blow on the head, immediately remove the uterus from
the body, suspend it in the chamber, and connect one horn of
the uterus to the lever with a thread. If necessary, weigh the
lever provided that the mass is not changed throughout the
assay. Start the assay after 15 to 30 minutes when the uterus
is completely relaxed. Administer the same quantities, 0.1 to
0.5 mL, of the standard solution and the sample solution to
the Magnus' chamber alternately twice with regular intervals
of between 10 and 20 minutes to contract the uterus, finally
administer the standard solution in a quantity which is 25%
larger than the preceding doses, and measure the height of
every contraction. The mean height of uterus contraction
caused by the standard solution is equal to or higher than that
caused by the sample solution. The height of contraction
caused by the increased dose of the standard solution is dis-
tinctly higher than those caused by the preceding doses of the
standard solution.
Extractable volume <6.05> It meets the requirement.
Assay (i) Test animals: Use healthy male rats weighing be-
tween 200 g and 300 g.
(ii) Standard stock solution: Dissolve 2000 Units of
Vasopressin Reference Standard, according to the labeled
Units, in exactly 100 mL of diluted acetic acid (100) (1 in
400). Pipet 1 mL of this solution, and add diluted acetic acid
(100) (1 in 400) to make exactly 10 mL. Store in a cold place,
avoiding freezing. Use within 6 months from the date of
preparation.
(iii) Standard solution: Dilute the standard stock solution
with isotonic sodium chloride solution so that 0.2 mL of the
obtained solution causes blood pressure increases of between
35 mmHg and 60 mmHg in test animals when injected ac-
cording to (vi), and designate this solution as the high-dose
standard solution (S H ). Then dilute this solution with isotonic
sodium chloride solution 1.5 to 2.0 times by volume, and
designate it as the low-dose standard solution (S L ).
(iv) Sample solution: Dilute an accurately measured
volume of Vasopressin Injection with isotonic sodium chlo-
ride solution so that the obtained solution contains the same
concentration in Units as the high-dose standard solution
based on the labeled Units, and designate it as the high-dose
sample solution (T H ). Then dilute this solution with isotonic
sodium chloride solution 1.5 to 2.0 times by volume, and
designate it as the low-dose sample solution (T L ). Make the
concentration ratio of S H to S L equal to the ratio of T H to T L .
When the sensitivity of an animal is changed, adjust the con-
centration of S H and T H before the next set of assay is started.
However, keep the same ratio of S H to S L and T H to T L as in
the primary set.
(v) Dose of injection: Although 0.2 mL of each solution
is usually injected, the dose of injection can be determined
based from preliminary tests or experiences. Inject the same
volume throughout a set of tests.
(vi) Procedure: Inject subcutaneously 0.7 mL of a solu-
tion of ethyl carbamate (1 in 4) per 100 g of body mass to
anesthetize the test animals and cannulate the trachea. Under
artificial respiration (about 60 strokes per minute), remove a
part of the second cervical vertebra, cut off the spinal cord
and destroy the brain through the foramen magnum. Insert a
cannula filled with isotonic sodium chloride solution into a
femoral vein. Through this cannula, inject the solution pre-
pared by dissolving 200 heparin Units of heparin sodium in
0.1 mL of isotonic sodium chloride solution, and then im-
mediately inject 0.3 mL of isotonic sodium chloride solution.
Insert a cannula into a carotid artery, and connect the cannu-
la to a manometer for blood pressure measurement with a
vinyl tube. The cannula and the vinyl tube have previously
been filled with isotonic sodium chloride solution. Inject the
standard and the sample solutions at regular intervals of 10 to
15 minutes into the femoral vein through the cannula fol-
lowed by 0.3 mL of the isotonic solution when the blood
pressure increases caused by each solution returns to the
original level. Measure the height of blood pressure increases
within 1 mmHg on the kymogram. Maintain a constant tem-
perature between 20°C and 25 °C during the assay. In ad-
vance, make four pairs from S H , S L , T H , T L as follows. Ran-
domize the order of injection for pairs, but keep the order of
injection within pairs as indicated.
Pairl: S H , T L Pair 2: S L , T H Pair 3: T H , S L Pair 4:
T L , S h
Carry out this assay using the same animals throughout a
set of four pairs of observations. Perform this assay with two
sets. If necessary, however, use the different animals for both
sets of tests.
(vii) Calculation: Subtract increases of blood pressure
caused by the low dose from those caused by the high dose in
the Pair 1 , 2, 3 and 4 of each set, and obtain the responses y u
y 2 , yi and y 4 , respectively. Sum up y lt for each set to obtain
Y lt and obtain Y 2 , Y } and Y 4 in the same way.
Units in each mL of Vasopressin Injection
JP XV
Official Monographs / Verapamil Hydrochloride 1225
= antilog M x (Units in each mL of the S H ) x {b/a)
M = (IYJY h )
I = log (Sh/S l ) = log (T H /T L )
y. = - y, + y, + y, - y 4
Y h =Y i +Y 1 +Y i + Y 4
a: Volume (mL) of Vasopressin Injection sampled.
b: Total volume (mL) of the high-dose sample solution pre-
pared by diluting with isotonic sodium chloride solution.
Compute i (P = 0.95) by the following equation, and con-
firm L to be 0.15 or less. If L exceeds 0.15, repeat the test,
improving the conditions of the assay or increasing the num-
ber of sets until L reaches 0.15 or less.
L = 2^j(C - 1)(CAF + P)
f: Number of sets
S 2 = [Ti yi - (j/f) - (Y'/4) + (Y b 2 /4f)]/n
Ey 1 : The sum of the squares of y u y 2 , y$ and y 4 .
y = y 2 + y 2 2 + y, 2 + r 4 2
Y'\ The sum of the squares of the sum of y lt y 2 , yi
and y 4 in each set.
n = 3(/ - 1)
t 2 : Value shown in the following table against n for
which s 2 is calculated.
n
fi = F 1
n
fl = F i
n
fl = F i
1
161.45
13
AMI
25
4.242
2
18.51
14
4.600
26
4.225
3
10.129
15
4.543
27
4.210
4
7.709
16
4.494
28
4.196
5
6.608
17
4.451
29
4.183
6
5.987
18
4.414
30
4.171
7
5.591
19
4.381
40
4.085
8
5.318
20
4.351
60
4.001
9
5.117
21
4.325
120
3.920
10
4.965
22
4.301
oo
3.841
11
4.844
23
4.279
12
4.747
24
4.260
Containers and storage Containers — Hermetic containers.
Storage — In a cold place, and avoid freezing.
Expiration date 36 months after preparation.
Verapamil Hydrochloride
Iproveratril Hydrochloride
C 27 H 38 N 2 04.HC1: 491.06
(2i?5')-5-[(3,4-Dimethoxyphenethyl)methylamino]-2-
(3,4-dimethoxyphenyl)-2-(l-methylethyl)pentanenitrile
monohydrochloride [152-11-4]
Verapamil Hydrochloride, when dried, contains not
less than 98.5% of C 27 H 38 N 2 4 .HC1.
Description Verapamil Hydrochloride occurs as a white,
crystalline powder. It is odorless.
It is freely soluble in methanol, in acetic acid (100) and in
chloroform, soluble in ethanol (95) and in acetic anhydride,
sparingly soluble in water, and practically insoluble in diethyl
ether.
Identification (1) To 2 mL of a solution of Verapamil
Hydrochloride (1 in 50) add 5 drops of Reinecke salt TS: a
light red precipitate is produced.
(2) Determine the absorption spectrum of a solution of
Verapamil Hydrochloride in 0.01 mol/L hydrochloric acid
TS (1 in 50,000) as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wavelengths.
(3) Determine the infrared absorption spectrum of Ver-
apamil Hydrochloride, previously dried, as directed in the
potassium chloride disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(4) A solution of Verapamil Hydrochloride (1 in 50)
responds to the Qualitative Tests <1.09> for chloride.
Melting point <2.60> 141 - 145 °C
pH <2.54> Dissolve 1.0 g of Verapamil Hydrochloride in 20
mL of freshly boiled and cooled water by warming, and cool:
the pH of this solution is between 4.5 and 6.5.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Verapamil Hydrochloride in 20 mL of water by warming: the
solution is clear and colorless.
(2) Heavy metals <1.07> — Proceed with 1.0 g of Ver-
apamil Hydrochloride according to Method 2, and perform
the test. Prepare the control solution with 2.0 mL of Stan-
dard Lead Solution (not more than 20ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Verapamil Hydrochloride according to Method 3, and per-
form the test (not more than 2 ppm).
(4) Related substances — Dissolve 0.50 g of Verapamil
Hydrochloride in exactly 10 mL of chloroform, and use this
solution as the sample solution. Pipet 1 mL of the sample so-
lution, add chloroform to make exactly 100 mL, and use this
solution as the standard stock solution. Pipet 5 mL of the
standard stock solution, add chloroform to make exactly 100
mL, and use this solution as the standard solution (1).
Separately, pipet 5 mL of standard stock solution, add chlo-
roform to make exactly 50 mL, and use this solution as the
standard solution (2). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL each of the sample solution and standard solutions (1) and
(2) on two plates of silica gel for thin-layer chromatography.
With the one plate, develop the plate with a mixture of cyclo-
hexane and diethylamine (17:3) to a distance of about 15 cm,
air-dry the plate, heat at 110°C for 1 hour, and cool. Exa-
mine immediately after spraying evenly iron (III) chloride-io-
1226 Verapamil Hydrochloride Tablets / Official Monographs
JP XV
dine TS on the plate: the three spots, having more intense
color in the spots other than the principal spot and the origi-
nal point from the sample solution, are not more intense than
the spot from the standard solution (2) in color. The remain-
ing spots from the sample solution are not more intense than
the spot from the standard solution (1) in color. With another
plate, develop the plate with a mixture of toluene, methanol,
acetone and acetic acid (100) (14:4:1:1), and perform the test
in the same manner.
Loss on drying <2.41> Not more than 1.0% (1 g, 105°C,
2 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.7 g of Verapamil
Hydrochloride, previously dried, dissolve in 50 mL of a mix-
ture of acetic anhydride and acetic acid (100) (7:3), and titrate
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
titration). Perform a blank determination, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 49.11 mg of C 27 H 38 N 2 4 .HC1
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Verapamil Hydrochloride Tablets
Verapamil Hydrochloride Tablets contain not less
than 95.0% and not more than 105. 0% of the labeled
amount of verapamil hydrochloride (C 2 7H3 g N 2 04.HCl:
491.06).
Method of preparation Prepare as directed under Tablets,
with Verapamil Hydrochloride.
Identification (1) To a quantity of pulverized Verapamil
Hydrochloride Tablets, equivalent to 0.2 g of Verapamil
Hydrochloride according to the labeled amount, add 70 mL
of 0.02 mol/L hydrochloric acid TS, and shake occasionally
in a water bath at 60°C. After cooling, add 0.02 mol/L
hydrochloric acid TS to make 100 mL, and filter. To 3 mL of
the filtrate add several drops of Reinecke's salt TS: a light red
precipitate is formed.
(2) To 2 mL of the filtrate obtained in (1) add 0.02 mol/L
hydrochloric acid TS to make 100 mL, and determine the ab-
sorption spectrum of this solution as directed under Ultrav-
iolet-visible Spectrophotometry <2.24>: it exhibits maxima
between 227 nm and 231 nm, and between 276 nm and 280
nm.
Uniformity of dosage unit <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Verapamil Hydrochloride Tablets add 70
mL of 0.02 mol/L hydrochloric acid TS, disintegrate the
tablet by occasional shaking in a water bath at 60°C for
about 30 minutes, and then shake for 5 minutes. After cool-
ing, add 0.02 mol/L hydrochloric acid TS to make exactly
100 mL, and filter. Discard the first 20 mL of the filtrate,
take exactly KmL of the subsequent filtrate, add 0.02 mol/L
hydrochloric acid TS to make exactly V so that each mL
contains about 40 mg of verapamil hydrochloride (C 27 H 38 N 2
4 .HC1), and use this solution as the sample solution. Here-
after, proceed as directed in the Assay.
Amount (mg) of verapamil hydrochloride (C 27 H 38 N 2 4 .HC1)
= W s x (A T /A S ) x(V'/V)x (1/25)
W s : Amount (mg) of verapamil hydrochloride for assay
Assay To 10 tablets of Verapamil Hydrochloride Tablets
add 140 mL of 0.02 mol/L hydrochloric acid TS, disintegrate
the tablets by occasional shaking in a water bath at 60°C for
about 30 minutes, and then shake for 5 minutes. After cool-
ing, add 0.02 mol/L hydrochloric acid TS to make exactly
200 mL, and filter. Discard the first 20 mL of the filtrate, take
a volume of the subsequent filtrate, equivalent to about 4 mg
of verapamil hydrochloride (C 27 H 38 N 2 4 .HC1), add 0.02
mol/L hydrochloric acid TS to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 0.1 g of verapamil hydrochloride for assay,
previously dried at 105°C for 2 hours, and dissolve in 70 mL
of 0.02 mol/L hydrochloric acid TS by occasional shaking in
a water bath at 60°C. After cooling, add 0.02 mol/L
hydrochloric acid TS to make exactly 100 mL. Pipet 4 mL of
this solution, add 0.02 mol/L hydrochloric acid TS to make
exactly 100 mL, and use this solution as the standard solu-
tion. Determine the absorbances, A T and A s , of the sample
solution and the standard solution at 278 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of verapamil hydrochloride (C 27 H 38 N 2 4 .HC1)
= W s x (A T /A S ) x(V'/V)x (1/25)
W s : Amount (mg) of verapamil hydrochloride for assay
Containers and storage Containers — Tight containers.
Vinblastine Sulfate
£>it = 7 7.3 L >mm&
C 4 6H5 8 N 4 9 .H 2 S04: 909.05
Methyl (3a.R,4.R,5S,5a.R, lObR, 13afl )-4-acetoxy-3a-ethyl-
9-[(5 5,7 5,9 S)-5-ethyl-5-hydroxy-9-methoxycarbonyl-
1,4,5,6,7, 8,9, 10-octahydro-3,7-methano-3-
azacycloundecino [5,4-b ]indol-9-yl]-5-hydroxy-8-methoxy-
6-methyl-3a,4,5,5a,6,ll,12,13a-octahydro-l//-
indolizino[8,l-cd]carbazole-5-carboxylate monosulfate
[143-67-9]
Vinblastine Sulfate contains not less than 96.0% and
not more than 102.0% of C 46 H5 8 N40 9 .H 2 S04, calculat-
ed on the dried basis.
JPXV
Official Monographs / Vinblastine Sulfate for Injection 1227
Description Vinblastine Sulfate occurs as a white to pale
yellow powder.
It is soluble in water, sparingly soluble in methanol, and
practically insoluble in ethanol (99.5).
It is hygroscopic.
Optical rotation [a]o- -28- -35° (0.20 g calculated on
the dried basis, methanol, 10 mL, 100 mm).
Identification (1) Determine the absorption spectrum of a
solution of Vinblastine Sulfate (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum or the spectrum of
a solution of Vinblastine Sulfate Reference Standard pre-
pared in the same manner as the sample solution: both spec-
tra exhibit similar intensities of absorption at the same
wavelengths.
(2) Determine the infrared absorption spectrum of
Vinblastine Sulfate as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum or the spec-
trum of Vinblastine Sulfate Reference Standard: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) A solution of Vinblastine Sulfate (1 in 100) responds
to the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 15 mg of Vinblastine Sulfate in 10 mL
of water: the pH of this solution is between 3.5 and 5.0.
Purity (1) Clarity and color of solution — Dissolve 50 mg
of Vinblastine Sulfate in 10 mL of water: the solution is clear
and colorless.
(2) Related substances — Dissolve about 4 mg of Vin-
blastine Sulfate in 10 mL of water, and use this solution as
the sample solution. Pipet 1 mL of the sample solution, add
water to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with exactly 200 //L each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine each peak area of these solu-
tions by the automatic integration method: the area of peak
other than the main peak is not larger than 1/4 of the peak
area of vinblastine from the standard solution, and the total
area of the peaks other than the main peak is not larger than
3/4 of the peak area of vinblastine from the standard
solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 4 times as long as the
retention time of vinblastine beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 2.5 mL of the
standard solution add water to make exactly 100 mL.
Confirm that the peak area of vinblastine obtained from
200 /uL of this solution is equivalent to 1.7 to 3.3% of that
from 200 /xh of the standard solution.
System performance: Proceed as directed in the system
suitability in the Assay.
System repeatability: When the test is repeated 5 times with
200 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
vinblastine is not more than 1.5%.
Loss on drying Perform the test with about 10 mg of
Vinblastine Sulfate as directed in Method 2 under the Ther-
mal Analysis <2.52> according to the following conditions:
not more than 15.0%.
Operating conditions —
Heating rate: 5°C/minute
Temperature range: room temperature to 200°C
Atmospheric gas: dried Nitrogen
Flow rate of atmospheric gas: 40 mL/minute
Assay Weigh accurately about 10 mg each of Vinblastine
Sulfate and Vinblastine Sulfate Reference Standard (previ-
ously determine the loss on drying in the same manner as Vin-
blastine Sulfate), dissolve in water to make exactly 25 mL,
and use these solutions as the sample solution and the stan-
dard solution, respectively. Perform the test with exactly 20
,uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of vinblastine.
Amount (mg) of C 4 6H5 8 N40 9 .H 2 S0 4 = W s x (A T /A S )
W s : Amount (mg) of Vinblastine Sulfate Reference
Standard, calculated on the dried basis
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 262 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: To 7 mL of diethylamine add water to make
500 mL, and adjust the pH to 7.5 with phosphoric acid. To
380 mL of this solution add 620 mL of a mixture of methanol
and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
vinblastine is about 8 minutes.
System suitability —
System performance: Dissolve 10 mg each of Vinblastine
Sulfate and vincristine sulfate in 25 mL of water. When the
procedure is run with 20 /xL of this solution under the above
operating conditions, vincristine and vinblastine are eluted in
this order with the resolution between these peaks being not
less than 4.
System repeatability: When the test is repeated 5 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
vinblastine is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, at not exceeding -20°C.
Vinblastine Sulfate for Injection
>±i7ffl \l '•> -i 7 x ^ > mm&
Vinblastine Sulfate for Injection is a preparation for
injection, which is dissolved before use.
It contains not less than 90.0% and not more than
1228 Vincristine Sulfate / Official Monographs
JP XV
110.0% of the labeled amount of vinblastine sulfate
(C 46 H 58 N 4 9 .H 2 S0 4 : 909.05).
Method of preparation Prepare as directed under Injec-
tions, with Vinblastine Sulfate.
Description Vinblastine Sulfate for Injection occurs as
white to pale yellow, light masses or powder.
It is freely soluble in water.
The pH of a solution (1 in 1000) is 3.5 - 5.0.
Identification Proceed as directed in the Identification (1)
under Vinblastine Sulfate.
Purity Related substances — Dissolve 4 mg of Vinblastine
Sulfate for Injection in 10 mL of water, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add water to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with exactly 200 fih each
of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: the area of the peak other than the
main peak from the sample solution is not larger than 1/2 of
the peak area of vinblastine from the standard solution, and
the total area of the peaks other than the main peak is not
larger than 2 times the peak area of vinblastine from the stan-
dard solution.
Operating conditions—
Perform as directed in the operating conditions in Purity
(2) under Vinblastine Sulfate.
System suitability —
Perform as directed in the system suitability in Purity (2)
under Vinblastine Sulfate.
Bacterial endotoxins <4.01> Less than lOEU/mg.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Dissolve 1 Vinblastine Sulfate for Injection in water to
make exactly FmL so that each mL contains about 0.4 mg of
vinblastine sulfate (C 46 H5 8 N409.H 2 S04) according to the la-
beled amount, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of Vinblastine Sul-
fate Reference Standard (previously determine the loss on
drying in the same manner as Vinblastine Sulfate), dissolve in
water to make exactly 25 mL, and use this solution as the
standard solution. Proceed as directed in the Assay under
Vinblastine Sulfate.
Amount (mg) of vinblastine sulfate (C4 6 H 58 N 4 09.H 2 S0 4 )
= W s x(Aj/A s )x(25/V)
W s : Amount (mg) of Vinblastine Sulfate Reference
Standard, calculated on the dried basis
Foreign insoluble matter <6.06> Perform the test according
to Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
filtration method: it meets the requirement.
Assay Take an amount of Vinblastine Sulfate for Injection,
equivalent to 0.10 g of vinblastine sulfate
(C 46 H 58 N 4 9 .H 2 S0 4 ), dissolve each content with a suitable
amount of water, transfer into a 100-mL volumetric flask,
wash each container with water, transfer the washings into
the volumetric flask, and add water to make exactly 100 mL.
Pipet 10 mL of this solution, add water to make exactly 25
mL, and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of Vinblastine Sulfate Refer-
ence Standard (previously determine the loss on drying in the
same manner as Vinblastine Sulfate), dissolve in water to
make exactly 25 mL, and use this solution as the standard so-
lution. Proceed as directed in the Assay under Vinblastine
Sulfate.
Amount (mg) of vinblastine sulfate (C 46 H 58 N 4 09.H 2 S0 4 )
= W s x (A T /A S ) x 10
W s : Amount (mg) of Vinblastine Sulfate Reference
Standard, calculated on the dried basis
Containers and storage Containers — Hermetic containers,
and colored containers may be used.
Storage — Light-resistant, at 2 to 8 °C.
Vincristine Sulfate
HO
• HfSOa
C 46 H 56 N 4 Oi .H 2 SO 4 : 923.04
Methyl (3ai?,4i?,5S , ,5ai?, 10b/?, 13a/? )-4-acetoxy-3a-ethyl-
9-[(5 5,7 S,9 S)-5-ethyl-5-hydroxy-9-methoxycarbonyl-
1,4,5,6,7, 8,9, 10-octahydro-3,7-methano-
3-azacycloundecino [5,4-b ]indol-9-yl]-6-f ormyl-5-hydroxy-
8-methoxy-3a,4,5,5a,6,ll,12,13a-octahydro-l//-
indolizino[8,l-crf]carbazole-5-carboxylate monosulfate
[2068-78-2]
Vincristine Sulfate contains not less than 95.0% and
not more than 105.0% of C 46 H 56 N 4 O 10 .H 2 SO 4 , calcu-
lated on the dried basis.
Description Vincristine Sulfate occurs as a white to light
yellowish white powder.
It is very soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
It is hygroscopic.
Optical rotation [a]™: +28.5 - +35.5° (0.2 g, calculated
on the dried basis, water, 10 mL, 100 mm).
Identification (1) Dissolve 5 mg of Vincristine Sulfate in 2
mL of cerium (IV) tetraammonium sulfate-phosphoric acid
TS: a blue-purple color develops.
(2) Determine the absorption spectrum of a solution of
Vincristine Sulfate (1 in 50,000) as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum: both spectra exhibit similar in-
JPXV
Official Monographs / Vitamin A Oil 1229
tensities of absorption at the same wavelengths.
(3) Dissolve 0.02 g of Vincristine Sulfate in 10 mL of so-
dium chloride TS, adjust the pH to between 9 and 10 with
ammonia TS, and extract with two 5-mL portions of chlo-
roform. Wash the combined chloroform extracts with a small
quantity of sodium chloride TS, add a small quantity of an-
hydrous sodium sulfate, and allow to stand for several
minutes. Filter through a pledget of absorbent cotton,
evaporate the filtrate to dryness under reduced pressure, and
dissolve the residue in a small quantity of chloroform. Deter-
mine the infrared absorption spectrum of the solution as
directed in the solution method under Infrared Spectrophoto-
metry <2.25>, and compare the spectrum with the Reference
Spectrum: both spectra exhibit similar intensities of absorp-
tion at the same wave numbers.
(4) A solution of Vincristine Sulfate (1 in 100) responds
to the Qualitative Tests <1.09> for sulfate.
pH <2.54> Dissolve 10 mg of Vincristine Sulfate in 10 mL of
water: the pH of this solution is between 3.5 and 4.5.
Purity (1) Clarity and color of solution — Dissolve 25 mg
of Vincristine Sulfate in 10 mL of water: the solution is clear
and colorless.
(2) Related substances — Dissolve 25 mg of Vincristine
Sulfate in 10 mL of water, and use this solution as the sample
solution. Pipet 1 mL of the sample solution, add water to
make exactly 20 mL, and use this solution as the standard so-
lution. Perform the test with exactly 20 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine each peak area of these solutions by the au-
tomatic integration method: the total area of the peaks other
than the principal peak of the sample solution is not larger
than the peak area of vincristine from the standard solution,
and the area of any peak other than the principal peak of the
sample solution is not larger than 2/5 of the peak area of vin-
cristine from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 297 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 25 cm in length, packed with octylsilanized
silica gel for liquid chromatography (5 fim in particle di-
ameter).
Column temperature: Room temperature.
Mobile phase: Use methanol as the mobile phase A, and a
mixture of water and diethylamine (197:3) adjusted with
phosphoric acid to a pH of 7.5 as the mobile phase B. Run a
mixture of the mobile phase A and the mobile phase B (3 1 : 1 9)
for 24 minutes after injection of the sample, and run a mix-
ture of the mobile phase A and the mobile phase B for subse-
quent 30 minutes, increasing the composition ratio of the mo-
bile phase A by 1% per minute. For subsequent 4 minutes,
run a mixture of the mobile phase A and the mobile phase B,
decreasing the composition ratio of the mobile phase A by
7.5% per minute, then continue running a mixture of the mo-
bile phase A and the mobile phase B (31:19).
Flow rate: Adjust the flow rate so that the retention time of
vincristine is about 19 minutes.
Selection of column: Dissolve 10 mg each of Vincristine
Sulfate and vinblastine sulfate in 100 mL of water. Proceed
with 20 /XL of this solution under the above operating condi-
tions, and calculate the resolution. Use a column giving elu-
tion of vincristine and vinblastine in this order with the reso-
lution between these peaks being not less than 4.
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of vincristine from 20 iiL of the standard
solution is between 5 mm and 15 mm.
Time span of measurement: About 3 times as long as the
retention time of vincristine beginning after the solvent peak.
Loss on drying <2.41> Not more than 12.0% (50 mg, in
vacuum, 105°C, 2 hours).
Assay Weigh accurately about 10 mg of Vincristine Sulfate,
dissolve in acetic acid-sodium acetate buffer solution, pH
5.0, to make exactly 50 mL. Pipet 5 mL of this solution, add
acetic acid-sodium acetate buffer solution, pH 5.0, to make
exactly 50 mL. Determine the absorbance A of this solution
at the maximum wavelength at about 296 nm as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>.
Amount (mg) of C 46 H5 6 N40 1 o.H 2 S04
= (,4/177) x 5000
Containers and storage Containers — Hermetic containers.
Storage — Light-resistant, and in a cold place.
Vitamin A Oil
Vitamin A Oil is synthetic vitamin A esters diluted
with fixed oils.
It contains not less than 30,000 vitamin A Units per
g-
It may contain suitable antioxidants.
It contains not less than 90.0% and not more than
120.0% of the labeled amount of vitamin A.
Description Vitamin A Oil is a yellow to yellow-brown,
clear or slightly turbid oil. It is odorless or has a faint, char-
acteristic odor.
It is decomposed upon exposure to air or light.
Identification Dissolve Vitamin A Oil, Retinol Acetate
Reference Standard and Retinol Palmitate Reference Stan-
dard, equivalent to 15,000 Units, in 5 mL of petroleum ether,
and use these solutions as the sample solution, the standard
solution (1) and the standard solution (2), respectively. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 fiL each of the sample so-
lution and standard solutions (1) and (2) on a plate of silica
gel for thin-layer chromatography. Develop with a mixture of
cyclohexane and diethyl ether (12:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly antimony (III) chlo-
ride TS: the principal spot obtained from the sample solution
has the same color tone and the same i?f value with the blue
spot obtained from the standard solution (1) or the standard
solution (2).
Purity (1) Acidity — Dissolve 1.2 g of Vitamin A Oil in 30
mL of a mixture of neutralized ethanol and diethyl ether
(1:1), boil gently for 10 minutes under a reflux condenser,
cool, and add 5 drops of phenolphthalein TS and 0.60 mL of
0.1 mol/L sodium hydroxide VS: a red color develops.
(2) Rancidity — No unpleasant odor of rancid oil is per-
ceptible by warming Vitamin A Oil.
1230 Vitamin A Oil Capsules / Official Monographs
JP XV
Assay Proceed as directed in Method 1-1 under Vitamin A
Assay <2.55>.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and almost well-filled, or under
Nitrogen atmosphere.
Vitamin A Oil Capsules
Vitamin A Capsules
Vitamin A Oil Capsules contain not less than 90.0%
and not more than 130.0% of the labeled Units of vita-
min A.
Method of preparation Prepare as directed under Capsules,
with Vitamin A Oil.
Description The content of Vitamin A Oil Capsules con-
forms to the requirements of Description under Vitamin A
Oil.
Identification Proceed the test with the content of Vitamin
A Oil Capsules as directed in the Identification under Vitamin
A Oil.
Assay Weigh accurately 20 Vitamin A Oil Capsules, and
open the capsules to take out the content. Wash the capsules
well with a small amount of diethyl ether, allow the capsules
to stand at ordinal temperature to vaporize the diethyl ether,
and weigh accurately. Perform the test with the content as
directed under Vitamin A Assay <2.55>, and calculate the u-
nits of vitamin A per capsule. Before applying Method 1-1, it
is necessary to know which the sample is, retinol acetate or
retinol palmitate.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Compound Vitamin B Powder
Method of preparation
Thiamine Nitrate
Riboflavin
Pyridoxine Hydrochloride
Nicotinamide
Starch, Lactose Hydrate or
their mixture
10g
10g
10 g
100 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Compound Vitamin B Powder is orange-yellow
in color. It has a slighly bitter taste.
It is slowly affected by light.
Identification (1) Shake 2 g of Compound Vitamin B
Powder with 100 mL of water, filter, and to 5 mL of the
filtrate add 2.5 mL of sodium hydroxide TS and 0.5 mL of
potassium hexacyanoferrate (III) TS. Then add 5 mL of 2-
methyl-1-propanol, shake the mixture vigorously for 2
minutes, allow to stand, and observe under ultraviolet light:
the 2-methyl-l-propanol layer shows a blue-purple fluores-
cence. This fluorescence disappears when the mixture is acidi-
fied, but reappears when it is again made alkaline (thiamine).
(2) Shake 0.1 g of Compound Vitamin B Powder with
100 mL of water, and filter. Perform the following tests with
the filtrate (riboflavin).
(i) The filtrate is light yellow-green in color and has an in-
tense yellow-green fluorescence. This color and fluorescence
of the solution disappears upon the addition of 0.02 g of so-
dium hydrosulfite to 5 mL of the filtrate, and again appears
by shaking the mixture in air. This fluorescence disappears
upon the addition of dilute hydrochloric acid or sodium
hydroxide TS.
(ii) To 10 mL of the filtrate placed in a glass-stoppered
test tube add 1 mL of sodium hydroxide TS, after illuminat-
ing with a fluorescence lamp of 10 to 30 watts at 20-cm dis-
tance for 30 minutes between 20°C and 40°C, acidify with
0.5 mL of acetic acid (31), and shake thoroughly with 5 mL
of chloroform: the chloroform layer shows yellow-green
fluorescence.
(3) Shake 1 g of Compound Vitamin B Powder with 100
mL of diluted ethanol (7 in 10), filter, and to 5 mL of the
filtrate add 2 mL of sodium hydroxide TS and 40 mg of man-
ganese dioxide. Heat on a water bath for 30 minutes, cool,
and filter. Add 5 mL of 2-propanol to 1 mL of the filtrate,
and use the solution as the sample solution. To 3 mL of the
sample solution add 2 mL of bartibal buffer solution, 4 mL
of 2-propanol and 2 mL of a freshly prepared solution of 2,6-
dibromo-N-chloro-l,4-benzoquinone monoimine in ethanol
(95) (1 in 4000) prepared when required for use: a blue color
develops. To 1 mL of the sample solution add 1 mL of a satu-
rated boric acid solution, and proceed as directed in the same
manner as above: no blue color develops (pyridoxine).
(4) Shake 0.5 g of Compound Vitamin B Powder with 10
mL of ethanol (95), filter, and evaporate 1 mL of the filtrate
on a water bath to dryness. Add 0.01 g of 2,4-
dinitrochlorobenzen to the residue, heat gently for 5-6 se-
conds to fuse, and after cooling, add 4 mL of potassium
hydroxide-ethanol TS: a red color develops (nicotinamide).
(5) Shake 1 g of Compound Vitamin B Powder with 5
mL of diluted ethanol (7 in 10), filter, and use the filtrate as
the sample solution. Separately, dissolve 0.01 g each of thia-
mine mononitrate, riboflavin, pyridoxine hydrochloride and
nicotinamide in 1 mL, 50 mL, 1 mL and 1 mL of water,
respectively, and use these solutions as standard solutions (1),
(2), (3) and (4). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 2 fiL
each of the sample solution and standard solutions (1), (2),
(3) and (4) on a plate of silica gel with fluorescent indicator
for thin-layer chromatography. Develop the plate with a mix-
ture of chloroform, ethanol (95) and acetic acid (100)
(100:50:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (broad spectrum
wavelength): four spots from the sample solution show the
same color tone and the same Rf value as the corresponding
spots from standard solutions (1), (2), (3) and (4).
Containers and storage Containers — Well-closed contain-
JPXV
Official Monographs / Voglibose 1231
ers.
Storage — Light-resistant.
Voglibose
Ci H 2 iNO 7 : 267.28
3,4-Dideoxy-4-[2-hydroxy-l-(hydroxymethyl)ethylamino]-
2-C-(hydroxymethyl)-D-epz-inositol
[83480-29-9]
Voglibose contains not less than 99.5% and not
more than 101.0% of C 10 H 21 NO 7 , calculated on the an-
hydrous basis.
Description Voglibose occurs as white crystals or crystalline
powder.
It is very slightly soluble in water, freely soluble in acetic
acid (100), slightly soluble in methanol, and very slightly
soluble in ethanol (99.5).
It dissolves in 0.1 mol/L hydrochloric acid TS.
Identification (1) Determine the infrared absorption spec-
trum of Voglibose as directed in the potassium bromide disk
method under Infrared Spectrophotometry <2.25>, and com-
pare the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(2) Determine the spectrum of a solution of Voglibose in
heavy water for nuclear magnetic resonance spectroscopy (3
in 70) as directed under Nuclear Magnetic Resonance Spec-
troscopy <2.21> ('H), using sodium 3-trimethylsilyl-
propionate-d 4 for nuclear magnetic resonance spectroscopy
as an internal reference compound: it exhibits 2 double sig-
nals A at about 5 1.5 ppm, 2 double signals B at about 5 2.1
ppm, a multiple signal C at about 5 2.9 ppm, and a multiple
signal D between 6 3.4 ppm and 5 3.9 ppm. The area intensity
ratio of each signal, A:B:C:D, is about 1:1:1:10.
Optical rotation <2.49> [a]™: +45 - +48° (0.2 g calculated
on the anhydrous basis, 0.1 mol/L hydrochloric acid TS, 20
mL, 100 mm).
pH <2.54> Dissolve 1.0 g of Voglibose in 10 mL of water:
the pH of the solution is between 9.8 and 10.4.
Melting point <2.60> 163 - 168°C
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Voglibose according to Method 1, and perform the test. Ad-
just the pH of the test solution between 3.0 and 3.5 with di-
lute hydrochloric acid instead of dilute acetic acid. Prepare
the control solution with 1.0 mL of Standard Lead Solution
(not more than 10 ppm).
(2) Related substances — Dissolve 50 mg of Voglibose in
50 mL of the mobile phase, and use this solution as the sam-
ple solution. Pipet 1 mL of the sample solution, add the mo-
bile phase to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 50,mL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing operating conditions, and determine each peak area
by the automatic integration method: the total area of the
peaks other than voglibose is not larger than 1/5 times the
peak area of voglibose from the standard solution. For the
calculate of the total area, use the area of the peaks, having
the relative retention time of about 1.7, about 2.0 and about
2.3 after multiplying by their relative response factors, 2, 2
and 2.5, respectively.
Operating conditions —
Apparatus: Use an apparatus consisting of 2 pumps for the
mobile phase and reaction reagent transportation, sample in-
jection port, column, reaction coil, cooling coil, detector and
recording device, and the reaction coil and cooling coil main-
tained at a constant temperature.
Detector: Fluorophotometer (excitation wavelength: 350
nm, fluorescence wavelength: 430 nm)
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with pentaethylenehex-
aaminated polyvinyl alcohol polymer bead for liquid chro-
matography.
Column temperature: A constant temperature of about
25°C.
Reaction coil: A polytetrafluoroethylene tube 0.5 mm in
inside diameter and 20 m in length
Cooling coil: A polytetrafluoroethylene tube 0.3 mm in in-
side diameter and 2 m in length
Mobile phase: To 1.56 g of sodium dihydrogen phosphate
dihydrate add water to make 500 mL. To this solution add a
solution, prepared by dissolving 3 .58 g of disodium hydrogen
phosphate dodecahydrate in water to make 500 mL, to adjust
to pH 6.5. To 370 mL of this solution add 630 mL of acetoni-
trile.
Reaction reagent: Dissolve 6.25 g of taurine and 2.56 g of
sodium periodate in water to make 1000 mL.
Reaction temperature: A constant temperature of about
100°C
Cooling temperature: A constant temperature of about
15°C
Flow rate of the mobile phase: Adjust the flow rate so that
the retention time of voglibose is about 20 minutes.
Flow rate of the reaction reagent: Same as the flow rate of
the mobile phase
Time span of measurement: About 2.5 times as long as the
retention time of voglibose, beginning after the solvent peak
System suitability —
Test for required detectability: Pipet 10 mL of the standard
solution, and add the mobile phase to make 100 mL. Confirm
that the peak area of voglibose obtained from 50 /uL of this
solution is equivalent to 7 to 13% of that of voglibose ob-
tained from 50 /uL of the standard solution.
System performance: When the procedure is run with 50
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of voglibose are not less than 7000 and be-
tween 0.8 and 1.2, respectively.
System repeatability: When the test is repeated 6 times with
50 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
voglibose is not more than 3.0%.
1232 Voglibose Tablets / Official Monographs
JP XV
Water <2.48> Not more than 0.2% (0.5 g, coulometric titra-
tion).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.4 g of Voglibose, dissolve
in 80 mL of acetic acid (100), and titrate <2.50> with 0.1
mol/L perchloric acid VS (potentiometric titration). Perform
a blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L perchloric acid VS
= 26.73 mg of C 10 H 21 NO 7
Containers and storage Containers — Tight containers.
Voglibose Tablets
Voglibose Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of vogli-
bose (C 10 H 21 NO 7 : 267.28).
Method of preparation Prepare as directed under Tablets,
with Voglibose.
Identification Shake vigorously an amount of pulverized
Voglibose Tablets, equivalent to 5 mg of Voglibose according
to the labeled amount, with 40 mL of water, and centrifuge.
Transfer the supernatant liquid to a chromatographic column
[prepared by pouring 1.0 mL of strongly acidic ion-exchange
resin (H type) for column chromatography (100 to 200 ,wm in
particle diameter) into a chromatographic column 8 mm in
inside diameter and 130 mm in height], and allow to flow at a
rate of about 5 mL per minute. Then wash the column with
200 mL of water, and allow to flow with 10 mL of diluted
ammonia TS (1 in 4) at a rate of about 5 mL per minute.
Filter the effluent solution two times through a membrane
filter with pore size of not more than 0.22 fim. Evaporate the
filtrate to dryness at 50°C under reduced pressure, dissolve
the residue with 0.5 mL of a mixture of water and methanol
(1:1), and use this solution as the sample solution. Separate-
ly, dissolve 20 mg of voglibose for assay in 2 mL of the mix-
ture of water and methanol (1:1), and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of acetone, ammonia water (28) and
water (5:3:1) to a distance of about 12 cm, air-dry the plate,
and allow to stand in iodine vapors: the principal spot from
the sample solution and the spot from the standard solution
show a yellow-brown color, and the same Rf value.
Uniformity of dosage unit <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Voglibose Tablets add exactly KmL of the
mobile phase so that the solution contains about 40 /ug of
voglibose (C 10 H 2 iNO 7 ) per mL according to the labeled
amount, disintegrate the tablet completely by shaking, and
centrifuge. Filter the supernatant liquid through a membrane
filter with pore size of not more than 0.45 fim. Discard the
first 1 mL of the filtrate, and use the subsequent filtrate as the
sample solution. Hereinafter, proceed as directed in the As-
say.
Amount (g) of voglibose (C 10 H 2 iNO 7 )
= W s x(A T /A s )x(V/500)
W s : Amount (mg) of voglibose for assay, calculated on the
anhydrous basis
Assay To 20 tables of Voglibose Tablets add 80 mL of the
mobile phase, and completely disintegrate by shaking. To an
exact volume of the solution, equivalent to about 4 mg of
voglibose (C 10 H 2 iNO 7 ) according to the labeled amount, add
the mobile phase to make exactly 100 mL, and centrifuge.
Filter the supernatant liquid through a membrane filter with
pore size of not more than 0.45 //m. Discard the first 1 mL of
the filtrate, and use the subsequent filtrate as the sample solu-
tion. Separately, weigh accurately about 20 mg of voglibose
for assay (previously determine the water <2.48> in the same
manner as Voglibose), and dissolve in the mobile phase to
make exactly 25 mL. Pipet 5 mL of this solution, add the mo-
bile phase to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with exactly 50,mL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of voglibose.
Amount (mg) of voglibose (C I0 H 21 NO 7 )
= W s x(A 1 /A s )x (1/500)
W s : Amount of voglibose for assay, calculated on the
dried basis
Operating conditions —
Apparatus: Use an apparatus consisting of 2 pumps for the
mobile phase and reaction reagent transportation, sample in-
jection port, column, reaction coil, cooling coil, detector and
recording device, and the reaction coil and cooling coil main-
tained at a constant temperature.
Detector: Fluorophotometer (excitation wavelength: 350
nm, fluorescence wavelength: 430 nm)
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with aminopropylsilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
25°C
Reaction coil: A polytetrafluoroethylene tube 0.5 mm in
inside diameter and 20 m in length
Cooling coil: A polytetrafluoroethylene tube 0.3 mm in in-
side diameter and 2 m in length
Mobile phase: To 1.56 g of sodium dihydrogen phosphate
dihydrate add water to make 500 mL. To this solution add a
solution, prepared by dissolving 3 .58 g of disodium hydrogen
phosphate dodecahydrate in water to make 500 mL, to adjust
to pH 6.5. To 300 mL of this solution add 600 mL of acetoni-
trile.
Reaction reagent: Dissolve 6.25 g of taurine and 2.56 g of
sodium periodate in water to make 1000 mL.
Reaction temperature: A constant temperature of about
100°C
Cooling temperature: A constant temperature of about
15°C
Flow rate of mobile phase: Adjust the flow rate so that the
retention time of voglibose is about 20 minutes.
JPXV
Official Monographs / Warfarin Potassium 1233
Flow rate of reaction reagent: Same as the flow rate of the
mobile phase
System suitability —
System performance: Dissolve 2 mg of voglibose for assay
and 0.2 g of lactose monohydrate in 5 mL of water, and add
the mobile phase to make 50 mL. When the procedure is run
with 50 fiL of this solution under the above operating condi-
tions, lactose and voglibose are eluted in this order with the
resolution between these peaks being not less than 4.
System repeatability: When the test is repeated 6 times with
50 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
voglibose is not more than 2.0%.
Containers and storage Containers — Tight containers.
Warfarin Potassium
and enanliomer
Ci 9 H 15 K0 4 : 346.42
Monopotassium (1 i?S)-2-oxo-3-(3-oxo-
l-phenylbutyl)chromen-4-olate [2610-86-8]
Warfarin Potassium, when dried, contains not less
than 98.0% and not more than 102.0% of Ci 9 H 15 K0 4 .
Description Warfarin Potassium occurs as a white, crystal-
line powder.
It is very soluble in water, and freely soluble in ethanol
(95).
It dissolves in sodium hydroxide TS.
The pH of a solution prepared by dissolving 1.0 g of
Warfain Potassium in 100 mL of water is 7.2 - 8.3.
It is colored to light yellow by light.
A solution of Warfarin Potassium (1 in 10) shows no opti-
cal rotation.
Identification (1) Determine the absorption spectrum of a
solution of Warfarin Potassium in 0.02 mol/L potassium
hydroxide TS (1 in 100,000) as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and compare the spectrum
with the Reference Spectrum or the spectrum of a slolution of
Warfarin Potassium Reference Standard prepared in the
same manner as the sample solution: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of
Warfarin Potassium, previously dried, as directed in the
potassium bromide disk method under Infrared Spec-
trophotometry <2.25>, and compare the spectrum with the
Reference Spectrum or the spectrum of Warfarin Potassium
Reference Standard: both spectra exhibit similar intensities
of absorption at the same wave numbers.
(3) A solution of Walfarin Potassium (1 in 250) responds
to the Qualitative Tests <1.09> (1) for potassium salt.
Purity (1) Alkaline colored substances — Dissolve 1.0 g of
Warfarin Potassium in a solution of sodium hydroxide (1 in
20) to make exactly 10 mL, and determine the absorbance at
385 nm within 15 minutes as directed under Ultraviolet-visi-
ble Spectrophotometry <2.24>, using a solution of sodium
hydroxide (1 in 20) as a blank: it does not exceed 0.20.
(2) Heavy metals <1.07> — Dissolve 2.0 g of Warfarin
Potassium in 30 mL of ethanol (95), add 2 mL of dilute acetic
acid and ethanol (95) to make 50 mL. Perform the test using
this solution as the test solution. Prepare the control solution
with 2.0 mL of Standard Lead Solution, 2 mL of dilute acetic
acid and ethanol (95) to make 50 mL (not more than 10
ppm).
(3) Related substances — Dissolve 0.10 g of Warfarin
Potassium in 100 mL of a mixture of water and methanol
(3:1), and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add the mixture of water and
methanol (3:1) to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly 20 /xL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine each peak area by the auto-
matic integration method: each peak area other than warfa-
rin is not larger than 1/10 times the peak area of warfarin ob-
tained with the standard solution, and the total area of the
peaks other than warfarin is not larger than 1/2 times the
peak area of warfarin with the standard solution.
Operating conditions —
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay.
Time span of measurement: About 2 times as long as the
retention time of warfarin beginning after the solvent peak.
System suitability —
Test for required detectability: To exactly 1 mL of the stan-
dard solution add the mixture of water and methanol (3:1) to
make exactly 20 mL. Confirm that the peak area of warfarin
obtained with 20 /xL of this solution is equivalent to 3.5 to
6.5% of that with 20 /xL of the standard solution.
System performance: Dissolve 20 mg of propyl para-
hydroxybenzoate in 50 mL of methanol, and add water to
make 200 mL. To 5 mL of this solution add 4 mL of a solu-
tion of Warfarin Potassium in the mixture of water and
methanol (3:1) (1 in 2000), and add the mixture of water and
methanol (3:1) to make 100 mL. When the procedure is run
with 20 /xL of this solution under the above operating condi-
tions, propyl parahydroxybenzoate and warfarin are eluted
in this order with the resolution between these peaks being
not less than 7.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
warfarin is not more than 2.0%.
Loss on drying <2.41> Not more than 4.5% (1 g, 105 °C, 3
hours).
Assay Weigh accurately about 25 mg each of Warfarin
Potassium and Warfarin Potassium Reference Standard,
previously dried, and dissolve separately in the mixture of
water and methanol (3:1) to make exactly 50 mL. Pipet 10
mL each of these solutions, add the mixture of water and
methanol (3:1) to make exactly 50 mL, and use these solu-
tions as the sample solution and the standard solution. Per-
form the test with exactly 20 /uL each of the sample solution
and standard solution as directed under Liquid Chro-
1234 Warfarin Potassium Tablets / Official Monographs
JP XV
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of warfarin.
Amount (mg) of C 19 H 15 K0 4 = W s x(A T /A s )
W s : Amount (mg) of Warfarin Potassium Reference Stan-
dard
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 260 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with cyanopropyl-
silanized silica gel for liquid chromatography (5 jum in parti-
cle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (68:32:1).
Flow rate: Adjust the flow rate so that the retention time of
warfarin is about 10 minutes.
System suitability —
System performance: When the procedure is run with 20
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of warfarin are not less than 8000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
warfarin is not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Warfarin Potassium Tablets
Warfarin Potassium Tablets contain not less than
95.0% and not more than 105.0% of the labeled
amount of warfarin potassium (C 19 H 15 K0 4 : 346.42).
Method of preparation Prepare as directed under Tablets,
with Warfarin Potassium.
Identification (1) Determine the absorption spectrum of
the solution T 2 obtained in the Assay, using 0.02 mol/L
potassium hydroxide TS as the blank, as directed under
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits a
maximum between 306 nm and 310 nm, and a minimum be-
tween 258 nm and 262 nm. Separately, determine the absorp-
tion spectrum of the solution T t obtained in the Assay, using
0.02 mol/L hydrochloric acid TS as the blank, as directed un-
der Ultraviolet-visible Spectrophotometry <2.24>: it exhibits
maxima between 281 nm and 285 nm and between 303 nm
and 307 nm, and a minimum between 243 nm and 247 nm.
(2) Weigh a quantity of Warfarin Potassium Tablets,
equivalent to 0.01 g of Warfarin Potassium according to the
labeled amount, add 10 mL of acetone, shake, and filter.
Heat the filtrate on a water bath to evaporate the acetone. To
the residue add 10 mL of diethyl ether and 2 mL of dilute
hydrochloric acid, and shake: the aqueous layer responds to
the Qualitative Tests <1.09> (1) for potassium salt.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
Powder 1 tablet of Warfarin Potassium Tablets, add 40
mL of water, and shake vigorouly for 30 minutes. Add water
to make exactly KmL of this solution containing about 20 /ug
of warfarin potassium (C 19 H 15 K0 4 ) per ml. Filter this solu-
tion, discard the first 5 mL of the filtrate, and use the subse-
quent filtrate as the sample solution. Separately, weigh ac-
curately about 40 mg of Warfarin Potassium Reference Stan-
dard, previously dried at 105°C for 3 hours, and dissolve in
water to make exactly 100 mL. Pipet 5 mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Pipet 20 mL each of the sample solu-
tion and the standard solution, add 0.05 mol/L hydrochloric
acid TS to make exactly 25 mL, and use these solutions as the
solution T t and the solution Si, respectively. Separately,
pipet 20 mL each of the sample solution and the standard so-
lution, add 0.05 mol/L potassium hydroxide TS to make ex-
actly 25 mL, and use these solutions as the solution T 2 and
the solution S 2 , respectively. Determine the absorbances, A T
and A s , of the solution Tj and the solution Si at 272 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using the solution T 2 and the solution S 2 as the blank, respec-
tively.
Amount (mg) of warfarin potassium (C19H15KO4)
= W s x(A T /A s )x (K/2000)
W s : Amount (mg) of Warfarin Potassium Reference
Standard
Assay Weigh accurately and powder not less than 20
Warfarin Potassium Tablets. Weigh accurately a portion of
the powder, equivalent to about 4 mg of warfarin potassium
(Ci 9 Hi 5 K0 4 ), add 80 mL of water, shake vigorously for 15
minutes, and add water to make exactly 100 mL. Filter this
solution, discard the first 10 mL of the filtrate, and use the
subsequent filtrate as the sample solution. Separately, weigh
accurately about 80 mg of Warfarin Potassium Reference
Standard, previously dried at 105°C for 3 hours, and dissolve
in water to make exactly 100 mL. Pipet 5 mL of this solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Pipet 10 mL each of the sample solu-
tion and the standard solution, add 0.02 mol/L hydrochloric
acid TS to make exactly 20 mL, and use these solutions as the
solution Ti and the solution Si, respectively. Separately,
pipet 10 mL each of the sample solution and the standard so-
lution, add 0.02 mol/L potassium hydroxide TS to make ex-
actly 20 mL, and use these solutions as the solution T 2 and
the solution S 2 , respectively. Determine the absorbances, A T
and A s , of the solution Ti and the solution Si at 272 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>,
using the solution T 2 and the solution S 2 as the blank, respec-
tively.
Amount (mg) of warfarin potassium (Ci 9 Hi 5 K0 4 )
= W s x(Aj/A s )x(l/20)
W s : Amount (mg) of Warfarin Potassium Reference Stan-
dard
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
JPXV
Official Monographs / Water for Injection 1235
Water
H 2 0: 18.02
Water meets the quality standards of water supplies
under Article 4 of the Water Supply Law (the Ministry
of Health, Labour and Welfare Ministerial Ordinance
No. 101, May 30, 2003), and also meets the following
requirement:
Purity Ammonium <1.02> — Perform the test with 30 mL of
Water as directed under Ammonium Limit Test. Prepare the
control solution as follows: to 0.15 mL of Standard Ammo-
nium Solution add purified water for ammonium limit test to
make 30 mL (not more than 0.05 mg/L).
Water for Injection
ai<fffl7k
Water for Injection is water either prepared by distil-
lation of Water or Purified Water, or by the Reverse
Osmosis-Ultranltration (a reverse osmosis membrane,
an ultrafiltration membrane or a combined purification
system using these membranes) of Purified Water, and
used for the preparation of injections, or for an alter-
native usage as a packed Water for Injection, which is
preserved in suitable containers and sterilized.
When Water for Injection is prepared by the Reverse
Osmosis-Ultranltration, take precaution against
microbial contamination of the purifying system to get
comfortable quality being equivalent to that of water
prepared by distillation.
Water for Injection for the preparation of injections
must be used immediately after preparation. However,
it may be stored for a certain period of time, if the
purifying system of water is established for avoiding
microbial contamination and its growth in preserved
period.
Water for Injection preserved in containers and
sterilized is used mainly as solvent for injections to be
dissolved or suspended before use.
Water for Injection prepared by distillation and
packed in containers as sterilized products may be la-
beled "Distilled Water for Injection" as a commonly
used name.
Purity (1) Acidity or alkalinity — To 20 mL of Water for
Injection add 0.1 mL of methyl red TS for acid or alkali test:
a yellow to orange color develops. Separately, to 20 mL of
Water for Injection add 0.05 mL of bromothymol blue TS:
no blue color develops.
(2) Chloride— To 50 mL of Water for Injection add 3
drops of nitric acid and 0.5 mL of silver nitrate TS: no
change occurs.
(3) Sulfate— To 50 mL of Water for Injection add 0.5
mL of barium chloride TS: no change occurs.
(4) Nitrogen from nitrate — Transfer 2.0 mL of Water for
Injection to a 50-mL beaker, add 1 mL of sodium salicylate-
sodium hydroxide TS, 1 mL of a solution of sodium chloride
(1 in 500) and 1 mL of a solution of ammonium amidosulfate
(1 in 1000), and evaporate on a water bath to dryness. Cool,
dissolve in 2 mL of sulfuric acid, allow to stand for 10
minutes with occasional shaking, add 10 mL of water, and
transfer to a Nessler tube. Cool, add 10 mL of a solution of
sodium hydroxide (2 in 5) slowly, and add water to make 25
mL: no yellow color develops.
(5) Nitrogen from nitrite — Transfer 10 mL of Water for
Injection to a Nessler tube, and add 1 mL of a solution of sul-
fanilamide in dilute hydrochloric acid (1 in 100) and 1 mL of
N,N-diethyl-N' -1-naphthylethylenediamine oxalate TS: no
pale red color develops.
(6) Ammonium <1.02> — Perform the test with 30 mL of
Water for Injection as the test solution. Prepare the control
solution as follows: to 0.15 mL of Standard Ammonium So-
lution add purified water for ammonium limit test to make 30
mL (not more than 0.05 mg/L).
(7) Heavy metals — To 40 mL of Water for Injection add
2 mL of dilute acetic acid and 1 drop of sodium sulfide TS : no
change occurs.
(8) Potassium permanganate-reducing substances — To
100 mL of Water for Injection add 10 mL of dilute sulfuric
acid, boil, add 0.10 mL of 0.02 mol/L potassium perman-
ganate VS, and boil again for 10 minutes: the red color does
not disappear.
(9) Residue on evaporation — Evaporate 100 mL of
Water for Injection on a water bath to dryness, and dry the
residue at 105 °C for 1 hour: the mass of the residue is not
more than 1.0 mg.
For Water for Injection prepared by the Reverse Osmosis-
Ultrafiltration for the preparation of injections, perform the
test for (8) Total organic carbon described below, instead of
(8) Potassium permanganate-reducing substances. For Water
for Injection preserved in containers and sterilized, perform
the test for (1) Acid or alkali, (2) Chloride, (6) Ammonium
and (9) Residue on evaporation according to the following
methods:
(1) Acidity or alkalinity — Shake gently 20 mL of Water
for Injection with 0.05 mL of phenol red TS and 0.13 mL of
0.01 mol/L sodium hydroxide VS, and allow to stand for 30
seconds: a red color develops. Separately, shake gently 20 mL
of Water for Injection with 0.05 mL of bromothymol blue
TS and 0.13 mL of 0.01 mol/L hydrochloric acid VS, and al-
low to stand for 30 seconds: a yellow color develops.
(2) Chloride — For Water for Injection in containers
holding a volume not more than 10 mL, add 2.0 mL of dilute
nitric acid to 15 mL of Water for Injection, and use this solu-
tion as the test solution. Separately, to 0.20 mL of 0.001 mol
/L hydrochloric acid VS add water to make 15 mL, then add
2.0 mL of dilute nitric acid, and use this solution as the con-
trol solution. Mix the test solution and the control solution
separately with 0.30 mL each of silver nitrate TS, allow to
stand for 5 minutes under the protection from sunlight, and
compare the turbidity of the solutions on a black back-
ground: the turbidity of the test solution is not thicker than
that of the control solution (not more than 0.00005%). For
Water for Injection in containers holding a volume exceeding
10 mL, add 3 drops of nitric acid and 0.5 mL of silver nitrate
TS to 50 mL of Water for Injection: the solution remains un-
1236 Purified Water / Official Monographs
JP XV
changed.
(6) Ammonium <1.02> — Perform the test with 30 mL of
Water for Injection as the test solution. Prepare the control
solution as follows: To 0.6 mL of Standard Ammonium So-
lution for Water for Injection in containers holding a volume
not more than 10 mL, and 0.3 mL of Standard Ammonium
Solution for Water for Injection in a volume exceeding 10
mL, add purified water for ammonium limit test to make 30
mL (not more than 0.2 mg/L for Water for Injection in a
volume not more than 10 mL, and not more than 0.1 mg/L
for that exceeding 10 mL).
(8) Total organic carbon <2.59> — Apply the test to Water
for Injection prepared by the Reverse Osmosis-Ultrafiltration
for the preparation of injections: it contains not more than
0.50 mg/L of total organic carbon.
(9) Residue on evaporation — Evaporate 100 mL of
Water for Injection, and dry the residue at 105°C for 1 hour:
the residue weighs not more than 4.0 mg for Water for Injec-
tion in a volume not more than 10 mL, and not more than 3.0
mg for that exceeding 10 mL.
Bacterial endotoxins <4.01> Less than 0.25 EU/mL.
Extractable volume <6.05> It meets the requirement.
Sterility <4.06> Apply the test to Water for Injection
preserved in containers as sterilized products: it meets the re-
quirement.
Containers and storage Containers — (1) For the prepara-
tion of injections, suitable containers, protected from
microbial contamination.
(2) Hermetic containers for Water for Injection
preserved in the containers as sterilized products. Plastic con-
tainers for aqueous infusions may be used.
Purified Water
mm*
Purified Water is purified Water by hyperfiltration
(reverse osmosis, ultrafiltration), ion-exchange treat-
ment, distillation or combination of these methods.
When prepare Purified Water, be careful to prevent
microbial contamination.
Use immediately after purification. It may be stored
for a certain period, if it is in suitable containers
preventing microbial growth.
Description Purified Water is a clear, colorless liquid. It is
odorless and tasteless.
Purity (1) Acidity or alkalinity — To 20 mL of Purified
Water add 0.1 mL of methyl red TS for acid or alkali test: a
yellow to orange color develops. To 20 mL of Purified Water
add 0.05 mL of bromothymol blue TS: no blue color de-
velops.
(2) Chloride— To 50 mL of Purified Water add 3 drops
of nitric acid and 0.5 mL of silver nitrate TS: no change oc-
curs.
(3) Sulfate— To 50 mL of Purified Water add 0.5 mL of
barium chloride TS: no change occurs.
(4) Nitrogen from nitrate — Transfer 2.0 mL of Purified
Water to a 50-mL beaker, add 1 mL of sodium salicylate-so-
dium hydroxide TS, 1 mL of a solution of sodium chloride (1
in 500) and 1 mL of a solution of ammonium amidosulfate (1
in 1000), and evaporate on a water bath to dryness. Cool, dis-
solve in 2 mL of sulfuric acid, allow to stand for 10 minutes
with occasional shaking, add 10 mL of water, and transfer to
a Nessler tube. Cool, add 10 mL of a solution of sodium
hydroxide (2 in 5) slowly, and add water to make 25 mL: no
yellow color develops.
(5) Nitrogen from nitrite — Transfer 10 mL of Purified
Water to a Nessler tube, and add 1 mL of a solution of sul-
fanilamide in dilute hydrochloric acid (1 in 100) and 1 mL of
A r -(l-naphthyl)-/V'-diethylethylenediamine oxalate TS: no
pale red color develops.
(6) Ammonium <1.02> — Perform the test with 30 mL of
Purified Water as the test solution. Prepare the control solu-
tion as follows: to 0.15 mL of Standard Ammonium Solution
add purified water for ammonium limit test to make 30 mL
(not more than 0.05 mg/L).
(7) Heavy metals — The 40 mL of Purified Water add 2
mL of dilute acetic acid and 1 drop of sodium sulfide TS: no
change occurs.
(8) Potassium permanganate-reducing substances — To
100 mL of Purified Water add 10 mL of dilute sulfuric acid,
boil, add 0.10 mL of 0.02 mol/L potassium permanganate
VS, and boil again for 10 minutes: the red color does not dis-
appear.
(9) Residue on evaporation — Evaporate 100 mL of Puri-
fied Water on a water bath to dryness, and dry the residue at
105 °C for 1 hour: the amount of the residue is not more than
l.Omg.
Containers and storage Containers — Tight containers.
Sterile Purified Water
McffiifitS*
Sterile Purified Water is sterilized Purified Water.
Description Sterile Purified Water is a clear, colorless liq-
uid. It is odorless and tasteless.
Purity (1) Acidity or alkalinity — To 20 mL of Sterile
Purified Water add 0. 1 mL of methyl red TS for acid or alkali
test: a yellow to orange color develops. To 20 mL of Sterile
Purified Water add 0.05 mL of bromothymol blue TS: no
blue color develops.
(2) Chloride— To 50 mL of Sterile Purified Water add 3
drops of nitric acid and 0.5 mL of silver nitrate TS: no
change occurs.
(3) Sulfate— To 50 mL of Sterile Purified Water add 0.5
mL of barium chloride TS: no change occurs.
(4) Nitrogen from nitrate — Transfer 2.0 mL of Sterile
Purified Water to a 50-mL beaker, add 1 mL of sodium
salicylate-sodium hydroxide TS, 1 mL of a solution of sodi-
um chloride (1 in 500) and 1 mL of a solution of ammonium
amidosulfate (1 in 1000), and evaporate on a water bath to
dryness. Cool, dissolve in 2 mL of sulfuric acid, allow to
stand for 10 minutes, with occasional shaking, add 10 mL of
water, and transfer to a Nessler tube. Cool, add 10 mL of a
solution of sodium hydroxide (2 in 5) slowly, and add water
to make 25 mL: no yellow color develops.
JP XV
Official Monographs / Wheat Starch 1237
(5) Nitrogen from nitrite — Transfer 10 mL of Sterile
Purified Water to a Nessler tube, and add 1 mL of a solution
of sulfanilamide in dilute hydrochloric acid (1 in 100) and 1
mL of Af-(1-Naphthyl)-/V' -diethylethylenediamine oxalate
TS: no pale red color develops.
(6) Ammonium <1.02> — Perform the test with 30 mL of
Sterile Purified Water as the test solution. Prepare the control
solution as follows: to 0.15 mL of Standard Ammonium So-
lution add purified water for ammonium limit test to make 30
mL (not more than 0.05 mg/L).
(7) Heavy metals — To 40 mL of Sterile Purified Water
add 2 mL of dilute acetic acid and 1 drop of sodium sulfide
TS: no change occurs.
(8) Potassium permanganate-reducing substances — To
100 mL of Sterile Purified Water add 10 mL of dilute sulfuric
acid, boil, add 0.10 mL of 0.02 mol/L potassium perman-
ganate VS, and boil again for 10 minutes: the red color does
not disappear.
(9) Residue on evaporation — Evaporate 100 mL of
Sterile Purified Water on a water bath to dryness, and dry the
residue at 105°C for 1 hour: the mass of the residue is not
more than 1.0 mg.
Sterility <4.06> Take 500 mL of Sterile Purified Water, and
perform the test by the Membrane filtration method: it meets
the requirements.
Containers and storage Containers — Hermetic containers.
Prastic containers for aqueous injections may be used.
Weil's Disease and Akiyami
Combined Vaccine
7 -f limfXV&MS 1 >7 1- >
Weil's Disease and Akiyami Combined Vaccine is a
liquid for injection containing inactivated Weil's dis-
ease leptospira, Akiyami A leptospira, Akiyami B lep-
tospira and Akiyami C leptospira. The product lacking
more than a kind of Akiyami leptospira may be pre-
pared, if necessary.
It conforms to the requirements of Weil's Disease
and Akiyami Combined Vaccine in the Minimum Re-
quirements for Biological Products.
Description Weil's Disease and Akiyami Combined Vac-
cine is a white-turbid liquid.
Wheat Starch
Amylum Tritici
This monograph is harmonized with the European
Pharmacopoeia and the U.S. Pharmacopeia. The parts
of the text that are not harmonized are marked with
symbols (* ♦)
Wheat Starch consists of the starch granules
obtained from the seeds of Triticum aestivum Linne
(Gramineae).
♦Description Wheat Starch occurs as white masses or
powder.
It is practically insoluble in water and in ethanol (99.5). ♦
Identification (1) Under a microscope <5.01>, Wheat
Starch, preserved in a mixture of water and glycerin (1:1), ap-
pears as large and small sized simple grains, or quite rarely
median sized simple grains; usually, large sized grains about
10-60//m in diameter, from upper view, disc like or quite
rarely reniform, centric hilum and striation indistinct or
hardly distinct, often cleft on marginal portion visible; from
lateral view, narrowly ellipsoid or fusiform, hilum recognized
as a long and slender cleft along with long axis; small sized
grains 2 - 10 /xm in diameter, spherical or polygonal; a black
cross, its intersection point on hilum, is observed when grains
are put between two nicol prisms fixed at right angle to each
other.
(2) To 1 g of Wheat Starch add 50 mL of water, boil for 1
minute, and allow to cool: a subtle white-turbid, pasty liquid
is formed.
(3) To 1 mL of the pasty liquid obtained in (2) add 0.05
mL of diluted iodine TS (1 in 10): a deep blue color is
formed, and the color disappears by heating.
pH <2.54> Put 5.0 g of Wheat Starch in a non-metal vessel,
add 25.0 mL of freshly boiled and cooled water, mix gently
for 1 minute, and allow to stand for 15 minutes: the pH of
the solution is between 4.5 and 7.0.
Purity (1) Iron— To 1 .5 g of Wheat Starch add 1 5 mL of 2
mol/L hydrochloric acid TS, mix, filter, and use the filtrate as
the test solution. To 2.0 mL of Standard Iron Solution add
water to make 20 mL, and use as the control solution. Put 10
mL each of the test solution and the control solution in test
tubes, add 2 mL of a solution of citric acid (2 in 10) and 0.1
mL of mercapto acetic acid, and mix. Alkalize with ammonia
solution (28) to litmus paper, add water to make 20 mL, and
mix. Transfer 10 mL each of these solutions into test tubes,
allow to stand for 5 minutes, and compare the color of these
solutions against a white background: the color of the test so-
lution is not more intense than that of the control solution
(not more than 10 ppm).
(2) Oxidizing substances — To 4.0 g of Wheat Starch add
50.0 mL of water, shake for 5 minutes, and centrifuge. To
30.0 mL of the supernatant liquid add 1 mL of acetic acid
(100) and 0.5 to 1.0 g of potassium iodide, shake, and allow
to stand for 25 to 30 minutes at a dark place. Add 1 mL of
starch TS, and titrate <2.50> with 0.002 mol/L sodium
thiosulfate VS until the color of the solution disappears. Per-
form a blank determination and make any necessary correc-
tion: the volume of 0.002 mol/L sodium thiosulfate VS con-
sumed is not more than 1.4 mL (not more than 20 ppm, cal-
culated as hydrogen peroxide).
(3) Sulfur dioxide —
(i) Apparatus Use as shown in the figure.
(ii) Procedure Introduce 150 mL of water into the boil-
ing flask, close the tap of the funnel, and pass carbon dioxide
through the whole system at a rate of 100 ±5 mL per minute.
Pass cooling water through the condenser, and place 10 mL
of hydrogen peroxide-sodium hydroxide TS in the test-tube.
After 15 minutes, remove the funnel without interrupting the
stream of carbon dioxide, and introduce through the opening
into the flask about 25 g of Wheat Starch, accurately
1238 White Ointment / Official Monographs
JP XV
Ai Boiling flask (!>00 ml)
B: Funnel (10() mL)
C: Condenser
D: Tcst-lubc
E:Tap
The figures arc in mm.
weighed, with the aid of 100 mL of water. Apply tap grease
to the outside of the connection part of the funnel, and load
the funnel. Close the tap of the funnel, pour 80 mL of 2
mol/L hydrochloric acid TS into the funnel, open the tap to
introduce the hydrochloric acid into the flask, and close the
tap while several mL of the hydrochloric acid remains, in
order to avoid losing sulfur dioxide. Place the flask in a water
bath, and heat the mixture for 1 hour. Transfer the contents
of the test-tube with the aid of a little water to a wide-necked
conical flask. Heat in a water bath for 15 minutes, and cool.
Add 0.1 mL of bromophenol blue TS, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS until the color changes from
yellow to violet-blue lasting for at least 20 seconds. Perform a
blank determination and make any necessary correction. Cal-
culate the amount of sulfur dioxide by applying the following
formula: it is not more than 50 ppm.
Amount (ppm) of sulfur dioxide = ( VI W) x 1000x3.203
W: Amount (g) of the sample
V: Amount (mL) of 0.1 mol/L sodium hydroxide VS
consumed
Loss on drying <2.41> Not more than 15.0% (1 g, 130°C, 90
minutes).
Residue on ignition <2.44> Not more than 0.6% (1 g).
♦Containers and storage Containers — Well-closed contain-
ers. ♦
White Ointment
Method of preparation
White Beeswax 50 g
Sorbitan Sesquioleate 20 g
White Petrolatum a sufficient quantity
To make 1000 g
Prepare as directed under Ointments, with the above
materials.
Description White Ointment is white in color. It has a
slight, characteristic odor.
Containers and storage Containers — Tight containers.
Whole Human Blood
Whole Human Blood is a liquid for injection which
is prepared by mixing human blood cells and an an-
ticoagulant solution for storage.
It conforms to the requirements of Whole Human
Blood in the Minimum Requirements for Biological
Products.
Description Whole Human Blood is a deep red liquid from
which the erythrocytes settle upon standing, leaving a yellow
supernatant layer. A gray layer which mainly consists of leu-
cocytes may appear on the surface of the settled erythrocyte
layer. The supernatant layer may become turbid in the
presence of fat, or may show the faint color of hemoglobin.
Wine
7* K ^
Wine is an alcoholic liquid obtained by fermenting
the juice of the fruits of Vitis vinifera Linne (Vitaceae)
or allied plants.
It contains not less than 11 vol% and not more than
14 vol% of ethanol (C 2 H 6 0: 46.07) (by specific gravi-
ty), and not less than 0.10 w/v% and not more than
0.40 w/v% of L-tartaric acid (C 4 H 6 6 : 150.09).
It contains no artificial sweetener and no artificial
coloring agent.
Description Wine is a light yellow or reddish purple to red-
purple liquid. It has a characteristic and aromatic odor. It has
a slightly astringent and faintly irritating taste.
Specific gravity <2.56>
dl : 0.990 ■
1.010
Optical rotation <2.49> Boil 160 mL of Wine, neutralize
with potassium hydroxide TS, and concentrate to 80 mL on a
water bath. Cool, dilute with water to 160 mL, add 16 mL of
lead subacetate TS, shake well, and filter. To 100 mL of the
filtrate add 10 mL of a saturated solution of sodium sulfate
decahydrate, shake well, filter, and use the filtrate as the sam-
ple solution. Allow 20 mL of the sample solution to stand for
24 hours, add 0.5 g of activated charcoal, shake, stopper,
and allow to stand for 10 minutes. Filter, and observe the op-
tical rotation of the filtrate in a 200-mm cell. Multiply the op-
tical rotation observed by 1.21, and designate as the optical
rotation of Wine: it is between —0.3° and +0.3°.
Purity (1) Total acid [as L-tartaric acid (C 4 H 6 6 )] — To ex-
actly 10 mL of Wine add 250 mL of freshly boiled and cooled
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
VS (indicator: 1 mL of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide VS
= 7.504 mg of C 4 H 6 6
JPXV
Total acid is not less than 0.40 w/v% and not more than
0.80 w/v%.
(2) Volatile acid [as acetic acid (C 2 H 4 2 : 60.05)] — Trans-
fer 100 mL of Wine to a beaker, add 1 mL of 1 mol/L sodi-
um hydroxide VS and the same volume of 1 mol/L sodium
hydroxide VS as that of 0.1 mol/L sodium hydroxide VS
titrated in (1) to make the solution alkaline, and concentrate
to 50 mL on a water bath. Cool, add water to make 100 mL,
transfer to a 1000-mL distillation flask, containing previously
added 100 g of sodium chloride. Wash the beaker with 100
mL of water, and combine the washings in the distillation
flask. Add 5 mL of a solution of L-tartaric acid (3 in 20), and
distil with steam cautiously to maintain the volume of the so-
lution in the flask until 450 mL of the distillate is obtained for
45 minutes. Dilute the distillate to exactly 500 mL with water,
and use this solution as the sample solution. Titrate <2.50> a
250-mL portion of the sample solution with 0.1 mol/L sodi-
um hydroxide VS (indicator: 5 drops of phenolphthalein TS).
Perform a blank determination, and make any necessary cor-
rection.
Each mL of 0.1 mol/L sodium hydroxide VS
= 6.005 mg of C 2 H 4 2
The volatile acid is not more than 0.15 w/v%.
(3) Sulfur dioxide — Stopper a 750-mL round-bottomed
flask with a stopper having two holes. Through one hole, in-
sert a glass tube A extending nearly to the bottom of the
flask. Through the other hole, insert a glass tube B ending to
the neck of the flask. Connect the tube B to a Liebig's con-
denser, and the end of the condenser to a joint of which inner
diameter is 5 mm at the lower end. Connect the other end of
the joint with a holed rubber stopper to a U tube having three
bulbs as shown in the Figure. Pass carbon dioxide washed
with a solution of potassium permanganate (3 in 100)
through the tube A. Displace the air in the apparatus by car-
bon dioxide, and place 50 mL of a freshly prepared and dilut-
ed starch TS (1 in 5) and 1 g of potassium iodide in the U
tube. From the other end of the U tube, add 1 to 2 drops of
0.01 mol/L iodine VS from a burette. While passing carbon
dioxide, remove the stopper of the flask a little, add exactly
25 mL of Wine, 180 mL of freshly boiled and cooled water,
0.2 g of tannic acid, and 30 mL of phosphoric acid, and stop-
per again. Pass carbon dioxide for further 15 minutes, heat
the distillation flask with caution so that 40 to 50 drops of the
distillate may be obtained in 1 minute. When the color of
starch TS in the U tube is discharged, add 0.01 mol/L iodine
VS dropwise from a burette so that the color of the starch TS
remains light blue to blue during the distillation. Read the
volume of 0.01 mol/L iodine VS consumed when exactly 60
minutes have passed after the beginning of distillation. In this
case, however, the coloration of starch TS produced by 1
drop of 0.01 mol/L iodine VS should persist at least for 1
minute.
Each mL of 0.01 mol/L iodine VS = 0.6406 mg of S0 2
The amount of sulfur dioxide (S0 2 : 64.06) does not exceed
7.5 mg.
(4) Total sulfuric acid — Transfer 10 mL of Wine to a
beaker, boil, and add 50 mL of a solution prepared by dis-
solving 5.608 g of barium chloride dihydrate in 50 mL of
hydrochloric acid and water to make 1000 mL. Cover the
beaker, and heat on a water bath for 2 hours, supplying the
Official Monographs / Wine 1239
■
water lost by distillation. Cool, centrifuge, and decant the su-
pernatant liquid in another beaker. To this solution add 1 to
2 drops of dilute sulfuric acid, and allow to stand for 1 hour:
a white precipitate is formed.
(5) Arsenic <1.11> — Evaporate 10 mL of Wine on a water
bath to dryness. Prepare the test solution with the residue ac-
cording to Method 3, and perform the test (not more than 0.2
ppm).
(6) Glycerin — Pipet 100 mL of Wine into a 150-mL por-
celain dish, and concentrate on a water bath to 10 mL. Add 1
g of sea sand (No. 1), and make the solution strongly alkaline
by adding a solution prepared by dissolving 4 g of calcium
hydroxide in 6 mL of water. Heat on a water bath with con-
stant stirring and pushing down any material adhering to the
wall of the dish until the contents of the dish become soft
masses. Cool, add 5 mL of ethanol (99.5), and grind to a
grue-like substance. Heat on a water bath, add 10 to 20 mL
of ethanol (99.5) while agitating, boil, and transfer to a
100-mL volumetric flask. Wash the dish with seven 10-mL
portions of hot ethanol (99.5), combine the washings with the
contents of the flask, cool, and add ethanol (99.5) to make
exactly 100 mL. Filter through a dry filter paper, evaporate
90 mL of the filtrate on a water bath, taking care not to boil
the solution during the evaporation. Dissolve the residue in a
small amount of ethanol (99.5), transfer to a 50-mL glass-
stoppered volumetric cylinder, wash with several portions of
ethanol (99.5), and add the washings to the solution in the
cylinder to make 15 mL. Add three 7.5-mL portions of de-
hydrated diethyl ether, shake vigorously each time, and allow
to stand. When the solution becomes quite clear, transfer to a
tared, flat weighing bottle. Wash the volumetric cylinder with
5 mL of a mixture of dehydrated diethyl ether and ethanol
(99.5) (3:2). Transfer the washings to the weighing bottle,
and evaporate carefully on a water bath. When the liquid
becomes sticky, dry at 105°C for 1 hour, and cool in a desic-
cator (silica gel), and weigh: the mass of the residue is not less
than 0.45 g and not more than 0.90 g.
(7) Reducing sugars — To a 25-mL portion of the sample
solution obtained in the Optical rotation add 50 mL of boil-
ing Fehling's TS, and heat for exactly 2 minutes. Filter the
separated precipitates by a tared glass filter by suction, wash
successively with hot water, with ethanol (95) and with
diethyl ether, and continue to dry the precipitates by suction.
Heat the filter gently at first, and then strongly until the
precipitates become completely black. Cool the precipitates
in a desiccator (silica gel), and weigh as copper (II) oxide: the
mass of cupric oxide does not exceed 0.325 g.
(8) Sucrose — Transfer a 50-mL portion of the sample so-
lution obtained in the Optical rotation to a 100-mL flask,
neutralize with diluted hydrochloric acid (1 in 30), followed
by further addition of 5 mL of diluted hydrochloric acid (1 in
1240 Xylitol / Official Monographs
JP XV
30). Heat in a water bath for 30 minutes, cool, neutralize with
a solution of potassium hydroxide (1 in 100), add 4 drops of
sodium carbonate TS, filter into a 100-mL volumetric flask,
wash with water, combine the washings with the filtrate, and
add water to make 100 mL. To 25 mL of this solution add 50
mL of boiling Fehling's TS, and proceed as directed in (7),
and weigh as copper (II) oxide. From the number obtained by
multiplying the mass (g) of copper (II) oxide by 2, deduct the
amount (g) of copper (II) oxide determined in (7), and mul-
tiply again the number so obtained by 1.2: the number ob-
tained does not exceed 0.104 (g).
(9) Benzoic acid, cinnamic acid and salicylic acid — Tran-
sfer exactly 50 mL of the sample solution obtained in (2) to a
separator, add 10 g of sodium chloride and 2 mL of dilute
hydrochloric acid, and extract with three 10-mL portions of
diethyl ether. Combine the diethyl ether extracts, wash with
two 5-mL portions of water, and extract with three 10-mL
portions of 0.1 mol/L sodium hydroxide VS. Combine the
alkaline extracts, evaporate the diethyl ether by warming on a
water bath, cool, neutralize with 1 mol/L hydrochloric acid
VS, and add 5 mL of potassium chloride-hydrochloric acid
buffer solution and water to make exactly 50 mL. Perform
the test as directed under Ultraviolet-visible Spectrophoto-
metry <2.24> with this solution, using a solution prepared in
the same manner instead of the sample solution as the blank:
the absorbance does not exceed 0.15 at a wavelength between
220 nm and 340 nm.
(10) Boric acid — Transfer 50 mL of Wine to a porcelain
dish, add 5 mL of sodium carbonate TS, evaporate on a
water bath to dryness, and ignite: a half portion of the
residue does not respond to Qualitative Tests <1.09> (1) for
borate. Dissolve another half portion of the residue in 5 mL
of hydrochloric acid: it does not respond to Qualitative Tests
<l.09> (2) for borate.
(11) Methanol — Wine meets the requirements of the
Methanol Test <1.12>, when proceeding with exactly 1 mL of
ethanol layer obtained by Method 1 of the Alcohol Number
Determination <1.01> and distilling without adding water af-
ter shaking with 0.5 g of calcium carbonate.
(12) Formaldehyde — To 25 mL of Wine add 5 g of sodi-
um chloride and 0.2 g of L-tartaric acid, distil, and obtain 15
mL of the distillate. To 5 mL of the distillate add 5 mL of
acetyl acetone TS, mix, and heat on a water bath for 10
minutes: the solution has no more color than that of the fol-
lowing control solution.
Control solution: Using 5 mL of water instead of the distil-
late, perform the test in the same manner.
Extract content 1.9-3.5w/v% Pipet 25 mL of Wine
to a 200-mL tared beaker containing 10 g of sea sand (No. 1),
previously dried at 105°C for 2.5 hours, and evaporate to
dryness on a water bath. Dry the residue at 105°C for 2
hours, cool in a desiccator (silica gel), and weigh.
Total ash 0.13 - 0.40 w/v% Pipet 50 mL of Wine to a
tared porcelain dish, and evaporate to dryness on a water
bath. Ignite the residue to the constant mass, cool, and
weigh.
Assay (1) Ethanol — Pipet Wine into a 100-mL volumetric
flask at 15°C, transfer to a 300- to 500-mL flask, and wash
this volumetric flask with two 15-mL portions of water. Add
the washings to the sample in the flask, connect the flask to a
distillation tube having a trap, and distil using the volumetric
flask as a receiver. When about 80 mL of the distillate is ob-
tained (it takes about 20 minutes), stop the distillation, allow
to stand in water at 15°C for 30 minutes, and add water to
make exactly 100 mL. Shake well, and determine the specific
gravity at 15°C according to Method 3 under Specific Gravity
<2.56>: the specific gravity d\\ is between 0.982 and 0.985.
(2) L-Tartaric acid — Pipet 100 mL of Wine, add 2 mL of
acetic acid (100), 0.5 mL of a solution of potassium acetate (1
in 5) and 15 g of powdered potassium chloride, and shake
vigorously to dissolve as much as possible. Add 10 mL of
ethanol (95), rub the inner wall of the beaker strongly for 1
minute to induce the crystallization, and allow to stand be-
tween 0°C and 5°C for more than 15 hours. Filter the crystals
by suction, wash successively the beaker and the crystals with
3-mL portions of a solution prepared by dissolving 15 g of
powdered potassium chloride in 120 mL of diluted ethanol (1
in 6), and repeat the washings five times. Transfer the crystals
together with the filter paper to a beaker, wash the filter with
50 mL of hot water, combine the washings in the beaker, and
dissolve the crystals by heating. Titrate <2.50> the solution
with 0.2 mol/L sodium hydroxide VS immediately (indica-
tor: 1 mL of phenolphthalein TS). The number obtained by
adding 0.75 to the amount (mL) of 0.2 mol/L sodium
hydroxide VS consumed represents the amount (mL) of 0.2
mol/L sodium hydroxide VS consumed.
Each mL of 0.2 mol/L sodium hydroxide VS
= 30.02 mg of C 4 H 6 6
Containers and storage Containers — Tight containers.
Xylitol
*->U Y-)l
H OH
HO H H OH
C 5 H 12 5 : 152.15
meso-Xylitol [87-99-0]
Xylitol, when dried, contains not less than 98.0% of
C 5 H 12 O s .
Description Xylitol occurs as white crystals or powder. It is
odorless and has a sweet taste.
It is very soluble in water, slightly soluble in ethanol (95).
It is hygroscopic.
Identification (1) To 1 mL of a solution of Xylitol (1 in 2)
add 2 mL of iron (II) sulfate TS and 1 mL of a solution of so-
dium hydroxide (1 in 5): blue-green color is produced without
turbidity.
(2) Determine the infrared absorption spectrum of
Xylitol, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
pH <2.54> Dissolve 5.0 g of Xylitol in 10 mL of freshly
boiled and cooled water: the pH of this solution is between
5.0 and 7.0.
JPXV
Official Monographs / Dried Yeast 1241
Melting point <2.60> 93.0 - 95.0°C
Purity (1) Clarity and color of solution — Dissolve 5 g of
Xylitol in 10 mL of water: the solution is clear and colorless.
(2) Chloride <1.03>— Perform the test with 2.0 g of
Xylitol. Prepare the control solution with 0.30 mL of 0.01
mol/L hydrochloric acid VS (not more than 0.005%).
(3) Sulfate <1.14>— Perform the test with 4.0 g of Xylitol.
Prepare the control solution with 0.50 mL of 0.005 mol/L
sulfuric acid VS (not more than 0.006%).
(4) Heavy metals <1.07>— Proceed with 4.0 g of Xylitol
according to Method 1, and perform the test. Prepare the
control solution with 2.0 mL of Standard Lead Solution (not
more than 5 ppm).
(5) Nickel — Dissolve 0.5 g of Xylitol in 5 mL of water,
add 3 drops of dimethylglyoxime TS and 3 drops of ammonia
TS, and allow to stand for 5 minutes: no red color is
produced.
(6) Arsenic <1.11> — Prepare the test solution with 1.5 g
of Xylitol according to Method 1, and perform the test (not
more than 1.3 ppm).
(7) Sugars — Dissolve 5.0 g of Xylitol in 15 mL of water,
add 4.0 mL of dilute hydrochloric acid, and heat in a water
bath for 3 hours under a reflux condenser. After cooling,
neutralize with sodium hydroxide TS (indicator: 2 drops of
methyl orange TS). Then add water to make 50 mL, transfer
10 mL of this solution to a flask, add 10 mL of water and 40
mL of Fehling's TS, boil gently for 3 minutes, and allow to
stand to precipitate copper (I) oxide. Remove the supernatant
liquid through a glass filter (G4), and wash the precipitate
with warm water until the last washing does not show alkalin-
ity. Filter these washings through the glass filter mentioned
above. Dissolve the precipitate in the flask in 20 mL of iron
(III) sulfate TS, filter the solution through the glass filter
mentioned above, wash with water, combine the washings
with the filtrate, heat at 80°C, and titrate <2.50> with 0.02
mol/L potassium permanganate VS: not more than 1.0 mL
of 0.02 mol/L potassium permanganate VS is consumed.
Loss on drying <2.41> Not more than 1.0% (1 g, in vacuum,
phosphorus (V) oxide, 24 hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.2 g of Xylitol, previously
dried, dissolve in water to make exactly 100 mL. Pipet 10 mL
of this solution into an iodine flask, add 50 mL of potassium
periodate TS exactly, and heat in a water bath for 15 minutes.
After cooling, add 2.5 g of potassium iodide, stopper, shake
well, allow to stand for 5 minutes in a dark place, and titrate
<2.50> with 0.1 mol/L sodium thiosulfate VS (indicator: 3
mL of starch TS). Perform a blank determination.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.902 mg of C 5 H 12 5
Containers and storage Containers — Tight containers.
Xylitol Injection
Xylitol Injection is an aqueous solution for injec-
tion.
It contains not less than 95% and not more than
105% of the labeled amount of xylitol (C 5 H 12 5 :
152.15).
Method of preparation Prepare as directed under Injec-
tions, with Xylitol.
No preservative may be added.
Description Xylitol Injection is a clear, colorless liquid. It
has a sweet taste.
Identification Measure a volume of Xylitol Injection,
equivalent to 0.1 g of Xylitol according to the labeled
amount, add water to make 10 mL, and use this solution as
the sample solution. Separately, dissolve 0.1 g of xylitol in 10
mL of water, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 2 juL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of ethanol (95), ammonia solution (28) and water (25:4:3) to
a distance of about 10 cm, and air-dry the plate. Spray evenly
silver nitrate-ammonia TS, and dry at 105°C for 15 minutes:
the spots from the sample solution and the standard solution
show a blackish brown color and the same Rf value.
pH <2.54> 4.5-7.5
Bacterial endotoxins <4.01> Less than 0.50EU/mL.
Extractable volume <6.05> It meets the requirement.
Assay Measure exactly a volume of Xylitol Injection,
equivalent to about 5 g of xylitol (C 5 H I2 05) according to the
labeled amount, and add water to make exactly 250 mL.
Measure exactly 10 mL of this solution, and add water to
make exactly 100 mL. Then, pipet 10 mL of this solution into
an iodine flask, and proceed as directed in the Assay under
Xylitol.
Each mL of 0.1 mol/L sodium thiosulfate VS
= 1.902 mg of C 5 H, 2 5
Containers and storage Containers — Hermetic containers.
Plastic containers for aqueous injections may be used.
Dried Yeast
Dried Yeast is dried and powdered cells of yeast be-
longing to Saccharomyces.
It contains not less than 400 mg of protein and not
less than 100 fig of thiamine compounds [as thiamine
chloride hydrochloride (C 12 H 17 ClN 4 OS.HCl: 337.27)]
in each 1 g.
Description Dried Yeast occurs as a light yellowish white to
brown powder. It has a characteristic odor and taste.
Identification Dried Yeast, when examined under a micro-
scope <5.01>, shows isolated cells, spheroidal or oval in
shape, and 6 to 12 /um in length.
Purity (1) Rancidity — Dried Yeast is free from any un-
pleasant or rancid odor or taste.
1242 Zaltoprofen / Official Monographs
JP XV
(2) Starch — Add iodine TS to Dried Yeast, and examine
microscopically <5.01>: no or only a few granules are tinted
blackish purple.
Loss on drying <2.41> Not more than 8.0% (1 g, 100°C,
8 hours).
Total ash <J.07> Not more than 9.0% (1 g).
Assay (1) Protein — Weigh accurately about 50 mg of
Dried Yeast and perform the test as directed under Nitrogen
Determination <1.08>.
Amount (mg) of protein in 1 g of Dried Yeast
= Nx 6.25 x (\/W)
N: Amount (mg) of nitrogen (N)
W: Amount (g) of sample
(2) Thiamine — Weigh accurately about 1 g of Dried
Yeast, add 1 mL of dilute hydrochloric acid and 80 mL of
water, and heat in a water bath at 80°C to 85°C for 30
minutes with occasional shaking. After cooling, add water to
make exactly 100 mL, and centrifuge for 10 minutes. Pipet 4
mL of the supernatant liquid, add exactly 5 mL of acetic
acid-sodium acetate TS and exactly 1 mL of enzyme TS, and
allow to stand at 45 °C to 50°C for 3 hours. Place exactly 2
mL of this solution onto a chromatographic column prepared
by pouring 2.5 mL of a weakly acidic CM-bridged cellulose
cation exchanger (H type) (40 to HO^m in particle diameter)
into a chromatographic tube about 1 cm in inside diameter
and about 17 cm in length, and elute at the flow rate of about
0.5 mL per minute. Wash the upper part of the column with a
small amount of water, and wash the column with two 10-mL
portions of water at the flow rate of about 1 mL per minute.
Elute the column with two 2.5-mL portions of diluted phos-
phoric acid (1 in 50) at the flow rate of about 0.5 mL per
minute, and combine the eluate. To the eluate add exactly 1
mL of the internal standard solution and 0.01 g of sodium 1-
octanesulfonate, and after dissolving, use this solution as the
sample solution. Separately, weigh accurately about 15 mg of
Thiamine Chloride Hydrochloride Reference Standard
(previously determine the water <2.48> in the same manner as
Thiamine Chloride Hydrochloride), dissolve in 0.001 mol/L
hydrochloric acid TS to make exactly 100 mL. Pipet 1 mL of
this solution, and add the mobile phase to make exactly 100
mL. Pipet 1 mL of this solution, add exactly 1 mL of the in-
ternal standard solution and 3 mL of the mobile phase, and
use this solution as the standard solution. Perform the test
with 200 iuL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and calculate the ratios,
Qj and Q s , of the peak area of thiamine to that of the inter-
nal standard.
Amount (fig) of thiamine in 1 g of Dried Yeast
= (W s xW T )x(Q J /Q s )xi2.S
W s : Amount (mg) of Thiamine Chloride Hydrochloride
Reference Standard, calculated on the anhydrous ba-
sis
W T : Amount (g) of the sample
Internal standard solution — Dissolve 0.01 g of phenacetin in
acetonitrile to make 100 mL, and to 1 mL of this solution add
diluted acetonitrile (1 in 5) to make 100 mL.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 //m in
particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 2.7 g of potassium dihydrogen-
phosphate in 1000 mL of water, and adjust the pH to 3.5 with
diluted phosphoric acid (1 in 10). Dissolve 1.6 g of sodium 1-
octanesulfonate in 800 mL of this solution, and add 200 mL
of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
thiamine is about 8 minutes.
Selection of column: Proceed with 200 /iL of the standard
solution under the above operating conditions, and calculate
the resolution. Use a column giving elution of thiamine and
the internal standard in this order with the resolution between
these peaks being not less than 8.
Containers and storage Containers — Tight containers.
Zaltoprofen
+f\IUh7 p Q7x>
C 17 H 14 3 S: 298.36
(2i?S)-2-(10-Oxo-10,ll-dihydrodibenzo[dJ]thiepin-
2-yl)propanoic acid
[74711-43-6]
Zaltoprofen, when dried, contains not less than 99.0% and
not more than 101.0% of C 17 H 14 3 S.
Description Zaltoprofen occurs as white to light yellow,
crystals or crystalline powder.
It is freely soluble in acetone, soluble in methanol and in
ethanol (99.5), and practically insoluble in water.
It is gradually decomposed by light.
A solution of Zaltoprofen in acetone (1 in 10) shows no op-
tical rotation.
Identification (1) To 0.2 g of Zaltoprofen add 0.5 g of so-
dium hydroxide, heat gradually to melt, and then carbonize.
After cooling, add 5 mL of diluted hydrochloric acid (1 in 2):
the gas evolved darkens moisten lead (II) acetate paper.
(2) Determine the absorption spectrum of a solution of
Zaltoprofen in ethanol (99.5) (1 in 100,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra ex-
hibit similar intensities of absorption at the same
wavelengths.
(3) Determine the infrared absorption spectrum of Zal-
toprofen as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>, and compare the
spectrum with the Reference Spectrum: both spectra exhibit
similar intensities of absorption at the same wave numbers.
Melting point <2.60> 135 - 139°C
JPXV
Official Monographs / Zaltoprofen Tablets 1243
Purity (1) Heavy metals <1.07> — Proceed with 2.0 g of
Zaltoprofen according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Zaltoprofen according to Method 3, using 10 mL of a so-
lution of magnesium nitrate hexahydrate in ethanol (95) (2 in
25), and perform the test (not more than 2 ppm).
(3) Related substances — Dissolve 50 mg of Zaltoprofen
in 50 mL of the mobile phase, and use this solution as the
sample solution. Pipet 1 mL of the sample solution, and add
the mobile phase to make exactly 50 mL. Pipet 1 mL of this
solution, add the mobile phase to make exactly 20 mL, and
use this solution as the standard solution. Perform the test
with exactly 20 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine each peak
area by the automatic integration method: the area of the
peak other than zaltoprofen and other than the peak having
the relative retention time of about 0.7 with respect to zal-
toprofen from the sample solution is not larger than the peak
area of zaltoprofen from the standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetonitrile, water and acetic
acid (100) (300:200:1)
Flow rate: Adjust the flow rate so that the retention time of
zaltoprofen is about 4 minutes.
Time span of measurement: About 15 times as long as the
retention time of zaltoprofen beginning after the solvent
peak.
System suitability —
Test for required detectability: To exactly 2 mL of the stan-
dard solution add the mobile phase to make exactly 20 mL.
Confirm that the peak area of zaltoprofen obtained with 20
/xL of this solution is equivalent to 8 to 12% of that with 20
/xL of the standard solution.
System performance: Dissolve 25 mg of zaltoprofen and 50
mg of isopropyl benzoate in 100 mL of ethanol (99.5). Pipet 1
mL of this solution, and add the mobile phase to make ex-
actly 50 mL. When the procedure is run with 20 /xL of this so-
lution under the above operating conditions, zaltoprofen and
isopropyl benzoate are eluted in this order with the resolution
between these peaks being not less than 6.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
zaltoprofen is not more than 2.0%.
Loss on drying <2.41> Not more than 0.5% (1 g, 105 °C, 4
hours).
Residue on ignition <2.44> Not more than 0.1% (1 g).
Assay Weigh accurately about 0.5 g of Zaltoprofen, dis-
solve in 50 mL of methanol, and titrate <2.50> with 0. 1 mol/L
sodium hydroxide VS (potentiometric titration). Perform a
blank determination in the same manner, and make any
necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
= 29.84 mg of C 17 Hi 4 3 S
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Zaltoprofen Tablets
Zaltoprofen Tablets contain not less than 95.0% and
not more than 105.0% of the labeled amount of zal-
toprofen (C 17 H 14 3 S: 298.36).
Method of preparation Prepare as directed under Tablets,
with Zaltoprofen.
Identification Powder a suitable amount of Zaltoprofen
Tablets. To a portion of the powder, equivalent to 80 mg of
Zaltoprofen, add 30 mL of ethanol (99.5), shake well, and
centrifuge. To 1 mL of the supernatant liquid add ethanol
(99.5) to make 20 mL. To 2 mL of this solution add ethanol
(99.5) to make 25 mL, and determine the absorption spec-
trum of this solution as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits maxima between 227
nm and 231 nm and between 329 nm and 333 nm, and a
shoulder between 241 nm and 245 nm.
Uniformity of dosage units <6.02> Perform the test accord-
ing to the following method: it meets the requirement of the
Content uniformity test.
To 1 tablet of Zaltoprofen Tablets add 4 mL of water, and
shake to disintegrate. Add a suitable amount of ethanol (95),
shake, then add ethanol (95) to make exactly KmL so that
each mL contains about 4 mg of zaltoprofen (C 17 H 14 3 S),
and centrifuge. Pipet 2 mL of the supernatant liquid, add ex-
actly 10 mL of the internal standard solution and ethanol (95)
to make 50 mL, and use this solution as the sample solution.
Proceed as directed in the Assay.
Amount (mg) of zaltoprofen (C 17 H 14 3 S)
= W s x(Q T /Q s )x(V/20)
W s : Amount (mg) of zaltoprofen for assay
Internal standard solution — A solution of benzyl benzoate in
acetonitrile (1 in 1000).
Dissolution <6.10> Perform the test according to the follow-
ing method: it meets the requirement.
Perform the test with 1 tablet of Zaltoprofen Tablets at 50
revolutions per minute according to the Paddle method, us-
ing 900 mL of 2nd fluid for dissolution test as the dissolution
medium. Withdraw not less than 20 mL of the dissolved solu-
tion 30 minutes after start of the test, and filter through a
membrane filter with pore size of not more than 0.45 //m.
Discard the first 10 mL of the filtrate, pipet KmL of the sub-
sequent filtrate, add 2nd fluid for dissolution test to make ex-
actly V mL so that each mL contains about 44 /xg of zal-
toprofen (C 17 H 14 3 S) per mL according to the labeled
amount, and use this solution as the sample solution.
Separately, weigh accurately about 22 mg of zaltoprofen for
assay, previously dried at 105°C for 4 hours, dissolve in 20
1244 Zinc Chloride / Official Monographs
JP XV
mL of ethanol (99.5), and add 2nd fluid for dissolution test
to make exactly 100 mL. Pipet 4 mL of this solution, add 2nd
fluid for dissolution test to make exactly 20 mL, and use this
solution as the standard solution. Determine the absor-
bances, A T and A s , of the sample solution and standard solu-
tion at 340 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>, using 2nd fluid for dissolution test as
the control. The dissolution rate in 30 minutes is not less than
75%.
Dissolution rate (%) with respect to the labeled amount of
zaltoprofen (C 17 H I4 3 S)
= W s x (Aj/As) x (V'/V)X (1/Q x 180
W s : Amount (mg) of zaltoprofen for assay
C: Labeled amount (mg) of zaltoprofen for assay in 1
tablet
Assay To 10 tablets of Zaltoprofen Tablets add 40 mL of
water, shake to disintegrate, then add a suitable amount of
ethanol (95), shake, add ethanol (95) to make exactly 200
mL, and centrifuge. Pipet an amount of the supernatant liq-
uid, equivalent to about 8 mg of zaltoprofen (Ci 7 H 14 3 S),
add exactly 10 mL of the internal standard solution and
ethanol (95) to make 50 mL, and use this solution as the sam-
ple solution. Separately, weigh accurately about 80 mg of zal-
toprofen for assay, previously dried at 105°C for 4 hours,
add 4 mL of water and ethanol (95) to make exactly 20 mL.
Pipet 2 mL of this solution, add exactly 10 mL of the internal
standard solution and ethanol (95) to make 50 mL, and use
this solution as the standard solution. Perform the test with 5
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the ratios, Qt and
Q s , of the peak area of zaltoprofen to that of the internal
standard.
Amount (mg) of zaltoprofen (C 17 H 14 3 S)
= w r s x(Q r /e s )x(i/io)
fV s : Amount (mg) of zaltoprofen for assay
Internal standard solution — A solution of benzyl benzoate in
acetonitrile (1 in 1000)
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
25°C.
Mobile phase: A mixture of acetonitrile, water and acetic
acid (100) (300:200:1).
Flow rate: Adjust the flow rate so that the retention time of
zaltoprofen is about 4 minutes.
System suitability —
System performance: When the procedure is run with 5 juL
of the standard solution under the above operating condi-
tions, zaltoprofen and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 10.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the ratio of the
peak area of zaltoprofen to that of the internal standard is
not more than 1.0%.
Containers and storage Containers — Tight containers.
Zinc Chloride
ZnCl 2 : 136.32
Zinc Chloride contains not less than 97.0% of
ZnCl 2 .
Description Zinc Chloride occurs as white, crystalline pow-
der, rods, or masses. It is odorless.
It is very soluble in water, and freely soluble in ethanol
(95), and its solution may sometimes be slightly turbid. The
solution becomes clear on addition of a small amount of
hydrochloric acid.
The pH of an aqueous solution of Zinc Chloride (1 in 2) is
between 3.3 and 5.3.
It is deliquescent.
Identification An aqueous solution of Zinc Chloride (1 in
30) responds to the Qualitative Tests <1.09> for zinc salt and
chloride.
Purity (1) Clarity and color of solution — Dissolve 1 .0 g of
Zinc Chloride in 10 mL of water and 2 drops of hydrochloric
acid: the solution has no color, and is clear.
(2) Sulfate <1.14>— Perform the test with 2.0 g of Zinc
Chloride. Prepare the control solution with 0.40 mL of 0.005
mol/L sulfuric acid VS (not more than 0.010%).
(3) Ammonium — Dissolve 0.5 g of Zinc Chloride in 5 mL
of water, and warm with 10 mL of a solution of sodium
hydroxide (1 in 6): the evolving gas does not change
moistened red litmus paper to blue.
(4) Heavy metals — Dissolve 0.5 g of Zinc Chloride in 5
mL of water in a Nessler tube, shake thoroughly with 15 mL
of potassium cyanide TS, add 1 drop of sodium sulfide TS,
allow to stand for 5 minutes, and immediately observe from
the top downward against a white background: the solution
has no more color than the following control solution.
Control solution: To 2.5 mL of Standard Lead Solution
add 3 mL of water and 15 mL of potassium cyanide TS,
shake thoroughly, and add 1 drop of sodium sulfide TS (not
more than 50 ppm).
(5) Alkali earth metals and alkali metals — Dissolve 2.0 g
of Zinc Chloride in 120 mL of water, add ammonium sulfide
TS to complete precipitation, add water to make 200 mL,
shake thoroughly, and filter through dry filter paper. Discard
the first 20 mL of the filtrate, take the following 100 mL of
the filtrate, evaporate with 3 drops of sulfuric acid to dryness,
and heat the residue strongly at 600°C to constant mass: the
mass is not more than 10.0 mg.
(6) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Zinc Chloride according to Method 1 , and perform the test
(not more than 5 ppm).
(7) Oxychloride — Shake gently 0.25 g of Zinc Chloride
with 5 mL of water and 5 mL of ethanol (95), and add 0.3 mL
of 1 mol/L hydrochloric acid VS: the solution is clear.
Assay Weigh accurately about 0.3 g of Zinc Chloride, add
JPXV
Official Monographs / Zinc Oxide Oil 1245
0.4 mL of dilute hydrochloric acid and water to make exactly
200 mL. Measure exactly 20 mL of the solution, add 80 mL
of water, 2 mL of ammonia-ammonium chloride buffer solu-
tion, pH 10.7, and titrate <2.50> with 0.01 mol/L disodium
dihydrogen ethylenediamine tetraacetate VS (indicator: 0.04
g of eriochrome black T-sodium chloride indicator).
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 1.363 mg of ZnCl 2
Containers and storage Containers — Tight containers.
Zinc Oxide
ZnO: 81.41
Zinc Oxide, when ignited, contains not less than
99.0% of ZnO.
Description Zinc Oxide occurs as a white, amorphous pow-
der. It is odorless and tasteless.
It is practically insoluble in water, in ethanol (95), in acetic
acid (100) and in diethyl ether.
It dissolves in diute hydrochloric acid and in sodium
hydroxide TS.
It gradually absorbs carbon dioxide from air.
Identification (1) Heat Zinc Oxide strongly: a yellow
color develops on strong heating, and disappears on cooling.
(2) A solution of Zinc Oxide in dilute hydrochloric acid
(1 in 10) responds to the Qualitative Tests <1.09> for zinc salt.
Purity (1) Carbonate, and clarity and color of
solution — Mix 2.0 g of Zinc Oxide with 10 mL of water, add
30 mL of dilute sulfuric acid, and heat on a water bath with
stirring: no effervescence occurs, and the solution obtained is
clear and colorless.
(2) Alkalinity— To 1.0 g of Zinc Oxide add 10 mL of
water, and boil for 2 minutes. Cool, filter through a glass
filter (G3), and to the filtrate add 2 drops of phenolphthalein
TS and 0.20 mL of 0.1 mol/L hydrochloric acid VS: no color
develops.
(3) Sulfate <1.14>— Shake 0.5 g of Zinc Oxide with 40 mL
of water, and filter. Take 20 mL of the filtrate, add 1 mL of
dilute hydrochloric acid and water to make 50 mL, and per-
form the test using this solution as the test solution. Prepare
the control solution with 0.50 mL of 0.005 mol/L sulfuric
acid VS (not more than 0.096%).
(4) Iron — Dissolve 1.0 g of Zinc Oxide in 50 mL of dilut-
ed hydrochloric acid (1 in 2), dissolve 0.1 g of ammonium
peroxodisulfate in this solution, and extract with 20 mL of 4-
methyl-2-pentanone. Add 30 mL of acetic acid-sodium
acetate buffer solution for Iron Limit Test, pH 4.5, to the 4-
methyl-2-pentanone layer, extract again, and use the layer of
the buffer solution as the test solution. Separately, perform
the test in the same manner with 1 .0 mL of Standard Iron So-
lution, and use the layer so obtained as the control solution.
Add 2 mL each of L-ascorbic acid solution for Iron Limit
Test (1 in 100) to the test solution and the control solution,
respectively, mix, allow to stand for 30 minutes, add 5 mL of
an ethanol (95) solution of a,a'-dipyridyl (1 in 200) and
water to make 50 mL. After allowing to stand for 30 minutes,
compare the color of the both liquids against a white back:
the color of the liquid from the test solution is not stronger
than that from the control solution (not more than 10 ppm).
(5) Lead — To 2.0 g of Zinc Oxide add 20 mL of water,
then add 5 mL of acetic acid (100) with stirring, and heat on a
water bath until solution is complete. Cool, and add 5 drops
of potassium chromate TS: no turbidity is produced.
(6) Arsenic <1.11> — Dissolve 0.5 g of Zinc Oxide in 5 mL
of dilute hydrochloric acid, use this solution as the test solu-
tion, and perform the test (not more than 4 ppm).
Loss on ignition <2.43> Not more than 1.0% (1 g, 850°C,
1 hour).
Assay Weigh accurately about 0.8 g of Zinc Oxide, previ-
ously ignited at 850°C for 1 hour, dissolve in 2 mL of water
and 3 mL of hydrochloric acid, and add water to exactly 100
mL. Pipet 10 mL of this solution, add 80 mL of water, then
add a solution of sodium hydroxide (1 in 50) until a slight
precipitate is produced. Add 5 mL of ammonia-ammonium
chloride buffer solution, pH 10.7, and titrate <2.50> with 0.05
mol/L disodium dihydrogen ethylenediamine tetraacetate VS
(indicator: 0.04 g of eriochrome black T-sodium chloride in-
dicator).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.071 mg of ZnO
Containers and storage Containers — Tight containers.
Zinc Oxide Oil
T><7$
Zinc Oxide Oil contains not less than 45.0% and not
more than 55.0% of zinc oxide (ZnO: 81.41).
Method of preparation
Zinc Oxide
Fixed oil
500 g
a sufficient quantity
To make 1000 g
Mix the above ingredients. An appropriate quantity of
Castor Oil or polysorbate 20 may be used partially in place of
fixed oil.
Description Zinc Oxide Oil is a white to whitish, slimy sub-
stance, separating a part of its ingredients when stored for a
prolonged period.
Identification Mix thoroughly, and place 0.5 g of Zinc
Oxide Oil in a crucible, heat gradually raising the tempera-
ture until the mass is thoroughly charred, and then ignite it
strongly: a yellow color is produced, and disappears on cool-
ing. To the residue add 10 mL of water and 5 mL of dilute
hydrochloric acid, shake well, and filter. To the filtrate add 2
to 3 drops of potassium hexacyanoferrate (II) TS: a white
precipitate is formed (zinc oxide).
Assay Weigh accurately about 0.8 g of Zinc Oxide Oil, mix-
ed well, place in a crucible, heat gradually raising the temper-
1246 Zinc Oxide Ointment / Official Monographs
JP XV
ature until the mass is thoroughly charred, and then ignite
until the residue becomes yellow, and cool. Dissolve the
residue in 1 mL of water and 1.5 mL of hydrochloric acid,
and add water to make exactly 100 mL. Pipet 20 mL of this
solution, add 80 mL of water, and add a solution of sodium
hydroxide (1 in 50) until a small amount of precipitates be-
gins to form in the solution. Add 5 mL of ammonia-ammoni-
um chloride buffer solution, pH 10.7, and titrate <2.50> with
0.05 mol/L disodium dihydrogen ethylenediamine tetraa-
cetate VS (indicator: 0.04 g of eriochrome black T-sodium
chloride indicator).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.071 mg of ZnO
Containers and storage Containers — Tight containers.
Zinc Oxide Ointment
Zinc Oxide Ointment contains not less than 18.5%
and not more than 21.5% of zinc oxide (ZnO: 81.41).
Method of preparation
Zinc Oxide
Liquid Paraffin
White Ointment
200 g
30 g
a sufficient quantity
To make 1000 g
Prepare as directed under Ointments, with the above in-
gredients.
Description Zinc Oxide Ointment is white in color.
Identification Place 1 g of Zinc Oxide Ointment in a cruci-
ble, melt by warming, heat gradually raising the temperature
until the mass is thoroughly charred, and then ignite it
strongly: a yellow color is produced, and disappears on cool-
ing. To the residue add 10 mL of water and 5 mL of dilute
hydrochloric acid, shake well, and filter. To the filtrate add 2
to 3 drops of potassium hexacyanoferrate (II) TS: a white
precipitate is formed (zinc oxide).
Purity Calcium, magnesium and other foreign inorganic
matters — Place 2.0 g of Zinc Oxide Ointment in a crucible,
melt by warming, and heat gradually raising the temperature,
until the mass is thoroughly charred. Ignite the mass strongly
until the residue becomes uniformly yellow, and cool. Add 6
mL of dilute hydrochloric acid, and heat on a water bath for
5 to 10 minutes: the solution is colorless and clear. Filter the
solution, add 10 mL of water to the filtrate, and add ammo-
nia TS until the precipitate first formed redissolves. Add 2
mL each of ammonium oxalate TS and disodium hydrogen-
phosphate TS to this solution: the solution remains un-
changed or becomes very slightly turbid within 5 minutes.
Assay Weigh accurately about 2 g of Zinc Oxide Ointment,
place in a crucible, melt by warming, heat gradually raising
the temperature until the mass is thoroughly charred, and
then ignite until the residue becomes uniformly yellow, and
cool. Dissolve the residue in 1 mL of water and 1.5 mL of
hydrochloric acid, and add water to make exactly 100 mL.
Add 80 mL of water to exactly 20 mL of this solution, and
add a solution of sodium hydroxide (1 in 50) until a small
amount of precipitates begins to form in the solution. Add 5
mL of ammonia-ammonium chloride buffer solution, pH
10.7, and titrate <2.50> with 0.05 mol/L disodium dihydro-
gen ethylenediamine tetraacetate VS (indicator: 0.04 g of
eriochrome black T-sodium chloride indicator).
Each mL of 0.05 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 4.071 mg of ZnO
Containers and storage Containers — Tight containers.
Zinc Oxide Starch Powder
tmT>-f>
Method of preparation
Zinc Oxide
Starch
500 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description
powder.
Zinc Oxide Starch Powder occurs as a white
Identification (1) Place 1 g of Zinc Oxide Starch Powder
in a crucible, heat gradually, raising the temperature until it is
charred, and then ignite strongly: a yellow color develops,
and disappears on cooling. To the residue add 10 mL of
water and 5 mL of dilute hydrochloric acid, shake well, and
filter. To the filtrate add 2 to 3 drops of potassium hexac-
yanoferrate (II) TS: a white precipitate is formed (zinc
oxide).
(2) Shake well 1 g of Oxide Starch Powder with 10 mL of
water and 5 mL of dilute hydrochloric acid, and filter. Boil
the residue on a filter paper with 10 mL of water, cool, and
add 1 drop of iodine TS: a dark blue-purple color is produced
(starch).
Containers and storage Containers — Tight containers.
Zinc Sulfate Hydrate
ZnS0 4 .7H 2 0: 287.58
Zinc Sulfate Hydrate contains not less than 99.0%
and not more than 102.0% of ZnS0 4 .7H 2 0.
Description Zinc Sulfate Hydrate occurs as colorless crys-
tals or a white, crystalline powder. It is odorless, and has an
astringent, characteristic taste.
It is very soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
The pH of a solution of Zinc Sulfate Hydrate (1 in 20) is
between 3.5 and 6.0.
It effloresces in dry air.
JPXV
Official Monographs / Zinostatin Stimalamer 1247
Identification A solution of Zinc Sulfate Hydrate (1 in 20)
responds to the Qualitative Tests <1.09> for zinc salt and for
sulfate.
Purity (1) Acidity — Dissolve 0.25 g of Zinc Sulfate Hy-
drate in 5 mL of water, and add 1 drop of methyl orange TS:
no red color develops.
(2) Heavy metals <1.07> — Dissolve 1.0 g of Zinc Sulfate
Hydrate in 10 mL of water contained in a Nessler tube. Add
20 mL of potassium cyanide TS, and mix well. Add 2 drops
of sodium sulfide TS, and allow the mixture to stand for 5
minutes. Observe vertically against a white background, the
color of the solution is not more intense than the following
control solution.
Control solution: To 1.0 mL of Standard Lead Solution
add 10 mL of water and 20 mL of potassium cyanide TS, and
mix well. Add 2 drops of sodium sulfide TS (not more than
10 ppm).
(3) Alkali earth metals and alkali metals — Dissolve 2.0 g
of Zinc Sulfate Hydrate in 150 mL of water, add a suitable
amount of ammonium sulfide TS to complete the precipita-
tion, and add water to make exactly 200 mL. Shake well, and
filter through a dry filter paper. Discard the first 20 mL of
the filtrate, take exactly 100 mL of the subsequent filtrate,
evaporate to dryness, and ignite as directed under Residue on
Ignition <2.44>: the mass of the residue is not more than 5.0
mg.
(4) Arsenic <1.11> — Prepare the test solution with 1.0 g
of Zinc Sulfate Hydrate according to Method 1, and perform
the test (not more than 2 ppm).
Assay Weigh accurately about 0.3 g of Zinc Sulfate Hy-
drate, and dissolve in water to make exactly 100 mL. Measure
exactly 25 mL of this solution, add 100 mL of water and 2
mL of ammonia-ammonium chloride buffer solution, pH
10.7, and titrate <2.50> with 0.01 mol/L disodium dihydoro-
gen ethylenediamine tetraacetate VS (indicator: 0.04 g of
eriochrome black T-sodium chloride indicator).
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.876 mg of ZnS0 4 .7H 2
Containers and storage Containers — Tight containers.
Zinc Sulfate Ophthalmic Solution
Prepare as directed under Ophthalmic Solution, with the
above ingredients.
Description Zinc Sulfate Ophthalmic Solution is a clear,
colorless liquid.
Identification (1) Zinc Sulfate Ophthalmic Solution
responds to the Qualitative Tests <1.09> for zinc salt.
(2) Zinc Sulfate Ophthalmic Solution responds to the
Qualitative Tests <1.09> for borate.
(3) Zinc Sulfate Ophthalmic Solution responds to the
Qualitative Tests <1.09> for chloride.
Assay Pipet accurately 25 mL of Zinc Sulfate Ophthalmic
Solution, add 100 mL of water and 2 mL of ammonia-ammo-
nium chloride buffer solution, pH 10.7, and titrate <2.50>
with 0.01 mol/L disodium dihydrogen ethylenediamine
tetraacetate VS (indicator: 0.04 g of eriochrome black T-sodi-
um chloride indicator).
Each mL of 0.01 mol/L disodium dihydrogen
ethylenediamine tetraacetate VS
= 2.876 mg of ZnS0 4 .7H 2
Containers and storage Containers — Tight containers.
Zinostatin Stimalamer
V
VX^f> Xf?77-
Chromophore moiety
imw®&m&
(4S,6i?,ll^,12i?)-ll-[a-D-2,6-Dideoxy-2-(methylamino)-
galactopyranosyloxy]-4-[(4/?)-2-oxo-l,3-dioxolan-4-yl]-
5-oxatricyclo[8.3.0.0 4 ' 6 ]trideca-l(13),9-diene-2,7-diyn-12-yl
2-hydroxy-7-methoxy-5-methylnaphthalene-l-
carboxylate
[123760-07-6]
Zinc Sulfate Ophthalmic Solution contains not less
than 0.27 w/v% and not more than 0.33 w/v% of zinc
sulfate hydrate (ZnS0 4 .7H 2 0: 287.58).
Method of preparation
Zinc Sulfate Hydrate
Boric Acid
Sodium Chloride
Fennel Oil
Purified Water
3 g
20 g
5 g
2 mL
a sufficient quantity
To make 1000 mL
1248 Zinostatin Stimalamer / Official Monographs
JP XV
Apoprotein moiely bonded lo Htyrone-maleio atad alto mats «)fx>lymer
R- Ala- Ala-Pro-Th r-Ala-T hr- Val-Thr-Pro-Ser-Ser-Gly- Leu -Ser-Asp-G ly-Th r- Val-
Val- Lys-Val- Ala-G!y-Ala-G ly-Uu-GIri- Ala-Gly-Thr- Ala-Tyr- Asp- Va l-Gly-G I n-
Cys-Ala-Trp-Val-Asp-Thr-Gly-Val-Leu-Ala-Cys-Asn-Pra-Ala-Asp-Phe-Ser-
Ser- Val-Thf- Ala- Asp- Ala- Asp- Gly-Ser- Ala -Ser-Thr-Ss r-Leu-Thr- Val-Arg-
Arg-Se r-Phe-G I u-Gly- Phe- Leu-Phe- Asp-Gly-Tlir- Arg-Trp-Gly-T fir- Val-Asp-
Cys-Thr-Tlir- Ala- Al a- Cys-G I n - Val-Gly- Le u- Ser-Asp- Ala- Ala-G ly- Asri -G ly-
Pro-G lu -G ly-Val- Ala-J le-Ser-Prie- Asn
OR 2 OR 12
R = "W-^a. *- -\ j y%.
R' O R' 1
R and FT*, and R' 1 and R' F are different each other as lollows. respectively
A-HorNtt,
A . A =H, M l 4 or C 4 I i» (no C4I Id appears at Ihe same lime al A and A )
Avorrjgo m+n-aboLjt5.ri
Zinostatin Stimalamer consists 1 molecule of
zinostatin, consisting of chromophore and apoprotein
(polypeptide consisting of 113 amino acid residues) and
2 molecules of partially butyl-esterified styrene-maleic
acid alternate copolymer, and has average molecular
mass of about 15,000. The alternate copolymer is
bound an amido bond to a-amino group of alanine of
N-terminal and to e-amino group of lysine 20 of the
apoprotein.
It contains not less than 900 /ug (potency) and not
more than 1080 fig (potency) per mg, calculated on the
anhydrous basis. The potency of Zinostatin
Stimalamer is expressed as mass (potency) of zinostatin
stimalamer.
Description Zinostatin Stimalamer occurs as a pale yellow
powder.
It is freely soluble in water, and practically insoluble in
ethanol (95) and in diethyl ether.
Identification (1) Dissolve 10 mg of Zinostatin
Stimalamer in 1 mL of sodium hydroxide TS, and add a drop
of copper (II) sulfate TS: a purple color develops.
(2) Dissolve 1 mg of Zinostatin Stimalamer in 1 mL of
0.05 mol/L phosphate buffer solution, pH 7.0, add 0.5 mL
of a solution of trichloroacetic acid (1 in 5), and shake: a
white precipitate is formed.
(3) Determine the absorption spectra of solutions of
Zinostatin Stimalamer and Zinostatin Stimalamer Reference
Standard in 0.05 mol/L phosphate buffer solution, pH 7.0 (1
in 2500) as directed under Ultraviolet-visible Spectrophoto-
metry <2.24>, and compare these spectra: both spectra exhibit
similar intensities of absorption at the same wavelengths.
(4) Determine the infrared absorption spectra of Zinosta-
tin Stimalamer and Zinostatin Stimalamer Reference Stan-
dard as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>, and compare these spec-
tra: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Absorbance <2.24> E\ 0/ ° m (268 nm): 15.5 - 18.5 (4 mg calcu-
lated on the anhydrous basis, 0.05 mol/L phosphate buffer
solution, pH 7.0, 10 mL).
Optical rotation <2.49> [a]£°: -30.0- -38.0° (20 mg cal-
culated on the anhydrous basis, 0.05 mol/L phosphate buffer
solution, pH 7.0, 5 mL, 100 mm).
pH <2.54> Dissolve 10 mg of Zinostatin Stimalamer in 1 mL
of water: the pH of the solution is between 4.5 and 5.5.
Purity (1) Clarity and color of solution — Being specified
separately.
(2) Heavy metals <1.07> — Weigh accurately 40 mg of
Zinostatin Stimalamer, place in a crucible, carbonize and in-
cinerate according to Method 2, add 2 mL of hydrochloric
acid, and evaporate on a water bath to dryness. After cool-
ing, weigh the residue W T g. Then, moisten the residue with
0.1 mL of diluted hydrochloric acid (1 in 5), add 1 mL of
water, 85 fiL of diluted ammonia TS (1 in 2) and 0.1 mL of
dilute acetic acid, and add water so that the mass is W T + 2.0
g. Adjust the pH of this solution to 3.2 to 3.4 with diluted
ammonia TS (1 in 20) or diluted hydrochloric acid (1 in 50),
add water so that the mass is W T + 2.5 g, and use this solu-
tion as the test solution. Separately, prepare the blank solu-
tion in the same manner without the sample. Separately, take
2 mL of nitric acid, 5 drops of sulfuric acid and 2 mL of
hydrochloric acid, and evaporate to dryness according to
Method 2. After cooling, weigh the residue W s g. Then,
moisten the residue with 0.1 mL of diluted hydrochloric acid
(1 in 5), and proceed in the same manner as directed in the
preparation of the test solution. After adjusting the pH of the
solution so obtained to 3.2 to 3.4, add 80 fiL of Standard
Lead Solution, and add water so that the mass is W s + 2.5 g,
and use this solution as the control solution. Add 10 fiL each
of diluted sodium sulfide TS (1 in 6) to the test solution, the
blank solution and the control solution, mix, and allow to
stand for 5 minutes. Determine the absorbances, A T , A and
A s of the test solution, the blank solution and the control so-
lution at 400 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: A-y — A is not larger than A s - A
(not more than 20 ppm).
(3) Related substances — Being specified separately.
(4) Inorganic salts of manufacturing process
origin — Being specified separately.
Water <2.48> Not more than 12.0% (10 mg, coulometric
titration).
Assay Perform the test according to the Cylinder-plate
method as directed under Microbial Assay for Antibiotics
<4.02> according to the following conditions. Perform the
procedures of (iii), (iv) and (v) without exposure to direct or
indirect sunlight.
(i) Test organism — Micrococcus luteus ATCC 9341
(ii) Culture medium — Use the medium i in 3) Medium for
other organisms under (1) Agar media for seed and base lay-
er. Adjust the pH of the medium so that it will be 7.9 to 8.1
after sterilization.
(iii) Standard solutions — Weigh accurately an amount of
Zinostatin Stimalamer Reference Standard equivalent to
about 20 mg (potency), dissolve in 0.1 mol/L phosphate
buffer solution, pH 8.0 to make exactly 50 mL, and use this
solution as the high concentration standard solution. Pipet 5
mL of the high concentration standard solution, add 0.1 mol
/L phosphate buffer solution, pH 8.0, to make exactly 20
mL, and use this solution as the low concentration standard
JP XV Official Monographs / Zinostatin Stimalamer 1249
solution.
(iv) Sample solutions — Weigh accurately an amount of
Zinostatin Stimalamer equivalent to about 20 mg (potency),
dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0 to
make exactly 50 mL, and use this solution as the high concen-
tration sample solution. Pipet 5 mL of the high concentration
sample solution, add 0.1 mol/L phosphate buffer solution,
pH 8.0, to make exactly 20 mL, and use this solution as the
low concentration sample solution.
(v) Procedure — Allow to stand at 3 to 5°C for 2 hours be-
fore incubation.
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and not exceeding -20°C.
Crude Drugs
Acacia
Gummi Arabicum
Acacia is the secretions obtained from the stems and
branches of Acacia Senegal Willdenow or other species
of the same genus (Leguminosae).
Description Colorless or light yellow-brown, translucent or
somewhat opaque spheroidal tears, or angular fragments
with numerous fissures on the surface; very brittle; the frac-
tured surface glassy and occasionally iridescent.
Odorless; tasteless, but produces a mucilaginous sensation
on the tongue.
Pulverized Acacia (1.0 g) dissolves almost completely in
2.0 mL of water, and the solution is acid.
It is practically insoluble in ethanol (95).
Identification To 1 g of powdered Acacia add 25 mL of
water and 1 mL of sulfuric acid, and heat under a reflux con-
denser in a boiling water bath for 60 minutes. After cooling,
add gently 2.0 g of anhydrous sodium carbonate. To 1 mL of
this solution add 9 mL of methanol, mix well, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 10 mg of D-galactose in 1 mL water, add methanol to
make 10 mL, and use this solution as the standard solution
(1). Proceed with L-arabinose and with L-rhamnose monohy-
drate in the same manner as for the preparation of the stan-
dard solution (1), and use so obtained solutions as the stan-
dard solution (2) and the standard solution (3), respectively.
Perform the test with these solutions as directed under Thin-
layer chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solutions (1), (2) and (3) on a plate of
silica gel for thin-layer chromatography. Develop the plate
with a mixture of acetone and water (9:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly 1-naphthol-
sulfuric acid TS on the plate, and heat at 105 °C for 5
minutes: the three spots from the sample solution are the
same with the spots of D-galactose, L-arabinose and L-rham-
nose from the standard solution in the color tone and the Rf
value, respectively.
Purity (1) Insoluble residue — To 5.0 g of pulverized Aca-
cia add 100 mL of water and 10 mL of dilute hydrochloric
acid, and dissolve by gentle boiling for 15 minutes with
swirling. Filter the warm mixture through a tared glass filter
(G3), wash the residue thoroughly with hot water, and dry at
105 °C for 5 hours: the mass of the residue does not exceed
10.0 mg.
(2) Tannin-bearing gums — To 10 mL of a solution of
Acacia (1 in 50) add 3 drops of iron (III) chloride TS: no dark
green color is produced.
(3) Glucose — Dissolve 10 mg of glucose in 1 mL of water,
add methanol to make 10 mL, and use this solution as the
standard solution. Proceed with the sample solution obtained
in the Identification and the standard solution obtained here
as directed in the Identification: any spot at the Rf value cor-
responding to glucose from the standard solution does not
appear from the sample solution.
Loss on drying <5.01> Not more than 17.0% (6 hours).
Total ash <5.07> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Powdered Acacia
Gummi Arabicum Pulveratum
Powdered Acacia is the powder of Acacia.
Description Powdered Acacia occurs as a white to light
yellowish white powder. It is odorless, tasteless, but produces
a mucilaginous sensation on the tongue.
Under a microscope <5.01>, Powdered Acacia, immersed in
olive oil or liquid paraffin, reveals colorless, angular frag-
ments or nearly globular grains. Usually starch grains or
vegetable tissues are not observed or very trace, if any.
Powdered Acacia (1.0 g) dissolves almost completely in 2.0
mL of water, and the solution is acid.
It is practically insoluble in ethanol (95).
Identification To 1 g of Powdered Acacia add 25 mL of
water and 1 mL of sulfuric acid, and heat under a reflux con-
denser in a boiling water bath for 60 minutes. After cooling,
add gently 2.0 g of anhydrous sodium carbonate. To 1 mL of
this solution add 9 mL of methanol, mix well, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 10 mg of D-galactose in 1 mL water, add methanol to
make 10 mL, and use this solution as the standard solution
(1). Proceed with L-arabinose and with L-rhamnose monohy-
drate in the same manner as for the preparation of the stan-
dard solution (1), and use so obtained solutions as the stan-
dard solution (2) and the standard solution (3), respectively.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solutions (1), (2) and (3) on a plate of
silica gel for thin-layer chromatography. Develop the plate
with a mixture of acetone and water (9:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly 1-naphthol-
sulfuric acid TS on the plate, and heat at 105°C for 5
minutes: the three spots from the sample solution are the
same with the spots of D-galactose, L-arabinose and L-rham-
nose from the standard solution in the color tone and the Rf
value, respectively.
Purity (1) Insoluble residue — To 5.0 g of Powdered Aca-
cia add 100 mL of water and 10 mL of dilute hydrochloric
acid, and dissolve by gentle boiling for 15 minutes with
swirling. Filter the warm mixture through a tared glass filter
1251
1252 Achyranthes Root / Crude Drugs
JP XV
(G3), wash the residue thoroughly with hot water, and dry at
105 °C for 5 hours: the mass of the residue does not exceed
10.0 mg.
(2) Tannin-bearing gums — To 10 mL of a solution of
Powdered Acacia (1 in 50) add 3 drops of iron (III) chloride
TS: no dark green color is produced.
(3) Glucose — Dissolve 10 mg of glucose in 1 mL of water,
add methanol to make 10 mL, and use this solution as the
standard solution. Proceed with the sample solution obtained
in the Identification and the standard solution obtained here
as directed in the Identification: any spot at the Ri value cor-
responding to glucose from the standard solution does not
appear from the sample solution.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.01> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Containers and storage Containers — Tight containers.
Achyranthes Root
Achyranthis Radix
=f->7
Achyranthes Root is the root of Achyranthes fauriei
Leveille et Vaniot or Achyranthes bidentata Blume
(Amaranthaceae).
Description Main root or main root with some lateral roots,
with or without short remains of rhizome at the crown; main
root, long cylindrical and sometimes somewhat tortuous, 15
- 90 cm in length, 0.3 - 0.7 cm in diameter; externally grayish
yellow to yellow-brown, with numerous longitudinal wrin-
kles, and with scattering scars of lateral roots. Fractured
surface is flat; grayish white to light brown on the circumfer-
ence, and with yellowish white xylem in the center. Hard and
brittle, or flexible.
Odor, slight; taste, slightly sweet, and mucilaginous.
Under a microscope <5.01>, a transverse section reveals a
rather distinct cambium separating the cortex from the xy-
lem; small protoxylem located at the center of the xylem, and
surrounded by numerous vascular bundles arranged on sever-
al concentric circles; parenchyma cells containing sand crys-
tals of calcium oxalate; starch grains absent.
Identification Shake vigorously 0.5 g of pulverized
Achyranthes Root with 10 mL of water: a lasting fine foam is
produced.
Purity (1) Stem — The amount of stems contained in
Achyranthes Root does not exceed 5.0%.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Achyranthes Root according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Achyranthes Root according to Method 4, and
perform the test (not more than 5 ppm).
(4) Foreign matter <5.01> — The amount of foreign matter
other than stems contained in Achyranthes Root does not ex-
ceed 1.0%.
Loss on drying <5.01> Not more than 17.0% (6 hours).
Total ash <J.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Agar
Agar
Agar is the solid residue obtained by freezing
dehydration of a mucilage derived from Gelidium
amansii Lamouroux, other species of the same genus
(Gelidiaceae), or other red algae (Rhodophyta).
Description White, translucent rectangular column, string
or flakes. Rectangular column about 26 cm in length, 4 cm
square in cross section; a string of about 35 cm in length and
about 3 mm in width; flakes about 3 mm in length; external-
ly, with wrinkles and somewhat lustrous, light and pliable.
Odorless; tasteless and mucilagenous.
It is practically insoluble in organic solvents.
A boiling solution of Agar (1 in 100) is neutral.
Identification (1) To a fragment of Agar add dropwise
iodine TS: a dark blue to reddish purple color develops.
(2) Dissolve 1 g of Agar in 65 mL of water by boiling for
10 minutes with constant stirring, and add a sufficient
amount of hot water to make up the water lost by evapora-
tion: the solution is clear. Cool the solution between 30°C
and 39°C: the solution forms a firm, resilient gel, which does
not melt below 85 °C.
Purity (1) Sulfuric acid — Dissolve 1.0 g of Agar in 100
mL of water by boiling: the solution is not acidic.
(2) Sulfurous acid and starch — To 5 mL of the solution
obtained in (1) add 2 drops of iodine TS: the solution does
not decolorize immediately, and does not show a blue color.
(3) Insoluble matter — To 7.5 g of Agar add 500 mL of
water, boil for 15 minutes, and add water to make exactly 500
mL. Measure exactly 100 mL of the solution, add 100 mL of
hot water, heat to boiling, filter while hot through a tared
glass filter (G3), wash the residue with a small amount of hot
water, and dry the residue at 105 °C for 3 hours: the mass of
the residue is not more than 15.0 mg.
(4) Water absorption — To 5.0 g of Agar add water to
make 100 mL, shake well, allow to stand at 25°C for 24
hours, and filter through moistened glass wool in a 100-mL
graduated cylinder: the volume of the filtrate is not more than
75 mL.
Loss on drying <5.01> Not more than 22.0% (6 hours).
Total ash <J.07> Not more than 4.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
JPXV
Crude Drugs / Powdered Alisma Rhizome 1253
Powdered Agar
Agar Pulveratum
Powdered Agar is the powder of Agar.
Description Powdered Agar appears as a white powder, is
odorless, and is tasteless and mucilagenous.
Under a microscope <5.01>, Powdered Agar, immersed in
olive oil or liquid paraffin, reveals angular granules with stria-
tions or nearly spheroidal granules 5 to 60 //m in diameter.
It becomes transparent in chloral hydrate TS.
It is practically insoluble in organic solvents.
A boiling solution of Powdered Agar (1 in 100) is neutral.
Identification (1) To a part of Powdered Agar add drop-
wise iodine TS: a dark blue to reddish purple color develops.
(2) Dissolve 1 g of Powdered Agar in 65 mL of water by
boiling for 10 minutes with constant stirring, and add a
sufficient amount of hot water to maintain the original
volume lost by evaporation: the solution is clear. Cool the
solution between 30°C and 39°C: the solution forms a firm,
resilient gel, which does not melt below 85 °C.
Purity (1) Sulfuric acid — Dissolve 1.0 g of Powdered
Agar in 100 mL of water by boiling: the solution is not acid.
(2) Sulfurous acid and starch — To 5 mL of the solution
obtained in (1) add 2 drops of iodine TS: the solution is not
decolorized immediately, and does not show a blue color.
(3) Insoluble matter — To 7.5 g of Powdered Agar add
500 mL of water, boil for 15 minutes, and add water to make
exactly 500 mL. Take exactly 100 mL of the solution, add 100
mL of hot water, heat to boiling, filter while hot through a
tared glass filter (G3), wash the residue with a small amount
of hot water, and dry the residue at 105°C for 3 hours: the
mass of the residue is not more than 15.0 mg.
(4) Water absorption — To 5.0 g of Powdered Agar add
water to make 100 mL, shake well, allow to stand at 25 °C for
24 hours, and filter through moistened glass wool in a
100-mL graduated cylinder: the volume of the filtrate is not
more than 75 mL.
Loss on drying <5.01> Not more than 22.0% (6 hours).
Total ash <J.07> Not more than 4.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Containers and storage Containers — Tight containers.
Akebia Stem
Akebiae Caulis
Akebia Stem is the climbing stem of Akebia quinata
Decaisne or Akebia trifoliata Koidzumi
(Lardizabalaceae), usually cut transversely.
Description Circular or ellipsoidal sections 0.2 - 0.3 cm in
thickness, and 1-3 cm in diameter; phloem on both frac-
tured surfaces is dark grayish brown; zylem reveals light
brown vessel portions and grayish white medullary rays lined
alternately and radially; pith light grayish yellow, and dis-
tinct; flank grayish brown, and with circular or transversely
elongated elliptical lenticels.
Almost odorless; slightly acrid taste.
Under a microscope <5.01>, a transverse section reveals
ring layers mainly consisting of fiber bundles with crystal cells
and stone cell groups and surrounding the outside of the
phloem in arc shape. Medullary rays of the phleom consisting
of sclerenchymatous cells containing solitary crystals; por-
tion near cambium is distinct; cells around the pith remarka-
bly thick-walled; xylem medullary rays and parenchymatous
cells around the pith contain solitary crystals of calcium oxa-
late and starch grains less than 8 /um in diameter.
Identification To 0.5 g of pulverized Akebia Stem add 10
mL of water, boil, allow to cool, and shake vigorously: last-
ing fine foams are produced.
Total ash <5.07> Not more than 10.0%.
Alisma Rhizome
Alismatis Rhizoma
Alisma Rhizome is the tuber of Alisma orientale
Juzepczuk (Alismataceae), from which periderm has
been usually removed.
Description Spherical or conical tubers, 3-8 cm in length,
3-5 cm in diameter, sometimes a 2- to 4-branched irregular
tuber; externally light grayish brown to light yellow-brown,
and slightly annulate; many remains of root appearing as
small warty protrusions; fractured surface nearly dense, the
outer portion grayish brown, and the inner part white to light
yellow-brown in color; rather light in texture and difficult to
break.
Slight odor and taste.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
pulverized Alisma Rhizome according to Method 3, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Alisma Rhizome according to Method 4, and
perform the test (not more than 5 ppm).
Total ash <J.07> Not more than 5.0%.
Acid-insoluble ash <5.0I> Not more than 0.5%.
Powdered Alisma Rhizome
Alismatis Rhizoma Pulveratum
Powdered Alisma Rhizome is the powder of Alisma
1254 Aloe / Crude Drugs
JP XV
Rhizome.
Description Powdered Alisma Rhizome occurs as a light
grayish brown powder, and has a slight odor and taste.
Under a microscope <5.01>, Powdered Alisma Rhizome
reveals mainly starch grains, fragments of parenchyma con-
taining them, parenchyma cells containing yellow contents,
and fragments of vascular bundles. Starch grains, spheroidal
to ellipsoidal simple grains, 3 - 15 /xm in diameter.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Powdered Alisma Rhizome according to Method 3, and per-
form the test. Prepare the control solution with 2.0 mL of
Standard Lead Solution (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 0.40 g
of Powdered Alisma Rhizome according to Method 3, and
perform the test (not more than 5 ppm).
Total ash <5.01> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Aloe
Aloe
7ni
Aloe is the dried juice of the leaves mainly of Aloe
ferox Miller, or of hybrids of the species with Aloe
africana Miller or Aloe spicata Baker (Liliaceae).
It contains not less than 4.0% of barbaloin, calculat-
ed on the basis of dried material.
Description Aloe occurs as blackish brown to dark brown,
irregular masses; sometimes the external surface covered with
a yellow powder; the fractured surface smooth and glassy.
Odor, characteristic; taste, extremely bitter.
Identification (1) Dissolve 0.5 g of pulverized Aloe in 50
mL of water by warming. After cooling, add 0.5 g of silice-
ous earth, and filter. Perform the following tests using the
filtrate as the sample solution.
(i) Dissolve 0.2 g of sodium tetraborate decahydrate in 5
mL of the sample solution by warming in a water bath. Add a
few drops of this solution into 30 mL of water, and shake: a
green fluorescence is produced.
(ii) Shake 2 mL of the sample solution with 2 mL of nitric
acid: a yellow-brown color which changes gradually to green
is produced. Then warm this colored solution in a water bath:
the color of the solution changes to red-brown.
(2) To 0.2 g of pulverized Aloe add 10 mL of methanol,
shake for 5 minutes, filter, and use the filtrate as the sample
solution. Separately, dissolve 1 mg of barbaloin for thin-
layer chromatography in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 ^L each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, acetone, water and acetic acid (100) (20:5:2:2) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 365 nm): one spot
among several spots from the sample solution and a red
fluorescent spot from the standard solution show the same
color tone and the same Rf value.
Purity (1) Resin — Warm 0.5 g of pulverized Aloe with 10
mL of diethyl ether on a water bath, and filter. Wash the
residue and the filter paper with 3 mL of diethyl ether. Com-
bine the filtrate and the washing, and evaporate the diethyl
ether solution: the mass of the residue is not more than 5.0
mg.
(2) Ethanol-insoluble substances — Boil 1.0 g of pulver-
ized Aloe with 50 mL of ethanol (95) on a water bath for 30
minutes under a reflux condenser. Filter the warm mixture
through a tared glass filter (G4), and wash the residue on the
filter with ethanol (95) until the last washing becomes color-
less. Dry the residue at 105°C for 5 hours, and weigh: the
mass of the residue is not more than 0.10 g.
Loss on drying <5.01> Not more than 12.0%.
Total ash <5.07> Not more than 2.0%.
Extract content <5.01> Water-soluble extract: not less than
40.0%.
Component determination Weigh accurately about 0.1 g of
pulverized Aloe, add 40 mL of methanol, and heat under a
reflex condenser on a water bath for 30 minutes. After cool-
ing, filter, and add methanol to the filtrate to make exactly 50
mL. Pipet 5 mL of the solution, add methanol to make ex-
actly 10 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of barbaloin for
component determination, previously dried in a desiccator
(in vacuum, phosphorus (V) oxide) for 24 hours, add 40 mg
of oxalic acid dihydrate, and dissolve in methanol to make
exactly 100 mL. Pipet 5 mL of the solution, add methanol to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with exactly 5 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and measure the peak areas of barbaloin, A T and A s ,
of both solutions.
Amount (mg) of barbaloin = W s x (A T /A S ) x (1/2)
W s : Amount (mg) of barbaloin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 360 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (74:26:1).
Flow rate: Adjust the flow rate so that the retention time of
barbaloin is about 12 minutes.
System suitability —
System performance: Dissolve 10 mg of barbaloin for
component determination add 40 mg of oxalic acid dihy-
drate, in methanol to make exactly 100 mL. Pipet 5 mL of
the solution, add 1 mL of a solution of ethenzamide in
methanol (1 in 2000) and methanol to make exactly 10 mL.
When the procedure is run with 5 fiL of this solution under
the above operating conditions except the wavelength of 300
JPXV
Crude Drugs / Powdered Aloe 1255
nm, barbaloin and ethenzamide are eluted in this order with
the resolution between these peaks being not less than 2.0.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
barbaloin is not more than 1.5%.
Powdered Aloe
Aloe Pulverata
7niJ
Powdered Aloe is the powder of Aloe.
It contains not less than 4.0% of barbaloin, calculat-
ed on the basis of dried material.
Description Powdered Aloe occurs as a dark brown to yel-
lowish dark brown powder. It has a characteristic odor and
an extremely bitter taste.
Under a microscope <5.01>, Powdered Aloe, immersed in
olive oil or liquid paraffin, reveals greenish yellow to reddish
brown, angular or rather irregular fragments.
Identification (1) Dissolve 0.5 g of Powdered Aloe in 50
mL of water by warming. After cooling, add 0.5 g of siliceous
earth, and filter. Perform the following tests with the filtrate
as the sample solution.
(i) Dissolve 0.2 g of sodium tetraborate decahydrate in 5
mL of the sample solution by warming in a water bath. Add a
few drops of this solution into 30 mL of water, and shake: a
green fluorescence is produced.
(ii) Shake 2 mL of the sample solution with 2 mL of nitric
acid: a yellow-brown color which changes gradually to green
is produced. Then warm this colored solution in a water bath:
the color of the solution changes to red-brown.
(2) To 0.2 g of Powdered Aloe add 10 mL of methanol,
shake for 5 minutes, filter, and use the filtrate as the sample
solution. Separately, dissolve 1 mg of barbaloin for thin-lay-
er chromatography in 1 mL of methanol, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, acetone, water
and acetic acid (100) (20:5:2:2) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 365 nm): one spot among several spots from the
sample solution has the same color tone and the same Rf
value with the red fluorescent spot from the standard solu-
tion.
Purity (1) Resin — Warm 0.5 of Powdered Aloe with 10
mL of diethyl ether on a water bath, and filter. Wash the
residue and the filter paper with 3 mL of diethyl ether. Com-
bine the filtrate and the washing, and evaporate the diethyl
ether: the mass of the residue does not exceed 5.0 mg.
(2) Ethanol-insoluble substances — Boil 1.0 g of Pow-
dered Aloe with 50 mL of ethanol (95) on a water bath for 30
minutes under a reflux condenser. Filter the warm mixture
through a tared glass filter (G4), and wash the residue on the
filter with ethanol (95) until the last washing becomes color-
less. Dry the residue at 105°C for 5 hours, and weigh: the
mass of the residue is not more than 0.10 g.
Loss on drying <5.0]> Not more than 12.0%.
Total ash <J.07> Not more than 2.0%.
Extract content <5.01> Water-soluble extract: not less than
40.0%.
Component determination Weigh accurately about 0.1 g of
Powdered Aloe, add 40 mL of methanol, and heat under a
reflex condenser on a water bath for 30 minutes. After cool-
ing, filter, and add methanol to the filtrate to make exactly 50
mL. Pipet 5 mL of the solution, add methanol to make ex-
actly 10 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of barbaloin for
component determination, previously dried in a desiccator
(in vacuum, phosphorus (V) oxide) for 24 hours, add 40 mg
of oxalic acid dihydrate, and dissolve in methanol to make
exactly 100 mL. Pipet 5 mL of the solution, add methanol to
make exactly 10 mL, and use this solution as the standard so-
lution. Perform the test with exactly 5 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas of barbaloin, A T and A s ,
of both solutions.
Amount (mg) of barbaloin = W s x (A T /A S ) x (1/2)
W s : Amount (mg) of barbaloin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 360 nm).
Column: A stainless steel column about 6 mm in inside
diameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /am in parti-
cle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (74:26:1).
Flow rate: Adjust the flow rate so that the retention time of
barbaloin is about 12 minutes.
System suitability —
System performance: To about 10 mg of barbaloin for
component determination add 40 mg of oxalic acid dihy-
drate, and dissolve in methanol to make exactly 100 mL.
Pipet 5 mL of the solution, add 1 mL of a solution of ethen-
zamide in methanol (1 in 2000) and methanol to make exactly
10 mL. When the procedure is run with 5 fiL of this solution
under the above operating conditions except the wavelength
of 300 nm, barbaloin and ethenzamide are eluted in this order
with the resolution between these peaks being not less than
2.0.
System repeatability: When the test is repeated 6 times with
5 fiL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak area of
barbaloin is not more than 1.5%.
Containers and storage Containers — Tight containers.
1256 Alpinia Officinarum Rhizome / Crude Drugs
JP XV
Alpinia Officinarum Rhizome
Alpiniae officinari Rhizoma
'J 3^7+3^7
Alpinia Officinarum Rhizome is the rhizome of Al-
pinia officinarum Hance (Zingiberaceae).
Description Alpinia Officinarum Rhizome is a slightly
curved and cylindrical rhizome, sometimes branched; 2 to 8
cm in length, 6 to 15 mm in diameter; externally red-brown to
dark brown with fine striped lines, grayish white nodes and
several traces of rootlet; hard to break; fracture surface, light
brown in color and thickness of cortex is approximately the
same as that of stele.
Odor, characteristic; taste, extremely pungent.
Under a microscope <5.01>, transverse section reveals
epidermal cells often containing resin-like substances; cortex,
endodermis and stele present beneath the epidermis; cortex
and stele divided by endodermis; vascular bundles surround-
ed by fibers, scattered throughout the cortex and stele, cortex
and stele composed of parenchyma interspersed with oil cells;
parenchymatous cells containing solitary crystals of calcium
oxalate and starch grains, starch grains generally simple
(sometimes 2- to 8-compound), ovate, oblong or narrowly
ovate, 10 - 40 fim in diameter and with an eccentric navel.
Identification To 0.5 g of pulverized Alpinia Officinarum
Rhizome add 5 mL of acetone, shake for 5 minutes, and
filter. Perform the test with the filtrate as directed under
Thin-layer Chromatography <2.03>. Spot 5 [iL of the filtrate
on a plate of silica gel for thin-layer chromatography, de-
velop the plate with a mixture of cyclohexane, ethyl acetate
and acetic acid (100) (12:8:1) to a distance of about 10 cm,
and air-dry the plate: two yellow-brown spots appear at
around Rf 0.4-0.5.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.01> Not more than 7.5%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01>
14.0%.
Dilute ethanol-extract: not less than
Amomum Seed
Amomi Semen
-> ZL *7 V -Y
Amomum Seed is the seed mass of Amomum xan-
thioides Wallich (Zingiberaceae).
Description Approximately spherical or ellipsoidal mass,
1-1.5 cm in length, 0.8 - 1 cm in diameter; externally
grayish brown to dark brown, and with white powder in
those dried by spreading lime over the seeds; the seed mass is
divided into three loculi by thin membranes, and each loculus
contains 10 to 20 seeds joining by aril; each seed is polygonal
and spherical, 0.3 - 0.5 cm in length, about 0.3 cm in di-
ameter, externally dark brown, with numerous, fine protru-
sions; hard tissue; under a magnifying glass, a longitudinal
section along the raphe reveals oblong section, with deeply
indented hilum and with slightly indented chalaza; white
perisperm covering light yellow endosperm and long embryo.
Characteristic aroma when cracked, and taste acrid.
Total ash <5.07> Not more than 9.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Essential oil content <5.01> Perform the test with 30.0 g of
pulverized Amomum Seed: the volume of essential oil is not
less than 0.6 mL.
Powdered Amomum Seed
Amomi Semen Pulveratum
Powdered Amomum Seed is the powder of Amo-
mum Seed.
Description Powdered Amomum seed occurs as a grayish
brown powder, and has a characteristic aroma and an acrid
taste.
Under a microscope <5.01>, Powdered Amomum Seed rev-
eals fragments of wavy perisperm cells filled with starch
grains and containing in each cell a calcium oxalate crystal;
yellow and long epidermal cells of seed coat and fragments of
thin-walled tissue perpendicular to them; fragments of
groups of brown, thick-walled polygonal stone cells.
Total ash <5.07> Not more than 9.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Essential oil content <5.01> Perform the test with 30.0 g of
Powdered Amomum Seed: the volume of essential oil is not
less than 0.4 mL.
Containers and storage Containers — Tight containers.
Anemarrhena Rhizome
Anemarrhenae Rhizoma
Anemarrhena Rhizome is the rhizome of Anemar-
rhena asphodeloides Bunge (Liliaceae).
Description Rather flat and cord-like rhizome, 3-15 cm in
length, 0.5 - 1.5 cm in diameter, slightly bent and branched;
externally yellow-brown to brown; on the upper surface, a
longitudinal furrow and hair-like remains or scars of leaf
sheath forming fine ring-nodes; on the lower surface, scars of
root appearing as numerous round spot-like hollows; light
and easily broken. Under a magnifying glass, a light yellow-
brown transverse section reveals an extremely narrow cortex;
stele porous, with many irregularly scattered vascular bun-
dles.
JPXV
Crude Drugs / Apricot Kernel Water 1257
Odor, slight; taste, slightly sweet and mucous, followed by
bitterness.
Identification (1) Shake vigorously 0.5 g of pulverized
Anemarrhena Rhizome with 10 mL of water in a test tube: a
lasting fine foam is produced. Filter the mixture, and to 2 mL
of the filtrate add 1 drop of iron (III) chloride TS: a dark
green precipitate is produced.
(2) Warm 0.5 g of pulverized Anemarrhena Rhizome
with 2 mL of acetic anhydride on a water bath for 2 minutes
while shaking, then filter, and to the filtrate add carefully 1
mL of sulfuric acid to make two layers: a red-brown color
develops at the zone of contact.
Purity Foreign matter <5.01> — The amount of fiber,
originating from the dead leaves, and other foreign matters
contained in Anemarrhena Rhizome is not more than 3.0%.
Total ash <5.01> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Angelica Dahurica Root
Angelica Dahurica Root is the root of Angelica da-
hurica Bentham et Hooker (Umbelliferae).
Description Main root from which many long roots are
branched out and nearly fusiform and conical in whole
shape, 10 - 25 cm in length; externally grayish brown to dark
brown, with longitudinal wrinkles, and with numerous scars
of rootlets laterally elongated and protruded. A few remains
of leaf sheath at the crown and ring-nodes closely protruded
near the crown. In a transverse section, the outer region is
grayish white in color, and the central region is sometimes
dark brown in color.
Odor, characteristic; taste, slightly bitter.
Identification To 0.2 g of pulverized Angelica Dahurica
Root add 5 mL of ethanol (95), allow to stand for 5 minutes
with shaking, and filter. Examine the filtrate under ultraviolet
light (main wavelength: 365 nm): a blue to blue-purple
fluorescence develops.
Purity (1) Leaf sheath — The amount of leaf sheath con-
tained in Angelica Dahurica Root does not exceed 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than leaf sheath contained in Angelica Dahurica Root
is not more than 1.0%.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 25.0%.
Apricot Kernel
Armeniacae Semen
Apricot Kernel is the seed of Prunus armeniaca
Linne or Prunus armeniaca Linne var. ansu Max-
imowicz (Rosaceae).
Description Flattened, somewhat asymmetric ovoid seed,
1.1 - 1.8 cm in length, 0.8 - 1.3 cm in width, 0.4 -0.7 cm in
thickness; sharp at one end and rounded at the other end
where chalaza situated; seed coat brown and its surface being
powdery with rubbing easily detachable stone cells of epider-
mis; numerous vascular bundles running from chalaza
throughout the seed coat, appearing as thin vertical furrows;
seed coat and thin semitransparent white albumen easily
separate from cotyledon when soaked in boiling water;
cotyledon, white in color.
Almost odorless; taste, bitter and oily.
Under a microscope <5.01>, surface of epidermis reveals
stone cells on veins protruded by vascular bundles, forming
angular circle to ellipse and approximately uniform in shape,
with uniformly thickened walls, and 60 - 90 //m in diameter;
in lateral view, stone cell appearing obtusely triangular and
its wall extremely thickened at the apex.
Identification To 1 .0 g of ground Apricot Kernel add 10 mL
of methanol, immediately heat under a reflux condenser on a
water bath for 10 minutes, cool, filter, and use the filtrate as
the sample solution. Separately, dissolve 2 mg of amygdalin
for thin-layer chromatography in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, methanol and water (7:3:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly dilute sulfuric acid
upon the plate, and heat at 105 °C for 10 minutes: one spot
among the spots from the sample solution and a brown to
dark green spot from the standard solution show the same
color tone and the same Rf value.
Purity (1) Rancidity — Grind Apricot Kernel with hot
water: no unpleasant odor of rancid oil is perceptible.
(2) Foreign matter <5.01> — Apricot Kernel does not con-
tain fragments of endocarp and other foreign matter.
Apricot Kernel Water
Apricot Kernel Water contains not less than 0.09
w/v% and not more than 0.11 w/v% of hydrogen
cyanide (HCN: 27.03).
Method of preparation Prepare by one of the following
methods.
(1) To Apricot Kernels, previously crushed and pressed
to remove fixed oils as much as possible, add a suitable
amount of Water or Purified Water, and carry out steam dis-
tillation. Determine the amount of hydrogen cyanide in the
distillate by the method as directed in the Assay, and carry on
the distillation until the content of hydrogen cyanide in the
distillate is about 0.14 w/v%. To the distillate add Ethanol in
about 1/3 of the volume of the distillate, and dilute with a
mixture of Purified Water and Ethanol (3:1) until the content
of hydrogen cyanide meets the specification.
1258 Areca / Crude Drugs
JP XV
(2) Dissolve 7.5 mL of freshly prepared mandelonitrile in
1000 mL of a mixture of Purified Water and Ethanol (3:1),
mix well, and filter. Determine the amount of hydrogen
cyanide in the solution as directed in the Assay, and, if the
amount is more than that specified above, dilute the solution
to the specified concentration by the addition of the mixture
of Purified Water and Ethanol (3:1).
Description Apricot Kernel Water is a clear, colorless or
pale yellow liquid. It has an odor of benzaldehyde and a
characteristic taste.
pH: 3.5-5.0
Identification To 2 mL of Apricot Kernel Water add 1 mL
of ammonia TS, and allow to stand for 10 minutes: a slight
turbidity is produced. Allow to stand for 20 minutes: the tur-
bidity is intensified.
Specific gravity <2.56> df : 0.968 - 0.978
Purity (1) Sulfate <1.14>— Add a few drops of 0.1 mol/L
sodium hydroxide VS to 5.0 mL of Apricot Kernel Water to
make slightly alkaline, evaporate on a water bath to dryness,
and ignite between 450°C and 550°C. Dissolve the residue in
1 .0 mL of dilute hydrochloric acid, and add water to make 50
mL. Perform the test using this solution as the test solution.
Prepare the control solution with 0.50 mL of 0.005 mol/L
sulfuric acid VS (not more than 0.005%).
(2) Heavy metals <1.07> — Evaporate 50 mL of Apricot
Kernel Water on a water bath to dryness, ignite between
450°C and 550°C, dissolve the residue in 5 mL of dilute acet-
ic acid with warming, add water to make exactly 50 mL, and
filter. Remove the first 10 mL of the filtrate, dilute the subse-
quent 20 mL to 50 mL with water, and perform the test using
this solution as the test solution. Prepare the control solution
as follows: to 2.0 mL of Standard Lead Solution add 2 mL of
dilute acetic acid and water to make 50 mL (not more than 1
ppm).
(3) Free hydrogen cyanide — To 10 mL of Apricot Kernel
Water add 0.8 mL of 0.1 mol/L silver nitrate VS and 2 to 3
drops of nitric acid at 15°C, filter, and add 0.1 mol/L silver
nitrate VS to the filtrate: no change occurs.
(4) Residue on evaporation — Evaporate 5.0 mL of
Apricot Kernel Water to dryness, and dry the residue at
105 °C for 1 hour: the mass of the residue is not more than 1.0
mg.
Assay Measure exactly 25 mL of Apricot Kernel Water,
add 100 mL of water, 2 mL of potassium iodide TS and 1 mL
of ammonia TS, and titrate <2.50> with 0.1 mol/L silver ni-
trate VS until a yellow turbidity persists.
Each mL of 0.1 mol/L silver nitrate VS
= 5.405 mg of HCN
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Areca
Arecae Semen
Areca is the seed of Areca catechu Linne (Palmae).
Description Rounded-conical or flattened nearly spherical
seed 1.5 - 3.5 cm high and 1.5-3 cm in diameter; hilum at
the center of its base and usually forming a dent; externally
grayish red-brown to grayish yellow-brown, with a network
of pale lines; hard in texture; cross section dense in texture,
exhibiting a marbly appearance of grayish brown seed coat
alternating with white albumen; center of the seed often hol-
low.
Odor, slight; taste, astringent and slightly bitter.
Identification Weigh 3 g of pulverized Areca in a glass-stop-
pered centrifuge tube, and add 30 mL of diethyl ether and 5
mL of sodium hydroxide TS, stopper tightly, shake for 5
minutes, centrifuge, and separate the diethyl ether layer.
Evaporate the diethyl ether on a water bath, dissolve the
residue in 1.5 mL of methanol, filter, and use the filtrate as
the sample solution. Separately, dissolve 5 mg of arecoline
hydrobromide for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer chromatography <2.03>. Spot 5 /xL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of acetone, water and acetic acid (100) (10:6:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly iodine TS
on the plate: one spot among the spots from the sample solu-
tion and a red-brown spot from the standard solution show
the same color tone and the same Rf value.
Purity (1) Pericarp — The amount of pericarp contained in
Areca is not more than 2.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the pericarp contained in Areca does not exceed
1.0%.
Total ash <J.07> Not more than 2.5%.
Artemisia Capillaris Flower
Artemisiae Capillaris Flos
Artemisia Capillaris Flower is the capitulum of
Artemisia capillaris Thunberg (Compositae).
Description Capitulum of ovoid to spherical, capitula,
about 1.5 -2 mm in length, about 2 mm in diameter, with
linear leaves, peduncles, and thin stem. Outer surface of
capitulum, light green to light yellowish brown in color;
peduncle, greenish brown to dark brown in color. Under a
magnifying glasses, the capitulum; involucral scale, in 3 - 4
succubous rows, outer scale of ovate with obtuse, inner scale
of elliptical, 1.5 mm in length, longer than outer one, with
keel midrib and thin membranous margin. Floret; tubular,
marginal flower of female, disk flower of hermaphrodite.
Achene of obovoid, 0.8 mm in length. Light in texture.
Odor, characteristic, slight; taste, slightly acrid, which
gives slightly numbing sensation to the tongue.
Identification To 0.5 g of pulverized Artemisia Capillaris
Flower add 10 mL of methanol, shake for 3 minutes, filter,
and use the filtrate as the sample solution. Perform the test
JP XV
Crude Drugs / Asparagus Tuber 1259
with the sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 5 [iL of the sample solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of acetone and n-hexane (1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultraviolet light (main wavelength: 365 nm): a principal spot
with a blue fluorescence appears at the Rf value of about 0.5.
Purity Stem — Artemisia Capillaris Flower does not contain
any stem less than 2 mm in diameter.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 9.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 15.0%.
Asiasarum Root
Asiasari Radix
1H'»
Asiasarum Root is the rhizome and root of Asiasa-
rum sieboldii F. Maekawa or Asiasarum hetero-
tropoides F. Maekawa var. mandshuricum F. Maeka-
wa (Aristolochiaceae).
Description Asiasarum Root is a nearly cylindrical rhizome
with numerous thin and long roots, externally light brown to
dark brown. The root, about 15 cm in length, about 0.1 cm in
diameter, with shallow longitudinal wrinkles on the surface,
and brittle. The rhizome, 2-4 cm in length, 0.2 - 0.3 cm in
diameter, often branched, with longitudinal wrinkles on the
surface; internode short; each node has several scars of peti-
ole and peduncle, and several thin and long roots.
Odor, characteristic; taste, acrid, with some sensation of
numbness on the tongue.
Purity (1) Terrestrial part — Any terrestrial parts are not
found.
(2) Foreign matter <5.01> — The amount of foreign matter
other than terrestrial part contained in Asiasarum Root does
not exceed 1.0%.
(3) Aristolochic acid I — To exactly 2.0 g of pulverized
Asiasarum Root add exactly 50 mL of diluted methanol (3 in
4), shake for 15 minutes, filter, and use the filtrate as the sam-
ple solution. Separately, dissolve exactly 1.0 mg of
aristolochic acid I for crude drugs purity test in diluted
methanol (3 in 4) to make exactly 100 mL. Pipet 1 mL of this
solution, add diluted methanol (3 in 4) to make exactly 25
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /iL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01>, according to the following conditions: the sample so-
lution shows no peak at the retention time corresponding to
aristolochic acid I from the standard solution. If the sample
solution shows such a peak, repeat the test under different
conditions to confirm that the peak in question is not
aristolochic acid I.
Operating conditions —
Detector: An ultraviolet or visible absorption photometer
(wavelength: 400 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of a solution prepared by dissolv-
ing 7.8 g of sodium dihydrogen phosphate dihydrate and 2
mL of phosphoric acid in water to make 1000 mL and
acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
aristolochic acid I is about 15 minutes.
System suitability —
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add diluted methanol (3 in 4) to
make exactly 10 mL. Confirm that the ratio, S/N, of the sig-
nal (S) and noise (N) of aristolochic acid I obtained from 20
fiL of this solution is not less than 3. In this case, S means the
peak height on the chromatogram not including noise ob-
tained by drawing an average line of the detector output, and
N is 1/2 of the difference between the maximum and mini-
mum output signals of the baseline around the peak in the
range of 20 times the width at half-height of the peak.
System repeatability: When the test is repeated 6 times with
20 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
aristolochic acid I is not more than 5.0%.
(4) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Total ash <J.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Essential oil content <5.01> Perform the test with 30.0 g of
pulverized Asiasarum Root: the volume of essential oil is not
less than 0.6 mL.
Asparagus Tuber
Asparagi Tuber
Asparagus Tuber is the tuber of Asparagus cochin-
chinensis Merrill (Liliaceae), from which most of the
cork layer is removed, usually, after being steamed.
Description Asparagus Tuber is a fusiform to cylindrical
tuber, 5 to 15 cm in length, 5 to 20 mm in diameter; external-
ly light yellow-brown to light brown, translucent and often
with longitudinal wrinkles; flexible, or hard and easily broken
in texture; fractured surface, grayish yellow, glossy and
horny.
Odor, characteristic; taste, sweet at first, followed by a
slightly bitter aftertaste.
Under a microscope <5.01>, a transverse section of Aspara-
gus Tuber reveals stone cells and bundles of them on outer
layer of cortex; mucilaginous cells containing raphides of cal-
cium oxalate in the parenchyma cells of cortex and central
cylinder; no starch grains.
Identification To 1 g of coarsely cut Asparagus Tuber add 5
1260 Astragalus Root / Crude Drugs
JP XV
mL of a mixture of 1-butanol and water (40:7), shake for 30
minutes, filter, and use the filtrate as the sample solution.
Perform the test with the sample solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xL of the sample
solution on a plate of silica gel for thin-layer chro-
matography, develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (10:6:3) to a distance of about 10
cm, and air-dry the plate. Spray evenly dilute sulfuric acid on
the plate, and heat at 105°C for 2 minutes: the spot of a red-
brown at first then changes to brown color appears at around
Rf 0.4.
Loss on drying <5.0I> Not more than 18.0% (6 hours).
Total ash <J.07> Not more than 3.0%.
Astragalus Root
Astragali Radix
Astragalus Root is the root of Astragalus mem-
branaceus Bunge, Astragalus mongholicus Bunge
(Leguminosae).
Description Nearly cylindrical root, 30 - 100 cm in length,
0.7 - 2 cm in diameter, with small bases of lateral root dis-
persed on the surface, twisted near the crown; externally light
grayish yellow to light yellow-brown, and covered with
irregular, dispersed longitudinal wrinkles and horizontal
lenticel-like patterns; difficult to break; fractured surface
fibrous. Under a magnifying glass, a transverse section rev-
eals an outer layer composed of periderm; cortex light yel-
lowish white, xylem light yellow, and zone near the cambium
somewhat brown in color; thickness of cortex from about
one-third to one-half of the diameter of xylem; white
medullary ray from xylem to cortex in thin root, but often
appearing as radiating cracks in thick root; usually pith un-
observable.
Odor, slight; taste, sweet.
Purity (1) Root of Hedysarum species and others — Under
a microscope <5.01>, a vertical section of Astragalus Root
reveals no crystal fiber containing solitary crystals of calcium
oxalate outside the fiber bundle.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Astragalus Root according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(3) Arsenic </.//> — Prepare the test solution with 0.40 g
of pulverized Astragalus Root according to Method 4, and
perform the test (not more than 5 ppm).
(4) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.01> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Atractylodes Lancea Rhizome
Atractylodis Lanceae Rhizoma
Atractylodes Lancea Rhizome is the rhizome of
Atractylodes lancea De Candolle or of Atractylodes
chinensis Koidzumi (Compositae).
Description Irregularly curved, cylindrical rhizome, 3-10
cm in length, 1 - 2.5 cm in diameter; externally dark grayish
brown to dark yellow-brown; a transverse section nearly
orbicular, with light brown to red-brown secretes as fine
points.
Often white cotton-like crystals produced on its surface.
Odor, characteristic; taste, slightly bitter.
Under a microscope <5.01>, a transverse section usually
reveals periderm with stone cells; parenchyma of cortex,
usually without any fiber bundle; oil sacs, containing light
brown to yellow-brown substances, located at the end region
of medullary rays; xylem exhibits vessels surrounded by fiber
bundles and arranged radially on the region adjoining the
cambium; pith and medullary rays exhibit the same oil sacs as
in the cortex; parenchyma cells contain spherocrystals of inu-
lin and fine needle crystals of calcium oxalate.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Atractylodes Lancea Rhizome according to
Method 3, and perform the test. Prepare the control solution
with 3.0 mL of Standard Lead Solution (not more than 10
ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Atractylodes Lancea Rhizome according to
Method 4, and perform the test (not more than 5 ppm).
(3) Atractylodes rhizome — Macerate 0.5 g of pulverized
Atractylodes Lancea Rhizome with 5 mL of ethanol (95) by
warming in a water bath for 2 minutes, and filter. To 2 mL of
the filtrate add 0.5 mL of vanillin-hydrochloric acid TS, and
shake immediately: no red to red-purple color develops
within 1 minute.
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Atractylodes Lancea Rhizome: the volume of
essential oil is not less than 0.7 mL.
Powdered Atractylodes Lancea
Rhizome
Atractylodis Lanceae Rhizoma Pulveratum
Powdered Atractylodes Lancea Rhizome is the
powder of Atractylodes Lancea Rhizome.
Description Powdered Atractylodes Lancea Rhizome oc-
curs as a yellow-brown powder. It has a characteristic odor,
JPXV
Crude Drugs / Powdered Atractylodes Rhizome 1261
and a slightly bitter taste.
Under a microscope <5.01>, Powdered Atractylodes Lan-
cea Rhizome reveals mainly parenchyma cells, spherocrystals
of inulin, fragments of parenchyma cells containing fine nee-
dle crystals of calcium oxalate as their contents; and further
fragments of light yellow thick-walled fibers, stone cells and
cork cells; a few fragments of reticulate and scalariform ves-
sels, and small yellow-brown secreted masses or oil drops;
starch grains absent.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Atractylodes Lancea Rhizome according to
Method 3, and perform the test. Prepare the control solution
with 3.0 mL of Standard Lead Solution (not more than 10
ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Atractylodes Lancea Rhizome according to
Method 4, and perform the test (not more than 5 ppm).
(3) Powdered atractylodes rhizome — To 0.5 g of Pow-
dered Atractylodes Lancea Rhizome add 5 mL of ethanol
(95), macerate by warming in a water bath for 2 minutes, and
filter. To 2 mL of the filtrate add 0.5 mL of vanillin-
hydrochloric acid TS, and shake immediately: no red to red-
purple color develops within 1 minute.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Atractylodes Lancea Rhizome: the volume of
essential oil is not less than 0.5 mL.
Containers and storage Containers — Tight containers.
Atractylodes Rhizome
Atractylodis Rhizoma
Atractylodes Rhizome is the rhizome of Atrac-
tylodes japonica Koidzumi ex Kitamura (Wa-
byakujutsu), or is the rhizome of Atractylodes ovata
De Candolle (Kara-byakujutsu) (Compositae).
Description (1) Wa-byakujutsu — Periderm-removed rhi-
zome is irregular masses or irregularly curved cylinder, 3-8
cm in length, 2-3 cm in diameter; externally light grayish
yellow to light yellowish white, with scattered grayish brown
parts. The rhizome covered with periderm is externally
grayish brown, often with node-like protuberances and
coarse wrinkles. Difficult to break, and the fractured surface
is fibrous. A transverse section, with fine dots of light yellow-
brown to brown secrete.
Odor, characteristic; taste, somewhat bitter.
Under a microscope <5.01>, a transverse section reveals
periderm with stone cell layers; fiber bundles in the parenchy-
ma of the cortex, often adjoined to the outside of the
phloem; oil sacs containing light brown to brown substances,
situated at the outer end of medullary rays; in the xylem,
radially lined vessels, surrounding large pith, and distinct
fiber bundle surrounding the vessels; in pith and in medullary
rays, oil sacs similar to those in cortex, and in parenchyma,
crystals of inulin and small needle crystals of calcium oxalate.
(2) Kara-byakujutsu — Irregularly enlarged mass, 4-8
cm in length, 2-5 cm in diameter; externally grayish yellow
to dark brown, having sporadic, knob-like small protrusions.
Difficult to break; fractured surface has a light brown to dark
brown xylem remarkably fibrous.
Odor, characteristic; taste, somewhat sweet, but followed
by slight bitterness.
Under a microscope <5.01>, a transverse section usually
reveals periderm with stone cells, absence of fibers in the cor-
tex; oil sacs containing yellow-brown contents in phloem ray
and also at the outer end of it; xylem with radially lined ves-
sels surrounding large pith, and distinct fiber bundle sur-
rounding the vessels; pith and medullary ray exhibit oil sacs
as in cortex; parenchyma contains crystals of inulin and small
needle crystals of calcium oxalate.
Identification Macerate 0.5 g of pulverized Atractylodes
Rhizome with 5 mL of ethanol (95) by warming in a water
bath for 2 minutes, and filter. To 2 mL of the filtrate add 0.5
mL of vanillin-hydrochloric acid TS, and shake immediately:
a red to red-purple color develops and persists.
Purity Atractylodes lancea rhizome — To 2.0 g of pulver-
ized Atractylodes Rhizome add exactly 5 mL of hexane,
shake for 5 minutes, filter, and use this filtrate as the sample
solution. Perform the test with this solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL of the solu-
tion on a plate of silica gel for thin-layer chromatography.
Develop the plate with a mixture of hexane and acetone (7:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly 4-dimethylaminobenzaldehyde TS for spraying on the
plate, and heat at 100°C for 5 minutes: no green to grayish
green spot appears between Rf 0.3 and 0.6.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Atractylodes Rhizome: the volume of essential oil
is not less than 0.5 mL.
Powdered Atractylodes Rhizome
Atractylodis Rhizoma Pulveratum
Powdered Atractylodes Rhizome is the powder of
Atractylodes Rhizome.
Description Powdered Atractylodes Rhizome occurs as a
light brown to yellow-brown powder, and has a characteristic
odor and a slightly bitter or slightly sweet taste, followed by a
slightly bitter aftertaste.
Under a microscope <5.01>, Powdered Atractylodes Rhi-
zome reveals mainly parenchyma cells, crystals of inulin and
fragments of parenchyma cells containing small needle crys-
tals of calcium oxalate; fragments of light yellow thick-
walled fibers, stone cells and cork cells; a few fragments of
reticulate and scalariform vessels; small yellow-brown secrete
masses or oil droplets; starch grains absent.
1262 Bear Bile / Crude Drugs
JP XV
Identification Macerate 0.5 g of Powdered Atractylodes
Rhizome with 5 mL of ethanol (95) by warming in a water
bath for 2 minutes, and filter. To 2 mL of the filtrate add 0.5
mL of vanillin-hydrochloric acid TS, and shake immediately:
a red to red-purple color develops and persists.
Purity Atractylodes lancea rhizome — To 2.0 g of Powdered
Atractylodes Rhizome add exactly 5 mL of hexane, shake for
5 minutes, filter, and use this filtrate as the sample solution.
Perform the test with the sample solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL of the solu-
tion on a plate of silica gel for thin-layer chromatography.
Develop the plate with a mixture of hexane and acetone (7:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly 4-dimethylaminobenzaldehyde TS for spraying on the
plate, and heat at 100°C for 5 minutes: no green to grayish
green spot appears at the Rf value of between 0.3 and 0.6.
Total ash <5.01> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Atractylodes Rhizome: the volume of essential oil
is not less than 0.4 mL.
Containers and storage <5.01> Containers — Tight contain-
ers.
Bear Bile
Fel Ursi
Bear Bile is the dried bile of Ursus arctos Linne or
allied animals (Ursidae).
Description Indefinite small masses; externally yellow-
brown to dark yellow-brown; easily broken; fractured sur-
face has a glassy luster, and is not wet; usually in a gall sac,
occasionally taken out, the gall sac consists of a fibrous and
strong membrane, 9-15 cm in length and 7-9 cm in width;
externally dark brown and translucent.
Odor, slight and characteristic; taste, extremely bitter.
Identification Warm 0.3 g of pulverized Bear Bile with 50
mL of petroleum ether under a reflux condencer on a water
bath for about 1 hour, and filter. To 20 mg of the residue add
0.5 mL of hydrochloric acid, 2 mL of acetic anhydride and 2
mL of chloroform, shake the mixture vigorously for 2
minutes, and filter. To the filtrate add carefully 0.5 mL of sul-
furic acid: a red color develops at the zone of contact, then
changes to reddish brown, and the upper layer acquires a
somewhat red color. Shake gently to mix the two layers
together, and allow to stand: a persistent reddish brown color
is produced.
Bearberry Leaf
Uvae Ursi Folium
Bearberry Leaf is the leaf of Arctostaphylos uva-ursi
(Linne) Sprengel (Ericaceae).
It contains not less than 7.0% of arbutin.
Description Obovate to spatulate leaves, 1 - 3 cm in length,
0.5 -1.5 cm in width; upper surface yellow-green to dark
green; lower surface light yellow-green; margin entire; apex
obtuse or round, sometimes retuse; base cuneate; petiole very
short; lamina thick with characteristic reticular vein, and
easily broken.
Odor, slight; taste, slightly bitter and astringent.
Under a microscope <5.01>, the transverse section reveals
thick cuticula; parenchyma cells of palisade tissue and sponge
tissue being similar in form; in the vascular bundle, medulla-
ry ray consisting of 2 to 7 rows of one-cell line, appearing as
bones of Japanese fan; polygonal solitary crystals and
clustered crystals of calcium oxalate present sparsely in cells
on both outer and inner sides of the vascular bundle, but no
crystals in mesophyll.
Identification (1) Macerate 0.5 g of pulverized Bearberry
Leaf with 10 mL of boiling water, shake the mixture for a few
minutes, allow to cool, and filter. Place 1 drop of the filtrate
on filter paper, and add 1 drop of iron (III) chloride TS: a
dark purple color appears.
(2) To 0.2 g of pulverized Bearberry Leaf add 10 mL of a
mixture of ethanol (95) and water (7:3), shake for 5 minutes,
filter, and use the filtrate as the sample solution. Separately,
dissolve 1 mg of arbutin for thin-layer chromatography in 1
mL of a mixture of ethanol (95) and water (7:3), and use this
solution as the standard solution. Perform the test with the
sample solution and standard solution as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL each of these
solutions on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl for-
mate, formic acid and water (8:1:1) to a distance of about 15
cm, and air-dry the plate. Spray evenly diluted sulfuric acid
(1 in 2) upon the plate, and heat at 105°C for 10 minutes: one
spot among several spots from the sample solution and that
from the standard solution show a yellow-brown to blackish
brown color and the same Ri value.
Purity (1) Twig — The amount of twigs contained in
Bearberry Leaf does not exceed 4.5%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than twigs contained in Bearberry Leaf does not exceed
2.0%.
Total ash <5.07> Not more than 4.0%.
Acid-insoluble ash <5.0]> Not more than 1.5%.
Component determination Weigh accurately about 0.5 g of
pulverized Bearberry Leaf in a glass-stoppered centrifuge
tube, add 40 mL of water, shake for 30 minutes, centrifuge,
and separate the supernatant liquid. To the residue add
40 mL of water, and proceed in the same manner. To the
JP XV
Crude Drugs / Belladonna Root 1263
combined extracts add water to make exactly 100 mL, and
use this solution as the sample solution. Separately, weigh
accurately about 40 mg of arbutin for component determina-
tion, previously dried for 12 hours (in vacuum, silica gel), dis-
solve in water to make exactly 100 mL, and use this solution
as the standard solution. Perform the test with exactly lO/iL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions. Determine the peak areas, A T and A s , of
arbutin in each solution.
Amount (mg) of arbutin = W s x (A T /A S )
W s : Amount (mg) of arbutin for component determination
Operating conditions —
Detector: An ultraviolet spectrophotometer (wavelength:
280 nm).
Column: A stainless steel column 4-6 mm in inside
diameter and 15 -25 cm in length, packed with octadecyl-
silanized silica gel (5 - 10 /xm in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, methanol and 0.1 mol/
L hydrochloric acid TS (94:5:1).
Flow rate: Adjust the flow rate so that the retention time of
arbutin is about 6 minutes.
Selection of column: Dissolve 0.05 g each of arbutin for
component determination, hydroquinone and gallic acid in
water to make 100 mL. Proceed with 10 fiL of this solution
under the above operating conditions, and calcutate the
resolution. Use a column giving elution of arbutin, hydroqui-
none and gallic acid in this order, and clearly dividing each
peak.
System repeatability: Repeat the test five times with the
standard solution under the above operating conditions: the
relative standard deviation of the peak area of arbutin is not
more than 1.5%.
Belladonna Extract
Extractum Belladonnae
Belladonna Extract contains not less than 0.85% and
not more than 1.05 % of hyoscyamine
(C l7 H 23 N0 3 : 289.37).
Method of preparation To 1000 g of a coarse powder of
Belladonna Root add 4000 mL of 35 vol% ethanol, and
digest for 3 days. Press the mixture, add 2000 mL of 35 vol%
ethanol to the residue, and digest again for 2 days. Combine
all the extracts, and allow to stand for 2 days. Filter, and
prepare the viscous extract as directed under Extracts. May
be prepared with an appropriate quantity of Ethanol and
Purified Water.
Description Belladonna Extract has a dark brown color, a
characteristic odor and a bitter taste.
Identification Mix 0.5 g of Belladonna Extract with 30 mL
of ammonia TS in a flask, transfer the mixture to a separator,
then add 40 mL of ethyl acetate, and shake the mixture.
Drain off the ethyl acetate layer, add 3 g of anhydrous sodi-
um sulfate to the ethyl acetate, shake, and filter after the
ethyl acetate becomes clear. Evaporate the filtrate to dryness
under reduced pressure, dissolve the residue in 1 mL of
ethanol (95), and use this solution as the sample solution.
Proceed as directed in the Identification under Belladonna
Root.
Assay Weigh accurately about 0.4 g of Belladonna Extract,
place in a glass-stoppered centrifuge tube, add 15 mL of am-
monia TS, and shake. Add 25 mL of diethyl ether, stopper
tightly, shake for 15 minutes, centrifuge, and separate the
diethyl ether layer. Repeat this procedure twice with the
water layer, using 25 mL each of diethyl ether. Combine the
extracts, and evaporate the diethyl ether on a water bath. Dis-
solve the residue in 5 mL of the mobile phase, add exactly 3
mL of the internal standard solution, and add the mobile
phase to make exactly 25 mL. Proceed as directed under
Belladonna Root.
Amount (mg) of hyoscyamine (Q7H23NO3)
= W s x (Q T /Qs) x (1/5) x 0.8551
W s : Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
Internal standard solution — A solution of brucine dihydrate
in the mobile phase (1 in 2500).
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Belladonna Root
Belladonnae Radix
Belladonna Root is the root of Atropa belladonna
Linne (Solanaceae).
Belladonna Root, when dried, contains not less than
0.4% of hyoscyamine (C n H 23 N0 3 : 289.37).
Description Cylindrical root, usually 10 - 30 cm in length,
0.5 - 4 cm in diameter; often cut crosswise or lengthwise;
externally grayish brown to grayish yellow-brown, with lon-
gitudinal wrinkles; periderm often removed; fractured sur-
face is light yellow to light yellow-brown in color and is
powdery.
Almost odorless; taste, bitter.
Identification Place 2.0 g of pulverized Belladonna Root in
a glass-stoppered centrifuge tube, add 30 mL of ammonia
TS, and centrifuge after irradiation of ultrasonic waves for 5
minutes. Transfer the supernatant liquid to a separator, add
40 mL of ethyl acetate, and shake. Drain off the ethyl acetate
layer, add 3 g of anhydrous sodium sulfate to the ethyl
acetate, shake, and filter after the ethyl acetate becomes
clear. Evaporate the filtrate to dryness under reduced pres-
sure, dissolve the residue in 1 mL of ethanol (95), and use this
solution as the sample solution. Separately, dissolve 2 mg of
Atropine Sulfate Reference Standard in 1 mL of ethanol (95),
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
1264 Benincasa Seed / Crude Drugs
JP XV
and standard solutions on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ace-
tone, water and ammonia water (28) (90:7:3) to a distance of
about 10 cm, and dry the plate at 80°C for 10 minutes. After
cooling, spray evenly Dragendorff's TS for spraying on the
plate: the principal spot from the sample solution is the same
in color tone and Ri value with a yellow-red spot from the
standard solution.
Purity (1) Stem and crown — The amount of stems and
crowns contained in Belladonna Root does not exceed
10.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than stems and crowns contained in Belladonna Root
does not exceed 2.0%.
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.0I> Not more than 4.0%.
Assay Weigh accurately about 0.7 g of pulverized Belladon-
na Root, previously dried at 60°C for 8 hours, place in a
glass-stoppered centrifuge tube, and moisten with 15 mL of
ammonia TS. To this add 25 mL of diethyl ether, stopper the
centrifuge tube tightly, shake for 15 minutes, centrifuge, and
separate the diethyl ether layer. Repeat this procedure twice
with the residue using 25-mL portions of diethyl ether. Com-
bine all the extracts, and evaporate the diethyl ether on a
water bath. Dissolve the residue in 5 mL of the mobile phase,
add exactly 3 mL of the internal standard solution, and add
the mobile phase to make exactly 25 mL. Filter this solution
through a filter of a porosity of not more than 0.8 fim, dis-
card the first 2 mL of the filtrate, and use the subsequent
filtrate as the sample solution. Separately, weigh accurately
about 25 mg of Atropine Sulfate Reference Standard (pre-
viosly determine the loss on drying <2.41> in the same manner
as Atropine Sulfate Hydrate), dissolve in the mobile phase to
make exactly 25 mL, and use this solution as the standard
stock solution. Pipet 5 mL of the standard stock solution,
add exactly 3 mL of the internal standard solution, then add
25 mL of the mobile phase, and use this solution as the stan-
dard solution. Perform the test with 10 /xL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine the ratios, Q T and Q s , of the peak area of
hyoscyamine (atropine), to that of the internal standard in
each solution.
Amount (mg) of hyoscyamine (Q7H23NO3)
= W s x (Qj/Qs) x (1/5) x 0.8551
WS: Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
Internal standard solution — A solution of brucine dihydrate
in the mobile phase (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption spectrometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 jum in parti-
cle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen
phosphate in 900 mL of water, add 10 mL of triethylamine,
adjust with phosphoric acid to pH 3.5, and add water to
make 1000 mL, and mix this solution with acetonitrile (9:1).
Flow rate: Adjust the flow rate so that the retention time of
atropine is about 14 minutes.
Selection of column: Proceed with 10 /uL of the standard
solution under the above operating conditions, and deter-
mine the resolution. Use a column giving elution of atropine
and the internal standard in this order with the resolution
between these peaks being not less than 4.
Benincasa Seed
Benincasae Semen
Benincasa seed is the seed of Benincasa cerifera Savi
(1) or Benincasa cerifera Savi forma emarginata K.
Kimura et Sugiyama (2) (Cucurbitaceae).
Description (1) Flattened, ovate to orbicular- ovate seed,
10 - 13 mm in length, 6-7 mm in width, about 2 mm in
thickness; slightly acute at base; hilum and germ pore form
two protrusions; externally light grayish yellow to light yel-
lowish brown; prominent band along with marginal edge of
seed; under a magnifying glass, surface of the seed is with fine
wrinkles and minute hollows.
(2) Flattened, ovate to ellipsoidal seed, 9-12 mm in
length, 5-6 mm in width, about 2 mm in thickness; hilum
and germ pore form two protrusions as in (1); externally light
grayish yellow, smooth, no prominent band along with mar-
ginal edge of seed.
Both (1) and (2) odorless; bland taste and slightly oily.
Under a microscope <5.01>, a transverse section of (1) rev-
eals the outermost layer of seed coat composed of a single-
layered and palisade like epidermis, the epidermis obvious at
prominent band along with marginal edge of seed; a trans-
verse section of (2) reveals the outermost layer composed of a
single-layered epidermis coated with cuticle, often detached;
hypodermis of (1) and (2) composed of slightly sclerified
parenchyma beneath epidermis; inside of the parenchyma
several layers of stone cells lie; the innermost layer of seed
coat composed of parenchyma several cells thick; perisperm
coated with cuticle, composed of parenchyma several cells
thick; endosperm composed of a row of compressed cells;
cotyledon contains oil drops and aleurone grains, occasional-
ly starch grains.
Identification To about 0.5 g of pulverized Benincasa Seed
add 10 mL of a mixture of methanol and water (4:1), shake
for 10 minutes, filter, and use the filtrate as the sample solu-
tion. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 20,mL of the
sample solution on a plate of silica gel for thin-layer chro-
matography, develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (8:6:3) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): two blue-white spots appear
around Ri 0.4, and the spot having the smaller Ri value
shows more intense fluoresence.
Purity Foreign matter <5.01> — It contains not more than
JPXV
Crude Drugs / Bitter Orange Peel 1265
2.0%.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <5.01> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 3.0%.
brown to dark brown in color, and hard in texture.
Odor, characteristic; taste, slightly bitter.
Total ash <J.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Bitter Cardamon: the volume of essential oil is not
less than 0.4 mL.
Benzoin
Benzoinum
Benzoin is the resin obtained from Styrax benzoin
Dryander or other species of the same genus
(Styracaceae).
Description Benzoin occurs as grayish brown to dark red-
brown blocks varying in size; the fractured surface exhibiting
whitish to light yellow-red grains in the matrix; hard and brit-
tle at ordinary temperature but softened by heat.
Odor, characteristic and aromatic; taste, slightly pungent
and acrid.
Identification (1) Heat a fragment of Benzoin in a test
tube: it evolves an irritating vapor, and a crystalline subli-
mate is produced.
(2) Digest 0.5 g of Benzoin with 10 mL of diethyl ether,
decant 1 mL of the diethyl ether into a porcelain dish, and
add 2 to 3 drops of sulfuric acid: a deep red-brown to deep
red-purple color develops.
Purity Ethanol-insoluble substances — Boil gently 1.0 g of
Benzoin with 30 mL of ethanol (95) on a water bath for 15
minutes under a reflux condenser. After cooling, collect the
insoluble substances through a tared glass filter (G3), and
wash with three 5-mL portions of ethanol (95). Dry the
residue at 105°C for 4 hours: the mass of the residue does not
exceed 0.30 g.
Total ash <J.07> Not more than 2.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Bitter Cardamon
Alpiniae Fructus
Bitter Cardamon is the fruit of Alpinia oxyphylla
Miquer (Zingiberaceae).
Description Spherical to fusiform fruit, with both ends
somewhat pointed; 1-2 cm in length, 0.7 - 1 cm in width;
externally brown to dark brown, with numerous longitudi-
nal, knob-like protruding lines; pericarp 0.3 - 0.5 mm in
thickness, closely adhering to the seed mass, and difficult to
separate; inside divided vertically into three loculi by thin
membranes, each loculus containing 5 to 8 seeds adhering by
aril; seeds irregularly polygonal, about 3.5 mm in diameter,
Bitter Orange Peel
Aurantii Pericarpium
tot
Bitter Orange Peel is the pericarp of the ripe fruit of
Citrus aurantium Linne or Citrus aurantium Linne var.
daidai Makino (Rutaceae).
Description Usually quartered sections of a sphere, some-
times warped or flattened, 4-8 cm in length, 2.5 - 4.5 cm in
width and 0.5 - 0.8 cm in thickness; the outer surface is dark
red-brown to grayish yellow-brown, with numerous small
dents associated with oil sacs; the inner surface is white to
light grayish yellow-red, with irregular indented reticulation
left by vascular bundles; light and brittle in texture.
Odor, characteristic aroma; taste, bitter, somewhat
mucilaginous and slightly pungent.
Identification To 1.0 g of Bitter Orange Peel add 10 mL of
ethanol (95), allow to stand for 30 minutes with occasional
shaking, filter, and use the filtrate as the sample solution.
Separately, dissolve 10 mg of naringin for thin-layer chro-
matography in 10 mL of ethanol (95), and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate, ethanol (99.5) and water
(8:2:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly dilute 2,6-dibromo-/V-chloro-l,4-benzoquinone
monoimine TS on the plate, and allow to stand in ammonia
gas: a spot from the sample solution and a grayish green spot
from the standard solution show the same color tone and the
same Rf value.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.07> Not more than 5.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Essential oil content <5.01> Perform the test with 50 g of
pulverized Bitter Orange Peel provided that 1 mL of silicon
resin is previously added to the test sample in the flask: the
volume of essential oil is not less than 0.2 mL.
1266 Bitter Tincture / Crude Drugs
JP XV
Bitter Tincture
Tinctura Amara
Bupleurum Root
Bupleuri Radix
+M=i
Method of preparation
Bitter Orange Peel, in coarse
powder
50 g
Swertia Herb, in coarse powder
5g
Zanthoxylum Fruit, in coarse
powder
5g
70 vol% Ethanol
a sufficient quantity
To make 1000 mL
Prepare as directed under Tinctures, with the above in-
gredients. An appropriate quantity of Ethanol and Purified
Water may be used in place of 70 vol% Ethanol.
Description Bitter Tincture is a yellow-brown liquid. It has
a characteristic aroma and a bitter taste.
Specific gravity df : about 0.90
Identification (1) To 1 mL of Bitter Tincture add 5 mL of
methanol, then add 0.1 g of magnesium in ribbon form and 1
mL of hydrochloric acid, and allow to stand: the solution is
red-purple in color.
(2) Use Bitter Tincture as the sample solution. Separate-
ly, to 5.0 g of pulverized Bitter Orange Peel add 100 mL of
diluted ethanol (7 in 10), stopper the vessel tightly, shake for
30 minutes, filter, and use the filtrate as the standard solution
(1). Proceed with 0.5 g each of pulverized Swertia Herb and
Zanthoxylum Fruit in the same manner, and use the solutions
so obtained as the standard solution (2) and the standard so-
lution (3). Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 10 /xL each
of the sample solution and standard solutions (1), (2) and (3)
on the plate of silica gel with complex fluorescent indicator
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate, ethanol (95) and water (8:2:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine the
plate under ultraviolet light (broad spectrum wavelength):
three of the several spots from the sample solution show the
same color tone and Rf value as those of the upper spot of the
two bright blue to purple spots among the several spots from
the standard solution (1), appearing close to each other at an
Rf value of about 0.4, and a bright red spot from the stan-
dard solution (2), appearing at an Rf value of about 0.35, and
a bright grayish red to red spot from the standard solution
(3), appearing at an Rf value of about 0.7.
Alcohol number <7.07> Not less than 6.9 (Method 2).
Containers and storage Containers — Tight containers.
Bupleurum Root is the root of Bupleurum falcatum
Linne (Umbelliferae).
It contains not less than 0.35% of the total saponin
(saikosaponin a and saikosaponin d), calculatd on the
basis of dried material.
Description Single or branched root of long cone or column
shape, 10 - 20 cm in length, 0.5 -1.5 cm in diameter; oc-
casionally with remains of stem on the crown; externally light
brown to brown and sometimes with deep wrinkles; easily
broken, and fractured surface somewhat fibrous.
Odor, characteristic, and taste, slightly bitter.
Under a microscope <5.01>, a transverse section reveals the
thickness of cortex reaching 1/3 ~ 1/2 of the radius, tangen-
tially extended clefts in cortex; and cortex scattered with a
good many intercellular schizogenous oil canals 15 - 35 //m in
diameter; in xylem, vessels lined radially or stepwise, and
fiber groups scattered; in the pith at the crown, the same oil
canals as in the cortex; parenchyma cells containing starch
grains and oil droplets. Starch grains composed of simple
grains, 2 - 10 /xm in diameter, or compound grains.
Identification (1) Shake vigorously 0.5 g of pulverized
Bupleurum Root with 10 mL of water: lasting fine foams are
produced.
(2) To 2.0 g of pulverized Bupleurum Root add 10 mL of
methanol, boil gently under a reflux condenser on a water
bath for 15 minutes, cool, filter, and use the filtrate as the
sample solution. Separately, dissolve 1 mg of saikosaponin a
for thin-layer chromatography in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chlo-
roform, methanol and water (30:10:1) to a distance of about
10 cm, and air-dry the plate. Spray evenly a mixture of sul-
furic acid and ethanol (95) (1:1) on the plate, and warm at 50
°C for 5 minutes: one spot among the several spots from the
sample solution and the blue spot from the standard solution
show the same Rf value, and the color tone is blue to blue-
purple.
Purity (1) Stem and leaf — The amount of the stems and
leaves contained in Bupleurum Root does not exceed 10.0%.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Bupleurum Root according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than lOppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Bupleurum Root according to Method 4, and
perform the test (not more than 5 ppm).
(4) Foreign matter <5.01> — The amount of foreign matter
other than stems and leaves contained in Bupleurum Root
does not exceed 1.0%.
Loss on drying <5.01> Not more than 12.5% (6 hours).
JPXV
Crude Drugs / Calumba 1267
Component determination Weigh accurately about 1 g of
pulverized Bupleurum Root, transfer in a glass-stoppered
centrifuge tube, add 20 mL of diluted methanol (9 in 10),
shake for 15 minutes, centrifuge, and separate the super-
natant liquid. Perform the same procedure with the
precipitate using two 15-mL potions of diluted methanol (9 in
10), combine whole supernatant liquids, and add diluted
methanol (9 in 10) to make exactly 50 mL. Pipet 5 mL of this
solution, add 2.5 mL of dilute sodium hydroxide TS, heat in
a water bath at 50°C for 1 hour, and add 7.5 mL of phos-
phate buffer solution for component determination of
bupleurum root. Allow this solution to flow through a chro-
matographic column [about 10 mm inside diameter contain-
ing 0.36 g of octadecylsilanized silica gel for pretreatment (55
to 105 //m in particle diameter), conditioned with 10 mL of
methanol then 10 mL of water just before use]. Wash the
column with 10 mL of diluted methanol (7 in 20), then flow
with methanol to get exactly 10 mL of effluent solution, and
use this as the sample solution. Separately, weigh accurately
each about 10 mg of saikosaponin a for component determi-
nation and saikosaponin d for component determination,
previously dried in a desiccator (silica gel) for 24 hours, dis-
solve in methanol to make exactly 200 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 20
/nL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A TA
and A SA , of saikosaponin a and A TD and A SD , of saikosapo-
nin d. Calculate the amount of saikosaponin a and sai-
kosaponin d by the following equation.
Amount (mg) of saikosaponin a = W S \ x G4ta/^4sa) x (1/2)
W SAl : Amount (mg) of saikosaponin a for component de-
termination
Amount (mg) of saikosaponin d= W SD x ^4 T d/^4sd) x (1/2)
W SD : Amount (mg) of saikosaponin d for component de-
termination
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 206 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel (5 /xm in particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of water and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
saikosaponin a is about 8 minutes.
System suitability —
System performance: When the procedure is run with 20
fiL of the standard solution under the above operating condi-
tions, saikosaponin a and saikosaponin d are eluted in this
order, and the numbers of theoretical plates and the symmet-
ry factors of their peaks are not less than 4000 and not more
than 1.4, respectively.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviations of the peak area
of saikosaponin a and saikosaponin d are not more than
1.5%, respectively.
Total ash <5.01> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 11.0%.
Burdock Fruit
Arctii Fructus
Burdock Fruit is the fruit of Arctium lappa Linne
(Compositae).
Description Burdock Fruit is slightly curved, long obovate
achene, 5 to 7 mm in length, 2.0 to 3.2 mm in width, 0.8 to
1 .5 mm in thickness; externally grayish brown to brown, with
black spots; hollow about 1 mm in diameter at one broad
end; flat, indistinct, longitudinal ridge at the other narrow
end. 100 fruits weighing 1.0 to 1.5 g.
Practically odorless; taste, bitter and oily.
Under a microscope <5.01>, transverse section reveals an
exocarp of single-layered epidermal tissue, mesocarp of
slightly sclerified parenchyma, and endocarp of a single layer
of stone cells; seed coat composed of radially elongated,
sclerified epidermis, and parenchyma several cells thick;
parenchymatous cells of the mesocarp contain a brown sub-
stance; stone cells of endocarp contain solitary, discrete crys-
tals of calcium oxalate; cotyledons with starch grains, oil
drops, aleurone grains, and minute crystals of calcium oxa-
late.
Identification To 0.5 g of pulverized Burdock Fruit add 20
mL of methanol, shake for 10 minutes, filter, and use filtrate
as the sample solution. Perform the test with the sample solu-
tion as directed under Thin-layer Chromatography <2.03>.
Spot 5 [iL of the sample solution on a plate of silica gel for
thin-layer chromatography, develop the plate with a mixture
of acetone, ethyl acetate and water (15:10:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly dilute sulfur-
ic acid on the plate, and heat at 105°C for 5 minutes: a red-
purple spot appears at around Rf 0.4.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.0I> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-extract: not less than
15.0%.
Calumba
Calumbae Radix
zip >tK'
Calumba is the cross-sectioned root of Jateorhiza
columba Miers (Menispermaceae).
Description Disk-like slices, 0.5 -2 cm in thickness, 3-8
cm in diameter; mostly with concave center and slightly
waved; side surface grayish brown in color, with irregular
1268 Powdered Calumba / Crude Drugs
JP XV
wrinkles; cut surface light yellow and powdery, with pale and
dark radiating stripes; cortex rather yellowish; cambium and
its neighborhood light grayish brown, warty protrusions in
the center; hard in texture, but brittle.
Odor characteristic; taste, bitter.
Identification To 3 g of pulverized Calumba add 30 mL of
water, allow to stand for 5 minutes with occasional shaking,
and filter. To 2 mL of the filtrate add gently 1 mL of sulfuric
acid, and, after cooling, add carefully chlorine TS to make
two layers: a light red to red color develops at the zone of
contact.
Total ash <J.07> Not more than 7.5%.
Powdered Calumba
Calumbae Radix Pulverata
Zl P >7f^
Powdered Calumba is the powder of Calumba.
Description Powdered Calumba occurs as a grayish yellow
powder, and has a characteristic odor and a bitter taste.
Under a microscope <5.01>, Powdered Calumba reveals
numerous starch grains, fragments of parenchyma cells con-
taining them; fragments of cork cells, stone cells, fibers, sub-
stitute fibers, vessels, tracheids, and also solitary crystals of
calcium oxalate; starch grains consisting of solitary grains or
2- to 3-compound grains; hilum, unevenly scattered, usually
25 - 50 /xm, but up to 90 ^m in diameter.
Identification To 3 g of Powdered Calumba add 30 mL of
water, allow to stand for 5 minutes with occasional shaking,
and filter. To 2 mL of the filtrate add gently 1 mL of sulfuric
acid, and after cooling, add carefully chlorine TS to make
two layers: a light red to red color develops at the zone of
contact.
Total ash <J.07> Not more than 7.5%.
Capsicum
Capsici Fructus
Capsicum is the fruit of Capsicum annuum Linne
(Solanaceae).
Capsicum contains not less than 0.10% of total cap-
saicins (capsaicin and dihydrocapsaicin), calculated on
the basis of dried material.
Description Elongated conical to fusiform fruit, often bent,
3-10 cm in length, about 0.8 cm in width; outer surface lus-
trous and dark red to dark yellow-red; interior of pericarp
hollow and usually divided into two loculi, containing
numerous seeds nearly circular and compressed, light yellow-
red, about 0.5 cm in diameter; usually with remains of calyx
and peduncle.
Odor, slight and characteristic; taste, hot and acrid.
Identification To 2.0 g of pulverized Capsicum add 5 mL of
ethanol (95), warm on a water bath for 5 minutes, cool, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 1 mg of capsaicin for thin-layer chro-
matography in 1 mL of ethanol (95), and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 iuL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of diethyl ether and methanol (19:1) to a
distance of about 12 cm, and air-dry the plate. Spray evenly
2,6-dibromo-7V-chloro-l,4-benzoquinone monoimine TS on
the plate, and allow to stand in ammonia gas: a spot from the
sample solution and a blue spot from the standard solution
show the same color tone and the same R f value.
Purity Foreign matter <5.01> — The amount of foreign mat-
ter contained in Capsicum does not exceed 1.0%.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.07> Not more than 8.0%.
Acid-insoluble ash <5.01> Not more than 1.2%.
Component determination Weigh accurately about 0.5 g of
medium powder of Capsicum in a glass-stoppered centrifuge
tube, add 30 mL of methanol, shake for 15 minutes, cen-
trifuge, and separate the supernatant liquid. To the residue
add 10 mL of methanol, shake for 5 minutes, centrifuge, and
separate the supernatant liquid. Repeat this procedure again,
combine the extracts, add methanol to make exactly 50 mL,
and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of capsaicin for component de-
termination, previously dried in a desiccator (in vacuum,
phosphorus (V) oxide, 40°C) for 5 hours, and dissolve in
methanol to make exactly 50 mL . Pipet 2 mL of this solution,
add methanol to make exactly 25 mL, and use this solution as
the standard solution. Perform the test with exactly 20 fiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A TC and
A TD , of capsaicin and dihydrocapsaicin (the relative retention
time to capsaicin is about 1.3) in the sample solution, and the
peak area, A s , of capsaicin in the standard solution.
Amount (mg) of total capsaicins
= W s x {(A TC + A JD )/A S } xo.08
W s : Amount (mg) of capsaicin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 281 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with phenylated silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
capsaicin is about 20 minutes.
System suitability —
System performance: Dissolve 1 mg each of capsaicin for
component determination and 4-hydroxy-3-methoxybenzyl
nonylic acid amide in methanol to make 50 mL. When the
JPXV
Crude Drugs / Capsicum Tincture 1269
procedure is run with 20 fiL of this solution under the above
operating conditions, 4-hydroxy-3-methoxybenzyl nonylic
acid amide and capsaicin are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of capsaicin is not more than 1.5%.
Powdered Capsicum
Capsici Fructus Pulveratus
Powdered Capsicum is the powder of Capsicum.
Powdered Capsicum contains not less than 0.10% of
total capsaicins (capsaicin and dihydrocapsaicin), cal-
culated on the basis of dried material.
Description Powdered Capsicum occurs as a yellow-red
powder. It has a slight, characteristic odor and a hot, acrid
taste.
Under a microscope <5.01>, Powdered Capsicum reveals
fragments of parenchyma containing oil droplets and yellow-
red chromoplasts; fragments of outer pericarp with thick
cuticle; fragments of stone cells from inner surface of
pericarp, with wavy curved side walls; fragments of thin ves-
sels; fragments of seed coat with thick wall, and fragments of
parenchyma consisting of small cells of endosperm contain-
ing fixed oil and aleuron grains.
Identification To 2.0 g of Powdered Capsicum add 5 mL of
ethanol (95), warm on a water bath for 5 minutes, cool, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 1 mg of capsaicin for thin-layer chro-
matography in 1 mL of ethanol (95), and use this solution as
the standard solution. Perform the test with these solutions
as directed under Thin-layer Chromatography <2.03>. Spot
10 /xL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of diethyl ether and methanol (19:1) to a
distance of about 12 cm, and air-dry the plate. Spray evenly
2,6-dibromo-N-chloro-l,4-benzoquinone monoimine TS on
the plate, and allow to stand in ammonia gas: a spot from the
sample solution and blue spot from the standard solution
show the same in color tone and Rf value.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 8.0%.
Acid-insoluble ash <5.01> Not more than 1.2%.
Component determination Weigh accurately about 0.5 g of
medium powder of Powdered Capsicum in a glass-stoppered
centrifuge tube, add 30 mL of methanol, shake for 15
minutes, centrifuge, and separate the supernatant liquid. To
the residue add 10 mL of methanol, shake for 5 minutes, cen-
trifuge, and separate the supernatant liquid. Repeat this
procedure again, combine the extracts, add methanol to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 10 mg of capsaicin
for component determination, previously dried in a desicca-
tor (in vacuum, phosphorus (V) oxide, 40°C) for 5 hours,
and dissolve in methanol to make exactly 50 mL. Pipet 2 mL
of this solution, add methanol to make exactly 25 mL, and
use this solution as the standard solution. Perform the test
with exactly 20 /xL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01>
according to the following conditions, and determine the
peak areas, A JC and A JD , of capsaicin and dihydrocapsaicin
(the relative retention time to capsaicin is about 1.3) in the
sample solution, and the peak area, A s , of capsaicin in the
standard solution.
Amount (mg) of total capsaicins
= W s x {(A TC +A TD )/A S ) X0.08
W s : Amount (mg) of capsaicin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength : 281 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with phenylated silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
capsaicin is about 20 minutes.
System suitability —
System performance: Dissolve 1 mg each of capsaicin for
component determination and 4-hydroxy-3-methoxybenzyl
nonylic acid amide in methanol to make 50 mL. When the
procedure is run with 20 /xL of this solution under the above
operating conditions, 4-hydroxy-3-methoxybenzyl nonylic
acid amide and capsaicin are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of capsaicin is not more than 1.5%.
Capsicum Tincture
Capsicum Tincture contains not less than 0.010
w/v% of total capsaicins (capsaicin and dihydrocap-
saicin).
Method of preparation
Capsicum, in medium cutting 100 g
Ethanol a sufficient quantity
To make lOOOmL
Prepare as directed under Tinctures, with the above in-
gredients.
Description Capsicum Tincture is a yellow-red liquid. It has
a burning, pungent taste.
Specific gravity d 2 2 ° : about 0.82
Identification Proceed as directed in the Identification un-
1270 Capsicum and Salicylic Acid Spirit / Crude Drugs
JP XV
der Capsicum, using Capsicum Tincture as the sample solu-
tion. Spot 20 /uL each of the sample solution and the standard
solution.
Alcohol number <7.07> Not less than 9.7 (Method 2).
Component determination Pipet 2 mL of Capsicum Tin-
cture, add methanol to make exactly 20 mL, and use this so-
lution as the sample solution. Separately, weigh accurately
about 10 mg of capsaicin for component determination,
previously dried in a desiccator (in vacuum, phosphorus (V)
oxide, 40°C) for 5 hours, dissolve in methanol to make exact-
ly 50 mL. Pipet 2 mL of this solution, add methanol to make
exactly 25 mL, and use this solution as the standard solution.
Perform the test with exactly 20 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A TC and A TD , of capsaicin and
dihydrocapsaicin (the relative retention time to capsaicin is
about 1.3) in the sample solution, and the peak area, A s , of
capsaicin in the standard solution.
Amount (mg) of total capsaicins
= W s x {(A TC +A TD )/A S } x 0.032
fV s : Amount (mg) of capsaicin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 281 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 25 cm in length, packed with phenylated silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
capsaicin is about 20 minutes.
System suitability-
System performance: Dissolve 1 mg each of capsaicin for
component determination and 4-hydroxy-3-methoxybenzyl
nonylic acid amide in methanol to make 50 mL. When the
procedure is run with 20 /uL of this solution under the above
operating conditions, 4-hydroxy-3-methoxybenzyl nonylic
acid amide and capsaicin are eluted in this order with the
resolution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of capsaicin is not more than 1.5%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Capsicum and Salicylic Acid Spirit
Method of preparation
Capsicum Tincture
Salicylic Acid
Liquefied Phenol
Castor Oil
aromatic substance
Ethanol
40 mL
50 g
20 mL
100 mL
a suitable quantity
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients.
Description Capsicum and Salicylic Acid Spirit is a light
brown-yellow liquid.
Specific gravity df : about 0.84
Identification (1) Shake 10 mL of Capsicum and Salicylic
Acid Spirit with 15 mL of sodium hydrogen carbonate TS
and 10 mL of diethyl ether, and separate the water layer. To 1
mL of the solution add hydrochloric acid-potassium chloride
buffer solution, pH 2.0, to make 200 mL, and to 5 mL of this
solution add 5 mL of a solution of iron (III) nitrate enneahy-
drate (1 in 200): a red-purple color is produced (salicylic
acid).
(2) To 0.5 mL of Capsicum and Salicylic Acid Spirit add
20 mL of water and 5 mL of dilute hydrochloric acid, extract
with 20 mL of diethyl ether, wash the diethyl ether extract
with two 5-mL portions of sodium hydrogen carbonate TS,
and then extract with 20 mL of dilute sodium hydroxide TS.
To 1 mL of the extract add 1 mL of sodium nitrite TS and 1
mL of dilute hydrochloric acid, shake, and allow to stand for
10 minutes. Add 3 mL of sodium hydroxide TS: a yellow
color is produced (phenol).
(3) To 0.2 mL of Capsicum and Salicylic Acid Spirit add
5 mL of dilute hydrochloric acid, extract with 5 mL of chlo-
roform, and use the extract as the sample solution. Dissolve
0.01 g of salicylic acid and 0.02 g of phenol in 5 mL and 25
mL of chloroform, respectively, and use both solutions as the
standard solution (1) and the standard solution (2). Perform
the test with the sample solution and standard solutions (1)
and (2) as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of these solutions on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of chloroform, acetone and acetic
acid (100) (45:5:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): two spots from the sample solution exhibit the same
Rf values as those from standard solution (1) and standard
solution (2). Spray evenly iron (III) chloride TS upon the
plate: the spot from standard solution (1) and the cor-
responding spot from the sample solution reveal a purple
color.
Alcohol number <7.07> Not less than 8.1 (Method 2). Pre-
pare the sample solution as follows: Pipet 5 mL of Capsicum
and Salicylic Acid Spirit at 15 ± 2°C into a glass-stoppered,
conical flask containing exactly 45 mL of water while shaking
JPXV
Crude Drugs / Chrysanthemum Flower 1271
vigorously, allow to stand, and filter the lower layer. Discard
the first 15 mL of the filtrate. Pipet 25 mL of the subsequent
filtrate, add exactly 10 mL of the internal standard solution,
and add water to make exactly 100 mL.
Containers and storage Containers — Tight containers.
Cardamon
Cardamomi Fructus
Cardamon is the fruit of Elettaria cardamomum
Maton (Zingiberaceae). The capsules are removed
from the seeds before use.
Description Nearly ellipsoidal, 1-2 cm in length, 0.5 - 1
cm in diameter; externally, light yellow with three blunt
ridges and many longitudinal lines; 0.1 - 0.2-cm beak at one
end; pericarp thin, light and fibrous; interior longitudinally
divided into three loculi by thin membranes, each loculus
containing 3 to 7 seeds joining by aril; seed irregularly angu-
lar ovoid, 0.3 - 0.4 cm in length, dark brown to blackish
brown; the dorsal side convex, the ventral side longitudinally
grooved; external surface coarsely tuberculated.
Seed has a characteristic aroma, and pungent, slightly bit-
ter taste; pericarp, odorless and tasteless.
Total ash <J.07> Not more than 6.0% (seed).
Acid-insoluble ash <5.01> Not more than 4.0% (seed).
Essential oil content <5.01> Perform the test with 30.0 g of
the pulverized seeds of Cardamon: the volume of essential oil
is not less than 1.0 mL.
Cassia Seed
Cassiae Semen
Cassia Seed is the seed of Cassia obtusifolia Linne or
Cassia tora Linne (Leguminosae).
Description Short cylindrical seed, 3-6 mm in length, 2 -
3.5 mm in diameter; acuminate at one end and flat at the
other; externally green-brown to brown and lustrous, with
light yellow-brown longitudinal lines or bands on both sides;
hard in texture; cross section round or obtuse polygonal; un-
der a magnifying glass, albumen enclosing a bent, dark-
colored cotyledon.
When ground, characteristic odor and taste.
Identification Place 0.1 g of pulverized Cassia Seed, previ-
ously dried in a desiccator (silica gel) for 48 hours, on a slide
glass, put a glass ring 10 mm in both internal diameter and
height on it, then cover with moistened filter paper, and heat
gently the slide glass over a small flame. Take off the filter
paper when a yellow color has developed on the upper sur-
face of it, and place 1 drop of potassium hydroxide TS on the
surface of the filter paper where a sublimate is present: a red
color appears.
Purity Foreign matter <5.0I> — The amount of foreign mat-
ter contained in Cassia Seed does not exceed 1.0%.
Total ash <5.07> Not more than 5.0%.
Catalpa Fruit
Catalpae Fructus
Catalpa Fruit is the fruit of Catalpa ovata G. Don or
Catalpa bungei C. A. Meyer (Bignoniaceae).
Description Slender stick-like fruit, 30 - 40 cm in length
and about 0.5 cm in diameter; externally, dark brown; inner
part contains numerous seeds; seed compressed or semitubu-
lar, about 3 cm in length and about 0.3 cm in width, external-
ly grayish brown; hairs, about 1 cm in length, attached to
both ends of seed; pericarp, thin and brittle.
Almost odorless; taste, slightly astringent.
Identification To 1.0 g of pulverized Catalpa Fruit add 20
mL of water, warm on a water bath for 5 minutes, and filter
immediately. Transfer the filtrate to a separator, and extract
with two 20-mL portions of 1-butanol. Combine the extracts,
evaporate to dryness under reduced pressure on a water bath,
dissolve the residue in 1 mL of methanol, and use this solu-
tion as the sample solution. Separately, dissolve 1 mg of
parahydroxybenzoic acid in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel with fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, ethanol (99.5) and water (20:2:1) to a dis-
tance of about 10 cm, and air-dry the plate. Examine under
ultra-violet light (main wavelength: 254 nm): one spot among
the spots from the sample solution and a dark purple spot
from the standard solution show the same color tone and the
same Rf value. Prescribe that the moving distance of the spot
corresponding to parahydroxybenzoic acid from the sample
solution is 1: a dark purple spot develops at the relative mov-
ing distance of about 0.3.
Purity Peduncle — The amount of peduncles contained in
Catalpa Fruit does not exceed 5.0%.
Total ash <5.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 8.0%.
Chrysanthemum Flower
Chrysanthemi Flos
Chrysanthemum Flower is the capitulum of 1)
Chrysanthemum morifolium Ramatulle or 2) Chrysan-
1272 Cimicifuga Rhizome / Crude Drugs
JP XV
themum indicum Linne (Compositae).
Description 1) Chrysanthemum Flower is capitulum, 15
to 40 mm in diameter; involucre consisting of 3 to 4 rows of
involucral scales; the outer involucral scale linear to lanceo-
late, inner involucral scale narrow ovate to ovate; ligulate
flowers are numerous, white to yellow; tubular flowers in
small number, light yellow-brown; tubular flowers oc-
casionally degenerate; outer surface of involucre green-
brown to brown; light in texture and easy to break.
Odor, characteristic; taste, slightly bitter.
2) Chrysanthemum Flower is capitulum, 3 to 10 mm in
diameter; involucre consisting of 3 to 5 rows of involucral
scales; the outer involucral scale linear to lanceolatae, inner
involucral scale narrow ovate to ovate; ligulate flower is sin-
gle, yellow to light yellow-brown; tubular flowers in
numerous, light yellow-brown; outer surface of involucre yel-
low-brown to brown; light in texture and easy to break.
Odor, characteristic; taste, slightly bitter.
Identification To 1 g of pulverized Chrysanthemum Flower
add 20 mL of methanol, shake for 10 minutes, and filter.
Evaporate the filtrate to dryness, dissolve the residue in 1 mL
of methanol, and use this as the sample solution. Separately,
dissolve 1 mg of luteolin for thin-layer chromatography in 1
mL of methanol, and use this as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography, develop the plate with a mixture of
ethyl acetate, 2-butanone, water and formic acid (25:3:1:1) to
a distance of about 10 cm, and air-dry the plate. Spray evenly
iron (III) chloride-methanol TS on the plate: one of several
spots obtained from the sample solution has the same color
tone and the same Rf value with the dark green spot obtained
from the standard solution.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.01> Not more than 8.5%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 30.0%.
Cimicifuga Rhizome
Cimicifugae Rhizoma
^t
Cimicifuga Rhizome is the rhizome of Cimicifuga
simplex Wormskjord, Cimicifuga dahurica (Turcz.)
Maximmowicz, Cimicifuga foetida Linne or Cimicifu-
ga heracleifolia Komarov (Ranunculaceae).
Description Knotted, irregularly shaped rhizome, 6-18 cm
in length, 1 - 2.5 cm in diameter; externally dark brown to
blackish brown, with many remains of roots, often with scars
of terrestrial stems; the center of the scar dented, and the cir-
cumference being pale in color and showing a radial pattern;
fractured surface fibrous; pith dark brown in color and often
hollow; light and hard in texture.
Almost odorless; taste, bitter and slightly astringent.
Purity Rhizome of Astilbe thunbergii Miquel — Under a
microscope <5.01>, powdered Cimicifuga Rhizome does not
contain crystal druses in the parenchyma.
Total ash <5.07> Not more than 9.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 18.0%.
Cinnamon Bark
Cinnamomi Cortex
Cinnamon Bark is the bark of the trunk of Cin-
namomum cassia Blume (Lauraceae), or such bark
from which a part of the periderm has been removed.
Description Usually semi-tubular or tubularly rolled pieces
of bark, 0.1 - 0.5 cm in thickness, 5-50 cm in length, 1.5-5
cm in diameter; the outer surface dark red-brown, and the in-
ner surface red-brown and smooth; brittle; the fractured sur-
face is slightly fibrous, red-brown, exhibiting a light brown,
thin layer.
Characteristic aroma; taste, sweet and pungent at first,
later rather mucilaginous and slightly astringent.
Under a microscope <5.01>, a transverse section of Cinna-
mon Bark reveals a primary cortex and a secondary cortex
divided by an almost continuous ring consisting of stone
cells; nearly round bundles of fibers in the outer region of the
ring; wall of each stone cell often thickened in a U-shape;
secondary cortex lacking stone cells, and with a small number
of sclerenchymatous fibers coarsely scattered; parenchyma
scattered with oil cells, mucilage cells and cells containing
starch grains; medullary rays with cells containing fine nee-
dles of calcium oxalate.
Identification To 2.0 g of pulverized Cinnamon Bark add
10 mL of diethyl ether, shake for 3 minutes, filter, and use the
filtrate as the sample solution. Perform the test with this solu-
tion as directed under Thin-layer Chromatography <2.03>.
Spot 10 [iL of the sample solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of hexane and ethyl acetate (2:1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): a purple spot
develops at the Rf value of about 0.4. Spray evenly 2,4-
dinitrophenylhydrazine TS upon the spot: a yellow-orange
color develops.
Purity Total BHC's and total DDT's <5.07>— Not more
than 0.2 ppm, respectively.
Loss on drying <5.01> Not more than 15.5% (6 hours).
Total ash <5.07> Not more than 6.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Cinnamon Bark provided that 1 mL of silicon re-
sin is previously added to the sample in the flask: the volume
of essential oil is not less than 0.5 mL.
JPXV
Crude Drugs / Citrus Unshiu Peel 1273
Powdered Cinnamon Bark
Cinnamomi Cortex Pulveratus
Powdered Cinnamon Bark is the powder of Cinna-
mon Bark.
Description Powdered Cinnamon Bark is red-brown to
brown in color. It has a characteristic aroma and a sweet,
pungent taste with a slightly mucilaginous and astringent af-
tertaste.
Under a microscope <5.01>, Powdered Cinnamon Bark
reveals starch grains, fragments of parenchyma cells contain-
ing them; fragments of fibers, oil cells containing yellow-
brown oil droplets, stone cells, cork stone cells, cork tissue,
and fine crystals of calcium oxalate. Starch grains are simple
and compound grains 6 to 15^m in diameter.
Identification To 2.0 g of Powdered Cinnamon Bark add 10
mL of diethyl ether, shake for 3 minutes, filter, and use the
filtrate as the sample solution. Perform the test with this solu-
tion as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL of the sample solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of hexane and ethyl acetate (2: 1) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (main wavelength: 254 nm): a purple spot
develops at the Rf value of about 0.4. Spray 2,4-
dinitrophenylhydrazine TS upon the spot: a yellow orange
color develops.
Purity (1) Petiole — Under a microscope <5.01>, Powdered
Cinnamon Bark does not reveal epidermal cells, hairs, cells
containing chlorophyll granules, and fragments of vascular
bundle.
(2) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <J.07> Not more than 6.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Cinnamon Bark provided that 1 mL of silicon re-
sin is previously added to the sample in the flask: the volume
of essential oil is not less than 0.35 mL.
Containers and storage Containers — Tight containers.
Cinnamon Oil
Oleum Cinnamomi
Cinnamon Oil is the essential oil distilled with steam
from the leaves and twigs or bark of Cinnamomum
cassia Blume or from the bark of Cinnamomum zey-
lanicum Nees (Lauraceae).
It contains not less than 60 vol% of the total alde-
hydes.
Description Cinnamon Oil is a yellow to brown liquid. It
has a characteristic, aromatic odor and a sweet, pungent
taste.
It is clearly miscible with ethanol (95) and with diethyl
ether.
It is practically insoluble in water.
It is weakly acidic. Upon aging or long exposure to air, it
darkens and becomes viscous.
Specific gravity df : 1.010-1.065
Identification Shake 4 drops of Cinnamon Oil with 4 drops
of nitric acid: the mixture forms white to light yellow crystals
at a temperature below 5°C.
Purity (1) Rosin — Mix 1.0 mL of Cinnamon Oil with 5
mL of ethanol (95), then add 3 mL of freshly prepared, satu-
rated ethanol solution of lead (II) acetate trihydrate: no
precipitate is produced.
(2) Heavy metals <1.07> — Proceed with 1.0 mL of Cinna-
mon Oil according to Method 2, and perform the test. Pre-
pare the control solution with 4.0 mL of Standard Lead Solu-
tion (not more than 40 ppm).
Assay Pipet 5.0 mL of Cinnamon Oil into a cassia flask,
add 70 mL of sodium hydrogensulfite TS, and heat the mix-
ture in a water bath with frequent shaking to dissolve com-
pletely. To this solution add sodium hydrogensulfite TS to
raise the lower level of the oily layer within the graduate por-
tion of the neck. Allow to stand for 2 hours, and measure the
volume (mL) of the separated oily layer.
Total aldehydes (vol%)
= [5.0 — (volume of separated oily layer)] x20
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Citrus Unshiu Peel
Aurantii Bobilis Pericarpium
Citrus Unshiu Peel is the pericarp of the ripe fruit of
Citrus unshiu Markovich or Citrus reticulata Blanco
(Rutaceae).
Description Irregular pieces of pericarp, about 2 mm in
thickness; externally yellow-red to dark yellow-brown, with
numerous small dents associated with oil sacs; internally
white to light grayish yellow-brown; light and brittle in tex-
ture.
Odor, characteristic aroma; taste, bitter and slightly pun-
gent.
Identification To 0.5 g of pulverized Citrus Unshiu Peel
add 10 mL of methanol, warm on a water bath for 2 minutes,
and filter. To 5 mL of the filtrate add 0.1 g of magnesium in
ribbon-form and 1 mL of hydrochloric acid, and allow to
stand: a red-purple color develops.
Purity Total BHC's and total DDT's <5.01>— Not more
than 0.2 ppm, respectively.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.07> Not more than 4.0%.
1274 Clematis Root / Crude Drugs
JP XV
Extract content <5.01>
less than 30.0%.
Dilute ethanol-soluble extract: not
Essential oil content <5.0I> Perform the test with 50.0 g of
pulverized Citrus Unshiu Peel provided that 1 mL of silicon
resin is previously added to the sample in the flask: the
volume of essential oil is not less than 0.2 mL.
Clematis Root
Clematidis Radix
Clematis Root is the root and rhizome of Clematis
chinensis Osbeck, Clematis manshurica Ruprecht, or
Clematis hexapetala Pallas (Ranunculaceae).
Description Clematis Root consists of short rhizome and
numerous slender roots. The root, 10 to 20 cm in length, 1 to
2 mm in diameter, externally brown to blackish brown, with
fine longitudinal wrinkles, brittle. The cortex easily separable
from central cylinder; root, grayish white to light brown in
the transverse section, light grayish yellow to yellow in the
central cylinder; under a magnifying glass, central cylinder
almost round, slight 2 to 4 sinuses on xylem. The rhizome, 2
to 4 cm in length, 5 to 20 mm in diameter, externally light
grayish brown to grayish brown; cortex peeled off and
fibrous, often with rising node; apex having the residue of lig-
nified stem.
Odor, slight; practically tasteless.
Under a microscope, <5.01> transverse section of root rev-
eals a uni-layered epidermis in the outermost layer; with ex-
odermis lying just inside of the epidermis; cortex and stele
divided by endodermis; cortex composed of parenchymatous
tissue; xylem with 2-4 small concavities where phloem is
present; parenchymatous cells contain both simple and 2- to
8-compound starch grains.
Identification (1) To 0.5 g of pulverized Clematis Root
add 10 mL of water, and boil for 2 to 3 minutes. After cool-
ing, shake vigorously: lasting fine foams appear.
(2) To 0.5 g of pulverized Clematis Root add 3 mL of
acetic anhydride, warm on a water bath for 2 minutes, and
filter. To the filtrate add 1 mL of sulfuric acid gently: a brown
color appears at the zone of contact.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.01> Not more than 8.5%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 15.0%.
Clove
Caryophylli Flos
Clove is the flowering bud of Syzygium aromaticum
Merrill et Perry (Eugenia caryophyllata Thunberg)
(Myrtaceae).
Description Dark brown to dark red buds, 1-1.8 cm in
length, consisting of slightly compressed and four-sided
receptacle, crowned by 4 thick sepals and 4 nearly spherical,
membranous, imbricated petals, enclosing numerous stamens
and a single style.
Odor, strong and characteristic; taste, pungent, followed
by a slight numbness of the tongue.
Identification Mix 0.1 mL of the mixture of essential oil
and xylene, obtained in the Essential oil content, with 2 mL
of ethanol (95), and add 1 to 2 drops of iron (III) chloride TS:
a green to blue color develops.
Purity (1) Stem — The amount of the stem contained in
Clove does not exceed 5.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the stem contained in Clove does not exceed 1.0%.
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Essential oil content <5.01> Perform the test with 10.0 g of
pulverized Clove: the volume of essential oil is not less than
1.6 mL.
Powdered Clove
Caryophylli Flos Pulveratus
fa 1 )-/*
Powdered Clove is the powder of Clove.
Description Powdered Clove occurs as a dark brown pow-
der. It has a strong, characteristic odor and a pungent taste,
followed by slight numbness of the tongue.
Under a microscope <5.01>, Powdered Clove reveals
epidermal tissue with stomata, collenchyma, parenchyma
with oil sacs, and spongy parenchyma or its fragments; fur-
thermore, a few fusiform thick-walled fibers, spiral vessels 6
- 10 /um in diameter, anther and pollen grains, and rosette ag-
gregates of calcium oxalate 10- 15 /xm in diameter. Epider-
mis of anther shows characteristically reticulated walls;
pollen grains tetrahedral 10 - 20 /um in diameter; rosette ag-
gregates of calcium oxalate arranged in crystal cell rows, or
contained in collenchyma cells and parenchyma cells.
Identification Mix 0.1 mL of a mixture of essential oil and
xylene, obtained in the Essential oil content, with 2 mL of
ethanol (95), and add 1 to 2 drops of iron (III) chloride TS: a
green to blue color develops.
Purity Foreign matter <5.01> — Under a microscope, Pow-
dered Clove does not contain stone cells or starch grains.
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.0]> Not more than 0.5%.
Essential oil content <5.01> Perform the test with 10.0 g of
Powdered Clove: the volume of essential oil is not less than
1.3 mL.
Containers and storage Containers — Tight containers.
JPXV
Crude Drugs / C iridium Rhizome 1275
Clove Oil
Oleum Caryophylli
Clove Oil is the volatile oil distilled with steam from
the flower buds or leaves of Syzygium aromaticum
Merrill et Perry (Eugenia caryophyllata Thunberg)
(Myrtaceae).
It contains not less than 80.0 vol% of total eugenol.
Description Clove Oil is a colorless or light yellow-brown,
clear liquid. It has a characteristic aroma and a burning taste.
It is miscible with ethanol (95) and with diethyl ether.
It is slightly soluble in water.
It acquires a brown color upon aging or by air.
Identification (1) To 5 drops of Clove Oil add 10 ml of
calcium hydroxide TS, and shake vigorously: the oil forms a
flocculent mass, and a white to light yellow color develops.
(2) Dissolve 2 drops of Clove Oil in 4 mL of ethanol (95),
and add 1 to 2 drops of iron (III) chloride TS: a green color is
produced.
Refractive index <2.45> n™\ 1.527 - 1.537
Optical rotation <2.49> c?°: - - 1.5° (100 mm).
Specific gravity <1.I3> d™: 1.040 - 1.068
Purity (1) Clarity of solution — Dissolve 1.0 mL of Clove
Oil in 2.0 mL of diluted ethanol (7 in 10): the solution is
clear.
(2) Water-soluble phenols — To 1.0 mL of Clove Oil add
20 mL of boiling water, shake vigorously, filter the aqueous
layer after cooling, and add 1 to 2 drops of iron (III) chloride
TS: a yellow-green, but no blue or violet, color develops.
(3) Heavy metals <1.07> — Proceed with 1.0 mL of Clove
Oil according to Method 2, and perform the test. Prepare the
control solution with 4.0 mL of Standard Lead Solution (not
more than 40 ppm).
Assay Take 10.0 mL of Clove Oil in a Cassia flask, add 70
mL of sodium hydroxide TS, shake for 5 minutes and warm
for 10 minutes in a water bath with occasional shaking, add
sodium hydroxide TS to the volume after cooling, and allow
to stand for 18 hours. Measure the volume (mL) of the sepa-
rated oily layer.
Total eugenol (vol%)
= [10 -(volume of separated oily layer)] x 10
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Cnidium Monnieri Fruit
Cnidii Monnieris Fructus
>■ -v -> a v
Cnidium Monnieri Fruit is the fruit of Cnidium
monnieri Cusson (Umbelliferae).
Description Elliptical cremocarp, often each mericarp sepa-
rated; 2-3 mm in length, 1-2 mm in width; externally light
brown to brown, each mericarp usually with five winged lon-
gitudinal ridges; inner surface of mericarp almost flat.
Odor, characteristic; it gives characteristic aroma, later a
slight sensation of numbness on chewing.
Under a microscope <5.01>, a transverse section reveals one
oil canal between longitudinal ridges, usually two oil canals
in the inner part of mericarp facing to gynophore; longitudi-
nal ridges composed of slightly lignified parenchymatous
cells, with vascular bundles in the base; epidermal cells and
parenchymatous cells of longitudinal ridges contain solitary
crystals of calcium oxalate; parenchymatous cells of albumen
contain oil drops and aleurone grains, and occasionally
starch grains.
Identification To 1 g of pulverized Cnidium Monnieri Fruit
add 10 mL of ethyl acetate, shake for 10 minutes, filter, and
use the filtrate as the sample solution. Separately, dissolve 1
mg of osthole for thin-layer chromatography in 2 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xh each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography, develop the plate with a mixture of
hexane and ethyl acetate (2:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 365 nm): one of the spot among the several spots
from the sample solution has the same color tone and the Rf
value with the blue-white fluorescent spot from the standard
solution.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.07> Not more than 17.0%.
Acid-insoluble ash <5.01> Not more than 6.0%.
Extract content <5.01>
less than 8.0%.
Dilute ethanol-soluble extract: not
Cnidium Rhizome
Cnidii Rhizoma
Cnidium Rhizome is the rhizome of Cnidium
officinale Makino (Umbelliferae), usually passed
through hot water.
Description Irregular massive rhizome, occasionally cut
lengthwise; 5-10 cm in length, and 3-5 cm in diameter; ex-
ternally grayish brown to dark brown, with gathered nodes,
and with knobbed protrusions on the node; margin of the
vertical section irregularly branched; internally grayish white
to grayish brown, translucent and occasionally with hollows;
dense and hard in texture.
Odor, characteristic; taste, slightly bitter.
Under a microscope <5.01>, a transverse section reveals
cortex and pith with scattered oil canals; in the xylem, thick-
walled and lignified xylem fibers appear in groups of various
sizes; starch grains usually gelatinized, but rarely remaining
as grains of 5 - 25 /um in diameter; crystals of calcium oxalate
1276 Powdered Cnidium Rhizome / Crude Drugs
JP XV
not observable.
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Powdered Cnidium Rhizome
Cnidii Rhizoma Pulveratum
Powdered Cnidium Rhizome is the powder of Cnidi-
um Rhizome.
Description Powdered Cnidium Rhizome occurs as a gray
to light grayish brown powder. It has a characteristic odor
and a slightly bitter taste.
Under a microscope <5.01>, Powdered Cnidium Rhizome
reveals colorless and gelatinized starch masses, and frag-
ments of parenchyma containing them; fragments of
scalariform and reticulate vessels 15-30//m in diameter;
fragments of thick-walled and lignified xylem fibers 20 - 60
fim in diameter; fragments of yellow brown cork tissue; frag-
ments of secretory tissue.
Purity Foreign matter <5.01> — Under a microscope, Pow-
dered Cnidium Rhizome does not contain a large quantity of
starch grains, stone cells, crystals of calcium oxalate or other
foreign matter.
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Coix Seed
Coicis Semen
Coix Seed is the seed of Coix lachryma-jobi Linne
var. ma-yuen Stapf (Gramineae), from which the seed
coat has been removed.
Description Ovoid or broad ovoid seed, about 6 mm in
length, and about 5 mm in width; with a slightly hollowed
apex and base; dorsal side distended; ventral side longitudi-
nally and deeply furrowed in the center; dorsal side mostly
white in color and powdery; in the furrow on the ventral sur-
face, attached brown, membranous pericarp and seed coat.
Under a magnifying glass, the cross section reveals light yel-
low scutellum in the hollow of the ventral side. Hard in tex-
ture.
Odor, slight; taste, slightly sweet; adheres to the teeth on
chewing.
Identification To a cross-section of Coix Seed add iodine
TS dropwise: a dark red-brown color develops in the en-
dosperm, and a dark gray color develops in the scutellum.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.07> Not more than 3.0%.
Powdered Coix Seed
Coicis Semen Pulveratum
Powdered Coix Seed is the powder of Coix Seed.
Description Powdered Coix Seed occurs as a brownish,
grayish white to grayish yellow-white powder, and has a
slight odor and a slightly sweet taste.
Under a microscope <5.01>, Powdered Coix Seed reveals
starch grains, and fragments of endosperm containing them;
fragments of tissue accompanied with epidermal cells of
pericarp composed of yellowish and oblong cells, and frag-
ments of parenchyma cells containing fixed oil, aleuron
grains and starch grains; a very few fragments of spiral ves-
sels. Starch grains are simple and 2-compound grains, simple
grain nearly equidiameter to obtuse polygon, 10-20//m in
diameter, and have a stellate cleft-like hilum in the center.
Spherical starch grains, coexisting with aleuron grains, are
spherical simple grains, 3-7 fim in diameter.
Identification Place a small amount of Powdered Coix Seed
on a slide glass, add dropwise iodine TS, and examine under
a microscope <5.01>: nearly equidiameter and obtuse poly-
gonal simple starch grains, usually 10- 15 /xm in diameter,
and compound starch grains have a reddish brown color.
Small spheroidal starch grains, coexisting with fixed oil and
with aleuron grains in parenchymatous cells, have a blue-pur-
ple color.
Purity Foreign matter — Under a microscope <5.01>, Pow-
dered Coix Seed reveals no fragments of tissue having silici-
fied cell wall, no stone cells, no fragments of other thick-
walled and lignified cells, no fragments of reticulate,
scalariform and pitted vessels, no fragments of fibers and
hairs, and no large starch grains, more than 10 ^m in di-
ameter, appearing blue-purple upon addition of iodine TS.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 3.0%.
Containers and storage Containers — Tight containers.
Condurango
Condurango Cortex
Condurango is the bark of the trunk of Marsdenia
cundurango Reichenbach fil. (Asclepiadaceae).
Description Tubular or semi-tubular pieces of bark, 0.1 -
0.6 cm in thickness, 4-15 cm in length; outer surface grayish
brown to dark brown, nearly smooth and with numerous len-
ticels, or more or less scaly and rough; inner surface light
grayish brown and longitudinally striate; fractured surface fi-
brous on the outer region and generally granular in the inner
JPXV
Crude Drugs / Coptis Rhizome 1277
region.
Odor, slight; taste, bitter.
Under a microscope <5.01>, a transverse section reveals a
cork layer composed of several layers of thin-walled cells;
primary cortex with numerous stone cell groups; scondary
cortex with phloem fiber bundles scattered inside the starch
sheath consisting of one-cellular layer; articulate latex tubes
scattered in both cortices; parenchyma cells containing starch
grains or rosette aggregates of calcium oxalate; starch grain 3
- 20 //m in diameter.
Identification Digest 1 g of pulverized Condurango in 5 mL
of water, and filter: the clear filtrate becomes turbid on heat-
ing, but becomes clear again upon cooling.
Purity Foreign matter <5.01> — The xylem and other foreign
matter contained in Condurango do not exceed 2.0%.
Total ash <5.01> Not more than 12.0%.
Condurango Fluidextract
Method of preparation Take medium powder of Con-
durango, and prepare the fluidextract as directed under
Fluidextracts using a suitable quantity of a mixture of Puri-
fied Water, Ethanol and Glycerin (5:3:2) as the first solvent,
and a suitable quantity of a mixture of Purified Water and
Ethanol (3:1) as the second solvent.
Description Condurango Fluidextract is a brown liquid. It
has a characteristic odor and a bitter taste.
Identification Mix 1 mL of Condurango Fluidextract with 5
mL of water, filter, if necessary, and heat the clear solution:
turbidity is produced. However, it becomes almost clear
upon cooling.
Containers and storage Containers — Tight containers.
Coptis Rhizome
Coptidis Rhizoma
Coptis Rhizome is the rhizome of Coptis japonica
Makino, Coptis chinensis Franchet, Coptis deltoidea
C.Y. Cheng et Hsiao or Coptis teeta Wallich (Ranun-
culaceae), from which the roots have been removed
practically.
It contains not less than 4.2% of berberine [as ber-
berine chloride (C 20 H 18 ClNO 4 : 371.81)], calculated on
the basis of dried material.
Description Irregular, cylindrical rhizome, 2 to 4 cm, rarely
up to 10 cm in length, 0.2 - 0.7 cm in diameter, slightly
curved and often branched; externally grayish yellow-brown,
with ring nodes, and with numerous remains of rootlets;
generally remains of petiole at one end; fractured surface
rather fibrous; cork layer light grayish brown, cortex and pith
are yellow-brown to reddish yellow-brown, xylem is yellow to
reddish yellow in color.
Odor, slight; taste, extremely bitter and lasting; it colors
the saliva yellow on chewing.
Under a microscope <5.01>, a transverse section of Coptis
Rhizome reveals a cork layer composed of thin-walled cork
cells; cortex parenchyma usually exhibiting groups of stone
cells near the cork layer and yellow phloem fibers near the
cambium; xylem consisting chiefly of vessels, tracheae and
wood fibers; medullary ray distinct; pith large; in pith, stone
cells or stone cells with thick and lignified cells are sometimes
recognized; parenchyma cells contain minute starch grains.
Identification (1) To 0.5 g of pulverized Coptis Rhizome
add 10 mL of water, allow to stand for 10 minutes with oc-
casional shaking, and filter. To 2 to 3 drops of the filtrate add
1 mL of hydrochloric acid and 1 to 2 drops of hydrogen
peroxide TS, and shake: a red-purple color develops.
(2) To 0.5 g of pulverized Coptis Rhizome add 20 mL of
methanol, shake for 2 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 1 mg of Berberine
Chloride Reference Standard in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (7:2:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): one spot among the spots from
the sample solution and a spot from the standard solution
with yellow to yellow-green fluorescence show the same color
tone and the same Rf value.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <J.07> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Assay Weigh accurately about 0.5 g of pulverized Coptis
Rhizome, add 30 mL of a mixture of methanol and dilute
hydrochloric acid (100:1), heat under a reflux condenser on a
water bath for 30 minutes, cool, and filter. Repeat the above
procedure twice with the residue, using 30-mL and 20-mL
portions of a mixture of methanol and dilute hydrochloric
acid (100:1). To the last residue add 10 mL of methanol,
shake well, and filter. Combine the whole filtrates, add
methanol to make exactly 100 mL, and use this solution as
the sample solution. Separately, weigh accurately about 10
mg of Berberine Chloride Reference Standard (previously de-
termine the water <2.48> in the same manner as Berberine
Chloride Hydrate), dissolve in methanol to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of berberine in each solution.
Amount (mg) of berberine [as berberine chloride
(C 20 H 18 ClNO 4 )]
= W s x(A T /A s )
W s : Amount (mg) of Berberine Chloride Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
1278 Powdered Coptis Rhizome / Crude Drugs
JP XV
(wavelength: 345 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 /um in particle diameter).
Column temperature: A constant temperature of about
45°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen-
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
Selection of column: Dissolve 1 mg each of Berberine
Chloride Reference Standard and palmatine chloride in 10
mL of methanol. Proceed with 20 iiL of this solution under
the above operating conditions. Use a column giving elution
of palmatine and berberine in this order, and clearly dividing
each peak.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative deviation of the peak area of berberine is not
more than 1.5%.
Powdered Coptis Rhizome
Coptidis Rhizoma Pulveratum
Powdered Coptis Rhizome is the powder of Coptis
Rhizome.
It contains not less than 4.2% of berberine [as ber-
berine chloride (C 20 H 18 ClNO 4 : 371.81)], calculated on
the basis of dried material.
Description Powdered Coptis Rhizome occurs as a yellow-
brown to grayish yellow-brown powder. It has a slight odor
and an extremely bitter, lasting taste, and colors the saliva
yellow on chewing.
Under a microscope <5.01>, almost all elements are yellow
in color; it reveals mainly fragments of vessels, tracheids and
xylem fibers; parenchyma cells containing starch grains; poly-
gonal cork cells. Usually, round to obtuse polygonal stone
cells and their groups, and phloem fibers, 10-20,Mm in di-
ameter, and fragments of their bundles. Sometimes, poly-
gonal and elongated epidermal cells, originated from the peti-
ole, having characteristically thickened membranes. Starch
grains are single grains 1 - 7//m in diameter.
Identification (1) To 0.5 g of Powdered Coptis Rhizome
add 10 mL of water, allow to stand for 10 minutes with oc-
casional shaking, and filter. To 2 to 3 drops of the filtrate add
1 mL of hydrochloric acid and 1 to 2 drops of hydrogen
peroxide TS, and shake: a red-purple color develops.
(2) To 0.5 g of Powdered Coptis Rhizome add 20 mL of
methanol, shake for 2 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 1 mg of Berberine
Chloride Reference Standard in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 fiL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (7:2:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): one spot among the spots from
the sample solution and a spot from the standard solution
with yellow to yellow-green fluorescence show the same color
tone and the same Rf value.
Purity (1) Phellodendron bark — Under a microscope
<5.01>, crystal cell rows or mucilage masses are not observa-
ble. Stir 0.5 g of Powdered Coptis Rhizome with 2 mL of
water: the solution does not become gelatinous.
(2) Curcuma — Place Powdered Coptis Rhizome on a
filter paper, drop diethyl ether on it, and allow to stand. Re-
move the powder from the filter paper, and drop 1 drop of
potassium hydroxide TS: no red-purple color develops. Un-
der a microscope <5.01>, Powdered Coptis Rhizome does not
contain gelatinized starch or secretory cells containing yel-
low-red resin.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <5.07> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Assay Weigh accurately about 0.5 g of Powdered Coptis
Rhizome, add 30 mL of a mixture of methanol and dilute
hydrochloric acid (100:1), heat under a reflux condenser on a
water bath for 30 minutes, cool, and filter. Repeat the above
procedure twice with the residue, using 30-mL and 20-mL
portions of a mixture of methanol and dilute hydrochloric
acid (100:1). To the last residue add 10 mL of methanol,
shake well, and filter. Combine the whole filtrates, add
methanol to make exactly 100 mL, and use this solution as
the sample solution. Separately, weigh accurately about 10
mg of Berberine Chloride Reference Standard (previously de-
termine the water <2.48> in the same manner as Berberine
Chloride Hydrate), dissolve in methanol to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 20 iiL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of berberine in each solution.
Amount (mg) of berberine [as berberine chloride
(C 20 H 18 ClNO 4 )]
= W s x(A T /A s )
W s : Amount (mg) of Berberine Chloride Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 345 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 mm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen-
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
Selection of column: Dissolve 1 mg each of Berberine
Chloride Reference Standard and palmatine chloride in 10
mL of methanol. Proceed with 20 fiL of this solution under
JP XV
Crude Drugs / Corydalis Tuber 1279
the above operating conditions. Use a column giving elution
of palmatine and berberine in this order, and clearly dividing
each peak.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative deviation of the peak area of berberine is not
more than 1.5%.
Cornus Fruit
Corni Fructus
H*>VnZL
Cornus Fruit is the sarcocarp of the pseudocarp of
Cornus officinalis Siebold et Zuccarini (Cornaceae).
Description Flattened oblong, 1.5 - 2 cm in length, about 1
cm in width; externally dark red-purple to dark purple, lus-
trous, and with coarse wrinkles; a crack-like scar formed by
removal of true fruit; a scar of calyx at one end, and a scar of
peduncle at the other; soft in texture.
Odor, slight; taste, acid and slightly sweet.
Identification To 1 g of coarse cuttings of Cornus Fruit add
10 mL of methanol, shake for 5 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve 1 mg of
loganin for thin-layer chromatography in 2 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot lO^L each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, water and formic acid (6:1:1) to a distance of about
10 cm, and air-dry the plate. Spray evenly 4-methoxybenzal-
dehyde-sulfuric acid TS on the plate, and heat at 105 °C for 5
minutes: one of the spots from the sample solution is the
same with a red-purple spot from the standard solution in
color tone and Ri value.
Purity (1) Foreign matter <5.01> — The amount of its
peduncles and other foreign matter contained in Cornus Fruit
does no exceed 2.0%.
(2) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Total ash <5.01> Not more than 5.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 35.0%.
Corydalis Tuber
Corydalis Tuber
x>=f-b-<7
Corydalis Tuber is the tuber of Corydalis
turtschaninovii Basser forma yanhusuo Y. H. Chou et
C. C. Hsu (Papaveraceae).
It contains not less than 0.08% of dehydrocoryda-
line (as dehydrocorydaline nitrate), calculated on the
basis of dried material.
Description Nearly flattened spherical, 1-2 cm in di-
ameter, and with stem scar at one end; externally grayish yel-
low to grayish brown; hard in texture; fractured surface is
yellow and smooth or grayish yellow-green in color and
granular.
Almost odorless; taste, bitter.
Identification To 0.5 g of pulverized Corydalis Tuber add
10 mL of dilute acetic acid, heat on a water bath for 3
minutes with occasional shaking, cool, and filter. To 5 mL of
the filtrate add 2 drops of Dragendorff's TS: immediately, an
orange-yellow precipitate is produced.
Loss on drying <5.01> Not more than 15.0%.
Total ash <5.07> Not more than 3.0%.
Component determination Weigh accurately about 1 g of
powdered Corydalis Tuber, add 30 mL of a mixture of
methanol and dilute hydrochloric acid (3:1), heat under a
reflux condenser on a water bath for 30 minutes, and filter af-
ter cooling. To the residue add 15 mL of a mixture of
methanol and dilute hydrochloric acid (3:1), and repeat the
above procedure. Combine the filtrates so obtained, add a
mixture of methanol and dilute hydrochloric acid (3:1) to
make exactly 50 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 10 mg of de-
hydrocorydaline nitrate for component determination, previ-
ously dried in a desiccator (silica gel) for not less than 1 hour,
dissolve in a mixture of methanol and dilute hydrochloric
acid (3:1) to make exactly 200 mL, and use this solution as
the standard solution. Perform the test with exactly 5 [iL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
dehydrocorydaline in each solution.
Amount (mg) of dehydrocorydaline [as dehydrocorydaline
nitrate C 22 H 24 N 2 7 )]
= W s x(A 1 /A s )x(l/4)
W s : Amount (mg) of dehydrocorydaline nitrate for com-
ponent determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 340 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about 40
°C.
Mobile phase: Dissolve 17.91 g of disodium hydrogen
phosphate dodecahydrate in 970 mL of water, and adjust to
pH 2.2 with phosphoric acid. In this solution add 14.05 g of
sodium perchlorate, dissolve, and add water to make exactly
1000 mL. To this solution add 450 mL of acetonitrile, then
dissolve 0.20 g of sodium lauryl sulfate.
Flow rate: Adjust the flow rate so that the retention time of
dehydrocorydaline is about 24 minutes.
System suitability —
System performance: Dissolve 1 mg each of de-
hydrocorydaline nitrate for component determination and
berberine chloride in 20 mL of a mixture of water and
acetonitrile (20:9). When the procedure is run with 5 /uL of
1280 Cyperus Rhizome / Crude Drugs
JP XV
this solution under the above operating conditions, berberine
and dehydrocorydaline are eluted in this order with the reso-
lution between these peaks being not less than 1.5.
System repeatability: When the test is repeated 6 times with
5 /uL of the standard solution under the above operating con-
ditions, the relative standard deviation of the peak areas of
dehydrocorydaline is not more than 1.5%.
Cyperus Rhizome
Cyperi Rhizoma
Cyperus Rhizome is the rhizome of Cyperus rotun-
dus Linne (Cyperaceae).
Description Fusiform rhizome, 1.5- 2.5 cm in length, 0.5 -
1 cm in diameter; externally grayish brown to grayish black-
ish brown, with 5 to 8 irregular ring nodes, and with hair-like
fiber bundles on each node; hard in texture. The transverse
section red-brown to light yellow in color, with waxy luster;
thickness of cortex approximately equal to or slightly smaller
than the diameter of stele. Under a magnifying glass, a trans-
verse section reveals fiber bundles as brown spots lined in
rings along circumference; here and there in the cortex, vas-
cular bundles appear as red-brown spots, and numerous
secretory cells scattered as minute yellow-brown spots; in the
stele, numerous vascular bundles scattered as spots or lines.
Characteristic odor and taste.
Total ash <J.07> Not more than 3.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Cyperus Rhizome, provided that 1 mL of silicon
resin is previously added on the sample in the flask: the
volume of essential oil is not less than 0.3 mL.
Powdered Cyperus Rhizome
Cyperi Rhizoma Pulveratum
Powdered Cyperus Rhizome is the powder of Cype-
rus Rhizome.
Description Powdered Cyperus Rhizome occurs as a light
red-brown powder, and has a characteristic odor and taste.
Under a microscope <5.01>, Powdered Cyperus Rhizome
reveals fragments of polygonal parenchyma cells, scalariform
vessels, and seta-like fibers; a large quantity of starch, mostly
gelatinized; an extremely small number of stone cells.
Purity Foreign matter — Under a microscope <5.01>, Pow-
dered Cyperus Rhizome does not show extremely lignified
cells, except stone cells, or crystals.
Total ash <5.01> Not more than 3.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Cyperus Rhizome provided that 1 mL of silicon re-
sin is previously added on the sample in the flask: the volume
of essential oil is not less than 0.2 mL.
Containers and storage Containers — Tight containers.
Digenea
Digenea
Digenea is the whole algae of Digenea simplex C.
Agardh (Rhodomelaceae) .
Description Rounded, string-like algae, 2-3 mm in di-
ameter; externally, dark red-purple to dark grayish red or
grayish brown; a few branched rods irregularly forked, co-
vered with short hairy twigs; calcified weeds and other small
algae often attached.
Odor, seaweed-like; taste, disagreeable and slightly salty.
Identification To 5 g of Digenea add 50 mL of water,
macerate between 50°C and 60 C C for 1 hour, and filter while
warm. Add 50 mL of water to the residue, macerate again be-
tween 50°C and 60°C for 1 hour, and filter while warm.
Evaporate all the filtrate on a water bath to about 25 mL, and
use this solution as the sample solution. Separately, dissolve
0.05 g of kainic acid in 10 mL of water, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with the upper layer of a mixture of water, 1-
butanol and acetic acid (100) (5:4:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly a water-saturated so-
lution of ninhydrin in 1-butanol (1 in 500) upon the plate,
and heat at 90°C for 10 minutes: the spots obtained from the
sample solution and the standard solution show a light yellow
color and the same Rf values.
Purity Foreign matter <5.01> — The amount of other algae
in Digenea does not exceed 20.0%.
Loss on drying <5.01> Not more than 22.0%.
Acid-insoluble ash <5.01> Not more than 8.0%.
Daiokanzoto Extract
Daiokanzoto Extract contains not less than 3.5 mg
and not more than 10.5 mg of sennoside A (C 42 H3 8 02o:
862.74) and not less than 9 mg and not more than 27
mg (for preparation prescribed 1 g of Glycyrrhiza) or
not less than 18 mg and not more than 54 mg (for
preparation prescribed 2 g of Glycyrrhiza) of glycyr-
rhizic acid (C 42 H 62 16 : 822.93) per a dried extract pre-
pared as directed in the Method of preparation.
Method of preparation Prepare a dried extract as directed
under Extracts, with 4 g of Rhubarb and 1 g of Glycyrrhiza,
or with 4 g of Rhubarb and 2 g of Glycyrrhiza.
JPXV
Crude Drugs / Daiokanzoto Extract 1281
Description Daiokanzoto Extract occurs as a brown pow-
der. It has a characteristic odor and an astringent first then
slightly sweet taste.
Identification (1) Rhubarb — To 1.0 g of Daiokanzoto Ex-
tract add 10 mL of water, shake, then add 10 mL of diethyl
ether, shake, centrifuge, and use the supernatant liquid as the
sample solution. Separately, dissolve 1 mg of rhein for thin-
layer chromatography in 1 mL of acetone, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): one of the spot among the several spots from the sample
solution has the same color tone and Ri value with the orange
fluorescent spot from the standard solution.
(2) Glycyrrhiza — To 0.5 g of Daiokanzoto Extract add 10
mL of water, shake, then add 10 mL of 1-butanol, shake,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of liquiritin for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105 °C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the yellow-brown spot from the standard.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1.0 g of Daiokanzoto Extract as directed in (4) in Ex-
tracts under the General Rules for Preparations, and perform
the test (not more than 30 ppm).
(2) Arsenic </.//> — Prepare the test solution with 0.67 g
of Daiokanzoto Extract according to Method 3, and perform
the test (not more than 3 ppm).
Loss on drying <2.41> Not more than 7.0% (1 g, 105°C, 5
hours).
Total ash <J.07> Not more than 10.0%.
Assay (1) Sennoside A — Weigh accurately about 0.2 g of
Daiokanzoto Extract, add 20 mL of ethyl acetate and 10 mL
of water, shake for 10 minutes, centrifuge, and remove the
upper layer. To the water layer add 20 mL of ethyl acetate,
shake for 10 minutes, centrifuge, and remove the upper layer.
To the water layer add 10 mL of methanol, shake for 30
minutes, centrifuge, and take the supernatant liquid. To the
residue add 20 mL of diluted methanol (1 in 2), shake for 5
minutes, centrifuge, and take the supernatant liquid. Com-
bine these supernatant liquids, add diluted methanol (1 in 2)
to make exactly 50 mL, and use this solution as the sample
solution. Separately, weigh accurately about 5 mg of Senno-
side A Reference Standard (separately determine the water),
dissolve in diluted methanol (1 in 2) to make exactly 200 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of sennoside A.
Amount (mg) of sennoside A (C 4 2H 38 02o)
= W s x(A T /A s )x(l/4)
W s : Amount (mg) of Sennoside A Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 340 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (2460:540:1).
Flow rate: 1.0 mL/min (the retention time of sennoside A
is about 14 minutes.)
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of sennoside A are not less than 5000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sennoside A is not more than 1.5%.
(2) Glycyrrhizic acid — Use the sample solution obtained
in the Assay (1) as the sample solution. Separately, weigh
accurately about 10 mg of Glycyrrhizic Acid Reference Stan-
dard (separately determine the water), dissolve in diluted
methanol (1 in 2) to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of glycyrrhizic acid.
Amount (mg) of glycyrrhizic acid (C 4 2H 62 I6 )
= W s x(A T /A s )x (1/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7)
Flow rate: l.OmL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
1282 Dioscorea Rhizome / Crude Drugs
JP XV
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
Dioscorea Rhizome
Dioscoreae Rhizoma
Dioscorea Rhizome is the rhizome (rhizophore) of
Dioscorea japonica Thunberg or Dioscorea batatas
Decaisne (Dioscoreaceae), from which the periderm
has been removed.
Description Cylindrical or irregular cylindrical rhizome, 5 -
15 cm in length, 1-4 cm in diameter, occasionally longitudi-
nally split or transversely cut; externally whitish to yellowish
white; fractured surface, whitish, smooth and powdery; hard
in texture but breakable.
Practically odorless and tasteless.
Identification (1) To the cut surface of Dioscorea Rhi-
zome add dilute iodine TS dropwise: a dark blue color de-
velops.
(2) To 0.2 g of pulverized Dioscorea Rhizome add 2 mL
of acetic anhydride, warm on a water bath for 2 minutes, and
filter. To 1 mL of the filtrate add 0.5 mL of sulfuric acid care-
fully to make two layers: a red-brown to purple-brown color
appears at the zone of contact.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Powdered Dioscorea Rhizome
Dioscoreae Rhizoma Pulveratum
Powdered Dioscorea Rhizome is the powder of Di-
oscorea Rhizome.
Description Powdered Dioscorea Rhizome occurs as nearly
yellowish white to white; odorless and tasteless.
Under a microscope <5.01>, Dioscorea rhizome powder
reveals starch grains; fragments of parenchyma cells contain-
ing starch grains; raphides of calcium oxalate, 100 to 200 /xm
in length and its containing mucilage cells; ring and
scalariform vessels, 15 to 35 /um in diameter; starch grain
isosceles deltoid or oblong, solitary, 18 to 35 ^m, hilum and
striation being distinct.
Identification To 0.2 g of Powdered Dioscorea Rhizome
add 2 mL of acetic anhydride, warm on a water bath for 2 to
3 minutes, and filter. To the filtrate add 0.5 mL of acetic an-
hydride, shake, and add carefully 0.5 mL of sulfuric acid to
make two layers: a red-brown to purple-brown color de-
velops at the zone of contact.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Containers and storage Containers — Tight containers.
Dolichos Seed
Dolichi Semen
Dolichos Seed is the seed of Dolichos lablab Linne
(Leguminosae).
Description Flattened ellipsoidal to flattened orbicular-o-
vate seed, 9-14 mm in length, 6-10 mm in width, 4-7 mm
in thickness; externally light yellowish white to light yellow,
smooth and somewhat lustrous; caruncle white, like a half-
moon, protrudent at one side; hard in texture.
Almost odorless; taste, slightly sweet and acid.
Under a microscope <5.01>, a transverse section reveals the
outermost layer of seed coat composed of a single layer of
palisade like epidermal cells coated with cuticle; beneath
epidermis a single layer of sclerenchymatous and sandglass
like cells; inside of the layer mentioned above parenchyma
lie, the innermost portion of the parenchyma decayed;
cotyledons occur inside of the seed coat; the outermost layer
of cotyledon composed of a single layer of epidermal cells,
inner part of cotyledon mainly parenchyma, containing aleu-
rone grains and oil drops, and occasionally starch grains.
Identification Put about 3 g of pulverized Dolichos Seed in
a centrifuge tube, add 30 mL of methanol, shake for 10
minutes, centrifuge, and take the supernatant liquid.
Evaporate the solvent of the supernatant liquid, add 30 mL
of water and 50 mL of ethyl acetate to the residue, shake, and
take the ethyl acetate layer. To the ethyl acetate add 10 g of
anhydrous sodium sulfate, shake, and filter. Evaporate the
solvent of the filtrate, add 1 mL of ethyl acetate to the
residue, and use this solution as the sample solution. Perform
the test with the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 20 /xL of the sample solution
on a plate of silica gel for thin-layer chromatography, de-
velop the plate with a mixture of ethyl acetate and acetic acid
(100) (100:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): a blue-white fluorescent spot appears around Rf 0.4.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 4.5%.
Extract content <5.0I>
less than 9.0%.
Dilute ethanol-soluble extract: not
JPXV
Crude Drugs / Ephedra Herb 1283
Eleutherococcus Senticosus
Rhizome
Eleutherococci senticosi Rhizoma
Eleutherococcus Senticosus Rhizome is the rhizomes
of Eleutherococcus senticosus (Ruprecht et Max-
imowicz) Maximowicz (Acanthopanax senticosus
(Ruprecht et Maximowicz) Harms) (Araliaceae), often
with root.
Description Slightly curved subcolumnar rhizome, 15-30
cm in length, 1 - 2.5 cm in diameter; externally grayish
brown and slightly rough; transversely cut surface light
brown, cortex thin, xylem thick with a pith in center; ex-
tremely hard in texture.
Odor, slightly characteristics; tasteless or slightly sweet, as-
tringency.
Under a microscope <5.01>, a transverse section reveals the
outermost layer consisting of a cork layer 3-7 cells thick; oil
canals scattered in parenchyma; fiber bundles lined stepwise
in phloem; phloem and xylem separated clearly by cambium;
xylem composed of vessels, xylem fibers and xylem parenchy-
ma; ray composed of 2 - 6 rows of cells; pith composed of
parenchyma; parenchyma of cortex and ray contain ag-
gregate crystals of calcium oxalate; occasionally starch grains
in ray, parenchyma of cortex and xylem.
Identification To about 0.5 g of pulverized Eleutherococcus
Senticosus Rhizome add 20 mL of diluted methanol (1 in 2),
shake for 15 minutes, centrifuge, and use the supernatant
liquid as the sample solution. Separately, dissolve 1 mg of
eleutheroside B for liquid chromatography in diluted
methanol (1 in 2) to make 20 mL. To 2 mL of this solution
add diluted methanol (1 in 2) to make 20 mL, and use this so-
lution as the standard solution. Perform the test with 10 iiL
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions: the peak correspond to eleutheroside B
shows the same retention time with that obtained with the
standard solution.
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 265 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of water and acetonitrile (9:1).
Flow rate: Adjust the flow rate so that the retention time of
eleutheroside B is about 10 minutes.
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of eleutheroside B are not less than 5000 and
not more than 1.5, respectively.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 2.5%.
Ephedra Herb
Ephdrae Herba
Ephedra Herb is the terrestrial stem of Ephedra sini-
ca Stapf, Ephedra intermedia Schrenk et C.A. Meyer
or Ephedra equisetina Bunge (Ephedraceae).
Ephedra Herb, when dried, contains not less than
0.7% of total alkaloids [as ephedrine (C 10 H 15 NO:
165.23) and pseudoephedrine (C 10 H 15 NO: 165.23)].
Description Thin cylindrical or ellipsoidal cylinder, 0.1 -
0.2 cm in diameter; 3-5 cm in length of internode; light
green to yellow-green; numerous parallel vertical furrows on
the surface; scaly leaves at the node portion; leaves, 0.2 - 0.4
cm in length, light brown to brown in color, usually being op-
posite at every node, adhering at the base to form a tubular
sheath around the stem. Under a magnifying glass, the trans-
verse section of the stem appears as circle and ellipse, the out-
er portion grayish green to yellow-green in color, and the cen-
ter filled with a red-purple substance or hollow. When frac-
tured at internode, the outer part is fibrous and easily split
vertically.
Odor, slight; taste, astringent and slightly bitter, giving a
slight sensation of numbness on the tongue.
Identification To about 0.5 g of pulverized Ephedra Herb
add 10 mL of methanol, shake for 2 minutes, filter, and use
the filtrate as the sample solution. Perform the test with this
solution as directed under Thin-layer Chromatography
<2.03>. Spot 10 fiL of the sample solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-butanol, water and acetic acid (100) (7:2:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly a
solution of ninhydrin in ethanol (95) (1 in 50), and heat the
plate at 105 °C for 5 minutes: a red-purple spot appears near
i?f 0.35.
Purity (1) Woody stem — The amount of the woody stems
contained in Ephedra Herb does not exceed 5.0%.
(2) Foreign matter <5.01> — Ephedra Herb does not con-
tain stems of Equisetaceae or Gramineae plants, or any other
foreign matter.
Total ash <J.07> Not more than 11.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Assay Weigh accurately about 0.5 g of medium powder of
Ephedra Herb, previously dried in a desiccator (silica gel) for
24 hours, in a glass-stoppered centrifuge tube, add 20 mL of
diluted methanol (1 in 2), shake for 30 minutes, centrifuge,
and separate the supernatant liquid. Repeat this procedure
twice with the residue using 20-mL portion of diluted
1284 Epimedium Herb / Crude Drugs
JP XV
methanol (1 in 2). Combine all the extracts, add diluted
methanol (1 in 2) to make exactly 100 mL, and use this solu-
tion as the sample solution. Separately, weigh accurately
about 50 mg of ephedrine hydrochloride for assay, previous-
ly dried at 105°C for 3 hours, and dissolve in diluted
methanol (1 in 2) to make exactly 20 mL. Pipet 2 mL of the
solution, add diluted methanol (1 in 2) to make exactly 100
mL, and use this solution as the standard solution. Pipet 10
HL each of the sample solution and standard solution, and
perform the test as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine the
peak areas, A TE and A TP , of ephedrine and pseudoephedrine
(the relative retention time to ephedrine is about 0.9) in the
sample solution, and the peak area, A s , of ephedrine in the
standard solution.
Amount (mg) of total alkaloids (ephedrine and pseudoephe-
drine)
= W s x l(A 1E +A TF )/A s } x (1/10) x 0.819
W s : Amount (mg) of ephedrine hydrochloride for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to lO^m in
particle diameter).
Column temperature: A constant temperature of about
45°C.
Mobile phase: A mixture of a solution of sodium lauryl
sulfate (1 in 128), acetonitrile and phosphoric acid (640:
360:1).
Flow rate: Adjust the flow rate so that the retention time of
ephedrine is about 14 minutes.
Selection of column: Dissolve 1 mg of ephedrine
hydrochloride for assay and 4 mg of Atropine Sulfate in
diluted methanol (1 in 2) to make 100 mL. Perform the test
with 10,mL of this solution under the above operating condi-
tions. Use a column giving elution of ephedrine and atropine
in this order, clearly separating each peak.
System repeatability: Repeat the test 6 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of ephedrine is not
more than 1.5%.
Epimedium Herb
Epimedii Herba
Epimedium Herb is the terrestrial part of Epimedi-
um pubescens Maximowicz, Epimedium brevicornum
Maximo wicz, Epimedium wushanense T. S. Ying,
Epimedium sagittatum Maximowicz, Epimedium
koreanum Nakai, Epimedium grandiflorum Morren
var. thunbergianum Nakai or Epimedium sempervi-
rens Nakai (Berberidaceae).
Description Epimedium Herb is composed of a stem and a
ternate to triternate compound leaf; leaflet ovate to broadly
ovate or ovate-lanceolate, 3-20 cm in length, 2-8 cm in
width, petiolule 15 -70 mm in length, apex of leaflet
acuminate, needle hair on margin 0.1 - 0.2 cm in length, base
of leaflet cordate to deeply cordate, lateral leaflet asymmetry;
upper surface green to greenish brown, sometimes lustrous,
lower surface light green, often pilose, especially on vein den-
sely pilose, papery or coriaceous; petiole and stem cylindri-
cal, light yellowish brown to slightly purplish and light green-
ish brown, easily broken.
Odor, slight; taste, slightly bitter.
Under a microscope <5.01>, a transverse section of the leaf
reveals 3-6 vascular bundles in midvein; mesophyll
composed of upper epidermis, single-layered palisade,
spongy tissue and lower epidermis; leaf margins orbicular or
oblong, sclerenchymatous; multi-cellular hairs on epidermis;
8-20 vascular bundles in petiole and 6-15 vascular bundles
in petiolule. Under a microscope <5.01>, a transverse section
of the stem reveals a single to several-layered hypodermis,
cortex of 4 - 10 layers of sclerenchymatous cells, vascular
bundle 13 - 30 in number, oblong to obovate.
Identification To 2 g of pulverized Epimedium Herb add 20
mL of methanol, shake for 15 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve 1 mg of
icariin for thin-layer chromatography in 1 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, ethanol (99.5) and water
(8:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
one of the spot among the several spots from the sample solu-
tion has the same color tone and Rf value with the dark pur-
ple spot from the standard solution.
Loss on drying <5.01> Not more than 12.5% (6 hours).
Total ash <J.07> Not more than 8.5%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 17.0%.
Eucommia Bark
Eucommiae Cortex
Eucommia Bark is the bark of Eucommia ulmoides
Oliver (Eucommiaceae).
Description Eucommia Bark is a semi-tubular or plate-like
bark, 2 to 6 mm in thickness; externally pale grayish brown to
grayish brown, and rough in texture, sometimes reddish-
brown due to the cork layer falling off; internally dark violet,
smooth and covered with a linear pattern that runs longitudi-
nally, silk-like threads of gutta-percha (a thermoplastic rub-
ber-like substance) appearing when broken.
Odor faint but distinctive; taste slightly sweet.
Under a microscope <5.01>, transverse section reveals
JP XV
Crude Drugs / Powdered Fennel 1285
parenchymatous cells containing gutta-percha; phloem with
stone-cell and fiber layers; rays in rows of 2 - 3 cells; calcium
oxalate crystals absent.
Identification Put 1 g of pulverized Eucommia Bark in a
glass-stoppered centrifuge tube, add 10 mL of water and
20 mL of diethyl ether, shake for 15 minutes, and centrifuge.
Take the diethyl ether layer so obtained, evaporate the
diethyl ether on a water bath, and add 1 mL of ethanol (99.5)
to the residue: colloidal substances appear.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 8.0%.
Acid-insoluble ash <5.01> Not more than 5.0%.
Extract content <5.01>
less than 7.0%.
Dilute ethanol-soluble extract: not
Evodia Fruit
Evodiae Fructus
zfvnZL
Evodia Fruit is the fruit of Evodia rutaecarpa Ben-
tham or Evodia officinalis Dode or Evodia bodinieri
Dode (Rutaceae).
Description Flattened spheroidal or globular fruit, 2-5
mm in diameter; externally dark brown to grayish brown,
with many oil sacs appearing as hollow pits, and often with
peduncle, 2-5 mm in length, covered densely with hairs; ma-
tured pericarp split to reveal five loculi, and each loculus con-
taining obovoid or globular seeds of a lustrous brown to
blackish brown or bluish black color.
Odor, characteristic; taste, acrid, followed by a lasting bit-
terness.
Identification To 1.0 g of pulverized Evodia Fruit add 20
mL of methanol, heat for 5 minutes on a water bath, cool,
and filter. Evaporate the filtrate to dryness, add 3 mL of di-
lute acetic acid to the residue, warm for 2 minutes on a water
bath, cool, and filter. Perform the following tests using the
filtrate as the sample solution.
(1) Spot one drop of the sample solution on a filter paper,
air-dry, spray Dragendorff's TS for spraying, and allow to
stand: a yellow-red color develops.
(2) To 0.2 mL of the sample solution add 0.8 mL of di-
lute acetic acid. To this solution add gently 2 mL of 4-
dimethylaminobenzaldehyde TS, and warm in a water bath: a
purple-brown ring develops at the zone of contact.
Purity (1) Peduncle — The amount of peduncles contained
in Evodia Fruit does not exceed 5.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than peduncles contained in Evodia Fruit does not ex-
ceed 1.0%.
Total ash <J.07> Not more than 8.0%.
Fennel
Foeniculi Fructus
Fennel is the fruit of Foeniculum vulgare Miller
(Umbelliferae).
Description Cylindrical cremocarp, 3.5 - 8 mm in length, 1
- 2.5 mm in width; externally grayish yellow-green to grayish
yellow; two mericarps closely attached with each other, and
with five longitudinal ridges; cremocarp often with pedicel 2 -
10 mm in length.
Characteristic odor and taste.
Under a microscope <5.01>, ridges near the bentral side are
far protruded than those on the dorsal side; one large oil
canal between each ridge, and two oil canals on the bentral
side.
Identification To 0.5 g of pulverized Fennel add 10 mL of
hexane, allow to stand for 5 minutes with occasional shaking,
filter, and use the filtrate as the sample solution. Perform the
test with this solution as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL of the sample solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of hexane
and ethyl acetate (20:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): a main spot with a dark purple color
appears at the Rf value of about 0.4.
Purity (1) Peduncle — The amount of peduncles contained
in Fennel does not exceed 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the peduncle contained in Fennel does not exceed
1.0%.
Total ash <5.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Fennel: the volume of essential oil is not less than
0.7 mL.
Powdered Fennel
Foeniculi Fructus Pulveratus
Powdered Fennel is the powder of Fennel.
Description Powdered Fennel occurs as a greenish pale
brown to greenish brown, and is a characteristic odor and
taste.
Under a microscope <5.01>, Powdered Fennel reveals frag-
ments of parenchyma cells of perisperm containing aleurone
grain, fragments of parenchyma cells of endosperm contain-
ing fatty oil, fragments of sclerenchyma with characteristic
single pits, fragments of oil canal within yellowish brown
material, fragments of endocarp shown scalariform, spiral
1286 Fennel Oil / Crude Drugs
JP XV
vessels, epidermis, stomata.
Identification To 0.5 g of Powdered Fennel add 10 mL of
hexane, allow to stand for 5 minutes with occasional shaking,
filter, and use the filtrate as the sample solution. Perform the
test with the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 5 /xL of the sample solution on
a plate prepared with silica gel with fluorescent indicator for
thin-layer chromatography. Then develop the plate with a
mixture of hexane and ethyl acetate (20:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): a main spot with dark pur-
ple color appears at the Rf value of about 0.4.
Total ash <J.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Fennel: the volume of essential oil is not less than
0.45 mL.
Containers and storage Containers — Tight containers.
Fennel Oil
Oleum Foeniculi
Fennel Oil is the essential oil distilled with steam
from the fruit of Foeniculum vulgare Miller (Umbel-
liferae) or of Illicium verum Hooker fil. (Illiciaceae).
Description Fennel Oil is a colorless to pale yellow liquid. It
has a characteristic, aromatic odor and a sweet taste with a
slight, bitter aftertaste.
It is miscible with ethanol (95) and with diethyl ether.
It is practically insoluble in water.
When cold, white crystals or crystalline masses may often
separate from the oil.
Identification Dissolve 0.30 g of Fennel Oil in 20 mL of
hexane, pipet 1 mL of this solution, add hexane to make ex-
actly 10 mL, and use this solution as the sample solution.
Perform the test with this solution as directed under Thin-
layer Chromatography <2.03>. Spot 5 /uL of the sample solu-
tion on a plate of silica gel with fluorescent indicator for thin-
layer chromatography. Develop the plate with a mixture of
hexane and ethyl acetate (20:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): a main spot with a dark purple color
appears at the Rf value of about 0.4.
Refractive index <2.45> n 2 °: 1.528 - 1.560
Specific gravity <7.75> df : 0.955 - 0.995
Purity (1) Clarity of solution — To 1.0 mL of Fennel Oil
add 3 mL of ethanol (95): the solution is clear. To this solu-
tion add 7 mL of ethanol (95): the solution remains clear.
(2) Heavy metals <1.07> — Proceed with 1 .0 mL of Fennel
Oil according to Method 2, and perform the test. Prepare the
control solution with 4.0 mL of Standard Lead Solution (not
more than 40 ppm).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Foeniculated Ammonia Spirit
T>=E-T-0^^3^m
Method of preparation
Ammonia Water
Fennel Oil
Ethanol
170 mL
30 mL
a sufficient quantity
To make 1000 mL
Prepare as directed under Medicated Spirits, with the
above ingredients. A sufficient quantity of ammonia solution
(28) and Purified Water may be used in place of Ammonia
Water.
Description Foeniculated Ammonia Spirit is a colorless to
yellow liquid, having a characteristic odor. It has a slightly
sweet, pungent taste.
Specific gravity df : about 0.85
Alcohol number <7.07> Not less than 7.8 (Method 2).
Containers and storage Containers — Tight containers.
Forsythia Fruit
Forsythiae Fructus
Forsythia Fruit is the fruit of Forsythia suspensa
Vahl or Forsythia viridissima Lindley (Oleaceae).
Description Ovoid to long ovoid capsule, 1.5 -2.5 cm in
length, 0.5 - 1 cm in width, with acute apex, and sometimes
with a peduncle at the base; externally light gray to dark
brown, scattered with light gray and small ridged dots, and
with two longitudinal furrows; a capsule dehiscing along the
longitudinal furrows has the apexes bent backward; the inner
surface of dehisced pericarp is yellow-brown in color, with a
longitudinal partition-wall in the middle; seeds, slender and
oblong, 0.5 - 0.7 cm in length, and usually with a wing.
Odor, slight; tasteless.
Identification (1) To 0.2 g of pulverized Forsythia Fruit
add 2 mL of acetic anhydride, shake well, allow to stand for 2
minutes, and filter. To 1 mL of the filtrate add gently 0.5 mL
of sulfuric acid to form two layers: a red-purple color de-
velops at the zone of contact.
(2) To 1 g of pulverized Forsythia Fruit add 10 mL of
methanol, warm on a water bath for 2 minutes, and filter. To
5 mL of the filtrate add 0.1 g of magnesium in ribbon form
and 1 mL of hydrochloric acid, and allow to stand: a light red
to yellow-red color develops.
Purity (1) Branchlet — The amount of branchlets con-
tained in Forsythia Fruit does not exceed 5.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than branchlets contained in Forsythia Fruit does not
exceed 1.0%.
JPXV
Crude Drugs / Powdered Gambir 1287
Total ash <5.01> Not more than 5.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 10.0%.
Fritillaria Bulb
Fritillariae Bulbus
Fritillaria Bulb is the bulb of Fritillaria verticillata
Willdenow var. thunbergii Baker (Liliaceae).
Description Fritillaria Bulb is a depressed spherical bulb, 2
to 3 cm in diameter, 1 to 2 cm in height, consisting of 2 thick-
ened scaly leaves often separated; externally and internally
white to light yellow-brown in color; inside base is in a slight-
ly dark color; the bulb sprinkled with lime before drying is
dusted with white powder; fractured surface, white in color
and powdery.
Odor, slight and characteristic; taste, bitter.
Under the microscope <5.01>, a transverse section reveals
the outermost layer (epidermis) to be composed of a single
layer of cells; numerous vascular bundles scattered through-
out the parenchyma inside of the epidermis; parenchyma
filled with starch grains; starch grains are mainly simple (rare-
ly 2 - 3 composite), 5-50 /um in diameter, narrowly ovate to
ovate or triangular to obovate, stratiform figure obvious;
epidermal cells and parenchymatous cells near the vessels
contain solitary crystals of calcium oxalate.
Identification Put 2 g of pulverized Fritillaria Bulb in a
glass-stoppered centrifuge tube, add 10 mL of ammonia TS
and 20 mL of a mixture of ethyl acetate and diethyl ether
(1:1), shake for 20 minutes, and centrifuge. Take the upper
layer, add 20 g of anhydrous sodium sulfate to the layer,
shake, and filter. Evaporate the filtrate to dryness, dissolve
the residue in 1 mL of ethanol (99.5), and use this solution as
the sample solution. Perform the test with the sample solu-
tion as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL of the sample solution on a plate of silica gel for
thin-layer chromatography, develop the plate with a mixture
of ethyl acetate, methanol and ammonia solution (28)
(17:2:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly Dragendorff's TS for spraying on the plate: two
spots of a yellow-red color appear at around Rf 0.4 and at
around Rf 0.6.
Loss on drying <5.01> Not more than 16.0% (6 hours).
Total ash <5.01> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 8.0%.
Gambir
Gambir
Gambir is the dried aqueous extract prepared from
the leaves and young twigs of Uncaria gambir Rox-
burgh (Rubiaceae).
Description Brown to dark brown, brittle mass; inside light
brown.
Odor, slight; taste, extremely astringent and bitter.
Identification (1) To 0.2 g of pulverized Gambir add 10
mL of water, warm in a water bath for 5 minutes with oc-
casional shaking, and filter. Cool the filtrate, and add 2 to 3
drops of gelatin TS: a white turbidity or precipitate is
produced.
(2) Shake 0.1 g of pulverized Gambir with 20 mL of di-
lute ethanol for 2 minutes, and filter. Mix 1 mL of the filtrate
with 9 mL of dilute ethanol, and to the solution add 1 mL of
vanillin-hydrochloric acid TS: a light red to red-brown color
develops.
Total ash <5.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01>
less than 70.0%.
Dilute ethanol-soluble extract: not
Powdered Gambir
Gambir Pulveratum
7ir>V?S
Powdered Gambir is the powder of Gambir.
Description Powdered Gambir occurs as a red-brown to
dark brown powder. It has a slight odor, and an extremely as-
tringent and bitter taste.
Under a microscope <5.01>, Powdered Gambir, immersed
in olive oil or liquid paraffin, consists of needle crystalline
masses or yellow-brown to red-brown angular fragments,
and reveals epidermal tissue and thick-walled hairs.
Identification (1) To 0.2 g of Powdered Gambir add 10
mL of water, warm in a water bath for 5 minutes with oc-
casional shaking, and filter. Cool the filtrate, and add 2 to 3
drops of gelatin TS: a white turbidity or precipitate is
produced.
(2) Shake 0.1 g of Powdered Gambir with 20 mL of di-
lute ethanol for 2 minutes, and filter. Mix 1 mL of the filtrate
with 9 mL of dilute ethanol, and to the solution add 1 mL of
vanillin-hydrochloric acid TS: a light red to red-brown color
develops.
Total ash <5.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
1288 Gardenia Fruit / Crude Drugs
less than 70.0%.
Gardenia Fruit
Gardeniae Fructus
t>yy
Gardenia Fruit is the fruit of Gardenia jasminoides
Ellis (Rubiaceae).
It contains not less than 3.0% of geniposide, calcu-
lated on the basis of dried material.
Description Nearly long ovoid to ovoid fruit, 1-5 cm in
length, 1-1.5 cm in width; usually having 6, rarely 5 or 7,
markedly raised ridges; calyx or its scar at one end, and
sometimes peduncle at the other end; inner surface of
pericarp yellow-brown, smooth and lustrous; internally
divided into two loculi, containing a mass of seeds in yellow-
red to dark red placenta; seed nearly circular, flat, about 0.5
cm in major axis, blackish brown or yellow-red.
Odor, slight; taste, bitter.
Identification (1) To 1.0 g of pulverized Gardenia Fruit,
previously dried in a desiccator (silica gel) for 24 hours, add
100 mL of hot water, warm the mixture between 60°C and
70°C for 30 minutes with frequent shaking, and filter after
cooling. To 1.0 mL of the filtrate add water to make 10 mL:
the color of the resulting solution is yellow and is not lighter
than that of the following control solution.
Control solution: Dissolve 9.8 mg of carbazochrome sodi-
um sulfonate for component determination in water to make
exactly 10 mL. Pipet 1 mL of this solution, and add water to
make exactly 50 mL.
(2) To 1.0 g of pulverized Gardenia Fruit add 20 mL of
methanol, warm for 3 minutes on a water bath, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of geniposide for thin-layer chromatography in 1
mL of methanol, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate and methanol (3 : 1) to a distance of about
10 cm, and air-dry the plate. Spray evenly 4-methoxybenzal-
dehyde-sulfuric acid TS on the plate, and heat at 105°C for
10 minutes: one spot among the spots from the sample solu-
tion and a dark purple spot from the standard solution show
the same color tone and the same Rf value.
Loss on drying <5.0]> Not more than 13.0%.
Total ash <5.01> Not more than 6.0%.
Component determination Weigh accurately about 0.5 g of
pulverized Gardenia Fruit, transfer into a glass-stoppered
centrifuge tube, add 40 mL of diluted methanol (1 in 2),
shake for 15 minutes, centrifuge, and take the supernatant
liquid. To the residue add 40 mL of diluted methanol (1 in 2),
and repeat the same procedure as above. Combine the ex-
tracts so obtained, and add diluted methanol (1 in 2) to make
exactly 100 mL. Pipet 5 mL of the solution, add methanol to
make exactly 20 mL, use this solution as the sample solution.
Separately, weigh accurately about 10 mg of geniposide for
JP XV
component determination, previously dried in a desiccator
(in vacuum, phosphorus (V) oxide) for 24 hours, and dissolve
in methanol to make exactly 100 mL. Pipet 5 mL of the solu-
tion, add methanol to make exactly 10 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
,uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and measure the peak areas of
geniposide, A T and A s , of both solutions.
Amount (mg) of geniposide =W s x (A T /A S ) x 2
W s : Amount (mg) of geniposide for component determi-
nation
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 240 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5/«n in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (22:3).
Flow rate: Adjust the flow rate so that the retention time of
geniposide is about 15 minutes.
System suitability —
System performance: Dissolve 1 mg each of geniposide for
component determination and caffeine in methanol to make
15 mL. When the procedure is run with 10 /xh of this solution
under the above operating conditions, caffeine and genipo-
side are eluted in this order with the resolution between these
peaks being not less than 3.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
geniposide is not more than 1.5%.
Powdered Gardenia Fruit
Gardeniae Fructus Pulveratus
t>yyj
Powdered Gardenia Fruit is the powder of Gardenia
Fruit.
It contains not less than 3.0% of geniposide, calcu-
lated on the basis of dried material.
Description Powdered Gardenia Fruit occurs as a yellow-
brown powder, and has a slight odor and a bitter taste.
Under a microscope <5.01>, Powdered Gardenia Fruit rev-
eals fragments of yellow-brown epidermis consisting of poly-
gonal epidermal cells in surface view; unicellular hairs, spiral
and ring vessels, stone cells often containing crystals of calci-
um oxalate; fragments of thin-walled parenchyma containing
yellow pigments, oil drops and rosette aggregates of calcium
oxalate (the above elements from fruit receptacle and
pericarp); fragments of large and thick-walled epidermis of
seed coat, containing a red-brown substance; fragments of
endosperm filled with aleuron grains (the above elements
from seed).
Identification (1) To 1.0 g of Powdered Gardenia Fruit,
JP XV
Crude Drugs / Gastrodia Tuber 1289
previously dried in a desiccator (silica gel) for 24 hours, add
100 mL of hot water, warm the mixture between 60°C and 70
°C for 30 minutes with frequent shaking, and filter after cool-
ing. To 1.0 mL of the filtrate add water to make 10 mL: the
color of the resulting solution is yellow and is not lighter than
that of the following control solution.
Control solution: Dissolve 9.8 mg of carbazochrome
sodium sulfonate for component determination in water to
make exactly 10 mL. Pipet 1 mL of this solution, and add
water to make exactly 50 mL.
(2) To 1.0 g of Powdered Gardenia Fruit add 20 mL of
methanol, warm for 3 minutes on a water bath, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of geniposide for thin-layer chromatography in 1
mL of methanol, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate and methanol (3 : 1) to a distance of about
10 cm, and air-dry the plate. Spray evenly 4-methoxybenzal-
dehyde-sulfuric acid TS on the plate, and heat at 105°C for
10 minutes: one spot among the spots from the sample solu-
tion and a dark purple spot from the standard solution show
the same in color tone and Rf value.
Loss on drying <5.01> Not more than 13.0%.
Total ash <5.01> Not more than 6.0%.
Component determination Weigh accurately about 0.5 g of
Powdered Gardenia Fruit, transfer into a glass-stoppered
centrifuge tube, add 40 mL of diluted methanol (1 in 2),
shake for 15 minutes, centrifuge, and take the supernatant
liquid. To the residue add 40 mL of diluted methanol (1 in 2),
and repeat the same procedure as above. Combine the ex-
tracts so obtained, and add diluted methanol (1 in 2) to make
exactly 100 mL. Pipet 5 mL of the solution, add methanol to
make exactly 20 mL, use this solution as the sample solution.
Separately, weigh accurately about 10 mg of geniposide for
component determination, previously dried in a desiccator
(in vacuum, phosphorus (V) oxide) for 24 hours, and dissolve
in methanol to make exactly 100 mL. Pipet 5 mL of the solu-
tion, add methanol to make exactly 10 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
iiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and measure the peak areas of
geniposide, A T and A s , of both solutions.
Amount (mg) of geniposide = W s x (A T /A S ) x 2
W s : Amount (mg) of geniposide for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 ^m in particle diameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (22:3).
Flow rate: Adjust the flow rate so that the retention time of
geniposide is about 15 minutes.
System suitability —
System performance: Dissolve 1 mg each of geniposide for
component determination and caffeine in methanol to make
15 mL. When the procedure is run with 10 iiL of this solution
under the above operating conditions, caffeine and genipo-
side are eluted in this order with the resolution between these
peaks being not less than 3.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
geniposide is not more than 1.5%.
Gastrodia Tuber
Gastrodiae Tuber
Gastrodia Tuber is the steamed tuber of Gastrodia
elata Blume (Orchidaceae).
Description Gastrodia Tuber is an irregularly curved and
flattened cylindrical to flattened fusiform tuber, 5 to 15 cm in
length, 2 to 5 cm in diameter, 1 to 2 cm in thickness; external-
ly light yellow-brown to light yellowish white; with ring
nodes, and irregular longitudinal wrinkles; hard in texture;
fractured surface, dark brown to yellow-brown in color, with
luster, horny and gluey.
Odor, characteristic; practically tasteless.
Under a microscope <5.01>, a transverse section reveals
parenchyma cells containing needle raphides of calcium oxa-
late; starch grain absent.
Identification To 1 g of pulverized Gastrodia Tuber add 5
mL of methanol, shake for 15 minutes, and filter. Evaporate
the filtrate to dryness, dissolve the residue in 1 mL of
methanol, and use this solution as the sample solution. Per-
form the test with the sample solution as directed under Thin-
layer Chromatography <2.03>. Spot 10 fiL of the sample solu-
tion on a plate of silica gel for thin-layer chromatography,
develop the plate with a mixture of ethyl acetate, methanol
and water (8:2:1) to a distance of about 10 cm, and air-dry
the plate. Spray evenly dilute sulfuric acid on the plate, and
heat at 105°C for 1 minutes: a red-purple spot appears at
around Rf 0.4.
Loss on drying <5.01> Not more than 16.0% (6 hours).
Total ash <5.07> Not more than 4.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 16.0%.
1290 Gentian / Crude Drugs
JP XV
Gentian
Gentianae Radix
Gentian is the root and rhizome of Gentiana lutea
Linne (Gentianaceae) .
Description Nearly cylindrical pieces, 10 - 50 cm in length,
2-4 cm in daiameter; externally dark brown; the rhizome
short, with fine, transverse wrinkles, and sometimes with
buds and remains of leaves at the upper edge. The root lon-
gitudinally and deeply wrinkled, and more or less twisted;
fractured surface yellow-brown and not fibrous, and a cambi-
um and its neighborhood tinged dark brown.
Odor, characteristic; taste, sweet at first, later persistently
bitter.
Under a microscope <5.01>, a transverse section of the root
reveals several layers of collenchyma adjoined internally to 4
to 6 layers of thin-walled cork; secondary cortex of the paren-
chyma with irregularly distributed phloem; xylem consisting
chiefly of parenchyma, with individual or clustered vessels
and tracheids, and exhibiting some sieve tubes of xylem;
parenchyma of the xylem and the cortex containing oil
droplets, minute needle crystals of calcium oxalate and very
rarely starch grains 10 - 20 tun in diameter.
Identification (1) Place 0.1 g of pulverized Gentian, previ-
ously dried in a desiccator (silica gel) for 48 hours, on a slide
glass, put a glass ring 10 mm in both inside diameter and in
height on it, then cover with another slide, and heat gently
and gradually: pale yellow crystals are sublimed on the upper
slide. The crystals are insoluble in water and in ethanol (95),
and soluble in potassium hydroxide TS.
(2) To 0.5 g of pulverized Gentian add 10 mL of
methanol, shake for 5 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 1 mg of gentiopicro-
side for thin-layer chromatography in 1 mL of methanol, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10,mL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, ethanol (99.5) and water
(8:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
one spot among the spots from the sample solution and a
dark purple spot from the standard solution show the same
color tone and the same Rf value.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Powdered Gentian
Gentianae Radix Pulverata
Powdered Gentian is the powder of Gentian.
Description Powdered Gentian occurs as a yellowish brown
powder, and has a characteristic odor. It has a sweet taste at
first, which later becomes persistently bitter.
Under a microscope <5.01>, Powdered Gentian reveals
parenchyma cells containing oil droplets and minute needle
crystals, vessels, tracheids, cork tissues, and crystals of calci-
um oxalate. Vessels are chiefly reticulate vessels and
scalariform vessels, 20 - 80 /um in diameter. Starch grains are
observed very rarely, in simple grains about 10 - 20 iim in di-
ameter.
Identification (1) Place 0.1 g of Powdered Gentian, previ-
ously dried in a desiccator (silica gel) for 48 hours, on a slide
glass, put a glass ring 10 mm in both inside diameter and in
height on it, then cover with another slide glass, and heat
gently and gradually: light yellow crystals are sublimed on the
upper glass. The crystals are insoluble in water and in ethanol
(95), and soluble in potassium hydroxide TS.
(2) To 0.5 g of Powdered Gentian add 10 mL of
methanol, shake for 5 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 1 mg of gentiopicro-
side for thin-layer chromatography in 1 mL of methanol, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 fiL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, ethanol (99.5) and water
(8:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 254 nm):
one spot among the spots from the sample solution and a
dark purple spot from the standard solution show the same
color tone and the same Rf value.
Purity Foreign matter — Under a microscope <5.01>, no
stone cell or fiber is observed.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Containers and storage Containers — Tight containers.
Gentian and Sodium Bicarbonate
Powder
*f>f-Ti-
Method of preparation
Powdered Gentian
Sodium Bicarbonate
300 g
700 g
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Gentian and Sodium Bicarbonate Powder oc-
curs as a light yellow-brown powder, and has a bitter taste.
Identification (1) To 2 g of Gentian and Sodium Bicar-
bonate Powder add 10 mL of water, stir, and filter: the
filtrate responds to the Qualitative Tests <1.09> (1) for bicar-
bonate.
JPXV
Crude Drugs / Ginger 1291
(2) To 1.5 g of Gentian and Sodium Bicarbonate Powder
add 10 mL of methanol, shake for 5 minutes, filter, and use
the filtrate as the sample solution. Separately, dissolve 1 mg
of gentiopicroside for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (99.5) and
water (8:2:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): one spot among the spots from the sample solution and
a dark purple spot from the standard solution show the same
color tone and the same Rf value.
Containers and storage Containers — Well-closed contain-
ers.
Geranium Herb
Geranii Herba
f > J v a O =i
Geranium Herb is the terrestrial part of Geranium
thunbergii Siebold et Zuccarini (Geraniaceae).
Description Stem with leaves opposite; stem, slender and
long, green-brown; stem and leaf covered with soft hairs; leaf
divided palmately into 3 to 5 lobes, and 2-4 cm in length,
grayish yellow-green to grayish brown; each lobe oblong to
obovate, and its upper margin crenate.
Odor, slight; taste, astringent.
Identification Boil 0.1 g of Geranium Herb with 10 mL of
water, filter, and to the filtrate add 1 drop of iron (III) chlo-
ride TS: a dark blue color develops.
Purity Foreign matter <5.01> — The amount of the root and
other foreign matter contained in Geranium Herb does not
exceed 2.0%.
Total ash <5.01> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 15.0%.
Powdered Geranium Herb
Geranii Herba Pulverata
Powdered Geranium Herb is the powder of Gerani-
um Herb.
Description Powdered Geranium Herb occurs as a grayish
green to light yellow-brown powder. It has a slight odor and
an astringent taste.
Under a microscope <5.01>, Powdered Geranium Herb rev-
eals mainly fibers, spiral vessels, pitted vessels, and unicellu-
lar hairs; furthermore, multicellular glandular hairs, epider-
mis with stomata, fragments of palisade tissue, rosette ag-
gregates of calcium oxalate, and starch grains. Fiber is thick-
walled, with somewhat distinct pits; unicellular hair shows
small point-like protrusions on the surface; palisade tissue
consisting of circular parenchyma cells in surface view, each
cell containing one rosette aggregate of calcium oxalate
which is about 20 /xm in diameter. Starch grains consisting of
simple grains but rarely of 2-compound grains, ovoid to
spherical, 5 - 30 ^m in diameter, with distinct hilum.
Identification Boil 0.1 g of Powdered Geranium Herb with
10 mL of water, filter, and to the filtrate add 1 drop of iron
(III) chloride TS: a dark blue color develops.
Purity Foreign matter — Under a microscope <5.01>, Pow-
dered Geranium Herb reveals no stone cells.
Total ash <5.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 15.0%.
Ginger
Zingiberis Rhizoma
-> a ^ + a ^
Ginger is the rhizome of Zingiber officinale Roscoe
(Zingiberaceae).
Description Irregularly compressed and often branched
massive rhizome or a part of it; the branched parts are slight-
ly curved ovoid or oblong-ovoid, 2-4 cm in length, and 1-2
cm in diameter; external surface grayish white to light grayish
brown, and often with white powder; fractured surface is
somewhat fibrous, powdery, light yellowish brown; under a
magnifying glass, a transverse section reveals cortex and stele
distinctly divided; vascular bundles and secretes scattered all
over the surface as small dark brown dots.
Odor, characteristic; taste, extremely pungent.
Identification To 2 g of pulverized Ginger add 5 mL of
diethyl ether, shake for 10 minutes, filter, and use the filtrate
as the sample solution. Separately, dissolve 1 mg of [6]-gin-
gerol for thin-layer chromatography in 2 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 fiL of the sample solution and
standard solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 4-dimethylaminobenzaldehyde
TS on the plate, heat at 105 °C for 5 minutes, and allow to
cool: one of the spots from the sample solution and a green
spot from the standard solution show the same color tone
and Rf value.
Purity (1) Heavy metals <l.07> — Proceed with 3.0 g of
pulverized Ginger according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 10 ppm).
1292 Powdered Ginger / Crude Drugs
JP XV
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Ginger according to Method 4, and perform the
test (not more than 5 ppm).
Total ash <J.07> Not more than 8.0%
Ginseng
Ginseng Radix
Powdered Ginger
Zingiberis Rhizoma Pulveratum
Powdered Ginger is the powder of Ginger.
Description Powdered Ginger occurs as a light grayish
brown to light grayish yellow powder. It has a characteristic
odor and an extremely pungent taste.
Under a microscope <5.01>, Powdered Ginger reveals
mainly starch grains and parenchyma cells containing them;
also, parenchyma cells containing yellow-brown to dark
brown resinous substances or single crystals of calcium oxa-
late; fragments of fibers with distinct pits; fragments of
spiral, ring and reticulate vessels, and rarely fragments of
cork tissue; starch grains composed of simple, compound or
half-compound grains, spherical, ovoid or globular, with
abaxial hilum, usually 20 - 30 iim. in long axis.
Identification To 2 g of Powdered Ginger add 5 mL of
diethyl ether, shake for 10 minutes, filter, and use the filtrate
as the sample solution. Separately, dissolve 1 mg of [6]-gin-
gerol for thin-layer chromatography in 2 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 iiL of the sample solution and
standard solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 4-dimethylaminobenzaldehyde
TS on the plate, heat at 105 °C for 5 minutes, and allow to
cool: one of the spots from the sample solution and a green
spot from the standard solution show the same color tone
and Ri value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Ginger according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Ginger according to Method 4, and perform the
test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Ginger does not show stone cells, lignified parenchyma
cells and other foreign matter.
Total ash <5.01> Not more than 8.0%.
Containers and storage Containers — Tight containers.
Ginseng is the root of Panax ginseng C. A. Meyer
(Panax schinseng Nees) (Araliaceae), from which root-
lets have been removed, or the root that has been
quickly passed through hot water.
It contains not less than 0.10% of ginsenoside Rgj
(C 42 H 72 14 : 801.01) and not less than 0.20%, of gin-
senoside Rbj (C54H92O23: 1109.29), calculated on the
basis of dried material.
Description Thin and long cylindrical to fusiform root,
often branching 2 to 5 lateral roots from the middle; 5-20
cm in length, main root 0.5 - 3 cm in diameter; externally
light yellow-brown to light grayish brown, with longitudinal
wrinkles and scars of rootlets; sometimes crown somewhat
constricted and with short remains of rhizome; fractured sur-
face practically flat, light yellow-brown in color, and brown
in the neighborhood of the cambium.
Odor, characteristic; taste, at first slightly sweet, followed
by a slight bitterness.
Identification (1) On a section of Ginseng add dilute io-
dine TS dropwise: a dark blue color is produced on the sur-
face.
(2) To 2.0 g of pulverized Ginseng add 20 mL of
methanol, boil gently under a reflux condenser on a water
bath for 15 minutes, cool, filter, and use the filtrate as the
sample solution. Separately, dissolve 1 mg of Ginsenoside
Rg t Reference Standand in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 11L each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with the lower layer of a mix-
ture of chloroform, methanol and water (13:7:2) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 110°C for 5 minutes: one
of the spots from the sample solution and a red-purple spot
from the standard solution show the same color tone and the
same Rf value.
Purity (1) Foreign matter <5.01> — The amount of stems
and other foreign matter contained in Ginseng does not ex-
ceed 2.0%.
(2) Heavy metals <1.07> — Proceed with 1.0 g of pulver-
ized Ginseng according to Method 4, and perform the test.
Prepare the control solution with 1.5 mL of Standard Lead
Solution (not more than 15 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of pulverized Ginseng according to Method 4, and perform
the test (not more than 2 ppm).
(4) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.07> Not more than 4.2%.
Extract content <5.0I> Dilute ethanol-soluble extract: not
JPXV
Crude Drugs / Powdered Ginseng 1293
less than 14.0%.
Assay (1) Ginsenoside Rgj — Weigh accurately about 1 .0 g
of pulverized Ginseng, put in a glass-stoppered centrifuge
tube, add 30 mL of diluted methanol (3 in 5), shake for 15
minutes, centrifuge, and separate the supernatant liquid.
Repeat the procedure with the residue using 15 mL of diluted
methanol (3 in 5), combine the supernatant liquids, and add
diluted methanol (3 in 5) to make exactly 50 mL. Pipet 10 mL
of this solution, add 3 mL of dilute sodium hydroxide TS, al-
low to stand for 30 minutes, add 3 mL of 0.1 mol/L
hydrochloric acid TS and diluted methanol (3 in 5) to make
exactly 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of Ginsenoside Rg t
Reference Standard (previously determine the water) dissolve
in diluted methanol (3 in 5) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 /uL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of ginsenoside Rg t .
Amount (mg) of ginsenoside Rgj (C4 2 H 7 20 14 ) = W s x (A T /A S )
W s : Amount (mg) of Ginsenoside Rgi Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rgi is about 25 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rgj Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 fiL of this solution under the above operating condi-
tions, ginsenoside Rg! and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rg! is not more than 1.5%.
(2) Ginsenoside Rbi — Use the sample solution obtained
in (1) as the sample solution. Separately, weigh accurately
about 10 mg of Ginsenoside Rbi Reference Standard (previ-
ously determine the water) dissolve in diluted methanol (3 in
5) to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 10 fiL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and determine the peak areas, A T and A s , of ginseno-
side Rbi,
Amount (mg) of ginsenoside Rbi (C 5 4H 92 023) = W s x (^4 T A4 S )
W s : Amount (mg) of Ginsenoside Rb t Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rbi is about 20 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rbi Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 /xL of this solution under the above operating condi-
tions, ginsenoside Rbi and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rbi is not more than 1.5%.
Powdered Ginseng
Ginseng Radix Pulverata
— >i/>~%.
Powdered Ginseng is the powder of Ginseng.
It contains not less than 0.10% of ginsenoside Rg!
(C4 2 H 72 14 : 801.01) and not less than 0.20% of gin-
senoside Rbi (C54H92O23: 1109.29), calculated on the
basis of dried material.
Description Powdered Ginseng occurs as a light yellowish
white to light yellowish-brown powder. It has characteristic
odor and is a slight sweet taste followed by a slight bitterness.
Under a microscope <5.01>, Powdered Ginseng reveals
round to rectangular parenchyma cells containing starch
grains, occasionally gelatinized starch, vessels, secretory cell,
sclerenchyma cell, big and thin-walled cork cell; crystals of
calcium oxalate and starch. Vessel are reticulate vessel, 45 /um
in diameter; scalariform vessel and spiral vessel, 15 to 40,Mm
in diameter. Secretory cell containing a mass of yellow
glistened contents; rosette aggregate of calcium oxalate, 20 to
50,wm in diameter, and 1 to 5 /xm in diameter, rarely 10 /xm,
in diameter. Starch grains are observed in simple grain and 2
to 4-compound grain, simple grain, 3 to 15//m in diameter.
Identification To 2.0 g of Powdered Ginseng add 20 mL of
methanol, boil gently under a reflux condenser on a water
bath for 15 minutes, cool, filter, and use the filtrate as the
sample solution. Separately, dissolve 1 mg of Ginsenoside
Rgi Reference Standard in 1 mL of methanol, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with the lower layer of a mix-
1294 Glehnia Root / Crude Drugs
JP XV
ture of chloroform, methanol and water (13:7:2) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 110°C for 5 minutes: one
of the spots from the sample solution and a red-purple spot
from the standard solution show the same color tone and the
same Ri value.
Purity (1) Heavy metals <1.07> — Proceed with 1.0 g of
Powdered Ginseng according to Method 4, and perform the
test. Prepare the control solution with 1.5 mL of Standard
Lead Solution (not more than 15 ppm).
(2) Arsenic </.//> — Prepare the test solution with 1.0 g
of Powdered Ginseng according to Method 4, and perform
the test (not more than 2 ppm).
(3) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.01> Not more than 4.2%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Extract content <5.01> Dilute ethanol-soluble extract; not
less than 14.0%.
Assay (1) Ginsenoside Rgi — Weigh accurately about 1 .0 g
of Powdered Ginseng, put in a glass-stoppered centrifuge
tube, add 30 mL of diluted methanol (3 in 5), shake for 15
minutes, centrifuge, and separate the supernatant liquid.
Repeat the procedure with the residue using 15 mL of diluted
methanol (3 in 5), combine the supernatant liquids, and add
diluted methanol (3 in 5) to make exactly 50 mL. Pipet 10 mL
of this solution, add 3 mL of dilute sodium hydroxide TS,
allow to stand for 30 minutes, add 3 mL of 0.1 mol/L
hydrochloric acid TS and diluted methanol (3 in 5) to make
exactly 20 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of Ginsenoside Rgi
Reference Standard (previously determine the water) dissolve
in diluted methanol (3 in 5) to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
exactly 10 fiL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions, and determine the peak
areas, A T and A s , of ginsenoside Rg!.
Amount (mg) of ginsenoside Rgi (C42H72O14) = W s x (,4 T A4 S )
W s : Amount (mg) of Ginsenoside Rg] Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rgi is about 25 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rg! Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 fiL of this solution under the above operating condi-
tions, ginsenoside Rgi and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 1.5.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rgi is not more than 1.5%.
(2) Ginsenoside Rb t — Use the sample solution obtained
in (1) as the sample solution. Separately, weigh accurately
about 10 mg of Ginsenoside Rbi Reference Standard,
separately determined its water content, dissolve in diluted
methanol (3 in 5) to make exactly 100 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
fiL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of ginsenoside Rb,.
Amount (mg) of ginsenoside Rbi (C54H92O23) = IV S x (^4 T A4 S )
W s : Amount (mg) of Ginsenoside Rb! Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 ftm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rbi is about 20 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rbi Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 fiL of this solution under the above operating condi-
tions, ginsenoside Rbi and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rbi is not more than 1.5%.
Containers and storage Containers — Tight containers.
Glehnia Root
Glehniae Radix cum Rhizoma
Glehnia Root is the root and rhizome of Glehnia lit-
toralis Fr. Schmidt ex Miquel (Umbelliferae).
Description Cylindrical to long conical root or rhizome, 10
- 20 cm in length, 0.5 - 1.5 cm in diameter; externally light
yellow-brown to red-brown. Rhizome short, with fine ring
nodes; roots having longitudinal wrinkes and numerous,
dark red-brown, warty protrusions or transversely elongated
JP XV
Crude Drugs / Glycyrrhiza 1295
protuberances. Brittle and easily breakable. A transverse sec-
tion white and powdery, and under a magnifying glass, oil
canals scattered as brown dots.
Odor, slight; taste, slightly sweet.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Glycyrrhiza
Glycyrrhizae Radix
Glycyrrhiza is the root and stolon, with (impeded)
or without (peeled) the periderm, of Glycyrrhiza ura-
lensis Fisher or Glycyrrhiza glabra Linne (Legumino-
sae).
It contains not less than 2.5% of glycyrrhizic acid
(C 42 H 62 16 : 822.93), calculated on the basis of dried
material.
Description Nearly cylindrical pieces, 0.5 -3.0 cm in di-
ameter, over 1 m in length. Glycyrrhiza is externally dark
brown to red-brown, longitudinally wrinkled, and often has
lenticels, small buds and scaly leaves; peeled Glycyrrhiza is
externally light yellow and fibrous. The transverse section rev-
eals a rather clear border between phloem and xylem, and a
radial structure which often has radiating splits; a pith in
Glycyrrhiza originated from stolon, but no pith from root.
Odor, slight; taste, sweet.
Under a microscope <5.01>, the transverse section reveals
several layers of yellow-brown cork layers, and 1- to 3-cellu-
lar layer of cork cortex inside the cork layer; the cortex ex-
hibiting medullary rays and obliterated sieve portions radiat-
ed alternately; the phloem exhibiting groups of phloem fibers
with thick but incompletely lignified walls and surrounded by
crystal cells; peeled Glycyrrhiza some times lacks periderm
and a part of phloem; the xylem exhibiting large yellow ves-
sels and medullary rays in 3 to 10 rows radiated alternately;
the vessels accompanied with xylem fibers surrounded by
crystal cells, and with xylem parenchyma cells; the paren-
chymatous pithonly in Glycyrrhiza originated from stolon.
The parenchyma cells contain starch grains and often solitary
crystals of calcium oxalate.
Identification To 2 g of pulverized Glycyrrhiza add 10 mL
of a mixture of ethanol (95) and water (7:3), heat by shaking
on a water bath for 5 minutes, cool, filter, and use the filtrate
as the sample solution. Separately, dissolve 5 mg of Glycyr-
rhizinic Acid Reference Standard in 1 mL of a mixture of
ethanol (95) and water (7:3), and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 2 /uL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (7:2:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): one spot among the spots from
the sample solution and a dark purple spot from the standard
solution show the same color tone and the same Ri value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Glycyrrhiza according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than lOppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Glycyrrhiza according to Method 4, and per-
form the test (not more than 5 ppm).
(3) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 25.0%.
Assay Weigh accurately about 0.5 g of pulverized Glycyr-
rhiza in a glass-stoppered centrifuge tube, add 70 mL of di-
lute ethanol, shake for 15 minutes, centrifuge, and separate
the supernatant liquid. To the residue add 25 mL of dilute
ethanol, and proceed in the same manner. Combine all the
extracts, add dilute ethanol to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Glycyrrhizic Acid Reference Stan-
dard (previously determine the water), dissolve in dilute
ethanol to make exactly 100 mL, and use this solution as the
standard solution. Pipet 20 fiL each of the sample solution
and standard solution, and perform the test as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine the peak areas, A r and A s , of glycyr-
rhizic acid of each solution.
Amount (mg) of glycyrrhizic acid (C 42 H 62 Oi 6 )
= W s x(A T /A s )
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: Use a column 4 to 6 mm in inside diameter and 15
to 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 to 10 mL in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
glycyrrhizic acid is about 10 minutes.
Selection of column: Dissolve 5 mg of Glycyrrhizic Acid
Reference Standard and 1 mg of propyl parahydroxybenzo-
ate in dilute ethanol to make 20 mL. Proceed with 20 /xh of
this solution under the above operating conditions. Use a
column giving elution of glycyrrhizic acid and propyl para-
hydroxybenzoate in this order, and clearly dividing each
peak.
System repeatability: Repeat the test 5 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of glycyrrhizic acid is
not more than 1.5%.
1296 Powdered Glycyrrhiza / Crude Drugs
JP XV
Powdered Glycyrrhiza
Glycyrrhizae Radix Pulverata
Powdered Glycyrrhiza is the powder of Glycyrrhiza.
It contains not less than 2.5% of glycyrrhizic acid
(C 42 H 62 16 : 822.93), calculated on the basis of dried
material.
Description Powdered Glycyrrhiza is light yellow-brown or
light yellow to grayish yellow (powder of peeled Glycyrrhiza)
in color. It has a slight odor and a sweet taste.
Under a microscope <5.01>, Powdered Glycyrrhiza reveals
mainly yellow sclerenchymatous fiber bundles accompanied
with crystal cell rows; vessels, 80 to 200 fim in diameter, with
pitted, reticulate and scalariform pits, and with round perfo-
rations; parenchyma cells, containing starch grains and soli-
tary crystals of calcium oxalate, their fragments, and cork tis-
sues; but powder of peeled Glycyrrhiza shows no cork tissue;
if any, a very few. Starch grains are simple grains, 2-20 /xm in
diameter; simple grains of calcium oxalate, 10-30 /xm in a di-
ameter.
Identification To 2 g of Powdered Glycyrrhiza add 10 mL
of a mixture of ethanol (95) and water (7:3), heat by shaking
on a water bath for 5 minutes, cool, filter, and use the filtrate
as the sample solution. Separately, dissolve 5 mg of Glycyr-
rhizinic Acid Reference Standard in 1 mL of a mixture of
ethanol (95) and water (7:3), and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 2 /uL each
of the sample solution and standard solution on a plate of sil-
ica gel with fluorescent indicator for thin-layer chro-
matography. Develop the plate with a mixture of 1-butanol,
water and acetic acid (100) (7:2:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): one spot among the spots from
the sample solution and a dark purple spot from the standard
solution show the same color tone and the same Rf value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Glycyrrhiza according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Glycyrrhiza according to Method 4, and per-
form the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Glycyrrhiza shows no stone cells.
(4) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 25.0%.
Assay Weigh accurately about 0.5 g of Powdered Glycyr-
rhiza in a glass-stoppered centrifuge tube, add 70 mL of di-
lute ethanol, shake for 15 minutes, centrifuge, and separate
the supernatant liquid. To the residue add 25 mL of dilute
ethanol, and proceed in the same manner. Combine all the
extracts, add dilute ethanol to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 25 mg of Glycyrrhizic Acid Reference Stan-
dard (separately determine the water), dissolve in dilute
ethanol to make exactly 100 mL, and use this solution as the
standard solution. Pipet 20 fiL each of the sample solution
and standard solution, and perform the test as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine the peak areas, A T and A s , of glycyr-
rhizic acid of each solution.
Amount (mg) of glycyrrhizic acid (C 4 2H 62 Oi 6 )
= W s x(A T /A s )
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: Use a column 4 to 6 mm in inside diameter and 15
to 25 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 to 10 mL in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (3:2).
Flow rate: Adjust the flow rate so that the retention time of
glycyrrhizic acid is about 10 minutes.
Selection of column: Dissolve 5 mg of Glycyrrhizic Acid
Reference Standard and 1 mg of propyl parahydroxybenzo-
ate in dilute ethanol to make 20 mL. Proceed with 20 /xh of
this solution under the above operating conditions. Use a
column giving elution of glycyrrhizic acid and propyl para-
hydroxybenzoate in this order, and clearly dividing each
peak.
System repeatability: Repeat the test 5 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of glycyrrhizic acid is
not more than 1.5%.
Glycyrrhiza Extract
*j>7^i+z
Glycyrrhiza Extract contains not less than 4.5% of
glycyrrhizic acid (C 42 H 62 16 : 822.93).
Method of preparation To 1 kg of fine cuttings of Glycyr-
rhiza or the root and stolon of Glycyrrhiza glabra Linne
(Leguminosae) which meets the requirement of Glycyrrhiza
add 5 L of Water or Purified Water, and macerate for 2 days.
Filter the macerated solution through a cloth filter. Add 3 L
of Water or Purified Water to the residue, macerate again for
12 hours, and filter through a cloth filter. Evaporate the com-
bined filtrates until the whole volume becomes 3 L. After
cooling, add 1 L of Ethanol, and allow to stand in a cold
place for 2 days. Filter, and evaporate the filtrate to a viscous
extract.
JPXV
Crude Drugs / Gypsum 1297
Description Glycyrrhiza Extract is a brown to blackish
brown, viscous extract, and has a characteristic odor and a
sweet taste.
It dissolves in water, forming a clear solution, or with a
slight turbidity.
Identification To 0.8 g of Glycyrrhiza Extract add 10 mL of
a mixture of ethanol (95) and water (7:3), shake for 2
minutes, centrifuge, and use the supernatant liquid as the
sample solution. Proceed as directed in the Identification un-
der Glycyrrhiza.
Purity Insoluble matter — Dissolve 2.0 g of Glycyrrhiza Ex-
tract in 18 mL of water, and filter. To 10 mL of the filtrate
add 5 mL of ethanol (95): a clear solution results.
Assay Weigh accurately about 0.15 g of Glycyrrhiza Ex-
tract, place in a glass-stoppered centrifuge tube, add 25 mL
of dilute ethanol, and heat at 50°C for 30 minutes with oc-
casional shaking. Cool, centrifuge, and separate the super-
natant liquid. To the residue add 20 mL of dilute ethanol,
and proceed in the same manner. Combine the extracts, add
dilute ethanol to make exactly 100 mL, and use this solution
as the sample solution. Separately, weigh accurately about 20
mg of Glycyrrhizic Acid Reference Standard (priviously de-
termine the water), dissolve in dilute ethanol to make exactly
100 mL, and use this solution as the standard solution. Pro-
ceed as directed in Assay under Glycyrrhiza.
Amount (mg) of glycyrrhizic acid (C 42 H 62 16 )
= W s x(A T /A s )
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
verized Crude Glycyrrhiza Extract with 100 mL of water. Af-
ter cooling, filter the mixture through tared filter paper, wash
with water, and dry the residue at 105°C for 5 hours: the
mass of the residue is not more than 1.25 g.
(2) Foreign matter — The filtrate obtained in (1) does not
have a strong bitter taste.
(3) Starch — To about 1 g of pulverized Crude Glycyrrhi-
za Extract add water to make 20 mL, shake the mixture
thoroughly, and filter. Examine the insoluble substance on
the filter paper under a microscope: the residue contains no
starch grains.
Total ash <5.07> Not more than 12.0% (1 g).
Assay Weigh accurately about 0.15 g of Crude Glycyrrhiza
Extract, place in a glass-stoppered centrifuge tube, add 25
mL of dilute ethanol, and heat at 50°C for 30 minutes with
occasional shaking. Cool, centrifuge, and separate the super-
natant liquid. To the residue add 20 mL of dilute ethanol,
and proceed in the same manner. Combine the extracts, add
dilute ethanol to make exactly 100 mL, and use this solution
as the sample solution. Separately, weigh accurately about 20
mg of Glycyrrhizic Acid Reference Standard (separately de-
termine the water), dissolve in dilute ethanol to make exactly
100 mL, and use this solution as the standard solution. Pro-
ceed as directed in Assay under Glycyrrhiza.
Amount (mg) of glycyrrhizic acid (C 42 H 62 16 )
= W s x(A T /A s )
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Containers and storage Containers — Tight containers.
Containers and storage Containers — Tight containers.
Crude Glycyrrhiza Extract
Gypsum
Gypsum Fibrosum
-tz ••/ =i ^
Glycyrrhiza Extract contains not less than 6.0% of
glycyrrhizic acid (C 42 H 62 16 : 822.93).
Method of preparation Boil coarse powder of Glycyrrhiza
or the root and stolon of Glycyrrhiza glabra Linne
(Leguminosae) which meets the requirement of Glycyrrhiza
with Water or Purified Water, filter the solution under pres-
sure, and evaporate the filtrate.
Description Crude Glycyrrhiza Extract occurs as lustrous,
dark yellow-red to blackish brown plates, rods or masses. It
is comparatively brittle when cold, and the fractured surface
is dark yellow-red, shell-like, and lustrous. It softens when
warmed.
It has a characteristic odor and a sweet taste.
It dissolves in water with turbidity.
Identification To 0.6 g of Crude Glycyrrhiza Extract add 10
mL of a mixture of ethanol (95) and water (7:3), dissolve by
warming if necessary, cool, centrifuge, and use the super-
natant liquid as the sample solution. Proceed as directed in
the Identification under Glycyrrhiza.
Purity (1) Water-insoluble substances — Boil 5.0 g of pul-
Gypsum is natural hydrous calcium sulfate. It possi-
bly corresponds to the formula CaS0 4 .2H 2 0.
Description Gypsum occurs as lustrous, white, heavy, fi-
brous, crystalline masses, which easily split into needles or
very fine crystalline powder.
It is odorless and tasteless.
It is slightly soluble in water.
Identification To 1 g of pulverized Gypsum add 20 mL of
water, allow to stand with occasional shaking for 30 minutes,
and filter: the filtrate responds to the Qualitative Tests <1.09>
(2) and (3) for calcium salt and to the Qualitative Tests <1.09>
for sulfate.
Purity (1) Heavy metals <1.07> — Boil 4.0 g of pulverized
Gypsum with 4 mL of acetic acid (100) and 96 mL of water
for 10 minutes, cool, add water to make exactly 100 mL, and
filter. Perform the test using 50 mL of the filtrate as the test
solution. Prepare the control solution as follows: to 4.0 mL
of Standard Lead Solution add 2 mL of dilute acetic acid and
water to make 50 mL (not more than 20 ppm).
(2) Arsenic </.//> — Prepare the test solution with 0.40 g
of pulverized Gypsum according to Method 2, and perform
1298 Exsiccated Gypsum / Crude Drugs
JP XV
the test (not more than 5 ppm).
Containers and storage Containers — Well-closed contain-
ers.
Exsiccated Gypsum
Gypsum Exsiccatum
jft-tz •» =1 *j
Exsiccated Gypsum possibly corresponds to the for-
mula CaS0 4 .>/ 2 H 2 0.
Description Exsiccated Gypsum occurs as a white to grayish
white powder. It is odorless and tasteless.
It is slightly soluble in water, and practically insoluble in
ethanol (95).
It absorbs moisture slowly on standing in air to lose its
solidifying property.
When it is heated to yield an anhydrous compound at a
temperature above 200°C, it loses its solidifying property.
Identification Shake 1 g of Exsiccated Gypsum with 20 mL
of water for 5 minutes, and filter: the filtrate responds to the
Qualitative Tests <1.09> (2) and (3) for calcium salt and to the
Qualitative Tests <1.09> for sulfate.
Purity Alkalinity — Take 3.0 g of Exsiccated Gypsum in a
glass-stoppered test tube, add 10 mL of water and 1 drop of
phenolphthalein TS, and shake vigorously: no red color de-
velops.
Solidification To 10.0 g of Exsiccated Gypsum add 10 mL
of water, stir immediately for 3 minutes, and allow to stand:
the period until water no longer separates, when the material
is pressed with a finger, is not more than 10 minutes from the
time when the water was added.
Containers and storage Containers — Tight containers.
Hemp Fruit
Cannabis Fructus
-7-> — >
Hemp Fruit is the fruit of Cannabis sativa Linne
(Moraceae).
Description Hemp Fruit is a slightly compressed void fruit,
4 to 5 mm in length, 3 to 4 mm in diameter; externally grayish
green to grayish brown; pointed at one end, a scar of gyno-
phore at the other end, and crest lines on both sides; outer
surface lustrous with white mesh-like pattern; slightly hard
pericarp; seed, slightly green in color and internally has
grayish white albumen; 100 fruits weighing 1.6 to 2.7 g.
Practically odorless, aromatic on chewing; taste, mild and
oily.
Under a microscope <5.01>, a transverse section reveals the
exocarp to be a single-layered epidermis; mesocarp composed
of parenchyma, a pigment cell layer and rows of short, small
cells; endocarp made up of a layer of radially elongated stone
cells; seed coat comprises a tubular cell layer and spongy tis-
sue. Inside of the seed; exosperm consists of one layer of
parenchymatous cells, endosperm of one to several layers of
parenchymatous cells; most of the embryo composed of
parenchyma, vascular bundles occurring in the center of
hypocotyls and cotyledons; embryo parenchyma contains
aleurone grains and oil drops.
Identification To 0.3 g of pulverized Hemp Fruit add 3 mL
of methanol, shake for 10 minutes, centrifuge, and use the
supernatant liquid as the sample solution. Perform the test
with the sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL of the sample solution on a
plate of silica gel for thin-layer chromatography, develop the
plate with a mixture of hexane and ethyl acetate (9:2) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly
vanillin-sulfuric acid TS on the plate, and heat at 105°C for 5
minutes: a dark blue-purple spot appears at around Rf 0.6.
Purity Bract — Hemp Fruit does not contain bract.
Loss on drying <5.01> Not more than 9.0% (6 hours).
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Hochuekkito Extract
Hochuekkito Extract contains not less than 16 mg
and not more than 48 mg of hesperidin, not less than
0.3 mg and not more than 1.2 mg (for preparation
prescribed 1 g of Bupleurum Root) or not less than 0.6
mg and not more than 2.4 mg (for preparation
prescribed 2 g of Bupleurum Root) of saikosaponin b 2 ,
and not less than 12 mg and not more than 36 mg of
glycyrrhizic acid (C 42 H 62 16 : 822.93) per a dried extract
prepared as directed in the Method of preparation.
Method of preparation Prepare a dried extract as directed
under Extracts, with 4 g of Ginseng, 4 g of Atractylodes Rhi-
zome or Atractylodes Lancea Rhizome, 4 g of Astragalus
Root, 3 g of Japanese Angelica Root, 2 g of Citrus Unshiu
Peel, 2 g of Jujube, 2 g of Bupleurum Root, 1.5 g of Glycyr-
rhiza, 0.5 g of Ginger and 1 g of Cimicifuga Rhizome, or
with 4 g of Ginseng, 4 g of Atractylodes Rhizome or Atrac-
tylodes Lancea Rhizome, 4 g of Astragalus Root, 3 g of
Japanese Angelica Root, 2 g of Citrus Unshiu Peel, 2 g of
Jujube, 1 g of Bupleurum Root, 1.5 g of Glycyrrhiza, 0.5 g
of Ginger and 0.5 g of Cimicifuga Rhizome, or with 4 g of
Ginseng, 4 g of Atractylodes Rhizome, 3 g of Astragalus
Root, 3 g of Japanese Angelica Root, 2 g of Citrus Unshiu
Peel, 2 g of Jujube, 2 g of Bupleurum Root, 1.5 g of Glycyr-
rhiza, 0.5 g of Ginger and 1 g of Cimicifuga Rhizome, or
with 4 g of Ginseng, 4 g of Atractylodes Rhizome, 4 g of As-
tragalus Root, 3 g of Japanese Angelica Root, 2 g of Citrus
Unshiu Peel, 2 g of Jujube, 1 g of Bupleurum Root, 1.5 g of
Glycyrrhiza, 0.5 g of Processed Ginger and 0.5 g of Cimicifu-
ga Rhizome.
Description Hochuekkito Extract occurs as a light brown to
brown powder. It has a slight odor, and a sweet and bitter
taste.
Identification (1) Ginseng — To 2.0 g of Hochuekkito Ex-
JPXV
Crude Drugs / Hochuekkito Extract 1299
tract add 30 mL of water, shake, then add 50 mL of 1-
butanol, and shake. Take the 1-butanol layer, evaporate the
layer under reduced pressure, add 3 mL of methanol to the
residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of Ginsenoside Rb, Reference Stan-
dard in 1 mL of methanol, and use this solution as the stan-
dard solution. Perform the test with these solutions as direct-
ed under Thin-layer Chromatography <2.03>. Spot 5 [iL each
of the sample solution and standard solution on a plate of sil-
ica gel for thin-layer chromatography, develop the plate with
a mixture of ethyl acetate, 1-propanol, water and acetic acid
(100) (7:5:4:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly vanillin-sulfuric acid TS on the plate, heat
at 105 °C for 5 minutes, and allow to cool: one of the spot
among the several spots from the sample solution has the
same color tone and Ri value with the purple spot from the
standard solution.
(2) Atractylodes rhizome (for preparation prescribed
Atractylodes Rhizome) — To 3.0 g of Hochuekkito Extract
add 30 mL of water, shake, then add 50 mL of diethyl ether,
shake, and take the diethyl ether layer. Evaporate the diethyl
ether layer under reduced pressure, add 1 mL of diethyl ether
to the residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of atractylenolide III for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL of the sample solution and 10 /uL of the standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 1-naphthol-sulfuric acid TS on
the plate, heat at 105 °C for 5 minutes, and allow to cool: one
of the spot among the several spots from the sample solution
has the same color tone and Ri value with the red spot from
the standard solution.
(3) Atractylodes lancea rhizome (for preparation
prescribed Atractylodes Lancea Rhizome) — To 2.0 g of
Hochuekkito Extract add 10 mL of water, shake, then add 25
mL of hexane, shake, and take the hexane layer. To the
hexane layer add anhydrous sodium sulfate to dry, filter,
evaporate the filtrate under reduced pressure, add 2 mL of
hexane to the residue, and use this solution as the sample so-
lution. Perform the test with the sample solution as directed
under Thin-layer Chromatography <2.03>. Spot 20 fih of the
sample solution on a plate of silica gel with fluorescent indi-
cator for thin-layer chromatography. Develop the plate with
a mixture of hexane and acetone (7:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): a dark purple spot appears
around Ri 0.4, which shows a greenish brown color after
spraying 4-dimethylaminobenzaldehyde TS for spraying,
heating at 105 °C for 5 minutes and allowing to cool.
(4) Astragalus root — To 3.0 g of Hochuekkito Extract
add 40 mL of a solution of potassium hydroxide in methanol
(1 in 50), shake for 15 minutes, centrifuge, and evaporate the
supernatant liquid under reduced pressure. Add 30 mL of
water to the residue, then add 20 mL of diethyl ether, shake,
and take the water layer. To the water layer add 20 mL of 1-
butanol, shake, and take the 1-butanol layer. To the 1-
butanol layer add 20 mL of water, shake, take the 1-butanol
layer, evaporate the layer under reduced pressure, add 1 mL
of methanol to the residue, and use this solution as the sam-
ple solution. Separately, dissolve 1 mg of astragaloside IV for
thin-layer chromatography in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 [iL each of the sample solution and standard
solution on a plate of octadecylsilanized silica gel for thin-
layer chromatography. Develop the plate with a mixture of
methanol, water, 1-butanol and acetic acid (100) (60:30:10:1)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly 4-dimethylaminobenzaldehyde TS for spraying on the
plate, and heat at 105 °C for 5 minutes: one of the spot
among the several spots from the sample solution has the
same color tone and Ri value with the red-brown spot from
the standard solution.
(5) Japanese angelica root — To 3.0 g of Hochuekkito Ex-
tract add 30 mL of water, shake, then add 50 mL of diethyl
ether, shake, and take the diethyl ether layer. Evaporate the
diethyl ether under reduced pressure, add 1 mL of diethyl
ether to the residue, and use this solution as the sample solu-
tion. Separately, dissolve 1 mg of (Z)-ligustilide for thin-layer
chromatography in 10 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate and hexane
(1 : 1) to a distance of about 10 cm, and air-dry the plate. Exa-
mine under ultraviolet light (main wavelength: 365 nm): one
of the spot among the several spots from the sample solution
has the same color tone and Ri value with the blue-white
fluorescent spot from the standard solution.
(6) Citrus unshiu peel — To 2.0 g of Hochuekkito Extract
add 30 mL of water, shake, then add 50 mL of 1-butanol,
shake, and take the 1-butanol layer. Evaporate the layer
under reduced pressure, add 3 mL of methanol to the residue,
and use this solution as the sample solution. Separately, dis-
solve 1 mg of hesperidin for thin-layer chromatography in 2
mL of methanol, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 2^L of the sample
solution and 20 /xL of the standard solution on a plate of sili-
ca gel for thin-layer chromatography. Develop the plate with
a mixture of ethyl acetate, acetone, water and acetic acid
(100) (10:6:3:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly 2,6-dibromo-,/V-chloro-l,4-benzoquinone
monoimine TS on the plate, and expose to ammonia vapor:
one of the spot among the several spots from the sample solu-
tion has the same color tone and Ri value with the blue spot
from the standard solution.
(7) Bupleurum root — To 2.0 g of Hochuekkito Extract
add 30 mL of water, shake, then add 50 mL of 1-butanol,
shake, and take the 1-butanol layer. Evaporate the layer un-
der reduced pressure, add 3 mL of methanol to the residue,
and use this solution as the sample solution. Separately, dis-
solve 1 mg of saikosaponin b 2 for thin-layer chromatography
in 1 mL of methanol, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL of the sample
solution and 2 /xL of the standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethyl acetate, ethanol (99.5) and water (8:2:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
4-dimethylaminobenzaldehyde TS on the plate: one of the
1300 Hochuekkito Extract / Crude Drugs
JP XV
spot among the several spots from the sample solution has
the same color tone and Ri value with the red spot from the
standard solution.
(8) Glycyrrhiza — To 2.0 g of Hochuekkito Extract add
30 mL of water, shake, then add 50 mL of 1-butanol, and
take the 1-butanol layer. Evaporate the layer under reduced
pressure, add 3 mL of methanol to the residue, and use this
solution as the sample solution. Separately, dissolve 1 mg of
liquiritin for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /uL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
ethyl acetate, methanol and water (20:3:2) to a distance of
about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 105 °C for 5 minutes: one
of the spot among the several spots from the sample solution
has the same color tone and Rf value with the yellow-brown
spot from the standard solution.
(9) Ginger (for preparation prescribed Ginger) — To 3.0 g
of Hochuekkito Extract add 30 mL of water, shake, then add
50 mL of diethyl ether, shake, and take the diethyl ether lay-
er. Evaporate the diethyl ether under reduced pressure, add 1
mL of diethyl ether to the residue, and use this solution as the
sample solution. Separately, dissolve 1 mg of [6]-gingerol for
thin-layer chromatography in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 4-dimethylaminobenzaldehyde
TS for spraying on the plate, heat at 105°C for 5 minutes,
and allow to cool: one of the spot among the several spots
from the sample solution has the same color tone and Rf
value with the blue-green spot from the standard solution.
(10) Processed ginger (for preparation prescribed Proc-
essed Ginger) — Put 10 g of Hochuekkito Extract in a 300-mL
hard-glass flask, add 100 mL of water and 1 mL of silicone
resin, connect an apparatus for essential oil determination,
and heat to boil under a reflux condenser. The graduated tube
of the apparatus is to be previously filled with water to the
standard line, and 2 mL of hexane is added to the graduated
tube. After heating under reflux for about 1 hour, separate
the hexane layer, and use this as the sample solution.
Separately, dissolve 1 mg of [6]-shogaol for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 60
fiL of the sample solution and 10 fiL of the standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of cyclohexane and ethyl
acetate (2:1) to a distance of about 10 cm, and air-dry the
plate. Spray evenly 4-dimethylaminobenzaldehyde TS for
spraying on the plate, heat at 105°C for 5 minutes, and allow
to cool: one of the spot among the several spots from the
sample solution has the same color tone and Ri value with the
blue-green spot from the standard solution.
(11) Cimicifuga rhizome — To 2.0 g of Hochuekkito Ex-
tract add 30 mL of water, shake, then add 50 mL of 1-
butanol, and take the 1-butanol layer. Evaporate the layer
under reduced pressure, add 3 mL of methanol to the residue,
and use this solution as the sample solution. Use 3-(3-hydrox-
y-4-methoxyphenyl)-2-(£)-propenic acid-(£)-ferulic acid TS
for thin-layer chromatography as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xL of the sample solution
and 2 /uL of the standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, acetone and water (20:12:3) to a distance of
about 10 cm, and air-dry the plate. Spray evenly sulfuric acid
on the plate, heat at 105°C for 5 minutes, and examine under
ultraviolet light (main wavelength: 365 nm): one of the spot
among the several spots from the sample solution has the
same color tone and Rf value with the yellow fluorescent spot
from the standard solution.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1.0 g of Hochuekkito Extract as directed in (4) in Ex-
tracts, and perform the test (not more than 30 ppm).
(2) Arsenic <7.77> — Prepare the test solution with 0.67 g
of Hochuekkito Extract according to Method 3, and perform
the test (not more than 3 ppm).
Loss on drying <2.41> Not more than 11.5% (1 g, 105°C, 5
hours).
Total ash <5.07> Not more than 9.0%.
Assay (1) Hesperidin — Weigh accurately about 0.1 g of
Hochuekkito Extract, add exactly 50 mL of diluted tetra-
hydrofuran (1 in 4), shake for 30 minutes, centrifuge, and use
the supernatant liquid as the sample solution. Separately,
weigh accurately about 10 mg of hesperidin for component
determination, previously dried in a desiccator (silica gel) for
not less than 24 hours, and dissolve in methanol to make ex-
actly 100 mL. Pipet 10 mL of this solution, add diluted tetra-
hydrofuran (1 in 4) to make exactly 100 mL, and use this so-
lution as the standard solution. Perform the test with exactly
10 /uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of peoniflorin.
Amount (mg) of hesperidin = W s x (A T /A S ) x (1/20)
W s : Amount (mg) of hesperidin for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 285 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (82:18:1)
Flow rate: 1 .0 mL/min. (the retention time of hesperidin is
about 15 minutes.)
System suitability —
System performance: Dissolve 1 mg each of hesperidin for
component determination and naringin for thin-layer chro-
matography in diluted methanol (1 in 2) to make 100 mL.
When the procedure is run with 10 fiL of this solution under
the above operating conditions, naringin and hesperidin are
JPXV
Crude Drugs / Honey 1301
eluted in this order with the resolution between these peaks
being not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
hespiridin is not more than 1.5%.
(2) Saikosaponin b 2 — Weigh accurately about 0.5 g of
Hochuekkito Extract, add exactly 50 mL of diluted methanol
(1 in 2), shake for 15 minutes, filter, and use the filtrate as the
sample solution. Separately, weigh accurately about 10 mg of
saikosaponin b 2 for component determination, previously
dried in a desiccator (silica gel) for not less than 24 hours, and
dissolve in diluted methanol (1 in 2) to make exactly 100 mL.
Pipet 10 mL of this solution, add diluted methanol (1 in 2) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 /xL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of sai-
kosaponin b 2 .
Amount (mg) of saikosaponin b 2 = W s x (A T /A S ) x (1/20)
W s : Amount (mg) of saikosaponin b 2 for component de-
termination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L sodium dihydro-
gen phosphate TS and acetonitrile (5:3).
Flow rate: l.OmL/min. (the retention time of saikosapo-
nin b 2 is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of saikosaponin b 2 are not less than 5000 and
not more than 1.5%, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
saikosaponin b 2 is not more than 1.5%.
(3) Glycyrrhizic acid — Weigh accurately about 0.5 g of
Hochuekkito Extract, add exactly 50 mL of diluted methanol
(1 in 2), shake for 15 minutes, filter, and use the filtrate as the
sample solution. Separately, weigh accurately about 10 mg of
Glycyrrhizic Acid Reference Standard (separately determine
the water), dissolve in diluted methanol (1 in 2) to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of glycyrrhizic
acid.
Amount (mg) of glycyrrhizic acid (C 42 H 62 16 )
= W s x(Aj/A s )x(l/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7).
Flow rate: 1.0 mL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10,mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
Honey
Mel
Honey is the saccharine substances obtained from
the honeycomb of Apis mellifera Linne or Apis indica
Radoszkowski (Apidae).
Description Honey is a light yellow to light yellow-brown,
syrupy liquid. Usually it is transparent, but often opaque
with separated crystals.
It has a characteristic odor and a sweet taste.
Specific gravity <2.56> Mix 50.0 g of Honey with 100 mL of
water: the specific gravity of the solution is not less than d^:
1.111.
Purity (1) Acidity — Mix 10 g of Honey with 50 mL of
water, and titrate <2.50> with 1 mol/L potassium hydroxide
VS (indicator: 2 drops of phenolphthalein TS): not more
than 0.5 mL is required.
(2) Sulfate— Mix 1.0 g of Honey with 2.0 mL of water,
and filter. To the filtrate add 2 drops of barium chloride TS:
the solution does not show any change immediately.
(3) Ammonia-coloring substances — Mix 1.0 g of Honey
with 2.0 mL of water, and filter. To the filtrate add 2 mL of
ammonia TS: the solution does not show any change immedi-
ately.
(4) Resorcinol-coloring substances — Mix well 5 g of
Honey with 15 mL of diethyl ether, filter, and evaporate the
diethyl ether solution at ordinary temperature. To the residue
add 1 to 2 drops of resorcinol TS: a yellow-red color may de-
velop in the solution of resorcinol and in the residue, and a
red to red-purple color which does not persist more than 1
1302 Houttuynia Herb / Crude Drugs
JP XV
hour.
(5) Starch or dextrin — (i) Shake 7.5 g of Honey with 15
mL of water, warm the mixture on a water bath, and add 0.5
mL of tannic acid TS. After cooling, filter, and to 1.0 mL of
the filtrate add 1 .0 mL of ethanol (99.5) containing 2 drops of
hydrochloric acid: no turbidity is produced.
(ii) To 2.0 g of Honey add 10 mL of water, warm in a
water bath, mix, and allow to cool. Shake 1.0 mL of the mix-
ture with 1 drop of iodine TS: no blue, green or red-brown
color develops.
(6) Foreign matter — Mix 1.0 g of Honey with 2.0 mL of
water, centrifuge the mixture, and examine the precipitate
microscopically <5.01>: no foreign substance except pollen
grains is observable.
Total ash <J.07> Not more than 0.4%.
Containers and storage Containers — Tight containers.
Houttuynia Herb
Houttuyniae Herba
Houttuynia Herb is the terrestrial part of Houttuy-
nia cordata Thunberg (Saururaceae), collected during
the flowering season.
Description Stem with alternate leaves and spikes; stem
light brown, with longitudinal furrows and protruded nodes;
when soaked in water and smoothed out, leaves wide ovate
and cordate, 3-8 cm in length, 3-6 cm in width; light
green-brown; margin entire, apex acuminate; petiole long,
and membranous stipule at the base; spike, 1 - 3 cm in
length, with numerous light yellow-brown achlamydeous
florets, and the base enclosed by 4 long ovate, light yellow to
light yellow-brown involucres.
Odor, slight; tasteless.
Identification Boil 2 g of pulverized Houttuynia Herb with
20 mL of ethyl acetate under a reflux condenser on a water
bath for 15 minutes, and filter. Evaporate the filtrate to dry-
ness, add 10 mL of water to the residue, warm the mixture on
a water bath for 2 minutes, and, after cooling, filter. Shake
well the filtrate with 20 mL of ethyl acetate in a separator,
take 15 mL of ethyl acetate solution, and evaporate the solu-
tion on a water bath to dryness. Dissolve the residue in 5 mL
of methanol, add 0.1 g of magnesium ribbon and 1 mL of
hydrochloric acid, and allow the mixture to stand: a light red
to red color develops.
Purity Foreign matter <5.01> — The amount of the rhizome,
roots and other foreign matter contained in Houttuynia Herb
is not more than 2.0%.
Total ash <J.07> Not more than 14.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 10.0%.
Immature Orange
Aurantii Fructus Immaturus
Immature Orange is the immature fruit or the fruit
cut crosswise of Citrus aurantium Linne var. daidai
Makino, Citrus aurantium Linne or Citrus natsu-
daidai Hayata (Rutaceae).
Description Nearly spherical fruit, 1-2 cm in diameter, or
semispherical, 1.5 -4.5 cm in diameter; external surface,
deep green-brown to brown, and without luster, with
numerous small dents associated with oil sacs; the outer por-
tion of transverse section exhibits pericarp and mesocarp
about 0.4 cm in thickness, yellow-brown in color in the
region contacting epidermis, and light grayish brown color in
the other parts; the central portion is radially divided into 8
to 16 small loculi; each loculus is brown and indented, often
containing immature seeds.
Odor, characteristc; taste, bitter.
Identification To 0.5 g of pulverized Immature Orange add
10 mL of methanol, boil gently for 2 minutes, and filter. To 5
mL of the filtrate add 0.1 g of magnesium ribbon and 1 mL
of hydrochloric acid, and allow to stand: a red-purple color
develops.
Total ash <5.07> Not more than 7.0%.
Imperata Rhizome
Imperatae Rhizoma
7K' r t7=l>
Imperata Rhizome is the rhizome of Imperata cylin-
drica Beauvois (Gramineae), from which rootlets and
scale leaves have been removed.
Description Long and thin cylindrical rhizome, 0.3 - 0.5
cm in diameter; sometimes branched; externally yellowish
white, with slight longitudinal wrinkles, and with nodes at 2 -
3-cm intervals; difficult to break; fractured surface fibrous.
Cross section irregularly round; thickness of cortex is slightly
smaller than the diameter of the stele; pith often forms a hol-
low. Under a magnifying glass, a transverse section reveals
cortex, yellowish white, and with scattered brown spots;
stele, yellow-brown in color.
Odorless, and tasteless at first, but later slightly sweet.
Identification To 1 g of pulverized Imperata Rhizome add
20 mL of hexane, allow the mixture to stand for 30 minutes
with occasional shaking, and filter. Evaporate the hexane of
the filtrate under reduced pressure, dissolve the residue in 5
mL of acetic anhydride, place 0.5 mL of this solution in a test
tube, and add carefully 0.5 mL of sulfuric acid to make two
layers: a red-brown color develops at the zone of contact, and
the upper layer acquires a blue-green to blue-purple color.
Purity (1) Rootlet and scaly leaf — The amount of the
JP XV
Crude Drugs / Powdered Ipecac 1303
rootlets and scaly leaves contained in Imperata Rhizome is
not more than 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than rootlets and scaly leaves contained in Imperata
Rhizome is not more than 1.0%.
Total ash <5.01> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Ipecac
Ipecacuanhae Radix
Ipecac is the root and rhizome of Cephaelis ipecacu-
anha (Broterol) A. Richard or Cephaelis acuminata
Karsten (Rubiaceae).
It contains not less than 2.0% of the total alkaloids
(emetine and cephaeline), calculated on the basis of
dried material.
Description Slender, curved, cylindrical root, 3-15 cm in
length, 0.3 - 0.9 cm in diameter; mostly twisted, and some-
times branched; outer surface gray, dark grayish brown, red-
dish brown in color and irregularly annulated; when root
fractured, cortex easily separable from the xylem; the cortex
on the fractured surface is grayish brown, and the xylem is
light brown in color: thickness of cortex up to about two-
thirds of radius in thickened portion. Scales in rhizome oppo-
site.
Odor, slight; powder irritates the mucous membrane of the
nose; taste, slightly bitter and unpleasant.
Under a microscope <5.01>, the transverse section of Ipecac
reveals a cork layer, consisting of brown thin-walled cork
cells; in the cortex, sclerenchyma cells are absent; in the xy-
lem, vessels and tracheids arranged alternately; parenchyma
cells filled with starch grains and sometimes with raphides of
calcium oxalate.
Identification To 0.5 g of pulverized Ipecac add 2.5 mL of
hydrochloric acid, allow to stand for 1 hour with occasional
shaking, and filter. Collect the filtrate into an evaporating
dish, and add a small pieces of chlorinated lime: circumfer-
ence of it turns red.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Component determination Weigh accurately about 0.5 g of
pulverized Ipecac, in a glass-stoppered centrifuge tube, add
30 mL of 0.01 mol/L hydrochloric acid TS, shake for 15
minutes, centrifuge, and separate the supernatant liquid.
Repeat this procedure twice with the residue using 30-mL
portions of 0.01 mol/L hydrochloric acid TS. Combine all
the extracts, add 0.01 mol/L hydrochloric acid TS to make
exactly 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of emetine
hydrochloride for component determination, previously
dried in a desiccator (reduced below 0.67 kPa, phosphorus
(V) oxide, 50°C) for 5 hours, dissolve in 0.01 mol/L
hydrochloric acid TS to make exactly 100 mL, and use this
solution as the standard solution. Pipet 10 fiL of the sample
solution and standard solution, and perform the test as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine the peak areas, A TE and
Aj C , of emetine and cephaeline in the sample solution, and
the peak area, A SE , of emetine in the standard solution.
Amount (mg) of total alkaloids
(emetine and cephaeline)
= W s x[ {A TE + (A TC x 0.971)} /A SE ] x 0.868
W s : Amount (mg) of emetine hydrochloride for compo-
nent determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength : 283 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 10 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 /xm in
particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 2.0 g of sodium 1-heptane sul-
fonate in 500 mL of water, adjust the pH 4.0 with acetic acid
(100), and add 500 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
emetine is about 14 minutes.
Selection of column: Dissolve 1 mg each of emetine
hydrochloride for component determination and cephaeline
hydrobromide in 10 mL of 0.01 mol/L hydrochloric acid TS.
Perform the test with 10 /xh of this solution under the above
operating conditions. Use a column giving elution of cephae-
line and emetine in this order, and clearly separating each
peak.
System repeatability: Repeat the test 6 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of emetine is not
more than 1.5%.
Powdered Ipecac
Ipecacuanhae Radix Pulverata
r-=i>*
Powdered Ipecac is the powder of Ipecac or its
powder diluted with Potato Starch.
It contains not less than 2.0% and not more than
2.6% of the total alkaloids (emetine and cephaeline).
Description Powdered Ipecac occurs as a light grayish yel-
low to light brown powder. It has a slight odor, which is ir-
ritating to the nasal mucosa, and has a somewhat bitter and
unpleasant taste.
Under a microscope <5.01>, Powdered Ipecac reveals
starch grains and needle crystals of calcium oxalate; frag-
ments of parenchyma cells containing starch grains or the
needle crystals; substitute fibers, thin-walled cork tissue; ves-
sels and tracheids with simple or bordered pits; a few wood
fibers and wood parenchyma. Starch grains inherent in
Ipecac, mainly 2 - 8-compound grains, rarely simple grains 4
1304 Ipecac Syrup / Crude Drugs
JP XV
- 22 //m in diameter; and needle crystals of calcium oxalate
25 - 60 //m in length.
Identification To 0.5 g of Powdered Ipecac add 2.5 mL of
hydrochloric acid, allow to stand for 1 hour with occasional
shaking, and filter. Collect the filtrate into an evaporating
dish, and add a small pieces of chlorinated lime: circumfer-
ence of it turns red.
Purity Foreign matter — Under a microscope <5.01>, groups
of stone cells and thick-walled fibers are not observed.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Component determination Weigh accurately about 0.5 g of
Powdered Ipecac, transfer into a glass-stoppered centrifuge
tube, add 30 mL of 0.01 mol/L hydrochloric acid TS, shake
for 15 minutes, centrifuge, and separate the supernatant liq-
uid. Repeat this procedure twice with the residue using 30-mL
portions of 0.01 mol/L hydrochloric acid TS. Combine all
the extracts, add 0.01 mol/L hydrochloric acid TS to make
exactly 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg of emetine
hydrochloride for component determination, previously
dried in a desiccator (reduced below 0.67 kPa, phosphorus
(V) oxide, 50°C) for 5 hours, dissolve in 0.01 mol/L
hydrochloric acid TS to make exactly 100 mL, and use this
solution as the standard solution. Pipet 10,mL of the sample
solution and standard solution, and perform the test as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine the peak areas, A TE and
A TC , of emetine and cephaeline in the sample solution, and
the peak area, A SE , of emetine in the standard solution.
Amount (mg) of total alkaloids
(emetine and cephaeline)
= W s x[ U TE + (A TC x 0.971)} A4 SE ] x 0.868
W s : Amount (mg) of emetine hydrochloride for compo-
nent determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength : 283 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 10 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to lO^m in
particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 2.0 g of sodium 1-heptane sul-
fonate in 500 mL of water, adjust the pH 4.0 with acetic acid
(100), and add 500 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
emetine is about 14 minutes.
Selection of column: Dissolve 1 mg each of emetine
hydrochloride for component determination and cephaeline
hydrobromide in 10 mL of 0.01 mol/L hydrochloric acid TS.
Perform the test with 10 /uL of this solution under the above
operating conditions. Use a column giving elution of cephae-
line and emetine in this order, and clearly separating each
peak.
System repeatability: Repeat the test 6 times with the stan-
dard solution under the above operating conditions: the rela-
tive standard deviation of the peak area of emetine is not
more than 1.5%.
Ipecac Syrup
Syrupus Ipecacuanha
h=i>->n •■/"/
Ipecac Syrup is a syrup containing not less than
0.12 g and not more than 0.15 g of the total alkaloids
(emetine and cephaeline) per 100 mL.
Method of preparation Take coarse powder of Ipecac, pre-
pare the fluidextract as directed under Fluidextracts using a
mixture of Ethanol and Purified Water (3:1), and evaporate
the mixture under reduced pressure or add a suitable amount
of Ethanol or Purified Water if necessary to get a solution
containing 1.7 to 2.1 g of the total alkaloids (emetine and
cephaeline) per 100 mL. To 70 mL of this solution add 100
mL of Glycerin and Simple Syrup to make 1000 mL, as
directed under Syrups.
Description Ipecac Syrup is a yellow-brown, viscous liquid.
It has a sweet taste and a bitter aftertaste.
Identification Take 2 mL of Ipecac Syrup into an evaporat-
ing dish, mix with 1 mL of hydrochloric acid, and add small
pieces of chlorinated lime: circumference of it turns orange.
Purity Ethanol — Take exactly 5 mL of Ipecac Syrup, add 5
mL of the internal standard solution and water to make 50
mL, and use this solution as the sample solution. Separately,
pipet 5 mL of ethanol (99.5), and add water to make exactly
100 mL. To exactly 5 mL of this solution add exactly 5 mL of
the internal standard solution and water to make 50 mL, and
use this solution as the standard solution. Perform the test
with 2 jXL each of the sample solution and standard solution
as directed under Gas Chromatography <2.02> according to
the following conditions, and determine the rate of peak
height of ethanol to that of the internal standard, Q T and Q s :
Qt is not larger than Q s .
Internal standard solution — A solution of acetonitrile (1 in
20).
Operating conditions —
Detector: A hydrogen flame-ionization detector.
Column: A glass-column about 3 mm in inside diameter
and about 1.5 m in length, packed with ethylvinylbenzene-
divinylbenzene porous co-polymer for gas chromatography
(150 to 180 /um in particle diameter).
Column temperature: A constant temperature of between
105°C and 115°C.
Carrier gas: Nitrogen
Flow rate: Adjust the flow rate so that the retention time of
ethanol is 5 to 10 minutes.
Selection of column: Proceed with 2 /xh of the standard so-
lution under the above operating conditions. Use a column
giving elution of ethanol and the internal standard in this
order, and clearly separating each peak.
Component determination Take exactly 5 mL of Ipecac
Syrup, add 0.01 mol/L hydrochloric acid TS to make exactly
50 mL, and use the solution as the sample solution. Separate-
JPXV
Crude Drugs / Powdered Japanese Angelica Root 1305
ly, weigh accurately about 10 mg of emetine hydrochloride
for component determination, previously dried in a desicca-
tor (reduced pressure under 0.67 kPa, phosphorus (V) oxide,
50°C) for 5 hours, dissolve in 0.01 mol/L hydrochloric acid
TS to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with 10 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions. Determine the peak areas, A TE and A TC , of emetine
and cephaeline in the sample solution, and the peak area,
A SE , of emetine in the standard solution.
Amount (mg) of total alkaloids (emetine and cephaeline)
= W s x[ {A TE + (A TC x 0.971)} /A SB ] x (1/2) x 0.868
W s : Amount (mg) of emetine hydrochloride for component
determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 283 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 10 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 /im in
particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 2.0 g of sodium 1-heptane sulfonate
in 500 mL of water, adjust the pH to 4.0 with acetic acid
(100), and add 500 mL of methanol.
Flow rate: Adjust the flow rate so that the retention time of
emetine is about 14 minutes.
Selection of column: Dissolve 1 mg each of emetine
hydrochloride for component determination and cephaeline
hydrobromide in 10 mL of 0.01 mol/L hydrochloric acid TS.
Perform the test with 10,mL of this solution under the above
operating conditions. Use a column giving elution of cephae-
line and emetine in this order, and clearly separating each
peak.
System repeatability: When the test is repeated 6 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak area of emetine is
not more than 1.5%.
Microbial limit <4.05> Proceed with Ipecac Syrup: the total
viable aerobic microbial count is not more than 1000 per mL,
and the total count of fungi and yeast is not more than 100
per mL. Escherichia coli, Salmonella, Pseudomonas aer-
uginosa and Staphylococcus aureus should not be observed.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Japanese Angelica Root
Angelicae Radix
Japanese Angelica Root is the root of Angelica
acutiloba Kitagawa or Angelica acutiloba Kitagawa
var. sugiyamae Hikino (Umbelliferae), usually after
being passed through hot water.
branched roots, nearly fusiform; 10-25 cm in length; exter-
nally dark brown to red-brown, with longitudinal wrinkles
and horizontal protrusions composed of numerous scars of
fine rootlets; fractured surface is dark brown to yellow-
brown in color, and smooth; and with a little remains of leaf
sheath at the crown.
Odor, characteristic; taste, slightly sweet, followed by
slight pungency.
Under a microscope <5.01>, a transverse section reveals 4 to
10 layers of cork, with several layers of collenchyma inside of
the layer; the cortex exhibits many oil canals surrounded by
secretory cells and often large hollows appear; boundary of
phloem and xylem is distinct; in the xylem, numerous vessels
radiate alternately with medullary rays; vessels in the outer
part of the xylem are singly or in several groups, and disposed
rather densely in a cuneiform pattern, but vessels in the
region of the center are scattered very sparsely; starch grains
are simple grains, not more than 20 fim in diameter, and rare-
ly 2- to 5-compound grains, up to 25 fim in diameter; starch
grains often gelatinized.
Purity (1) Leaf sheath — The amount of leaf sheath con-
tained in Japanese Angelica Root does not exceed 3.0%.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Japanese Angelica Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Japanese Angelica Root according to Method
4, and perform the test (not more than 5 ppm).
(4) Foreign matter <5.01> — The amount of foreign matter
other than leaf sheath contained in Japanese Angelica Root
does not exceed 1.0%.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 35.0%.
Powdered Japanese Angelica Root
Angelicae Radix Pulverata
Powdered Japanese Angelica Root is the powder of
Japanese Angelica Root.
Description Powdered Japanese Angelica Root occurs as a
light grayish brown powder. It has a characteristic odor and a
slight, sweet taste with a slightly pungent aftertaste.
Under a microscope <5.01>, Powdered Japanese Angelica
Root reveals starch grains or masses of gelatinized starch,
and fragments of parenchyma containing them; fragments of
light yellow-brown cork tissue; fragments of rather thick-
walled collenchyma and phloem tissue; fragments of resin
duct surrounded by secretory cells; fragments, 20 - 60,Mm in
diameter, of scalariform and reticulate vessels with simple
perforation; starch grains composed of simple grains not
more than 20 fim in diameter, and rarely 2- to 3-compound
grains.
Description Thick and short main root, with numerous Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
1306 Japanese Gentian / Crude Drugs
JP XV
Powdered Japanese Angelica Root according to Method 3,
and perform the test. Prepare the control solution with 3.0
mL of Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Japanese Angelica Root according to Method 4,
and perform the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Japanese Angelica Root does not show remarkably lig-
nified sclerenchymatous cells.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01>
less than 35.0%.
Dilute ethanol-soluble extract: not
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Japanese Gentian
Gentianae Scabrae Radix
'J 3.^9>
Japanese Gentian is the root and rhizome of Gen-
tiana scabra Bunge, Gentiana manshurica Kitagawa or
Gentiana triflora Pallas (Gentianaceae).
Description Irregular, cylindrical, short rhizome with
numerous, slender roots around, and externally yellow-
brown to grayish yellow-brown. The root is 10 to 15 cm in
length, about 0.3 cm in diameter, and has longitudinal,
coarse wrinkles on the outer surface; flexible; fractured sur-
face, smooth and yellow-brown in color. The rhizome is
about 2 cm in length, about 0.7 cm in diameter, and has buds
or short remains of stems at the top.
Odor, slight; taste, extremely bitter and lasting.
Under a microscope <5.01>, a transverse section of the
young root reveals epidermis, exodermis and a few layers of
primary cortex; usually, the outermost layer is endodermis
consisting of characteristic cells divided into a few daughter
cells, often with collenchyma of 1 to 2 layers contacting the
inner side; secondary cortex having rents here and there, and
irregularly scattered sieve tubes; vessels arranged rather radi-
ally in xylem, sieve tubes existing in xylem; the rhizome has a
large pith, rarely with sieve tubes; parenchyma cells contain
needle, plate or sand crystals of calcium oxalate and oil
drops; starch grains usually absent.
Identification To 0.5 g of Powdered Japanese Gentian add
10 mL of methanol, shake for 20 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve 1 mg of
gentiopicroside for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (99.5) and
water (8:2:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): one spot among the spots from the sample solution and
a dark purple spot from the standard solution show the same
color tone and the same Rf value.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Powdered Japanese Gentian
Gentianae Scabrae Radix Pulverata
Powdered Japanese Gentian is the powder of
Japanese Gentian.
Description Powdered Japanese Gentian occurs as a
grayish yellow-brown powder. It has a slight odor and a last-
ing, extremely bitter taste.
Under a microscope <5.01>, Powdered Japanese Gentian
reveals fragments of parenchyma cells containing oil droplets
and fine crystals, fragments of endodermis and exodermis
divided into daughter cells with suberized membrane, and
fragments of vessels. Vessels mainly consist of reticulate ves-
sels and scalariform vessels, 20 - 30 /um in diameter.
Identification To 0.5 g of Powdered Japanese Gentian add
10 mL of methanol, shake for 20 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve 1 mg of
gentiopicroside for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (99.5) and
water (8:2:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 254
nm): one spot among the spots from the sample solution and
a dark purple spot from the standard solution show the same
color tone and the same Rf value.
Purity Foreign matter — Under a microscope <5.01>, Pow-
dered Japanese Gentian usually reveals no stone cells and
fibers. No starch grains; if any, very few.
Total ash <5.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Japanese Valerian
Valerianae Radix
Japanese Valerian is the root and rhizome
Valeriana fauriei Briquet (Valerianaceae).
of
Description Obovoid, short rhizome with numerous, fine
and long roots; externally dark brown to grayish brown. The
root, 10 - 15 cm in length, 0.1 - 0.3 cm in diameter; external-
ly, with fine longitudinal wrinkles; brittle. The rhizome, 1-2
cm in length, 1-2 cm in diameter, with buds and remains of
JPXV
Crude Drugs / Jujube Seed 1307
stem at the crown; hard in texture and difficult to break; flank
of rhizome sometimes accompanied with stolons having thick
and short or thin, long and extremely small, scaly leaves. Un-
der a magnifying glass, the transverse section reveals a thick,
light grayish brown cortical layer, and a grayish brown stele.
Odor, strong and characteristic; taste, slightly bitter.
Total ash <5.01> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 5.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Japanese Valerian provided that 1 mL of silicon
resin is previously added to the sample in the flask: the
volume of essential oil is not less than 0.3 mL.
Containers and storage Containers — Tight containers.
Powdered Japanese Valerian
Valerianae Radix Pulverata
Powdered Japanese Valerian is the powder of
Japanese Valerian.
Description Powdered Japanese Valerian occurs as a dark
grayish brown powder. It is somewhat moist to the touch. It
has a strong, characteristic odor and a slightly bitter taste.
Under a microscope <5.01>, Powdered Japanese Valerian
reveals starch grains and fragments of parenchyma cells con-
taining them; fragments of pitted vessels, reticulate vessels,
ring vessels, and spiral vessels; fragments of exodermis con-
taining oil droplets and composed of cells suberized and
divided into daughter cells; fragments of yellow stone cells
from the rhizome and the stolon; and very rarely, some frag-
ments of epidermis and phloem fibers. Starch grains, simple
grains 10 - 20 /um in diameter and 2- to 4-compound grains;
oil droplets stained red with sudan III TS.
Total ash <5.01> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 5.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
Powdered Japanese Valerian provided that 1 mL of silicon
resin is previously added to the sample in the flask: the
volume of essential oil is not less than 0.2 mL.
Containers and storage Containers — Tight containers.
Jujube
Zizyphi Fructus
Jujube is the fruit of Zizyphus jujuba Miller var. in-
ermis Rehder (Rhamnaceae).
Description Ellipsoidal or broad ovoid fruit, 2-3 cm in
length, 1-2 cm in diameter; externally reddish brown with
coarse wrinkles, or dark grayish red with fine wrinkles, and
both lustrous; both ends slightly dented, with a scar of style
on one end and a scar of peduncle on the other; epicarp thin
and leather; mesocarp thick, dark grayish brown in color,
spongy, soft and adhesive; endocarp extremely hard,
fusiform, and divided into two loculi; seeds flat and ovoid.
Odor, slight and characteristic; taste, sweet.
Purity (1) Rancidity — Jujube has no unpleasant, rancid
odor and taste.
(2) Total BHC's and total DOT's <5.07> Not more than
0.2 ppm, respectively.
Total ash <5.07> Not more than 3.0%.
Jujube Seed
Zizyphi Semen
Jujube Seed is the seed of Zizyphus jujuba Miller
var. spinosa (Bunge) Hu ex H. F. Chou (Rhamnaceae).
Description Jujube Seed is a compressed ovate to orbicular,
lenticular seed, 5 to 9 mm in lengh, 4 to 6 mm in width, 2 to 3
mm in thickness, externally brown to dark red-brown, glossy;
hilum at one end, charaza at the other end; seed coat sightly
flexible, covering, milky white endosperm and light yellow
embryo. 100 seeds weighing 3.0 to 4.5 g.
Odor, slightly oily; taste, mild and slightly oily.
Under a microscope <5.01>, transverse section reveals seed
coat composed of an upper epidermis, parenchyma and lower
epidermis; upper epidermal cells sclerified and elongated in
radial direction; lower epidermis covered with cuticle; en-
dosperm composed of parenchyma, containing aggregated
crystals of calcium oxalate, aleurone grains and starch grains;
cotyledons composed of parenchyma that contains aleurone
grains, starch grains and oil drops.
Identification To 2 g of pulverized Jujube Seed add 10 mL
of methanol, and heat under a reflux condenser for 10
minutes. After cooling, filter, and use the filtrate as the sam-
ple solution. Perform the test with the sample solution as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL of the sample solution on a plate of silica gel with fluores-
cent indicator for thin-layer chromatography, develop the
plate with a mixture of acetone, ethyl acetate, water and acet-
ic acid (100) (10:10:3:1) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): a purple spot appears at around Rf 0.3,
which shows a yellow-green to grayish green color after
spraying 1-naphthol-sulfuric acid TS on the plate and heating
at 105°C for 5 minutes.
Purity Foreign matter <5.01> — Jujube Seed contains not
more than 1.0% of the endocarp and other foreign matters.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <5.07> Not more than 5.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 9.0%.
1308 Kakkonto Extract / Crude Drugs
JP XV
Kakkonto Extract
Kakkonto Extract contains not less than 9 mg and
not more than 27 mg (for preparation prescribed 3 g of
Ephedra Herb) or not less than 12 mg and not more
than 36 mg (for preparation prescribed 4 g of Ephedra
Herb) of total alkaloids [ephedrine (C 10 H 15 NO: 165.23)
and pseudoephedrine (CioH 15 NO: 165.23)], not less
than 14 mg and not less than 42 mg (for preparation
prescribed 2 g of Peony Root) or not less than 21 mg
and not more than 63 mg (for preparation prescribed 3
g of Peony Root) of peonifiorin (C^HzgOn: 480.46),
and not less than 19 mg and not more than 57 mg of
glycyrrhizic acid (C 42 H 62 16 : 822.93) per a dried extract
prepared as directed in the Method of preparation.
Method of preparation Prepare a dried extract as directed
under Extracts, with 8 g of Pueraria Root, 4 g of Ephedra
Herb, 4 g of Jujube, 3 g of Cinnamon Bark, 3 g of Peony
Root, 2 g of Glycyrrhiza and 1 g of Ginger, or with 4 g of
Pueraria Root, 4 g of Ephedra Herb, 3 g of Jujube, 2 g of
Cinnamon Bark, 2 g of Peony Root, 2 g of Glycyrrhiza and 1
g of Ginger, or with 4 g of Pueraria Root, 3 g of Ephedra
Herb, 3 g of Jujube, 2 g of Cinnamon Bark, 2 g of Peony
Root, 2 g of Glycyrrhiza and 1 g of Ginger, or with 4 g of
Pueraria Root, 3 g of Ephedra Herb, 3 g of Jujube, 2 g of
Cinnamon Bark, 2 g of Peony Root, 2 g of Glycyrrhiza and 2
g of Ginger.
Description Kakkonto Extract occurs as a light brown to
brown powder. It has a characteristic odor, and a sweet first,
then hot, and slightly bitter taste.
Identification (1) Pueraria root — To 1.0 g of Kakkonto
Extract add 10 mL of water, shake, then add 10 mL of 1-
butanol, shake, centrifuge, and use the supernatant liquid as
the sample solution. Separately, dissolve 1 mg of Puerarin
Reference Standard in 1 mL of methanol, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): one of the spot among the several spots from the sample
solution has the same color tone and Rf value with the blue-
white fluorescent spot from the standard solution.
(2) Ephedra herb — To 1.0 g of Kakkonto Extract add 10
mL of water, shake, then add 10 mL of 1-butanol, shake,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of ephedrine hydrochloride in
1 mL of methanol, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of 1-butanol, water and acetic acid (100) (7:2:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly nin-
hydrin TS on the plate, and heat at 105°C for 5 minutes: one
of the spot among the several spots from the sample solution
has the same color tone and Rf value with the red-purple spot
from the standard solution.
(3) Cinnamon bark — Put 10 g of Kakkonto Extract in a
300-mL hard-glass flask, add 100 mL of water and 1 mL of
silicone resin, connect the apparatus for essential oil determi-
nation, and heat to boil under a reflux condenser. The grad-
uated tube of the apparatus is to be previously filled with
water to the standard line, and 2 mL of hexane is added to the
graduated tube. After heating under reflux for about 1 hour,
separate the hexane layer, and use this as the sample solution.
Separately, dissolve 1 mg of cinnamaldehyde for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 20 /uL of the sample solution and 2 /uL the standard so-
lution on a plate of silica gel for thin-layer chromatography.
Develop the plate with a mixture of hexane and ethyl acetate
(2:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly 2,4-dinitrophenylhydrazine TS on the plate: one
of the spot among the several spots from the sample solution
has the same color tone and Rf value with the yellow-orange
spot from the standard solution.
(4) Peony Root— To 1.0 g of Kakkonto Extract add 10
mL of water, shake, then add 10 mL of 1-butanol, shake,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of Peonifiorin Reference
Standard in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, methanol and water (20:3:2)
to a distance of about 10 cm, and air-dry the plate. Spray
evenly 4-methoxybezaldehyde-sulfuric acid TS on the plate,
and heat at 105°C for 5 minutes: one of the spot among the
several spots from the sample solution has the same color
tone and Rf value with the purple spot from the standard so-
lution.
(5) Glycyrrhiza — To 1.0 g of Kakkonto Extract add 10
mL of water, shake, then add 10 mL of 1-butanol, shake,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of liquiritin for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105°C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the yellow-brown spot from the standard solu-
tion.
(6) Ginger — To 1.0 g of Kakkonto Extract add 10 mL of
water, shake, then add 25 mL of diethyl ether, shake, cen-
trifuge, and take the diethyl ether layer. Evaporate the
diethyl ether under reduced pressure, dissolve the residue in 2
mL of diethyl ether, and use the solution as the sample solu-
tion. Separately, dissolve 1 mg of [6]-gingerol for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
JPXV
Crude Drugs / Kakkonto Extract 1309
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 /xL of the sample solution and 5 /uL of the standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 4-dimethylaminobenzaldehyde
TS for spraying on the plate, heat at 105°C for 5 minutes,
and allow to cool: one of the spot among the several spots
from the sample solution has the same color tone and Rf
value with the blue-green spot from the standard solution.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1.0 g of Kakkonto Extract as directed in (4) in Extracts
under the General Rules for Preparations, and perform the
test (not more than 30 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.67 g
of Kakkonto Extract according to Method 3, and perform
the test (not more than 3 ppm).
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C, 5
hours).
Total ash <J.07> Not more than 10.0%.
Assay (1) Total alkaloids (ephedrine and pseudo-
ephedrine) — Weigh accurately about 0.5 g of Kakkonto Ex-
tract, add exactly 50 mL of diluted methanol (1 in 2), shake
for 15 minutes, filter, and use the filtrate as the sample solu-
tion. Separately, weigh accurately about 10 mg of ephedrine
hydrochloride for assay, previously dried at 105°C for 3
hours, and dissolve in diluted methanol (1 in 2) to make ex-
actly 100 mL. Pipet 10 mL of this solution, add diluted
methanol (1 in 2) to make exactly 50 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions. Determine the peak areas, A TE and
Aj P , of ephedrine and pseudoephedrine with the sample solu-
tion, and the peak area, A s , of ephedrine with the standard
solution.
Amount (mg) of total alkaloids [ephedrine (C 10 H 15 NO) and
pseudoephedrine (C 10 H I5 NO)]
= W s x {(A JE +A TP )/A S } x 0.819 x (1/10)
W s : Amount (mg) of ephedrine hydrochloride for assay
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of a solution of sodium lauryl
sulfate (1 in 130), acetonitrile and phosphoric acid
(650:350:1).
Flow rate: 1.0 mL/min. (the retention time of ephedrine is
about 27 minutes.)
System suitability —
System performance: Dissolve 1 mg each of ephedrine
hydrochloride for assay and pseudoephedrine hydrochloride
in diluted methanol (1 in 2) to make 10 mL. When the proce-
dure is run with 10 /xL of this solution under the above oper-
ating conditions, pseudoephedrine and ephedrine are eluted
in this order with the resolution between these peaks being
not less than 1.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ephedrine is not more than 1.5%.
(2) Peoniflorin — Weigh accurately about 0.5 g of Kak-
konto Extract, add exactly 50 mL of diluted methanol (1 in
2), shake for 15 minutes, and filter. Pipet 5 mL of the filtrate,
flow through in a column packed with 2 g of polyamide for
column chromatography, elute with water to make exactly 20
mL of eluate, and use this as the sample solution. Separately,
weigh accurately about 10 mg of Peoniflorin Reference Stan-
dard (separately determine the water) and dissolve in diluted
methanol (1 in 2) to make exactly 100 mL. Pipet 5 mL of this
solution, add diluted methanol (1 in 2) to make exactly 20
mL, and use this solution as the standard solution. Perform
the test with exactly 10 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of peoniflorin.
Amount (mg) of peoniflorin (C 2 3H 28 Oii)
= W s x(A T /A s )x(l/2)
W s : Amount (mg) of Peoniflorin Reference Standard, cal-
culated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (850:150:1)
Flow rate: 1.0 mL/min. (the retention time of peoniflorin
is about 9 minutes.)
System suitability —
System performance: Dissolve 1 mg each of Peoniflorin
Reference Standard and albiflorin in diluted methanol (1 in 2)
to make 10 mL. When the procedure is run with 10 /uL of this
solution under the above operating conditions, albiflorin and
peoniflorin are eluted in this order with the resolution be-
tween these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
peoniflorin is not more than 1.5%.
(3) Glycyrrhizic acid — Weigh accurately about 0.5 g of
Kakkonto Extract, add exactly 50 mL of diluted methanol (1
in 2), shake for 15 minutes, filter, and use the filtrate as the
sample solution. Separately, weigh accurately about 10 mg of
Glycyrrhizic Acid Reference Standard (separately determine
the water), dissolve in diluted methanol (1 in 2) to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10,mL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of glycyrrhizic
1310 Kamishoyosan Extract / Crude Drugs
JP XV
acid.
Amount (mg) of glycyrrhizic acid (C 42 H 62 16 )
= W s x(A T /A s )x (.1/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7).
Flow rate: 1.0 mL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
not more than 1.5%, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
Kamishoyosan Extract
Kamishoyosan Extract contains not less than 28 mg
and not more than 84 mg of peoniflorin (C23H 2 80 n :
480.46), not less than 25 mg and not more than 75 mg
of geniposide, and not less than 12 mg and not more
than 36 mg (for preparation prescribed 1.5 g of Glycyr-
rhiza) or not less than 16 mg and not more than 48 mg
(for preparation prescribed 2 g of Glycyrrhiza) of
glycyrrhizic acid (C 42 H 62 16 : 822.93) per a dried extract
prepared as directed in the Method of preparation.
Method of preparation Prepare a dried extract as directed
under Extracts, with 3 g of Japanese Angelica Root, 3 g of
Peony Root, 3 g of Atractylodes Rhizome or Atractylodes
Lancea Rhizome, 3 g of Poria Sclerotium, 3 g of Bupleurum
Root, 2 g of Moutan Bark, 2 g of Gardenia Fruit, 2 g of
Glycyrrhiza, 1 g of Ginger and 1 g of Mentha Herb, or with 3
g of Japanese Angelica Root, 3 g of Peony Root, 3 g of
Atractylodes Rhizome or Atractylodes Lancea Rhizome, 3 g
of Poria Sclerotium, 3 g of Bupleurum Root, 2 g of Moutan
Bark, 2 g of Gardenia Fruit, 1.5 g of Glycyrrhiza, 1 g of Gin-
ger and 1 g of Mentha Herb, or with 3 g of Japanese Angelica
Root, 3 g of Peony Root, 3 g of Atractylodes Rhizome, 3 g of
Poria Sclerotium, 3 g of Bupleurum Root, 2g of Moutan
Bark, 2 g of Gardenia Fruit, 1.5 g of Glycyrrhiza, 1.5 g of
Ginger and 1 g of Mentha Herb, or with 3 g of Japanese An-
gelica Root, 3 g of Peony Root, 3 g of Atractylodes Rhizome,
3 g of Poria Sclerotium, 3 g of Bupleurum Root, 2 g of Mou-
tan Bark, 2 g of Gardenia Fruit, 1.5 g of Glycyrrhiza, 0.5 g of
Ginger and 1 g of Mentha Herb.
Description Kamishoyosan Extract occurs as a yellow-
brown to brown powder. It has slightly a characteristic odor,
and a sweet, slightly hot, then bitter taste.
Identification (1) Japanese angelica root — To 2.0 g of
Kamishoyosan Extract add 10 mL of water, shake, then add
5 mL of diethyl ether, shake, centrifuge, and use the super-
natant liquid as the sample solution. Separately, dissolve 1
mg of (Z)-ligustilide for thin-layer chromatography in 10 mL
of methanol, and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /uL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate and hexane (1:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): one of the spot among the sever-
al spots from the sample solution has the same color tone and
Rf value with the blue-white fluorescent spot from the stan-
dard solution.
(2) Peony root — To 2.0 g of Kamishoyosan Extract add
10 mL of water, shake, then add 5 mL of 1-butanol, shake,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of albiflorin in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /uL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
ethyl acetate, methanol and ammonia solution (28) (6:3:2) to
a distance of about 10 cm, and air-dry the plate. Spray evenly
4-methoxybenzaldehyde-sulfuric acid TS on the plate, heat at
105 °C for 5 minutes, and examine under ultraviolet light
(main wavelength: 365 nm): one of the spot among the sever-
al spots from the sample solution has the same color tone and
Rf value with the orange fluorescent spot from the standard
solution.
(3) Atractylodes rhizome (for preparation prescribed
Atractylodes Rhizome) — To 2.0 g of Kamishoyosan Extract
add 10 mL of water, shake, then add 5 mL of diethyl ether,
shake, centrifuge, and use the supernatant liquid as the sam-
ple solution. Separately, dissolve 1 mg of atractylenolide III
for thin-layer chromatography in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate and hexane (1:1) to a distance of about 10 cm, and
air-dry the plate. Spray evenly 1-naphthol-sulfuric acid TS on
the plate, heat at 105°C for 5 minutes, and allow to cool: one
of the spot among the several spots from the sample solution
has the same color tone and Rf value with the red spot from
the standard solution.
(4) Atractylodes lancea rhizome (for preparation
prescribed Atractylodes Lancea Rhizome) — To 2.0 g of
Kamishoyosan Extract add 10 mL of water, shake, then add
25 mL of hexane, and shake. Take the hexane layer, add an-
hydrous sodium sulfate to dry, and filter. Evaporate the
filtrate under reduced pressure, add 2 mL of hexane to the
JPXV
Crude Drugs / Kamishoyosan Extract 1311
residue, and use this solution as the sample solution. Perform
the test with the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 20 [iL of the sample solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of hexane
and acetone (7:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): a dark purple spot is observed at around Rf 0.4. The
spot shows a greenish brown color after being sprayed 4-
dimethylaminobenzaldehyde TS for spraying, heated at
105 °C for 5 minutes, and allowed to cool.
(5) Bupleurum root — To 2.0 g of Kamishoyosan Extract
add 10 mL of sodium hydroxide TS, shake, then add 5 mL of
1-butanol, shake, centrifuge, and use the supernatant liquid
as the sample solution. Separately, dissolve 1 mg of sai-
kosaponin b 2 for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL of the sample solu-
tion and 2 [iL of the standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate, ethanol (99.5) and water (8:2:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
dimethylaminobenzaldehyde TS on the plate: one of the spot
among the several spots from the sample solution has the
same color tone and Rf value with the red spot from the stan-
dard solution.
(6) Moutan bark — To 2.0 g of Kamishoyosan Extract
add 10 mL of water, shake, then add 15 mL of diethyl ether,
and shake. Take the diethyl ether layer, evaporate the layer
under reduced pressure, add 1 mL of diethyl ether to the
residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of peonol for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/uL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography, develop the
plate with a mixture of hexane and diethyl ether (5:3) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
methoxybenzaldehyde-sulfuric acid TS on the plate, and heat
at 105 °C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the orange spot from the standard solution.
(7) Gardenia fruit — To 2.0 g of Kamishoyosan Extract
add 10 mL of water, shake, then add 5 mL of 1-butanol,
shake, centrifuge, and use the supernatant liquid as the sam-
ple solution. Separately, dissolve 1 mg of geniposide for thin-
layer chromatography in 1 mL of methanol, and use this so-
lution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, methanol and ammonia solution (28) (6:3:2) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
methoxybenzaldehide-sulfuric acid TS on the plate, and heat
at 105 °C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the purple spot from the standard solution.
(8) Glycyrrhiza — To 2.0 g of Kamishoyosan Extract add
10 mL of water, shake, then add 5 mL of 1-butanol, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 1 mg of liquiritin for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL of the sample solution and 5 /uL of the standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105°C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the yellow-brown spot from the standard solu-
tion.
(9) Ginger — To 2.0 g of Kamishoyosan Extract add 10
mL of water, shake, then add 5 mL of diethyl ether, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 1 mg of [6]-gingerol for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>, Spot 10
/iL each of the sample solution and standard solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate and hexane (1 : 1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
dimethylaminobenzaldehyde TS for spraying on the plate,
heat at 105°C for 5 minutes, and allow to cool: one of the
spot among the several spots from the sample solution has
the same color tone and Rf value with the blue-green spot
from the standard solution.
(10) Mentha herb — To 2.0 g of Kamishoyosan Extract
add 10 mL of diluted phosphoric acid (1 in 30), shake, then
add 15 mL of ethyl acetate, shake, centrifuge, and use the su-
pernatant liquid as the sample solution. Separately, shake 0.2
g of pulverized Mentha Herb with 10 mL of diluted phos-
phoric acid (1 in 30), then add 15 mL of ethyl acetate, shake,
centrifuge, and use the supernatant liquid as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /xL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate, water and formic acid (10:1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly
vanillin-sulfuric acid TS on the plate, heat at 105 °C for 5
minutes, and allow to cool: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the red-purple spot (around Rf 0.4) from the
standard solution.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1.0 g of Kamishoyosan Extract as directed in (4) in Ex-
tracts, and perform the test (not more than 30 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.67 g
of Kamishoyosan Extract according to Method 3, and per-
form the test (not more than 3 ppm).
Loss on drying <2.41> Not more than 9.0% (1 g, 105 °C, 5
hours).
Total ash <J.07> Not more than 10.0%.
Assay (1) Peoniflorin — Weigh accurately about 0.5 g of
Kamishoyosan Extract, add exactly 50 mL of diluted
methanol (1 in 2), shake for 15 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of Peoniflorin Reference Standard (separately
1312 Kamishoyosan Extract / Crude Drugs
JP XV
determine the water), dissolve in diluted methanol (1 in 2) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 liL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of peoniflo-
jH?<jOi
W s x(A T /A s )x(l/2)
W s : Amount (mg) of Peoniflorin Reference Standard, cal-
culated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (850:150:1)
Flow rate: 1.0 mL/min. (the retention time of peoniflorin
is about 9 minutes.)
System suitability —
System performance: Dissolve 1 mg each of Peoniflorin
Reference Standard and albiflorin in diluted methanol (1 in 2)
to make 10 mL. When the procedure is run with 10 fiL of this
solution under the above operating conditions, albiflorin and
peoniflorin are eluted in this order with the resolution be-
tween these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
10 iiL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
peoniflorin is not more than 1.5%.
(2) Geniposide — Weigh accurately about 0.5 g of
Kamishoyosan Extract, add exactly 50 mL of diluted
methanol (1 in 2), shake for 15 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of geniposide for component determination,
previously dried in a desiccator (in vacuum, phosphorous (V)
oxide) for 24 hours, dissolve in diluted methanol (1 in 2) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 iiL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of genipo-
side.
Amount (mg) of geniposide = W s x (A T /A S ) X (1/2)
W s : Amount (mg) of geniposide for component determi-
nation
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 240 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (900:100:1).
Flow rate: 1 .0 mL/min. (the retention time of geniposide is
about 10 minutes.)
System suitability —
System performance: When the procedure is run with 10
liL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of geniposide are not less than 5000 and not
more than 1.5%, respectively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
geniposide is not more than 1.5%.
(3) Glycyrrhizic acid — Weigh accurately about 0.5 g of
Kamishoyosan Extract, add exactly 50 mL of diluted
methanol (1 in 2), shake for 15 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of Glycyrrhizic Acid Reference Standard
(separately determine the water), dissolve in diluted methanol
(1 in 2) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 10 iiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
glycyrrhizic acid.
Amount (mg) of glycyrrhizic acid (C 4 2H 62 16 )
= W s x(A T /A s )x (1/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7).
Flow rate: 1.0 mL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
liL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
not more than 1.5%, respectively.
System repeatability: When the test is repeated 6 times with
10 /nL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
JPXV
Crude Drugs / Longgu 1313
Lindera Root
Linderae Radix
Lindera Root is the root of Lindera strychnifolia
Fernandez- Villar (Lauraceae).
Description Fusiform or rosary-like root, 10 -15 cm in
length, 10 -25 mm in diameter; externally yellowish brown
to brown, with a few scars of rootlets; a transverse section
reveals cortex brown, xylem light yellowish brown, concen-
tric circles and radially arranged lines brown; dense and hard
in texture.
Odor, camphor-like; taste, bitter.
Under a microscope <5.01>, a transverse section of the root
with periderm reveals a cork layer several cells thick, partially
consisting of cork stone cells; cortex parenchyma sometimes
contains oil cells and fibers; in xylem, vessels-xylem fibers and
rays are arranged alternately; parenchyatous cells of cortex
and xylem contain sandy and columnar crystals of calcium
oxalate, simple starch grains 1-15 /xm in diameter, and 2- to
4- compound starch grains.
Identification To 3 g of pulverized Lindera Root add 40 mL
of hexane, and warm under a reflux condenser on a water
bath for 30 minutes. After cooling, filter, to the residue add
10 mL of ammonia TS and 30 mL of a mixture of ethyl
acetate and diethyl ether (1:1), shake vigorously for 20
minutes, and centrifuge. Separate the supernatant liquid, add
10 g of anhydrous sodium sulfate, shake, and filter.
Evaporate the filtrate, dissolve the residue with 0.5 mL of
ethanol (99.5), and use this solution as the sample solution.
Perform the test with this solution as directed under Thin-
layer Chromatography <2.03>. Spot 20 fiL of the sample
solution on a plate of silica gel for thin-layer chro-
matography, develop the plate with a mixture of ethyl
acetate, methanol and ammonia water (28) (10:2:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
Dragendorff's TS for spraying on the plate: a yellow-brown
spot appears at around Ri 0.4.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 2.5%.
Extract content <J.07>
less than 6.0%.
Dilute ethanol-soluble extract: not
Lithospermum Root
Lithospermi Radix
Lithospermum Root is the root of Lithospermum
erythrorhizon Siebold et Zuccarini (Boraginaceae).
Description Rather slender conical root, often branched,
6-10 cm in length, 0.5 - 1.5 cm in diameter; externally dark
purple, coarse in texture, thin and easily peeled; mostly with
twisted and deep longitudinal furrows, which sometimes
reach to xylem; sometimes remains of stem at the crown; ea-
sily broken; fractured surface granular and with many clefts.
Under a magnifying glass, a transverse section reveals a dark
purple color at the outer portion of cortex, and light brown
inner portion making irregular wave; xylem yellowish in
color; the center of the crown is often cracked, and the sur-
rounding part red-purple.
Odor, slight; taste, slightly sweet.
Identification (1) Heat 0.5 g of pulverized Lithospermum
Root in a test tube: red vapor evolves, which condenses on
the wall of the upper part of the tube into red-brown oil
drops.
(2) Shake 0.5 g of pieces or powder of Lithospermum
Root with 1 mL of ethanol (95), and to the red solution there-
by obtained add 1 drop of sodium hydroxide TS: the red
color changes to blue-purple. To this solution add 1 to 2
drops of dilute hydrochloric acid: the color turns red again.
(3) To 0.5 g of pulverized Lithospermum Root add 5 mL
of ethanol (95), shake for 30 minutes, filter, and evaporate
the filtrate at a temperature not higher than 40°C under
reduced pressure. Add 1 mL of ethanol (95) to the residue,
and use this solution as the sample solution. Perform the test
with this solution as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL of the sample solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of ethyl acetate and ethanol (95) (3:1) to
a distance of about 10 cm, and air-dry the plate: a red-purple
spot appears around the Rf 0.75.
Total ash <J.07> Not more than 11.0%.
Acid-insoluble ash <5.01> Not more than 3.5%.
Longgu
Fossilia Ossis Mastodi
Longgu is the ossified bone of large mammal, and is
mainly composed of calcium carbonate.
Description Irregular masses or fragments, occasionally
cylindrical masses; externally light grayish white, sometimes
with grayish black or yellow-brown spots here and there; the
outer part consists of a layer 2-10 mm in thickness, and is
minute in texture, surrounding the light brown, porous por-
tion; heavy and hard, but somewhat fragile in texture; when
crushed, it changes into pieces and powder.
Odorless, tasteless, and strongly adhesive to the tongue on
licking.
Identification (1) Dissolve 0.5 g of pulverized Longgu in
10 mL of dilute hydrochloric acid: it evolves a gas, and forms
a slightly brownish and turbid solution. Pass the gas evolved
through calcium hydroxide TS: a white precipitate is
produced.
(2) The turbid solution, obtained in (1), has a characteris-
tic odor. Filter this solution, and neutralize with ammonia
TS: the solution responds to the Qualitative test <1.09> for
calcium salt.
(3) Dissolve 0.1 g of pulverized Longgu in 5 mL of nitric
1314 Lonicera Leaf and Stem / Crude Drugs
JP XV
acid by warming, and add hexaammonium heptamolybdate
TS: a yellow precipitate is produced.
Purity (1) Heavy metals <1.07> — To 2.0 g of pulverized
Longgu add 5 mL of water, shake to mix, add carefully 6 mL
of hydrochloric acid, and evaporate on a water bath to dry-
ness. Dissolve the residue in 50 mL of water, and filter. To 25
mL of the filtrate add 2 mL of dilute acetic acid, 1 drop of
ammonia TS and water to make 50 mL. Perform the test us-
ing this solution as the test solution. Prepare the control solu-
tion as follows: Evaporate 3 mL of hydrochloric acid on a
water bath to dryness, add 2 mL of dilute acetic acid and 2.0
mL of Standard Lead Solution, and add water to make 50
mL (not more than 20 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.20 g
of pulverized Longgu according to Method 2, and perform
the test (not more than 10 ppm).
several spots from the sample solution has the same color
tone and Rf value with the blue-white fluorescent spot from
the standard solution (1). Spray evenly 4-methoxybenzalde-
hyde-sulfuric acid TS on the plate, and heat at 105°C for 5
minutes: one of the spot among the several spots from the
sample solution has the same color tone and Rf value with the
red-purple spot from the standard solution (2).
Purity Stem — Lonicera Leaf and Stem does not contains
the stems larger than 5 mm in diameter.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.07> Not more than 9.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 12.0%.
Lonicera Leaf and Stem
Lonicerae Folium Cum Caulis
Loquat Leaf
Eriobotryae Folium
Lonicera Leaf and Stem is the leaves and stems of
Lonicera japonica Thunberg (Caprifoliaceae).
Description Leaves and opposite leaves on short stem; leaf,
ovate and entire, with short petiole, 3-7 cm in length, 1-3
cm in width; upper surface greenish brown, lower surface
light grayish green; under a magnifying glass, both surfaces
pubescent. Stem, 1-4 mm in diameter; externally grayish
yellow-brown to purplish brown, a transverse section of
stem, round and hollow.
Almost odorless; taste, slightly astringent, followed by a
litter bitterness.
Under a microscope <5.01>, a transverse section of leaf rev-
eals the outermost layer of upper and lower surfaces to be
composed of a single-layered epidermis, uni-cellular non-
glandular hairs and multi-cellular glandular hairs on epider-
mis; in midvein, several-layered collenchyma present beneath
the epidermis and vascular bundles in the center; in
mesophyll, palisade layer adjacent to upper epidermis,
spongy tissue adjacent to lower epidermis; glandular hairs
contain brown secretion, parenchymatous cells contain ag-
gregate crystals of calcium oxalate, and occasionally starch
grains.
Identification To 1 g of pulverized Lonicera Leaf and Stem
add 5 mL of methanol, shake for 5 minutes, centrifuge, and
use the supernatant liquid as the sample solution. Separately,
dissolve 1 mg of chlorogenic acid for thin-layer chro-
matography in 2 mL of methanol, and use this solution as the
standard solution (1). Separately, dissolve 1 mg of loganin
for thin-layer chromatography in 2 mL of methanol, and use
this solution as the standard solution (2). Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 [iL each of the sample solution
and standard solutions (1) and (2) on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, water and formic acid (6:1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 365 nm): one of the spot among the
Loquat Leaf is the leaf of Eriobotrya japonica Lin-
dley (Rosaceae).
Description Loquat Leaf is an oblong to wide lanceolate
leaf, 12 to 30 cm in length, 4 to 9 cm in width; pointed at the
apex and wedge-shaped at the base; roughly serrate leaf with
short petiole; occasionally being cut into strips 5 to 10 mm in
shorter diameter and several cm in longer diameter; upper
surface green to green-brown in color, lower surface light
green-brown with light brown woolly hairs; vein, light yel-
low-brown in color, raised out on the lower surface of the
leaf.
Odor, slight; practically tasteless.
Under a microscope <5.01>, a transverse section of Loquat
Leaf reveals thick cuticle on both surfaces; palisade tissue,
mostly 4 to 5 layers with several large cells without chlo-
roplast; at main vein, ring of collateral bundle partly cut by
intruding fundamental tissue at xylem side, and group of
fiber attaching to phloem; solitary and clustered crystals of
calcium oxalate in mesophyll; woolly hair, unicellular and
curved, about 25 jum in thickness, and up to 1.5 mm in
length.
Identification To 0.3 g of pulverized Loquat Leaf add 10
mL of methanol, warm on a water bath for 5 minutes with
occasional shaking, cool, filter, and use the filtrate as the
sample solution. Perform the test with this solution as direct-
ed under Thin-layer Chromatography <2.03>. Spot 5 /uL of
the sample solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of water and
acetonitrile (3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105 °C for 10 minutes: a red-purple principal spot appears
at around Rf 0.5.
Purity Total BHC's and total DDT's <5.07>— Not more
than 0.2 ppm, respectively.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.07> Not more than 10.0%.
JPXV
Crude Drugs / Magnolia Bark 1315
Extract content <5.0I> Dilute ethanol-soluble extract: not
less than 16.0%.
Lycium Bark
Lycii Cortex
Lycium Bark is the root bark of Lycium chinense
Miller or Lycium barbarum Linne (Solanaceae).
Description Tubular to semitubular bark, 1 - 6 mm in
thickness; externally light brown to light yellowish brown,
periderm peeled easily as scale; internally grayish brown, lon-
gitudinally striate; brittle in texture; fractured surface,
grayish white, not fibrous.
Odor, weak and characteristic; taste, slightly sweet at first.
Under a microscope <5.01>, a transverse section reveals
periderm composed of a cork layer of several layers of thin
walled cork cells; in cortex parenchymatous cells containing
sandy crystals of calcium oxalate sparsely distributed, oc-
casionally a few fibers observed; parenchymatous cells con-
tain starch grains, 1 - lO^m in diameter; stone cells very
rare.
Identification To 1.0 g of pulverized Lycium Bark add 10
mL of methanol, shake for 15 minutes, filter, and use the
filtrate as the sample solution. Perform the test with this
solution as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL of the sample solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-butanol, water, pyridine and acetic acid (100)
(3:1:1:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly Dragendorff's TS for spraying on the plate, heat
at 105°C for 3 minutes, then spray evenly sodium nitrite TS:
a dark brown principal spot appears at around Rf 0.5.
Loss on drying <5.01> Not more than 11.5% (6 hours).
Total ash <5.07> Not more than 20.0%.
Acid-insoluble ash <5.01> Not more than 3.0%.
Extract content <J.07>
less than 10.0%.
Dilute ethanol-soluble extract: not
Lycium Fruit
Lycii Fructus
Lycium Fruit is the fruit of Lycium chinense Miller
or Lycium barbarum Linne (Solanaceae).
Description Fusiform fruit with acute apex, 6-20 mm in
length, 3-8 mm in diameter, pericarp red to dark red,
externally roughly wrinkled; under a magnifying glass, a
transverse section of fruit reveals two locules containing
numerous seeds; seed light brown to light yellowish brown,
about 2 mm in a diameter, compressed reniform.
Odor, characteristic; taste, sweet, later slightly bitter.
Identification To 1.0 g of powdered Lycium Fruit add 5 mL
of ethyl acetate, shake for 15 minutes, filter, and use the
filtrate as the sample solution. Perform the test with this
solution as directed under Thin-layer Chromatography.
<2.03> Spot 20 /uL of the sample solution on a plate of silica
gel for thin-layer chromatography, develop the plate with a
mixture of hexane and ethyl acetate (10:1) to a distance of
about 10 cm, and air-dry the plate: a yellow principal spot ap-
pears at around Rf 0.6.
Purity Foreign matter <5.01> — It contains not more than
2.0% of foreign matter such as peduncle or others.
Total ash <J.07> Not more than 8.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 35.0%.
Magnolia Bark
=l^7T<?
Magnolia Bark is the bark of the trunk of Magnolia
obovata Thunberg, Magnolia officinalis Rehder et Wil-
son, Magnolia officinalis Rehder et Wilson var. biloba
Rehder et Wilson (Magnoliaceae).
It contains not less than 0.8% of magnolol.
Description Plate-like or semi-tubular bark, 2-7 mm in
thickness; externally grayish white to grayish brown, and
rough, sometimes cork layer removed, and externally red-
brown; internally light brown to dark purplish brown; cut
surface extremely fibrous, and light red-brown to purplish
brown.
Odor, slight; taste, bitter.
Under a microscope <5.01>, a transverse section reveals a
thick cork layer or several thin cork layers, and internally ad-
joining the circular tissue of stone cells of approximately e-
qual diameter; primary cortex thin; fiber groups scattered in
the pericycle; phloem fibers lined stepwise between medullary
rays in the secondary cortex, and then these tissues show a
latticework; oil cells scattered in the primary and secondary
cortex, but sometimes observed in the narrow medullary
rays.
Identification To 1.0 g of pulverized Magnolia Bark add 10
mL of methanol, stir for 10 minutes, centrifuge, and use the
supernatant liquid as the sample solution. Perform the test
with this solution as directed under Thin-layer Chro-
matography <2.03>. Spot 20,mL of the sample solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, water and acetic acid (100)
(4:2:1) to a distance of about 10 cm, and air-dry the plate,
spray evenly the plate with Dragendorff's TS: a yellow spot
appears at the Rf value of around 0.3.
Total ash <J.07> Not more than 6.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 11.0%.
Component determination Weigh accurately about 0.5 g of
1316 Powdered Magnolia Bark / Crude Drugs
JP XV
pulverized Magnolia Bark, add 40 mL of diluted methanol (7
in 10), heat under a reflux condenser on a water bath for 20
minutes, cool, and filter. Repeat the above procedure with
the residue, using 40 mL of diluted methanol (7 in 10). Com-
bine the whole filtrates, add diluted methanol (7 in 10) to
make exactly 100 mL, and use this solution as the sample so-
lution. Separately, dry magnolol for component determina-
tion in a desiccator (silica gel) for 1 hour or more. Weigh ac-
curately about 10 mg of it, dissolve in diluted methanol (7 in
10) to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with 10 iiL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A$, of magnolol
in each solution.
Amount (mg) of magnolol
= W s x(A T /A s )
W s : Amount (mg) of magnolol for component
determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 289 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 /um in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (50:50:1).
Flow rate: Adjust the flow rate so that the retention time of
magnolol is about 14 minutes.
Selection of column: Dissolve 1 mg each of magnolol for
component determination and honokiol in 10 mL of diluted
methanol (7 in 10). Proceed with 10 ^L of this solution under
the above operating conditions. Use a column giving elution
of honokiol and magnolol in this order with the resolution
between these peaks being not less than 5.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative deviation of the peak area of magnolol is not
more than 1.5%.
Powdered Magnolia Bark
Magnoliae Cortex Pulveratus
Powdered Magnolia Bark is the powder of Magnolia
Bark.
It contains not less than 0.8% of magnolol.
Description Powdered Magnolia Bark occurs as a yellow-
brown powder, and has a slight odor and a bitter taste.
Under a microscope <5.01>, Powdered Magnolia Bark rev-
eals starch grains and parenchyma cells containing them;
stone cells of various sizes or its groups; fibers 12 to 25 /urn in
diameter; yellowish red-brown cork tissue; oil cells contain-
ing a yellow-brown to red-brown substance. Simple starch
grains about 10 /um in diameter and 2- to 4-compound starch
grains.
Identification To 1.0 g of Powdered Magnolia Bark add 10
mL of methanol, stir for 10 minutes, centrifuge, and perform
the test with the supernatant liquid as the sample solution as
directed under Thin-layer Chromatography <2.03>. Spot 20
iiL of the sample solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (4:2:1) to a distance of
about 10 cm, and air-dry the plate, and spray evenly with
Dragendorff's TS on the plate: a yellow spot appears at the
Ri value of around 0.3.
Total ash <J.07> Not more than 6.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 11.0%.
Component determination Weigh accurately about 0.5 g of
Powdered Magnolia Bark, add 40 mL of diluted methanol (7
in 10), heat under a reflux condenser on a water bath for 20
minutes, cool, and filter. Repeat the above procedure with
the residue, using 40 mL of diluted methanol (7 in 10). Com-
bine the whole filtrates, add diluted methanol (7 in 10) to
make exactly 100 mL, and use this solution as the sample so-
lution. Separately, dry magnolol for component determina-
tion in a desiccator (silica gel) for 1 hour or more. Weigh ac-
curately about 10 mg of it, dissolve in diluted methanol (7 in
10) to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with 10 /uL each of the sample
solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of magnolol
in each solution.
Amount (mg) of magnolol
= W s x(A T /A s )
W s : Amount (mg) of magnolol for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 289 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 fim in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and acetic
acid (100) (50:50:1).
Flow rate: Adjust the flow rate so that the retention time
of magnolol ia about 14 minutes.
Selection of column: Dissolve 1 mg each of magnolol for
component determination and honokiol in 10 mL of diluted
methanol (7 in 10). Proceed with 10 LiL of this solution under
the above operating conditions. Use a column giving elution
of honokiol and magnolol in this order with the resolution
between these peaks being not less than 5.
System repeatability: When the test is repeated 5 times
with the standard solution under the above operating condi-
tions, the relative deviation of the peak area of magnolol is
not more than 1.5%.
Containers and storage Containers — Tight containers.
JPXV
Crude Drugs / Mentha Herb 1317
Magnolia Flower
Magnoliae Flos
Magnolia Flower is the flower bud of Magnolia
salicifolia Maximowicz, Magnolia kobus De Candolle,
Magnolia biondii Pampanini, Magnolia sprengeri
Pampanini or Magnolia denudata Desrousseaux (Mag-
no liaceae).
Description Magnolia Flower is a fusiform flower bud, 15
to 45 mm in length, 6 to 20 mm in diameter of central part;
often having ligneous peduncles on base; usually 3 bracts, ex-
ternally with sparse hairs; brown to dark brown; or with
dense hairs, grayish white to light yellow-brown, and the in-
ner surface smooth and dark brown in color; interior
perianth of 9 pieces or 12 pieces, same size or exterior three
pieces are smaller; 50 to 100 stamens and numerous pistils.
Brittle in texture.
Odor, characteristic; taste, acrid and slightly bitter.
Identification To 1 g of pulverized Magnolia Flower add 10
mL of methanol, shake for 15 minutes, filter, and use the
filtrate as the sample solution. Perform the test with the
sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 20 /xL of the sample solution on a
plate of silica gel for thin-layer chromatography, develop the
plate with a mixture of ethyl acetate, acetone, water and for-
mic acid (5:3:1:1) to a distance of about 10 cm, and air-dry
the plate. Spray evenly Dragendorff's TS for spraying on the
plate: a yellow-red spot appears at around Rf 0.3.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.01> Not more than 5.5%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <J.07>
13.0%.
Dilute ethanol-extract: not less than
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Magnolia Flower: the volume of essential oil is not
less than 0.5 mL.
Mallotus Bark
Malloti Cortex
Mallotus Bark is the bark of Mallotus japonica
Mueller Arg. (Euphorbiaceae).
Description Mallotus Bark is fiat or semitubular pieces of
bark, 1 to 3 mm in thickness; externally green-gray to brown-
gray brow in color, with a vertically striped shape gathering
numerous lenticels; internal surface light yellowish brown to
grayish brown in color, and smooth with numerous fine
striped lines; easy to break; slightly fibrous at fracture sur-
face.
Mallotus Bark has a slight odor, a bitter taste and slightly
astringent.
Identification To 0.5 g pulverized Mallotus Bark add 10 mL
of methanol, warm on a water bath for 5 minutes, filter, and
use the filtrate as the sample solution. Separately, dissolve 1
mg of bergenin for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /uL each of the sample so-
lution and standard solution on a plate of silica gel with
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (95) and
water (100:17:13) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): a principal spot with a dark blue color which ap-
pears at Rf about 0.5 from the sample solution is the same as
the spot from the standard solution in color and the Rf.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 12.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Extract content <5.01>
less than 11.0%.
Dilute ethanol-soluble extract: not
Mentha Herb
Menthae Herba
Mentha Herb is the terrestrial part of Mentha arven-
sis Linne var. piperascens Malinvaud (Labiatae).
Description Stem with opposite leaves; stem, square, light
brown to red-purple in color, and with fine hairs; when
smoothed by immersing in water, leaf, ovate to oblong, with
acute apex and base, 2 - 8 cm in length, 1 - 2.5 cm in width,
margin irregularly serrated; the upper surface, light brown-
yellow to light green-yellow, and the lower surface, light
green to light green-yellow in color; petiole 0.3 - 1 cm in
length. Under a magnifying glass, leaf reveals hairs, glandu-
lar hairs and scales.
It has a characteristic aroma and gives a cool feeling on
keeping in the mouth.
Identification To 1 mL of the mixture of essential oil and
xylene, obtained in the Essential oil content, add carefully 2
mL of sulfuric acid to make two layers: a deep red to red-
brown color develops at the zone of contact.
Purity Foreign matter <5.01> — The amount of roots and
other foreign matter contained in Mentha Herb does not ex-
ceed 2.0%.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <J.07> Not more than 11.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Mentha Herb after adding 1 mL of silicone resin
to the sample in the flask: the volume of essential oil is not
1318 Mentha Oil / Crude Drugs
less than 0.4 mL.
Mentha Oil
Oleum Menthae Japonicae
Mentha Oil is the essential oil which is distilled with
steam from the terrestrial parts of Mentha arvensis
Linne var. piperascens Malinvaud (Labiatae), and
from which solids are removed after cooling.
It contains not less than 30.0% of menthol
(C 10 H 20 O: 156.27).
Description Mentha Oil is a colorless or pale yellow, clear
liquid. It has a characteristic, pleasant aroma and has a pun-
gent taste, followed by a cool aftertaste.
It is miscible with ethanol (95), with ethanol (99.5), with
warm ethanol (95), and with diethyl ether.
It is practically insoluble in water.
Refractive index <2.45>
"d-
1.455 - 1.467
Optical rotation <2.49> a™: - 17.0 - - 36.0° (100 mm).
Specific gravity <7.73> d\\: 0.885 - 0.910
Acid value <1.13> Not more than 1.0.
Purity (1) Clarity and color of solution — To 1.0 mL of
Mentha Oil add 3.5 mL of diluted ethanol (7 in 10), and
shake: Mentha Oil dissolves clearly. To the solution add 10
mL of ethanol (95): the solution is clear or has no more tur-
bidity, if any, than the following control solution.
Control solution: To 0.70 mL of 0.01 mol/L hydrochloric
acid VS add 6 mL of dilute nitric acid and water to make 50
mL, add 1 mL of silver nitrate TS, and allow to stand for 5
minutes.
(2) Heavy metals <1.07> — Proceed with 1.0 mL of Men-
tha Oil according to Method 2, and perform the test. Prepare
the control solution with 4.0 mL of Standard Lead Solution
(not more than 40 ppm).
Assay Weigh accurately about 5 g of Mentha Oil, and dis-
solve in ethanol (95) to make exactly 20 mL. Pipet 10 mL of
this solution, add exactly 10 mL of the internal standard so-
lution, and use this solution as the sample solution. Separate-
ly, weigh accurately about 10 g of /-menthol for assay, and
dissolve in ethanol (95) to make exactly 100 mL. Pipet 10 mL
of this solution, add exactly 10 mL of the internal standard
solution, and use this solution as the standard solution. Per-
form the test with 1 /xL each of the sample solution and stan-
dard solution as directed under Gas Chromatography <2.02>
according to the following conditions. Calculate the ratios,
Qj and Q s , of the peak area of menthol to that of the internal
standard.
Amount (mg) of Ci H 2 oO
= W s x (g T /e s )
W s : Amount (mg) of /-menthol for assay
Internal standard solution — A solution of «-ethyl caprylate
in ethanol (95) (1 in 25).
Operating conditions —
JP XV
Detector: A hydrogen flame-ionization detector.
Column: A glass column about 3 mm in inside diameter
and about 2 m in length, packed with 25% of polyethylene
glycol 6000 for gas chromatography supported on acid-
washed 180 - 250 /um siliceous earth for gas chromatography.
Column temperature: A constant temperature of about
150°C.
Carrier gas: Nitrogen.
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is about 10 minutes.
Selection of column: Proceed with 1 /xh of the standard so-
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of the internal
standard and /-menthol in this order with the resolution be-
tween these peaks being not less than 5.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Mentha Water
Method of preparation
Mentha Oil
Purified Water
2 mL
a sufficient quantity
To make 1000 mL
Prepare as directed under Aromatic Waters, with the
above ingredients.
Description Mentha Water is a clear, colorless liquid, hav-
ing the odor of mentha oil.
Containers and storage Containers — Tight containers.
Moutan Bark
Moutan Cortex
Moutan Bark is the root bark of Paeonia suffrutico-
sa Andrews (Paeonia moutan Sims) (Paeoniaceae).
It contains not less than 1.0% of paeonol.
Description Tubular to semi-tubular bark, about 0.5 cm in
thickness, 5-8 cm in length, 0.8 - 1.5 cm in diameter; exter-
nally dark brown to purplish brown, with small and transver-
sely elongated ellipsoidal scars of lateral roots, and with lon-
gitudinal wrinkles; internally, light grayish brown to purplish
brown and smooth; fractured surface coarse; white crystals
often attached on the internal and fractured surfaces.
Odor, characteristic; taste, slightly pungent and bitter.
Identification To 2.0 g of pulverized Moutan Bark add 10
mL of hexane, shake for 3 minutes, filter, and use the filtrate
as the sample solution. Separately, dissolve 1 mg of paeonol
for thin-layer chromatography in 10 ml of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL of the sample solution and standerd solu-
JPXV
Crude Drugs / Powdered Moutan Bark 1319
tion on a plate of silica gel with fluorescent indicator for thin-
layer chromatography. Develop the plate with a mixture of
hexane and ethyl acetate (1:1) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): a spot among several spots from the
sample solution is similar with the spot from the standard so-
lution in color tone and Ri value.
Purity (1) Xylem — The amount of the xylem contained in
Moutan Bark is not more than 5.0%.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Moutan Bark according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Moutan Bark according to Method 4, and per-
form the test (not more than 5 ppm).
(4) Foreign matter <5.01> — The amount of foreign matter
other than xylem contained in Moutan Bark is not exceed
1.0%.
(5) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Component determination Weigh accurately about 0.3 g of
pulverized Moutan Bark, add 40 mL of methanol, heat under
a reflux condenser on a water bath for 30 minutes, cool, and
filter. Repeat the above procedure with the residue, using 40
mL of methanol. Combine the whole filtrates, add methanol
to make exactly 100 mL, then pipet 10 mL of this solution,
add methanol to make exactly 25 mL, and use this solution as
the sample solution. Separately, dry paeonol for component
determination in a desiccator (calcium chloride for drying)
for more than 1 hour. Weigh accurately about 10 mg of it,
dissolve in methanol to make exactly 100 mL, then pipet 10
mL of this solution, add methanol to make exactly 50 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography <
2.01> according to the following conditions, and determine
the peak areas, A T and A s , of paeonol in each solution.
Amount (mg) of paeonol
= W s x(A T /A s )x(l/2)
W s : Amount (mg) of paeonol for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 /um in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile, and acetic
acid (100) (65:35:2).
Flow rate: Adjust the flow rate so that the retention time of
paeonol is about 14 minutes.
Selection of column: Dissolve 1 mg of paeonol for compo-
nent determination and 5 mg of butyl parahydroxybenzoate
in 25 mL of methanol. Proceed with 10 fiL of this solution
under the above operating conditions, and calculate the reso-
lution. Use a column giving elution of paeonol and butyl
parahydroxybenzoate in this order, with the resolution be-
tween these peaks being not less than 2.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative deviation of the peak area of paeonol is not more
than 1.5%.
Powdered Moutan Bark
Moutan Cortex Pulveratus
Powdered Moutan Bark is the powder of Moutan
Bark.
It contains not less than 0.7% of paeonol.
Description Powdered Moutan Bark occurs as a light
grayish yellow-brown powder. It has a characteristic odor
and a slight, pungent and bitter taste.
Under a microscope <5.01>, Powdered Moutan Bark rev-
eals starch grains and fragments of parenchyma containing
them; fragments of cork tissue containing tannin; fragments
of somewhat thick-walled collenchyma, medullary rays, and
phloem parenchyma; rosette aggregates of calcium oxalate
and also fragments of parenchyma cells containing them.
Starch grains are simple or 2- to 10-compound grains, 10 - 25
/um in diameter; rosette aggregates are 20-30//m in di-
ameter.
Identification (1) To 2.0 g of Powdered Moutan Bark add
10 mL of hexane, shake for 3 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve lmg of
peonol for thin-layer chromatography in 10 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 nL of the sample solution and
standard solution on a plate of silica gel with fluorescent indi-
cator for thin-layer chromatography. Develop the plate with
a mixture of hexane and ethyl acetate (1:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): a spot among several spots
from the sample solution is similar with the spot from the
standard solution in color tone and Ri value.
(2) Evaporate to dryness 1 mL of the sample solution ob-
tained in (1), dissolve the residue in ethanol (95) to make 50
mL, and determine the absorption spectrum of this solution
as directed under Ultraviolet-visible Spectrophotometry
<2.24>: it exhibits maxima at around 228 nm, 274 nm and
313 nm.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Moutan Bark according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Moutan Bark according to Method 4, and per-
form the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, usually
vessels and other thick-walled cells are not observable.
(4) Total BHC's and total DDT's <5.01>— Not more than
1320 Mulberry Bark / Crude Drugs
JP XV
0.2 ppm, respectively.
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Component determination Weigh accurately about 0.5 g of
Powdered Moutan Bark, add 40 mL of methanol, heat under
a reflux condenser on a water bath for 30 minutes, cool, and
filter. Repeat the above procedure with the residue, using 40
mL of methanol. Combine the whole filtrates, add methanol
to make exactly 100 mL, then pipet 10 mL of this solution,
add methanol to make exactly 25 mL, and use this solution as
the sample solution. Separately, dry paeonol for component
determination in a desiccator (calcium chloride for drying)
for more than 1 hour. Weigh accurately about 10 mg of it,
dissolve in methanol to make exactly 100 mL, then pipet 10
mL of this solution, add methanol to make exactly 50 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 /uL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of paeonol in each solution.
Amount (mg) of paeonol
= W s x(A T /A s )x(l/2)
W s : Amount (mg) of paeonol for component determina-
tion
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 274 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 /um in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile, and acetic
acid (100) (65:35:2).
Flow rate: Adjust the flow rate so that the retention time of
paeonol is about 14 minutes.
Selection of column: Dissolve 1 mg of paeonol for compo-
nent determination and 5 mg of butyl parahydroxybenzoate
in 25 mL of methanol. Proceed with 10 /iL of this solution
under the above operating conditions, and calculate the reso-
lution. Use a column giving elution of paeonol and butyl
parahydroxybenzoate in this order, with the resolution be-
tween these peaks being not less than 2.
System repeatability: When the test is repeated 5 times with
the standard solution under the above operating conditions,
the relative deviation of the peak area of paeonol is not more
than 1.5%.
Containers and storage Containers — Tight containers.
Mulberry Bark
Mori Cortex
Mulberry Bark is the root bark of Morus alba Linne
(Moraceae).
Description Tubular, semi-tubular or cord-like bark, 1-6
mm thick, often in fine lateral cuttings; externally, white to
yellow-brown; in the case of the bark with periderm, its
periderm is yellow-brown in color, easy to peel, with
numerous longitudinal, fine wrinkles and numerous red-pur-
ple lenticels laterally elongated; inner surface, dark yellow-
brown in color and flat; cross section, white to light brown in
color, and fibrous.
Odor, slight; taste, slight.
Under a microscope <5.01>, a transverse section of bark
with periderm reveals 5 to 12 layers of cork cells in the outer
portion; phloem fibers or their bundles scattered in the cor-
tex, arranged alternately and stepwise with phloem parenchy-
ma; lactiferous tubes; solitary crystals of calcium oxalate;
starch grains as spheroidal or ellipsoidal, simple or com-
pound grains, simple grain 1-7 /um in diameter.
Identification Heat 1 g of pulverized Mulberry Bark with 20
mL of hexane under a reflux condenser on a water bath for 15
minutes, and filter. Evaporate the hexane of the filtrate under
reduced pressure, dissolve the residue in 10 mL of acetic an-
hydride, place 0.5 mL of the solution in a test tube, and add
carefully 0.5 mL of sulfuric acid to make two layers: a red-
brown color develops at the zone of contact.
Purity Foreign matter <5.01> — The amount of the root xy-
lem and other foreign matter contained in Mulberry Bark
does not exceed 1.0%.
Total ash <J.07> Not more than 11.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Nelumbo Seed
Nelumbis Semen
U> = ?
Nelumbo Seed is the seed of Nelumbo nucifera Gaer-
tner (Nymphaeaceae), usually with the endocarp,
sometime being removed the embryo.
Description Ovoid to ellipsoidal seed, at the base a papillate
protuberance surrounded with shallow depression, 1.0-1.7
cm in length, 0.5 - 1.2 cm in width; externally light reddish
brown to light yellowish brown; projection part dark reddish
brown; endocarp not lustrous and hardly peeled off; en-
dosperm yellowish white, a green embryo in the center.
Almost odorless; taste, slightly sweet and oily, embryo is
extremely bitter.
Under a microscope <5.01>, a transverse section of the seed
at central portion reveals endocarp composed of parenchyma
or endocarp occasionally left out; seed coat composed of
epidermis and parenchyma of compressed cells; vascular
bundles scattered in parenchyma; endosperm composed of
epidermis and parenchyma; aggregate crystals of calcium ox-
alate and tannin-like substances contained in endocarp
remained; parenchymatous cells of seed coat contain tannin-
like substances; parenchyma of endosperm contain starch
grains.
Identification To 0.5 g of pulverized Nelumbo Seed add 5
mL of water, shake for 5 minutes, and centrifuge. To 0.5 mL
of the supernatant liquid add 1 drop of a solution of 1-
JP XV
Crude Drugs / Nux Vomica 1321
naphthol in ethanol (99.5) (1 in 5), mix, then add gently 1 mL
of sulfuric acid: the solution shows a purple color.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <5.01> Not more than 5.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 14.5%.
less than 20.0°
Nuphar Rhizome
Nupharis Rhizoma
-tz>=i7
Notopterygium Rhizome
Notopterygii Rhizoma
Notopterygium Rhizome is the rhizome and root of
Notopterygium incisum Ting ex H. T. Chang or
Notopterygium forbesii Boissieu (Umbelliferae).
Description Notopterygium Rhizome is slightly curved,
cylindrical to conical, 3 to 10 cm in length, 5 to 20 mm in di-
ameter; rhizome occasionally branched; externally yellow-
brown to dark brown. The rhizome with nearly orbicular,
hollowed stem scars at the apex, sometimes having short
residue of stem; externally node rising, internode short; root
scars in warty processes on the node; externally root has
coarse longitudinal wrinkles and lateral root scars in warty
processes; light and slightly brittle in texture, easy to break.
The transverse section of the rhizome reveals numerous radi-
al cracks; cortex yellow-brown to brown; xylem light yellow
to light grayish yellow; pith grayish white to light brown. Un-
der a magnifying glass, the rhizome reveals brown, fine
points of resin canals in the cortex and pith.
Odor, characteristic; taste, slightly acid at first, followed
by a slightly pungent and slightly numbing aftertaste.
Under a microscope <5.01>, transverse section shows the
outermost layer to be composed of a cork layer several to a
dozen or so cells thick; collenchyma just inside of the cork
layer; oil canals scattered in cortex, large ones more than 300
[im in diameter; intercellular space occurring in radial direc-
tion in cortex; oil canals scattered in pith, large ones more
than 500 /xm in diameter; parenchymatous cells contain sim-
ple and 2- to 3-compound starch grains.
Identification To 0.3 g of pulverized Notopterygium Rhi-
zome add 3 mL of hexane in a glass-stoppered centrifuge
tube, shake for 10 minutes, centrifuge, and use the super-
natant liquid as the sample solution. Perform the test with
the sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL of the sample solution on a
plate of octadecylsilanized silica gel with fluorescent indicator
for thin-layer chromatography, develop the plate with a mix-
ture of methanol and water (9:1) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): a bluish white fluorescent spot
appears at around Rf 0.5, which shows a dark purple color
under ultraviolet light (main wavelength: 254 nm).
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
Nuphar Rhizome is the longitudinally split rhizome
of Nuphar japonicum De Candolle.
Description Usually, longitudinally split irregular column,
twisted, bent or somewhat pressed, 20 - 30 cm in length,
about 2 cm in width; the outer surface, dark brown, and the
cross section, white to grayish white in color; one side shows
nearly round to blunt triangular scars of petiole about 1 cm in
diameter, and the other side numerous scars of roots less than
0.3 cm in diameter; light, spongy in texture, and easily
broken; fractured surface flat and powdery. Under a mag-
nifying glass, a transverse section reveals a black outer por-
tion, and porous tissue with scattered vascular bundles in the
inner portion.
Odor, slight; taste, slightly bitter and unpleasant.
Identification Boil 1 g of pulverized Nuphar Rhizome with
20 mL of methanol under a reflux condenser on a water bath
for 15 minutes, cool, and filter. Evaporate the filtrate to dry-
ness, warm the residue with 5 mL of dilute acetic acid on a
water bath for 1 minute, cool, and filter. Spot 1 drop of the
filtrate on a piece of filter paper, air-dry the paper, spray
Dragendorff's TS for spraying on it, and allow it to stand: a
yellow-red color appears.
Purity (1) Petiole — The amount of its petioles contained
in Nuphar Rhizome does not exceed 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than petiole contained in Nuphar Rhizome does not ex-
ceed 1.0%.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <J.07> Not more than 10.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Nux Vomica
Strychni Semen
Nux Vomica is the seed of Strychnos nux-vomica
Linne (Loganiaceae).
When dried, it contains not less than 1.07% of
strychnine (C 2 iH 22 N 2 2 : 334.41).
Description Disk, often slightly bent, 1-3 cm in diameter,
0.3 - 0.5 cm in thickness; externally light grayish yellow-
green to light grayish brown, covered densely with lustrous
appressed hairs radiating from the center to the circumfer-
ence; on both sides, the margin and the central part bulged a
little; the dot-like micropyle situated at one point on the mar-
gin, and from which usually a raised line runs to the center on
one side; extremely hard in texture; when cracked upon soak-
1322 Nux Vomica Extract / Crude Drugs
JP XV
ing in water, the seed coat thin, the interior consisting of two
horny, light grayish yellow endosperms, and leaving a central
narrow cavity at the center; a white embryo, about 0.7 cm in
length, situated at one end between the inner surfaces of the
endosperms.
Odorless; taste, very bitter and persisting.
Identification (1) To 3 g of pulverized Nux Vomica add 3
mL of ammonia TS and 20 mL of chloroform, macerate for
30 minutes with occasional shaking, and filter. Remove most
of the chloroform from the filtrate by warming on a water
bath, add 5 mL of diluted sulfuric acid (1 in 10), and warm
on a water bath while shaking well until the odor of chlo-
roform is no longer perceptible. After cooling, filter through
a pledget of absorbent cotton, and add 2 mL of nitric acid to
1 mL of the filtrate: a red color develops.
(2) To the remaining filtrate obtained in (1) add 1 mL of
potassium dichromate TS, and allow to stand for 1 hour: a
yellow-red precipitate is produced. Collect the precipitate by
filtration, and wash with 1 mL of water. Transfer a part of
the precipitate to a small test tube, add 1 mL of water, dis-
solve by warming, cool, and add 5 drops of sulfuric acid
dropwise carefully along the wall of the test tube: the layer of
sulfuric acid shows a purple color which turns immediately
red to red-brown.
Total ash <J.07> Not more than 3.0%.
Assay Weigh accurately about 1 g of pulverized Nux Vomi-
ca, previously dried at 60°C for 8 hours, place in a glass-stop-
pered centrifuge tube, and moisten with 1 mL of ammonia
solution (28). To this solution add 20 mL of diethyl ether,
stopper the centrifuge tube tightly, shake for 15 minutes, cen-
trifuge, and separate the supernatant liquid. Repeat this
procedure three times with the residue using 20-mL portions
of diethyl ether. Combine all the extracts, and evaporate the
diethyl ether on a water bath. Dissolve the residue in 10 mL
of the mobile phase, add exactly 10 mL of the internal stan-
dard solution, and add the mobile phase to make exactly 100
mL. Filter this solution through a membrane filter with a
porosity not more than 0.8 /urn, discard the first 2 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 75 mg of strychnine ni-
trate for assay (priviously determine the loss on drying), and
dissolve in the mobile phase to make exactly 50 mL. Pipet 10
mL of this solution, add exactly 10 mL of the internal stan-
dard solution, then add the mobile phase to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with 5 fiL each of the sample solution and standard
solution as directed under Liquid Chromatography <2.01> ac-
cording to the following conditions. Determine the ratio, Qj
and Q s , of the peak area of strychnine to that of the internal
standard in each solution.
Amount (mg) of strychnine (C21H22N2O2)
= ^ s x(g T /g s )x (1/5) x 0.8414
fV s : Amount (mg) of strychnine nitrate for assay, calculat-
ed on the dried basis
Internal standard solution — A solution of barbital sodium in
the mobile phase (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /um in parti-
cle diameter).
Column temperature: Room temperature.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen-
phosphate in water to make 1000 mL, and mix with acetoni-
trile and triethylamine (45:5:1), and adjust the mixture with
phosphoric acid to pH 3.0.
Flow rate: Adjust the flow rate so that the retention time of
Strychnine is about 17 minutes.
Selection of column: Proceed with 5 fiL of the standard so-
lution under the above operating conditions. Use a column
giving elution of the internal standard and strychnine in this
order, and clearly separating each peak.
Nux Vomica Extract
tK$71X + X
Nux Vomica Extract contains not less than 6.15%
and not more than 6.81% of strychnine (C21H22N2O2:
334.41).
Method of preparation After defatting 1000 g of coarse
powder of Nux Vomica with hexane, extract with the percola-
tion method, using a mixture of 750 mL of Ethanol, 10 mL
of Acetic Acid and 240 mL of Purified Water as the first sol-
vent, and 70 vol% ethanol as the second solvent. Combine
the extracts, and prepare the dry extract as directed under Ex-
tracts. Where, an appropriate quantity of Ethanol and Puri-
fied Water may be used instead of 70 vol% ethanol.
Description Nux Vomica Extract occurs as yellow-brown to
brown powder. It has a slight characteristic odor, and an ex-
tremely bitter taste.
Identification Extract 0.5 g of Nux Vomica Extract with 0.5
mL of ammonia TS and 10 mL of chloroform with oc-
casional shaking. Filter the chloroform extract, evaporate the
filtrate on a water bath until most of the chloroform is re-
moved, and proceed as directed in the Identification under
Nux Vomica.
Assay Weigh accurately about 0.2 g of Nux Vomica Ex-
tract, place in a glass-stoppered centrifuge tube, add 15 mL
of ammonia TS, and shake. Add 20 mL of diethyl ether,
stopper tightly, shake for 15 minutes, centrifuge to disperse
the diethyl ether layer. Repeat this procedure three times with
the water layer, using 20-mL portions of diethyl ether. Com-
bine the extracts, and evaporate the diethyl ether on a water
bath. Dissolve the residue in 10 mL of the mobile phase, add
exactly 10 mL of the internal standard solution, and add the
mobile phase to make exactly 100 mL. Proceed as directed in
the Assay under Nux Vomica.
Amount (mg) of strychnine (C21H22N2O2)
= W s x(Q T /Q s )x (1/5) x 0.8414
W s : Amount (mg) of strychnine nitrate for assay, calculat-
ed on the dried basis
Internal standard solution — A solution of barbital sodium in
the mobile phase (1 in 500).
Containers and storage Containers — Tight containers.
JPXV
Storage — Light-resistant.
Nux Vomica Extract Powder
Nux Vomica Extract Powder contains not less than
0.61% and not more than 0.68% of strychnine
(C 21 H 22 N 2 2 : 334.41).
Method of preparation
Nux Vomica Extract
Starch, Lactose Hydrate or
their mixture
100 g
a sufficient quantity
To make
1000 g
To Nux Vomica Extract add 100 mL of Purified Water,
then warm, and soften with stirring. Cool, add 800 g of
Starch, Lactose Hydrate or their mixture little by little, and
mix well. Dry, preferably at a low temperature, and dilute
with a sufficient additional quantity of Starch, Lactose or
their mixture to make 1000 g of the homogeneous powder.
Description Nux Vomica Extract Powder occurs as a yel-
low-brown to grayish brown powder. It has a slight, charac-
teristic odor and a bitter taste.
Identification (1) To 3 g of Nux Vomica Extract Powder
add 3 mL of ammonia TS and 20 mL of chloroform, macer-
ate for 30 minutes with occasional shaking, and filter. Re-
move most of the chloroform from the filtrate by warming on
a water bath, add 5 mL of diluted sulfuric acid (1 in 10), and
warm on a water bath while shaking well until the odor of
chloroform is no longer perceptible. After cooling, filter
through a pledget of absorbent cotton, and add 2 mL of
nitric acid to 1 mL of the filtrate: a red color develops.
(2) To the remaining filtrate obtained in (1) add 1 mL of
potassium dichromate TS, and allow to stand for 1 hour: a
yellow-red precipitate is produced. Collect the precipitate by
filtration, and wash with 1 mL of water. Transfer a part of
the precipitate to a small test tube, add 1 mL of water, dis-
solve by warming, cool, and add 5 drops of sulfuric acid
dropwise carefully along the wall of the test tube: the layer of
sulfuric acid shows a purple color which turns immediately
red to red-brown.
Assay Weigh accurately about 2.0 g of Nux Vomica Extract
Powder, place in a glass-stoppered centrifuge tube, add 15
mL of ammonia TS, and shake. Add 20 mL of diethyl ether,
stopper tightly, shake for 15 minutes, centrifuge to separate
the diethyl ether layer. Repeat this procedure three times with
the water layer, using 20-mL portions of diethyl ether. Com-
bine the extracts, and evaporate the diethyl ether on a water
bath. Dissolve the residue in 10 mL of the mobile phase, add
exactly 10 mL of the internal standard solution, and add the
mobile phase to make exactly 100 mL. Filter this solution
through a membrane filter with a porosity not more than 0.8
jum, discard the first 2 mL of the filtrate, and use the subse-
quent filtrate as the sample solution. Separately, weigh ac-
curately about 75 mg of strychnine nitrate for assay (previ-
ously determine the loss on drying), and dissolve in the mo-
bile phase to make exactly 50 mL. Pipet 10 mL of this solu-
Crude Drugs / Nux Vomica Tincture 1323
tion, add exactly 10 mL of the internal standard solution,
then add the mobile phase to make exactly 100 mL, and use
this solution as the standard solution. Perform the test with
the sample solution and standard solution as directed under
Liquid Chromatography <2.01> according to the following
conditions. Determine the ratio, Q T and Q s , of the peak area
of strychnine to that of the internal standard in each solution.
Amount (mg) of strychnine (C21H22N2O2)
= W s x (Q T /Qs) x (1/5) x 0.8414
W s : Amount (mg) of strychnine nitrate for assay, calculat-
ed on the dried basis
Internal standard solution — A solution of barbital sodium in
the mobile phase (1 in 500).
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: Room temperature.
Mobile phase: A mixture of a solution of potassium di-
hydrogenphosphate (6.8 in 1000), acetonitrile and triethyla-
mine (45:5:1), adjusted the pH to 3.0 with phosphoric acid.
Flow rate: Adjust the flow rate so that the retention time of
strychnine is about 17 minutes.
Selection of column: Proceed with 5 /xL of the standard so-
lution under the above operating conditions. Use a column
giving elution of the internal standard and strychnine in this
order, and clearly dividing each peak.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Nux Vomica Tincture
Nux Vomica Tincture contains not less than 0.097
w/v% and not more than 0.116 w/v% of strychnine
(C 21 H 22 N 2 2 : 334.41).
Method of preparation
Nux Vomica, in coarse powder 100 g
70 vol% Ethanol a sufficient quantity
To make
1000 mL
Prepare as directed under Tinctures, with the above in-
gredients. May be prepared with an appropriate quantity of
Ethanol and Purified Water.
Description Nux Vomica Tincture is a yellow-brown liquid.
It has an extremely bitter taste.
Specific gravity d\l: about 0.90
Identification Heat 20 mL of Nux Vomica Tincture on a
water bath to remove ethanol, cool, transfer to a separator,
add 2 mL of ammonia TS and 20 mL of chloroform, and
shake well for 2 to 3 minutes. Filter the chloroform layer
through a pledget of absorbent cotton, warm the filtrate on a
water bath to remove most of chloroform, and proceed as
1324 Ophiopogon Tuber / Crude Drugs
JP XV
directed in the Identification under Nux Vomica.
Alcohol number <7.07> Not less than 6.7 (Method 2).
Assay Pipet 3 mL of Nux Vomica Tincture into a glass-
stoppered centrifuge tube, add 10 mL of ammonia TS and 20
mL of diethyl ether, stopper tightly, shake for 15 minutes,
centrifuge to separate the diethyl ether layer. Repeat this
procedure twice with the water layer, using 20-mL portions
of diethyl ether. Combine the extracts, and evaporate the
diethyl ether on a water bath. Dissolve the residue with 10 mL
of the mobile phase, add exactly 5 mL of the internal stan-
dard solution, and add the mobile phase to make exactly 50
mL. Filter the solution through a membrane filter with a pore
size not exceeding 0.8-//m, discard the first 2 mL of the
filtrate, and use the subsequent filtrate as the sample solution.
Separately, weigh accurately about 75 mg of strychnine ni-
trate for assay (previously determine the loss on drying), and
dissolve in the mobile phase to make exactly 100 mL. Pipet 5
mL of this solution, add exactly 5 mL of the internal stan-
dard solution, add the mobile phase to make exactly 50 mL,
and use this solution as the standard solution. Proceed with
the sample solution and the standard solution as directed in
the Assay under Nux Vomica.
Amount (mg) of strychnine (C21H22N2O2)
= W s x (Q T /Qs) x (1/20) x 0.8414
W s : Amount (mg) of strychnine nitrate for assay, calculat-
ed on the dried basis
Internal standard solution — A solution of barbital sodium in
the mobile phase (1 in 500).
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Ophiopogon Tuber
Ophiopogonis Tuber
Ophiopogon Tuber is the enlarged part of the root of
Ophiopogon japonicus Ker-Gawler (Liliaceae).
Description Fusiform root, 1 - 2.5 cm in length, 0.3 - 0.5
cm in diameter, somewhat sharp at one end, and somewhat
rounded at the other; externally light yellow to light yellow-
brown, with longitudinal wrinkles of various sizes; when
fractured, cortex flexible and friable, stele strong; fractured
surface of cortex light yellow-brown in color, slightly translu-
cent and viscous.
Odor, slight; taste, slightly sweet and mucous.
Under a microscope <5.01>, a transverse section reveals
brown, 4- to 5-layer velamen internally adjoining the epider-
mis; a single-layer exodermis inside the velamen, and cortex
of parenchyma cells inside the exodermis; endodermis is dis-
tinct; about 20 protoxylems in actionstele; cortex parenchy-
ma contains columnar crystals and needle raphides of calci-
um oxalate; oil drops in the exodermis.
Purity (1) Rootlets — The amount of the rootlets con-
tained in Ophiopogon Tuber is not exceed 1.0%.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Ophiopogon Tuber according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than lOppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Ophiopogon Tuber according to Method 4, and
perform the test (not more than 5 ppm).
Total ash <J.07> Not more than 3.0%.
Opium Ipecac Powder
Dover's Powder
7^>- r-=l>g[
Opium Ipecac Powder contains not less than 0.90%
and not more than 1.10% of morphine (C 17 H 19 N0 3 :
285.34).
Method of preparation
Powdered Opium 100 g
Powdered Ipecac 100 g
Starch or a suitable ingredient a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients. Lactose Hydrate should not be used.
Description Opium Ipecac Powder occurs as a light brown
powder.
Identification (1) Proceed with 1 g of Opium Ipecac Pow-
der as directed in the Identification (1) under Powdered Opi-
um.
(2) Proceed with 1 g of Opium Ipecac Powder as directed
in the Identification (2) under Powdered Opium.
(3) Shake frequently a mixture of 3 g of Opium Ipecac
Powder and 5 mL of hydrochloric acid, and allow to stand
for 1 hour. Filter the solution into an evaporating dish. Add 5
mg of chlorinated lime to the filtrate: an orange color is
produced at the circumference of the chlorinated lime (eme-
tine).
Assay Weigh accurately about 50 g of Opium Ipecac Pow-
der in a glass stoppered flask, add 250 mL of dilute ethanol,
warm in a water bath at 40°C for 1 hour with stirring, and
filter through a glass filter (G3). Transfer the residue on the
filter to the first glass-stoppered flask, add 50 mL of dilute
ethanol, warm in a water bath at 40°C for 10 minutes with
stirring, and filter through the glass filter. Repeat the extrac-
tion with three 50-mL portions of dilute ethanol. Combine all
the filtrates in a mortar, evaporate on a water bath to dry-
ness, add 10 mL of ethanol (99.5) to the residue, and
evaporate again. After cooling, triturate the residue with an
exactly measured 10 mL of water, add 2 g of calcium
hydroxide and an exactly measured 40 mL of water, stir the
mixture for 20 minutes, and filter. To 30 mL of the filtrate
add 0.1 g of magnesium sulfate heptahydrate, shake for 1
minute, then add 0.3 g of calcium hydroxide, shake for 1
minute, allow to stand for 1 hour, and filter. To an exactly
measured 20 mL of the filtrate add 5 mL of sodium
hydroxide TS, and adjust the pH to between 9.0 and 9.2 with
ammonium chloride. Extract the solution successively with
60 mL, 40 mL and 30 mL of a mixture of chloroform and
JPXV
Crude Drugs / Oriental Bezoar 1325
ethanol (95) (3:1). Combine all the extracts, distil, then
evaporate off the solvent on a water bath. Dissolve the
residue in 20 mL of dilute sodium hydroxide TS and 10 mL
of diethyl ether with shaking, add 0.5 g of ammonium chlo-
ride, shake vigorously with caution, and proceed as directed
in the Assay under Powdered Opium.
Each mL of 0.05 mol/L sulfuric acid VS
= 28.53 mg of C, 7 H 19 N0 3
Containers and storage Containers — Tight containers.
Orange Peel Syrup
r- -> t v P ••/ ~f
Method of preparation
Orange Peel Tincture
Simple Syrup
200 mL
a sufficient quantity
To make 1000 mL
Prepare as directed under Syrups, with the above in-
gredients. An appropriate quantity of Sucrose and Purified
Water may be used in place of Simple Syrup.
Description Orange Peel Syrup is a brownish yellow to red-
dish brown liquid. It has a characteristic odor, a sweet taste
and a bitter aftertaste.
Specific gravity d^: about 1.25
Identification To 25 mL of Orange Peel Syrup add 50 mL
of ethyl acetate, shake for 5 minutes, allow to stand until
clear ethyl acetate layer separate, and take the ethyl acetate
layer, and evaporate on a water bath to dryness. Dissolve the
residue in 10 mL of ethanol (95), filter if necessary, and use
this solution as the sample solution. Separately, dissolve 10
mg of naringin for thin-layer chromatography in 10 mL of
ethanol (95), and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, ethanol (99.5) and water (8:2:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute 2,6-
dibromo-/V-chloro-l,4-benzoquinone monoimine TS on the
plate, and allow to stand in ammonia gas: a spot from the
sample solution and a grayish green spot from the standard
solution show the same color tone and the same Rf value.
Containers and storage Containers — Tight containers.
Orange Peel Tincture
Method of preparation
Bitter Orange Peel, in coarse powder 200 g
70 vol% Ethanol a sufficient quantity
To make 1000 mL
Prepare as directed under Tinctures, with the above in-
gredients. An appropriate quantity of Ethanol and Purified
Water may be used in place of 70 vol% Ethanol.
Description Orange Peel Tincture is a yellowish brown liq-
uid. It has a characteristic odor, and a bitter taste.
Specific gravity df : about 0.90
Identification To 5.0 mL of Orange Peel Tincture add 5 mL
of ethanol (95), filter if necessary, and use the filtrate as the
sample solution. Proceed as directed in the Identification un-
der Bitter Orange Peel.
Alcohol number <7.07> Not less than 6.6 (Method 2).
Containers and storage Containers — Tight containers.
Oriental Bezoar
Bezoar Bo vis
Oriental Bezoar is a stone formed in the gall sac of
Bos taurus Linne var. domesticus Gmelin (Bovidae).
Description Spherical or massive stone, l - 4 cm in di-
ameter; externally yellow-brown to red-brown; light, fragile
and easily broken. Fractured surface shows yellow-brown to
red-brown annular rings, often containing white granular
substances or thin layers in these annular rings.
Odor, slight; taste, slightly bitter, followed by slight sweet-
ness.
Identification (1) Shake 0.1 g of pulverized Oriental
Bezoar with 10 mL of petroleum ether for 30 minutes, filter,
and wash the residue with 10 mL of petroleum ether. Shake
0.01 g of the residue with 3 mL of acetic anhydride for 1 to 2
minutes, add a mixture of 0.5 mL of acetic anhydride and 2
drops of sulfuric acid, and shake: a yellow-red to deep red
color develops, and changes through dark red-purple to dark
red-brown.
(2) Shake well 0.01 g of Oriental Bezoar with 1 mL of
hydrochloric acid and 10 mL of chloroform, separate the
chloroform layer when it acquires a yellow-brown color, and
shake with 5 mL of barium hydroxide TS: a yellow-brown
precipitate is produced.
Purity (1) Synthetic dye — To 2 mg of pulverized Oriental
Bezoar add 1 mL dilute hydrochloric acid: no violet color de-
velops.
(2) Starch — To 5 mg of pulverized Oriental Bezoar add 2
mL of water, and heat on a water bath for 5 minutes. Cool
and add 2 to 3 drops of iodine TS: no blue-purple color de-
velops.
(3) Sucrose — To 0.02 g of pulverized Oriental Bezoar add
10 mL of water, shake for 15 minutes, and filter. To 1 mL of
the filtrate add 2 mL of anthrone TS, and shake: no deep
blue-green to dark green color develops.
Total ash <5.07> Not more than 10.0%.
Content of the active principle Weigh accurately about 0.5
g of pulverized Oriental Bezoar in a flask, add 50 mL of
petroleum ether, warm under a reflux condenser on a water
bath for 2 hours, and filter. Place the residue along with the
filter paper in the flask, add 2 mL of hydrochloric acid and 40
1326 Oyster Shell / Crude Drugs
JP XV
mL of chloroform, warm under a reflux condenser on a water
bath for 1 hour, and filter into a tared flask. Wash the filter
paper with a small quantity of chloroform, combine the
washings with the filtrate, and distil off the chloroform. Dry
the residue in a desiccator (silica gel) for 24 hours, and weigh:
the mass of the residue is not less than 12.0%.
Oyster Shell
Ostreae Testa
Oyster Shell is the shell of Ostrea gigas Thunberg
(Ostreidae).
Description Irregularly curved, foliaceous or lamellated
broken pieces. The unbroken oyster shell forms a bivalve 6 -
10 cm in length and 2-5 cm in width. The upper valve is flat
and the lower one is somewhat hollow. Both the upper and
lower edges of the valve are irregularly curved and bite with
each other. The surface of the valve is externally light green-
ish gray-brown and internally milky in color.
Odorless and tasteless.
Identification (1) Dissolve 1 g of sample pieces of Oyster
Shell in 10 mL of dilute hydrochloric acid by heating: it
evolves a gas, and forms a very slightly red, turbid solution in
which a transparent, thin suspended matter remains. Pass the
evolved gas through calcium hydroxide TS: a white
precipitate is produced.
(2) The solution obtained in (1) has a slight, characteristic
odor. Filter this solution and neutralize with ammonia TS:
the solution responds to the Qualitative Tests <1.09> for calci-
um salt.
(3) Ignite 1 g of pulverized Oyster Shell: it turns blackish
brown in color at first, and evolves a characteristic odor. Ig-
nite it further: it becomes almost white.
Purity Barium — Dissolve 1 g of pulverized Oyster Shell in
10 mL of dilute hydrochloric acid: the solution does not
respond to the Qualitative Tests (1) <1.09> for barium salt.
Powdered Oyster Shell
Ostreae Testa Pulverata
Powdered Oyster Shell is the powder of Oyster Shell.
Description Powdered Oyster Shell occurs as a grayish
white powder. It is odorless and tasteless.
Identification (1) Dissolve 1 g of Powdered Oyster Shell in
10 mL of dilute hydrochloric acid by heating: it evolves a gas,
and forms a very slightly red, turbid solution. Pass the gas
evolved through calcium hydroxide TS: a white precipitate is
produced.
(2) The solution obtained in (1) has a slight, characteristic
odor. Filter this solution, and neutralize with ammonia TS:
the solution responds to the Qualitative Tests <1.09> for calci-
um salt.
(3) Ignite 1 g of Powdered Oyster Shell: it turns blackish
brown in color at first evolving a characteristic odor, and
becomes almost white by further ignition.
Purity (1) Water-soluble substances — Shake 3.0 g of
Powdered Oyster Shell with 50 mL of freshly boiled and
cooled water for 5 minutes, filter, and evaporate 25 mL of the
filtrate to dryness. Dry the residue at 105 C C for 1 hour, cool,
and weigh: the mass of the residue does not exceed 15 mg.
(2) Acid-insoluble substances — To 5.0 g of Powdered
Oyster Shell add 100 mL of water, and add hydrochloric acid
in small portions with stirring until the solution becomes
acid. Boil the acidic mixture with additional 1 mL of
hydrochloric acid. After cooling, collect the insoluble sub-
stance by filtration, and wash it with hot water until the last
washing no longer gives any reaction in Qualitative Tests
<1.09> (2) for chloride. Ignite the residue and weigh: the mass
of the residue does not exceed 25 mg.
(3) Barium — Dissolve 1 g of Powdered Oyster Shell in 10
mL of dilute hydrochloric acid: the solution does not
responds to the Qualitative Tests <1.09> (1) for barium salt.
Loss on drying <2.41> Not more than 4.0% (1 g, 180 C C,
4 hours).
Containers and storage Containers — Tight containers.
Panax Japonicus Rhizome
Panacis Japonici Rhizoma
Panax Japonicus Rhizome is the rhizome of Panax
japonicus C. A. Meyer (Araliaceae), usually after
being treated with hot water.
Description Irregularly cylidrical rhizome with distinct
nodes, 3-20 cm in length, 1-1.5 cm in diameter, internode
1-2 cm; externally light yellow-brown, with fine longitudi-
nal wrinkles; stem scars, hollowed at the center, protruding
on the upper surface, and root scars protruding as knobs on
internodes; easily broken; fractured surface approximately
flat, and light yellow-brown in color; horny in texture.
Odor, slight; taste, slightly bitter.
Identification Shake 0.5 g of pulverized Panax Japonicus
Rhizome with 10 mL of methanol for 10 minutes, filter, and
use the filtrate as the sample solution. Separately, dissolve 2
mg of chikusetsusaponin IV for thin-layer chromatography
in 1 mL of methanol, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate, water and formic acid (5:1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly di-
lute sulfuric acid on the plate, and heat the plate at 1 10°C for
5 minutes: one of several spots obtained from the sample so-
lution shows the same color and the same Rf value with the
purple-red spot from the standard solution.
Total ash <J.07> Not more than 5.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
JPXV
less than 30.0%.
Powdered Panax Japonicus
Rhizome
Panacis Japonici Rhizoma Pulveratum
Powdered Panax Japonicus Rhizome is the powder
of Panax Rhizome.
Description Powdered Panax Japonicus Rhizome occurs as
a light grayish yellow-brown powder, and has a slight odor
and a slightly bitter taste.
Under a microscope <5.01>, Powdered Panax Japonicus
Rhizome reveals mainly starch grains or gelatinized starch
masses, and fragments of parenchyma cells containing them;
also fragments of cork tissue, somewhat thick-walled collen-
chyma, phloem tissue, and reticulate vessels; rarely frag-
ments of scalariform vessels with a simple perforation, fibers
and fiber bundles, rosette aggregates of calcium oxalate, and
parenchyma cells containing them; yellow to orange-yellow
resin; starch grains consisting of simple grains or 2- to 4-com-
pound grains, simple grains, 3 - 18 /um in diameter; rosette
aggregates of calcium oxalate are 20 - 60 /um in diameter.
Identification Shake 0.5 g of Powdered Panax Japonicus
Rhizome with 10 mL of methanol for 10 minutes, filter, and
use the filtrate as the sample solution. Separately, dissolve 2
mg of chikusetsusaponin IV for thin-layer chromatography
in 1 mL of methanol, and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate, water and formic acid (5:1:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly di-
lute sulfuric acid on the plate, and heat the plate at 1 10°C for
5 minutes: one of several spots obtained from the sample so-
lution shows the same color tone and the same Ri value with
the purple-red spot from the standard solution.
Total ash <J.07> Not more than 5.0%.
Extract content <J.0/>
less than 30.0%.
Dilute ethanol-soluble extract: not
Peach Kernel
Persicae Semen
Peach Kernel is the seed of Prunus persica Batsch or
Prunus persica Batsch var. davidiana Maximowicz
(Rosaceae).
Description Flattened, asymmetric ovoid seed, 1.2 - 2.0 cm
in length, 0.6 - 1.2 cm in width, and 0.3 - 0.7 cm in thick-
ness; somewhat sharp at one end, and round at the other end
with chalaza; seed coat red-brown to light brown; externally,
Crude Drugs / Powdered Peach Kernel 1327
its surface being powdery by easily detachable stone cells of
epidermis; numerous vascular bundles running and rarely
branching from chalaza through the seed coat, and, appear-
ing as dented longitudinal wrinkles; when soaked in boiling
water and softened, the seed coat and thin, translucent, white
albumen easily separated from the cotyledone; cotyledone
white in color.
Almost odorless; taste, slightly bitter and oily.
Under a microscope <5.01>, the outer surface of seed coat
reveals polygonal, long polygonal, or obtuse triangular stone
cells on the protrusion from vascular bundles, shape of which
considerably different according to the position, and their
membranes almost equally thickened; in lateral view, appear-
ing as a square, rectangle or obtuse triangle.
Identification To 1.0 g of ground Peach Kernel add 10 mL
of methanol, immediately heat under a reflux condenser on a
water bath for 10 minutes, cool, filter, and use the filtrate as
the sample solution. Separately, dissolve 2 mg of amygdalin
for thin-layer chromatography in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /uL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, methanol and water (7:3:1) to a distance of about 10
cm, and air-dry the plate. Spray evenly dilute sulfuric acid
upon the plate, and heat at 105 °C for 10 minutes: one spot
among the spots from the sample solution and a brown to
dark green spot from the standard solution show the same
color tone and the same Rf value.
Purity (1) Rancidity — Grind Peach Kernel with boiling
water: no odor of rancid oil is perceptible.
(2) Foreign matter <5.01> — Peach Kernel does not con-
tain broken pieces of endocarp or other foreign matter.
Powdered Peach Kernel
Persicae Semen Pulveratum
Powdered Peach Kernel is the powder of the Peach
Kernel.
Description Powdered Peach Kernel occurs as a reddish-
light brown to light brown powder. It is almost odorless and
is oily and has slightly a bitter taste.
Under a microscope <5.01>, Powdered Peach Kernel frag-
ments of outer seed coat epidermis; elliptical to ovoid, con-
taining yellowish brown compound 50 to 80 /um in diameter
and stone cell; cap-like shape to ovoid, yellowish brown in
color. The stone cell is element of epidermis, 50 to 80 /um in
diameter and 70 to 80 /um in height, cell wall of the top, 12 to
25 ^m thickness, the base 4 /um in thickness, with obvious
and numerous pits. Inner seed coat, yellowish brown, irregu-
lar and somewhat long polygon, 1 5 to 30 /um in diameter; and
fragments of cotyledon and albumen containing aleurone
grains and fatted oil, Aleurone grains are almost spherical
grains, 5 to 10 /um in diameter.
Identification (1) Grind Powdered Peach Kernel with
1328 Peony Root / Crude Drugs
JP XV
water: the odor of benzaldehyde is perceptible.
(2) To 1.0 g of Powdered Peach Kernel add 10 mL of
methanol, and immediately heat under a reflux condenser on
a water bath for 10 minutes. After cooling, filter, and use the
filtrate as the sample solution. Separately, dissolve 2 mg of
amygdalin for thin-layer chromatography in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
ethyl acetate, methanol and water (7:3:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 105°C for 10 minutes: one
spot among the spots shows the same in color tone and Rf
value with the brown to dark green spot obtained with the
standard solution.
Loss on drying <5.01> Not more than 8.5% (6 hours).
Total ash <J.07> Not more than 3.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Containers and storage Containers — Tight containers.
Peony Root
Paeoniae Radix
-> -v (7 V (7
Peony Root is the root of Paeonia lactiflora Pallas
(Paeoniaceae).
It contains not less than 2.0% of peonifiorin,
(C^F^gOn: 480.46) calculated on the dried basis.
Description Cylindrical root, 7-20 cm in length, 1 - 2.5
cm in diameter; externally brown to light grayish brown, with
distinct longitudinal wrinkles, with warty scars of lateral
roots, and with laterally elongated lenticels; fractured surface
dense in texture, light grayish brown, and with light brown
radial lines in xylem.
Odor, characteristic; taste, slightly sweet at first, followed
by an astringency and a slight bitterness.
Identification (1) Shake 0.5 g of pulverized Peony Root
with 30 mL of ethanol (95) for 15 minutes, and filter. Shake 3
mL of the filtrate with 1 drop of iron (III) chloride TS : a blue-
purple to blue-green color is produced, and it changes to dark
blue-purple to dark green.
(2) To 2g of pulverized Peony Root add 10 mL of
methanol, warm on a water bath for 5 minutes, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of Paeonifiorin Reference Standard in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
acetone, ethyl acetate and acetic acid (100) (10:10:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
methoxybenzaldehyde-sulfuric acid TS upon the plate, and
heat at 105 °C for 5 minutes: one spot among the spots from
the sample solution and the purple-red spot from the stan-
dard solution show the same color tone and the same Rf
value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Peony Root according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.U> — Prepare the test solution with 0.40 g
of pulverized Peony Root according to Method 4, and per-
form the test (not more than 5 ppm).
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Assay Weigh accurately about 0.5 g of pulverized Peony
Root, add 50 mL of diluted methanol (1 in 2), heat under a
reflux condenser on a water bath for 30 minutes, cool, and
filter. To the residue add 50 mL of diluted methanol (1 in 2),
and proceed in the same manner. Combine the filtrates, add
diluted methanol (1 in 2) to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of Paeonifiorin Reference Standard
(previously determine the water) dissolve in diluted methanol
(1 in 2) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine the peak areas, A T and A s , of
paeonifiorin in each solution.
Amount (mg) of paeonifiorin (C23H 28 On) = W s x (,4 T A4 S )
W s : Amount (mg) of Paeonifiorin Reference Standard,
calculated on the anhydrous basis
Operating conditions-
Detector: An ultraviolet absorption photometer (wave-
length: 232 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (850:150:1).
Flow rate: Adjust the flow rate so that the retention time of
paeonifiorin is about 10 minutes.
System suitability —
System performance: Dissolve 1 mg each of Paeonifiorin
Reference Standard and albiflorin in diluted methanol (1 in 2)
to make 10 mL. When the procedure is run with 10 /uL of this
solution under the above operating conditions, albiflorin and
paeonifiorin are eluted in this order with the resolution be-
tween these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak area of paeonifio-
rin is not more than 1.5%.
JPXV
Crude Drugs / Perilla Herb 1329
Powdered Peony Root
Paeoniae Radix Pulverata
Powdered Peony Root is the powder of Peony Root.
It contains not less than 2.0% of paeonifiorin
(C 2 3H280 n : 480.46), calculated on the dried basis.
Description Powdered Peony Root occurs as a light grayish
brown powder, and has a characteristic odor and a slightly
sweet taste at first, followed by an astringency and a slight
bitterness.
Under a microscope <5.01>, Powdered Peony Root reveals
starch grains and fragments of parenchyma cells containing
them; fragments of cork cells, vessels, tracheids and xylem
fibers; rosette aggregates of calcium oxalate, and fragments
of rows of crystal cells containing them. Starch grains consist
of simple grains, 5-25 /um in diameter, occasionaly 2- to 3-
compound grains.
Identification (1) Shake 0.5 g of Powdered Peony Root
with 30 mL of ethanol (95) for 15 minutes, and filter. To 3
mL of the filtrate add 1 drop of iron (III) chloride TS, and
mix: a blue-purple to blue-green color is produced, and there-
after it changes to dark blue-purple to dark green.
(2) To 2 g of Powdered Peony Root add 10 mL of
methanol, warm on a water bath for 5 minutes, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of Paeonifiorin Reference Standard in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 10 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
acetone, ethyl acetate and acetic acid (100) (10:10:1) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly 4-
methoxybenzaldehyde-sulfuric acid TS on the plate, and heat
at 105°C for 5 minutes: one spot among the spots from the
sample solution and the purple spot from the standard solu-
tion show the same color tone and the same Rf value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Peony Root according to Method 3, and perform
the test. Prepare the control solution with 3.0 mL of Stan-
dard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Peony Root according to Method 4, and per-
form the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Peony Root does not show groups of light yellow stone
cells and fibers.
Loss on drying <5.01> Not less than 14.0% (6 hours).
Total ash <J.07> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Assay Weigh accurately about 0.5 g of Powdered Peony
Root, add 50 mL of diluted methanol (1 in 2), heat under a
reflux condenser on a water bath for 30 minutes, cool, and
filter. To the residue add 50 mL of diluted methanol (1 in 2),
and proceed in the same manner. Combine the filtrates, add
diluted methanol (1 in 2) to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of Paeonifiorin Reference Standard
(previously determine the water), dissolve in diluted
methanol (1 in 2) to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with
exactly 10 /xL each of the sample solution and standard solu-
tion as directed under Liquid Chromatography <2.01>
according to the following conditions. Determine the peak
areas, A T and A s , of paeonifiorin in each solution.
Amount (mg) of paeonifiorin (C23H 2 sOn)
= W s x(A T /A s )
W s : Amount (mg) of Paeonifiorin Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 232 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of water, acetonitrile and
phosphoric acid (850:150:1).
Flow rate: Adjust the flow rate so that the retention time of
paeonifiorin is about 10 minutes.
System suitability —
System performance: Dissolve 1 mg each of Paeonifiorin
Reference Standard and albiflorin in diluted methanol (1 in 2)
to make 10 mL. When the procedure is run with 10 fiL of this
solution under the above operating conditions, albiflorin and
paeonifiorin are eluted in this order with the resolution be-
tween these peaks being not less than 2.5.
System repeatability: When the test is repeated 6 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak area of paeonifio-
rin is not more than 1.5%.
Perilla Herb
Perillae Herba
Perilla Herb is the leaf and twig of Perilla frutescens
Britton var. acuta Kudo or Perilla frutescens Britton
var. crispa Decaisne (Labiatae).
Description Usually, contracted and wrinkled leaves, often
with thin stems. Both surfaces of the leaf are brownish pur-
ple, or the upper surface is grayish green to brownish green,
and the lower surface is brownish purple in color. When
smoothed by immersion in water, the lamina is ovate to ob-
cordate, 5-12 cm in length, 5-8 cm in width; the apex,
acuminate; the margin, serrate; the base, broadly cuneate;
petiole, 3 - 5 cm in length; cross sections of stem and petiole,
square. Under a magnifying glass, hairs are observed on both
surfaces of the leaf, but abundantly on the vein and sparsely
1330 Pharbitis Seed / Crude Drugs
JP XV
on other parts; small glandular hairs are observed on the low-
er surface.
Odor, characteristic; taste slightly bitter.
Identification To 0.3 mL of the mixture of essential oil and
xylene, obtained in Essential oil content, add 1 mL of acetic
anhydride, shake, and add 1 drop of sulfuric acid: a red-pur-
ple to dark red-purple color develops.
Purity (1) Stem — The amount of its stems, which are not
less than 3 mm in diameter, contained in Perilla Herb does
not exceed 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the stems contained in Perilla Herb does not ex-
ceed 1.0%.
(3) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 16.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Perilla Herb after adding 1 mL of silicone resin to
the sample in the flask: the volume of essential oil is not less
than 0.2 mL.
Pharbitis Seed
Pharbitidis Semen
Pharbitis Seed is the seed of Pharbitis nil Choisy
(Convolvulaceae).
Description Longitudinally quartered or sexpartite globe,
6-8 mm in length, 3-5 mm in width; externally black to
grayish red-brown or grayish white, smooth, but slightly
shrunken and coarsely wrinkled. The transverse section
almost fan-shaped, light yellow-brown to light grayish
brown, and dense in texture. Under a magnifying glass, the
surface of the seed coat reveals dense, short hairs; dented hi-
lum at the bottom of the ridge. Seed coat thin, the outer layer
dark gray, and the inner layer light gray; two irregularly fold-
ed cotyledons in the transverse section at one end; two thin
membranes from the center of the dorsal side to the ridge
separating cotyledons but unrecognizable in the transverse
section of the other end having hilum; dark gray secretory
pits in the section of the cotyledon. 100 seeds weighing about
4.5 g.
When cracked, odor, slight; taste, oily and slightly pun-
gent.
Total ash <J.07> Not more than 6.0%.
Phellodendron Bark
Phellodendri Cortex
Phellodendron Bark is the bark of Phellodendron
amurense Ruprecht or Phellodendron chinense
Schneider (Rutaceae), from which the periderm has
been removed.
It contains not less than 1.2% of berberine [as ber-
berine chloride (C 20 H 18 ClNO 4 : 371.81)], calculated on
the basis of dried material.
Description Flat or rolled semi-tubular pieces of bark, 2-4
mm in thickness; externally grayish yellow-brown to grayish
brown, with numerous traces of lenticel; the internal surface
yellow to dark yellow-brown in color, with fine vertical lines,
and smooth; fractured surface fibrous and bright yellow. Un-
der a magnifying glass, the transverse section of Phelloden-
dron Bark reveals a thin and yellow outer cortex, scattered
with stone cells appearing as yellow-brown dots; inner cortex
thick; primary medullary rays expanding its width towards
the outer end, the phloem appearing as a nearly triangular
part between these medullary rays in secondary cortex, and
many secondary medullary rays radiating and gathering to
the tip of the triangle; brown phloem fiber bundles lined in
tangential direction, crossed over the secondary medullary
rays, and then these tissues show a latticework.
Odor, slight; taste, extremely bitter; mucilaginous; it
colors the saliva yellow on chewing.
Identification (1) To 1 g of pulverized Phellodendron
Bark add 10 mL of diethyl ether, allow to stand for 10
minutes with occasional shaking, and filter to remove the
diethyl ether. Collect the powder on the filter paper, add 10
mL of ethanol (95), allow to stand for 10 minutes with oc-
casional shaking, and filter. To 2 to 3 drops of the filtrate add
1 mL of hydrochloric acid, add 1 to 2 drops of hydrogen
peroxide TS, and shake: a red-purple color develops.
(2) Use the filtrate obtained in (1) as the sample solution.
Separately, dissolve 1 mg of Berberine Chloride Reference
Standard in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /uL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of 1-butanol, water and acetic acid (100)
(7:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 365 nm):
one spot among the spots from the sample solution and a
spot with yellow to yellow-green fluorescence from the stan-
dard solution show the same color tone and the same Rf
value.
(3) Stir up pulverized Phellodendron Bark with water:
the solution becomes gelatinous owing to mucilage.
Loss on drying <5.01> Not more than 11.0% (105°C, 6
hours).
Total ash <J.07> Not more than 7.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Assay Weigh accurately about 0.5 g of pulverized Phel-
lodendron Bark, add 30 mL of a mixture of methanol and di-
lute hydrochloric acid (100:1), heat under a reflux condenser
on a water bath for 30 minutes, cool, and filter. Repeat the
above procedure twice with the residue, using 30-mL and
20-mL portions of a mixture of methanol and dilute
hydrochloric acid (100:1). To the last residue add 10 mL of
methanol, shake well, and filter. Combine the whole filtrates,
add methanol to make exactly 100 mL, and use this solution
JPXV
Crude Drugs / Powdered Phellodendron Bark 1331
as the sample solution. Separately, weigh accurately about 10
mg of Berberine Chloride Reference Standard (previously de-
termine the water <2.48> in the same manner as Berberine
Chloride Hydrate), dissolve in methanol to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 20 fiL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of berberine in each solution.
Amount (mg) of berberine [as berberine chloride
(C 20 H 18 ClNO 4 )]
= W s x (A T /A S )
W s : Amount (mg) of Berberine Chloride Reference Stan-
dards, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 345 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel (5 to 10 mm in particle diameter).
Column temperature: A constant temperature of about
40°C
Mobile phase: Dissolve 3.4 g of potassium dihydrogen-
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
Selection of column: Dissolve 1 mg each of Berberine
Chloride Reference Standard and palmatine chloride in 10
mL of methanol. Perform the test with 20 mL of this solution
under the above operating conditions. Use a column giving e-
lution of palmatine and berberine in this order, and clearly
separating each peak.
System repeatability: Repeat the test 5 times with the stan-
dard solution under the above operating conditions the rela-
tive deviation of the peak area of berberine is not more than
1.5%.
Powdered Phellodendron Bark
Phellodendri Cortex Pulveratus
Powdered Phellodendron Bark is the powder of
Phellodendron Bark.
It contains not less than 1.2% of berberine chloride
[as berberine chloride (C 2 oH 18 ClN0 4 : 371.81)], calcu-
lated on the basis of dried material.
Description Powdered Phellodendron Bark occurs as a
bright yellow to yellow powder. It has a slight odor and an
extremely bitter taste, is mucilaginous, and colors the saliva
yellow on chewing.
Under a microscope <5.01>, Powdered Phellodendron Bark
reveals fragments of yellow, thick-walled fiber bundles or
fibers, and fibers often accompanied by crystal cell rows; few-
er groups of stone cells together with idioblasts; fragments of
parenchyma cells containing starch grains and oil droplets;
fragments of medullary ray and phloem; mucilage cells and
mucilage masses. Numerous solitary crystals of calcium oxa-
late, 7-20 [im in diameter; starch grains, simple grains and
2- to 4-compound grains, simple grain, 2-6 /xm in diameter;
oil droplets, stained red with sudan III TS.
Identification (1) To 1 g of Powdered Phellodendron
Bark add 10 mL of diethyl ether, allow to stand for 10
minutes with occasional shaking, and filter to remove the
diethyl ether. Collect the powder on the filter paper, add 10
mL of ethanol (95), allow to stand for 10 minutes with oc-
casional shaking, and filter. To 2 to 3 drops of the filtrate add
1 mL of hydrochloric acid, add 1 to 2 drops of hydrogen
peroxide TS, and shake: a red-purple color develops.
(2) Use the filtrate obtained in (1) as the sample solution.
Separately, dissolve 1 mg of Berberine Chloride Reference
Standard in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fih
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of 1-butanol, water and acetic acid (100)
(7:2:1) to a distance of about 10 cm, and air-dry the plate.
Examine under ultraviolet light (main wavelength: 365 nm):
one spot among the spots from the sample solution and a
spot with yellow to yellow-green fluorescence from the stan-
dard solution show the same color tone and the same i?f
value.
(3) Stir up Powdered Phellodendron Bark with water: the
solution becomes gelatinous owing to mucilage.
Purity Curcuma — Place Powdered Phellodendron Bark on
filter paper, drop diethyl ether on it, and allow to stand. Take
the powder off the filter paper, and drip 1 drop of potassium
hydroxide TS: no red-purple color develops. Under a micro-
scope <5.01>, Powdered Phellodendron Bark does not con-
tain gelatinized starch or secretory cells containing yellow-red
resin.
Loss on drying <5.07> Not more than 11.0% (105°C 6
hours).
Total ash <5.07> Not more than 7.5%.
Acid-insoluble ash <5.01> Not more than 0.5%.
Assay Weigh accurately about 0.5 g of Powdered Phel-
lodendron Bark, add 30 mL of a mixture of methanol and di-
lute hydrochloric acid (100:1), heat under a reflux condenser
on a water bath for 30 minutes, cool, and filter. Repeat the
above procedure twice with the residue, using 30-mL and
20-mL portions of a mixture of methanol and dilute
hydrochloric acid (100:1). To obtained residue add 10 mL of
methanol, shake well, and filter. Combine the whole filtrates,
add methanol to make exactly 100 mL, and use this solution
as the sample solution. Separately, weigh accurately about 10
mg of Berberine Chloride Reference Standard (separately de-
termine the water <2.48> in the same manner as Berberine
Chloride Hydrate), dissolve in methanol to make exactly 100
mL, and use this solution as the standard solution. Perform
the test with exactly 20 /xL each of the sample solution and
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of berberine in each solution.
Amount (mg) of berberine ([as berberine chloride)
(C 20 H 18 ClNO 4 )]
1332 Compound Phellodendron Powder / Crude Drugs
JP XV
= W s x(A T /A s )
W s : Amount (mg) of berberine chloride for component de-
termination Berberine Chloride Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 345 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to lO^m in
particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 3.4 g of potassium dihydrogen-
phosphate and 1.7 g of sodium lauryl sulfate in 1000 mL of a
mixture of water and acetonitrile (1:1).
Flow rate: Adjust the flow rate so that the retention time of
berberine is about 10 minutes.
Selection of column: Dissolve 1 mg each of Berberine
Chloride Reference Standard and palmatine chloride in 10
mL of methanol. Proceed with 20 /xh of this solution under
the above operating conditions. Use a column giving elution
of palmatine and berberine in this order, and clearly dividing
each peak.
System repeatability: When repeat the test 5 times with the
standard solution under the above operating conditions, the
relative standard deviation of the peak area of berberine is
not more than 1.5%.
Compound Phellodendron Powder
for Cataplasm
Method of preparation
Powdered Phellodendron Bark
Powdered Gardenia Fruit
d- or (//-Camphor
dl- or /-Menthol
660)
325j
10j
5)
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Compound Phellodendron Powder for
Cataplasm occurs as a yellow-brown powder, having a
characteristic odor.
Identification Shake thoroughly 0.2 g of Compound Phel-
lodendron Powder for Cataplasm with 5 mL of methanol,
filter, and use the filtrate as the sample solution. Separately,
dissolve 1 mg of Berberine Chloride Reference Standard in 1
mL of methanol, and use the solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 5 /uL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of 1-butanol, water and acetic acid (100) (7:2:1) to a dis-
tance of about 10 cm, air-dry the plate. Examine under
ultraviolet light (main wavelength: 365 nm): one of the spot
among the several spots from the sample solution has the
same color tone and Rf value with the yellow to yellow-green
fluorescent spot from the standard solution (phellodendron
bark).
Containers and storage Containers — Tight containers.
Phellodendron, Albumin Tannate
and Bismuth Subnitrate Powder
Phellodendron, Albumin Tannate and Bismuth Sub-
nitrate Powder contains not less than 12.9% and not
more than 16.3% of bismuth (Bi: 208.98).
Method of preparation
Powdered Phellodendron Bark
300 g
Albumin Tannate
300 g
Bismuth Subnitrate
200 g
Scopolia Extract
10 g
Starch, Lactose Hydrate or
their mixture
a sufficient quantity
To make
1000 g
Prepare as directed under Powders, with the above in-
gredients. Scopolia Extract Powder may be used in place of
Scopolia Extract.
Description Phellodendron, Albumin Tannate and Bismuth
Subnitrate Powder is brownish yellow in color, and has a bit-
ter taste.
Identification (1) Shake thoroughly 0.1 g of Phelloden-
dron, Albumin Tannate and Bismuth Subnitrate Powder
with 5 mL of methanol, filter, and use the filtrate as the sam-
ple solution. Separately, dissolve 1 mg of Berberine Chloride
Reference Standard in 1 mL of methanol, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of 1-butanol, water and acetic
acid (100) (7:2:1) to a distance of about 10 cm, air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): one spot among the spots from the sample solution and
a spot with yellow to yellow-green fluorescence from the stan-
dard solution show the same color tone and the same Rf
value (phellodendron bark).
(2) To 0.3 g of Phellodendron, Albumin Tannate and
Bismuth Subnitrate Powder add 20 mL of ethanol (95), heat
in a water bath for 3 minutes with shaking, cool, and filter.
To 10 mL of the filtrate add 1 drop of iron (III) chloride TS:
a blue-green color is produced. Allow to stand: a bluish black
precipitate is produced (albumin tannate).
(3) To 0.3 g of Phellodendron, Albumin Tannate and
Bismuth Subnitrate Powder add 10 mL of diluted pyridine (1
in 5), warm in a water bath for 3 minutes with shaking, cool,
and filter. Add 1 mL of ninhydrin-ascorbic acid TS to the
filtrate, and heat in a water bath: a blue color is produced (al-
bumin tannate).
(4) To 0.5 g of Phellodendron, Albumin Tannate and
JPXV
Crude Drugs / Pinellia Tuber 1333
Bismuth Subnitrate Powder add 5 mL of dilute hydrochloric
acid and 10 mL of water, warm, shake thoroughly, and filter.
The filtrate responds to the Qualitative Tests <1.09> for bis-
muth salt.
Assay Weigh accurately about 0.7 g of Phellodendron, Al-
bumin Tannate and Bismuth Subnitrate Powder, shake well
with 10 mL of water and 20 mL of diluted nitric acid (1 in 3),
add water to make exactly 100 mL, and filter. Discard the
first 20 mL of the filtrate, pipet the subsequent 10 mL of the
filtrate, and add water to make exactly 100 mL. Pipet 25 mL
of this solution, add diluted nitric acid (1 in 100) to make ex-
actly 100 mL, and use this solution as the sample solution.
Separately, weigh accurately about 0.23 g of bismuth nitrate
pentahydrate, add 20 mL of diluted nitric acid (1 in 3) and
water to make exactly 100 mL. Pipet 10 mL of this solution,
and add water to make exactly 100 mL. Pipet 25 mL of this
solution, add diluted nitric acid (1 in 100) to make exactly 100
mL, and use this solution as the standard solution. Determine
the absorbances, A T and A s , of the sample solution and stan-
dard solution according to Atomic Absorption Spec-
trophotometry <2.23> under the following conditions. On the
other hand, determine the absorbance A of the solution pre-
pared in the same manner using 20 mL of diluted nitric acid
(1 in 3) instead of the standard solution.
Gas: Combustible gas — Acetylene
Supporting gas — Air
Lamp: A bismuth hollow-cathode lamp
Wavelength: 223.1 nm
Amount (mg) of bismuth (Bi)
= Wx l(A T -A )/(A s -A Q )} x 0.4308
W: Amount (mg) of bismuth nitrate pentahydrate
Containers and storage Containers — Well-closed contain-
ers.
Picrasma Wood
Picrasmae Lignum
-■ft*
Picrasma Wood is the wood of Picrasma quassioides
Bennet (Simaroubaceae).
Description Light yellow chips, slices or short pieces of
wood; a transverse section reveals distinct annual rings and
thin medullary rays; tissue dense in texture.
Odorless; taste, extremely bitter and lasting.
Under a microscope <5.01>, it reveals medullary rays con-
sisting of 1 - 5 cells wide for transverse section, and 5-50
cells high for longitudinal section; vessels of spring wood up
to about 150 /um in diameter, but those of autumn wood only
one-fifth as wide; vessels, single or in groups, scattered in the
xylem parenchyma; membrane of wood fibers extremely
thickened; medullary rays and xylem parenchyma cells con-
tain rosette aggregates of calcium oxalate and starch grains.
Vivid yellow or red-brown, resinous substance often present
in the vessels.
Purity Foreign matter <5.01> — The amount of foreign mat-
ter contained in Picrasma Wood does not exceed 1.0%.
Total ash <5.07> Not more than 4.0%.
Powdered Picrasma Wood
Picrasmae Lignum Pulveratum
Powdered Picrasma Wood is the powder of Picras-
ma Wood.
Description Powdered Picrasma occurs as a grayish white
to light yellow powder. It is odorless, and has an extremely
bitter and lasting taste.
Under a microscope <5.0I>, Powdered Picrasma Wood
reveals fragments of vessels of various sizes, xylem fibers and
xylem parenchyma cells; fragments of medullary rays con-
taining starch grains; all tissues lignified; a few crystals of cal-
cium oxalate observed. Starch grains are 5 to 15 /um in di-
ameter.
Total ash <J.07> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Pinellia Tuber
Pinelliae Tuber
)\>f
Pinellia Tuber is the tuber of Pinellia ternata
Breitenbach (Araceae), from which the cork layer has
been removed.
Description Slightly flattened spherical to irregular-shaped
tuber; 0.7 - 2.5 cm in diameter and 0.7 - 1.5 cm in height; ex-
ternally white to grayish white-yellow; the upper end dented,
where the stem has been removed, with root scars dented as
numerous small spots on the circumference; dense in texture;
cross section white and powdery.
Almost odorless; tasteless at first, slightly mucous, but
leaving a strong acrid taste.
Under a microscope <5.01>, a transverse section reveals
mainly tissue of parenchyma filled with starch grains, and
scattered with a few mucilage cells containing raphides of cal-
cium oxalate. Starch grains mostly 2- to 3-compound grains,
usually 10-15 /um in diameter, and simple grains, usually 3 -
7 /um in diameter; raphides of calcium oxalate 25 - 150 /um in
length.
Purity (1) Rhizome of Arisaema species and others — Un-
der a microscope <5.01>, no mucilage canal is revealed on the
outer layer of cortex.
(2) Heavy metals <1.07> — Proceed with 3.0 g of pulver-
ized Pinellia Tuber according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 10 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Pinellia Tuber according to Method 4, and per-
form the test (not more than 5 ppm).
Loss on drying <5.01> Not more than 14.0% (6 hours).
1334 Plantago Herb / Crude Drugs
JP XV
Total ash <5.01> Not more than 3.5%.
Plantago Herb
Plant aginis Herb a
*> + M > V ^
Plantago Herb is the entire plant of Plantago asiati-
ca Linne (Plantaginaceae), collected during the flower-
ing season.
Description Usually wrinkled and contracted leaf and
spike, grayish green to dark yellow-green in color; when
soaked in water and smoothed out, the lamina is ovate to or-
bicular-ovate, 4 - 15 cm in length, 3 - 8 cm in width; apex a-
cute, and base sharply narrowed; margin slightly wavy, with
distinct parallel veins; glabrous or nearly glabrous; petiole is
rather longer than the lamina, and its base is slightly expand-
ed with thin-walled leaf-sheath; scape is 10-50 cm in length,
one-third to one-half of the upper part forming the spike,
with dense florets; the lower part of inflorescence often shows
pyxidia; roots usually removed, but, if any, fine roots are
closely packed.
Odor, slight; tasteless.
Identification To 2.0 g of pulverized Plantago Herb add 10
mL of methanol, warm on a water bath for 3 minutes, cool,
filter, and use the filtrate as the sample solution. Perform the
test with this solution as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /xL of the sample solution on a
plate of silica gel for thin-layer chromatography. Develop the
plate with a mixture of 1-butanol, water and acetic acid (100)
(7:2:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly iron (III) chloride TS on the plate: a dark blue
spot appears at the Rf value about 0.5.
Total ash <J.07> Not more than 15.0%.
Acid-insoluble ash <5.0I> Not more than 4.0%.
Extract content <5.01>
less than 14.0%.
Dilute ethanol-soluble extract: not
Plantago Seed
Plantaginis Semen
Plantago Seed is the seed of Plantago asiatica Linne
(Plan taginaceae).
Description Flattened ellipsoidal seed, 2 - 2.5 mm in
length, 0.7 - 1 mm in width, 0.3 - 0.5 mm in thickness; exter-
nally brown to yellow-brown and lustrous. Under a mag-
nifying glass, the surface of the seed is practically smooth,
with the dorsal side protruding like a bow, and with the ven-
tral side somewhat dented; micropyle and raphe not observa-
ble. 100 seeds weigh about 0.05 g.
Odorless; taste, slightly bitter and mucous.
Under a microscope <5.01>, a transverse section reveals a
seed coat consisting of three layers of epidermis composed of
cells containing mucilage, a vegetative layer, and a pigment
layer of approximately equidiameter cells; in the interior, en-
dosperm thicker than seed coat, enclosing two cotyledons.
Identification (1) To 1 g of Plantago Seed add 2 mL of
warm water, and allow the mixture to stand for 10 minutes:
the seed coat swells to discharge mucilage.
(2) Boil gently 1 g of Plantago Seed with 10 mL of dilute
hydrochloric acid for 2 minutes, and filter. Neutralize the
filtrate with sodium hydroxide TS, to 3 mL of this solution
add 1 mL of Fehling's TS, and warm the mixture: a red
precipitate is produced.
Purity Foreign matter <5.01> — The amount of foreign mat-
ter contained in Plantago Seed does not exceed 2.0%.
Total ash <5.07> Not more than 5.5%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Platycodon Fluidextract
Method of preparation Take coarse powder of platycodon,
and prepare the fluidextract as directed under Fluidextracts
using 25 vol% ethanol. An appropriate quantity of Ethanol
and Purified Water may be used in place of 25 vol% ethanol.
Description Platycodon Fluidextract is a red-brown liquid.
It is miscible with water, producing slight turbidity. It has a
mild taste at first, followed by an acrid and bitter taste.
Identification (1) Shake vigorously 0.5 mL of Platycodon
Fluidextract with 10 mL of water: a lasting fine foam is
produced.
(2) Dissolve 1 drop of Platycodon Fluidextract in 2 mL
of acetic anhydride, and add gently 0.5 mL of sulfuric acid: a
red to red-brown color develops at the zone of contact.
Purity Starch — Mix 1 mL of Platycodon Fluidextract with
4 mL of water, and add 1 drop of dilute iodine TS: no purple
or blue color develops.
Content of the active principle Transfer exactly 5 mL of
Platycodon Fluidextract to a tared beaker, evaporate to dry-
ness on a water bath, and dry at 105 °C for 5 hours: the mass
of the residue is not less than 0.50 g.
Containers and storage Containers — Tight containers.
Storage — Light-resistant.
Platycodon Root
Platycodi Radix
**at7
Platycodon Root is the root of Platycodon gran-
diflorum A. De Candolle (Campanulaceae).
Description Irregular, somewhat thin and long fusiform to
conical root, often branched; externally grayish brown, light
brown or white; main root 10 - 15 cm in length, 1-3 cm in
diameter; the upper end, with dented scars of removed stems;
JPXV
Crude Drugs / Powdered Polygala Root 1335
the neighborhood, with fine lateral wrinkles and longitudinal
furrows and also slightly constricted; the greater part of the
root, except the crown, covered with coarse longitudinal
wrinkles, lateral furrows and lenticel-like lateral lines; hard in
texture, but brittle; fractured surface not fibrous, often with
cracks. Under a magnifying glass, a transverse section reveals
cambium and its neighborhood often brown in color; cortex
slightly thinner than xylem, almost white and with scattered
cracks; xylem white to light brown in color, and the tissue
slightly denser than cortex.
Odor, slight; tasteless at first, later acrid and bitter.
Identification (1) Boil 0.5 g of pulverized Platycodon
Root with 10 mL of water for a while, allow to cool, and
shake the mixture vigorously: a lasting fine foam is produced.
(2) Warm 0.2 g of pulverized Platycodon Root with 2 mL
of acetic anhydride on a water bath for 2 minutes, and filter.
To 1 mL of the filtrate add carefully 0.5 mL of sulfuric acid
to make two layers: a red to red-brown color develops at the
zone of contact, and the upper layer acquires a blue-green to
green color.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Platycodon Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic </.//> — Prepare the test solution with 0.40 g
of pulverized Platycodon Root according to Method 4, and
perform the test (not more than 5 ppm).
Total ash <5.01> Not more than 4.0%.
Extract content <5.01>
less than 25.0%.
Dilute ethanol-soluble extract: not
Powdered Platycodon Root
Platycodi Radix Pulverata
Powdered Platycodon Root is the powder of
Platycodon Root.
Description Powdered Platycodon Root occurs as a light
grayish yellow to light grayish brown powder. It has a slight
odor, and is tasteless at first, later acrid and bitter.
Under a microscope <5.01>, Powdered Platycodon Root
reveals numerous fragments of colorless parenchyma cells;
fragments of reticulate vessels and scalariform vessels; frag-
ments of sieve tubes and lactiferous tubes; fragments of cork
layer are sometimes observed. Usually, starch grains are not
observed, but very rarely simple grain.
Identification (1) Boil 0.5 g of Powdered Platycodon
Root with 10 mL of water for a while, allow to cool, and
shake the mixture vigorously: a lasting fine foam is produced.
(2) Warm 0.2 g of Powdered Platycodon Root with 2 mL
of acetic anhydride on a water bath for 2 minutes, and filter.
To 1 mL of the filtrate add carefully 0.5 mL of sulfuric acid
to make two layers: a red to red-brown color develops at the
zone of contact, and the upper layer acquires a blue-green to
green color.
Powdered Platycodon Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Platycodon Root according to Method 4, and
perform the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Platycodon Root does not show fibers, stone cells or
other foreign matter.
Total ash <5.07> Not more than 4.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 25.0%.
Polygala Root
Polygalae Radix
Polygala Root is the root of Polygala tenuifolia
Willdenow (Polygalaceae).
Description Thin, long and bent, cylindrical or tubular
root; main root, 10 - 20 cm in length, 0.2 - 1 cm in diameter,
sometimes with one to several lateral roots; externally light
grayish brown, with coarse longitudinal wrinkles, and with
deep lateral furrows cracked to some degree here and there;
brittle, and fractured surface not fibrous; margin of the
transverse section irregularly undulate; cortex, comparatively
thick, with large cracks here and there; xylem usually round
to elliptical, light brown in color, and often tears in a wedge-
like shape.
Odor, slight; taste, slightly acrid.
Identification (1) Shake vigorously 0.5 g of pulverized
Polygala Root with 10 mL of water: a lasting fine foam is
produced.
(2) To 0.5 g of pulverized Polygala Root add 2 mL of
acetic anhydride. After shaking well, allow to stand for 2
minutes, and filter. To the filtrate add carefully 1 mL of sul-
furic acid to make two layers: a red-brown color develops at
the zone of contact, and changes to dark green.
Purity (1) Stem — The amount of the stems contained in
Polygala Root does not exceed 10.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the stems contained in Polygala Root is not more
than 1.0%.
(3) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Total ash <5.07> Not more than 6.0%.
Powdered Polygala Root
Polygalae Radix Pulverata
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Polygala Root is the powder of Polygala
1336 Polygonatum Rhizome / Crude Drugs
JP XV
Root.
Description Powdered Polygala Root occurs as a light
grayish yellow-brown powder. It has a slight odor and a
slightly acrid taste.
Under a microscope <5.01>, Powdered Polygala Root rev-
eals fragments of cork layers, pitted vessels, reticulate vessels
and tracheids; fragments of xylem fibers and xylem parenchy-
ma cells with a small number of simple pits; fragments of
parenchyma cells containing substances such as oil droplets,
rosette aggregates and solitary crystals of calcium oxalate.
Oil drop-like contents stained red with sudan III TS.
Identification (1) Shake vigorously 0.5 g of Powdered
Polygala Root with 10 mL of water: a lasting fine foam is
produced.
(2) To 0.5 g of Powdered Polygala Root add 2 mL of
acetic anhydride. After shaking well, allow to stand for 2
minutes, and filter. To the filtrate add carefully 1 mL of sul-
furic acid to make two layers: a red-brown color develops at
the zone of contact, and changes to dark green later.
Purity (1) Foreign matter <5.01> — Under a microscope,
Powdered Polygala Root does not show stone cells or starch
grains.
(2) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Total ash <5.01> Not more than 6.0%.
Polygonatum Rhizome
Polygonati Rhizoma
Polygonatum Rhizome is the rhizome of Polygona-
tum falcatum A. Gray, Polygonatum sibiricum Red-
oute, Polygonatum kingianum Collett et Hemsley or
Polygonatum cyrtonema Hua (Liliaceae), usually after
being steamed.
Description Irregularly cylindrical rhizome, 3-10 cm in
length, 0.5 - 3 cm in diameter; or irregular massive rhizome,
5-10 cm in length, 2-6 cm in diameter, occasionally bran-
ched; both rhizomes with many cyclic nodes and longitudi-
nally striate; externally yellowish brown to dark brown; stem
scars, round, concave at their center, and protuberant on the
upper surface; root scars on the lower surface; cut surface flat
and horny.
Odor, slight; taste, slightly sweet.
Under a microscope <5.01>, a transverse section of the rhi-
zome reveals epidermis coated with cuticle; inside of epider-
mis parenchyma lie; numerous vascular bundles and
mucilage cells scattered in parenchyma; vascular bundles col-
lateral or amphivasal concentric; mucilage cells contain
raphides of calcium oxalate.
Identification (1) To 0.5 g of fine cutted Polygonatum
Rhizome add 2 mL of acetic anhydride, warm on a water
bath for 2 minutes, and filter. To 1 mL of the filtate add gent-
ly 0.5 mL of sulfuric acid: a red-brown color appears at the
zone of contact.
(2) To 1.0 g of fine cutted Polygonatum Rhizome add 10
mL of dilute hydrochloric acid, boil gently for 2 minutes, and
filter. Neutralize the filtrate with sodium hydroxide TS. To 3
mL of this solution add 1 mL of Fehling's TS, and warm: red
precipitates appear.
Total ash <5.07> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Polygonum Root
Polygoni Multiflori Radix
Polygonum Root is the root of Polygonum multiflo-
rum Thunberg (Polygonaceae), often being cut into
round slices.
Description Polygonum Root is nearly fusiform, 10 to 15
cm in length, 2 to 5 cm in diameter; externally red-brown to
dark brown; roughly wrinkled; a cross section light red-
brown or light grayish brown, with numerous abnormal vas-
cular bundles scattering irregularly around the large vascular
bundles near center; heavy and hard in texture.
Odor, slight and characteristic; taste, astringent and slight-
ly bitter.
Under a microscope <5.01>, transverse section reveals the
outermost layer to be several cells thick and composed of
cork; cork cells contain brown substances; cortex composed
of parenchyma; abnormal vascular bundles, exhibiting a ring
of cambium; xylem lies inside of the cambium, and phloem
outside; fibers lie outside the phloem; central portion of root
lignified; parenchymatous cells contain aggregated crystals of
calcium oxalate, and both simple and 2- to 8-compound
starch grains; navel of starch grain obvious.
Identification To 1 g of pulverized Polygonum Root add 10
mL of methanol, shake for 15 minutes, and filter. Evaporate
the filtrate to dryness, dissolve the residue in 2 mL of
methanol, and use this as the sample solution. Perform the
test with the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 5 [iL of the sample solution on
a plate of silica gel for thin-layer chromatography, develop
the plate with a mixture of ethyl acetate, water, methanol and
acetic acid (100) (200:10:10:3) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 365 nm): a fluorescent bluish white spot appears
at around Rf 0.3.
Loss on drying <5.01> Not more than 14.0% (6 hours).
Total ash <J.07> Not more than 5.5%.
Extract content <5.07>
less than 17.0%.
Dilute ethanol-soluble extract: not
Polyporus Sclerotium
Polyporus
Polyporus Sclerotium is the sclerotium of Polyporus
JPXV
Crude Drugs / Powdered Poria Sclerotium 1337
umbellatus Fries (Polyporaceae).
Description Irregularly shaped mass, usually 5-15 cm in
length; externally blackish brown to grayish brown, with
numerous dents and coarse wrinkles; breakable; fractured
surface rather soft and cork-like, and almost white to light
brown in color, and a white speckled pattern on the inner
region; light in texture.
Odorless and tasteless.
Identification Warm, while shaking, 0.5 g of pulverized
Polyporus Sclerotium with 5 mL of acetone on a water bath
for 2 minutes, filter, and evaporate the filtrate to dryness.
Dissolve the residue in 5 drops of acetic anhydride, and add 1
drop of sulfuric acid: a red-purple color develops, and im-
mediately changes to dark green.
Total ash <J.07> Not more than 16.0%.
Acid-insoluble ash <5.07> Not more than 4.0%.
Powdered Polyporus Sclerotium
Polyporus Pulveratus
Powdered Polyporus Sclerotium is the powder of the
Polyporus Sclerotium.
Description Powdered Polyporus Sclerotium occurs as a
light grayish brown to light brown powder. It has a slight
odor, and a slightly bitter and acrid taste, is gritty between
the teeth on chewing.
Under a microscope <5.01>, Powdered Polyporus Scleroti-
um reveals hypha, l to 2 iim, rarely up to 13 Lira in diameter,
and colorless transparent; granule strongly refracting light;
and a few mucilage plates; sometimes fragments of false tis-
sue consisting of them; somewhat brown false tissues; and
solitary crystal. Solitary crystal is 10 to 40 /um in diameter,
sometimes 100 iim in diameter.
Identification Warm, while shaking, 0.5 g of Powdered
Polyporus Sclerotium with 5 mL of acetone on a water bath
for 2 minutes, filter and evaporate the filtrate to dryness. Dis-
solve the residue in 5 drops of acetic anhydride, and add 1
drop of sulfuric acid: a red-purple color develops, and im-
mediately changes to dark green.
Total ash <5.01> Not more than 16.0%.
Acid-insoluble ash <5.0I> Not more than 4.0%.
Containers and storage Containers — Tight containers.
Poria Sclerotium
Poria
-?<7 'Ja9
Poria Sclerotium is the sclerotium of Poria cocos
Wolf (Polyporaceae), from which usually the external
layer has been mostly removed.
2 kg in mass; usually it appears as broken or chipped pieces;
white or slightly reddish white; sclerotium with remaining
outer layer is dark brown to dark red-brown in color, coarse,
which fissures; hard in texture, but brittle.
Almost odorless, tasteless, and slightly mucous.
Identification (1) Warm 1 g of pulverized Poria Scleroti-
um with 5 mL of acetone on a water bath for 2 minutes with
shaking, and filter. Evaporate the filtrate to dryness, dissolve
the residue in 0.5 mL of acetic anhydride, and add 1 drop of
sulfuric acid: a light red color develops, which changes im-
mediately to dark green.
(2) To a section or powder of Poria Sclerotium add 1
drop of iodine TS: a deep red-brown color is produced.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Poria Sclerotium according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than lOppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Poria Sclerotium according to Method 4, and perform the
test (not more than 5 ppm).
Total ash <J.07> Not more than 1.0%.
Powdered Poria Sclerotium
Poria Pulveratum
-1*7 U a *J%
Powdered Poria Sclerotium is the powder of Poria
Sclerotium.
Description Powdered Poria Sclerotium occurs as a white
to grayish white powder. It is almost odorless and tasteless,
but is slightly mucous.
Under a microscope <5.01>, Powdered Poria Sclerotium
reveals colorless and transparent hyphae strongly refracting
light, and fragments of false tissue consisting of granules and
mucilage plates. Thin hyphae, 2 - 4 iim in diameter; thick
ones, usually 10 - 20,um, up to 30,Mm.
Identification (1) Warm 1 g of Powdered Poria Scleroti-
um with 5 mL of acetone on a water bath for 2 minutes with
shaking, and filter. Evaporate the filtrate to dryness, dissolve
the residue in 0.5 mL of acetic anhydride, and add 1 drop of
sulfuric acid: a light red color develops, which changes im-
mediately to dark green.
(2) To Powdered Poria Sclerotium add 1 drop of iodine
TS: a deep red-brown color is produced.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Poria Sclerotium according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Poria Sclerotium according to Method 4, and
perform the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Poria Sclerotium does not show starch grains.
Total ash <5.07> Not more than 1.0%.
Description Mass, about 10 - 30 cm in diameter, up to 0.1
1338 Processed Aconite Root / Crude Drugs
JP XV
Processed Aconite Root
Processi Aconiti Radix
7~>
Processed Aconite Root is the tuberous root of
Aconitum carmichaeli Debeaux or Aconitum japoni-
cum Thunberg (Ranunculaceae) prepared by the fol-
lowing processes.
Process 1: Autoclaving. [Processed Aconite Root 1]
Process 2: Heating or autoclaving after rinsing in
salt or rock salt solution. [Processed
Aconite Root 2]
Process 3: Treating with lime after rinsing in salt so-
lution. [Processed Aconite Root 3]
Processed Aconite Root 1, Processed Aconite Root 2
and Processed Aconite Root 3 contain the total
alkaloid [as benzoyl aconin (C 32 H45NO 10 : 603.70)] of
not less than 0.7% and not more than 1.5%, not less
than 0.1% and not more than 0.6%, and not less than
0.5% and not more than 0.9%, calculated on the dried
bases, respectively.
The label indicates the treating process.
Description Processed Aconite Root 1 : Cut pieces irregular-
ly polygonal, less than 10 mm in diameter; externally dark
grayish brown to blackish brown; hard in texture; cut surface
flat, light brown to dark brown, usually horny and lustrous.
Odor, weak and characteristic.
Under a microscope <5.01>, transverse and longitudinal
sections reveal pitted, scaraliform, reticulate and spiral ves-
sels; starch grains in parenchymatous cells usually gelatinized
but sometimes not gelatinized; starch grains, simple, spheri-
cal or ellipsoid, 2-25 fim in diameter, or 2- to a dozen or so-
compound, hilum of starch grain distinct.
Processed Aconite Root 2: Nearly obconical root, 15-30
mm in length, 12 - 16 mm in diameter, slices cut longitudi-
nally or transversely, 20 - 60 mm in length, 15 -40 mm in
width, and 200 - 700 fim in thickness, or cut pieces irregular-
ly polygonal, less than 12 mm in diameter; externally light
brown to dark brown or yellowish brown; hard in texture,
usually without wrinkles; cut surface flat, light brown to dark
brown or yellowish white to light yellowish brown, usually
horny, semi-transparent and lustrous.
Odor, weak and characteristic.
Under a microscope <5.01>, transverse and longitudinal
sections reveal metaderm, primary cortex, endodermis, sec-
ondary cortex, cambium, and xylem; primary cortex contains
oblong to oblong-square sclerenchymatous cells, 30-75 fim
in short axis, 60- 150 ^m in long axis; endodermis single
layered, endodermal cells elongated in tangential direction;
cambium, star shaped or irregular polygons to orbicular; a
group of vessel in xylem v-shaped; sometimes isolated ring of
cambium appears in secondary cortex or in pith; vessels, pit-
ted, scaraliform, reticulate and spiral; starch grains in paren-
chymatous cells gelatinized.
Processed Aconite Root 3: Cut pieces irregularly polygonal,
less than 5 mm in diameter; externally grayish brown; hard in
texture; cut surface flat, light grayish brown to grayish white,
not lustrous.
Odor, weak and characteristic.
Under a microscope <5.01>, transverse and longitudinal
sections reveal pitted, scaraliform, reticulate and spiral ves-
sels; starch grains, simple, spherical or ellipsoid, 2-25 fim in
diameter, or 2- to a dozen or so- compound, hilum of starch
grain distinct.
Identification To 3 g of pulverized Processed Aconite Root
add 20 mL of diethyl ether and 2 mL of ammonia TS, shake
for 10 minutes, centrifuge, and take the ether layer.
Evaporate the ether layer to dryness under reduced pressure,
dissolve the residue in 1 mL of diethyl ether, and use this so-
lution as the sample solution. Separately, dissolve 1 mg of
benzoylmesaconine hydrochloride for thin-layer chro-
matography in 10 mL of ethanol (99.5), and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 10 fiL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, de-
velop the plate with a mixture of ethyl acetate, ethanol (99.5)
and ammonia water (28) (40:3:2) to a distance of about 10
cm, and air-dry the plate. Spray evenly DragendorfFs TS for
spraying on the plate, air-dry the plate, and spray evenly so-
dium nitrite TS: one of the spot among the several spots from
the sample solution has the same color tone and Rf value with
the yellow-brown spot from the standard solution.
Purity Aconitum diester alkaloids (aconitine, jesaconitine,
hypaconitine and mesaconitine) — Weigh accurately about 0.5
g of pulverized Processed Aconite Root, put in a glass-stop-
pered centrifuge tube, suspend in 3.0 mL of water by shak-
ing, and add 1.0 mL of ammonia TS and 20 mL of diethyl
ether. Stopper tightly the tube, shake for 30 minutes, cen-
trifuge, and separate the ether layer. To the residue add 1.0
mL of ammonia TS and 20 mL of diethyl ether, and repeat
the above process twice more. Combine all extracts,
evaporate to dryness under reduced pressure below 40°C,
and dissolve the residue with exactly 10 mL of a mixture of
the phosphate buffer solution for aconite root and acetoni-
trile (1:1). Centrifuge this solution, and use the supernatant
liquid as the sample solution. Perform the test with exactly 20
fiL each of the sample solution and aconitum diester
alkaloids standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the heights of the peaks corresponding to
aconitine, H TA and H SA , jesaconitine, H Ti and H sl ,
hypaconitine, H TH and H SH , and mesaconitine, H TM and
H SM , respectively, and calculate the amounts of them by the
following formulae: the amounts of aconitine, jesaconitine,
hypaconitine and mesaconitine per g calculated on the dried
basis are not more than 60 fig, 60 fig, 280 fig and 140 fig, re-
spectively, and the total amount of them is not more than 450
m-
Amount (pig) of aconitine (C34H 47 NOn)
= (C SA /^)X(// TA /// SA )X10
Amount (pig) of jesaconitine (CssL^NOn)
= (C S1 /W)X(H T ,/H S1 )X10
Amount (fig) of hypaconitine (C33H45NOK))
= (C Sil /W)x(H Til /H sa )xl0
Amount (fig) of mesaconitine (C 3 3H 45 NO u )
JPXV
Crude Drugs / Powdered Processed Aconite Root 1339
= (C SM /»0x(// TM /i/ SM )xio
C SA : Concentration (ug/mL) of aconitine for purity in the
aconitum diester alkaloids standard solution for puri-
ty
C SJ : Concentration (wg/mL) of jesaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
C SH : Concentration (Mg/mL) of hypaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
C SM : Concentration (Mg/mL) of mesaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
W: Amount (g) of the sample, calculated on the dried basis
Operating conditions—
Detector: An ultraviolet absorption photometer
(wavelength: 231 nm for aconitine, hypaconitine and
mesaconitine; 254 nm for jesaconitine).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of the phosphate buffer solution
for aconite root and tetrahydrofuran (183:17).
Flow rate: Adjust the flow rate so that the retention time of
mesaconitine is about 31 minutes.
System suitability —
System performance: When the procedure is run with
20 /xL of the aconitum diester alkaloids standard solution for
purity under the above operating conditions, using 254 nm,
mesaconitine, hypaconitine, aconitine and jesaconitine are
eluted in this order, and each resolution between their peaks
is not less than 1.5, respectively.
System repeatability: To 1 mL of aconitum diester
alkaloids standard solution for purity add a mixture of the
phosphate buffer solution for aconite root and acetonitrile
(1:1) to make 10 mL. When the test is repeated 6 times with
20 /xL of this solution under the above operating conditions,
using 231 nm, the relative standard deviation of the peak
height of mesaconitine is not more than 1.5%.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.01>
Processed Aconite Root 1: Not more than 4.0%.
Processed Aconite Root 2: Not more than 12.0%.
Processed Aconite Root 3: Not more than 19.0%.
Acid-insoluble ash <5.0I> Not more than 0.9%.
Assay Weigh accurately about 2 g of pulverized Processed
Aconite Root, put in a glass-stoppered centrifuge tube, and
add 1.6 mL of ammonia TS and 20 mL of diethyl ether. Stop-
per tightly the tube, shake for 30 minutes, centrifuge, and
separate the ether layer. To the residue add 0.8 mL of ammo-
nia TS and 20 mL of diethyl ether, and proceed as above.
Repeat this process more three times. Combine all extracts,
evaporate to dryness under reduced pressure, dissolve the
residue in 5 mL of ethanol (99.5), add 30 mL of freshly
boiled and cooled water, and titrate <2.50> with 0.01 mol/L
hydrochloric acid VS until the color of the solution changes
from green to gray-blue through blue-green (indicator: 3
drops of methyl red-methylene blue TS). Perform a blank de-
termination and make any necessary correction.
Each mL of 0.01 mol/L hydrochloric acid VS
= 6.037 mg of total alkaloid [as benzoylaconine
(C 32 H 45 NO 10 )]
Powdered Processed Aconite Root
Processi Aconiti Radix Pulverata
Powdered Processed Aconite Root is the powder of
Processed Aconite Root prepared by the process 1 or
process 2, the powder of Processed Aconite Root pre-
pared by process 1, or the powder of Processed Aconite
Root prepared by the process 1 to which Corn Starch
or Lactose Hydrate is added.
Process 1: Autoclaving. [Powdered Processed
Aconite Root 1]
Process 2: Heating or autoclaving after rinsing in
salt or rock salt solution. [Powdered
Processed Aconite Root 2]
Powdered Processed Aconite Root 1 and Powdered
Processed Aconite Root 2 contain the total alkaloid [as
benzoyl aconin (C 3 2H45NO 10 : 603.70)] of not less than
0.4% and not more than 1.2%, and not less than 0.1%
and not more than 0.3%, calculated on the dried bases,
respectively.
The label indicates the treating process.
Description Powdered Processed Aconite Root 1: Pow-
dered Processed Aconite Root 1 occurs as a light grayish
brown powder. It has a characteristic odor.
Under a microscope <5.01>, Powered Processed Aconite
Root 1 reveals gelatinized starch masses or starch grains and
parenchymatous cells containing them, fragments of reddish
brown metaderm, fragments of pitted, scaraliform, reticulate
and spiral vessels; also square to oblong-square scleren-
chymatous cells, 30- 150 /um in diameter, 100- 250 ^m in
length, cell wall of sclerenchymatous cells, 6-12 /xm in thick-
ness; starch grains of Aconitum carmichaeli Debeaux or
Aconitum japonicum Thunberg (Ranunculaceae) origin, sim-
ple, spherical or ellipsoid, 2-25 /xm in diameter, or 2- to a
dozen or so- compound, hilum of starch grain distinct.
Powdered Processed Aconite Root 2: Powdered Processed
Aconite Root 2 occurs as a light yellowish white powder. It
has a characteristic odor.
Under a microscope <5.01>, Powered Processed Aconite
Root 2 reveals gelatinized starch masses and parenchymatous
cells containing them, fragments of reddish brown
metaderm, fragments of pitted, scaraliform, reticulate and
spiral vessels; also square to oblong-square sclerenchymatous
cells, 30- 150 /xm in diameter, 100- 250 /xm in length, cell
wall of sclerenchymatous cells, 6 - 12 /xm in thickness.
Identification To 3 g of Powdered Processed Aconite Root
add 2 mL of ammonia TS and 20 mL of diethyl ether, shake
for 10 minutes, and centrifuge. Evaporate the ether layer to
dryness under reduced pressure, dissolve the residue in 1 mL
of diethyl ether, and use this solution as the sample solution.
Separately, dissolve 1 mg of benzoylmesaconine hydrochlo-
1340 Powdered Processed Aconite Root / Crude Drugs
JP XV
ride for thin-layer chromatography in 10 mL of ethanol
(99.5), and use this solution as the standard solution.
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution and standard solution on a plate of silica gel for
thin-layer chromatography, develop the plate with a mixture
of ethyl acetate, ethanol (99.5) and ammonia water (28)
(40:3:2) to a distance of about 10 cm, and air-dry the plate.
Spray evenly Dragendorff's TS for spraying on the plate,
air-dry the plate, and spray evenly sodium nitrite TS: one of
the spot among the several spots from the sample solution
has the same color tone and Rf value with the yellow-brown
spot from the standard solution.
Purity Aconitum diester alkaloids (aconitine, jesaconitine,
hypaconitine and mesaconitine) — Weigh accurately about 0.5
g of Powdered Processed Aconite Root, put in a glass-stop-
pered centrifuge tube, suspend in 3.0 mL of water by shak-
ing, and add 1.0 mL of ammonia TS and 20 mL of diethyl
ether. Stopper tightly the tube, shake for 30 minutes, cen-
trifuge, and separate the ether layer. To the residue add 1.0
mL of ammonia TS and 20 mL of diethyl ether, and repeat
the above process two times. Combine all extracts, evaporate
to dryness under reduced pressure at not more than 40°C,
and dissolve the residue with exactly 10 mL of a mixture of
phosphate buffer solution for aconite root and acetonitrile
(1:1). Centrifuge this solution, and use the supernatant liquid
as the sample solution. Perform the test with exactly 20 /xL
each of the sample solution and aconitum diester alkaloids
standard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the heights of the peaks corresponding to aconitine, H TA and
H SA , jesaconitine,// T j and H sl , hypaconitine, H TH and H sli ,
and mesaconitine, H TM and H SM , respectively, and calculate
the amounts of them by the following formulae: the amounts
of aconitine, jesaconitine, hypaconitine and mesaconitine per
g calculated on the dried basis are not more than 55 /xg, 40
/ug, 55 /ug and 120 //g, respectively, and the total amount of
them is not more than 230 /ug.
Amount (jug) of aconitine (C34H 47 NO n )
= (C sa /W0x(// ta ///sa)x10
Amount (jug) of jesaconitine (C35H49NCV)
= (C S! /W)x(H TS /H S! )x(0
Amount (jug) of hypaconitine (C33H45NOK))
= (C SH /W0X(// TH /// SH )X10
Amount (fig) of mesaconitine (C33H45NO11)
= (C SM /W)X(H TM /H SM )X10
C S a- Concentration (ug/mL) of aconitine for purity in the
aconitum diester alkaloids standard solution for puri-
ty
C SJ : Concentration (/ug/mL) of jesaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
C SH : Concentration (ag/mL) of hypaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
C SM : Concentration (Mg/mL) of mesaconitine for purity in
the aconitum diester alkaloids standard solution for
purity
W: Amount (g) of the sample, calculated on the dried basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 231 nm for aconitine, hypaconitine and
mesaconitine; 254 nm for jesaconitine).
Column: A stainless steel column 4.6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of the phosphate buffer solution
for aconite root and tetrahydrofuran (183:17).
Flow rate: Adjust the flow rate so that the retention time of
mesaconitine is about 31 minutes.
System suitability —
System performance: When the procedure is run with 20
,«L of the aconitum diester alkaloids standard solution for
purity under the above operating conditions, using 254 nm,
mesaconitine, hypaconitine, aconitine and jesaconitine are
eluted in this order, and each resolution between their peaks
is not less than 1.5, respectively.
System repeatability: To 1 mL of aconitum diester
alkaloids standard solution for purity add a mixture of
phosphate buffer solution for aconite root and acetonitrile
(1:1) to make 10 mL. When the test is repeated 6 times with
20 /xL of this solution under the above operating conditions,
using 231 nm, the relative standard deviation of the peak
height of mesaconitine is not more than 1.5%.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <J.07>
Powdered Processed Aconite Root 1: Not more than
4.0%.
Powdered Processed Aconite Root 2: Not more than
7.0%.
Acid-insoluble ash <5.01> Not more than 0.7%.
Assay Weigh accurately about 2 g of Powdered Processed
Aconite Root, put in a glass-stoppered centrifuge tube, and
add 1.6 mL of ammonia TS and 20 mL of diethyl ether. Stop-
per tightly the tube, shake for 30 minutes, centrifuge, and
separate the ether layer. To the residue add 0.8 mL of ammo-
nia TS and 20 mL of diethyl ether, and proceed as above.
Repeat this process more three times. Combine all extracts,
evaporate to dryness under reduced pressure, dissolve the
residue in 5 mL of ethanol (99.5), add 30 mL of freshly
boiled and cooled water, and titrate <2.50> with 0.01 mol/L
hydrochloric acid VS until the color of the solution changes
from green to gray-blue through blue-green (indicator: 3
drops of methyl red-methylene blue TS). Perform a blank de-
termination and make any necessary correction.
Each mL of 0.01 mol/L hydrochloric acid VS
= 6.037 mg of total alkaloid [as benzoylaconine
(C32H 45 NO 10 )]
JPXV
Crude Drugs / Pueraria Root 1341
Processed Ginger
Zingiberis Processum Rhizoma
Processed Ginger is the rhizome of Zingiber
officinale Roscoe (Zingiberaceae), after being passed
through hot water or being steamed.
Description Irregularly compressed and often branched
massive rhizome; branched parts slightly curved ovoid or ob-
long-ovoid, 2-4 cm in length, and 1-2 cm in diameter;
external surface grayish yellow to grayish yellow-brown, with
wrinkles and ring node; fractured surface brown to dark
brown, transparent and horny; under a magnifying glass, a
transverse section reveals cortex and stele distinctly divided;
vascular bundles scattered throughout the surface.
Odor, characteristic; taste, extremely pungent.
Under a microscope <5.01>, a transverse section reveals
cork layer, cortex and stele in this order from the outside;
cortex and stele, divided by a single-layered endodermis,
composed of parenchyma, vascular bundles scattered and
surrounded by fiber bundles; oil cells contain yellow oil-like
substances, scattered in parenchyma; parenchymatous cells
contain solitary crystals of calcium oxalate, and gelatinized
starch.
Identification To 2 g of pulverized Processed Ginger add 5
mL of diethyl ether, shake for 10 minutes, filter, and use the
filtrate as the sample solution (1). To the residue add 5 mL of
methanol, proceed in the same manner as above, and use so
obtained solution as the sample solution (2). Separately,
dissolve 1 mg of [6]-shogaol for thin-layer chromatography
in 2 mL of methanol, and use this solution as the standard so-
lution (1). Separately, dissolve 1 mg of Sucrose in 2 mL of
methanol, and use this solution as the standard solution (2).
Perform the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 10 /xL each of the sample
solution (1) and standard solution (1) on a plate of silica gel
for thin-layer chromatography. Develop the plate with a mix-
ture of ethyl acetate and hexane (1:1) to a distance of about
10 cm, and air-dry the plate. Spray evenly 4-
dimethylaminobenzaldehyde TS on the plate, heat at 105°C
for 5 minutes, and allow to cool: one of the spot among the
several spots from the sample solution (1) has the same color
tone and Ri value with the green spot from the standard
solution (1). Spot 10 juL each of the sample solution (2) and
standard solution (2) on a plate of silica gel for thin-layer
chromatography, develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (8:5:3) to a distance of
about 10 cm, and air-dry the plate. Spray evenly 1,3-
naphthalenediol TS on the plate, and heat at 105 °C for 5
minutes: one of the spot among the several spots from the
sample solution (2) has the same color tone and i?f value with
the red-purple spot from the standard solution (2).
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <5.01> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 8.0%.
Prunella Spike
Prunellae Spica
Prunella Spike is the spike of Prunella vulgaris Linne
var. lilacina Nakai (Labiatae).
Description Spikes in nearly cylindrical and wheat ear-like
shape, 3-6 cm in length, 1-1.5 cm in diameter, externally
grayish brown; spikes composed of a floral axis having
numerous bracts and calyxes; corollas often remaining on the
upper part; a calyx usually enclosing four mericarps; bract,
cordate to eccentric, and exhibiting white hairs on the vein, as
on the calyx; light in texture.
Almost odorless and tasteless.
Purity (1) Stem — The amount of the stems contained in
Prunella Spike does not exceed 5.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than the stems contained in Prunella Spike does not ex-
ceed 1.0%.
Total ash <5.07> Not more than 13.0%.
Acid-insoluble ash <5.01> Not more than 5.0%.
Pueraria Root
Puerariae Radix
Pueraria Root is the root of Pueraria lobata Ohwi
(Leguminosae), from which periderm has been re-
moved.
It contains not less than 2.0% of puerarin (C 2 iH 2 o0 9 :
416.38), calculated on the basis of dried material.
Description Usually cut into small pieces of irregular hexa-
gons of about 0.5 cm cube, or cut into longitudinally plate-
like pieces 20 - 30 cm in length, 5-10 cm in width, and about
1 cm in thickness; externally light grayish yellow to grayish
white; transverse section showing concentric annulate ring or
part of it formed by abnormal growth of cambium. Under a
magnifying glass, phloem light grayish yellow in color; in xy-
lem, numerous vessels appearing as small dots; medullary
rays slightly dented; vertical section showing longitudinal
patterns formed alternately by fibrous xylem and parenchy-
ma; easily breakable lengthwise, and its section extremely fi-
brous.
Odorless; taste, slightly sweet.
Under a microscope <5.0l>, a transverse section reveals
fiber bundles accompanied by crystal cells in phloem; distinct
vessels and xylem fibers in xylem; starch grains numerous in
parenchyma, mainly composed of polygonal simple grains,
rarely 2- to 3-compound grains, 2-18 /xm, mostly 8 - 12 /xm,
in size, with hilum or cleft in the center, and also with striae.
Identification To 2 g of pulverized Pueraria Root add 10
1342 Red Ginseng / Crude Drugs
JP XV
mL of methanol, shake for 3 minutes, filter, and use the
filtrate as the sample solution. Separately, dissolve 1 mg of
Puerarin Reference Standard in 1 mL of methanol, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 2 fXL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, methanol and water (12:2:1) to a distance of about
10 cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): one of the spot among the sever-
al spots from the sample solution has the same color tone and
Ri value with the blue-white fluorescent spot from the stan-
dard solution.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Pueraria Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Pueraria Root according to Method 4, and per-
form the test (not more than 5 ppm).
Loss on drying <5.01> Not less than 13.0% (6 hours).
Total ash <5.01> Not more than 6.0%.
Assay Weigh accurately about 0.3 g of pulverized Pueraria
Root, add 50 mL of diluted methanol (1 in 2), and heat under
a reflex condenser on a water bath for 30 minutes, cool, and
filter. To the residue add 50 mL of diluted methanol (1 in 2),
and perform as the same as above. Combine the filtrates, add
diluted methanol (1 in 2) to make exactly 100 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of Puerarin Reference Standard
(separately determine the water), add diluted methanol (1 in
2) to make exactly 100 mL, and use this solution as the stan-
dard solution. Perform the test with exactly 10 /uL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions, and measure the peak areas of puerarin, A T and A s ,
of each solution.
Amount (mg) of puerarin (C 2 iH 2 o0 9 ) = W s x (A T /A S )
W s : Amount (mg) of Puerarin Reference Standard, calcu-
lated on the anhydrous basis
Operating conditions -
Detector: An ultraviolet absorption photometer (wave-
length: 250 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle
diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L sodium dihydro-
gen phosphate TS and acetonitrile (9:1).
Flow rate: Adjust the flow rate so that the retention time of
puerarin is about 15 minutes.
System suitability -
System performance: When the procedure is run with
10 /xL of the standard solution under the above operating
conditions, the number of theoretical plates and the symmet-
ry coefficient of the peak of puerarin are not less than 3000
and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
puerarin is not more than 1.5%.
Red Ginseng
Ginseng Radix Rubra
Red Ginseng is the root of Panax ginseng C. A. Mey-
er (Panax schinseng Nees) (Araliaceae), after being
steamed.
It contains not less than 0.10% of ginsenoside Rgj
(C 4 2H 7 20 14 : 801.01) and not less than 0.20%, of gin-
senoside Rbi (C54H92O23: 1109.29), calculated on the
basis of dried material.
Description Thin and long cylindrical to fusiform root,
often branching out into 2 to 5 lateral roots from the middle;
5-25 cm in length, main root 0.5 - 3 cm in diameter; exter-
nally light yellow-brown to red-brown, and translucent and
with longitudinal wrinkles; crown somewhat constricted, and
sometimes with short remains of stem; fractured surface flat;
horny and hard in texture.
Odor, characteristic; taste, at first slightly sweet, followed
by a slight bitterness.
Identification (1) To 0.2 g of pulverized Red Ginseng add
2 mL of acetic anhydride, warm on a water bath for 2
minutes, and filter. To 1 mL of the filtrate add gently 0.5 mL
of sulfuric acid to make two layers: a red-brown color de-
velops at the zone of contact.
(2) To 2.0 g of pulverized Red Ginseng add 20 mL of
methanol, boil gently under a reflux condenser on a water
bath for 15 minutes, cool, filter, and use the filtrate as the
sample solution. Separately, dissolve 1 mg of Ginsenoside
Rg t Reference Standard in 1 mL of methanol, and use this
solution as the standard solution. Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with the lower layer of a mix-
ture of chloroform, methanol and water (13:7:2) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plate, and heat at 110°C for 5 minutes: one
spot among the spots from the sample solution and a red-pur-
ple spot from the standard solution show the same color tone
and the same Ri value.
Purity (1) Foreign matter <5.01> — The amount of stems
and other foreign matter contained in Red Ginseng does not
exceed 2.0%.
(2) Heavy metals <1.07> — Proceed with 1.0 g of pulver-
ized Red Ginseng according to Method 4, and perform the
test. Prepare the control solution with 1.5 mL of Standard
Lead Solution (not more than 15 ppm).
(3) Arsenic <1.11> — Prepare the test solution with 1.0 g
of pulverized Red Ginseng according to Method 4, and per-
form the test (not more than 2 ppm).
JPXV
Crude Drugs / Rehmannia Root 1343
(4) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 15.5% (6 hours).
Total ash <5.01> Not more than 4.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 18.0%.
Assay (1) Ginsenoside Rgi — Weigh accurately about 1 g
of pulverized Red Ginseng, put in a glass- stoppered cen-
trifuge tube, add 30 mL of diluted methanol (3 in 5), shake
for 15 minutes, centrifuge, and separate the supernatant liq-
uid. Repeat the procedure with the residue using 15 mL of
diluted methanol (3 in 5), combine the supernatant liquids,
and add diluted methanol (3 in 5) to make exactly 50 mL.
Pipet 10 mL of this solution, add 3 mL of dilute sodium
hydroxide TS, allow to stand for 30 minutes, add 3 mL of 0.1
mol/L hydrochloric acid TS and diluted methanol (3 in 5) to
make exactly 20 mL, and use this solution as the sample solu-
tion. Separately, weigh accurately about 10 mg of Ginseno-
side Rgi Reference Standard (separately determine the water)
dissolve in diluted methanol (3 in 5) to make exactly 100 mL,
and use this solution as the standard solution. Perform the
test with exactly 10 fiL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of ginsenoside Rgi.
Amount (mg) of ginsenoside Rgi (C 4 2H 7 20 14 ) = W s x (A T /A S )
W s : Amount (mg) of Ginsenoside Rgi Reference Standard,
calculated on the anhydrous basis
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
30°C.
Mobile phase: A mixture of water and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rgi is about 25 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rgi Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 fiL of this solution under the above operating condi-
tions, ginsenoside Rgi and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 1.5.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rgi is not more than 1.5%.
(2) Ginsenoside Rbi — Use the sample solution obtained
in (1) as the sample solution. Separately, weigh accurately
about 10 mg of Ginsenoside Rbi Reference Standard
(separately determine the water) dissolve in diluted methanol
(3 in 5) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 10 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
ginsenoside Rbj.
Amount (mg) of ginsenoside Rbi (C54H92O23) = fV s x (^4 T A4 S )
W s : Amount (mg) of Ginsenoside Rb! Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 203 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water and acetonitrile (7:3).
Flow rate: Adjust the flow rate so that the retention time of
ginsenoside Rbi is about 20 minutes.
System suitability —
System performance: Dissolve 1 mg each of Ginsenoside
Rbi Reference Standard and ginsenoside Re in diluted
methanol (3 in 5) to make 10 mL. When the procedure is run
with 10 fiL of this solution under the above operating condi-
tions, ginsenoside Rbi and ginsenoside Re are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
ginsenoside Rbi is not more than 1.5%.
Rehmannia Root
Rehmanniae Radix
Rehmannia Root is the root of Rehmannia glutinosa
Liboschitz var. purpurea Makino or Rehmania
glutinosa Liboschitz (Scrophulariaceae), with or
without the application of steaming.
Description Thin and long, usually, fusiform root, 5-10
cm in length, 0.5 - 1.5 cm in diameter, often broken or mar-
kedly deformed in shape; externally yellow-brown to blackish
brown, with deep, longitudinal wrinkles and constrictions;
soft in texture and mucous; cross section yellow-brown to
blackish brown, and cortex darker than xylem in color; pith
hardly observable.
Odor, characteristic; taste, slightly sweet at first, followed
by a slight bitterness.
Under a microscope <5.01>, a transverse section reveals 7 to
15 layers of cork; cortex composed entirely of parenchyma
cells; outer region of cortex with scattered cells containing
brown secretes; xylem practically filled with parenchyma;
vessels radially lined, mainly reticulate vessels.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Rehmannia Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
1344 Rhubarb / Crude Drugs
JP XV
of pulverized Rehmannia Root according to Method 4, and
perform the test (not more than 5 ppm).
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Rhubarb
Rhei Rhizoma
Rhubarb is usually the rhizome of Rheum palmatum
Linne, Rheum tanguticum Maximowicz, Rheum
officinale Baillon, Rheum coreanum Nakai or their
interspecific hybrids (Polygonaceae).
It contains not less than 0.25% of sennosides A
(C4 2 H3 8 2 o: 862.74), calculated on the basis of dried
material.
Description Ovoid, oblong-ovoid or cylindrical rhizome,
often cut crosswise or longitudinally, 4-10 cm in diameter, 5
- 15 cm in length. In the case of Rhubarb without most part
of cortex, the outer surface is flat and smooth, yellow-brown
to light brown in color, and sometimes exhibiting white, fine
reticulations; thick and hard in texture. In the case of
Rhubarb with cork layer, externally dark brown or reddish
black, and with coarse wrinkles; rough and brittle in texture.
The fractured surface of Rhubarb is not fibrous; transverse
section grayish brown, light grayish brown or brown in color,
having patterns of dark brown tissue complicated with white
and light brown tissues; near the cambium, the patterns often
radiate, and in pith, consist of whirls of tissues radiated from
the center of a small brown circle 1-3 mm in diameter and
arranged in a ring or scattered irregularly.
Odor, characteristic; taste, slightly astringent and bitter;
when chewed, gritty between the teeth, and coloring the sali-
va yellow.
Under a microscope <5.01>, the transverse section reveals
mostly parenchyma cells; small abnormal cambium-rings
scattered here and there in the pith; the cambium-rings
produce phloem inside and xylem outside, accompanied with
2 to 4 rows of medullary rays containing brown-colored sub-
stances, and the rays run radiately from the center of the ring
towards the outside forming whirls of tissues; parenchyma
cells contain starch grains, brown-colored substances or crys-
tal druses of calcium oxalate.
Identification To 2 g of pulverized Rhubarb add 40 mL of a
mixture of tetrahydrofuran and water (7:3), shake for 30
minutes, and centrifuge. Transfer the supernatant liquid to a
separator, add 13 g of sodium chloride, and shake for 30
minutes. Separate the water layer with undissolved sodium
chloride, and adjust the pH to 1.5 by adding 1 mol/L
hydrochloric acid TS. Transfer this solution to another sepa-
rator, add 30 mL of tetrahydrofuran, shake for 10 minutes,
separate the tetrahydrofuran layer, and use this solution as
the sample solution. Separately, dissolve 1 mg of Sennoside
A Reference Standard in 4 mL of a mixture of tetrahydrofu-
ran and water (7:3), and use this solution as the standard so-
lution. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 40,mL each of the
sample solution and standard solution on a plate of silica gel
for thin-layer chromatography at 10 mm along the initial
line. Develop the plate with a mixture of 1-propanol, ethyl
acetate, water and acetic acid (100) (40:40:30:1) to a distance
of about 15 cm, and air-dry the plate. Examine under ultra-
violet light (main wavelength: 365 nm): one of the spots from
the sample solution and a red fluorescent spot from the stan-
dard solution show the same color tone and the same Rf
value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Rhubarb according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Rhubarb according to Method 4, and perform
the test (not more than 5 ppm).
(3) Raponticin — To 0.5 g of pulverized Rhubarb add ex-
actly 10 mL of ethanol (95), heat on a water bath with a reflux
condenser for 10 minutes, and filter. Perform the test as
directed under Thin-layer Chromatography <2.03>, using the
filtrate as the sample solution. Spot 10,mL of the sample solu-
tion on a plate of silica gel for thin-layer chromatography
<2.03>. Develop the plate with a mixture of isopropyl ether, 1-
butanol and methanol (26:7:7) to a distance of about 10 cm,
and air-dry the plate. Examine under ultraviolet light (main
wavelength 365 nm): no spot with blue-purple fluorescence is
observed at an Rf value between 0.3 and 0.6, though a bluish
white fluorescence may appear.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 13.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 30.0%.
Assay Weigh accurately about 0.5 g of pulverized Rhubarb,
add exactly 50 mL of a solution of sodium hydrogen car-
bonate (1 in 1000), shake for 30 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of Sennoside A Reference Standard, (separately
determine the water) dissolve in a solution of sodium hydro-
gen carbonate (1 in 1000) to make exactly 50 mL. Pipet 5 mL
of this solution, add a solution of sodium hydrogen car-
bonate (1 in 1000) to make exactly 20 mL and use this solu-
tion as the standard solution. Perform the test with exactly 10
/xL of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
lowing conditions, and determine the peak areas, A T and A s ,
of sennoside A in each solution.
Amount (mg) of sennoside A (C 4 2H 38 02o)
= W s x(A T /A s )x (1/4)
W s : Amount (mg) of Sennoside A Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 340 nm).
Column: A stainless steel column 4-6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
JPXV
Crude Drugs / Powdered Rhubarb 1345
Mobile phase: A mixture of diluted acetic acid (100) (1 in
80) and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
sennoside A is about 15 minutes.
System suitability —
System performance: Dissolve 1 mg each of Sennoside A
Reference Standard and naringin for thin-layer chro-
matography in a solution of sodium hydrogen carbonate (1 in
1000) to make 10 mL. When the procedure is run with 20 juL
of this solution under the above operating conditions, senno-
side A and naringin are eluted in this order with the resolu-
tion between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sennoside A is not more than 1.5%.
Powdered Rhubarb
Rhei Rhizoma Pulveratum
Powdered Rhubarb is the powder of Rhubarb.
It contains not less than 0.25% of sennoside A
(C 4 2H 3g 02o: 862.74), calculated on the basis of dried
materials.
Description Powdered Rhubarb occurs as a brown powder.
It has a characteristic odor and a slightly astringent and bitter
taste; is gritty between the teeth and colors the saliva yellow
on chewing.
Under a microscope <5.01>, Powdered Rhubarb reveals
starch grains, dark brown substances or druses of calcium ox-
alate, fragments of parenchyma cells containing them, and
reticulate vessels. The starch grains are spherical, simple, or
2- to 4-compound grains. Simple grain, 3-l8^m in di-
ameter, rarely 30 /um; crystal druses of calcium oxalate, 30 -
60 fim in diameter, sometimes exceeding lOO/zm.
Identification To 2 g of Powdered Rhubarb add 40 mL of a
mixture of tetrahydrofuran and water (7:3), shake for 30
minutes, and centrifuge. Transfer the supernatant liquid to a
separator, add 13 g of sodium chloride, and shake for 30
minutes. Separate the water layer with undissolved sodium
chloride, and adjust the pH to 1.5 with 1 mol/L hydrochloric
acid TS. Transfer this solution to another separator, add 30
mL of tetrahydrofuran, shake for 10 minutes, separate the
tetrahydrofuran layer, and use this solution as the sample so-
lution. Separately, dissolve 1 mg of Sennoside A Reference
Standarad in 4 mL of a mixture of tetrahydrofuran and water
(7:3), and use this solution as the standard solution. Perform
the test with these solutions as directed under Thin-layer
Chromatography <2.03>. Spot 40 /xL each of the sample solu-
tion and standard solution on a plate of silica gel for thin-lay-
er chromatography at 10 mm along the initial line. Develop
the plate with a mixture of 1-propanol, ethyl acetate, water
and acetic acid (100) (40:40:30:1) to a distance of about 15
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): one of the spots from the sample
solution and a red fluorescent spot from the standard solu-
tion show the same color tone and the same Rf value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Rhubarb according to Method 3, and perform the
test. Prepare the control solution with 3.0 mL of Standard
Lead Solution (not more than lOppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Rhubarb according to Method 4, and perform
the test (not more than 5 ppm).
(3) Raponticin — To 0.5 g of Powdered Rhubarb add ex-
actly 10 mL of ethanol (95), heat on a water bath under a
reflux condenser for 10 minutes, and filter. Perform the test
as directed under Thin-layer Chromatography <2.03>, using
the filtrate as the sample solution. Spot 10 /xL of the sample
solution on a plate of silica gel for thin-layer chromatography
<2.03>. Develop the plate with a mixture of isopropyl ether,
methanol and 1-butanol (26:7:7) to a distance of about 10
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 365 nm): no spot with blue-purple fluores-
cence is observed at the Ri value of between 0.3 and 0.6,
though a bluish white fluorescence may appear.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.07> Not more than 13.0%.
Acid-insoluble ash <5.0I> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 30.0%.
Assay Weigh accurately about 0.5 g of Powdered Rhubarb,
add exactly 50 mL of a solution of sodium hydrogen car-
bonate (1 in 1000), shake for 30 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of Sennoside A Reference Standard, (separately
determine the water) dissolve in a solution of sodium hydro-
gen carbonate (1 in 1000) to make exactly 50 mL. Pipet 5 mL
of this solution, add a solution of sodium hydrogen car-
bonate (1 in 1000) to make exactly 20 mL, and use this solu-
tion as the standard solution. Perform the test with exactly 10
/uL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of sennoside A in each solution.
Amount (mg) of sennoside A (C^F^C^o)
= W s x(A T /A s )x(l/4)
W s : Amount (mg) of Sennoside A Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 340 nm).
Column: A stainless steel column about 4-6 mm in inside
diameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (100) (1 in
80) and acetonitrile (4:1).
Flow rate: Adjust the flow rate so that the retention time of
sennoside A is about 15 minutes.
System suitability —
System performance: Dissolve 1 mg each of Sennoside A
Reference Standard and naringin for thin-layer chro-
1346 Compound Rhubarb and Senna / Crude Drugs
JP XV
matography in a solution of sodium hydrogen carbonate (1 in
1000) to make 10 mL. When the procedure is run with 20 fiL
of this solution under the above operating conditions, senno-
side A and naringin are eluted in this order with the resolu-
tion between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sennoside A is not more than 1.5%.
Compound Rhubarb and Senna
Powder
W5¥4*<'j--£>1-ik
Method of preparation
Powdered Senna Leaves
Powdered Rhubarb
Sulfur
Magnesium Oxide
110 j
110 j
555j
225 I
To make
1000}
Prepare as directed under Powders, with the above in-
gredients.
Description Compound Rhubarb and Senna Powder occurs
as a yellow-brown powder, having a characteristic odor and a
bitter taste.
Identification To 2 g of Compound Rhubarb and Senna
Powder add 50 mL of water, warm on a water bath for 30
minutes, and filter. Add 2 drops of dilute hydrochloric acid
to the filtrate, shake with two 20-mL portions of diethyl
ether, and remove the diethyl ether layer. Add 5 mL of
hydrochloric acid to the aqueous layer, and heat it on a water
bath for 30 minutes. Cool, shake with 20 mL of diethyl ether,
take the diethyl ether layer, add 10 mL of sodium hydrogen
carbonate TS, and shake: the aqueous layer is red in color.
Containers and storage Containers — Well-closed contain-
ers.
Rice Starch
Amylum Oryzae
Rice Starch consists of the starch granules obtained
from the seeds of Oryza sativa Linne (Gramineae).
Description Rice Starch occurs as white masses or powder.
It is odorless and tasteless.
Under a microscope <5.01>, Rice Starch appears as poly-
hedral, simple grains 3 - 10//m, mostly 4-6//m, in size.
These simple grains often gather in ellipsoidal, compound
grains 50 - 100 //m in diameter. Hilum and striation are not
observable.
It is practically insoluble in water and in ethanol (95).
Identification (1) To 1 g of Rice Starch add 50 mL of
water, boil, and allow to cool: a turbid, neutral and pasty liq-
uid is formed.
(2) To a portion of Rice Starch add iodine TS: a dark
blue-purple color is produced.
Purity Foreign matter — Under a microscope <5.01>, Rice
Starch does not contain starch grains of any other origin. It
may contain a minute quantity, if any, of fragments of the
tissue of the original plant.
Loss on drying <5.01> Not more than 15.0% (6 hours).
Total ash <J.07> Not more than 1.0%.
Rose Fruit
Rosae Fructus
Rose Fruit is the pseudocarp of fruit of Rosa mul-
tiflora Thunberg (Rosaceae).
Description The pseudocarp, spherical, ellipsoidal or
spheroidal, 5 - 9.5 mm in length, 3.5 - 8 mm in diameter; the
external surface red to dark brown in color, smooth and lus-
trous; often with peduncle about 10 mm in length at one end,
and with pentagonal remains of calyx without sepal at the
other end; internal wall of receptacle covered densely with
silvery hairs; the interior containing 5-10 mature nuts; the
nut, irregularly angular ovoid, about 4 mm in length, about 2
mm in diameter; external surface, light yellow-brown; obtuse
at one end, and slightly acute at the other.
Odor, slight; taste of receptacle, sweet and acid, and of
nut, mucilaginous at first, later astringent, bitter and irrita-
tive.
Identification Boil gently 1 g of pulverized Rose Fruit with
20 mL of methanol for 2 minutes, and filter. To 5 mL of the
filtrate add 0.1 g of magnesium in ribbon form and 0.5 mL of
hydrochloric acid, and allow the mixture to stand: a light red
to red color develops.
Purity Foreign matter <5.01> — The amount of the peduncle
and other foreign matter contained in Rose Fruit is not more
than 1.0%.
Total ash <J.07> Not more than 6.0%.
Powdered Rose Fruit
Rosae Fructus Pulveratus
Powdered Rose Fruit is the powder of Rose Fruit.
Description Powdered Rose Fruit occurs as a grayish yel-
low-brown powder. It has a slight odor, and has a slightly
mucilaginous, astringent, bitter, and slightly acid taste.
Under a microscope <5.01>, Powdered Rose Fruit reveals
fragments of extremely thick-walled hairs 35 - 70 fim in di-
ameter, fragments of epidermis and hypodermis containing
brown tannin masses, fragments of thin-walled fundamental
JPXV
Crude Drugs / Ryokeijutsukanto Extract 1347
tissue containing grayish brown substances, fragments of fine
vessels, and solitary or twin crystals or rosette agregates of
calcium oxalate (components of receptacle); fragments of
sclerenchyma, fiber groups, fine vessels, and fragments of
epidermis containing brown tannin and mucilage (compo-
nents of pericarp); fragments of endosperm composed of
polygonal cells containing aleuron grains and fatty oil, frag-
ments of outer epidermis composed of polygonal cells con-
taining tannin, and fragments of inner epidermis composed
of elongated cells having wavy lateral walls (components of
seed).
Identification Boil gently 1 g of Powdered Rose Fruit with
20 mL of methanol for 2 minutes, and filter. To 5 mL of the
filtrate add 0. 1 g of magnesium in ribbon form and 0.5 mL of
hydrochloric acid, and allow the mixture of stand: a light red
to red color develops.
Total ash <J.07> Not more than 6.0%.
Rosin
Colophonium
Resina Pini
Rosin is the resin obtained from the exudation of
plants of Pinus species (Pinaceae) from which essential
oil has been removed.
Description Rosin occurs as a light yellow to light brown,
glassily transparent, brittle mass, the surfaces of which are
often covered with a yellow powder. The fractured surface is
shell-like and lustrous.
It has a slight odor.
It melts easily, and burns with a yellow-brown flame.
It is freely soluble in ethanol (95), in acetic acid (100) and
in diethyl ether.
A solution of Rosin in ethanol (95) is acidic.
Acid value <I.I3> 150-177
Total ash <5.07> Not more than 0.1%.
Ryokeijutsukanto Extract
^gTfW^I + X
Ryokeijutsukanto Extract contains not less than 1
mg and not more than 4 mg of (£)-cinnamic acid, and
not less than 21 mg and not more than 63 mg of glycyr-
rhizic acid (C 42 H 62 16 : 822.93) per a dried extract pre-
pared as directed in the Method of preparation.
Method of preparation Prepare a dried extract as directed
under Extracts, with 6 g of Poria Sclerotium, 4 g of Cinna-
mon Bark, 3 g of Atractylodes Rhizome or Atractylodes Lan-
cea Rhizome and 2 g of Glycyrrhiza.
Description Ryokeijutsukanto Extract occurs as a brown
powder. It has an odor, and a sweet first then bitter taste.
Identification (1) Cinnamon bark — To 1.0 g of
Ryokeijutsukanto Extract add 10 mL of water, shake, then
add 25 mL of diethyl ether, and shake. Take the diethyl ether
layer, evaporate the layer under reduced pressure, add 2 mL
of diethyl ether to the residue, and use this solution as the
sample solution. Separately, dissolve 1 mg of (E)-cinnamic
acid for thin-layer chromatography in 1 mL of methanol,
and use this solution as the standard solution. Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 5 /xL each of the sample solution
and standard solution on a plate of silica gel with fluorescent
indicator for thin-layer chromatography, develop the plate
with a mixture of hexane, ethyl acetate, formic acid and
water (60:40:4:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): one of the spot among the several spots from the
sample solution has the same color tone and Rf value with the
blue-purple spot from the standard solution.
(2) Atractylodes rhizome (for preparation prescribed
Atractylodes Rhizome) — To 1.0 g of Ryokeijutsukanto Ex-
tract add 10 mL of water, shake, then add 25 mL of diethyl
ether, and shake. Take the diethyl ether layer, evaporate the
layer under reduced pressure, add 2 mL of diethyl ether to the
residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of atractylenolide III for thin-layer
chromatography in 2 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /uL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography, de-
velop the plate with a mixture of ethyl acetate and hexane
(1:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly dilute sulfuric acid on the plat, heat at 105°C
for 5 minutes, and examine under ultraviolet light (main
wavelength: 365 nm): one of the spot among the several spots
from the sample solution has the same color tone and Rf
value with the blue-white fluorescent spot from the standard
solution.
(3) Atractylodes lancea rhizome (for preparation
prescribed Atractylodes Lancea Rhizome) — To 2.0 g of
Ryokeijutsukanto Extract add 10 mL of water, shake, then
add 25 mL of hexane, and shake. Take the hexane layer, add
anhydrous sodium sulfate to dry, and filter. Evaporate the
filtrate under reduced pressure, add 2mL of hexane to the
residue, and use this solution as the sample solution. Perform
the test with the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 20 /xL of the sample solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of hexane
and acetone (7:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): a dark purple spot is observed at around Rf 0.4. The
spot shows a greenish brown color after being sprayed 4-
dimethylaminobenzaldehyde TS for spraying, heated at
105 °C for 5 minutes, and allowed to cool.
(4) Glycyrrhiza — To 1.0 g of Ryokeijutsukanto Extract
add 10 mL of water, shake, then add 10 mL of 1-butanol,
centrifuge, and use the supernatant liquid as the sample solu-
tion. Separately, dissolve 1 mg of liquiritin for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
1348 Safflower / Crude Drugs
JP XV
Spot 5 juL each of the sample solution and standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105 °C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the yellow-brown spot from the standard solu-
tion.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1.0 g of Ryokeijutsukanto Extract as directed in (4) in
Extracts under the General Rules for Preparations, and per-
form the test (not more than 30 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.67 g
of Ryokeijutsukanto Extract according to Method 3, and
perform the test (not more than 3 ppm).
Loss on drying <2.41> Not more than 8.5% (1 g, 105 °C, 5
hours).
Total ash <J.07> Not more than 8.0%.
Assay (1) (£)-Cinnamic acid — Conduct this procedure
without exposure to daylight, using light-resistant vessels.
Weigh accurately about 0.5 g of Ryokeijutsukanto Extract,
add exactly 50 mL of diluted methanol (1 in 2), shake for 15
minutes, filter, and use the filtrate as the sample solution.
Separately, weigh accurately about 10 mg of (£)-cinnamic
acid for component determination, previously dried in a
desiccator (silica gel) for more than 24 hours, dissolve in
diluted methanol (1 in 2) to make exactly 100 mL. Pipet 10
mL of this solution, add diluted methanol (1 in 2) to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /xL each of the sample solu-
tion and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of (£)-cinnamic
acid.
Amount (mg) of (£)-cinnamic acid
= W s x(,4 T A4 s )x(l/20)
W s : Amount (mg) of (£)-cinnamic acid for component de-
termination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 273 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of water, acetonitrile and phos-
phoric acid (750:250:1)
Flow rate: 1 .0 mL/min. (the retention time of (£)-cinnamic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of (£)-cinnamic acid are not less than 5000
and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
(£)-cinnamic acid is not more than 1.5%.
(2) Glycyrrhizic acid — Weigh accurately about 0.5 g of
Ryokeijutsukanto Extract, add exactly 50 mL of diluted
methanol (1 in 2), shake for 15 minutes, filter, and use the
filtrate as the sample solution. Separately, weigh accurately
about 10 mg of Glycyrrhizic Acid Reference Standard
(separately determine the water), dissolve in diluted methanol
(1 in 2) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /xL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and ^4 S , of
glycyrrhizic acid.
Amount (mg) of glycyrrhizic acid (C 42 H 62 Oi 6 )
= W s x(A T /A s )x(l/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7).
Flow rate: 1.0 mL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
Safflower
Carthami Flos
Safflower is the tubulous flower of Carthamus tin-
ctorius Linne (Compositae) without any treatment or
with most of the yellow pigment removed, and pressed
into a fiat slab.
Description Red to red-brown corolla, yellow style and sta-
men, rarely mixed with immature ovary; total length about 1
cm; corolla, tubular and with 5 lobes; 5 stamens surrounding
long pistil; pollen grains yellow and approximately spherical,
about 50 /xm in diameter, with fine protrusions on the sur-
JPXV
Crude Drugs / Saireito Extract 1349
face. The pressed slab, about 0.5 cm in thickness, consists of
a collection of numerous corollas.
Odor, characteristic; taste, slightly bitter.
Identification Boil 0.2 g of Safflower with 10 mL of dilute
ethanol under a reflux condenser for 15 minutes, and after
cooling, filter. Place 3 mL of the filtrate in a small glass vessel
about 3 cm in both internal diameter and height, hang a piece
of filter paper, 20 mm by 300 mm, so that one end of the filter
paper reaches the bottom of the vessel, and allow the paper to
soak up the liquid for 1 hour. Transfer and immediately hang
the paper in another glass vessel of the same type, containing
3 mL of water, and allow the paper to soak up the water for 1
hour: most of the upper part of the paper is colored light yel-
low, and the lower portion, light red.
Purity Foreign matter <5.0I> — The amount of ovaries,
stems, leaves and other foreign matter contained in Safflower
does not exceed 2.0%.
Total ash <5.01> Not more than 18.0%.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Saffron
Crocus
Saffron is the stigma of Crocus sativus Linne
(Iridaceae).
Description Thin cord-like stigma, externally dark yellow-
red to red-brown, 1.5 -3.5 cm in length, tripartite or
separate; the end of partite part widened and the other end
narrowed gradually.
Odor, strong and characteristic; taste, bitter; colors the
saliva yellow on chewing.
Under a microscope <5.01>, when softened by immersion in
water, the upper end has numerous tubular protrusions about
150 /im in length, with a small number of pollen grains.
Identification Add 1 drop of sulfuric acid to Saffron: the
color changes to dark blue which gradually turns red-brown
through purple.
Purity (1) Aniline dyes — Shake 0.05 g of Saffron with 10
mL of chloroform: the solution is colorless, or only slightly
yellow.
(2) Glycerol, sugar or honey — Saffron has no sweet taste.
Press it between two pieces of paper: no spot is left on the
paper.
(3) Yellow style — The yellow style in Saffron does not ex-
ceed 10.0%.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 7.5%.
Content of the active principle Crocin — Dry Saffron in a
desiccator (silica gel) for 24 hours, and powder. To exactly
0.100 g of the powder add 150 mL of warm water, warm the
mixture between 60°C and 70°C for 30 minutes with frequent
shaking, cool, and filter. Pipet 1 mL of the filtrate, add water
to make exactly 10 mL, and use this solution as the sample
solution. Separately, dissolve exactly 98 mg of car-
bazochrome sodium sulfonate for content of the active prin-
ciple in water to make exactly 100 mL. Pipet 5 mL of this so-
lution, add water to make exactly 100 mL, and use this solu-
tion as the standard solution. Determine the absorbances of
the sample solution and standard solution at 438 nm as
directed under Ultraviolet-visible Spectrophotometry <2.24>:
the absorbance of the sample solution is larger than that of
the standard solution.
Containers and storage Containers — Well-closed contain-
ers.
Storage — Light-resistant.
Saireito Extract
Saireito Extract contains not less than 2 mg and not
more than 8 mg of saikosaponin b 2 , not less than 80 mg
and not more than 240 mg of baicalin (C 21 H 18 O n :
446.37), and not less than 17 mg and not more than 51
mg of glycyrrhizic acid (C 42 H 62 16 : 822.93) per a dried
extract prepared as directed in the Method of prepara-
tion.
Method of preparation Prepare a dried extract as directed
under Extracts, with 7 g of Bupleurum Root, 5 g of Pinellia
Tuber, 1 g of Ginger, 3 g of Scutellaria Root, 3 g of Jujube, 3
g of Ginseng, 2 g of Glycyrrhiza, 6 g of Alisma Rhizome, 4.5
g of Polyporus Sclerotium, 4.5 g of Poria Sclerotium, 4.5 g
of Atractylodes Rhizome and 3 g of Cinnamon Bark, or with
7 g of Bupleurum Root, 5 g of Pinellia Tuber, 1 g of Ginger,
3 g of Scutellaria Root, 3 g of Jujube, 3 g of Ginseng, 2 g of
Glycyrrhiza, 5 g of Alisma Rhizome, 3 g of Polyporus Sclero-
tium, 3 g of Poria Sclerotium, 3 g of Atractylodes Lancea
Rhizome and 2 g of Cinnamon Bark.
Description Saireito Extract occurs as a light yellow-brown
powder. It has slightly a characteristic odor, and a sweet,
then bitter taste.
Identification (1) Bupleurum root — To 2.0 g of Saireito
Extract add 10 mL of sodium hydroxide TS, shake, then add
5 mL of 1-butanol, shake, centrifuge, and use the super-
natant liquid as the sample solution. Separately, dissolve 1
mg of saikosaponin b 2 for thin-layer chromatography in 1
mL of methanol, and use this solution as the standard solu-
tion. Perform the test with these solutions as directed under
Thin-layer Chromatography <2.03>. Spot 10 /uL of the sample
solution and 2 /uL of the standard solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of ethyl acetate, ethanol (99.5) and water (8:2:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
4-dimethylaminobenzaldehyde TS on the plate: one of the
spot among the several spots from the sample solution has
the same color tone and Rf value with the red spot from the
standard solution.
(2) Ginger — To 1.0 g of Saireito Extract add 10 mL of
water, shake, then add 25 mL of diethyl ether, and shake.
Take the diethyl ether layer, evaporate the diethyl ether layer
1350 Saireito Extract / Crude Drugs
JP XV
under reduced pressure, add 2 mL of diethyl ether to the
residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of [6]-gingerol for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 15
fiL of the sample solution and 5 fiL of the standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate and hexane
(1:1) to a distance of about 10 cm, and air-dry the plate.
Spray evenly 4-dimethylaminobenzaldehyde TS for spraying
on the plate, heat at 105 °C for 5 minutes, and allow to cool:
one of the spot among the several spots from the sample solu-
tion has the same color tone and Ri value with the blue-green
spot from the standard solution.
(3) Scutellaria root — To 1.0 g of Saireito Extract add 10
mL of water, shake, then add 25 mL of diethyl ether, and
shake. Take the diethyl ether layer, evaporate the diethyl
ether layer under reduced pressure, add 2 mL of diethyl ether
to the residue, and use this solution as the sample solution.
Separately, dissolve 1 mg of wogonin for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 20
fiL of the sample solution and 2 fiL of the standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, hexane and
acetic acid (100) (10:10:1) to a distance of about 10 cm, air-
dry the plate, and spray evenly iron (III) chloride-methanol
TS on the plate: one of the spot among the several spots from
the sample solution has the same color tone and Ri value with
the yellow-brown spot from the standard solution.
(4) Ginseng — To 2.0 g of Saireito Extract add 10 mL of
sodium hydroxide TS, shake, then add 5 mL of 1-butanol,
shake, centrifuge, and use the supernatant liquid as the sam-
ple solution. Separately, dissolve 1 mg of Ginsenoside Rbj
Reference Standard in 1 mL of methanol, and use this solu-
tion as the standard solution. Perform the test with these so-
lutions as directed under Thin-layer Chromatography <2.03>.
Spot 10 fiL of the sample solution and 2^L of the standard
solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of ethyl
acetate, 1-propanol, water and acetic acid (100) (7:5:4:1) to a
distance of about 10 cm, and air-dry the plate. Spray evenly
vanillin-sulfuric acid TS on the plate, heat at 105 °C for 5
minutes, and allow to cool: one of the spot among the several
spots from the sample solution has the same color tone and
Rf value with the purple spot from the standard solution.
(5) Glycyrrhiza — To 2.0 g of Saireito Extract add 10 mL
of water, shake, then add 5 mL of 1-butanol, shake, cen-
trifuge, and use the supernatant liquid as the sample solution.
Separately, dissolve 1 mg of liquiritin for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 10
fiL of the sample solution and 2 fiL of the standard solution
on a plate of silica gel for thin-layer chromatography. De-
velop the plate with a mixture of ethyl acetate, methanol and
water (20:3:2) to a distance of about 10 cm, and air-dry the
plate. Spray evenly dilute sulfuric acid on the plate, and heat
at 105 °C for 5 minutes: one of the spot among the several
spots from the sample solution has the same color tone and
Ri value with the yellow-brown spot from the standard solu-
tion.
(6) Alisma rhizome — To 2.0 g of Saireito Extract add 10
mL of sodium carbonate TS, shake, then add 10 mL of
diethyl ether, shake, centrifuge, and use the supernatant liq-
uid as the sample solution. Separately, dissolve 1 mg of alisol
A for thin-layer chromatography in 1 mL of methanol, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 40 fiL of the sample solution and 2
fiL of the standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of
ethyl acetate, hexane and acetic acid (100) (10:10:3) to a dis-
tance of about 10 cm, and air-dry the plate. Spray evenly
vanillin-sulfuric acid TS on the plate, heat at 105 °C for 5
minutes, and allow to cool: one of the spot among the several
spots from the sample solution has the same color tone and
Ri value with the purple spot from the standard solution.
(7) Atractylodes rhizome (for preparation prescribed
Atractylodes Rhizome) — To 1.0 g of Saireito Extract add 10
mL of water, shake, then add 25 mL of diethyl ether, and
shake. Take the diethyl ether layer, evaporate the layer under
reduced pressure, add 2 mL of diethyl ether to the residue,
and use this solution as the sample solution. Separately, dis-
solve 1 mg of atractylenolide III for thin-layer chro-
matography in 2 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
each of the sample solution and standard solution on a plate
of silica gel for thin-layer chromatography, develop the plate
with a mixture of ethyl acetate and hexane (1:1) to a distance
of about 10 cm, and air-dry the plate. Spray evenly dilute sul-
furic acid on the plat, heat at 105°C for 5 minutes, examine
under ultraviolet light (main wavelength: 365 nm): one of the
spot among the several spots from the sample solution has
the same color tone and Ri value with the blue-white fluores-
cent spot from the standard solution.
(8) Atractylodes lancea rhizome (for preparation
prescribed Atractylodes Lancea Rhizome) — To 2.0 g of
Saireito Extract add 10 mL of water, shake, then add 25 mL
of hexane, and shake. Take the hexane layer, add anhydrous
sodium sulfate to dry, and filter. Evaporate the filtrate under
reduced pressure, add 2 mL of hexane to the residue, and use
this solution as the sample solution. Perform the test with the
sample solution as directed under Thin-layer Chro-
matography <2.03>. Spot 20 fiL of the sample solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of hexane
and acetone (7:1) to a distance of about 10 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
254 nm): a dark purple spot is observed at around Ri 0.4. The
spot shows a greenish brown color after being sprayed 4-
dimethylaminobenzaldehyde TS for spraying, heated at
105 °C for 5 minutes, and allowed to cool.
(9) Cinnamon bark — To 1.0 g of Saireito Extract add 10
mL of water, shake, then add 25 mL of diethyl ether, and
shake. Take the diethyl ether layer, evaporate the layer under
reduced pressure, add 2 mL of diethyl ether to the residue,
and use this solution as the sample solution. Separately, dis-
solve 1 mg of (£)-cinnamic acid for thin-layer chro-
matography in 1 mL of methanol, and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 40
fiL of the sample solution and 2 fiL of the standard solution
JPXV
Crude Drugs / Saireito Extract 1351
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography, develop the plate with a mixture of hexane,
ethyl acetate, formic acid and water (60:40:4:1) to a distance
of about 10 cm, and air-dry the plate. Examine under ultrav-
iolet light (main wavelength: 254 nm): one of the spot among
the several spots from the sample solution has the same color
tone and Ri value with the dark purple spot from the stan-
dard solution.
Purity (1) Heavy metals <1.07> — Prepare the test solution
with 1 .0 g of Saireito Extract as directed in (4) in Extracts un-
der the General Rules for Preparations, and perform the test
(not more than 30 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.67 g
of Saireito Extract according to Method 3, and perform the
test (not more than 3 ppm).
Loss on drying <2.41> Not more than 10.0% (1 g, 105°C, 5
hours).
Total ash <5.01> Not more than 9.0%.
Assay (1) Saikosaponin b 2 — Weigh accurately about 0.5 g
of Saireito Extract, add exactly 50 mL of diluted methanol (1
in 2), shake for 15 minutes, filter, and use the filtrate as the
sample solution. Separately, weigh accurately about 10 mg of
saikosaponin b 2 for component determination, previously
dried in a desiccator (silica gel) for more than 24 hours, dis-
solve in diluted methanol (1 in 2) to make exactly 100 mL.
Pipet 10 mL of this solution, add diluted methanol (1 in 2) to
make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 10 liL each of the sam-
ple solution and standard solution as directed under Liquid
Chromatography <2.01> according to the following condi-
tions, and determine the peak areas, A T and A s , of sai-
kosaponin b 2 .
Amount (mg) of saikosaponin b 2 = W s x (A T /A S ) x (1/20)
W s : Amount (mg) of saikosaponin b 2 for component de-
termination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 0.05 mol/L sodium dihydro-
gen phosphate TS and acetonitrile (5:3)
Flow rate: l.OmL/min. (the retention time of saikosapo-
nin b 2 is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of saikosaponin b 2 are not less than 5000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 liL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
saikosaponin b 2 is not more than 1.5%.
(2) Baicalin — Weigh accurately about 0.1 g of Saireito
Extract, add exactly 50 mL of diluted methanol (7 in 10),
shake for 15 minutes, filter, and use the filtrate as the sample
solution. Separately, weigh accurately about 10 mg of Baica-
lin Reference Standard (separately determine the water), dis-
solve in diluted methanol (7 in 10) to make exactly 200 mL,
and use this solution as the standard solution. Perform test
with exactly 10 liL each of the sample solution and the stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions, and determine
the peak areas, A T and A s , of baicalin.
Amount (mg) of baicalin (C 2 iH I8 O u )
= W s x(A T /A s )x(l/4)
W s : Amount (mg) of Baicalin Reference Standard, calcu-
lated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 277 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
200) and acetonitrile (19:6)
Flow rate: l.OmL/min. (the retention time of baicalin is
about 10 minutes.)
System suitability —
System performance: When the procedure is run with 10
liL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of baicalin are not less than 5000 and not
more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
baicalin is not more than 1.5%.
(3) Glycyrrhizic acid — Weigh accurately about 0.5 g of
Saireito Extract, add exactly 50 mL of diluted methanol (1 in
2), shake for 15 minutes, filter, and use the filtrate as the sam-
ple solution. Separately, weigh accurately about 10 mg of
Glycyrrhizic Acid Reference Standard (separately determine
the water), dissolve in diluted methanol (1 in 2) to make ex-
actly 100 mL, and use this solution as the standard solution.
Perform the test with exactly 10 /uL each of the sample solu-
tion and the standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, A T and A s , of glycyrrhizic
acid.
Amount (mg) of glycyrrhizic acid (C 42 H 62 16 )
= W s x(A T /A s )x(l/2)
W s : Amount (mg) of Glycyrrhizic Acid Reference Stan-
dard, calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 254 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 iim in particle di-
ameter).
1352 Saposhnikovia Root / Crude Drugs
JP XV
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted acetic acid (31) (1 in 15)
and acetonitrile (13:7).
Flow rate: 1.0 mL/min. (the retention time of glycyrrhizic
acid is about 12 minutes.)
System suitability —
System performance: When the procedure is run with 10
/uL of the standard solution under the above operating condi-
tions, the number of theoretical plates and the symmetry fac-
tor of the peak of glycyrrhizic acid are not less than 5000 and
not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
glycyrrhizic acid is not more than 1.5%.
Containers and storage Containers — Tight containers.
Saposhnikovia Root
Saposhnikoviae Radix
**777
Saposhnikovia Root is the root and rhizome of
Saposhnikovia divaricata Schischkin (Umbelliferae).
Description Long and narrow, conical rhizome and root, 15
- 20 cm in length, 0.7 - 1.5 cm in diameter; externally light
brown; rhizome reveals dense crosswise wrinkles like ring
nodes, and sometimes reveals brown and hair -like remains of
leaf sheath; the root reveals many longitudinal wrinkles and
scars of rootlets; in a cross section, cortex is grayish brown in
color and reveals many lacunae, and xylem is yellow in color.
Odor, slight; taste, slightly sweet.
Purity Foreign matter <5.01> — The amount of stems and
other foreign matter contained in Saposhnikovia Root is not
more than 2.0%.
Total ash <5.01> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.01>
less than 20.0%.
Dilute ethanol-soluble extract: not
Sappan Wood
Sappan Lignum
Sappan Wood is the duramen of Caesalpinia sappan
Linne (Leguminosae).
Description Chips, slices or short pieces of wood; yellowish
red to grayish yellow-brown, sometimes with light brown to
grayish white splint woods; hard in texture; a transverse sec-
tion shows a pattern like annual ring.
Almost odorless; almost tasteless.
Under a microscope <J.07>, a transverse section reveals ray
composed of 1 - 2 rows of slender and long cells; the area
between rays filled with fiber cells, and large and oblong
vessels scattered there; solitary crystals of calcium oxalate in
parenchymatous cells of the innermost of xylem.
Identification To 0.5 g of pulverized Sappan Wood add 10
mL of dilute ethanol, shake, and filter. To 5 mL of the filtrate
add 2 to 3 drops of sodium hydroxide TS: a dark red color
develops.
Purity Put a small piece of Sappan Wood in calcium
hydroxide TS: no purple-blue color develops.
Loss on drying <5.01> Not more than 11.5% (6 hours).
Total ash <J.07> Not more than 2.0%.
Extract content <5.01>
less than 7.0%.
Dilute ethanol-soluble extract: not
Saussurea Root
Saussureae Radix
=E ••/ =1 7
Saussurea Root is the root of Saussurea lappa Clarke
(Compositae).
Description Nearly cylindrical roots, 5-20 cm in length, 1
- 6 cm in diameter; some of them slightly bent, and some-
times longitudinally cut; scar of stem dented on the top of the
root with crown; externally yellow-brown to grayish brown,
with coarse longitudinal wrinkles and fine reticulate furrows,
and also with remains of lateral roots; sometimes root from
which periderm has been removed; hard and dense in texture,
and difficult to break. A cross section is yellow-brown to dark
brown, and cambium part has a dark color. Under a mag-
nifying glass, medullary rays distinct, here and there, large
clefts, and brown oil sacs scattered; in old root, pith existing
in the center, and often forming a hollow.
Odor, characteristic; taste, bitter.
Identification Warm 0.5 g of pulverized Saussurea Root
with 10 mL of ethanol (95) for 1 minute, cool, and filter.
Shake 1 mL of the filtrate with 0.5 mL of hydrochloric acid: a
purple color is produced.
Purity Foreign matter — Add iodine TS dropwise to a trans-
verse section: no blue-purple color develops.
Total ash <5.07> Not more than 4.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 17.0%.
Schisandra Fruit
Schisandrae Fructus
Schisandra Fruit is the fruit of Schisandra chinensis
Baillon (Schisandraceae).
Description Sap fruit of irregular sphere or spheroid, about
JPXV
Crude Drugs / Scopolia Extract 1353
6 mm in diameter; externally dark red to blackish brown in
color, with wrinkles, and occasionally with white powder;
seeds, kidney-shaped, externally yellow-brown to dark red-
brown, lustrous, with distinct raphe on the dorsal side; exter-
nal seed coat easily peeled but internal seed coat adhering
closely to the albumen.
Odor, slight; taste, acid, later astringent and bitter.
Identification To 1.0 g of pulverized Schisandra Fruit add
10 mL of methanol, warm on a water bath for 3 minutes with
shaking, cool, filter, and use the filtrate as the sample solu-
tion. Separately, dissolve 1 mg of schisandrin for thin-layer
chromatography in 1 mL of methanol, and use this solution
as the standard solution. Perform the test with these solu-
tions as directed under Thin-layer Chromatography <2.03>.
Spot 5 /xL each of the sample solution and standard solution
on a plate of silica gel with fluorescent indicator for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, hexane and acetic acid (100) (10:10:1) to a distance of
about 10 cm, and air-dry the plate. Examine under ultraviolet
light (main wavelength: 254 nm): one of the spots from the
sample solution and a blue-violet spot from the standard so-
lution show the same color tone and Rf value.
Purity Foreign matter <5.01> — The amount of receptacle,
peduncle and other foreign matter contained in Schisandra
Fruit is not more than 1.0%.
Total ash <J.07> Not more than 5.0%.
Schizonepeta Spike
Schizonepetae Spica
Schizonepeta Spike is the spike of Schizonepeta
tenuifolia Briquet (Labiatae).
Description Oblong spike, 5-10 cm in length, 0.5 - 0.8 cm
in diameter, purplish green-brown to green-brown in color.
Spike, 5-10 cm in length, with calyx-tubes containing small
labiate flower or often fruits; sometimes leaves under spike;
leaf, linear or small lanceolate; stem, prismatic, purple-
brown in color. Under a magnifying glass, it reveals short
hairs.
It has a characteristic aroma and slightly cool feeling on
keeping in the mouth.
Identification To 2 g of pulverized Schizonepeta Spike add
20 mL of water, shake well, and distill. To 3 mL of the distil-
late add 2 or 3 drops of 2,4-dinitrophenylhydrazine-ethanol
TS: an orange-red precipitate is formed.
Total ash <5.05> Not more than 11.0%.
Acid-insoluble ash <5.05> Not more than 3.0%.
Extract content <5.05> Dilute ethanol-soluble extract: not
less than 8.0%.
Scopolia Extract
P- r-I + X
Scopolia Extract contains not less than 0.90% and
not more than 1.09% of total alkaloids [hyoscyamine
(C 17 H 2 3N0 3 : 289.37) and scopolamine (C 17 H 21 N0 4 :
303.35)].
Method of preparation Extract the coarse powder of
Scopolia Rhizome with 35 vol% ethanol, Water, or Purified
Water, and prepare the viscous extract as directed under Ex-
tracts.
Description Scopolia Extract is brown to dark brown in
color. It has a characteristic odor, and a bitter taste.
It dissolves in water with a slight turbidity.
Identification (1) Dissolve 4 g of Scopolia Extract in 10
mL of water, add 8 mL of ammonia TS and 80 mL of diethyl
ether, stopper tightly, shake for 1 hour, add 2.5 g of pow-
dered tragacanth, shake vigorously, allow to stand for 5
minutes, and separate the diethyl ether layer into a porcelain
dish. Evaporate the diethyl ether on a water bath, add 5
drops of fuming nitric acid, and evaporate on a water bath to
dryness. After cooling, dissolve the residue in 1 mL of N,N-
dimethylformamide, and add 5 to 6 drops of tetraethylam-
monium hydroxide TS: a red-purple to purple color develops.
(2) Mix 0.5 g of Scopolia Extract with 30 mL of ammonia
TS in a flask, and transfer the mixture to a separator. Add 40
mL of ethyl acetate to the separator, and shake the mixture.
After drain off the ethyl acetate layer, add 3 g of anhydrous
sodium sulfate to the ethyl acetate, shake, and filter after the
ethyl acetate becomes clear. Evaporate the filtrate to dryness
under reduced pressure, dissolve the residue in 1 mL of
ethanol (95), and use this solution as the sample solution.
Proceed as directed in Identification (2) under Scopolia Rhi-
zome.
Assay Weigh accurately about 0.4 g of Scopolia Extract,
place in a glass-stoppered, centrifuge tube, add 15 mL of am-
monia TS, and shake. Add 25 mL of diethyl ether, stopper
tightly, shake for 15 minutes, centrifuge, and separate the
diethyl ether layer. Repeat this procedure twice with the
water layer, using 25 mL each of diethyl ether. Combine the
extracts, and evaporate the diethyl ether on a water bath. Dis-
solve the residue in 5 mL of the mobile phase, add exactly 3
mL of the internal standard solution, and add the mobile
phase to make 25 mL. Proceed as directed under Scopolia
Rhizome.
Amount (mg) of hyoscyamine (Q7H23NO3)
= W SA x (Q TA /Q SA ) x (1/5) x 0.8551
Amount (mg) of scopolamine (C 17 H 2 iN0 4 )
= ^ss x (Gts/Sss) x (1/25) x 0.7894
W SA : Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
W ss : amount (mg) of Scopolamine Hydrobromide Refer-
ence Standard, calculated on the dried basis
Internal standard solution — A solution of brucine dihydrate
1354 Scopolia Extract Powder / Crude Drugs
JP XV
in the mobile phase (1 in 2500).
Containers and storage Containers — Tight containers.
Storage — Light-resistant, and in a cold place.
Scopolia Extract Powder
Scopolia Extract Powder contains not less than
0.085% and not more than 0.110% of total alkaloids
[hyoscyamine (C17H23NO3: 289.37) and scopolamine
(C 17 H 21 N0 4 : 303.35)].
Method of preparation
Scopolia Extract
Starch, Lactose Hydrate or
their mixture
100 g
a sufficient quantity
To make
1000 g
To Scopolia Extract add 100 mL of Purified Water, then
warm and soften the mixture with stirring. Cool, add 800 g of
starch, Lactose Hydrate or their mixture little by little, and
mix well. Dry preferably at a low temperature, and dilute
with a sufficient additional quantity of starch, Lactose Hy-
drate or their mixture to make 1000 g of homogeneous pow-
der.
Description Scopolia Extract Powder is a brownish yellow
to grayish yellow-brown powder. It has a faint, characteristic
odor and a slightly bitter taste.
Identification (1) To 20 g of Scopolia Extract Powder add
15 mL of water and 8 mL of ammonia TS, mix homogene-
ously, add 100 mL of diethyl ether and 7 g of sodium chlo-
ride, stopper tightly, shake for 1 hour, add 5 g of Powdered
Tragacanth, and shake vigorously. Allow to stand for 5
minutes, take the clearly separated diethyl ether layer, and
filter. Proceed with the filtrate as directed in the Identification
(1) under Scopolia Extract.
(2) Place 5.0 g of Scopolia Extract Powder in a glass-
stoppered centrifuge tube, add 30 mL of ammonia TS, and
centrifuge after irradiation of ultrasonic waves for 5 minutes.
Transfer the supernatant liquid to a separator, add 40 mL of
ethyl acetate, and shake. Drain off the ethyl acetate layer, add
3 g of anhydrous sodium sulfate to the ethyl acetate, shake,
and filter after the ethyl acetate becomes clear. Evaporate the
filtrate to dryness under reduced pressure, dissolve the
residue in 1 mL of ethanol (95), and use this solution as the
sample solution. Proceed as directed in the Identification (2)
under Scopolia Rhizome.
Assay Weigh accurately about 4 g of Scopolia Extract Pow-
der, place in a glass-stoppered, centrifuge tube, add 15 mL of
ammonia TS, and shake. Add 25 mL of diethyl ether, stop-
per tightly, shake for 15 minutes, centrifuge to take the
diethyl ether layer. Repeat this procedure three times with the
water layer, using 25-mL portions of diethyl ether. Combine
the extracts, and evaporate the diethyl ether on a water bath.
Dissolve the residue in 5 mL of the mobile phase, add exactly
3 mL of the internal standard solution, and add the mobile
phase to make exactly 25 mL. Filter this solution through a
membrane filter with a pore size not exceeding 0.8 ^m, dis-
card the first 2 mL of the filtrate, and use the subsequent
filtrate as the sample solution. Separately, weigh accurately
about 25 mg of Atropine Sulfate Reference Standard
(separately determine the loss on drying <2.41> in the same
manner as Atropine Sulfate Hydrate), dissolve in the mobile
phase to make exactly 25 mL, and use this solution as stan-
dard stock solution A. Weigh accurately about 25 mg of
Scopolamine Hydrobromide Reference Standard (separately
determine the loss on drying <2.41> in the same manner as
Scopolamine Hydrobromide Hydrate), dissolve in the mobile
phase to make exactly 25 mL, and use this solution as stan-
dard stock solution B. Pipet 5 mL of the standard stock solu-
tion A and 1 mL of the standard stock solution B, add exactly
3 mL of the internal standard solution, then add the mobile
phase to make exactly 25 mL, and use this solution as the
standard solution. Perform the test with 10 /xL each of the
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con-
ditions. Determine the ratios, Qta and Q SA , of the peak area
of hyoscyamine (atropine), and ratios, Q TS and g ss , of the
peak area of scopolamine to that of the internal standard in
each solution, calculate the amounts of hyoscyamine and
scopolamine by the following equation, and designate the
total as the amount of total alkaloids.
Amount (mg) of hyoscyamine (C I7 H23N0 3 )
= W SA x (Q TA /Q SA ) x (1/5) x 0.8551
Amount (mg) of scopolamine (C 17 H 21 N0 4 )
= ^ss x (Gts/Qss) x (1/25) x 0.7894
W S a- Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
Wss' Amount (mg) of Scopolamine Hydrobromide Refer-
ence Standard, calculated on the dried basis
Internal standard solution — A solution of brucine dihydrate
in the mobile phase (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption spectrometer
(wavelength: 210 nm).
Column: A stainless steel column about 4 mm in inside di-
ameter and about 15 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 /urn in parti-
cle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: A mixture of a solution obtained by dissolv-
ing 6.8 g of potassium dihydrogenphosphate in 900 mL of
water, adding 10 mL of triethylamine, adjusting the pH to
3.5 with phosphoric acid, and adding water to make 1000
mL, and acetonitrile (9:1).
Flow rate: Adjust the flow rate so that the retention time of
scopolamine is about 8 minutes.
Selection of column: Proceed with 10 /uL of the standard
solution under the above operating conditions, and deter-
mine the resolution. Use a column giving elution of scopola-
mine, atropine and the internal standard in this order with
the resolution between the peaks of scopolamine and atro-
pine being not less than 11, and the resolution between the
peaks of atropine and the internal standard being not less
than 4.
Containers and storage Containers — Tight containers.
JPXV
Crude Drugs / Scopolia Extract and Ethyl 1355
Scopolia Extract and Carbon
Powder
Method of preparation
contains not less than 22.5% and not more than 27.5%
of ethyl aminobenzoate (C 9 H n N0 2 : 165.19).
Scopolia Extract
5g
Medicinal Carbon
550 g
Natural Aluminum Silicate
345 g
Starch, Lactose Hydrate or
their mixture
a sufficient quantity
To make 1000 g
Prepare before use as directed under Powders, with the
above ingredients. May be prepared with an equivalent
amount of Scopolia Extract Powder in place of Scopolia Ex-
tract.
Description Scopolia Extract and Carbon Powder is easily
dustable and black in color. It is tasteless.
Containers and storage Containers — Well-closed contain-
ers.
Compound Scopolia Extract and
Diastase Powder
W5U- HtX-/7^- £»
Method of preparation
Scopolia Extract
Diastase
Precipitate Calcium Carbonate
Sodium Bicarbonate
Magnesium Oxide
Powdered Gentian
Starch, Lactose Hydrate or
their mixture
200 g
300 g
250 g
100 g
50 g
a sufficient quantity
To make 1000 g
Prepare before use as directed under Powders, with the
above ingredients. May be prepared with an equivalent
amount of Scopolia Extract Powder in place of Scopolia Ex-
tract.
Description Compound Scopolia Extract and Diastase
Powder is light yellow in color. It has a bitter taste.
Containers and storage Containers — Well-closed contain-
ers.
Scopolia Extract and Ethyl
Aminobenzoate Powder
Scopolia Extract and Ethyl Aminobenzoate Powder
Method of preparation
Scopolia Extract
Ethyl Aminobenzoate
Magnesium Oxide
Sodium Bicarbonate
Starch, Lactose Hydrate or
their mixture
10 g
250 g
150 g
500 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients. May be prepared with an equivalent amount of
Scopolia Extract Powder in place of Scopolia Extract.
Description Scopolia Extract and Ethyl Aminobenzoate
Powder is slightly brownish white in color. It has a slightly
bitter taste, leaving a sensation of numbness on the tongue.
Identification (1) To 2 g of Scopolia Extract and Ethyl
Aminobenzoate Powder add 20 mL of diethyl ether, shake,
and filter through a glass filter (G4). Wash the residue with
three 10-mL portions of diethyl ether, combine the filtrate
and the washings, evaporate to dryness, and perform the fol-
lowing test with the residue (ethyl aminobenzoate).
(i) Dissolve 0.01 g of the residue in 1 mL of dilute
hydrochloric acid and 4 mL of water: the solution responds
to the Qualitative Tests <1.09> for primary aromatic amines.
(ii) Dissolve 0.1 g of the residue in 5 mL of water with the
aid of dilute hydrochloric acid added dropwise, and add io-
dine TS dropwise: a brown precipitate is produced.
(iii) Warm 0.05 g of the residue with 2 drops of acetic
acid (31) and 5 drops of sulfuric acid: the odor of ethyl
acetate is perceptible.
(2) To the diethyl ether-insoluble residue obtained in (1)
add 30 mL of water, shake gently, and filter: the filtrate
responds to the Qualitative Tests <1.09> for sodium salt and
for bicarbonate.
(3) To the water-insoluble residue obtained in (2) add 10
mL of dilute hydrochloric acid, shake, and filter: the filtrate
responds to the Qualitative Tests <1.09> for magnesium salt.
(4) Place 30 g of Scopolia Extract and Ethyl Aminoben-
zoate Powder in a glass-stoppered conical flask, add 100 mL
of water, shake for 30 minutes, and filter immediately by suc-
tion through a glass filter (G3). Transfer the residue in the
flask to the same glass filter with the filtrate, and filter the
residue by suction while pressing vigorously the residue on
the same glass filter. Place 75 mL of the filtrate in a 300-mL
beaker, and add cautiously 10 mL of diluted sulfuric acid (1
in 3). Add 0.2 mL of bromocresol green TS to this solution,
and add dilute sulfuric acid dropwise while shaking thor-
oughly, until the color of the solution changes from green to
yellow-green. After cooling, place this solution in a separa-
tor, wash with two 25-mL portions of a mixture of hexane
and diethyl ether (1:1) by shaking well, and place the water
layer in another separator. Make slightly alkaline with am-
monia TS, add immediately 30 mL of diethyl ether, and
shake well. Wash the diethyl ether layer with two 10-mL por-
tions of a saturated solution of sodium chloride, separate the
diethyl ether layer, add 3 g of anhydrous sodium sulfate,
shake, and filter through a pledget of cotton. Evaporate the
filtrate to dryness, dissolve the residue in 0.2 mL of ethanol
(95), and use this solution as the sample solution. Separately,
1356 Scopolia Extract, Papaverine / Crude Drugs
JP XV
dissolve 2 mg of Atropine Sulfate Reference Standard and 1
mg of Scopolamine Hydrobromide Reference Standard in 1
mL each of ethanol (95), and use these solutions as standard
solution (1) and standard solution (2). Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 fiL each of the sample solution and standard
solutions on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of acetone,
water and ammonia solution (28) (90:7:3) to a distance of
about 10 cm, and dry the plate at 80°C for 10 minutes. After
cooling, spray evenly Dragendorff's TS for spraying on the
plate: two principal spots from the sample solution show the
same color tone and the same Rf value with each yellow-red
spot from the standard solutions, respectively.
Assay Weigh accurately about 0.3 g of Scopolia Extract
and Ethyl Aminobenzoate Powder, transfer to a Soxhlet ex-
tractor, extract with 100 mL of diethyl ether for 1 hour, and
evaporate the diethyl ether on a water bath. Dissolve the
residue in 25 mL of 1 mol/L hydrochloric acid TS, and add
water to make exactly 100 mL. Pipet 5 mL of this solution,
add water to make exactly 250 mL, and use this solution as
the sample solution. Weigh accurately about 75 mg of Ethyl
Aminobenzoate Reference Standard, previously dried in a
desiccator (silica gel) for 3 hours, dissolve in 25 mL of 1
mol/L hydrochloric acid TS, and add water to make exactly
100 mL. Pipet 5 mL of this solution, add water to make ex-
actly 250 mL, and use this solution as the standard solution.
Pipet 5 mL each of the sample solution and standard solu-
tion, to each add 10 mL of 1 mol/L hydrochloric acid TS,
then add 1 mL of a solution of sodium nitrite (1 in 200), pre-
pared before use, and allow to stand for 5 minutes with oc-
casional shaking. Add 5 mL of ammonium amidosulfate TS,
shake well, and allow to stand for 10 minutes. Add 2 mL of
Af-TV-diethyl-ZV-l-naphthylethylenediamine oxalate-acetone
TS, mix immediately, and add water to make exactly 50 mL.
Allow to stand for 2 hours, determine the absorbances, A T
and A s , of these solutions at 550 nm, as directed under
Ultraviolet-visible Spectrophotometry <2.24> using a blank
prepared in the same manner with 5 mL of water in place of
the sample solution.
Amount (mg) of ethyl aminobenzoate (CgHnNO^)
= W s x(A T /A s )
W s : Amount (mg) of Ethyl Aminobenzoate Reference
Standard
Containers and storage Containers — Well-closed contain-
ers.
Scopolia Extract, Papaverine and
Ethyl Aminobenzoate Powder
Method of preparation
Scopolia Extract
Papaverine Hydrochloride
Ethyl Aminobenzoate
Starch, Lactose Hydrate or
their mixture
15 g
15 g
120 g
a sufficient quantity
hl + X-/\°/\ViJ >T^X9 i >l
Scopolia Extract, Papaverine and Ethyl Aminoben-
zoate Powder contains not less than 10.8% and not
more than 13.2% of ethyl aminobenzoate (C 9 H n N0 2 :
165.19).
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients. May be prepared with an equivalent amount of
Scopolia Extract Powder in place of Scopolia Extract.
Description Scopolia Extract, Papaverine and Ethyl
Aminobenzoate Powder is brownish yellow to grayish yel-
low-brown in color. It has a slightly bitter taste, leaving a sen-
sation of numbness on the tongue.
Identification (1) To 4 g of Scopolia Extract, Papaverine
and Ethyl Aminobenzoate Powder add 20 mL of diethyl
ether, shake, and filter through a glass filter (G4). Wash the
residue with three 10-mL portions of diethyl ether, combine
the filtrate and the washings, evaporate to dryness, and per-
form the following test with the residue (ethyl aminobenzo-
ate):
(i) Dissolve 0.01 g of the residue in 1 mL of dilute
hydrochloric acid and 4 mL of water: the solution respounds
to the Qualitative Tests <1.09> for primary aromatic amines.
(ii) Dissolve 0.1 g of the residue in 5 mL of water with the
aid of dilute hydrochloric acid added dropwise, and add io-
dine TS dropwise: a brown precipitate is produced.
(iii) Warm 0.05 g of the residue with 2 drops of acetic
acid (31) and 5 drops of sulfuric acid: the odor of ethyl
acetate is perceptible.
(2) To the diethyl ether-insoluble residue obtained in (1)
add 20 mL of chloroform, shake well, filter, and further wash
the residue with 10 mL of chloroform. Combine the filtrate
and the washing, transfer this solution to a separator, and
add 10 mL of 0.1 mol/L hydrochloric acid TS. After shak-
ing, separate the chloroform layer, add 2 g of anhydrous so-
dium sulfate, shake, and filter through a pledget of cotton.
Evaporate the filtrate to dryness, dry the residue at 105°C for
3 hours, and perform the following tests (papaverine
hydrochloride):
(i) To 1 mg of the residue add 1 drop of formaldehyde
solution-sulfuric acid TS: a colorless or light yellow-green
color, changing to red-purple, is produced.
(ii) Dissolve 1 mg of the residue in 3 mL of acetic anhy-
dride and 5 drops of sulfuric acid, heat in a water bath for 1
minute, and view under ultraviolet light: the solution shows a
yellow-green fluorescence.
(3) Place 20 g of Scopolia Extract, Paraverine and Ethyl
Aminobenzoate Powder in a glass-stopperd conical flask, add
80 mL of water, shake for 15 minutes, and filter by suction
through a glass filter (G3). Transfer 60 mL of the filtrate to a
separator, add 0.5 mL of 1 mol/L hydrochloric acid TS, and
extract with three 20-mL portions of chloroform by shaking.
Make the aqueous layer slightly alkaline with ammonia TS,
add immediately 30 mL of diethyl ether, and shake well.
Wash the diethyl ether layer with two 10-mL portions of a
saturated solution of sodium chloride, and separate the
diethyl ether layer. Add 3 g of anhydrous sodium sulfate,
shake, and filter through a pledget of cotton. Evaporate the
filtrate to dryness, dissolve the residue in 0.2 mL of ethanol
JPXV
Crude Drugs / Scopolia Rhizome 1357
(95), and use the solution as the sample solution. Dissolve 20
mg of atropine sulfate for thin-layer chromatography, 10 mg
of scopolamine hydrobromide and 20 mg of papaverine
hydrochloride in 10 mL each of ethanol (95), and use these
solutions as standard solutions (1), (2) and (3). Perform the
test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10,mL each of the sample solution
and standard solutions on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of chlo-
roform, methanol, acetone and ammonia solution (28)
(73:15:10:2) to a distance of about 10 cm, and dry the plate at
80°C for 20 minutes. After cooling, spray Dragendorff's TS
for spraying upon the plate evenly: three yellow-red principal
spots obtained from the sample solution and the correspond-
ing spots from standard solutions (1), (2) and (3) show the
same Rf values.
Assay Weigh accurately about 0.6 g of Scopolia Extract,
Papaverine and Ethyl Aminobenzoate Powder, transfer to a
Soxhlet extractor, and extract with 100 mL of diethyl ether
for 1 hour, and evaporate the diethyl ether on a water bath.
Dissolve the residue in 25 mL of 1 mol/L hydrochloric acid
TS, and add water to make exactly 100 mL. Pipet 5 mL of
this solution, add water to make exactly 250 mL, and use this
solution as the sample solution. Separately, weigh accurately
about 75 mg of Ethyl Aminobenzoate Reference Standard,
previously dried in a desiccator (silica gel) for 3 hours, dis-
solve in 25 mL of 1 mol/L hydrochloric acid TS, and add
water to make exactly 100 mL. Pipet 5 mL of this solution,
add water to make exactly 250 mL, and use this solution as
the standard solution. Pipet 5 mL each of the sample solution
and standard solution, add 10 mL of 1 mol/L hydrochloric
acid TS to each, then add 1 mL of a solution of sodium nitrite
(1 in 200) prepared before use, and allow to stand for 5
minutes with occasional shaking. Add 5 mL of ammonium
amidosulfate TS, shake well, and allow to stand for 10
minutes. Add 2 mL of iV-Af-diethyWV'-l-naphthyl-
ethylenediamine oxalate-acetone TS, mix immediately, and
add water to make exactly 50 mL. Allow to stand for 2 hours,
and determine the absorbances, A T and A s , of these solu-
tions at 550 nm as directed under Ultraviolet-visible Spec-
trophotometry <2.24> using a blank prepared in the same
manner with 5 mL of water in place of the sample solution.
Amount (mg) of ethyl aminobenzoate (C 9 H n N02)
= W s x(A T /A s )
W s : Amount (mg) of Ethyl Aminobenzoate Reference
Standard
Containers and storage Containers — Well-closed contain-
ers.
Scopolia Extract and Tannic Acid
Suppositories
□ - \-j:%-x-9>->ibkM
Method of preparation
Scopolia Extract 0.5 g
Tannic Acid 1 g
Cacao Butter or a suitable base a sufficient quantity
Prepare 10 suppositories as directed under Suppositories,
with the above ingredients.
Description Scopolia Extract and Tannic Acid Supposito-
ries are light brown in color.
Identification (1) To 2 Scopolia Extract and Tannic Acid
Suppositories add 20 mL of diethyl ether, and dissolve the
base of suppositories with shaking for 10 minutes. Shake
thoroughly the mixture with 15 mL of water, separate the
water layer, and filter. To the filtrate add 10 mL of chlo-
roform, shake well, and separate the chloroform layer. Take
5 mL of the chloroform solution, add 5 mL of ammonia TS,
shake, and allow to stand: the ammonia layer shows a blue-
green fluorescence.
(2) To 1 mL of the aqueous layer obtained in (1) after ex-
traction with diethyl ether, add 2 drops of iron (III) chloride
TS: a bluish-black color develops. Allow to stand: a bluish-
black precipitate is formed (tannic acid).
Containers and storage Containers — Well-closed contain-
ers.
Scopolia Rhizome
Scopoliae Rhizoma
p- r-=l>
Scopolia Rhizome is the rhizome and root of Scopo-
lia japonica Maximowicz, Scopolia carniolica Jacquin
or Scopolia parviflora Nakai (Solanaceae).
When dried, it contains not less than 0.29% of total
alkaloids [hyoscyamine (C17H23NO3: 289.37) and
scopolamine (C 17 H 2 iN0 4 : 303.35)].
Description Chiefly irregularly branched, slightly curved
rhizome, about 15 cm in length, about 3 cm in diameter, oc-
casionally longitudinally cut; externally grayish brown, with
wrinkles; constrictions make the rhizome appear nodular;
rarely, stem base at one end; stem scars at upper side of each
node; roots or root scars on both sides and lower surface of
rhizome; fractured surface granular, grayish white to light
brown in color, with lighter colored cortex.
Odor characteristic; taste sweet, later slightly bitter.
Under a microscope <5.01>, xylem reveals groups of vessels
arranged stepwise, and accompanied with xylem sieve tubes
in medullary rays; parenchyma cells contain starch grains,
and sometimes sand crystals of calcium oxalate.
Identification (1) To 1 g of pulverized Scopolia Rhizome
add 10 mL of diethyl ether and 0.5 mL of ammonia TS,
shake for 30 minutes, and filter. Wash the residue with 10 mL
of diethyl ether, transfer the filtrate and the washing to a
separator, add 20 mL of diluted sulfuric acid (1 in 50), shake
well, and drain off the acid extract into another separator.
Render the solution slightly alkaline with ammonia TS, add
10 mL of diethyl ether, shake well, transfer the diethyl ether
layer to a porcelain dish, and evaporate the diethyl ether on a
water bath. To the residue add 5 drops of fuming nitric acid,
and evaporate the mixture on a water bath to dryness. Cool,
dissolve the residue in 1 mL of A^A^-dimethylformamide, and
add 5 to 6 drops of tetraethylammonium hydroxide TS: a
1358 Scutellaria Root / Crude Drugs
JP XV
red-purple to purple color develops.
(2) Place 2.0 g of pulverized Scopolia Rhizome in a glass-
stoppered centrifuge tube, add 30 mL of ammonia TS, and
centrifuge after irradiation of ultrasonic waves for 5 minutes.
Transfer the supernatant liquid to a separator, add 40 mL of
ethyl acetate, and shake. Drain off the ethyl acetate layer, add
3 g of anhydrous sodium sulfate to the ethyl acetate, shake,
and filter after the ethyl acetate becomes clear. Evaporate the
filtrate to dryness under reduced pressure, dissolve the
residue in 1 mL of ethanol (95), and use this solution as the
sample solution. Separately, dissolve 2 mg of Atropine Sul-
fate Reference Standard and 1 mg of Scopolamine
Hydrobromide Reference Standard in 1 mL each of ethanol
(95), and use these solutions as standard solution (1) and
standard solution (2). Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 5 /xL
each of the sample solution and standard solutions on a plate
of silica gel for thin-layer chromatography. Develop the plate
with a mixture of acetone, water and ammonia water (28)
(90:7:3) to a distance of about 10 cm, and dry the plate at
80°C for 10 minutes. After cooling, spray evenly Dragendor-
ff's TS for spraying on the plate: two principal spots from the
sample solution and each yellow-red spot from the standard
solutions show the same color tone and the same Rf value.
(3) To 3 g of pulverized Scopolia Rhizome add 10 mL of
chloroform, shake thoroughly, and filter. To 5 mL of the
filtrate add 5 mL of ammonia TS, shake, and allow to stand:
the ammonia layer shows a blue-green fluorescence.
Total ash <5.01> Not more than 7.0%.
Assay Weigh accurately about 0.7 g of pulverized Scopolia
Rhizome, previously dried at 60°C for 8 hours, in a glass-
stoppered, centrifuge tube, and moisten with 15 mL of am-
monia TS. To this add 25 mL of diethyl ether, stopper the
centrifuge tube tightly, shake for 15 minutes, centrifuge, and
separate the diethyl ether layer. Repeat this procedure twice
with the residue using 25-mL portions of diethyl ether. Com-
bine all the extracts, and evaporate the diethyl ether on a
water bath. Dissolve the residue in 5 mL of the mobile phase,
add exactly 3 mL of the internal standard solution, and add
the mobile phase to make 25 mL. Filter this solution through
a filter of a porosity of not more than 0.8 [im, discard the first
2 mL of the filtrate, and use the subsequent filtrate as the
sample solution. Separately, weigh accurately about 25 mg of
Atropine Sulfate Reference Standard (previously determine
the loss on drying <2.41> in the same manner as Atropine Sul-
fate Hydrate), dissolve in the mobile phase to make exactly
25 mL, and use this solution as standard stock solution A.
Weigh accurately about 25 mg of Scopolamine Hydro-
bromide Reference Standard (previously determine the loss
on drying <2.41> in the same manner as Scopolamine
Hydrobromide Hydrate), dissolve in the mobile phase to
make exactly 25 mL, and use this solution as standard stock
solution B. Pipet 5 mL of standard stock solution A and 1
mL of standard stock solution B, add exactly 3 mL of the in-
ternal standard solution, then add 25 mL of the mobile
phase, and use this solution as the standard solution. Per-
form the test with 10 /xL each of the sample solution and stan-
dard solution as directed under Liquid Chromatography
<2.01> according to the following conditions. Determine the
ratios, Qta and g SA , of the peak area of hyoscyamine (atro-
pine), and the ratios, g T s and g ss , of the peak area of
scopolamine to that of the internal standard in each solution,
calculate the amounts of hyoscyamine and scopolamine by
the following equation, and designate the total as the amount
of total alkaloids.
Amount (mg) of hyoscyamine (C17H23NO3)
= ^sa><(Qta/Gsa)x (1/5) x 0.8551
Amount (mg) of scopolamine (C 17 H 2 iN0 4 )
= ^ss x (Gts/Sss) x (1/25) x 0.7894
W SA : Amount (mg) of Atropine Sulfate Reference Stan-
dard, calculated on the dried basis
JFss: amount (mg) of Scopolamine Hydrobromide Refer-
ence Standard, calculated on the dried basis
Internal standard solution — A solution of brucine dihydrate
in the mobile phase (1 in 2500).
Operating conditions —
Detector: An ultraviolet absorption spectrometer
(wavelength: 210 nm).
Column: A stainless steel column 4 mm in inside diameter
and 15 cm in length, packed with octadesilcylanized silica gel
for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
20°C.
Mobile phase: Dissolve 6.8 g of potassium dihydrogen-
phosphate in 900 mL of water, add 10 mL of triethylamine,
adjust with phosphoric acid to pH 3.5, and add water to
make 1000 mL. To 9 parts of this solution add 1 part of
acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
scopolamine is about 8 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, scopolamine, atropine and the internal standard are
eluted in this order with the resolution between the peaks of
scopolamine and atropine being not less than 11, and with the
resolution between the peaks of atropine and the internal
standard being not less than 4.
Scutellaria Root
Scutellariae Radix
Scutellaria Root is the root of Scutellaria baicalensis
Georgi (Labiatae), from which the periderm has been
removed.
It contains not less than 10.0% of baicalin
(C 2 iH 18 O n : 446.36), calculated on the basis of dried
material.
Description Cone-shaped, semitubular or flattened root, 5
- 20 cm in length, 0.5 - 3 cm in diameter; externally yellow-
brown, with coarse and marked longitudinal wrinkles, and
with scattered scars of lateral root and remains of brown
periderm; scars of stem or remains of stem at the crown; xy-
lem rotted in old roots, often forming a hollow; hard in tex-
ture and easily broken; fractured surface fibrous and yellow
in color.
Almost odorless; taste, slightly bitter.
JPXV
Crude Drugs / Powdered Scutellaria Root 1359
Identification (1) Boil gently 0.5 g of pulverized Scutellar-
ia Root with 20 mL of diethyl ether under a reflux condenser
on a water bath for 5 minutes, cool, and filter. Evaporate the
filtrate, dissolve the residue in 10 mL of ethanol (95), and to 3
mL of the solution add 1 to 2 drops of dilute iron (III) chlo-
ride TS: a grayish green color develops, and it changes to pur-
ple-brown.
(2) To 2 g of pulverized Scutellaria Root add 10 mL of
methanol, warm on a water bath for 3 minutes, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of Baicalin Reference Standard in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 fiL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (4:2:1) to a distance of
about 10 cm, and air-dry the plate. Spray evenly iron (III)
chloride-methanol TS on the plate: one spot among the spots
from the sample solution and a dark green spot from the
standard solution show the same color tone and the same Rf
value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Scutellaria Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Scutellaria Root according to Method 4, and
perform the test (not more than 5 ppm).
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 6.0%.
Assay Weigh accurately about 0.5 g of pulverized Scutellar-
ia Root, add 30 mL of the mobile phase, heat under a reflux
condenser on a water bath for 30 minutes, and cool. Transfer
the mixture of the pulverized Scutellaria Root and the mobile
phase into a glass-stoppered centrifuge tube, centrifuge after
shaking for 5 minutes, and separate the supernatant liquid.
Wash the vessel for the reflux extraction with 30 mL of the
mobile phase, transfer the washings to the glass-stoppered
centrifuge tube, centrifuge, and separate the supernatant liq-
uid. To the residue add 30 mL of the mobile phase, shake for
5 minutes, centrifuge, and separate the supernatant liquid.
Combine all the extracts, add the mobile phase to make ex-
actly 100 mL, then pipet 2 mL of the extract, add the mobile
phase to make exactly 20 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Baicalin Reference Standard (separately determine the water)
dissolve in methanol to make exactly 20 mL. Pipet 2 mL of
the solution, add the mobile phase to make exactly 20 mL,
and use this solution as the standard solution. Pipet 10 juL of
the sample solution and standard solution, and perform the
test as directed under Liquid Chromatography <2.01> accord-
ing to the following conditions. Determine the peak areas, A T
and A s , of baicalin in each solution.
Amount (mg) of baicalin (C 2 iH 18 O u )
= W s x (A T /A S ) x 5
fV s : Amount (mg) of Baicalin Reference Standard, calcu-
lated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 277 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 //m in
particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
146) and acetonitrile (18:7).
Flow rate: Adjust the flow rate so that the retention time of
baicalin is about 6 minutes.
Selection of column: Dissolve 1 mg of Baicalin Reference
Standard and 2 mg of methyl parahydroxybenzoate in
methanol to make 100 mL. Perform the test with 10 fiL of
this solution under the above operating conditions and calcu-
late the resolution. Use a column giving elution of baicalin
and methyl parahydroxybenzoate in this order with the reso-
lution between these peaks being not less than 3.
System repeatability: When the test is repeated 6 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak area of baicalin is
not more than 1.5%.
Powdered Scutellaria Root
Scutellariae Radix Pulverata
Powdered Scutellaria Root is the powder of Scutel-
laria Root.
It contains not less than 10.0% of baicalin
(C 2 iH 18 O n : 446.36), calculated on the basis of dried
material.
Description Powdered Scutellaria Root occurs as a yellow-
brown powder. It is almost odorless, and has a slight, bitter
taste.
Under a microscope <5.01>, Powdered Scutellaria Root
reveals fragments of parenchyma cells containing small
amount of starch grains, fragments of reticulate vessels,
tracheids and elongated stone cells; also a few fragments of
spiral vessels and xylem fibers are observed.
Identification (1) Boil gently 0.5 g of Powdered Scutellar-
ia Root with 20 mL of diethyl ether under a reflux condenser
on a water bath for 5 minutes, cool, and filter. Evaporate the
filtrate, dissolve the residue in 10 mL of ethanol (95), and to 3
mL of the solution add 1 to 2 drops of dilute iron (III) chlo-
ride TS: a grayish green color develops, and it changes to pur-
ple-brown later.
(2) To 2 g of Powdered Scutellaria Root add 10 mL of
methanol, warm on a water bath for 3 minutes, cool, filter,
and use the filtrate as the sample solution. Separately, dis-
solve 1 mg of Baicalin Reference Standard in 1 mL of
methanol, and use this solution as the standard solution. Per-
form the test with these solutions as directed under Thin-lay-
er Chromatography <2.03>. Spot 5 /xL each of the sample so-
lution and standard solution on a plate of silica gel for thin-
layer chromatography. Develop the plate with a mixture of 1-
butanol, water and acetic acid (100) (4:2:1) to a distance of
1360 Senega / Crude Drugs
JP XV
about 10 cm, and air-dry the plate. Spray evenly iron (III)
chloride-methanol TS on the plate: one spot among the spots
from the sample solution and dark green spot from the stan-
dard solution show the same color tone and the same Ri
value.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
Powdered Scutellaria Root according to Method 3, and per-
form the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of Powdered Scutellaria Root according to Method 4, and
perform the test (not more than 5 ppm).
(3) Foreign matter — Under a microscope <5.01>, Pow-
dered Scutellaria Root does not show crystals of calcium oxa-
late.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Assay Weigh accurately about 0.5 g of Powdered Scutellar-
ia Root, add 30 mL of the mobile phase, heat under a reflux
condenser on a water bath for 30 minutes, and cool. Transfer
the mixture to a glass-stoppered centrifuge tube, centrifuge,
and separate the supernatant liquid. Wash the vessel for the
reflux extraction with 30 mL of the mobile phase, transfer the
washings to the glass-stoppered centrifuge tube, centrifuge
after shaking for 5 minutes, and separate the supernatant liq-
uid. To the residue add 30 mL of the mobile phase, shake for
5 minutes, centrifuge, and separate the supernatant liquid.
Combine all the extracts, add the mobile phase to make ex-
actly 100 mL, then pipet 2 mL of the extract, add the mobile
phase to make exactly 20 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Baicalin Reference Standard (previously determine the water)
and dissolve in methanol to make exactly 20 mL. Pipet 2 mL
of the solution, add the mobile phase to make exactly 20 mL,
and use this solution as the standard solution. Pipet 10 /xL of
the sample solution and standard solution, and perform the
test as directed under Liquid Chromatography <2.01> accord-
ing to the following conditions. Determine the peak areas, A T
and A s , of baicalin in each solution.
Amount (mg) of baicalin (C 2 iH 18 On)
= W s x (A T /A S ) x 5
W s : Amount (mg) of Baicalin Reference Standard, calcu-
lated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 277 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 25 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 ^m in
particle diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
146) and acetonitrile (18:7).
Flow rate: Adjust the flow rate so that the retention time of
baicalin is about 6 minutes.
Selection of column: Dissolve 1 mg of Baicalin Reference
Standard and 2 mg of methyl parahydroxybenzoate in
methanol to make 100 mL. Perform the test with 10 ^L of
this solution under the above operating conditions and calcu-
late the resolution. Use a column giving elution of baicalin
and methyl parahydroxybenzoate in this order with the reso-
lution between these peaks being not less than 3.
System repeatability: When repeat the test 6 times with the
standard solution under the above operating conditions, the
relative standard deviation of the peak area of baicalin is not
more than 1.5%.
Senega
Senegae Radix
-tz*;tf
Senega is the root of Polygala senega Linne or Poly-
gala senega Linne var. latifolia Torrey et Gray
(Polygalaceae).
Description Slender, conical root often branched, 3-10 cm
in length; main root 0.5 - 1.5 cm in diameter; externally light
grayish brown to grayish brown; with many longitudinal
wrinkles and sometimes with twisted protruding lines;
tuberously enlarged crown, with remains of stems and red
buds; branched rootlets twisted; a transverse section reveals
grayish brown cortex and yellowish white xylem; usually
round, and sometimes cuneate to semicircular; cortex on the
opposite side is thickened.
Odor, characteristic, resembling the aroma of methyl
salicylate; taste, sweet at first but leaving an acrid taste.
Under a microscope <5.01>, a transverse section of the
main root reveals a cork layer consisting of several rows of
light brown cork cells; secondary cortex composed of paren-
chyma cells and sieve tubes, traversed by medullary rays, 1 to
3 cells wide; medullary rays on zylem not distinct. Its paren-
chyma cells contain oil droplets, but starch grains and calci-
um oxalate crystals are absent.
Identification (1) Shake vigorously 0.5 g of pulverized
Senega with 10 mL of water: a lasting fine foam is produced.
(2) Shake 0.5 g of pulverized Senega with 30 mL of water
for 15 minutes, and filter. Take 1 mL of the filtrate, mix with
50 mL of water, and determine the absorption spectrum of
the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum at about 317
nm.
Purity (1) Stem — The amount of stems contained in Sene-
ga is not more than 2.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than stems contained in Senega is not more than 1.0%.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.07> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 30.0%.
JPXV
Crude Drugs / Senna Leaf 1361
Powdered Senega
Senegae Radix Pulverata
Powdered Senega is the powder of Senega.
Description Powdered Senega occurs as a light brown pow-
der, and has a characteristic odor resembling the aroma of
methyl salicylate; taste, sweet at first, but later acrid.
Under a microscope <5.01>, Powdered Senega reveals frag-
ments of pitted vessels, reticulate vessels and tracheids; frag-
ments of xylem fibers with oblique pits; fragments of xylem
parenchyma cells with simple pits; fragments of phloem
parenchyma containing oily droplets; fragments of exoder-
mis often composed of cells suberized and divided into
daughter cells; oily droplets stained red by sudan III TS. The
parenchyma cells of Powdered Senega do not contain starch
grains and crystals of calcium oxalate.
Identification (1) Shake vigorously 0.5 g of Powdered
Senega with 10 mL of water: a lasting fine foam is produced.
(2) Shake 0.5 g of Powdered Senega with 30 mL of water
for 15 minutes, and filter. Take 1 mL of the filtrate, mix with
50 mL of water, and determine the absorption spectrum of
the solution as directed under Ultraviolet-visible Spec-
trophotometry <2.24>: it exhibits a maximum at about 317
nm.
Purity Foreign matter <5.01> — Under a microscope, stone
cells, starch grains or crystals of calcium oxalate are not ob-
servable.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <5.01> Not more than 5.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 30.0%.
Water. May be prepared with an appropriate quantity of
Ethanol and Purified Water in place of 10vol% Ethanol.
Description Senega Syrup is a yellow-brown, viscous liquid.
It has a characteristic odor resembling methyl salicylate and a
sweet taste.
Identification Add 5 mL of water to 1 mL of Senega Syrup,
and shake: lasting small bubbles are produced.
Containers and storage Containers — Tight containers.
Senna Leaf
Sennae Folium
iz>i-
Senna Leaf is the leaflets of Cassia angustifolia Vahl
or Cassia acutifolia Delile (Leguminosae).
It contains not less than 1.0% of total sennosides
[sennoside A (C 4 2H 38 02o: 862.74) and sennoside B
(C4 2 H 38 02o: 862.74)], calculated on the basis of dried
material.
Description Lanceolate to narrow lanceolate leaflets, 1.5 -
5 cm in length, 0.5 - 1.5 cm in width, light grayish yellow to
light grayish yellow-green in color; margin entire, apex acute,
base asymmetric, petiole short; under a magnifying glass,
vein marked, primary lateral veins running toward the apex
along the margin and joining the lateral vein above; lower
surface having slight hairs.
Odor slight; taste, bitter.
Under a microscope <5.01>, a transverse section of Senna
Leaf reveals epidermis with thick cuticle, with numerous
stomata, and with thick-walled, warty unicellular hairs;
epidermal cells are often separated into two loculi by a sep-
tum which is in parallel with the surface of the leaf, and con-
tain mucilage in the inner loculus; palisade of a single layer
under each epidermis; spongy tissue, consisting of 3 to 4 lay-
ers, and containing clustered or solitary crystals of calcium
oxalate; cells adjacent to vascular bundle, forming crystal cell
Senega Syrup
Syrupus Senegae
Method of preparation
Senega, in medium cutting
Sucrose
10 vol% Ethanol
Purified Water
40 g
780 g
a sufficient quantity
a sufficient quantity
To make 1000 mL
Add 400 mL of 10 vol% ethanol to Senega, and macerate
for one or two days. Filter the extract, wash the residue with
a small amount of 10 vol% Ethanol, filter, and combine the
filtrate of the extracts and washings until total volume meas-
ures about 500 mL. Dissolve Sucrose in the mixture, by
warming if necessary, and dilute to 1000 mL with Purified
Identification (1) Macerate 0.5 g of pulverized Senna Leaf
in 10 mL of diethyl ether for 2 minutes, and filter. Add 5 mL
of ammonia TS to the filtrate: a yellow-red color is produced
in the water layer. To the residue of maceration add 10 mL of
water, and macerate for 2 minutes. Filter, and add 5 mL of
ammonia TS: a yellow-red color is produced in the water lay-
er.
(2) To 2 g of pulverized Senna Leaf add 40 mL of a mix-
ture of tetrahydrofuran and water (7:3), shake for 30
minutes, and centrifuge. Transfer the supernatant liquid to a
separator, add 13 g of sodium chloride, and shake for 30
minutes. Separate the water layer with undissolved sodium
chloride, and adjust the pH to 1.5 by adding 1 mol/L
hydrochloric acid TS. Transfer this solution to another sepa-
rator, shake with 30 mL of tetrahydrofuran for 10 minutes,
separate the tetrahydrofuran layer, and use this solution as
the sample solution. Separately, dissolve 1 mg of Sennoside
A Reference Standarad in 1 mL of a mixture of tetrahydrofu-
ran and water (7:3), and use this solution as the standard so-
lution. Perform the test as directed under Thin-layer Chro-
1362 Powdered Senna Leaf / Crude Drugs
JP XV
matography <2.03> with the sample solution and standard so-
lution. Spot 10 [iL each of these solutions on a plate of silica
gel for thin-layer chromatography. Develop the plate with a
mixture of 1-propanol, ethyl acetate, water and acetic acid
(100) (40:40:30:1) to a distance of about 15 cm, and air-dry
the plate. Examine under ultraviolet light (main wavelength:
365 nm): one spot among the spots from the sample solution
and a red fluorescent spot from the standard solution show
the same color tone and the same Rf value.
Purity (1) Rachis and fruit — The amount of rachis and
fruits contained in Senna Leaf does not exceed 5.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than rachis and fruits contained in Senna Leaf does not
exceed 1.0%.
(3) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 12.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Assay Weigh accurately about 0.5 g of pulverized Senna
Leaf in a glass-stoppered centrifuge tube, add 25 mL of dilut-
ed methanol (7 in 10), shake for 30 minutes, centrifuge, and
separate the supernatant liquid. To the residue add 10 mL of
diluted methanol (7 in 10), shake for 10 minutes, centrifuge,
and separate the supernatant liquid. Repeat this procedure
once more, combine all the extracts, add diluted methanol (7
in 10) to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Sennoside A Reference Standard (separately determine the
water), dissolve in a solution of sodium hydrogen carbonate
(1 in 100) to make exactly 20 mL, and use this solution as
standard stock solution (1). Weigh accurately about 10 mg of
Sennoside B Reference Standard (separately determine the
water), dissolve in a solution of sodium hydrogen carbonate
(1 in 100) to make exactly 20 mL, and use this solution as
standard stock solution (2). Pipet 5 mL of the standard stock
solution (1) and 10 mL of the standard stock solution (2), add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with exactly 10 /uL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine the peak areas, ^4 Ta and y4 Sa , of
sennoside A, and the peak areas, A Tb and A sb , of sennoside B
in each solution, calculate the amounts of sennoside A and
sennoside B by the following equations, and designate the
total as the amount of total sennosides.
Amount (mg) of sennoside A (C4 2 H 38 02o)
= W Sa x(A Ta /A s Jx(l/4)
Amount (mg) of sennoside B (C 42 H 38 02o)
= W sb x(A Jb /A sb )x(l/2)
W S!i : Amount (mg) of Sennoside A Reference Standard,
calculated on the anhydrous basis
W sb : Amount (mg) of Sennoside B Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet aborption photometer (wave-
length: 340 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 [im in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 2.45 g of tetra-M-heptylammonium
bromide in 1000 mL of a mixture of diluted 1 mol/L acetic
acid-sodium acetate buffer solution, pH 5.0 (1 in 10) and
acetonitrile (17:8).
Flow rate: Adjust the flow rate so that the retention time of
sennoside A is about 26 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, sennoside B and sennoside A are eluted in this order
with the resolution between these peaks being not less than
15, and the number of theoretical plates of the peak of senno-
side A being not less than 8000.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sennoside A is not more than 1.5%.
Powdered Senna Leaf
Sennae Folium Pulveratum
Powdered Senna Leaf is the powder of Senna Leaf.
It contains not less than 1.0% of total sennosides
[sennoside A (C 42 H3 8 02o: 862.74) and sennoside B
(C42H 38 2 o: 862.74)], calculated on the basis of dried
material.
Description Powdered Senna Leaf occurs as a light yellow
to light grayish yellow-green powder. It has a slight odor and
a bitter taste.
Under a microscope <5.01>, Powdered Senna Leaf reveals
fragments of vessels and vein tissue accompanied with crystal
cell rows; fragments of thick-walled, bent, unicellular hairs;
fragments of palisade and spongy tissue; clustered and solita-
ry crystals of calcium oxalate, 10 to 20 /xm in diameter.
Identification (1) Macerate 0.5 g of Powdered Senna Leaf
in 10 mL of diethyl ether for 2 minutes, and filter. Add 5 mL
of ammonia TS to the filtrate: a yellow-red color is produced
in the water layer. To the residue of maceration add 10 mL of
water, and macerate for 2 minutes. Filter, and add 5 mL of
ammonia TS: a yellow-red color is produced in the water lay-
er.
(2) To 2 g of Powdered Senna Leaf add 40 mL of a mix-
ture of tetrahydrofuran and water (7:3), shake for 30
minutes, and centrifuge. Transfer the supernatant liquid to a
separator, add 13 g of sodium chloride, and shake for 30
minutes. Separate the water layer with undissolved sodium
chloride, and adjust the pH to 1.5 with 1 mol/L hydrochloric
acid TS. Transfer this solution to another separator, shake
with 30 mL of tetrahydrofuran for 10 minutes, separate the
tetrahydrofuran layer, and use this solution as the sample so-
lution. Separately, dissolve 1 mg of Sennoside A Reference
Standard in 1 mL of a mixture of tetrahydrofuran and water
JPXV
Crude Drugs / Sinomenium Stem 1363
(7:3), and use this solution as the standard solution. Perform
the test as directed under Thin-layer Chromatography <2.03>
with the sample solution and standard solution. Spot 10 /xL
each of these solutions on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of 1-
propanol, ethyl acetate, water and acetic acid (100)
(40:40:30:1) to a distance of about 15 cm, and air-dry the
plate. Examine under ultraviolet light (main wavelength: 365
nm): one spot among the spots from the sample solution and
a red fluorescent spot from the standard solution show the
same color tone and the same Rf value.
Purity (1) Foreign matter <5.01> — Under a microscope,
stone cells and thick fibers are not observable.
(2) Total BHC's and total DDT's <5.01>— Not more than
0.2 ppm, respectively.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <5.01> Not more than 12.0%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Assay Weigh accurately about 0.5 g of Powdered Senna
Leaf in a glass-stoppered centrifuge tube, add 25 mL of dilut-
ed methanol (7 in 10), shake for 30 minutes, centrifuge, and
separate the supernatant liquid. To the residue add 10 mL of
diluted methanol (7 in 10), shake for 10 minutes, centrifuge,
and separate the supernatant liquid. Repeat this procedure
once more, combine all the extracts, add diluted methanol (7
in 10) to make exactly 50 mL, and use this solution as the
sample solution. Separately, weigh accurately about 10 mg of
Sennoside A Reference Standard (separately determine the
water) dissolve in a solution of sodium hydrogen carbonate (1
in 100) to make exactly 20 mL, and use this solution as stan-
dard stock solution (1). Weigh accurately about 10 mg of
Sennoside B Reference Standard (separately determine the
water) dissolve in a solution of sodium hydrogen carbonate (1
in 100) to make exactly 20 mL, and use this solution as stan-
dard stock solution (2). Pipet 5 mL of the standard stock so-
lution (1) and 10 mL of the standard stock solution (2), add
methanol to make exactly 50 mL, and use this solution as the
standard solution. Perform the test with exactly 10 fiL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions. Determine the peak areas, A Ta and A Sa , of
sennoside A, and the peak areas, A Tb and A sb , of sennoside B
in each solution, calculate the amounts of sennoside A and
sennoside B by the following equations, and designate the
total as the amount of total sennoside.
Amount (mg) of sennoside A (C4 2 H 38 02o)
= ^saXG4 Ta A4sa)x(l/4)
Amount (mg) of sennoside B (C4 2 H 38 02o)
= W sb x(A Jb /A sb )x(l/2)
W Sa : Amount (mg) of Sennoside A Reference Standard,
calculated on the anhydrous basis
W sb : Amount (mg) of Sennoside B Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer (wave-
length: 340 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /um in particle
diameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: Dissolve 2.45 g of tetra-M-heptylammonium
bromide in 1000 mL of a mixture of diluted 1 mol/L acetic
acid-sodium acetate buffer solution, pH 5.0 (1 in 10) and
acetonitrile (17:8).
Flow rate: Adjust the flow rate so that the retention time of
sennoside A is about 26 minutes.
System suitability —
System performance: When the procedure is run with 10
/xL of the standard solution under the above operating condi-
tions, sennoside B and sennoside A are eluted in this order
with the resolution between these peaks being not less than
15, and the number of theoretical plates of the peak of senno-
side A being not less than 8000.
System repeatability: When the test is repeated 6 times with
10 [iL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area of
sennoside A is not more than 1.5%.
Sinomenium Stem
Sinomeni Caulis et Rhizoma
Sinomenium Stem is the climbing stem and rhizome
of Sinomenium acutum Rehder et Wilson (Menisper-
maceae).
Description Round or elliptic sections, 0.2 - 0.4 cm in
thickness, 1 - 4.5 cm in diameter; cortex on both fractured
surfaces, light brown to dark brown; in xylem, grayish brown
vessel portions and dark brown medullary rays lined alter-
nately and radially; flank, dark gray, with longitudinal wrin-
kles and warty protrusions.
Almost odorless; taste, bitter.
Under a microscope <5.01>, a transverse section reveals ex-
tremely thick-walled stone cells in primary cortex and pericy-
cle; irregular-sized vessels lined nearly stepwise in the vessel
portion; cells of medullary ray mostly not lignified, and ex-
tremely thick-walled and large stone cells scattered here and
there; primary cortex containing needle crystals of calcium
oxalate; medullary rays containing starch gains, simple grain,
3 - 10 /um in diameter, and small needle crystals of calcium
oxalate.
Identification To 0.5 g of pulverized Sinomenium Stem add
10 mL of dilute acetic acid, heat for 2 minutes on a water
bath with frequent shaking, cool, and filter. To 5 mL of the
filtrate add 2 drops of Dragendorff's TS: an orange-yellow
precipitate is immediately produced.
Total ash <J.07> Not more than 7.0%.
Acid-insoluble ash <5.01> Not more than 0.5%.
1364 Smilax Rhizome / Crude Drugs
JP XV
Smilax Rhizome
Smilacis Rhizoma
Sodium Bicarbonate and Bitter
Tincture Mixture
Smilax Rhizome is the tuber of Smilax glabra Rox-
burgh (Liliaceae).
Description Flattened and irregular cylindrical tuber, often
with node-like branches; usually 5-15 cm in length, 2-5 cm
in diameter; the outer surface grayish yellow-brown to yel-
low-brown, and the upper surface scattered with knotty
remains of stem; cross section irregular elliptical to obtuse
triangular, consisting of extremely thin cortical layer and
mostly of stele.
Odor, slight; almost tasteless.
Under a microscope <5.01>, a transverse section reveals a 2-
to 3-cell-wide cork layer, with extremely narrow cortical lay-
er, usually consisting of a 2- to 4-cell-wide, thick-walled
parenchyma cells, showing large mucilage cells here and
there; mucilage cell containing raphides of calcium oxalate;
stele consisting chiefly of parenchyma cells, and scattered
with vascular bundles; parenchyma cells containing starch
grains composed mostly of simple grains, 12 - 36 /um in di-
ameter, and sometimes mixed with 2- to 4-compound grains.
Total ash <5.01> Not more than 5.0%.
Powdered Smilax Rhizome
Smilacis Rhizoma Pulveratum
Powdered Smilax Rhizome is the powder of Smilax
Rhizome.
Description Powdered Smilax Rhizome occurs as a light
yellow-brown powder, and has a slight odor, and is practical-
ly tasteless.
Under a microscope <5.01>, Powdered Smilax Rhizome
reveals starch grains and fragments of parenchyma cells con-
taining them; fragments of raphides of calcium oxalate con-
tained in mucilage masses; fragments of lignified parenchyma
cells of cortical layer; fragments of cork cells and scalariform
vessels; starch grains composed mostly of simple grains, and
mixed with a few 2- to 4-compound grains 12 - 36 //m in di-
ameter.
Purity Foreign matter — Under a microscope <5.01>, Pow-
dered Smilax Rhizome does not show a large quantity of
stone cells or thick-walled fibers.
Total ash <5.01> Not more than 5.0%.
Method of preparation
Sodium Bicarbonate
Bitter Tincture
Water or Purified Water
30 g
20 mL
a sufficient quantity
To make 1000 mL
Prepare before use, with the above ingredients.
Description Sodium Bicarbonate and Bitter Tincture Mix-
ture is a clear, yellowish liquid, having a bitter taste.
Containers and storage Containers — Tight containers.
Sophora Root
Sophorae Radix
W>
Sophora Root is the root of Sophora flavescens Ai-
ton (Leguminosae) or often such root from which the
periderm has been removed.
Description Cylindrical root, 5-20 cm in length, 2-3 cm
in diameter; externally dark brown to yellow-brown, with
distinct longitudinal wrinkles, and with laterally extended
lenticels; root without periderm, externally yellowish white,
with somewhat fibrous surface; the transversely cut surface,
light yellow-brown; cortex, 0.1 - 0.2 cm in thickness, slightly
tinged with dark color near cambium, forming a crack be-
tween xylem.
Odor, slight; taste, extremely bitter and lasting.
Identification To 0.5 g of powdered Sophora Root add 10
mL of dilute acetic acid, heat on a water bath for 3 minutes
with occasional shaking, cool, and filter. To 5 mL of the
filtrate add 2 drops of Dragendorff's TS: an orange-yellow
precipitate is produced immediately.
Purity (1) Stem — The amount of its stems contained in
Sophora Root does not exceed 10.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than stems contained in Sophora Root does not exceed
1.0%.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Powdered Sophora Root
Sophorae Radix Pulverata
Powdered Sophora Root is the powder of Sophora
JPXV
Crude Drugs / Swertia Herb 1365
Root.
Description Powdered Sophora Root occurs as a light
brown powder. It has a slight odor, and an extremely bitter
and lasting taste.
Under a miscroscope <5.01>, Powdered Sophora Root rev-
eals mainly starch grains and fragments of parenchyma cells
containing them, fibers, bordered pitted vessels, reticulate
vessels; a few fragments of corky tissue and solitary crystals
of calcium oxalate. Starch grains usually composed of 2- to 4-
compound grains 15 - 20 ^m in diameter, and simple grains 2
- 5 /um in diameter.
Identification To 0.5 g of Powdered Sophora Root add 10
mL of dilute acetic acid, heat on a water bath for 3 minutes
while occasional shaking, cool, and filter. To 5 mL of the
filtrate add 2 drops of Dragendorff's TS: an orange-yellow
precipitate is produced immediately.
Not more than 6.0%.
Not more than 1.5%.
Total ash <5.07>
Acid-insoluble ash <5.01>
Sweet Hydrangea Leaf
Hydrangeae Dulcis Folium
7vft
Sweet Hydrangea Leaf is the leaf and twig of
Hydrangea macrophylla Seringe var. thunbergii Maki-
no (Saxifragaceae).
Description Usually wrinkled and contracted leaf, dark
green to dark yellow-green in color. When soaked in water
and smoothed out, it is lanceolate to acuminately ovate,
about 12 cm in length, about 5 cm in width; margin serrated,
base slightly wedged; coarse hair on both surfaces, especially
on the veins; lateral veins not reaching the margin but curving
upwards and connecting with each other; petiole short and
less than one-fifth of the length of lamina.
Odor, slight; taste, characteristically sweet.
Identification Mix 0.5 g of pulverized Sweet Hydrangea
Leaf with 8 mL of a mixture of diethyl ether and petroleum
ether (1:1), shake well, filter, and evaporate the filtrate to dry-
ness. Dissolve the residue in 1 mL of dilute ethanol, and add
1 drop of dilute iron (III) chloride TS: a red-purple color de-
velops, which disappears on the addition of 2 to 3 drops of
dilute sulfuric acid.
Purity (1) Stem — The amount of stems contained in
Sweet Hydrangea Leaf does not exceed 3.0%.
(2) Foreign matter <5.01> — The amount of foreign matter
other than stems contained in Sweet Hydrangea Leaf does
not exceed 1.0%.
Loss on drying <5.01> Not more than 13.0% (6 hours).
Total ash <J.07> Not more than 12.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Powdered Sweet Hydrangea Leaf
Hydrangeae Dulcis Folium Pulveratum
7?ft*
Powdered Sweet Hydrangea Leaf is the powder of
Sweet Hydrangea Leaf.
Description Powdered Sweet Hydrangea Leaf occurs as a
dark yellow-green powder, and has a faint odor and a charac-
teristic, sweet taste.
Under a microscope <5.01>, Powdered Sweet Hydrangea
Leaf reveals fragments of epidermis with wavy lateral mem-
brane; stomata with two subsidiary cells; unicellular and
thin-walled hair with numerous protrusions of the surface,
150- 300 ^m in length; fragments of palisade tissue and
spongy tissue; fragments of vascular bundle and mucilage
cells containing raphides of calcium oxalate 50 - 70 [im in
length.
Identification Mix 0.5 g of Powdered Sweet Hydrangea
Leaf with 8 mL of a mixture of diethyl ether and petroleum
ether (1:1), shake well, filter, and evaporate the filtrate to dry-
ness. Dissolve the residue in 1 mL of dilute ethanol, and add
1 drop of dilute iron (III) chloride TS: a red-purple color de-
velops, which disappears on the addition of 2 to 3 drops of
dilute sulfuric acid.
Purity Foreign matter <5.01> — Under a microscope, Pow-
dered Sweet Hydrangea Leaf does not show stone cells, a
large quantity of fibers or starch grains.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 12.0%.
Acid-insoluble ash <5.01> Not more than 2.5%.
Swertia Herb
Swertiae Herba
Swertia Herb is the whole herb of Swertia japonica
Makino (Gentianaceae) collected during the blooming
season.
It contains not less than 2.0% of swertiamarin
(C 16 H 22 O 10 : 374.34), calculated on the basis of dried
material.
Description Herb, 20 cm in length, having flowers, opposite
leaves, stems, and, usually, with short, lignified roots; stems
square, about 0.2 cm in diameter, often with branches; the
leaves and stems dark green to dark purple or yellow-brown
in color; the flowers white to whitish, and the roots yellow-
brown. When smoothed by immersing in water, leaves, linear
or narrow lanceolate, 1 - 4 cm in length, 0.1 -0.5 cm in
width, entire, and sessile; corolla split deeply as five lobes; the
lobes narrow, elongated ellipse shape, and under a mag-
nifying glass, with two elliptical nectaries juxtaposed at the
base of the inner surface; the margin of lobe resembles eye-
1366 Powdered Swertia Herb / Crude Drugs
JP XV
lashes; the five stamens grow on the tube of the corolla and
stand alternately in a row with corolla-lobes; peduncle dis-
tinct. Odor, slight; taste, extremely bitter and persisting.
Identification To 2 g of pulverized Swertia Herb add 10 mL
of ethanol (95), shake for 5 minutes, filter, and use the filtrate
as the sample solution. Separately, dissolve 2 mg of Swer-
tiamarin Refereance Standard in 1 mL of ethanol (95), and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /uL each of the sample solution
and standard solution on a plate of silica gel with complex
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, 1-propanol and
water (6:4:3) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (broad spectrum
wavelength): one spot among several spots from the sample
solution and a red spot from the standard solution show the
same color tone and the same Rf value.
Purity Foreign matter <5.01> — The amount of straw and
other foreign matters contained in Swertia Herb is not more
than 1.0%.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 6.5%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 20.0%.
Assay Weigh accurately about 1 g of medium powder of
Swertia Herb in a glass-stoppered centrifuge tube, add 40 mL
of methanol, shake for 15 minutes, centrifuge, and separate
the supernatant liquid. To the residue add 40 mL of
methanol, and proceed in the same manner. Combine the ex-
tracts, and add methanol to make exactly 100 mL. Pipet 5
mL of the solution, add the mobile phase to make exactly 20
mL, and use this solution as the sample solution. Separately,
weigh accurately about 10 mg of Swertiamarin Reference
Standard (separately determine the water), dissolve in
methanol to make exactly 20 mL. Pipet 5 mL of the solution,
add the mobile phase to make exactly 20 mL, and use this so-
lution as the standard solution. Perform the test with exactly
10 /xL each of the sample solution and standard solution as
directed under Liquid Chromatography <2.01> according to
the following conditions, and determine the peak areas, A T
and A s , of swertiamarin in each solution.
Amount (mg) of swertiamarin (C 16 H 2 2O 10 )
= J*'s x G4tA4s) x 5
W s : amount (mg) of Swertiamarin Reference Standard,
calculated on the anhydrous basis
Operating conditions-
Detector: An ultraviolet absorption photometer
(wavelength: 238 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of water and acetonitrile (91:9).
Flow rate : Adjust the flow rate so that the retention time
of swertiamarin is about 12 minutes.
System suitability —
System performance: Dissolve 1 mg each of Swertiamarin
Reference Standard and theophylline in the mobile phase to
make 10 mL. When the procedure is run with 10 ^L of this
solution under the above operating conditions, theophylline
and swertiamarin are eluted in this order with the resolution
of these peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /uL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of swertiamarin is not more than 1.5%.
Powdered Swertia Herb
Swertiae Herba Pulverata
Powdered Swertia Herb is the powder of Swertia
Herb.
It contains not less than 2.0% of swertiamarin
(C 16 H 22 O 10 : 374.34), calculated on the basis of dried
material.
Description Powdered Swertia Herb occurs as a grayish yel-
low-green to yellow-brown powder. It has a slight odor, and
extremely bitter, persistent taste.
Under a microscope <5.01>, Powdered Swertia Herb rev-
eals xylem tissues with fibers (components of stems and
roots); assimilation tissues (components of leaves and
calyces); striated epidermis (components of stems and pedun-
cles); tissues of corollas and filaments with spiral vessels; cells
of anthers and their inner walls; spherical pollen grains with
granular patterns (components of flowers), about 30 /xm in
diameter; starch grains are simple grain, about 6 /xm in di-
ameter, and very few.
Identification To 2 g of Powdered Swertia Herb add 10 mL
of ethanol (95), shake for 5 minutes, filter, and use the filtrate
as the sample solution. Separately, dissolve 2 mg of Swer-
tiamarin Reference Standard in 1 mL of ethanol (95), and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatography
<2.03>. Spot 10 /xL each of the sample solution and standard
solution on a plate of silica gel with complex fluorescent indi-
cator for thin-layer chromatography. Develop the plate with
a mixture of ethyl acetate, 1-propanol and water (6:4:3) to a
distance of about 10 cm, and air-dry the plate. Examine un-
der ultraviolet light (broad spectrum wavelength): one spot
among several spots from the sample solution and a red spot
from the standard solution show the same color tone and the
same Rf value.
Purity Foreign matter — Under a microscope <5.01>, crys-
tals of calcium oxalate, a large quantity of starch grains and
groups of stone cells are not observable.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 6.5%.
Acid-insoluble ash <5.01> Not more than 2.0%.
Extract content <5.01> Dilute ethanol-soluble extract: not
less than 20.0%.
JPXV
Crude Drugs / Termeric 1367
Assay Weigh accurately about 1 g of Powdered Swertia
Herb in a glass-stoppered centrifuge tube, add 40 mL of
methanol, shake for 15 minutes, centrifuge, and separate the
supernatant liquid. To the residue add 40 mL of methanol,
and proceed in the same manner. Combine the extracts, and
add methanol to make exactly 100 mL. Pipet 5 mL of the so-
lution, add the mobile phase to make exactly 20 mL, and use
this solution as the sample solution. Separately, weigh ac-
curately about 10 mg of Swertiamarin Reference Standard
(separately determine the water), dissolve in methanol to
make exactly 20 mL. Pipet 5 mL of the solution, add the mo-
bile phase to make exactly 20 mL, and use this solution as the
standard solution. Perform the test with exactly 10,mL each
of the sample solution and standard solution as directed un-
der Liquid Chromatography <2.01> according to the follow-
ing conditions, and determine the peak areas, A T and A s , of
swertiamarin in each solution.
Amount (mg) of swertiamarin (C I6 H 2 2O 10 )
= J^ S X(,4 T A4 S )X5
W s : Amount (mg) of Swertiamarin Reference Standard,
calculated on the anhydrous basis
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 238 nm).
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
50°C.
Mobile phase: A mixture of water and acetonitrile (91:9).
Flow rate: Adjust the flow rate so that the retention time of
swertiamarin is about 12 minutes.
System suitability —
System performance: Dissolve 1 mg each of Sweriamarin
Reference Standard and theophylline in the mobile phase to
make 10 mL. When the procedure is run with 10 ^L of this
solution under the above operating conditions, theophylline
and swertiamarin are eluted in this order with the resolution
of these peaks being not less than 10.
System repeatability: When the test is repeated 6 times with
10 /xL of the standard solution under the above operating
conditions, the relative standard deviation of the peak areas
of swertiamarin is not more than 1.5%.
Swertia and Sodium Bicarbonate
Powder
-tz>-f*)-w.m
Method of preparation
Powdered Swertia Herb
Sodium Bicarbonate
Starch, Lactose Hydrate or
their mixture
30 g
700 g
a sufficient quantity
To make 1000 g
Prepare as directed under Powders, with the above in-
gredients.
Description Swertia and Sodium Bicarbonate Powder oc-
curs as a light grayish yellow powder, having a bitter taste.
Identification (1) To 10 g of Swertia and Sodium Bicar-
bonate Powder add 10 mL of ethanol (95), shake for 15
minutes, filter, and use the filtrate as the sample solution.
Separately, dissolve 1 mg of Swertiamarin Reference Stan-
dard in 1 mL of ethanol (95), and use this solution as the
standard solution. Perform the test with these solutions as
directed under Thin-layer Chromatography <2.03>. Spot 30
fiL each of the sample solution and standard solution on a
plate of silica gel with fluorescent indicator for thin-layer
chromatography. Proceed as directed in the Identification
under Powdered Swertia Herb.
(2) To 0.5 g of Swertia and Sodium Bicarbonate Powder
add 10 mL of water. After stirring, centrifuge the mixture
with 500 revolutions per minute. Smear, using a small glass
rod, the slide glass with a small amount of the precipitate,
add 1 drop of a mixture of water and glycerin (1:1), and put a
cover glass on it so that the tissue section spreads evenly
without overlapping each other, taking precaution against in-
clusion of bubbles, and use this as the preparation for
microscopic examination. If the precipitate separates into
two layers, proceed with the upper layer in the same manner,
and use as the preparation for microscopic examination.
Heat the preparation for microscopic examination in a short
time: the preparation reveals the yellow-green to yellow-
brown, approximately spherical pollen grains with granular
patterns under a microscope <5.01>. The pollen grains are 25
- 34 /um in diameter.
(3) The supernatant liquid obtained in (2) by centrifuging
responds to the Qualitative Tests <1.09> (1) for bicarbonate.
Containers and storage Containers — Well-closed contain-
ers.
Termeric
Curcumae Rhizoma
Termeric is the rhizome or being removed the cork
layer from it of Curcuma longa Linne (Zingiberaceae),
usually after being passed through hot water.
Description Termeric is a main rhizome or a lateral rhi-
zome; main rhizome, nearly ovoid, about 3 cm in diameter,
about 4 cm in length; lateral rhizome, cylindrical, with round
tips, curved, about 1 cm in diameter, 2-6 cm in length; both
main and lateral rhizomes with cyclic nodes; rhizome with
cork layer, yellowish brown, lustrous; rhizome without cork
layer, dark yellowish red, with yellowish red powders on sur-
face; hard in texture, not easily broken; transversely cut sur-
face yellowish brown to reddish brown, lustrous like wax.
Odor, characteristic; taste, slightly bitter and stimulant, it
colors a saliva yellow on chewing.
Under a microscope <5.01>, a transverse section reveals the
outermost layer to be composed of a cork layer 4-10 cells
thick; sometimes a cork layer partly remains; cortex and
stele, divided by a single-layered endodermis, composed of
1368 Toad Venom / Crude Drugs
JP XV
parenchyma, vascular bundles scattered; oil cells scattered in
parenchyma; parenchymatous cells contain yellow sub-
stances, sandy and solitary crystals of calcium oxalate, and
gelatinized starch.
Identification To 0.5 g of pulverized Termeric add 20 mL of
methanol, shake for 15 minutes, filter and use the filtrate as
the sample solution. Perform the test with this solution as
directed under Thin-layer Chromatography <2.03>. Spot 5 fiL
of the sample solution on a plate of silica gel for thin-layer
chromatography. Develop the plate with a mixture of ethyl
acetate, hexane and acetic acid (100) (70:30:1) to a distance of
about 10 cm, and air-dry the plate: a yellow spot appears at
around Rf 0.4.
Loss on drying <5.01> Not more than 17.0% (6 hours).
Total ash <J.07> Not more than 7.5%.
Acid-insoluble ash <5.01> Not more than 1.0%.
Extract content <5.07> Not less than 9.0% (dilute ethanol-
soluble extract).
Toad Venom
Bufonis Venerium
•te>7
Toad Venom is the venomous secretion of Bufo bufo
gargarizans Cantor or Bufo melanostictus Schneider
(Bufonidae).
When dried, it contains not less than 5.8% of bufo
steroid.
Description A round disk with slightly dented bottom and
protuberant surface, about 8 cm in diameter, about 1.5 cm in
thickness, the mass of one disk being about 80 to 90 g; or a
round disk with almost flattened surfaces on both sides,
about 3 cm in diameter, and about 0.5 cm in thickness, the
mass of one disk being about 8 g; externally red-brown to
blackish brown, somewhat lustrous, approximately uniform
and horny, hard in texture, and difficult to break; fractured
surface nearly flat, and edges of broken pieces red-brown and
translucent.
Odorless; taste, bitter and irritating, followed a little later
by a lasting sensation of numbness.
Identification To 1 g of pulverized Toad Venom add 10 mL
of acetone, shake for 10 minutes, filter, and use the filtrate as
the sample solution. Separately, dissolve 5 mg of resibufoge-
nin for thin-layer chromatography in 5 mL of acetone, and
use this solution as the standard solution. Perform the test
with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 10 /uL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
chromatography, develop the plate with a mixture of cyclo-
hexane and acetone (3:2) to a distance of about 10 cm, and
air-dry the plate. Spray evenly dilute sulfuric acid on the
plate, and heat at 105 °C for 5 minutes: one of several spots
obtained from the sample solution has the same color tone
and the same Ri value with the blue-green spot obtained from
the standard solution.
Acid-insoluble ash <5.01> Not more than 2.0%.
Component determination Weigh accurately about 0.5 g of
pulverized Toad Venom, previously dried in a desiccator (sili-
ca gel) for 24 hours, add 50 mL of methanol, heat under a
reflux condenser on a water bath for 1 hour, cool, and filter.
Wash the residue with 30 mL of methanol, and combine the
washing and filtrate. To this solution add methanol to make
exactly 100 mL. Pipet 10 mL of this solution, add exactly 5
mL of the internal standard solution, add methanol to make
exactly 25 mL, and use this solution as the sample solution.
Separately, weigh accurately about 10 mg, about 20 mg and
about 20 mg of bufalin for component determination,
cinobufagin for component determination and resibufogenin
for component determination, respectively, previously dried
in a desiccator (silica gel) for 24 hours, and dissolve in
methanol to make exactly 100 mL. Pipet 10 mL of this solu-
tion, proceed in the same manner as the sample solution, and
use this solution as the standard solution. Perform the test
with 10 /uL each of the sample solution and standard solution
as directed under Liquid Chromatography <2.01> according
to the following conditions. Determine the ratios, Q TB and Q
sb, of the peak area of bufalin, Qtc and Q sc , of the peak area
of cinobufagin, and Qtr and Qsr, of the peak area of
resibufogenin, respectively, to that of the internal standard in
each solution, and designate the total amount as an amount
of bufosteroid.
Amount (mg) of bufalin = W SB x (g TB /Q SB )
Amount (mg) of cinobufagin = W sc x (Qj C /Qsc)
Amount (mg) of resibufogenin = W SR x (g TR /g SR )
W SB : Amount (mg) of bufalin for component determina-
tion
W sc '- Amount (mg) of cinobufagin for component deter-
mination
W SR : Amount (mg) of resibufogenin for component deter-
mination
Internal standard solution — A solution of indometacin in
methanol (1 in 4000).
Operating conditions —
Detector: An ultraviolet spectrophotometer (wavelength:
300 nm).
Column: A stainless steel column 4 to 6 mm in inside di-
ameter and 15 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 to 10 /xm in
particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of diluted phosphoric acid (1 in
1000) and acetonitrile (11:9).
Flow rate: Adjust the flow rate so that the retention time of
the internal standard is 16 to 19 minutes.
Selection of column: Proceed with 10,mL of the standard
solution under the above operating conditions. Use a column
giving elution of bufalin, cinobufagin, resibufogenin and the
internal standard in this order, and clearly dividing each
peak.
Total ash <5.W> Not more than 5.0%.
JPXV
Crude Drugs / Trichosanthes Root 1369
Tragacanth
Tragacantha
Tribulus Fruit
Tribuli Fructus
v7U v
Tragacanth is the exudation obtained from the
trunks of Astragalus gummifer Labillardiere or other
species of the same genus (Leguminosae).
Description Tragacanth occurs as curved, flattened or
lamellate fragments, 0.5 to 3 mm in thickness. It is white to
light yellow in color, translucent, and horny in texture. It is
easily broken, and swells in water.
Odorless; tasteless and mucilaginous.
Identification (1) To 1 g of powdered Tragacanth add 50
mL of water: a nearly uniform, slightly turbid mucilage is
formed.
(2) To pulverized Tragacanth add dilute iodine TS, and
examine the mixture microscopically <5.01>: a few blue-
colored starch grains are observable.
Purity Karaya gum — Boil 1 g of Tragacanth with 20 mL of
water until a mucilage is formed, add 5 mL of hydrochloric
acid, and again boil the mixture for 5 minutes: no light red to
red color develops.
Total ash <5.01> Not more than 4.0%.
Powdered Tragacanth
Tragacantha Pulverata
Powdered Tragacanth is the powder of Tragacanth.
Description Powdered Tragacanth occurs as a white to yel-
lowish white powder. It is odorless, tasteless and
mucilaginous.
Under a microscope <5.01>, it, immersed in olive oil or liq-
uid paraffin, reveals numerous angular fragments with a
small amount of the circular or irregular lamellae or of starch
grains. Starch grains are spherical to elliptical, mostly simple
and occasionally 2- to 4-compound grains, simple grain, 3 -
25 //m in diameter. The fragments are swollen and altered
with water.
Identification (1) To 1 g of Powdered Tragacanth add 50
mL of water: a nearly uniform, slightly turbid mucilage is
formed.
(2) To Powdered Tragacanth add dilute iodine TS, and
examine the mixture microscopically <5.01>: a few blue-
colored starch grains are observable.
Purity Karaya gum — Boil 1 g of Powdered Tragacanth with
20 mL of water until a mucilage is formed, add 5 mL of
hydrochloric acid, and again boil the mixture for 5 minutes:
no light red to red color develops.
Total ash <J.07> Not more than 4.0%.
Containers and storage Containers — Tight containers.
Tribulus Fruit is the fruit of Tribulus terrestris Linne
(Zygophyllaceae) .
Description Pentagonal star shaped fruit, composed of five
mericarps, 7-12 mm in diameter, often each mericarp
separated; externally grayish green to grayish brown; a pair
of longer and shorter spines on surface of each mericarp, the
longer spine 3-7 mm in length, the shorter one 2-5 mm in
length, numerous small processes on midrib; pericarp hard in
texture, cut surface light yellow; each mericarp contains 1-3
seeds.
Almost odorless; taste, mild at first, followed by bitterness.
Under a microscope <5.01>, a transverse section reveals
epicarp composed of a single-layered epidermis; mesocarp
composed of parenchyma and sclerenchyma layer; endocarp
composed of several-layered fiber cells; a single-layer of cell
between mesocarp and endocarp contain solitary crystals of
calcium oxalate; cotyledons of seed contain oil drops and
aleurone grains, and occasionally starch grains.
Identification To 2 g of pulverized Tribulus Fruit add 5 mL
of methanol, shake for 10 minutes, filter, and use the filtrate
as the sample solution. Perform the test with this solution as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL of the sample solution on a plate of silica gel for thin-layer
chromatography, develop the plate with a mixture of ethyl
acetate and water (40: 1) to a distance of about 10 cm, and air-
dry the plate. Spray evenly dilute sulfuric acid on the plate,
heat at 105°C for 5 minutes, and examine under ultraviolet
light (main wavelength: 365 nm): a blue-white fluorescent
spot appears at around Rf 0.4.
Purity (1) Peduncle — Not more than 4.0%.
(2) Foreign matters <5.01> — Not more than 1.0% of for-
eign matters other than peduncle.
Loss on drying <5.01> Not more than 11.0% (6 hours).
Total ash <5.07> Not more than 13.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Extract content <5.07>
less than 8.5%.
Dilute ethanol-soluble extract: not
Trichosanthes Root
Trichosanthis Radix
Trichosanthes Root is the root of Trichosanthes
kirilowii Maximowicz, Trichosanthes kirilowii Max-
imowicz var. Japonicum Kitamura or Trichosanthes
bracteata Voigt (Cucurbitaceae), from which the corti-
cal layer has been removed.
1370 Uncaria Hook / Crude Drugs
JP XV
Description Irregular cylindrical root 5-10 cm in length,
3-5 cm in diameter, often cut lengthwise; externally light
yellowish white, and with irregular pattern of vascular bun-
dles appearing as brownish yellow lines; fractured surface
somewhat fibrous and light yellow in color; under a mag-
nifying glass, the transverse section reveals wide medullary
rays and brownish yellow spots or small holes formed by ves-
sels.
Odorless; taste, slightly bitter.
Purity (1) Heavy metals <1.07> — Proceed with 3.0 g of
pulverized Trichosanthes Root according to Method 3, and
perform the test. Prepare the control solution with 3.0 mL of
Standard Lead Solution (not more than 10 ppm).
(2) Arsenic <1.11> — Prepare the test solution with 0.40 g
of pulverized Trichosanthes Root according to Method 4,
and perform the test (not more than 5 ppm).
Total ash <5.01> Not more than 4.0%.
Uncaria Hook
Uncariae Uncis Cum Ramulus
Uncaria Hook is, hook or the hook-bearing stem, of
Uncaria rhynchophylla Miquel, Uncaria sinensis
Haviland or Uncaria macrophylla Wallich (Rubia-
ceae).
Uncaria Hook contains not less than 0.03% of total
alkaloids (rhynchophylline and hirstine), calculated on
the dried basis.
Description Uncaria Hook is uncinate hook or short stem
with opposite or single hook; the hook, 1 to 4 cm in length,
curved and acuminate; externally red-brown to dark brown
or yellow-brown, some one with hairs, the transverse section
oblong to elliptical, light brown; stem thin and prismatic
square to cylindrical, 2 to 5 mm in diameter, externally, red-
brown to dark brown or yellow-brown; the transverse sec-
tion, square to elliptical; the pith light brown, square to ellip-
tical; hard in texture.
Odorless and practically tasteless.
Under a microscope <5.01>, a transverse section of the
hook reveals vascular bundles in the cortex, unevenly dis-
tributed and arranged in a ring. Parenchyma cells in the sec-
ondary cortex containing sand crystals of calcium oxalate.
Identification To 1 g of pulverized Uncaria Hook add 20
mL of methanol, boil under a reflux condenser on a water
bath for 5 minutes, and filter. Evaporate the filtrate to dry-
ness, add 5 mL of dilute acetic acid to the residue, warm the
mixture on a water bath for 1 minute, and filter after cooling.
Spot 1 drop of the filtrate on a filter paper, air-dry, spray
Dragendorff's TS for spraying on it, and allow to stand: a
yellow-red color develops.
Loss on drying <5.01> Not more than 12.0% (6 hours).
Total ash <J.07> Not more than 4.0%.
Extract content <5.01> Dilute ethanol-souble extract: not
less than 8.5%.
Component determination Weigh accurately about 0.2 g of
medium powder of Uncaria Hook, transfer into a glass-stop-
pered centrifuge tube, add 30 mL of a mixture of methanol
and dilute acetic acid (7:3), shake for 30 minutes, centrifuge,
and separate the supernatant liquid. To the residue add two
10-mL portions of a mixture of methanol and dilute acetic
acid (7:3), proceed in the same manner, and combine all of
the supernatant liquid. To the combined liquid add a mixture
of methanol and dilute acetic acid (7:3) to make exactly 50
mL, and use this as the sample solution. Separately, weigh
accurately about 5 mg of rhynchophylline for component de-
termination, previously dried in a desiccator (silica gel) for 24
hours, and dissolve in a mixture of methanol and dilute acetic
acid (7:3) to make exactly 100 mL. Pipet 1 mL of this solu-
tion, add a mixture of methanol and dilute acetic acid (7:3) to
make exactly 10 mL, and use this solution as the standard so-
lution (1). Separately, dissolve 1 mg of hirsutine in 100 mL of
a mixture of methanol and dilute acetic acid (7:3), and use
this solution as the standard solution (2). Perform the test
with exactly 20 /xL each of the sample solution and standard
solutions (1) and (2) as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and determine the peak areas, v4 Ta and A Tb , of rhynchophyl-
line and hirsutine obtained from the sample solution, and the
peak area, A s , of rhynchophylline from the standard solution
(!)■
Amount (mg) of total alkaloids (rhynchophylline and
hirstine)
= W s x {{A Ta +lA05A Th )/A s } x (1/20)
W s : Amount (mg) of rhynchophylline for component
determination
Operating conditions —
Detector: An ultraviolet absorption photometer
(wavelength: 245 nm).
Column: A stainless steel column 4.6 mm in inside
diameter and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (5 /xm in particle di-
ameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Dissolve 3.85 g of ammonium acetate in 200
mL of water, add 10 mL of acetic acid (100) and water to
make 1000 mL, and add 350 mL of acetonitrile.
Flow rate: Adjust the flow rate so that the retention time of
rhynchophylline is about 17 minutes.
System suitability —
System performance: Dissolve 5 mg of rhynchophylline for
component determination in 100 mL of a mixture of
methanol and dilute acetic acid (7:3). To 5 mL of this solu-
tion add 1 mL of ammonia solution (28), and reflux for 10
minutes or warm at about 50°C for 2 hours. After cooling, to
1 mL of the solution so obtained add a mixture of methanol
and dilute acetic acid (7:3) to make 5 mL. When the proce-
dure is run with 20 /uh of this solution under the above
operating conditions, the peak of isorhynchophylline is
appears in addition to the peak of rhynchophylline, and the
resolution between these peaks is not less than 1.5.
System repeatability: When the test is repeated 6 times with
20 [iL of the standard solution (1) under the above operating
conditions, the relative standard deviation of the peak areas
of rhynchophylline is not more than 1.5%.
JPXV
Crude Drugs / Powdered Zanthoxylum Fruit 1371
Uva Ursi Fluidextract
^^^JUv/lx + X
Uva Ursi Fluidextract contains not less than 3.0
w/v% of arbutin.
Method of preparation Prepare an infusion from Bearberry
Leaf, in coarse powder, as directed under Fluidextracts, us-
ing hot Purified Water. Remove a part of the accompanying
tannin, evaporate the mixture under reduced pressure, if
necessary, and add Purified Water to adjust the percentage.
It may contain an appropriate quantity of Ethanol.
Description Uva Ursi Fluidextract is a yellow-brown to
dark red-brown liquid, and has a bitter and astringent taste.
It is miscible with water and with ethanol (95).
Identification To 1 mL of Uva Ursi Fluidextract add 30 mL
of a mixture of ethanol (95) and water (7:3), shake, filter, and
use the filtrate as the sample solution. Proceed as directed in
the Identification (2) under Bearberry Leaf.
Component determination Pipet 1 mL of Uva Ursi Fluidex-
tract, add water to make exactly 100 mL, and use this solu-
tion as the sample solution. Proceed as directed in the Com-
ponent determination under Bearberry Leaf.
Amount (mg) of arbutin = W s x (A T /A S )
W s : Amount (mg) of arbutin for component determina-
tion
Containers and storage Containers — Tight containers.
Zanthoxylum Fruit
Zanthoxyli Fructus
Zanthoxylum Fruit is the pericarps of the ripe fruit
of Zanthoxylum piperitum De Candolle (Rutaceae),
from which the seeds separated from the pericarps have
been mostly removed.
Description Capsules of 2 or 3 flattened spheroidal
mericarps, which are dehiscent in 2 pieces about 5 mm in di-
ameter; the outer surface of pericarp, dark yellow-red to dark
red-brown, with numerous dented spots originated from oil
sacs; the inner surface, light yellowish white.
Odor, characteristically aromatic; taste, acrid, which gives
numbing sensation to the tongue.
Under a microscope <5.01>, transverse section of Zanthox-
ylum Fruit reveals the external epidermis and the adjoined
unicellular layer containing red-brown tannin; the pericarp
holds oil sacs being up to approximately 500 /xm in diameter
and sporadically vascular bundles consisting mainly of spiral
vessels; the endocarp consists of stone cell layers; inner
epidermal cells very small.
Identification To 0.5 g of pulverized Zanthoxylum Fruit
add 100 mL of diluted ethanol (7 in 10), stopper the vessel
tightly, shake for 30 minutes, filter, and use this filtrate as the
sample solution. Perform the test with the sample solution as
directed under Thin-layer Chromatography <2.03>. Spot 10
/xL of the sample solution on a plate of silica gel with complex
fluorescent indicator for thin-layer chromatography. Develop
the plate with a mixture of ethyl acetate, ethanol (95) and
water (8:2:1) to a distance of about 10 cm, and air-dry the
plate. Examine under ultraviolet light (broad spectrum
wavelength): one spot showing a grayish red to red color at
the Rf value of about 0.7 appears.
Purity (1) Seed — The amount of the seeds contained in
Zanthoxylum Fruit does not exceed 20.0%.
(2) Peduncle and twig — The amount of the peduncles and
twigs contained in Zanthoxylum Fruit does not exceed 5.0%.
(3) Foreign matter <5.01> — The amount of foreign matter
other than peduncles and twigs contained in Zanthoxylum
Fruit does not exceed 1.0%.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 30.0 g of
pulverized Zanthoxylum Fruit: the volume of essential oil is
not less than 1.0 mL.
Powdered Zanthoxylum Fruit
Zanthoxyli Fructus Pulveratus
Powdered Zanthoxylum Fruit is the powder of Zan-
thoxylum Fruit.
Description Powdered Zanthoxylum Fruit occurs as a dark
yellow-brown powder. It has a strong, characteristic aroma
and an acrid taste leaving a sensation of numbness on the
tongue.
Under a microscope <5.01>, Powdered Zanthoxylum Fruit
reveals fragments of inner tissue of pericarp consisting of
stone cells with membranes about 2.5 /um in thickness; frag-
ments of spiral and annular vessels 10 to 15 fxm. in diameter;
fragments of oil sacs containing essential oil or resin; frag-
ments of epidermal cells, polygonal in surface view, contain-
ing tannin; numerous oil drops; masses of tannin, colored red
by adding vanillin-hydrochloric acid TS.
Identification To 0.5 g of Powdered Zanthoxylum Fruit
add 100 mL of diluted ethanol (7 in 10), stopper the vessel
tightly, shake for 30 minutes, filter, and perform the test with
the filtrate as the sample solution as directed under Thin-layer
Chromatography <2.03>. Spot 10 /xL of the sample solution
on a plate of silica gel with complex fluorescent indicator for
thin-layer chromatography. Develop the plate with a mixture
of ethyl acetate, ethanol (95) and water (8:2:1) to a distance
of about 10 cm, and air-dry the plate. Examine under ultrav-
iolet light (broad spectrum wavelength): one spot showing a
grayish red to red color at the Rf value of about 0.7 appears.
Total ash <J.07> Not more than 6.0%.
Acid-insoluble ash <5.01> Not more than 1.5%.
Essential oil content <5.01> Perform the test with 30.0 g of
1372 Zedoary / Crude Drugs JP XV
Powdered Zanthoxylum Fruit: the volume of essential oil is
not less than 0.8 mL.
Containers and storage Containers — Tight containers.
Zedoary
Zedoariae Rhizoma
Hi* n 7
Zedoary is the rhizome of Curcuma zedoaria Roscoe
(Zingiberaceae), usually after being passed through hot
water.
Description Nearly ovoid rhizome, 4-6 cm in length, 2.5 -
4 cm in diameter; externally grayish yellow-brown to grayish
brown; nodes protruded as rings; internode of 0.5 - 0.8 cm,
with thin, longitudinal wrinkles, scars of removed roots, and
small protrusions of branched rhizomes; under a magnifying
glass, external surface covered with coarse hairs; horny in
texture and difficult to cut; cross section grayish brown in
color; cortex 2-5 mm in thickness, stele thick, a light grayish
brown ring separating them.
Odor, characteristic; taste, pungent, bitter and cooling.
Total ash <5.01> Not more than 7.0%.
Essential oil content <5.01> Perform the test with 50.0 g of
pulverized Zedoary, provided that 1 mL of silicon resin is
previously added to the sample in the flask: the volume of es-
sential oil is not less than 0.5 mL.
JPXV
Infrared Reference Spectra 1373
Acebutolol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Acetaminophen
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Acetohexamide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1374 Infrared Reference Spectra
Acetylcholine Chloride for Injection
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
l — ' — ' — ' — ' — r
1000.0 500.0
Aclarubicin Hydrochloride
-\ 1 1 [ 1 — ~t — i 1— — r — ] i 1 1 1 [ 1 1 1 1 1 — ~\ 1 1 r — p
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Acrinol Hydrate
100.0
75.0-
50.0
25.0-
0.0 ~~ I 1 r 1 1 1 r 1 1 1 1 1 1 1 1 [ 1 1 1 1 1 1 1 1 1 [~
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1375
Afloqualone
100.0
75.0 ~
50.0
25.0
"< — ' r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Alacepril
100.0
75.0-
50.0-
25.0-
4000.0 3000,0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Alprenolol Hydrochloride
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
— ' — r
1000.0
500.0
1376 Infrared Reference Spectra
JP XV
Alprostadil
100.0
75.0-
50.0-
25.0
0.0 -j i 1 1 1 1 ; i " — i r — | 1 1 1 ! ] r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
t I r r^ p
1000.0 500.0
Amantadine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Ambenonium Chloride
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1377
Amidotrizoic Acid
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Amikacin Sulfate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
i ! i i
500.0
Amoxapine
100.0 -t
75.0
50.0
25.0
0.0
■ r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1378 Infrared Reference Spectra
JP XV
Amoxicillin Hydrate
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Anhydrous Ampicillin
100.0
75.0-
50.0-
25.0-
0.0 -| — i — i — i — i — | — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Ampicillin Hydrate
100.0
75.0-
50.0
25.0
■J --[--■!■■■ | [■ ■■■!■■■ i — i 1 1 1 1 r — | r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
7 | I I I I |
1000.0
500.0
JPXV
Infrared Reference Spectra 1379
Ampicillin Sodium
100.0
75.0-
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Amyl Nitrite
100.0
75.0 -
50.0
25.0 -
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
L-Arginine
100.0-
75.0-
50.0-
25.0-
0.0
i 1 1 1 J 1 1 1 r
l | i i i i | i i i r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
1380 Infrared Reference Spectra
L-Arginine Hydrochloride
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Arotinolol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
L-Aspartic Acid
100.0-
75.0-
50.0 —
25.0
t 1 1 1 1 1 r
4000.0 3000.0
1 — i ■ r — i 1 1 1 1 1 1 — i 1 1 1 f
2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1381
Aspoxicillin Hydrate
100.0
75.0 -•
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Atenolol
100.0
75.0-
i — j — \ — i — i — i — j— i — i — i — i — \-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Azithromycin Hydrate
100.0
75.0-
50.0
25.0 —
1 | | ] j | i i i"" ■■ i" ■ i ■ r r " r i — i ~t ■ r i ~ ' r '-■ l :
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
1382 Infrared Reference Spectra
JP XV
Bacampicillin Hydrochloride
100.0
75.0
50.0
25.0
"■ ' r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
i — < — ' — ' — ■ — r
1000.0 500.0
Beclometasone Dipropionate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Benidipine Hydrochloride
25.0-
"i r— t — t ] r ■ ,, i" ■ t— -I-- j 1 1 1 ; 1 1 1 1 [ r t— i r- -r f-
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
JPXV
Infrared Reference Spectra 1383
Benserazide Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Benzbromarone
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Benzyl Alcohol
100.0
75.0-
50.0
25.0-
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1 I I I '"; [ I I I | 1 ! I T
1500.0 1000.0 500.0
1384 Infrared Reference Spectra
JP XV
Benzylpenicillin Benzathine Hydrate
100.0
75.0
50.0-
25.0
1 ! 1 1 1 1 1 r
4000.0 3000.0
"I 1 f 1 1 1 1 1 1 [ 1 1 ! r
2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Benzylpenicillin Potassium
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i 1 1 1 1 r
1500.0
1 — r ~~ 1 — ' i I
1000.0 500.0
Berberine Chloride Hydrate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
' — 1 " ' ' ■"
1000.0
500.0
JPXV
Infrared Reference Spectra 1385
Berberine Tannate
100.0
75.0-
50.0
25.0
0.0 ~i — i — i — ■ -
4000.0 3000.0
i i i r
T I 1 I t
2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0 500.0
Betahistine Mesilate
100.0-
0.0
l 1 1 1 1 1 1 1 1 1 1 1 1 [ 1 1 r 1 1 1 I 1 1 f
3000.0 2000.0 1500.0 1000.0 500.0
4000.0
Preparation of sample: Potassium bromide disk method
Betamethasone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1386 Infrared Reference Spectra
JP XV
Betamethasone Dipropionate
100.0 -r
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Betamethasone Sodium Phosphate
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Betamethasone Valerate
100.0
75.0
50.0
25.0
"i — •-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1387
Bethanechol Chloride
100.0
75.0 -'
50.0—-
25,0
-, 1 , , _! _p ■ p- ,
4000.0 3000.0 2000.0
Preparation of sample: Paste method
ill 1
1500.0
1000.0
500.0
Bezafibrate
100.0
75.0
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Bifonazole
100.0
75.0
50.0
25.0
-| r-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
1388 Infrared Reference Spectra
JP XV
Biperiden Hydrochloride
100.0
75.0
50.0
25.0
r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1 — "* — r
1 000.0 500.0
Bisacodyl
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Bleomycin Hydrochloride
100,0-
75.0 —
50.0-
25.0-
~i — i — i — i — ■)■ ■ i — i — i — r
4000.0 3000.0 2000.0
i | i r
1500.0
Preparation of sample: Potassium bromide disk method
i ' ' ' i r
1000.0 500.0
JP XV
Infrared Reference Spectra 1389
Bleomycin Sulfate
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Bromazepam
4000.0 3000.0 2000.0 1 500.0
Preparation of sample: Potassium bromide disk method
1 r
1000.0
Bromhexine Hydrochloride
100.0
75.0
50.0
25.0
i ■ ' ■ ' ■ r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
1390 Infrared Reference Spectra
JP XV
Bromocriptine Mesilate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
Bucillamine
100.0
75.0
50.0-
25.0 —
-r — | 1 1 1 r — | ( 1 1 1 1 1 1 1 1 j r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
Bucumolol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1391
Bufetolol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Bufexamac
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Bumetanide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1392 Infrared Reference Spectra
JP XV
Bunazosin Hydrochloride
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Bupranolol Hydrochloride
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Busulfan
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JPXV
Infrared Reference Spectra 1393
Butyl Parahydroxybenzoate
100.0
75.0-
50.0
25.0
~\ i i ^ | r
4000.0 3000.0
2000.0
Preparation of sample: Potassium bromide disk method
I 1 1 1 1 1 1 1 1 1 r
1500.0 1000.0
Calcium Folinate
100.0
75.0 '
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Calcium Para-aminosalicylate Hydrate
100.0 ■
i 1 1 1 1 1 1 1 1 1 1 r
1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
i 1 1 1 1 1 1 1 1 1 r
4000.0 3000.0 2000.0
1394 Infrared Reference Spectra
JP XV
Calcium Polystyrene Sulfonate
100.0 -
75.0
50.0
25.0
4000.0 3000.0 2000.0 J 500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Captopril
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Carbazochrome Sodium Sulfonate Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1395
Carbidopa Hydrate
100.0
75.0
50.0 "
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
L-Carbocisteine
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Carmofur
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1396 Infrared Reference Spectra
JP XV
Carteolol Hydrochloride
100.0
75.0
50.0 ~
25.0 ~
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Carumonam Sodium
100.0
75.0
50.0-
25.0 —
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Cefaclor
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1397
Cefadroxil
100.0
75.0
50.0
25.0
4000.0 3000.0 2000,0 1500.0
Preparation of sample: Potassium bromide disk method
Cefalexin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cefalotin Sodium
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1398 Infrared Reference Spectra
JP XV
Cefapirin Sodium
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 1 r-
1000.0 500.0
Cefatrizine Propylene Glycolate
100.0
75.0
50.0
25.0 ~
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cefazolin Sodium
100.0
75.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1399
Cefixime
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cefmenoxime Hydrochloride
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Cefmetazole Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1400 Infrared Reference Spectra
JP XV
Cefminox Sodium Hydrate
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cefodizime Sodium
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Cefotaxime Sodium
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1401
Cefotetan
100.0
75.0-
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Cefotiam Hydrochloride
100.0
75.0 "
50.0 -
25.0 ~
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Cefpodoxime Proxetil
100.0
75.0-
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1402 Infrared Reference Spectra
JP XV
Cefsulodin Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ceftazidime Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ceftizoxime Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1403
Cefuroxime Axetil
0.0
"i 1 1 1 1 1 r
i — i — | — s — i — t~~i — i — i — i — i — i — r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Cefuroxime Sodium
100.0
75.0
50.0
25.0
r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cellacefate
100.0
75.0
50.0 ~
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1404 Infrared Reference Spectra
JP XV
Cetraxate Hydrochloride
100.0
75.0
50.0
25.0^
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Chenodeoxycholic Acid
100.0-
75.0
50.0-
25.0-
0.0
ffl^SfT
— i 1 r — r— ] — ~t- — r — r
4000.0 3000.0
Preparation of sample: Potassium bromide disk method
~1 1 ' ~T— ■ I \~
2000.0 1500.0
i — i — | — i — i — i — i — | —
1000.0 500.0
Chloramphenicol
100.0
75.0-
50.0
25.0
-\ 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1405
Chloramphenicol Sodium Succinate
100.0
75.0-
50.0
25.0-
0.0 -f — i' ■ 'i ■ ■ i ■■■! — ■ ]■■ ■ i ■■ 'i — — i r~— i r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
1 r™^
1500.0
1 — i — i — i — r
1000.0 500.0
Chlordiazepoxide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Chlormadinone Acetate
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1406 Infrared Reference Spectra
JP XV
Chlorphenesin Carbamate
100.0
75.0
50.0 "
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Chlorpheniramine Maleate
100.0
75.0
50.0-
25.0-
1 1 1 1 1 1 1 1 1— ~ | 1— r — ,-■■■ - r - ~j~ - T - — i i r" j 1 1 1 1 j -
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Paste method
(/-Chlorpheniramine Maleate
100.0
75.0-
50.0-
25.0"
o.o-
i i r— ~-r
1 1 1 1 r
4000.0 3000.0 2000.0
Preparation of sample: Paste method
i — i — i — i — | — i — \ — i — i — | — i — i — i — i — r
1500.0 1000.0 500.0
JPXV
Infrared Reference Spectra 1407
Chlorpropamide
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cholecalciferol
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ciclacillin
100.0
75.0
50.0
25.0-
o.o — p — r~~i — i — i — | — * — i — i — i — | — i — i — i — i — | — i — i — i — i — \ — i — i — i — i — r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
1408 Infrared Reference Spectra
JP XV
Ciclosporin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cilastatin Sodium
100.0-
75.0-
50.0
25.0-
0.0
l 1 1 1 ] 1 1 1 1 1 1 1 1 r |— i 1 — t- — r— | r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
1 ' I
500.0
Cilostazol
100.0
75.0-
50.0-
25.0-
0.0
1 ■ ' ' ■ I r"
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1409
Cimetidine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cisplatin
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
t 1 1 r r~ — i r
1000.0 500.0
Anhydrous Citric Acid
100.0
75.0-
50.0-
25.0
— i 1 1 1 1 r
4000.0 3000.0
i — i — i — i — | — i — i — i — i — | — i — i — i — i — | —
1000.0 500.0
2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1410 Infrared Reference Spectra
JP XV
Citric Acid Hydrate
100.0-
75.0-
50.0-
25.0-
0.0 — I r 1 r" r ) 1 r — ~r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
) - •- 1 "" i — i i — i — i — | — i — i — i — i — r
1500.0 1000.0 500.0
Clindamycin Phosphate
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1 1 1 1 1 1 1 1 1 1 1 1 1 1 — |-
1500.0 1000.0 500.0
Clinofibrate
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1411
Clocapramine Hydrochloride Hydrate
100.0 -
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Clofedanol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Clofibrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
1412 Infrared Reference Spectra
JP XV
Clonazepam
100.0
75.0 -
50.0
25.0 "
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Clonidine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Cloperastine Hydrochloride
100.0 -
75.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JP XV
Infrared Reference Spectra 1413
Clotrimazole
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cloxacillin Sodium Hydrate
100.0
75.0
50.0 "
25.0
~" — ' — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cocaine Hydrochloride
100.0
75.0
50.0^
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1414 Infrared Reference Spectra
JP XV
Codeine Phosphate Hydrate
100.0
75.0-
50.0
25.0 ~
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Colchicine
100.0
75.0
50.0
25.0-
4000.0
3000.0
2000.0
1500.0
1000.0
Preparation of sample: Potassium bromide disk method [To 0.5 mL of a solution of Colchicine in methanol (1 in
50) add 1 g of potassium bromide, and dry under reduced pressure (80°C, 1 hour).]
Colistin Sodium Methanesulfonate
100.0
75.0 ~
50.0 i
25.0
— r r 1 r -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1415
Cortisone Acetate
100.0-
75.0-
50.0-
25.0-
0.0 — I 1 1 1 1 1 1 1— I 1 1 1 1 1 1 1 1 1 1 r [ ! — I — I — 1 — j~
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Croconazole Hydrochloride
100.0 -i
75.0 -
50.0 -
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Cyanamid
100.0
75.0-
50.0-
25.0
4000.0
3000.0
2000.0
1500.0
1000.0
500.0
Preparation of sample: Film method (Drop one or two drops of a solution in acetone (1 in 100) onto a potassium
bromide disk, and dry.)
1416 Infrared Reference Spectra
JP XV
Cyclopentolate Hydrochloride
100.0 3
75.0
50.0 "
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Cycloserine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Cytarabine
100.0
75.0"
50.0
25.0-
0.0 — I 1 1 1 1 f ' "i — i 1 1 j 1 1 1 1 1 1 1 1 r~ i r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
JPXV
Infrared Reference Spectra 1417
Dantrolene Sodium Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Deferoxamine Mesilate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Demethylchlortetracycline Hydrochloride
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
i 1 1 J i 1 r
1000.0 ;:::;:;
1418 Infrared Reference Spectra
JP XV
Dexamethasone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Dextromethorphan Hydrobromide Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
i 1 i r-
1000.0
500.0
Dibucaine Hydrochloride
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1419
Diclofenac Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Diclofenamide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
500.0
Dicloxacillin Sodium Hydrate
100.0
75.0 -
50.0 ~
25.0 ~
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1420 Infrared Reference Spectra
JP XV
Digoxin
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Dihydrocodeine Phosphate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
t — ~~i 1 p
1000.0
Dihydroergotamine Mesilate
100.0
75.0
50.0
25.0
—y ~i ■ 7 " —i 1"
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JPXV
Infrared Reference Spectra 1421
Dihydroergotoxine Mesilate
100.0
75.0
50.0
25.0
T
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 ' ' T- ~ -r— -]-
1000.0 500.0
Dilazep Hydrochloride Hydrate
100.0
75.0-
50.0 -
25,0 -
i — ' — ' — ' 1 — ! — ' — ■■ — ■ — ' — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Dimemorfan Phosphate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
1422 Infrared Reference Spectra
JP XV
Dimercaprol
100.0
75.0-
50.0-
25.0-
o.o-
t 1 1 r
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
] 1 ! 1 1 ] 1 1 1 r — f
1500.0 1000.0 500.0
Dimorpholamine
100.0
75.0-
50.0-
25.0-
0.0 I 1 i 1 1 j 1 1 1 1 1" 'T r ■-"i [ r — i 1 1 j— i 1 " c " 'f j""
4000.0 3000.0 2000.0 1500.O 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Dinoprost
.100.0
75.0
50.0-
25.0-
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
JPXV
Infrared Reference Spectra 1423
Diphenhydramine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Dipyridamole
100.0
75.0
50.0
25.0
0.0 "i
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Disopyramide
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1424 Infrared Reference Spectra
JP XV
Distigmine Bromide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Disulfiram
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Dobutamine Hydrochloride
100.0
75.0 ^
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JPXV
Infrared Reference Spectra 1425
Dopamine Hydrochloride
100.0
75.0
50.0
25.0-
•4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Doxapram Hydrochloride Hydrate
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1 500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Doxifluridine
100.0
~i i i i i i i i i i i i "j i"" 'i i i j r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
1426 Infrared Reference Spectra
JP XV
Doxorubicin Hydrochloride
100.0-
75.0-
50.0-
25.0
0.0
t~ — r — 1~ — i r — r 1 1 r~~~i r— i p — r
3000.0 2000.0 1500.0
4000.0
Preparation of sample: Potassium chloride disk method
r " r f ■■ r — ~r
1000.0
500.0
Doxycycline Hydrochloride Hydrate
100.0
75.0-
50.0
25.0-
i — ' — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Droperidol
100.0
75.0
50.0 -
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1427
Dydrogesterone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Enflurane
0.0 -f
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
Enoxacin Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1428 Infrared Reference Spectra
Eperisone Hydrochloride
JP XV
~\ 1 1 1 [ r 1 1 1 1 1 — n r — r~~~] — t
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1 1 1 1 1 1 r
1000.0 500.0
Ephedrine Hydrochloride
100.0
75.0
50.0 "
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Ergocalciferol
100.0
75.0
50.0-
25.0
~i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 — r
1000.0
JPXV
Infrared Reference Spectra 1429
Erythromycin
100.0 ■
75.0-
50.0-
25.0-
o.o-
] 1 1 1 1 J 1 1 1 1 1 r
3000.0 2000.0 1500.0
4000.0
Preparation of sample: Potassium bromide disk method
l 1 1 1 1 1 r
1000.0 500.0
Erythromycin Ethylsuccinate
100.0
75.0 -
50.0 -
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Erythromycin Stearate
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500,0
1430 Infrared Reference Spectra
JP XV
Estradiol Benzoate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Estriol
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
i ' : ' ' r
1000.0 500.0
Ethanol
100.0
75.0
50.0
25.0-
1 l l [ l l l ' l l l : l l l | I l ' " 1 I j
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Liquid film method
JPXV
Infrared Reference Spectra 1431
Anhydrous Ethanol
100.0
75.0
50.0-
25.0
1 — i — ' — i — ' — I — '
4000.0 3000.0
i 1 1 1 1 1 1 1 1 1 1 1 r
2000.0 1500.0 1000.0 500.0
Preparation of sample: Liquid film method
Ethionamide
100.0
75.0-
50.0-
25.0-
0.0
i — i — i — i — [ — i — i — i — i — (■
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Ethyl L-Cysteine Hydrochloride
100.0
75.0-
50.0
25.0-
4000.0 3000O 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1432 Infrared Reference Spectra
JP XV
Ethyl Icosapentate
100.0-
75.0-
50.0-
25.0-
0.0-
n 1 1 1 1 ! 1 r
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
i — i — i — i — | — i — i — ! — i — | — i — i — i — i — r
1500.0 1000.0 500.0
Ethylmorphine Hydrochloride Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ethyl Parahydroxybenzoate
100.0
75.0-
50.0-
25.0-
~\ 1 1 1 1 1 r~T 1 1 1 r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
1 1 1 T 1 1 1 1 1 1 1~
1500.0 1000.0 500.0
JPXV
Infrared Reference Spectra 1433
Etidronate Disodium
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000,0 1500.0
Preparation of sample: Potassium bromide disk method
Etilefrine Hydrochloride
100.0
75.0
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
Etizolam
100.0
75.0-
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1434 Infrared Reference Spectra
Etodolac
100.1
JP XV
75.0-
50.0-
25.0-
o.o-
t 1 1 r
n 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 1 1 1 T— -r-
1000.0 500.0
Etoposide
100.0
75.0-
50.0-
25.0
0,0 "~ I 1 1 1 1 j 1 1 1 r ]■■■" "r r r — t [ 1 r 1 — "i r— i 1 1 r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Famotidine
100.0
75.0
50.0
25.0 -I
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
T 1 1 f
1000.0
1 —
500.0
JPXV
Infrared Reference Spectra 1435
Fenbufen
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 1 1 T 1
1000.0 500.0
Fentanyl Citrate
100.0
75.0
50.0^
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Flavin Adenine Dinucleotide Sodium
1O0.O
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1436 Infrared Reference Spectra
JP XV
Flavoxate Hydrochloride
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Flomoxef Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Fludiazepam
100.0
75.0
50.0
25.0 "
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1437
Flunitrazepam
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Fluocinolone Acetonide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Fluorometholone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
— ■ — r
1000.0
1438 Infrared Reference Spectra
Fluoxymesterone
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Fluphenazine Enanthate
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
--, — |-
1000.0
500.0
Flurazepam Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1439
Flurbiprofen
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Formoterol Fumarate Hydrate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Fosfestrol
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
1440 Infrared Reference Spectra
JP XV
Fosfomycin Calcium Hydrate
100.0
75.0
50.0 -
25.0
T
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Fosfomycin Sodium
100.0
75.0
50.0 -j
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Fructose
100.0 -r
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
JPXV
Infrared Reference Spectra 1441
Furosemide
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Fursultiamine Hydrochloride
100.0
75.0 ~
50.0 ^
25.0 -
o.o-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Glibenclamide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1442 Infrared Reference Spectra
JP XV
Glutathione
100.0 ■
75.0-
50.0
25.0
0.0
~\ 1 1 r
~i 1 1 r
l 1 1 i 1 1 1 1 1 1 1 p
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Glycerin
J 00.0-
75.0-
50.0-
25.0- -
o.o-
i 1 1 r— i 1 1 1 r
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
~1 i i i i i i | i i "I I i
1500.0 1000.0 500.0
Concentrated Glycerin
100.0
75.0-
50.0
25.0
0.0— j 1 1 1 r — \ 1 i 1 1 1 1 1 1 i [ — i — ■ i i ■ ■ i ■■■ [- -i i 1 r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Liquid film method
JPXV
Infrared Reference Spectra 1443
Glycine
100.0
75.0
50.0
25.0
0.0
■ I 3
1 1 n 1 1 i— — — f— — ~i ■* ' -t r r 1 r~ ■'"■ i™ r*~ — T™ i 1 1 1 1 1 i
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Gonadrelin Acetate
100.0
75.0-
50.0
25.0-
i 1 1 1 1 1 1 1 1 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
T — i 1 1 1 1 r
1000.0 500.0
Griseofulvin
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1444 Infrared Reference Spectra
JP XV
Guaifenesin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Guanabenz Acetate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
"■ r
1000.0
500.0
Guanethidine Sulfate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1445
Haloperidol
0.0
i 1 1 1 j 1 1 1 r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i — t — | — i — i — i — i — i — i — r~ — t — i — r
1500.0 1000.0 500.0
Halothane
4000,0 3000.0 2000.0
Preparation of sample: Gas sampling method
1500.0
1000.0
500.0
Haloxazolam
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1446 Infrared Reference Spectra
JP XV
Homochlorcyclizine Hydrochloride
100.0
75.0
50.0
25.0
~r — i \ 1 1 1 1 — ~i 1 1 — ~i t r — i— — | — ■ f ■ i ■ — i 1 1 ■ 'i r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium chloride disk method
500.0
Hydralazine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Hydrocortisone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra \AA1
Hydrocortisone Butyrate
100.0
75.0
50.0
25.0
1 T . . 1 J"
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
Hydrocortisone Sodium Phosphate
100.0-
50.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
Hydrocortisone Sodium Succinate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
| r r ^t ~— r — " — ■ 1 1 1 \ r r — — t r— ~- — 1 r r- —-i— " — . 1 1- — r — ■ — r r— r
1000.0
500.0
1448 Infrared Reference Spectra
JP XV
Hydrocortisone Succinate
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Hydrocotarnine Hydrochloride Hydrate
100.0 -i —
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Hymecromone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1449
Hypromellose Phthalate (200713)
100.0
75.0
50.0
25.0
0.0
■ — r r 1 j i 1 1 1 1 1 1 1 1 i 1 - r ~™^ r - , ; r , , ■*-"- - \ ■
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Hypromellose Phthalate (220824)
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ibuprofen
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1450 Infrared Reference Spectra
Ifenprodil Tartrate
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Imipenem Hydrate
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Indenolol Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1451
Indometacin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Iodamide
100.0
75.0 - 4
50.0
25.0 ~
4000,0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
■ r
1.000.0
500.0
Iopamidol
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1452 Infrared Reference Spectra
JP XV
Iotalamic Acid
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Iotroxic Acid
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ipratropium Bromide Hydrate
100.0
75.0 -
50.0
25.0 "
o.o-
4000.0
3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1453
Isoflurane
t 1 1 r
3000.0
-\ 1 1 1 1 1 1 j 1 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Liquid film method
500.0
L-Isoleucine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Isoniazid
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
] 1 1 1 1 f-
1000.0 500.0
1454 Infrared Reference Spectra
JP XV
Isosorbide
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ketamine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ketoprofen
100.0
75.0-
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromid disk method
JPXV
Infrared Reference Spectra 1455
Ketotifen Fumarate
100.0
75.0-
50.0-
25.0-
0.0
1 1 1 1 1 1 i 1 ! 1 1 1 1 1 1 1 1 1 r
3000.0 2000.0 1500.0 1000.0 500.0
4000.0
Preparation of sample: Potassium bromide disk method
Kitasamycin Acetate
100.0
75.0 -
50.0 n
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Kitasamycin Tartrate
100.0 ■
75.0-
50.0-
25.0-
o.o—i — ' — ' — ' — ' — i — ' — ' — r ~~ ] — i — ' — ■ — ' — ' — I — ' — ' — ' — ! — I — ' — ' — ' — r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
1456 Infrared Reference Spectra
JP XV
Anhydrous Lactose
100.0 -■
75.0 "
50.0 ~
25.0
~\ — ' — i ~r -i™ — i f 1 r— - — t — — r p
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
■ r
1000.0
Lactose Hydrate
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Latamoxef Sodium
100.0-
75.0-
50.0-
25.0-
0.0 — I i 1 1 1 \ 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 1 1 r~~\ — n p
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1457
Lenampicillin Hydrochloride
100.0 -p—
75.0-
50.0-
25.0-
0.0
[ ■■ t " i ] i 1 1 1 |~~i 1 1 1 1 — ~i — i — n 1 1 1 r — [ — ~\ — ~]~
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
L-Leucine
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Levallorphan Tartrate
100.0
75.0 -■
50.0 ~
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1458 Infrared Reference Spectra
Lidocaine
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
i r
1000.0
500.0
Lincomycin Hydrochloride Hydrate
100.0
75.0
50.0-
25.0-
0.0 - 1 1 1 1 1 p - 1 1 1 1 1 1 :
4000.0 3000.0 2000.0
Preparation of sample: Paste method
l 1 1 1 1 1 [ r
1500.0 1000.0
~> — r
500.0
Lisinopril Hydrate
100.0-
75.0-
50.0-
25.0
0.0 ") — i — i — i — i — \ — i — ! — i — i — —] — i — i — i — ~i — j— i — i — i — i — I"
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Paste method
1 1-
500.0
JPXV
Infrared Reference Spectra 1459
Lorazepam
100.0
75.0 -
50.0 -
25.0
- 1 i 1 1 r-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Loxoprofen Sodium Hydrate
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i 1 1 1 1 1 r
1500.0 1000.0
L-Lysine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1460 Infrared Reference Spectra
JP XV
D-Mannitol
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Maprotiline Hydrochloride
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
-™ — r ", - " r~ 1 ! 1 1 1 1 1 1 i 1 1 r 1 1 1 1 i -*——-,-- r 1 j
500.0
Mefloquine Hydrochloride
100.0-
75.0-
50.0-
25.0-
0.0
i 1 1 1 1 1 1 1 1 r r~ " ' i — "i — 'i ■ ■ i 1 1 1 1— I — ~i 1 1 r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
JPXV
Infrared Reference Spectra 1461
Mefruside
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Menatetrenone
100.0
75.0 -■
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
Mepitiostane
100.0
75.0
50.0
25.0
40O0.O 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
' — r
1000.0
500.0
1462 Infrared Reference Spectra
Mepivacaine Hydrochloride
100.0
75.0 -,
JP XV
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Mequitazine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Mestranol
100.0
75.0
50.0 ~
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JP XV
Infrared Reference Spectra 1463
Metenolone Acetate
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Metformin Hydrochloride
100.0
75.0
50.0-
25.0
i — ' — ' ' i — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
L-Methionine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1464 Infrared Reference Spectra
JP XV
Methotrexate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Methyldopa Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
d/-Methylephedrine Hydrochloride
100.0
75.0-
50.0
25.0
"i — r - '" i — i — | 1 r
4000.0 3000.0
i — | — i — i — i — i — r-
2000.0 1500.0
Preparation of sample: Potassium chloride disk method
i 1 1 1 1 r
1000.0 500.0
JPXV
Infrared Reference Spectra 1465
Methyl Parahydroxybenzoate
100.0 ■
75.0-
50.0-
25.0-
0.0-
t 1 1 1 1 1 1 1 r
i p 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
l I 1 p i 1 i I |~
1000.0 500.0
Methylprednisolone Succinate
100.0
75.0
50.0-
25.0 —
"i i r™ '"I j i i r — t r
4000.0 3000.0 2000.0
I 1 ' ] I I I " V | I I ' I ' ■ I ■" T"
1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Methyltestosterone
100.0
75.0-
50.0-
25.0-
t 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1466 Infrared Reference Spectra
JP XV
Meticrane
100.0
75.0
50.0
25.0
■~f " -t — t — ~i — '-"T — r 1 1 1 1 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
t— -i— — i r
500.0
Metildigoxin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Metoprolol Tartrate
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1 1 J 1 r
1500.0
1000.0
500.0
JPXV
Infrared Reference Spectra 1467
Metronidazole
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Mexiletine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500,0
Miconazole
100.0
75.0
50.0 -
25.0
0.0 -4
4000.0
3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1468 Infrared Reference Spectra
JP XV
Midecamycin
100.0
75.0
50.0
25.0 "
0.0
~1 T ™-y— " j— J-
3000.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Midecamycin Acetate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Minocycline Hydrochloride
100.0 - j
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1469
Mitomycin C
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i — | — i — i — i — i — | — i f r" ■ i — r
1500.0 1000.0 500.0
Morphine Hydrochloride Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Nalidixic Acid
i 1 1 1 j 1 1 1 1 1 1~ — r — r — ■ r" ■ | 1 1 i i "j 1 1 1 1 f
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
1470 Infrared Reference Spectra
JP XV
Naloxone Hydrochloride
100.0
75.0-
50.0
25.0
i 1 1 1 1 1 r~~
4000.0 3000.0
l" — r~ — i — ~i 1 1 — " i "' 1 T 1 — I — I — I — I — |-
2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
Naproxen
100.0
75.0 -
50.0 -
25.0
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
1 1 T~ 1-
1500.0
1000.0
Neostigmine Methylsulfate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1471
Nicardipine Hydrochloride
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000,0
500.0
Nicergoline
i 1 r ' • i — ~| 1 1 r
4000.0 3000.0
i 1 1 1 1 1 1 r
2000.0 1500.0
Preparation of sample: Potassium bromide disk method
i 1 1 1 1 1 r
1000.0 500.0
Niceritrol
100.0
75.0
50.0 -t
25.0
4000.0 3000.0 2000.0 1 500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1472 Infrared Reference Spectra
JP XV
Nicomol
100.0
75.0
50.0
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 ' — T
1000.0
500.0
Nicorandil
100.0
75.0-
50.0-
25.0-
0.0 — I — i — i 1 — i — i 1 — i — i 1 — I — i — i r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
1 ' <"
1500.0
i 1 1 1 1 r
1000.0 500.0
Nifedipine
100.0 -
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1473
Nilvadipine
100.0-
75.0-
50.0-
25.0-
o.o "|— "t ■ -i - T — T — p-- ",■■ ■ | — i — , — j — -, — i — i — i — j — i — i — i — i — i — i — i — i — i — r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Nitrendipine
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Noradrenaline
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 1"™ t r— -y—-r~—T™ r~ r~ y
1000.0 500.0
1474 Infrared Reference Spectra
JP XV
Norfloxacin
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Norgestrel
100.0
75.0
50.0
25.0
-^ r — .-i-p. , p
4000.0 3000.0 2000.0 1500,0
Preparation of sample: Potassium bromide disk method
-|- 1— r— : r j-
1000.0 500.0
Nortriptyline Hydrochloride
100.0
50.0
"I r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1475
Noscapine
100.0
75.0
4000.0 3000.0 2000.0 I5O0.0
Preparation of sample: Potassium bromide disk method
500.0
Ofloxacin
~~\ — r - — n — i — f — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — t — i — p
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Oxapium Iodide
100.0
75.0
50.0
25.0
— i
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
1476 Infrared Reference Spectra
JP XV
Oxaprozin
100.0
75.0
50.0 "
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Oxethazaine
100.0
75.0 -
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
- ' — r
1000.0
500.0
Oxprenolol Hydrochloride
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra \A11
Oxycodone Hydrochloride Hydrate
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Oxymetholone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Pancuronium Bromide
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
— i [-
1000.0
1478 Infrared Reference Spectra
JP XV
Penbutolol Sulfate
100.0
50.0
0.0 4-^
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
Pentobarbital Calcium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Pentoxyverine Citrate
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
JPXV
Infrared Reference Spectra 1479
Pethidine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Phenethicillin Potassium
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
L-Phenylalanine
100.0
75.0-
50.0
25.0
~i — ■■— i — ■ — i < p
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1480 Infrared Reference Spectra
JP XV
Phytonadione
100.0
75.0
50.0-
25.0
0.0
f* r ^fH)
i ■ " i ) •) [ — r " • i " " i 1 j r
1500.0 1000.0
•T ,,, " 1 "
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
500.0
Pindolol
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Pipemidic Acid Hydrate
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JPXV
Infrared Reference Spectra 1481
Piperacillin Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Piperazine Adipate
100.0
75.0
50.0 - 1
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Piperazine Phosphate Hydrate
0.0
-, r-.y . .j™™^- - _
3000.0
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
T- ■ t — ■ — r- p— "i"" ■ t ■ ■ i — —ir— |—
1500.0 1000.0
500.0
1482 Infrared Reference Spectra
JP XV
Pirenoxine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Pirenzepine Hydrochloride Hydrate
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
Piroxicam
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1483
Pivmecillinam Hydrochloride
100.0
75.0-
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
i — i — i — i — i — r
1000.0 500.0
Potassium Canrenoate
100.0
75.0
50.0 "
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Potassium Clavulanate
100.0
-1 [ ] 1 ]■
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1484 Infrared Reference Spectra
JP XV
Povidone
100.0
75.0 "
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Pranoprofen
100.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Pravastatin Sodium
100.0
75.0
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 I i i | I r
1000.0
JPXV
Infrared Reference Spectra 1485
Prazepam
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Prednisolone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Prednisolone Acetate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1486 Infrared Reference Spectra
JP XV
Prednisolone Succinate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 J 500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Procaine Hydrochloride
100.0
75.0
50.0
25.0 -
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Procarbazine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1487
Procaterol Hydrochloride Hydrate
100.0
75.0^
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Progesterone
100.0
75.0
50.0
25,0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Proglumide
— r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1488 Infrared Reference Spectra
JP XV
Promethazine Hydrochloride
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Propranolol Hydrochloride
100.0
75.0
50.0
25.0-
0.0
i 1 1 1 1 r i i r 1 i 1 1 1 1 1 1 1 1 p
4000.0 3000.0 2000.0 15O0.0 1000.0
Preparation of sample: Potassium chloride disk method
1 ■ r
500.0
Propyl Parahydroxybenzoate
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1489
Protirelin
100.0
75.0
50.0
25.0
0.0
1 f-™~-j T — - — j r~~~i T^-^—t. ■* -T-— — [ ■ — 1 -t \ f 1 — I r ■ ■ i ^t r i i '" " "i
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000,0
500.0
Pyrantel Pamoate
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Pyrazinamide
100.0
75.0-
50.0
25.0-
-\ ! 1 1 j 1 1 1 ; 1 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
-i 1 1 1 1 r
1000.0 500.0
1490 Infrared Reference Spectra
JP XV
Pyridoxine Hydrochloride
100.0
75.0
50.0
25.0-
i — i — | — i — r — i — i — | 1 — i — '"! i — f
2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
~1 i ! I f— "~ t ~ 1 r
4000.0 3000.0
Pyrrolnitrin
100.0
75.0 -
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Quinine Ethyl Carbonate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
JPXV
Infrared Reference Spectra 1491
Quinine Sulfate Hydrate
100.0
75.0
50.0-
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ranitidine Hydrochloride
4000.0 3000.0 2000.0
Preparation of sample: Paste method
i 1 i 1 1 1 1 1 1 1 1 1 r
1500.0 1000.0 500.0
Reserpine
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
1492 Infrared Reference Spectra
JP XV
Rifampicin
100.0
75.0
50.0
0.0
■ [■■■■! , — i j 1 1 1 1 1 1 1 ■ i I 1 1 1 \ 1 1 1 1 1 j
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Ritodrine Hydrochloride
100.0
75.0-
50.0-
25.0-
t 1 1 1 1 1 1 1 1 1 — i — i — i — i — | — i — r ■■ ■ r — i — | — i — i — n — ~i — f
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium chloride disk method
Rokitamycin
100.0-
75.0-
50.0-
25.0-
o.o-
i 1 1 1 ] 1 1 1 r
4000.0 3000.0
Preparation of sample: Potassium bromide disk method
r ■-■[■■ ■ i 1 1 1 1 1 1 [ — l 1 1 1 — |-
2000.0 1500.0 1000.0 500.0
JPXV
Infrared Reference Spectra 1493
Roxatidine Acetate Hydrochloride
~\ 1 1 1 1 1 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
Roxithromycin
100.0
75.0 -
50.0
25.0
0.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Saccharin
n 1 1 1 1 r " " i " " 'I 1 1 — I — 1 — I — I — 1~
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
~| i i i r
3000.0 2000.0
1494 Infrared Reference Spectra
Saccharin Sodium Hydrate
JP XV
' i i i ; ~~ ~i I 1 I
1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
"i i i i | i i i r
4000.0 3000.0 2000.0
Salbutamol Sulfate
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Santonin
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1495
Scopolamine Butylbromide
100.0
75.0
50.0
25.0 i
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
r , r , , ,- -p
1000.0 500.0
Siccanin
100.0
75.0
50.0
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 r— r
500.0
Sodium Fusidate
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1496 Infrared Reference Spectra
JP XV
Sodium Picosulfate Hydrate
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sodium Polystyrene Sulfonate
100.0
75.0 -
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sodium Prasterone Sulfate Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
500.0
JPXV
Infrared Reference Spectra 1497
Sodium Salicylate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sodium Valproate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
Spiramycin Acetate
100.0
75.0
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1498 Infrared Reference Spectra
JP XV
Spironolactone
1O0.O -,
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sulbactam Sodium
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sulbenicillin Sodium
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1499
Sulfadiazine Silver
100.0
75.0
50.0 '
25.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
Sulfamethizole
100.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sulfamethoxazole
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1500 Infrared Reference Spectra
JP XV
Sulfamonomethoxine Hydrate
100.0
75.0-
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
500.0
Sulfinpyrazone
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Sulpiride
i 1 1 1 — \ 1 1 1 r~"| 1 1 — i 1 — [ — i 1 1 1 — i — i 1 — r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
JPXV
Infrared Reference Spectra 1501
Sultamicillin Tosilate Hydrate
100.0
50.0
4000.0 3000.0 2000.0
Preparation of sample: Paste method
1500.0
1000.0
500.0
Suxamethonium Chloride Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Talampicillin Hydrochloride
100.0
75.0
50.0-
25.0-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1502 Infrared Reference Spectra
JP XV
Tamsulosin Hydrochloride
100.0-
75.0-
50.0-
25.0-
0.0-
l 1 1 1 1 1 1 1 1 1 1 1 1 p — 1 1 1 r 1 — i — i — i—~i — 1~
3000.0 2000.0 1500.0 1000.0 500.0
4000.0
Preparation of sample: Potassium chloride disk method
Taurine
~i ^ i i | i i i i | r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
r | i 1 1 1 1 1 1 1 r
1500.0
1000.0
500.0
Tegafur
100.0 -r
75.0 -
50.0
25.0
i — <-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JP XV
Infrared Reference Spectra 1503
Testosterone Propionate
100.0
75.0
50.0-
25.0
i ! 1 1 1 1 1 ! 1 1 1 1 f 1 1 1 1 1 1 1 i 1 1 1 f
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Tetracycline Hydrochloride
100.0
75.0 -
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Theophylline
100.0
75.0-
50.0-
25.0-
0.0
i i i i i i i i i ] i i i r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
l i i f
~i 1 1 1 f
1000.0 500.0
1504 Infrared Reference Spectra
Thiamine Chloride Hydrochloride
JP XV
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Thiamylal Sodium
100.0
75.0
50.0
25.0-
0.0 I i 1 1 i j i 1 r 1 J— — i 1 1 1 [ 1 1 1 r — i 1 r— i — i p
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Thioridazine Hydrochloride
100.0
75.0 -
50.0
25.0-1
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1505
L-Threonine
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i r
1500.0
1000.0
500.0
Tiaramide Hydrochloride
100.0 -r-
75.0
50.0
25.0
0.0
~\ — <—
3000.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Ticlopidine Hydrochloride
100.0
75.0
50.0
25.0
i — ' ■■■ v
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1506 Infrared Reference Spectra
JP XV
Timepidium Bromide Hydrate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 , T ■' ■. 1 r
1000.0 500.0
Timolol Maleate
100.0-
75.0-
50.0-
25.0-
0.0 — I 1 1 1 1 j 1 ' ' • r — i r
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i i i i i j l r r l |~
1500.0 1000.0 500.0
Tinidazole
100.0-
75.0-
50.0-
25.0-
0.0
i 1 1 1 | 1 1 1 1 1 1 1 1 1 1 1 1 r i "i i 1 r— r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1507
Tipepidine Hibenzate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
Tizanidine Hydrochloride
100.0
75.0
50.0-
25.0-
-1 1 1 1 1 1 1 1 1 1 1 1 1 r 1 r— ~t — n r— | r—
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium chloride disk method
Tocopherol
100.0
75.0
50.0
25.0 -
0.0 -h-
4000.0
3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
1508 Infrared Reference Spectra
JP XV
Tocopherol Acetate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1 ' r "
1500.0 1000.0
500.0
Tocopherol Calcium Succinate
100.0
75.0
50.0
25.0
— p 1 r t T~"
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Liquid film method (measured as a solution of 0.08 g of Tocopherol Calcium Succinate in
0.2 mL of carbon tetrachloride)
Tocopherol Nicotinate
100.0
75.0
50.0
25.0 -1
4000.0 3000.0 2000.0
Preparation of sample: Liquid film method
1500.0
1000.0
500.0
JPXV
Infrared Reference Spectra 1509
Todralazine Hydrochloride Hydrate
1 00.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Tofisopam
100.0
75.0
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Tolazamide
100.0
75.0 -
50.0
25.0 -
0.0 -t
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1510 Infrared Reference Spectra
Tolnaftate
100.0
75.0
JP XV
50.0
25.0
0.0
4000,0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
I sS^^
¥1 /
1
1
I!
Iff
i — i —
if "i
■ I , A...
V
1 1 1 1 i 1
500.0
Tranexamic Acid
0.0
r" [ "i"' — t 1 1 ] 1 1 1 1 1 1 1 ! 1 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
Triamcinolone
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1511
Triamcinolone Acetonide
100.0
75.0
50.0
25.0
"T — ' ■ '' ' ' 1 ' ' ' 1— T
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
T r-~ 1 1 1 V
1000.0 500.0
Trichlormethiazide
100.0
75.0-
50.0
25.0
-\ r~ — i 1 1 f i i ■■ t 1"" ' i i r r~~ ~ ] 1 i~™t 1 1 1 1 r
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Potassium bromide disk method
500.0
Triclofos Sodium
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1512 Infrared Reference Spectra
JP XV
Trimebutine Maleate
100.0
75.0-
50.0-
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
Trimetazidine Hydrochloride
100.0
75.0-
50.0 -;
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
Trimethadione
100.0
50.0 -
0.0 -
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Solution method (measured as a solution in chloroform)
JPXV
Infrared Reference Spectra 1513
Trimetoquinol Hydrochloride Hydrate
100.0
75.0
50.0
25.0
4000.0 3000,0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
500.0
L-Tryptophan
100.0
75.0
50.0 -
25.0 -
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Tulobuterol Hydrochloride
100.0
75.0 "l
50.0
25.0
-f f 1 p , .^ r ._ — r — -^ — T -jn-^^ JT - r — . — r , j-
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
1514 Infrared Reference Spectra
JP XV
Ubidecarenone
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Urapidil
100.0
75.0-
50.0-
25.0-
0.0
i 1 1 1 1 1 1 1 1 1 1 1 1 1 j 1 1 r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 r
1000.0
i r
500.0
L-Valine
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
JPXV
Infrared Reference Spectra 1515
Vancomycin Hydrochloride
100.0
75.0
50.0
25.0
0.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Verapamil Hydrochloride
100.0
75.0
50.0 -
25.0
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium chloride disk method
1000.0
Vinblastine Sulfate
100.0
75.0-
50.0-
25.0-
0.0
i — i — i — i
i 1 1 1 1 i r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1 I 1 1 [ 1 1 I 1 " [
1000.0 500.0
1516 Infrared Reference Spectra
JP XV
Vincristine Sulfate
100.0
75.0
50.0
25.0
4000.0 3000.0 2000.0 1500.0 1000.0
Preparation of sample: Solution method (measured as a solution in chloroform)
500.0
Voglibose
100.0
75.0
50.0
25.0-
0.0
i — i — -i — i — r
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
~\ 1 1 1 ! 1 1 r
1000.0 500.0
Warfarin Potassium
100.1
0.0
i — i — i — i — i — i — i — i — i — i — \ — i — i — i — p — r — ■ — ' — ' — i — ' — ' — T ~~ ' — r
4000.0 3000.0 2000.0 1500.0 1000.0 500.0
Preparation of sample: Potassium bromide disk method
JPXV
Infrared Reference Spectra 1517
Xylitol
100.0
75.0 ~
50.0
25.0 -
0.0^
4000.0 3000.0 2000.0 1500.0
Preparation of sample: Potassium bromide disk method
1000.0
500.0
Zaltoprofen
100.0
75.0
50.0
25.0-
0.0
4000.0 3000.0 2000.0
Preparation of sample: Potassium bromide disk method
i 1 1 1 1 1 1 1 1 1 1 r
1500.0 1000.0 500.0
JP XV
Ultraviolet-visible Reference Spectra 1519
Acebutolol Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 f 1 i 1 1 1 r v ■ l ■ — i r t — ™i r-™i r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 100,000)
Acetohexamide 1
0.75 ~
0.25
- i 1 1 1 1 1 r r — t ■■] t 1 1 1 1 —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.10 g in 100 mL of methanol. To 5 mL of this solution add 20 mL of 0.5
mol/L hydrochloric acid TS and 75 mL of methanol (Sample solution 1).
Acetohexamide 2
1.0
-| — i — i — i — i — ] — r — i — i — i — i — i — i — i — i — prn — i — i — | — i — i — i — ■ — | — i — i — i — i — | — 1 — i — i — r-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 10 mL of Sample solution 1 add methanol to make 50 mL (Sample solution 2).
1520 Ultraviolet-visible Reference Spectra
JP XV
Aclarubicin Hydrochloride
2.0
-\ — i — i — i — i — i — i — i — i — | — i — r~i — i "i ■ i — i — i — i — | — i — i — r
200.0 300.0 400.0
A solution in diluted methanol (4 in 5) (3 in 100,000)
500.0
600.0
Acrinol Hydrate
1.0
0.75
0.5
0.25 —
0.0
r i i i i i [ i i T"i i i i i i | i i i i i i i i i | i i TTl i i i i
200.0 250.0 300.0
An aqueous solution (3 in 250,000)
350.0
400.0
450.0
500.0
Actinomycin D
1.0
0.75-
0.25-
i — i — i — i — i — i — i — i — I — | — I — I — I — I — I — i — i — I — I — |~i — i — i — r — l — i — i — i — i — | — i — i — i — i — r
200.0 300.0 400.0 500.0 550.0
A solution in methanol (3 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1521
Afloqualone
1,0
0.75
0.5
0.25
0.0
1 — i — l — i — r — r — r™i — i — i — n — < — i — r • ■ i ■ r t" ' — i — 1 — i — i — i — i — — i — i — i — r — 1 — i — i — i — r — 1 — i — i T i" "h — \ — i — i — i — i — i — t — i — i —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in ethanol (99.5) (1 in 150,000)
Alimemazine Tartrate
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0
An aqueous solution (1 in 100,000)
325.0 350.0 375.0 400.0
Allopurinol
1.0
0.75
0.5
0.25
0.0
T 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 200,000)
1522 Ultraviolet-visible Reference Spectra
JP XV
Alprazolam
1.0
0.75
0.5
0.25
0.0
0.75
0.5
0.0
r 1 : ' i r — ~r — t 1 i H — r 1 r^-\ 1 1 1 ^ i T — i 1 1 1 ■ 1 1 ■ r 1 1 1 r -t _ -t
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 200,000)
Alprenolol Hydrochloride
1.0
1" 1 1 1 i 1 1 1 1 r T 1 7
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 10,000)
Alprostadil
1.0
0.75-
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution prepared as follows: To 10 mL of a solution in ethanol (99.5) (1 in 100,000) add 1 mL of potassium
hydroxide-ethanol TS, and allow to stand for 15 minutes.
JP XV
Ultraviolet-visible Reference Spectra 1523
Alprostadil Alfadex
1.0
0.75
0.5
0.25
0.0
— 1 T" — I - ' 'i r 1 r 1 1 1 1 1 r 1 — ~r — t r~r 1 i 1 1 1 t r™i 1 — i i i i i i i ■ -f - ■ r = *r* :
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 10 mL of a solution in dilute ethanol (3 in 10,000) add 1 mL of potassium
hydroxide-ethanol TS, and allow to stand for 15 minutes.
Ambenonium Chloride
1.0
0.75
0.5
0.25
0.0
:
200.0 225.0 250.0 275.0
A solution in methanol (1 in 5000)
300.0
325.0 350.0
375.0
400.0
Amitriptyline Hydrochloride
1.0
0.75 -
0.5 -
0.25 - 1
0.0
r — i 1 1 1 1 1 1 1 1 1 — ■ r t
200.0 225.0 250.0
An aqueous solution (1 in 100,000)
— r 1 — ~i— i T I I — r"
275.0 300.0
-I i 1 1-
— i 1 1 r-
325.0 350.0
375.0
400.0
1524 Ultraviolet-visible Reference Spectra
JP XV
Amoxapine
1.0
0.75
0.25 -
-i i . i ■< -> — ■ — i — i — < — i — > — ' — ; — i ■ - ■ i — i — r
200.0 225.0 250.0 275.0 300.0 325.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 50,000)
350.0
375.0
400.0
Amphotericin B
-r— i i i i j— t i i i i i i i | i i i i | i i i i | i—i . p |-
250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 25 mg in 5 mL of dimethylsulfoxide, and add methanol to make 50 mL. To
1 mL of this solution add methanol to make 50 mL.
Arotinolol Hydrochloride
1.0
0.75
0.25
0.0
200.0 225.0 250.0 275.0
A solution in methanol (1 in 75,000)
300.0 325.0
350.0
375.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1525
Aspoxicillin Hydrate
1.0
0.75
0.5
0.25
0.0
T— | 1 1 1 1 1 -r- i 1 1 1 r—i 1 1 1 1 1 1 r
300.0 325.0 350.0 375.0 400.0
200.0 225.0 250.0 275.0
An aqueous solution (1 in 4000)
0.75
0.25 —
t — r
200.0 250.0
A solution in methanol (1 in 50,000)
300.0
350.0
400.0
Azathioprine
1.0
0.75
0.5
0.25
0.0
t — i — i — i - -i — i — i — i — i — I — t-~t — i — i — I — t — i — r — i — r-^i — i — i — i — i — i — i — i — i — i — i — i — f~i — i — i — i — i — i — i — i — l — i — i — r~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: To 5 mL of a solution in 2 mol/L hydrochloric acid TS (1 in 10,000) add water to
make 50 mL.
1526 Ultraviolet-visible Reference Spectra
JP XV
Aztreonam
1.0
0.75 -
0.25
1 1 1 I . 1 1 . 1 1 1 [ 1 1 1 r ; 1 1-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (3 in 100,000)
Bacampicillin Hydrochloride
1.0
0.5
0.25
0.0
1 r 1 r i — "f nn 1 r — i 1 1 r~H — ~-r 1 1 f 1 1 p 1 r — i r"n — -i-~-i , "'t T 1 — i I I i T 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 1000)
Baclofen
1.0
0.75
0.25
-| 1 . 1 1 1 1 1 1- ■ i - | r--| 1 . 1 i . 1 . J— 1 r— i 1 1 1 1 1 1 1 1 1 i-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 2000)
JP XV
Ultraviolet-visible Reference Spectra 1527
Bamethan Sulfate
1.0
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
Benidipine Hydrochloride
1.0
0.75-
0.5-
0.25-
0.0-
t — i — r " r ' i — r
200.0
A solution in methanol (1 in 100,000)
i — i — | — r i ' i ' t — i — i — r~i — i — | — r
250.0 300.0
i — r~i — r i" i — t — j t r "v
350.0
400.0
Benserazide Hydrochloride
1.0
0.75
0.5
0.0
1 1 r 1 i 1 1 1 r i r 1 1 1 i ■■ t ■ r i \1 i i 1 1 r 1 1 1 1 1 i " r 1 1 r " 'r T"
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 10,000)
1528 Ultraviolet-visible Reference Spectra
JP XV
Benzalkonium Chloride
1.0
0.75
0.5
0.25
0.0
r 1 1 r" — i 1 1 i ■ H 1 1 1 — ^" — r~i 1 ; ■■ ■ i 1 r t — ' i r r 1 1 i 1 1 1 1 i 1 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid (1 in 2000)
Benzbromarone
1.0
0.75-'
0.25-
A solution in 0.01 mol/L sodium hydroxide TS (1 in 100,000)
Benzethonium Chloride
1.0
0.75 -
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 5000)
JP XV
Ultraviolet-visible Reference Spectra 1529
Benzylpenicillin Benzathine Hydrate
1.0
0.75 —
0.5 —
0.25 -
0.0 — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0
A solution in methanol (1 in 2000)
1 — I | I I — I — I — I — I — I — I — r
350.0 400.0
Benzylpenicillin Potassium
1.0
0.75
0.5-
0.25-
0.0
T — I — I — I 1 — 1 — I — I — I — j — I — I — 1 — I II I I I | I I ! I I I I I I — f
i \ i i i i i i r
200.0 250.0
An aqueous solution (1 in 1000)
300.0
350.0
400.0
Berberine Chloride Hydrate
1.0
0.75
0.5
0.25
0.0
200.0 250.0 300.0
An aqueous solution (1 in 100,000)
350.0
400.0
450.0
500.0
1530 Ultraviolet-visible Reference Spectra
JP XV
Berberine Tannate
2.0
1.5 -
1.0 -
0.5
0.0
200.0
250.0
300.0
350.0
400.0
450.0
500.0
A solution prepared as follows: Dissolve 0.01 g in 10 mL of methanol and 0.4 mL of 1 mol/L hydrochloric acid TS,
and add water to make 200 mL. To 8 mL of this solution add water to make 25 mL.
Betahistine Mesilate
1.0
0.75 —
0.5 —
0.25 —
0.0 "I — i — i — i — i — i — i — i — i — i — I — r~r
200.0 250.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 50,000)
i ! — l — I — I — I — | — I — I — I — I — I — I — i — l — 1 — | — I — I — I — I — I — I — I — I — r
300.0 350.0 400.0
Betamethasone
1.0
0.75 -
0.25
250.0
300.0
350.0
400.0
450.0
500.0
550.0
A solution prepared as follows: Dissolve 1.0 mg in 10 mL of ethanol (95). To 2 mL of this solution, add 10 mL of
phenylhydrazine hydrochloride TS, shake, heat in a water bath at 60°C for 20 minutes, and allow it to cool.
Blank: A solution obtained by proceeding in the same manner with 2.0 mL of ethanol (95).
JP XV
Betamethasone Dipropionate
1.0
0.75
Ultraviolet-visible Reference Spectra 1531
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (3 in 200,000)
Bezafibrate
1.0
0.75
0.25-™
200.0
A solution in methanol (1 in 100,000)
l — ] — I — i — r
350.0
400.0
Bifonazole
1.0
0.75 -
0.5 —
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
1532 Ultraviolet-visible Reference Spectra
Biperiden Hydrochloride
1.0
JP XV
0.75 -
0.5 -
0.25
0.0
-i 1 1 1 1 r— -r
i — i — i — r — p
~i — i — r
T f 1 1 1 1 1 1 1 1 1 1 r™
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2000)
Bisacodyl
1.0
0.75
0.25 -
-. r I | r I I ■!■■■]■ i r— i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (3 in 100,000)
Bleomycin Hydrochloride
1.0
0.75 —
0.5 -
0.25
0.0 ■ [ [ ■■[■ -) — i — i — i — i — i — i — I — i — i — i — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i — i | I — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.O
A solution prepared as follows: To 4 mg add 5 /uL of copper (II) sulfate TS, and dissolve in water to make 100 mL.
JP XV
Ultraviolet-visible Reference Spectra 1533
Bleomycin Sulfate
1.0
0.75 —
0.5
0.25
0.0
A solution prepared as follows: To 4 mg add 5 /uL of copper (II) sulfate TS, and dissolve in water to make 100 mL.
Bromazepam
1.0
0.75
0.25 -■
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 200,000)
Bromhexine Hydrochloride
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0
A solution in 0.01 mol/L hydrochloric acid TS (3 in 100,000)
1 — ' — ' — ' — ' — I — i — ' — i — <~
350.0 375.0 400.0
1534 Ultraviolet-visible Reference Spectra
JP XV
Bromocriptine Mesilate
1.0
0.75
0.5
0.25
0.0
-
1 \ j
■ i -- i i j i ii r ■ — ! 1 i i i 7-1 i- r ,■
, ■ I I I | I" , I T -
1 << '
1 1 1 1
200.0 225.0 250.0 275.0
A solution in methanol (3 in 100,000)
300.0 325.0 350.0 375.0
400.0
Bucumolol Hydrochloride
1.0
200.0 225.0 250.0 275.0
An aqueous solution (1 in 60,000)
300.0 325.0 350.0 375.0
400.0
Bufetolol Hydrochloride
0.75
0.25
-| i i i i | i r i i j-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 20,000)
JP XV
Ultraviolet-visible Reference Spectra 1535
Bufexamac
1.0
0.75
0.5
0.25 -
0.0
-^ p 'J ■ t ■- i 1 1 f
200.0 225.0 250.0 275.0
A solution in ethanol (95) (1 in 100,000)
300.0 325.0 350.0 375.0
400.0
Bumetanide
1.0
0.75 —
0.25 -
200.0
225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.04 g in 100 mL of phosphate buffer solution, pH 7.0. To 10 mL of this
solution add water to make 100 mL.
Bupranolol Hydrochloride
1.0
0.75
0.5
0.25
0.0 ~~ I — ' — ' — ' — ' — i — ' — ' — ' — ' — i — ' — ' — ' — ' — r -1 — ■ — ' — ' — i — ' — ' — ' — ■ — i — ' — > — ' — ■ — i — ■ — ' — ■ — ■ r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 10,000)
1536 Ultraviolet-visible Reference Spectra
Butropium Bromide 1
1.0
0.75
JP XV
0.5
0.25
0.0
I i I i I
: : : :
• : : : : !
1 1 1 1 i 1 1 1 I — -^ ^i i i i** t fc — i 1 1 i 1 1 1 1 i 1 1 1 1 i — ■ * 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Butropium Bromide 2
1.0
0.75 ~
0.5
0.25
0.0
r — i — I--- i -[ — i — i 1 — -t — p ^i — 3 — i — i — | — i — i — T I | — r — i — i — r — | — 1 — r — t — i — p*-n — i — i 1 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 5000)
Calcium Folinate
1.0
0.75
0.25
-| 1 1 1 r 1 p 1 1 1 1 1 1 1 r J 1 r 1 1 j 1 — t— - 1 r 1 1 1 1 1 [-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
JP XV
Camostat Mesilate
1.0
0.75
Ultraviolet-visible Reference Spectra 1537
0.5
0.0
2
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
Carbamazepine
1.0
0.75
0.5
0.25
0.0
;/ \ | | ||
; ^""~\ \
i i i i i i i i i
, , -, , j , . , , i , . , , j , , . , j ■■ ■ r . , | . . i i i i i i i
200.0 225.0 250.0 275.0
The sample solution obtained in the Assay
300.0 325.0 350.0 375.0
400.0
Carbazochrome Sodium Sulfonate Hydrate
1.0
0.75
0.25 "
—] 1 1 1 1 1 "I" — t — t — t r
200.0 250.0 300.0 350.0
An aqueous solution (1 in 100,000)
400.0
450.0
500.0
550.0
1538 Ultraviolet-visible Reference Spectra
JP XV
Carbidopa Hydrate
1.0 1
0.75
0.5
0.25
0.0
- r 1 r r t j f r 1 r~
~t 1 1 1 1 1 1 r
i r- i -i-| ■-« -.■ . i 1— . i.i,.
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.01 g in 250 mL of a solution of hydrochloric acid in methanol (9 in 1000)
Carmofur
1.0
o.o -*-\
225.0 250.0
-r — l [ 1 1 1 1 1 1 1 1 1 1-
275.0 300.0 325.0 350.0 375.0 400.0
A solution in a mixture of methanol and phosphoric acid-acetic acid-boric acid buffer solution, pH 2.0 (9:1) (1 in
100,000)
Carteolol Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 1 1
....
... 1
1 — ' ' ■ '
....
1 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1539
Carumonam Sodium
1.0
0.75-
0.5-
0.25 —
0.0
i — i — i — i — i — i — i — i — | — i — i — r
200.0 250.0
An aqueous solution (3 in 100,000)
1 — I — I — I — I — | — I — I — I — I — I — I — I — I — I — | — I [ I I I — I — I — I — r
300.0 350.0 400.0
0.75-
025
i — i — rn — i — i — i — i — i — ) — i — l — i — i — i — i — i — i — i — | I I — i — i — i — i — i — i — i — | — i — i — i — r
200.0 250.0 300.0 350.0
An aqueous solution (1 in 50,000)
400.0
Cefadroxil
1.0
0.75
0.25
i ' ' ' ' i
200.0 225.0 250.0 275.0
An aqueous solution (1 in 50,000)
I — i — i 1 — | — i — i — i — i — r
300.0 325.0 350.0 375.0 400.0
1540 Ultraviolet-visible Reference Spectra
JP XV
Cefalexin
1.0
0.75
05
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (3 in 100,000)
Cefalotin Sodium
1.0
0.75-
0.25-
200.0
An aqueous solution (1 in 50,000)
Cefapirin Sodium
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (3 in 200,000)
JP XV
Cefatrizine Propylene Glycolate
1.0
Ultraviolet-visible Reference Spectra 1541
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Cefazolin Sodium
—y 1 1 1 1 1 t t ■ "i 1 1 1 1 1 1 1 1 1 1 1 1—
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Cefbuperazone Sodium
1.0
0.75-
0.25-
An aqueous solution (1 in 50,000)
1542 Ultraviolet-visible Reference Spectra
JP XV
Cefditoren Pivoxil
1.0
0.75
0.5
0.25 —
0.0 — 1~~ i — n — n — n — i — i | i — i — n — i — r~i — i i | i i — i — n — i — n — i | i — i — n — i — i i i — i ""["1 — n — i — n — i — r— r
200.0 250.0 300.0 350.0 400.0 450.0
A solution in methanol (1 in 50,000)
Cefixime
1.0
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0
A solution in 0.1 mol/L phosphate buffer solution, pH 7.0 (1 in 62,500)
' — ■ — r
375.0
400.0
Cefmenoxime Hydrochloride
1.0
0.75-
0.25-
0.0
T — f
210.0
-i — i — i" | r ™ i — {•■■•]■■ rn — i — r~\ — r-
250.0 300.0
i — i — i — i — | — i — n i i — i — r
350.0
A solution in 0.1 mol/L phosphate buffer solution, pH 6.8 (3 in 200,000)
400.0
JP XV
Cefmetazole Sodium
1.0
Ultraviolet-visible Reference Spectra 1543
0.75
0.5
0.25
0.0
-
."^^
I 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 40,000)
Cefminox Sodium Hydrate
1.0
0.75 -
0.25-
— i 1 r r 1 t— — r 1 — t r 1 1 — t r — T^~f — f — t — i-"T'
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Cefodizime Sodium
1.0
0.75 —
0.25
v r-pi — i — i — i —
200.0 250.0
An aqueous solution (1 in 50,000)
1 — I — I — j — I — I — I — I — I — I — 1 I T | I — 1 — I — I — [ — I — I — 1 — r
300.0
350.0
400.0
1544 Ultraviolet-visible Reference Spectra
JP XV
Cefoperazone Sodium
1.0
0.75
0.5
0.25
0.0
\ ->-~~-***.
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Cefotaxime Sodium
1.0
0.75
0.5 —
0.25 —
0.0
i — m — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — r
200.0 250.0 300.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 50,000)
i i i I I | — I — i — i — i — i — i — i — i — r
350.0 400.0
Cefotetan
1.0
~i — i — i — i — i — i — t — i — i — ] — t— r — i — i — i — i — i — i — i — i — i — i — i — i — r~i — i — r~~i — | — r
200.0 250.0 300.0 350.0
i — i — i — i — i — r
400.0
A solution in phosphate buffer solution for antibiotics, pH 6.5 (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1545
Cefotiam Hexetil Hydrochloride
1.0
0.75-
0.25-
~\ — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — j — i — i — i — i i I I — I — I — | — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 125,000)
Cefotiam Hydrochloride
1.0
0.75
0,25
— i 1 1 1 1 p r 1 [ 1 t 1 1 1 1" — i r — 1™ i 1 1 I I ^ ■ i — "-\ 1 1 p
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Cefpiramide Sodium
1.0
0.5
0.25
0.0
- V
1 — i — rr ■! ■■■! — i — i — i — j — i — r — i' '"i" i — i - i ■ i — i i — tt^t — i — l — i — i — i — i — j — i — i — i — i — i — i — i — i — i —
200.0 250.0 300.0 350.0 400.0
A solution in 0.05 mol/L phosphate buffer solution, pH 7.0 (1 in 50,000)
1546 Ultraviolet-visible Reference Spectra
JP XV
Cefpodoxime Proxetil
1.0
0.75 —
0.5 -,
0.25-
0.0 ' -| , 'T I T I I 1 111(11 II II I I I j I I I I' 1 T I ' I I | I I T T P I I I ] - } I I I II I I I I
200.0 250.0 300.0 350.0 400.0 450.0
A solution in acetonitrile (3 in 200,000)
Cefroxadine Hydrate
1.0
0.75-
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — r
200.0 250.0 300.0
A solution in 0.001 mol/L hydrochloric acid TS (1 in 50,000)
1 — I — I — I — I — r
350.0
400.0
Cefsulodin Sodium
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
JP XV
Ultraviolet-visible Reference Spectra 1547
Ceftazidime Hydrate
1.0
0.75
0.5
0.25
0.0
i ! : :
i ] : i i
t 1 — t — i i 1 1 1 [ r ' r 11 — i 1 1 f 1 1 1 1 1 1 1 1 1 — i r ^T""l 1 r 1 1 i 1 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in phosphate buffer solution, pH 6.0 (1 in 100,000)
Cefteram Pivoxil
1.0
0.75-
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i i i i — i — i — r
200.0 250.0 300.0 350.0
A solution in 0.05 mol/L hydrochloric acid-methanol TS (1 in 100,000)
i — i — i — i — i — r
400.0
Ceftizoxime Sodium
1.0
0.75
0.5
0.25
0.0
~ ! i !
1 1 "' i 1 i 1 1 1 i~H — n j 1 1 i 1 1 r — i " i r — i 1 1 i f 1 — -r^^ — I I i r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 63,000)
1548 Ultraviolet-visible Reference Spectra
JP XV
Ceftriaxone Sodium Hydrate
1.0
0.75
0.5 —
0.25 —
0.0 — ' — i — i — i — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i — i — : — i — i — i — i — i — i "1 i I I — i — i — i — r i -i — i — i — r
200.0 250.0 300.0 350.0 400.0
An aqueous solution (1 in 100,000)
Cefuroxime Axetil
1.0
0.75 —
0.25
-\ — i — i — i — i — i — i — i — i — | — i — r
200.0 250.0
A solution in methanol (3 in 200,000)
1 — I — I — I — I — | — r
350.0
400.0
Cefuroxime Sodium
1.0
0.75
0.5
0.25
0.0
1 1 1 1 1 1 1 1 f
' ' ' '
1 1 1 I
1 1 I 1
.11.
III.
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1549
Cetraxate Hydrochloride
1.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 2500)
Chloramphenicol
1.0
0.75 —
0.25-
"i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — r
200.0 250.0 300.0
The sample solution obtained in the Assay
Chloramphenicol Palmitate
1.0
0.75
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — | — r
200.0 250.0
i — i — i — i — i — i — | — i — i — i — i — i — i i i i | I I I I I — I — i — i — r
300.0
350.0
400.0
A solution in ethanol (99.5) (1 in 33,000)
1550 Ultraviolet-visible Reference Spectra
Chloramphenicol Sodium Succinate
0.75-
JP XV
0.25
0.0 — I — i — r
200.0
An aqueous solution (1 in 50,000)
1 — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — r^r
250.0 300.0 350.0
r~i — i — n~
400.0
Chlordiazepoxide
1.0
0.5
0.25
0.0
■ !
i i i
I I I j
—
^\ \ / \ : !
— ^v
1 1 — T ' i ' — j 1 1 1 1 j 1 r t ■ t j " t — t~*t r — j r— "H r — r ■■■ j ■ i ■■■ r — t — t
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 200,000)
Chlorphenesin Carbamate
1.0
0.75
0.5
0.25
0.0
1 1 — i — i j 1 — i 1 1 r — r r - r i — j — i — i 1 i — i — i — i — i — i 1 1 — i 1 — i j — i — i — i 1 — i — i — I
200.0 225.0 250.0 275.0
A solution in ethanol (95) (3 in 200,000)
300.0 325.0 350.0
375.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1551
Chlorpheniramine Maleate
1.0
0.75
0.25 -
t — n — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — ' — i - 1
200.0 250.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
1 — ( — | — 111 — I — I — I — I — I — r
350.0 400.0
(/-Chlorpheniramine Maleate
1.0
0.75 —
0.5 —
0.25 —
0.0
i — r~r — r~i — i — i — i — i — | — r— i — i — i — i — i — i — i — i f~"r i — I — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
350.0
400.0
Chlorpropamide
1.0 _
i
0.75 _
0.5 _
0.25 _
nr\
1 F < ■ | | | . ,- , , . , ,
— 1 . , T
.,. ., . , ,.
■ I r- r -t
. T . ! . .., ..,-. ..
— 1 1 r 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.08 g in 50 mL of methanol. To 1 mL of this solution add 0.01 mol/L
hydrochloric acid TS to make 200 mL.
1552 Ultraviolet-visible Reference Spectra
Cilostazol
1.0
0.75 -
JP XV
0.5
0.25 —
0.0
i — i — [ — i — i — i — i — i — i — i — i — i — i — i — i — i — i — rr — i — i — i — i — i — l — | — [ — r — i — i — \ — i — r
200.0 250.0 300.0 350.0
A solution in methanol (1 in 100,000)
400.0
Cisplatin
1.0
0.75
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r — i — i — r~-r — \ T"] i — i — i — i — t i -\ — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in a solution of sodium chloride in 0.01 mol/L hydrochloric acid TS (9 in 1000) (1 in 2000)
Clinofibrate
0.75 -
0.25
200.0 225.0 250.0 275.0
A solution in ethanol (99.5) (1 in 50,000)
400.0
JP XV
Ultraviolet-visible Reference Spectra 1553
Clocapramine Hydrochloride Hydrate
1.0
0.5
0.25
0.0
-
\j
-
\ \
1 1 1 1 j 1 1 1 1
,- '• ' — ■ — ' — i — i — ■ — ■ — i — i — i — r T*"n — — i — i — i — i — i — i — i — i — i — i — i — i — i — i —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 40,000)
Clofedanol Hydrochloride
1.0
0.75
0.5
0.25
0.0
;
11,1
I / \ 1
: I 1 \ I I
i i i i j i i i r j i l i i j r i l i t ' f l 1 P 1 1 f 1 l 1 l l 1 I I I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 2500)
Clofibrate 1
0.75
0.25
"T — r ~ — ■"" — 1 ~~ 1 — I "" ■ " ''■■ — r
225.0 250.0 275.0 300.0
A solution in ethanol (99.5) (1 in 100,000)
■[■■- ■ t — "T r r
275.0
400.0
1554 Ultraviolet-visible Reference Spectra
JP XV
Clofibrate 2
1.0
0.75
0.5
0.25
0.0
j j ! I
— ■ i \ ■
_ \ _j
i 1 1 1 1 1 1 1 r 1 i 1 1 i^^F -I 1 1 ^n ~-i 1 r r r i f ,'1111 — 1 T ^T
225.0 250.0 275.0 300.0
A solution in ethanol (99.5) (1 in 100,000)
325.0
350.0
375.0
400.0
Clomifene Citrate
1.0
0.75 -
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 50,000)
Clomipramine Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 t 1 1 j ■ ■ t — ■* ■■■ i 1 i 1 1 1 r j 1 r 1 1 j 1 1 1 1* I 1™" \ I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1555
Clonazepam
1.0 -i
0.75
0.5 -
0.25
f\f\ 1 T T~l j 1 1 1 1 j 1 1 1 1 j TT 1 T"T — i — i — i — i — j — i — i — i — i — 1 — I — I — I I 7 ^^^^^^^™F
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in methanol (1 in 100,000)
Clonidine Hydrochloride
1.0
0.75
0.5
0.25
0.0
, , >■ i
1 T IT" T" "T ■■"T " ] T 1 "
..it
, . . ,
i i i i
, . . ,
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (3 in 10,000)
Cloperastine Hydrochloride 1
1.0
0.75
0.25
-i 1 1 1 1 1 1 1 1 1 1 1 r — | r — i — " i ,- i 1 -, j
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 2500)
1556 Ultraviolet-visible Reference Spectra
JP XV
Cloperastine Hydrochloride 2
0.75
0.5
0.25
0.0
— — T , ; „_ j . ^ j j
1 1 1 1 r — i 1 1 — ii i t i ^7 ^— j t t t 1 1 — t 1 t 1 [ r 1 1 1 p^r — i -l r " i " j 1 1 1 r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 62,500)
Clotiazepam
1.0
0.75
0.25
0.0
t 1 ] 1 t r 1 r t 1 —
200.0 250.0 300.0 350.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
1 1 ~~ "
400.0
450.0
500.0
Clotrimazole
1.0
0.75
0.5
0.0
1 1 1 1 i 1 r — c 1 — H r^ — t^ — "t t ■ i t - t ■ t -'■ r Ii t r ■ 1 — ' > ■ " i
200.0 225.0 250.0 275.0
A solution in methanol (1 in 5000)
300.0 325.0 350.0 375.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1557
Cloxacillin Sodium Hydrate
1.0
0.75 —
0.5
0.25 —
0.0
i — il I I — i — r
300.0
n — r
t — i — i — | — i — r
230.0 250.0
A solution in methanol (1 in 2500)
~! — I — I — | — I — I — r
350.0
t — i — i — i — r
400.0
Cloxazolam
1.0
-| r-T-T i -j
200.0 225.0 250.0 275.0
A solution in ethanol (99.5) (1 in 100,000)
I r i — r ■' i ■ y - ■■!■■ — , — i — ] — [ — i — i — r ••t — |" r " t — i — r-
300.0 325.0 350.0 375.0 400.0
Cocaine Hydrochloride 1
1.0
0.75
0.5
0.25
0.0 ~~ I ' ' ' """i ' ' ' ' i ' ' ' ' i ' ' ' T ~~i ' ' l— ' I ' ' ■ ' i ' ' ' ' 1 ' ' ' '
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
1558 Ultraviolet-visible Reference Spectra
JP XV
Cocaine Hydrochloride 2
1.0
0.75
0.5
0.25
0.0
0.75
0.5
0.25
1 1 1 1 i 1 r—\ 1 1 — i 1 r 1 i 1 1 i*" I 1 1 1 p ■ i - " f ■ I I 1 i J I I I i 1 1 1 1 —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 50,000)
Codeine Phosphate Hydrate
1.0
0.0
r 1 r r— ^-j 1 — i t — r™i r™i 1 1 j 1 1 1 i — j 1 I i I I j I I i — I j — i I I ™i— r— i ' T"' T -n
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Colchicine
1.0
0.75 —
0.5-
0.25
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — r r t r i t'j — r~ i — i — i — i — i — r~i — r pci — r~r~i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in ethanol (95) (1 in 100,000)
JP XV
Cortisone Acetate
1.0
Ultraviolet-visible Reference Spectra 1559
0.75
0.5 —
0.25
0.0
-\ — i — i — i — i — i — i — i — i — | — i — i — i — r
200.0 250.0
A solution in methanol (1 in 50,000)
~\ — i — i — i — ) — r~t — i — i — i — i — i — i — i — | — i — i — i "i "i — i — i — i — r
300.0 350.0 400.0
Croconazole Hydrochloride
1.0
0.75
0.25 -
r ■> ■ ' — ' — ' — : — ' — ' — ' — ■ — i — ' — ■ — ■ — ' — i — ' — r— r=i — i — i — i — i — i — i — i — i— i — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 20,000)
Cyanocobalamin
1.0
0.75
0.5
0.25
f\ A — I r — l 1 r — I r — i 1 — r-~T"T~"r — r — t 1 1 1 — i — i 1 — i 1 1 1 — i 1 — i — t — i — r-
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
The sample solution obtained in the Assay
1560 Ultraviolet-visible Reference Spectra
Cyproheptadine Hydrochloride Hydrate
0.75 -;
JP XV
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 100,000)
Cytarabine
1.0
0.75'
0.25-
0.0
i — i — i — i — i — ! — i — i — i — | — i — i — i — i — i — i — i — i — i — J — i T I — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
Dantrolene Sodium Hydrate
1.0
0.75
0.5
0.25
0.0
_
i \
-
i
— ™i 1 1 1 j 1 1 1 1 j 1 1 1 1
1 t 1 1
1 1 f ■ T f 1
200.0 250.0 300.0
An aqueous solution (1 in 100,000)
350.0 400.0
450.0 500.0
JP XV
Ultraviolet-visible Reference Spectra 1561
Daunorubicin Hydrochloride
1.0
0.75-
0.25 —
200.0
A solution in methanol (1 in 100,000)
600.0
Demethylchlortetracycline Hydrochloride
1.0
0.75-
0.25-
n 1 — i 1 — i — i 1 1 — i — i 1 1 — i — i — r — r — i " - | — i — i — i 1 — i I I 1 — r
220.0 300.0 400.0 5O0.O
A solution prepared as follows: Dissolve 40 mg in 250 mL of water. To 10 mL of this solution add 85 mL of water
and 5 mL of a solution of sodium hydroxide (1 in 5).
Dexamethasone
1.0
0.75
0.5
0.25
0.0
1 i r— i . j 1 1 . 1 j 1 1 r r— j i i i i i r . i i |=1 I I I
250.0
300.0
350.0
400.0
450.0
500.0
550.0
A solution prepared as follows: Dissolve 1 mg in 10 mL of ethanol (95). To 2 mL of this solution add 10 mL of
phenylhydrazine hydrochloride TS, shake, warm in a water bath at 60°C for 20 minutes, and allow to cool.
Blank: A solution obtained by proceeding in the same manner with 2.0 mL of ethanol (95).
1562 Ultraviolet-visible Reference Spectra
Dextromethorphan Hydrobromide Hydrate
1.0
0.75
JP XV
0.25
0.0
1 1 1 1 1 1 1 1 1 1 1 r t — r — 'i 1 ■■t m — r 1 t^t 1™ — i 1 ' "T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Diazepam
1.0
0.75 -
0.25
™i — i — r^~i — | — t — i — i — i — | — i — p — i — i — | — r — r — 1 — i — | — i — r-
200.O 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 2 mg in 200 mL of a solution of sulfuric acid in ethanol (99.5) (3 in 1000).
Dibucaine Hydrochloride
1.0
0.75
0.5
0.25
0.0
~i 1 r — r— i 1 1 1 j 1 1 1 1 j-
225.0 250.0 275.0 300.0 325.0
A solution in 1 mol/L hydrochloric acid TS (1 in 100,000)
350.0
375.0
400.0
JP XV
Diclofenamide
0.75
Ultraviolet-visible Reference Spectra 1563
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.01 g in 100 mL of 0.01 mol/L sodium hydroxide TS. To 10 mL of this
solution add 0.1 mL of hydrochloric acid.
Dicloxacillin Sodium Hydrate
1.0
0.75
0.5
0.25
0.0
\
1 1 r t r 1 r— ~n 1 i f — i r — i ■ ! ■ — i 1 1 1 : 1 1 1 1 1 1 1 : 1 1 1 1 i i ^i" — r 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2500)
Dihydrocodeine Phosphate
1.0
0.75
0.25 ~
220.0 240.0
An aqueous solution (1 in 10,000)
1 1 J 1 r-
280.0
340.0
1564 Ultraviolet-visible Reference Spectra
JP XV
Dihydroergotamine Mesilate
1.0
0.75 -
0.5
0.25
0.0
t — r — i— i — [ — i — i — i 1 '. — i r— j — i — [ •■ i ■ t — i — i — ] — r^T — i — i — | — i — t — i — r — [ — r — i — i 1 — p
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 20,000)
Dilazep Hydrochloride Hydrate
1.0
0.75
0.5
0.25 -
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Diltiazem Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 1 1 — i 1 — -t — t" 'T — -i ■■ t" — i 1 1 i r — T ■ i 7 " 1 'i — *i 1 — l 1 r-~i 1 \ 1 i r"*T f — I I T t~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1565
Dimemorfan Phosphate
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 5000)
Dimorpholamine
1.0
0.75
0,25
:
200.0 225.0
An aqueous solution (1 in 50,000)
^"~i — r — i — l — j — i — r
250.0 275.0
"T 1 1 — ^T"
1 v j — i — i — i~-^r — | — i — r — i — r"| — i — i — i —
300.0 325.0 350.0 375.0 400.0
Dinoprost
1.0
0.75 -
0.5 —
0.25
0.0 I ■ ' ■ ■ ■ i j ■ i i— r- j— r -i- "T— . - i -i i . . j . i , i | ■■ ■■ r , -j i t . . j" i i i i | I I
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
A solution prepared as follows: Dissolve 1 mg in 50 mL of diluted sulfuric acid (7 in 10). Warm this solution in a
water bath at 50°C for 40 minutes, and allow to cool.
1566 Ultraviolet-visible Reference Spectra
Diphenhydramine Hydrochloride
JP XV
1.0
0.75
0.5
0.25
0.0
T" "■ 1 1 F 1 -
200.0 225.0 250.0
A solution in methanol (1 in 2000)
f
T
300.0 325.0 350.0 375.0
400.0
Dipyridamole
1.0
0.75
0.5
0.0
! i
— [ ' I I J...........
- s — \. s \
i 1 1 1 i 1 1 1 r j r t - ^ — t -i — ' — r t-~ — i r^" — r^ j r t-
200.0 250.0 300.0 350.0 400.0
A solution in a mixture of methanol and hydrochloric acid (99:1) (1 in 100,000)
450.0
500.0
Disopyramide
1.0
~n 1 1 1 ] 1 1 < 1 1 1 1 r r" "T "'l t r v ,- r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.05 mol/L sulfuric acid-methanol TS (1 in 25,000)
JP XV
Ultraviolet-visible Reference Spectra 1567
Distigmine Bromide
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0
An aqueous solution (1 in 25,000)
300.0 325.0 350.0 375.0 400.0
Disulfiram
1.0
0.75
0.5
0.25
0.0 1' "'""' — ' — ' — I — ' — ' — ' — ' — i — ' — ' — ' — ' — i — ' — ' — ' — ■ — i — ' — ' — r " r ' i " 7 ~ 1 ' ' i ' ' '' '' I ' ' r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 100,000)
Dopamine Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 — r — i t " — r — i — i — i i — i — i — r — i — t — i — i — i — it i — i — i — i — i — i — r— i — i i — i — i — i — i — i — i — tr
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 25,000)
1568 Ultraviolet-visible Reference Spectra
Doxapram Hydrochloride Hydrate
1.0
0.75
0.5
JP xv
0.25
0.0
1 1 ■ ■ l r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2500)
Doxifluridine
1.0
0.75 —
0.25 -
t — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | r~\ — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 50,000)
Doxorubicin Hydrochloride
1.0
0.75-
0.5-
0.25
0.0 — I — i — i — i — i — i — i — i — i — i — I — i — i — i — i — i — m — i — i 1 — i — i — i—t — i — i — i — i — j — i — r
200.0 300.0 400.0 500.0
riii
600.0
A solution in methanol (1 in 100,000)
JP XV
Droperidol
1.0
0.75
Ultraviolet-visible Reference Spectra 1569
0.5
0.25
0.0
1 1 ! ! I
j 1
vjl^4
|\
I
1
:
i\l
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0
400.0
A solution prepared as follows: Dissolve 0.03 g in 10 mL of 0.1 mol/L hydrochloric acid TS and ethanol (95) to
make 100 mL (in a brown volumetric flask). To 5 mL of this solution add 10 mL of 0. 1 mol/L hydrochloric acid TS
and ethanol (95) to make 100 mL (in a brown volumetric flask).
Dydrogesterone
1.0
0.75
0.25 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 200,000)
Edrophonium Chloride
1.0
0.75 -
0.5
0.25
0.0 i ' n ' "' i ' ' ■ • •' ' i ' ' ' ' I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 20,000)
1570 Ultraviolet-visible Reference Spectra
JP XV
Elcatonin
1.0
0.75
0.5
0.25
0.0
T 1 1 1 1 I — [ 1 1 1 1 1 [~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 5 mg in 5 mL of water.
Enoxacin Hydrate
1.0
0.75
0.25
0.0
! i I ! ! i
1 1 r — i — 4" — i 1 1 1 1 1 1 1 1 1 1 1 1 1 \ 1 j [ 1 1 [ 1 1 1 — t — ■ i - ■ t — i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 1 mL of a solution in dilute sodium hydroxide TS (1 in 2000) add water to make
100 mL.
Enviomycin Sulfate
1.0-
0.75-
0.5-
0.25 —
0.0
i — i — i — r
i — i — i — r~[ — r~r
250.0
200.0
An aqueous solution (1 in 100,000)
~rn — i- i i ■ ■] \ — r
300.0
~t — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
350.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1571
Eperisone Hydrochloride
1.0
0.75 -;
0.25-
200.0
A solution in methanol (1 in 100,000)
-\ — ryTTT'l — I — I — I — I — I — | — I — I — I — I — I — 1 — I — I — T
300.0 350.0 400.0
Ephedrine Hydrochloride
1.0
0.75
0.5
0.25
0.0
\ . — .. VTT . T , T „„L - j. L_,. ii _l iiiiiiiiiiiiiiiiiii J._.l iiiiiiiii .l iiiiii . i |__ — X. ..L.....^*
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2000)
Epirizole
1.0
0.75
0.5
0.25
0.0
I
]
]
1 1 1 T T T 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 200,000)
1572 Ultraviolet-visible Reference Spectra
JP XV
Epirubicin Hydrochloride
1.0
0.75-
0.5-^
0.25-
0.0
i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — r
200.0 300.0
A solution in methanol (3 in 200,000)
1 — | — f — I — I — I — I — I — 1 — I — I — | — I — ! — I — I — I I I I I
400.0
500.0
600.0
Estazolam
1.0
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0
A solution in 1 mol/L hydrochloric acid TS (1 in 100,000)
325.0
-A 1 r — i
400.0
i ' ' > > i — ' — ' — ■— "-"i — ■ - ■ > — r— i — i—i — i—i — i— ■— i — i— r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.01 mg in 100 mL of ethanol (95) (warming)
JP XV
Ultraviolet-visible Reference Spectra 1573
Etacrynic Acid
1.0
0.75
0.5
0.25
00
. i i i j i i , r j i i i i j . , i i j i i i i r i i . i j . i i i i i r i i I i i i i I
200.0 225.0 250.0 275.0 300.0 325.0 35O0 375.0 400.0 425.0 450.0
A solution in methanol (1 in 20,000)
Ethionamide
1.0
0.75
0.25-
A solution in methanol (3 in 160,000)
Ethosuximide
1.0
0.75
0.25
0.0
-
1
lJ
i
IH\
\
■■■■,- T T--T-
r i r i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 2000)
1574 Ultraviolet-visible Reference Spectra
Ethyl Icosapentate
1.0
0.75 —
JP XV
0.5
0,25
0.0
i — i — i — i — i — i — i — i — i — ] — i — i — i — i — i — i — i — i — i — J — i — i — i — i — i — i — i — i — i — | — i — i i I I — i — i — i — r
200.0 250.0 300.0 350.0 400.0
To 20 mg add 3 mL of a solution of potassium hydroxide in ethylene glycol (21 in 100), stopper tightly after passing
of Nitrogen, heat at 180°C for 15 minutes, and add methanol to make 100 mL after cooling. To 4 mL of this solu-
tion add methanol to make 100 mL.
Blank: A solution obtained by proceeding in the same manner with 3 mL of the solution of potassium hydroxide in
ethylene glycol (21 in 100).
Ethylmorphine Hydrochloride Hydrate
1.0
0.75
0.5
0.25
0.0
1 1 r — i i 1 1 1 1 i 1 1 - -i ■ i 1 — i 1 r — r — i — i^l I I I [—1 — t 1 — t — "r — T— r — l 1 r — I r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Etilefrine Hydrochloride
1.0
200.0 225.0 250.0 275.0 300.0 325.0
A solution prepared as follows: Dissolve 5 mg in 100 mL of diluted hydrochloric acid (1 in 1000)
400.0
JP XV
Ultraviolet-visible Reference Spectra 1575
Etizolam
1.0
0.75-
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — r~| — i — r~i — i — r
200.0 250.0
A solution in ethanol (99.5) (1 in 100,000)
tt~| — i ■■■[■■ i — i — r
300.0
T~\ — | — i — i — i — i — i — i — i — i — r
350.0 400.0
Etodolac
1.0
0.75 —
0.25
0.0
i — i — i — i — i — i — i — i — i — I — i — i — i — i — i — i — r
200.0 250.0
A solution in ethanol (99.5) (3 in 200,000)
1 — | T~\ — I — I — I — I — I — I — I — | — I — I — r~ i — i — i — i — i — r
300.0 350.0 400.0
Etoposide
1.0
0.75-
0.25-
t — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — i — i — r~i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in methanol (1 in 10,000)
1576 Ultraviolet-visible Reference Spectra
Famotidine
0.75
JP XV
0.25 -
-i 1 1 1 i . 1 1 j 1 r r— i ] r 1 1 r 1 , , I I 1 1 1 1 1 1 1 I 1 1-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.05 mol/L potassium dihydrogen phosphate TS (1 in 50,000)
Fenbufen
1.0
0.75
0.5 -
0.25 -
0.0 ~" I ' ' ' r— [— r— i 1 1 1 1 1 1 1 1 , <■■■ , - r p
200.0 225.0 250.0 275.0 300.0 325.0
A solution in ethanol (95) (1 in 200,000)
T ■ | ■ T' — I 1 1 -
350.0 375.0 400.0
Fentanyl Citrate
1.0
0.75
0.5
0.25
0.0
1 1 1 1 i — n 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.05 g in 10 mL of 0.1 mol/L hydrochloric acid TS and ethanol (95) to
make 100 mL.
JP XV
Ultraviolet-visible Reference Spectra 1577
Flavoxate Hydrochloride
1.0
0.75 -
0.5 -
0.25 -
0.0
t r 1 " i i ■ ■ n 1 1 1 1 1 1 1 1 1 1 1 r — i 1 — "i r — ! 1 ] 1 1 1 1 1 1 p 1 1 r~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 50,000)
Flomoxef Sodium
1.0
0.75-
0.25
t — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0
An aqueous solution (3 in 100,000)
i — i — i — i — i — i — | — i — i — i — i — i — i — i — ■ — r
350.0 400.0
Flopropione
1.0
0.75
0.5
0.25
0.0
1 1 — t p r — t 1 1 1 i 1 1 1 1 r — i — t 1 1 1 1 1 1 r j 1 1 1 1 i i i ■ n 1 i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 200,000)
1578 Ultraviolet-visible Reference Spectra
Flucytosine
1.0
0.75
JP XV
0.5
0.25
0.0
i i \ i I |
1 r 1 1 i rr — l 1 i 1 1 1 e i 1 1 r 1 i 1 1 r^T- - -i — t — T T 1 r r-~*i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 125,000)
Fludiazepam 1
1.0
0.75
0.5
0.25
0.0
: ! I I I I
t 1 1 1 1 1 1 1 1 t 1 1 1 t i r 1 — ■ v t "i 1 1 1 1 i 1 r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 200,000)
Fludiazepam 2
1.0
0.75
0.5
0.25
0.0
\ 1
3
j
,11,
. f t i j . r j t
i i
jiii
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 20,000)
JP XV
Ultraviolet-visible Reference Spectra 1579
Flunitrazepam
1.0
0.75 -
0.25 -
200,0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 100,000)
Fluocinonide
1.0
0.75
0.5 -
0.25
0.0 ~" I — ' — ■ — ■ — ' — i — ' — ' — ' — ' — r" 1 — ' — ' — i — r~ i — i — i — i — i — ' — i — ' — ' — i — i — ' — ' — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Fluorometholone
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0
A solution in methanol (1 in 100,000)
300.0 325.0 350.0 375.0 400.0
1580 Ultraviolet-visible Reference Spectra
JP XV
Fluorouracil
1.0
075 -;
0.25 "
-i — i — i — i — [-
200.0 225.0 250.0 275.0 300.0 325.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
I ' ' '
350.0 375.0 400.0
Fluoxymesterone
1.0
-i 1 r . f
200.0 225.0 250.0 275.0
A solution in ethanol (95) (1 in 100,000)
t — ■ — ■ — ' — ■ — i — i ■ i ■ i i ■ i — <—r
300.0 325.0 350.0 375.0 400.0
Fluphenazine Enanthate
1.0
0.75 ~
0.5
0.25
f\ f) — | t 1 1 t j — -i 1 1 1 j r 1 1 1 1 — ^ — — i r-^i — -j 1 r— i 1 j 1 1 1 1 j i i I I | r— *i 1 "i
200.0 225.0 250,0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 2 mg in 200 mL of a solution of hydrochloric acid in methanol (17 in
2000).
JP XV
Ultraviolet-visible Reference Spectra 1581
Flurazepam 1
1.0
0.75
0.5
0.25
0.0
1 1 [ 1 j 1 1 1 1 j 1 1 r" — l — ~j
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Flurazepam 2
1.0
0.75 -
0.25 -
-\ 1 r — i - i- i — i — i—i 1 — ] — i 1 1 — i 1 — i ' — i — i — ] 1- i - i ■■ i » l i — i — j-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 10,000)
Flurazepam Hydrochloride
1.0
0.75 —
0.5
0.25
0.0 "1 •-<"• '"I i ■ > ■ f i i i i- I i " i i I ' i i i I i i i i I i i i i'T~i-T'-r -i- j
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in sulfuric acid-ethanol TS (1 in 100,000)
1582 Ultraviolet-visible Reference Spectra
JP XV
Flurbiprofen
1.0
0.75
0.5
0.25
0.0
1 1 1 1 1 1 1 1 1 j — "i r~ — i 1 j 1 1 — i 1 " I — j r" i 1 1 r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 200,000)
Folic Acid
1.0
0.75
0.5
0.25
0.0
I ■ I ■ I I I I I I I I I'"! 'I |" I I - I ■, I . I V |
225.0 250.0 275.0 300.0 325.0 350.0
A solution in dilute sodium hydroxide TS (3 in 200,000)
I ' ' ' ' I
375.0 400.0 425.0 450.0
Formoterol Fumarate Hydrate
1.0
0.75
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 40,000)
JP XV
Ultraviolet-visible Reference Spectra 1583
Furosemide
1.0
0.75
0.25 -
A solution in dilute sodium hydroxide TS (1 in 125,000)
Gabexate Mesilate
1.0
0.75
0.5
0.25
0.0
-
—
—
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
/?-Galactosidase (Aspergillus)
1.0
0.75
0.5
0.25
0.0
! ■* i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.1 mg in 100 mL of water (Filter if necessary.).
1584 Ultraviolet-visible Reference Spectra
JP XV
Glibenclamide
1.0
-> — ■ — r
200.0 225.0 250.0 275.0
A solution in methanol (1 in 10,000)
400.0
Gonadorelin Acetate
1.0
0.75
0.25 —
400.0
A solution in methanol (1 in 10,000)
Gramicidin
1.0
0.75
0.5-
0.25-
0.0
r~i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — rn — i — i — i — \ — i — i — i — | — i — i — i — i — i — r
200.0 250.0 300.0 350.0
A solution in ethanol (95) (1 in 20,000)
400.0
JP XV
Ultraviolet-visible Reference Spectra 1585
Griseofulvin
1.0
0.75-
05-'
0.25 -
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0
A solution in ethanol (95) (1 in 100,000)
l — i — i — i — i — ! — | — i — I i I I — i — i — i — r
350.0 400.0
Guaifenesin
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Guanabenz Acetate
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
1586 Ultraviolet-visible Reference Spectra
JP XV
Haloperidol
1.0
0.75
0.5
0.25
0.0
225.0
400.0
A solution prepared as follows: Dissolve 30 mg in 100 mL of 2-propanol. To 5 mL of this solution add 10 mL of 0.1
mol/L hydrochloric acid TS and 2-propanol to make 100 mL.
Haloxazolam
1.0
0.75
0.5
0.0
1 1 1 r"H t — i 1 1 i 1 1 1 1 i 1 1 — i ■ f i s- — ^i — -t" — H — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Homochlorcyclizine Hydrochloride
1.0
0.75 -
0.5
0.25 —
0.0 — I — i — i — i — i — j — i — i — i — i i i i i I i — i — i — i — I — i — i — i — i — j — i — i — i — i — j — i — i — i — i — j — i — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
JP XV
Hydralazine Hydrochloride
0.75 ~
Ultraviolet-visible Reference Spectra 1587
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
Hydrochlorothiazide
1.0
0.75
0.25
oo -*— t
l 1 1 1 1 1 1 1 1 1 1 ] 1 j 1 1 1 1 1 1 1 p
225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 12 mg in 100 mL of sodium hydroxide TS. 10 mL of this solution add
water to make 100 mL.
Hydrocotarnine Hydrochloride Hydrate
1.0
0.75
0.25 -
0.0 ' ■ ■ ' i ' ' •~~' r r , '" 1 — ' — i — ■ — ' — ' — r " v " r ■'-■■' — i — ■ — ' — ■ — ■ — i — ' — ■— > ' r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
1588 Ultraviolet-visible Reference Spectra
JP XV
Hydroxocobalamin Acetate
1.0
0.75
0.5
0.25
0.0
1 r — i — i r — i — i — "i ,_ T"" t — "H 1 i ■ i 1 1 1 r - ■ r — i r~-H r — i — i ■ 1 — "™^t — t — i — "i — i ■ i i I I t r — I —
250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
A solution in acetic acid-sodium acetate buffer solution, pH 4.5 (1 in 50,000)
Hydroxyzine Hydrochloride
1.0
0.5
0.25
0.0
~~ :;;!;;
— ~~t ] 1 1 i 1 1 r 1 i i p i I i i — *t 1 i — r ' — i 1 i r 1 1 1 r r™i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Hydroxyzine Pamoate
0.75
0.25
-|— i— , I 1 i i | ■ r i i | - . i ^-|-
275.0 300.0 325.0 350.0 375.0
■100.0
A solution prepared as follows: Evaporate 2 mL of the sample solution obtained in the Identification (1) on a water
bath to dryness, and dissolve the residue in 0.1 mol/L hydrochloric acid TS to make 500 mL.
JP XV
Ultraviolet-visible Reference Spectra 1589
Hymecromone
1.0
0.75
0.5
0.25
0.0
1 — i — i — i — r — i — •]•• 'i ' i — i — i — i — i — i — f — i — i — i — i — i — i — i — i — i — 1 — i — i — i — i — i — i — i — i — i — i — i— — r- ^ == r == F™i"™r — I — r — i — i — i — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in ethanol (99.5) (1 in 250,000)
Ibuprofen
1.0
0.75
0.5
0.25
0.0
— ■ " — r — t 1 1 1 1 1 — -i 1 i 1 1 1 1 i r — i — i 1 1 — ^i 1 1 ■ 1 r
200.0 225.0 250.0 275.0 300.0
A solution in dilute sodium hydroxide TS (3 in 20,000)
325.0
350.0
375.0
400.0
Idarubicin Hydrochloride
1.0
0.75 —
0.25
t — i — i — i — r — i — i — i — i — | — i — i — i — i — i — i" i "'i""i — | — r~i — i — i — i — r— i — r'T ' | i — i — i i i i I i — r
200.0 300.0 400.0 500.0 600.0
A solution in methanol (1 in 100,000)
1590 Ultraviolet-visible Reference Spectra
JP XV
Idoxuridine
1.0
0.75
0.5
0.25
0.0
225.0 250.0 275.0 300.0 325.0
A solution in 0.01 mol/L sodium hydroxide TS (1 in 25,000)
350.0
1 —\- — t 1 1 i 1 1 1 j r~ — i" — r ■ ■■■[ r~ — r- — t 1 — -T 7 * 1 -i r™^ — ~r r- — 1 i 1 1 1 1 r
375.0
400.0
Ifenprodil Tartrate
1.0
0.75
0.25 -
0.0
t 1 1 1 1 1 1 1 1 1 1 1 1 1 r 1 — i 1 1 1 1 i ■■ j ■■ v — i— ^r 1 1 1 1 1 1 j—
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Imipenem Hydrate
1.0
0.75-
0.5-
0.25
0.0 1 — r — i — I — i — j — i — i — i — i — i — i — i — i — i — j — i — i — i — i — i — i — i 1 F | I — i — i — i — i — i — i — i — r
220.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L 3-(iV-morpholino)propanesulfonic acid buffer solution, pH 7.0 (1 in 50,000)
JP XV
Ultraviolet-visible Reference Spectra 1591
Imipramine Hydrochloride
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 5 mg in 250 mL of 0.01 mol/L hydrochloric acid TS
Indenolol Hydrochloride 1
1.0
0.75
0.5
0.25
0.0
■ \
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Indenolol Hydrochloride 2
1.0
0.75 -
0.5
0.25 -
0.0
^ 1 T" ■ i 1 1 1 1 1 1 r— i 1 — m t •• t — ^-r — r^ — i 1 t r 1 ^ 1 1 r — i 1 1 1 1 1 r ■■ r • • i r 1 1 r -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
1592 Ultraviolet-visible Reference Spectra
Indigocarmine
1.0
0.75
JP XV
0.5
025
0.0
-
:
1
i
-
-
1 ■ ' ' i ■ ■ ■ ■
l i l r | i i ,m t ■ t —
i i i i | , ,• , ,
,,,,,,,, i
..I.
[ ' '*^"
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0 700.0
A solution prepared as follows: Dissolve 0.1 g in 100 mL of an aqueous solution of ammonium acetate (1 in 650).
To 1 mL of this solution add the aqueous solution of ammonium acetate (1 in 650) to make 100 mL.
Indometacin
1.0
0.75
0.5
0.25
0.0
1 — i — i — i — i — i — i — r — i — i — r — l — i — r — H^t — i — rn — f — i — t — i — i — 1 — r ■ t ■ ■■! — i — i — i — i — r — rH — c — f — T" r 1 r f 1 — T — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 2 mg in 200 mL of methanol.
Ipratropium Bromide Hydrate
1.0
0.75
0.5
0.25
0.0
-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (3 in 2000)
JP XV
Ultraviolet-visible Reference Spectra 1593
0.75
0.25 -
t— | 1— i 1— i- r l -i ■■ - | I i - I | ■ I i . | ■■ I- I r |
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 5 mL of an aqueous solution (1 in 10,000) add 1 mL of 0.1 mol/L hydrochloric
acid TS and water to make 50 mL.
/-Isoprenaline Hydrochloride
1.0
0.75 "
0.5
0.25 "
f\ f) — | p 1 p 1 j 1 1 r — r— j 1 1 1 1 f 1 1 [ i^f 1 I ^^^r™"^^^^^~ I P I 1 |'
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 20,000)
Josamycin
1.0
0.75-
0.5-
025
0.0
l — r v pT I I ! l — r—i — i — i — | — i — i — i — i — i — i — [— i — r~y— t~i — r~ i — I — I — i — I — 1~
250.0 300.0 350.0 400.0
200.0
A solution in methanol (1 in 100,000)
1594 Ultraviolet-visible Reference Spectra
JP XV
Josamycin Propionate
1.0-
0.75-
0.5-
0.25
0.0
i — i — i — i — i — i — i — i — i — | — i— i — m — i — i — t— i — r~| — n — r
200.0 250.0 300.0
A solution in methanol (1 in 100,000)
-, — j — j—j — j — | — , — j — ! — ,__, — ! — ! — ! — r
350.0 400.0
Ketamine Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 1 I 1 T ■ T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 3000)
Ketoprofen
1.0
0.75
0.25
— i 1 1 1 1 1 1 1 1 ■ i r — t 1 1 1 1 1 1 1 1 1 1 r — I 1 1 p i t 1 i 1 1 — "i 1 1 1 r~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 200,000)
JP XV
Ketotifen Fumarate
0.75 —
Ultraviolet-visible Reference Spectra 1595
0.25 —
i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — r
200.0 250.0 300.0 350.0
A solution in methanol (1 in 50,000)
1 — I — I — i — i — i — T~
400.0
Kitasamycin
1.0
0.75
0.25
I 1 1 " T" ' I ' — I l~~
l — i — ■— ' — r ~i — ' "''■■ — ' — i — ' — r=F— ! ' — ' — ■— > — i — ' — ■— > — ' — r -1 — ' — ' ■ '■ ■ i '
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 40,000)
Kitasamycin Acetate
1.0
0.75
0.5
0.25
0.0
r i i l ■ ■ r t i ■■ 1— ■
i . i . i- ,,,,,,,,
i , , . j . , . ,
, i , ,
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 40,000)
1596 Ultraviolet-visible Reference Spectra
JP XV
Kitasamycin Tartrate
1.0
0.75-
0.25-
t — i — i — r~! — i — i — i — i — [ — i — i — i — i — i — i — i T" i | I i — i — i — i — i — \ — i — i — | — i — i — l — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in methanol (1 in 40,000)
Latamoxef Sodium
1.0
0.75-
0.5-
0.25-
0.0 1 — i — i — i — i — i — i — i — i — j — i — i — ! — i — i — i — i — i — i — j i~T I — i — i — i — i — i — r — p— t — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
An aqueous solution (3 in 100,000)
Levallorphan Tartrate
0.75
0.25
— i 1 1 1 r 1 r 1 1 1 i 1 v l — "j 1 1 1 1 ""I i 1 1 ■ 'i ■ ] 1 r — i t " '"J 1 T 1 1 1 1 1 r™
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
Ultraviolet-visible Reference Spectra 1597
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.001 mol/L hydrochloric acid TS (3 in 100,000)
Levothyroxine Sodium Hydrate
1.0
0.75
0.5
0.25 "
O0 ~ 1 i — ■ — ' — ,- ~ i — i — p — ■ — > — ' — t — r — ■ — ■ — ■ — i — ■ — ' — r — ■ — i — > — > — ■ — > — r
225.0 250.0 275.0 300.0 325.0 350.0
A solution in dilute sodium hydroxide TS (1 in 10,000)
375.0
400.0
Lidocaine
1.0
0.75
0.5
0.25
0.0
■ r ■ -i 1 1 r — r — i < 1 i 1 1 1 1 i — -r^ r — i—\ — t — -, i ■ • l — i j 1 r — i 1 j r — i— ^i 1 r
200.0
225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.04 g in 10 mL of 1 mol/L hydrochloric acid TS and water to make 100
mL.
1598 Ultraviolet-visible Reference Spectra
JP XV
Limaprost Alfadex
1.0
0.75 —
0.25 —
200.0
250.0
300.0
350.0
400.0
A solution prepared as follows: To 10 mL of a solution in dilute ethanol (3 in 10,000) add 1 mL of potassium
hydroxide-ethanol TS, and allow to stand for 15 minutes.
Liothyronine Sodium
1.0
0.5
0.25
0.0
1 1 r 1 i r — l 1 1 i 1 1 1 — i — H— — r~— i 1 1 r 1 — — r — ; — r ■ — »~ ~ t i i^^^^^^^T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 10,000)
Lisinopril Hydrate
1.0
0.75 —
0.5-
0.25 —
0.0
t — i — i — i — i — I — i — i — I — i — i — i — i i"~i — i — i — i — I — r
200.0 250.0
A solution in methanol (1 in 1000)
300.0
i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
350.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1599
Lorazepam
1.0
0.75
0.25
-| 1 — i 1 — r— | — r—. — i 1 — 1 — . — i 1 — i — | — i 1 — i — i — | — i — i — i—i — | — i — i — i 1 — p
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 200,000)
Loxoprofen Sodium Hydrate
1.0
0.75
0.5
0.25
0.0
- \
200.0 225.0 250.0 275.0
An aqueous solution (1 in 55,000)
300.0 325.0 350.0 375.0
400.0
Lysozyme Hydrochloride
1.0
0.75-
0.25-
A solution in acetate buffer solution, pH 5.4 (1 in 10,000)
1600 Ultraviolet-visible Reference Spectra
JP XV
Maprotiline Hydrochloride
1.0
0.75 -
0.25
~1 1 IT — I T 1 T 1 1 1 T " I T — ^T ' ' : ^ I 1 1 1 1 1 1 1 J 1 1 1 1 1 1 1 1 1" — I l~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 10,000)
Meclofenoxate Hydrochloride
1.0
0.75
0.5
0.25
0.0
1 1 1 1 1 1 1 1 1 j [ r 1 r r™n 1 1 — i* j i I I — T — j r" *n — *i 1 1 — T 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Mecobalamin 1
1.0
0.75 -
0.25
i ' ' ' ' i •— ,— ■ ' i ' ' : ' i ' r ' ' i r r r ' i ' ' • • i
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
A solution in hydrochloric acid-potassium chloride buffer solution, pH 2.0 (1 in 20,000)
JP XV
Ultraviolet-visible Reference Spectra 1601
Mecobalamin 2
1.0
0.75
0.25
T" 1 "' 1 ■ ■ | i i ■-■ |'T— i— , 1 -- , ■ -t— r ~i— r— |-
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
A solution in phosphate buffer solution, pH 7.0 (1 in 20,000)
Medazepam
1.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in methanol (1 in 100,000)
Mefenamic Acid
1.0
0.75
0.5
0.25
0.0
1 — i — i — r — i — i — i — r — i — f — t — r — r~~i — i — i — i — i — r — ■i ,,-, i , m i — r i — r — i — r — i — i — j — i — i — r- — i — » — f — t — i i^| r 1 I I i — i — T 1 i" ■ r
200.0 225.0 250.0 275.0 300.0 3250 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 7 mg in a solution of hydrochloric acid in methanol (1 in 1000) to make 500
mL.
1602 Ultraviolet-visible Reference Spectra
JP XV
Mefloquine Hydrochloride
1.0
0.75-
0.25
1 ' i i j i i i i i i i i i | i i r~ i r*"n i i i I n i i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in methanol (1 in 25,000)
Mefruside
1.0
0.75-
0.25-
-, , , , 1 , , , 1 [ , , , , 1 1 r— t r -J 1 1 1 1 1 1 . 1 1-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 40,000)
Melphalan
1.0
0.75
0.5
0.25
0.0
1 1 1 1 i 1 ! 1 r i 1 ! t t i t^-t — ~i < i 1 1 r i"" I i 1 r — i — ^i r — r 1 1 f 1 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 10,000)
JP XV
Ultraviolet-visible Reference Spectra 1603
Mepenzolate Bromide
1.0
0.75 -
0.25
- 1 1 r ■ l ■ ■ f ■ ■! r
225.0
i ' ' — i~'~r
275.0 300.0
200.0 225.0 250.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 2000)
325.0 350.0 375.0 400.0
Mepivacaine Hydrochloride
1.0
0.75
0.5
0.25
0.0
— ■ — i — "i — t — i i 1 i 1 1 j i 1 1 1 1 i — i" i — i 1 i 1 1 1 1 i 1 r — i 1 i 1 1 — t — i — i i I I — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2500)
Mequitazine
1.0
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 250,000)
1604 Ultraviolet-visible Reference Spectra
Mercaptopurine Hydrate
1.0
0.75
JP XV
0.25
0.0
■ : i
! 1 j j ! i ;
; :.::::
1 1 — i — i — j 1 — i — i 1 j — i — i — , 1 — i — i — i - i - i — j — i - i-"i — i — j— . — i — r— i j — i T~ 1 — i — j — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 200,000)
Mestranol
1.0
0.75 -
0.25
-i — i — r
200.0 225.0 250.0 275.0
A solution in ethanol (99.5) (1 in 10,000)
400.0
Metformin Hydrochloride
1.0
0.75
0.25-
200.0 250.0
An aqueous solution (1 in 100,000)
300.0
350.0
400.0
JP XV
Ultraviolet-visible Reference Spectra 1605
Methotrexate
1.0
-^ , T t j — ™, n
200.0 250.0 300.0 350.0 400.0 450.0 500.0
A solution prepared as follows: Dissolve 1 mg in 100 mL of 0.1 mol/L hydrochloric acid TS.
Methoxsalen
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 200,000)
Methyldopa Hydrate
1.0
0.75 -
0.5 -
0.25
0.0
t 1 1 i ' 1 1 : 1 1 1 r 1 r
200.0 225.0 250.0 275.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 25,000)
400.0
1606 Ultraviolet-visible Reference Spectra
JP XV
tf/-Methylephedrine Hydrochloride
1.0
0.75—-
0.5
0.25-
0.0
T i i i j i i — i i p~i i i i ] i i i i | i i i i i i i i i ( i i i i [ i i i r
200.0 225.0 250.0 275.0 300.0
An aqueous solution (1 in 2000)
325.0
350.0
375.0
400.0
Methylergometrine Maleate
1.0
0.75
0.5
0.25
0.0
j j I I :
_ : :
I
300.0 400.0 500.0
The colored solution obtained in the Assay
600.0
700.0
800.0
Methylprednisolone
1.0
0.75 -
0.5
0.25
0.0 — 1 — ' — ' — ' — ■ — i — ' — i — ' — ' — r— i — i — ~ r ~~< — i — ' — ' ~ i I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 100.0
A solution in methanol (1 in 100,000)
JP XV
Methylprednisolone Succinate
0.75 —
Ultraviolet-visible Reference Spectra 1607
0.25-
i — i — i — i — | — i — i — i — i — i — i — i — i i | i i i I
250.0 300.0
200.0
A solution in methanol (1 in 50,000)
i — I — i — i — j — i — i — i — i — i — i — i — i — r
350.0 400.0
Methyltestosterone
1.0
0.75
0.5-
0.25-
0.0 — j — i — i — i — ] — i — i — i — ] — i — I — i — i — i — i i I I — I — I — I — I — I — I — I — I — I — i — i — i — i — i — i — i — i — i — i — i — \ — r
200.0 250.0 300.0 350.0 400.0
A solution in ethanol (95) (1 in 100,000)
Meticrane
1.0
0.75
0.25
1 1 1 ■ r— | r— , , , [■
200.0 225.0 250.0 275.0 300.0
1 — ■ — <— ■ — r ~ r
325.0 350.0 375.0 400.0
A solution in methanol (3 in 10,000)
1608 Ultraviolet-visible Reference Spectra
JP XV
Metildigoxin
1.0
0.75
0.5
0.25
0.0
1 1 1 1 i 1 1 1 r |~ ' T ' r 1 r — i r ■- t t 1 r — r" — i 1 1 i i 1 1 1 j 1 1 r* — i f 1 1 — r ~i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
Metoclopramide
1.0
0.75 -
0.5
0.25 -
0.0
t — t 1 r— | 1 1 1 1 1— i r— t 1 1 1 1 1 1 1 1 1 r 1 1 ' — t— i 1 1 1 1 1 1 1-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: Dissolve 0.1 g in 1 mL of 1 mol/L hydrochloric acid TS, and add water to make 100
mL. To 1 mL of this solution add water to make 100 mL.
Metoprolol Tartrate
1.0
0.75
0.5
0.25
0.0
: j " " 'I""""" " " f~~ " "
1 — i — I — i — I — I — I — I — r ■(■ r ■ i — t— i — i — r — r — r 5 "! — t~~T — l ■■■;■-] — i — I — I — i — I — I — I — I — I — I — I — I — I — I — I —
200.0 250.0 300.0
A solution in ethanol (95) (1 in 10,000)
350.0
400.0
JP XV
Ultraviolet-visible Reference Spectra 1609
Metronidazole
1.0
0.75
0.5
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
Metyrapone
1.0
0.75
0.5
0.25
0.0
I I j I I I I I I E I I j
-t ■■ " r- -r — r-r 't — r~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.5 mol/L sulfuric acid TS (1 in 100,000)
Mexiletine Hydrochloride
1.0
-|— , ■■ .■ i T r-r ,■ [ •■ ' r~T— 1 ' -r f i T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 2000)
1610 Ultraviolet-visible Reference Spectra
JP XV
Miconazole
1.0
0.75 -
0.5 -
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 2500)
Miconazole Nitrate
1.0
0.75
0.5
0.25
0.0
- •
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 2500)
Midecamycin
1.0
0.75
0.5
0.25
0.0
\
/
•
-
1 — 1 — 1 — 1
, , , . 1 ■ , , , 1 1 , , I
1 1 1 1
■t ■■!■■ i r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
JP XV
Ultraviolet-visible Reference Spectra 1611
Midecamycin Acetate
1.0
0.75 ~
0.5
0.25 -
0.0 I ' ' ' ' 1 — ' ' ' i ■ — ' — l—l — r -1 — <— *—■ — ^ ■ H^ -1 — ' — ■ i ■ ' — i i j i i i i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
Minocycline Hydrochloride
1.0
0.75-
0.25-
r i i i i i i ■! i ] r rH'nTH-p : ! i i i i i i | i i i i m i i i | i rn i n i i
200.0 250.0 300.0 350.0 400.0 450.0
A solution in a solution of hydrochloric acid in methanol (19 in 20,000) (1 in 62,500)
Mitomycin C
1.0
0.75
0.25
200.0
An aqueous solution (1 in 100,000)
l~f
300.0
i — i — i — r
400.0
i i i i i i i i
500.0
1612 Ultraviolet-visible Reference Spectra
JP XV
Morphine Hydrochloride Hydrate 1
1.0
0.75 -
0.5
0.25
0.0
r- — 1~ — i r — T^*l ' — ~~*~
T — ■ — ■ — ■ — ' — r
225.0 250.0 275.0
An aqueous solution (1 in 10,000)
300.0
325.0
350.0
"i 1 1" — i ■ ,, m t f r
375.0 400.0
Morphine Hydrochloride Hydrate 2
1.0
0.75
0.5
0.25
0.0
225.0 250.0 275.0 300.0
A solution in dilute sodium hydroxide TS (1 in 10,000)
325.0
350.0
375.0
400.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 5000)
JP XV
Ultraviolet-visible Reference Spectra 1613
Nalidixic Acid
1.0
0.75
0.5
0.25
0.0
~"" ' " « '" 'i " " I 1 j n 1 1 1 j 1 1 1 1 j ■ r ■ i 1 f — 'j ■ r — i r r r — i r r r r 1 r" ta T 1 f — i r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L sodium hydroxide TS (1 in 200,000)
Naloxone Hydrochloride
1.0
0.75
0.5
0.0
r t < 1 i r 1 1 r'"T"T 1 1 r f 1 1 1 1 1 p 1 1 i i I I I 1 i 1 1 1 1 i 1 r t 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Naproxen
1.0
0.75
0.5
0.25
0.0
j i
\
|
....
\j / \
i i i i
■ ! 1 1 1 1 1 1 1
1 1 1 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 50,000)
1614 Ultraviolet-visible Reference Spectra
JP XV
Neostigmine Methylsulfate
1.0
0.75
0.25
i •~~' ■ — ' — i — ^~ ' — ' — ' — i > ' — ■ — ' — i — ' — ' — ' — ■ — i — ■ — ' — ' — ■ — i — 1 ~ 1 r ' r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2000)
Nicardipine Hydrochloride
1.0
0.75
0.5
0.25
0.0
j |
— " — t t r ■■-- r- 1 i 1 1 1 1 i 1 1 1 r— i 1 1 1 1 i < 1 1 1 i 1 1 ■ i ■ " i " — I 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 100,000)
Nicergoline
0.75-
0.25 —
200.0
A solution in ethanol (99.5) (1 in 100,000)
l j I I I I I I I I I j " '" i
250.0 300.0
350.0
400.0
JP XV
Niceritrol
0.75 —
Ultraviolet-visible Reference Spectra 1615
0.25
i ■ "■' '■ ' • — r-^ -1 — ' — ,— r^ — ' — <— ' — r -1 " 1 — r ^~n — ' ' ' ' — i < < < — <— r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
Nicomol
1.0
0.75
0.5
0.25
0.0
— ---- j - \ ;---. | - \ : ;
I I j
i i i i | i ■!- i — i i — i — . — i 1 r-^ 1 -! 1 — 1 — i 1 1 — i 1 — i 1 1 — i 1 i — i — i — i — i — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 1 mol/L hydrochloric acid TS (1 in 100,000)
Nicotinamide
1.0
0.75
0.5
0.25
0.0
1 1 1 1 j i : 1 1 j 1 1 T 1 ; ■ -r ' f '■ ■■ -t r — T 1 T" "I p — T T - — I — "T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
1616 Ultraviolet-visible Reference Spectra
JP XV
Nicotinic Acid
1.0
0.75 -
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Nifedipine
1.0
0.75 -
0.25
T-T ■ t i i i ■ I i i i i j ' i i i j ' r i i j i i i i | r - i i j i - i i j i i i . | I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution in methanol (1 in 100,000)
Nilvadipine
1.0
0.75-
0.5-
0.25
U.U i i i i i i i i i j i i i i i i i i i | i i i T" I I I i i | i i i i i i i i r
200.0 300.0 400.0 500.0 600.0
A solution in ethanol (99.5) (1 in 100,000)
JP XV
Nitrazepam
1.0
Ultraviolet-visible Reference Spectra 1617
0.75
0.5
0.25
0.0
-
|
-
-
... ,..,... , . ,. j , , , ,
1 ■ t ■'
-
1 1 1 1 j t 1 1 r ■■ r ,,, t ■ i 1" — r — -j 1 1 1 1 j 1 r- ■! ■ i — ~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 100,000)
Nitrendipine
1.0
0.75 —
0.25
0.0
I I I I [ I I I I | [ |-"T-T 11111(111 ~~ !"
200.0 250.0 300.0
A solution in methanol (1 in 80,000)
350.0
I 1 l | IT I I [ I I I I
400.0 450.0
Noradrenaline
1.0
0.75 —
0.25— ■-
1 — I — T~T — | — I — I — I — I — I 1 — I — I — \J I — I
200.0 250.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
1 — l — I — I — I — | — I — i — I — 1 — I — I — I — I — r
350.0 400.0
1618 Ultraviolet-visible Reference Spectra
JP XV
Norfloxacin
1.0
0.75 ~
0.25
225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in an aqueous solution of sodium hydroxide (1 in 250) (1 in 200,000)
Nortriptyline Hydrochloride
1.0
0.75
0.5
0.0
~- — t 1 ■ -[ 1 1 j 1 1 r 1 1 1 1 1 ii r i r— i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
Noscapine
1.0
0.75
0.5
0.25
0.0
1 1 1 1 r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 : 1 1 1 — i~ i — i 1 1 1 i 1 1 1 — t
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 20,000)
JP XV
Nystatin
Ultraviolet-visible Reference Spectra 1619
-
1
0.5 -
1 :
o.o -
. I I . i I I , I i ■ I ■ . | r . 1 I
l i r r
i > r i ; i i i i i i i i i
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: To 10 mg add 50.25 mL of a mixture of diluted methanol (4 in 5) and sodium
hydroxide TS (200:1), dissolve by warming at not exceeding 50°C, and add diluted methanol (4 in 5) to make 500
mL.
Ofloxacin
1.0
0.75-
0.5-
0.25 -■
0.0 f l' T 'l " I' T" 'l " I 'I j I 1 I 1 I I I I T | 1 I I I I 1 I I I | I I I I i TT I ) | I I I I I 1 I I I
200.0 250.0 300.0 350.0 400.0 450.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 150,000)
Orciprenaline Sulfate
1.0
0.75
0.25
— i 1 — ^i 1 [ 1 r 1 1 \ 1 r 1 — i — ] 1 1 i~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
1620 Ultraviolet-visible Reference Spectra
JP XV
Oxazolam
1.0
0.75
0.5
0.25
0.0
:| \ |
\
N— ^ \ !
i i . i
■ ■ ' = i ' ■ - ■ ■
1 1 i ! i
1 — i — i — i — r=
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 100,000)
Oxethazaine
1.0
0.5
0.25
0.0
1 1 ,- i " I T 1 1 1 1 T 1 T T T i 7 r~ t- — I 1 i E — "T— "T-^-i T — ^T 1 1
200.0 225.0
A solution in ethanol (95) (1 in 2500)
300.0 325.0
350.0 375.0
400.0
Oxybuprocaine Hydrochloride
1.0
-i , r
200.0 225.0 250.0 275.0
An aqueous solution (1 in 100,000)
300.0 325.0 350.0 375.0 400.0
JP XV
Ultraviolet-visible Reference Spectra 1621
Oxycodone Hydrochloride Hydrate
1.0
0.75
0.5
0.25
0.0
1 r — i 1 i 1 ; 1 1 i 1 1 1 1 t ■■ i —i 1 ? j. . . . ._.. . . — . I I — r^^\ 1 1 1 r— j n 1 1 1 —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 10,000)
Oxymetholone
1.5
1.0
0.5 -
0.0
200.0
375.0 400.0
A solution prepared as follows: To 5 mL of a solution in methanol (1 in 5000) add 5 mL of sodium
hydroxide-methanol TS and methanol to make 50 mL.
Oxytetracycline Hydrochloride
0.75-
0.25-
n — i — i — r~i — i — i — i — i — | — i — i — i — i — i — i — i — i — i" f"r
200.0 250.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 50,000)
1 — I — I — I — I — I — | — r
350.0
400.0
1622 Ultraviolet-visible Reference Spectra
JP XV
Oxytocin
1.0
0.75
0.5 —
0.25-
0.0 - ( — i — i — i — i — i — i — i — i — ! — j" T" i • i ■ i ■<■ i — i — r~\ — | i r !'
200.0 250.0 300.0
An aqueous solution (1 in 2000)
i — i — i — i — [— i — r- 1 — i — n — i — i — i — r
350.0 400.0
Penbutolol Sulfate
1.0
0.75
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 10,000)
Pentazocine
1.0
0.75 -
0.5 -f
0.25
0.0 — t— > — ' — ■• '- i — r~i — ' — <-~ i— ' — r— , — , — i — , — , — , 1 "1 i - i -i ^i — i — ! — I — I — I — | — I — I — I — : — | — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
■100.0
JP XV
Ultraviolet-visible Reference Spectra 1623
Peplomycin Sulfate
1.0
0.75-
0.25
A solution prepared as follows: To 4 mg add 5 fiL of copper (II) sulfate TS, and dissolve in water to make 100 mL.
Perphenazine 1
1.0
0.75
0.5
0.25
0.0
225.0 250.0 275.0 300.0 325.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 200,000)
350.0
375.0
400.0
Perphenazine 2
1.0
0.75
0.5
0.0
| j i I i ;
j , 1 1 , j 1 , 1 , I I ,,.,,, I I I , T 1—1 j 1 1 1 1 i "■■!-■ ■!■■■! I
225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution obtained by adding 10 mL of water to 10 mL of the solution for Perphenazine 1
1624 Ultraviolet-visible Reference Spectra
JP XV
Perphenazine Maleate 1
1.0
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 20,000)
Perphenazine Maleate 2
0.75
0.25 -
"F 1 1 1 1 1 1— r — p-n 1 1 1 j r 1 1 r j r r— r 1 j r~ — r 1 r — f ■ i f 1 — 1 f -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution obtained by adding 30 mL of water to 10 mL of the solution for Perphenazine Maleate 1
Pethidine Hydrochloride
1.0
0.75
0.5 -
0.25
0.0
\ f\
r — i — r — t — H— i — i — i — i i — "i 1 — i — i T™ i I i — 5 — i — ] — i — i — i — ~i — p — ! — r — i — -i — r — i — 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2000)
JP XV
Ultraviolet-visible Reference Spectra 1625
Phenethicillin Potassium
1.0
0.75 —
0.5
0.25-
0.0
j—\ — i — i — i — i — i — i — i — [ — i — i — i — i — i — i — r~i — i — | — i — [ — i — i — i — i — i — [ — i — ] — i — i — i — i — t — i — i — i — r
200.0 250.0 300.0 350.0 400.0
An aqueous solution (1 in 5000)
Phenolsulfonphthalein
1.0
0.75 -
0.5
0.25 -
0.0
250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0
A solution in diluted sodium carbonate TS (1 in 10) (1 in 400,000)
Phenylbutazone
1.0
0.75
0.5
0.25
0.0
1 1 1 1 r r 1 r 1 i 1 1 r r H — t ! i r— *T----"i I i 1 i 1 1 1 1 1 r 1 r 1 1 1 1
200.0 225.0 250.0
275.0 300.0 325.0 350.0 375.0
-100.0
A solution prepared as follows: Dissolve 1 mg in 10 mL of dilute sodium hydroxide TS, and add water to make 100
mL.
1626 Ultraviolet-visible Reference Spectra
Phytonadione 1
1.0
0.75
0.5
JP XV
0.25
0.0
; ! ;
: ■
-I 1 i :
— ! j :
1 r* H j 1 1 1 1 1 1 1 1 1 i ■ 1 — I 1 f " 'I 1 " T "' I 1 — I 1 1
250.0 300.0
A solution in isooctane (1 in 100,000)
350.0
400.0
450.0
500.0
Phytonadione 2
0.75
0.25
250.0 300.0
A solution in isooctane (1 in 10,000)
500.0
Pimaricin
1.0
0.75-
0.25-
"i — i — i — ] — i — i — i — i — i — r
230.0 250.0
A solution in a solution of acetic acid (100) in methanol (1 in 100) (1 in 200,000)
JP XV
Ultraviolet-visible Reference Spectra 1627
Pindolol
1.0
0.75
0.5
0.25
0.0
0.75 -
0.5 -
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
Pipemidic Acid Hydrate
1.0
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 0.1 g in 20 mL of sodium hydroxide TS, and add water to make 200 mL.
To 1 mL of this solution add water to make 100 mL.
Pirarubicin
1.0
0.75-
0.25-
T — I — i — I — I — I — I — I — I — | — i — I — I — I — I — 1 — ! — I — I — | — I — I — I — I — I — I — I — I — I — | — I — I — I — \ — I T I — i — r
200.0 300.0 400.0 500.0 600.0
A solution prepared as follows: Dissolve 10 mg in 80 mL of methanol and 6 mL of diluted hydrochloric acid (1 in
5000), and add water to make 100 mL. To 10 mL of this solution add diluted methanol (4 in 5) to make 100 mL.
1628 Ultraviolet-visible Reference Spectra
JP XV
Pirenoxine
1.0
0.75
0.25
-i 1 1 1 1 3 ! r 1 r-
200.0 250.0 300.0 350.0 400.0
A solution in phosphate buffer solution, pH 6.5 (1 in 200,000)
450.0 500.0 550.0
Pirenzepine Hydrochloride Hydrate
1.0
0.75-
0.25 —
200.0
An aqueous solution (1 in 40,000)
i — i — i — i— t~ r—r— r— i — j — i — i Tl I i I — r~! — j — I — I — i — I — I r _ r i — r
250.0 300.0 350.0 400.0
Piroxicam
1.0
0.75-
0.5
0.25-
0.0
i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i
200.0 250.0 300.0 350.0 400.0
A solution in a mixture of methanol and 0.5 mol/L hydrochloric acid TS (490:1) (1 in 200,000)
450.0
JP XV
Ultraviolet-visible Reference Spectra 1629
Potassium Canrenoate
1.0
0.75
_
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Potassium Clavulanate
1.0
0.75
0.25 -
An aqueous solution (1 in 50,000). To 1 mL of this solution add 5 mL of imidazole TS (Warm in a water bath at 30°
C for 12 minutes).
Potassium Guaiacolsulfonate
1.0
0.75
0.5
0.25
0.0
1 1 1 1 1 r t 1 1 i 1 1 r 1 — ^ 1 •: 1 ■ — ;^~~r" i 1 " t 1 — i 1 r 1 1 — H r — r^^i 1 i 1 1 f 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 10 mL of an aqueous solution (1 in 2000) add phosphate buffer solution, pH 7.0
to make 100 mL.
1630 Ultraviolet-visible Reference Spectra
JP XV
Pranoprofen
1.0
0.75
0.5
0.25 -I
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0
400.0
A solution prepared as follows: To 10 mL of a solution in 1 mol/L hydrochloric acid TS (1 in 5000) add water to
make 100 mL.
Pravastatin Sodium
1.0
0.75 -
0.5-
0.25 — ■
0.0 1 — i — i — i — i — i — i — i — i — I — i T - 1 — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i — f — l — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
An aqueous solution (1 in 100,000)
Prazepam
1.0
0.75 -
0.5-
0.25
0.0 ' I ■' ' ' ' i ' r ' r i ' v ' ' i ' ' ' ' i ' ' ' ' i ' ■ ' ' i ' ' ■ ' i ' ' ' ' i r ~ r ~' 1 i i ■ > ■
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 0.01 g in 1000 mL of a solution of sulfuric acid in ethanol (99.5) (3 in
1000).
JP XV
Ultraviolet-visible Reference Spectra 1631
Probenecid
1.0
0.75
0.25
-i — i — i — i — | — i — i — i — i — [ — i — i — ' — i — | — i — i — i — i — i — i — i — ' — ' — i — ' — ' — ' — i — r — i — i — i — i — ; — i — ■ — <~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 50,000)
Procaine Hydrochloride
1.0
0.75
0.25
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
Procarbazine Hydrochloride
1.0
0.75
0.25 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
1632 Ultraviolet-visible Reference Spectra
JP XV
Procaterol Hydrochloride Hydrate
1.0
0.5
0.25
0.0
200.0 225.0 250.0 275.0
An aqueous solution (7 in 1,000,000)
300.0
325.0 350.0 375.0
400.0
Promethazine Hydrochloride
1.0
0.75 -
0.5
0.25
0.0 ~~ t ' ' ' ■ i ' , ~™ 1 — ' — i — '"'' ' '""'■' i — ' — ' — ' — ' — i — p — ' — ' — ' — i — ' — ' ' ' I — ' — ' — ' — ' — i — ' — ■ — ■ — ■"
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
Propranolol Hydrochloride
1.0
0.75
0.25 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
JP XV
Ultraviolet-visible Reference Spectra 1633
Pyrantel Pamoate
1.0
0.75
0.5
0.25
0.0
250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 0.1 g in 50 mL of dimethylformamide, and add methanol to make 200 mL.
To 2 mL of this solution add a solution of hydrochloric acid in methanol (9 in 1000) to make 100 mL.
Pyrazinamide
1.0
0.75
0.5
0.25
0.0
1 1 1 1 i 1 1 1 1 j 1 1 t — t r — i 1 1 r—^r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
Pyridostigmine Bromide
1.0
0.25
- 1 1 1 — "i" — f
200.0 225.0
-i — i — i — r — i — r
250.0 275.0
300.0 325.0 350.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 30,000)
375.0
400.0
1634 Ultraviolet-visible Reference Spectra
JP XV
Pyridoxine Hydrochloride
1.0
0.75-
0.5-
0.25-
0.0— ) — i — i — i — rn — i — i — i — i — j — i — n — i — i — i — i — i — i — j — i — i T"1 — t — r~i — i — r~j — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 100,000)
Pyrrolnitrin
1.2
1.0-
0.75
0.5-
0.25-
0,0 1 — i — i — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i — i — pT - ! r- 1 i i i i i | p i i i i — i — i — i — r
200.0 250.0
A solution in ethanol (95) (1 in 100,000)
300.0
350.0
400.0
Quinine Ethyl Carbonate
1.0
0.75
0.5
0.25
0.0
1 1 1 ' ■ j 1 1 1 1 j 1 1 1" — I j T 1 1 1 1 ! 1 1 1 j 1 1 1 1 1 .. r ■ t^— | t j 1 1 — ~r 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 20,000)
JP XV
Quinine Sulfate Hydrate
1.0
Ultraviolet-visible Reference Spectra 1635
0.75
0.5
0.25
0.0
1: j |
I 1
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 20,000)
Ranitidine Hydrochloride
1.0
0.75-
0.25-
"1 — ! — I — T~T — I — I — |--|—j--f — | — f— t — i — i — i — i — i — i — i — ] — i — i — r
200.0 250.0 300.0
An aqueous solution (1 in 100,000)
i — i — i — [ — i — r
350.0
400.0
Reserpine
1.0
0.75-
0.25
0.0
i 1 r — j 1 1 1 1 — "j — T ■ t — "i 1 — *~\ 1 1 1 1 j r — r™i 1 — [ I 1 — "i r 1 1 r — i 1 f — I ' t '
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in acetonitrile (1 in 50,000)
1636 Ultraviolet-visible Reference Spectra
Riboflavin
1.0
0.75
JP XV
0.5
0.25
0.0
200.0 250.0 300.0 350.0 400.0
A solution in phosphate buffer solution, pH 7.0 (1 in 100,000)
450.0
500.0
550.0
Riboflavin Butyrate
1.0
0.75
0.5
0.25
0.0
250.0 300.0 350.0
The sample solution obtained in the Assay
" i : !
400.0
450.0
500.0
Riboflavin Sodium Phosphate
1.0
0.75
0.5
0.25
0.0
/ \ !/ \
r* — l r 1 1 1 — "t 1 1 1 1 1 1 r i r t r 1 i 1 r~ — i r
200.0 250.0 300.0 350.0 400.0
A solution in phosphate buffer solution, pH 7.0 (1 in 100,000)
450.0
500.0
550.0
JP XV
Ultraviolet-visible Reference Spectra 1637
Rifampicin
1.0-
0.75-
0.5-
~i — i — 1~-| — i — i — i — i — i — i — i — i — if i — i — i — i — i — i — i — i — i — | — i — i — i — i — i i I — i — r
300.0 400.0 500.0 600.0
T — I — I — r
200.0
A solution prepared as follows: To 5 mL of a solution in methanol (1 in 5000) add 0.05 mol/L phosphate buffer so-
lution, pH 7.0 to make 100 mL.
Ritodrine Hydrochloride
1.0
0.75-
0.25-
0.0
i — i — i — i — i — i — i — r r -\ ■] — l — l — i — i — i — i — m — | — i — i — i — i — i — i — r
200.0 250.0 300.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 20,000)
350.0
i — i — i — i — i — r
400.0
Rokitamycin
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 50,000)
1638 Ultraviolet-visible Reference Spectra
JP XV
Roxatidine Acetate Hydrochloride
1.0
0.75 —
0.5
0.25 -
0.0
i — i — i — i — i — r~T — i — i — | — i — i — i — i — i — i — i — i T™| — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — r
200.0 250.0
A solution in ethanol (99.5) (1 in 10,000)
300.0
350.0
400.0
Salazosulfapyridine
1.0
-1 I | . r I I | I I . I | I . r , [ , , , : p
250.0 300.0 350.0 400.0 450.0
A solution in dilute sodium hydroxide TS (1 in 100,000)
i ■ ■ ' ' r
500.0 550.0 600.0
Salbutamol Sulfate
1.0
0.75
0.5
0.25
0.0
1 1 m, ~T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 12,500)
JP XV
Ultraviolet-visible Reference Spectra 1639
Santonin
1.0
0.75 —
0.5-
0.25 -
0.0
i — i ■ r "r 'i — i — i — i — i — | — i — i — i — i — i — i — i — i \~\ — i — i — i — i — i — i — i — ! — i — | — i — i — ! — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in ethanol (95) (3 in 250,000)
Scopolamine Butylbromide
1.0
0.75
0.5
0.25
0.0
— i \ \— i
r 1 f f i [ 1 1 1 i 1 t — i r i" 1 ' T T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 1000)
0.75-
0.25
T — i — i — i — i — i — i — i — i — |~n — i — i — i — i — r — r~i — r^ — i — i — i — i — ! — r
200.0 250.0 300.0
A solution in ethanol (99.5) (1 in 10,000)
*i — I — pi — m — i— i — i — i v r
350.0
400.0
1640 Ultraviolet-visible Reference Spectra
Sodium Cromoglicate
1.0
JP XV
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in phosphate buffer solution, pH 7.4 (1 in 100,000)
Sodium Picosulfate Hydrate
1.0
0.75 -
0.5 -
0.25
0.0 r ' ■ ' i ' ■ r r r
200.0 225.0 250.0
An aqueous solution (1 in 25,000)
t 1 f 1 r t 1 "| — T — i r - r ■ -j - — 1 1 r — 1 j r~^ 1 1 j 1 1 1 r-
275.0 300.0 325.0 350.0 375.0 400.0
Spiramycin Acetate
1.0
0.75-
0.25-
200.0
A solution in methanol (1 in 50,000)
JP XV
Spironolactone
1.0
0.75
Ultraviolet-visible Reference Spectra 1641
0.5 -
0.25
0.0
i — i — i — i — r -t ■ i ■ ■ i- i — |— i — i — . — , — i — i — . — i — i — ] — i — i — i — i — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Sulfinpyrazone
1.0
0.75
0.5 -
0.25
QQ " — | — i — i — i — t — | — t — i 1 — i — i — i — i — i — i — i — \ — t— i — t"] — i I \ — i — ] r—\ — i — i f — i — t — i — r-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L sodium hydroxide TS (1 in 100,000)
— i 1 1 r—
Sulpiride
1.0
0.75 -
0.25
~r — t -■ i 1 r 1 1 1 1 1 r 1 r — i ^l — j 1 1 1 1 1 1 1 1 1 r~
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution prepared as follows: To 5 mL of a solution in 0.05 mol/L sulfuric acid TS (1 in 1000) add water to make
100 mL.
1642 Ultraviolet-visible Reference Spectra
JP XV
0.75 "I
0.25
00 "" I ' •~~' ' I ' ' ' ' ■ T '" ' ' ' — ' — ' — i — ' ~ n ~< — ' — I — ' — ' — ' — ' — I — ' — ' — ' — ' — I — ' — ' — ' — ' — i ' "' " 1 r "
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
Tamsulosin Hydrochloride
0.75-
0.25-
t — i — i — i — i — i — i — i — i — | — r
200.0 250.0
An aqueous solution (3 in 160,000)
400.0
Tegafur
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L sodium hydroxide TS (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1643
Terbutaline Sulfate
1.0
0.75 ^
0.25 —
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 10,000)
Testosterone Propionate
1.0
0.75-
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — rH — ! — i — i — i — I — i — i — i — i — i — i — i — i — i — | — i " t • i — i — i • i • i — j — r
200.0 250.0 300.0 350.0 400.0
A solution in ethanol (95) (1 in 100,000)
Tetracaine Hydrochloride
1.0
0.75
0.5
0.25
0.0
: : :
: : r
— -4 - i J i ™.-™™™j™ „„„„„„„,„„„„
r 1 1 1 1 1 1 1 i" 1^^t* == t— — r — i r — i r 1 1 1 r" — i — -i 1 — H 1 1 7*^ i 1 1 1 r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 200,000)
1644 Ultraviolet-visible Reference Spectra
JP XV
Tetracycline Hydrochloride
1.0
0.75 -
0.25
I ' « « ' | I ■ ' I 1 ' ■ ' ' | ■ ■ ' ' [ ■ 1 1 r [ i I r r | I I I I | I I
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
An aqueous solution (1 in 62,500)
Theophylline
1.0
0.75
0.25-
200.0 250.0 300.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 200,000)
350.0
400.0
Thiamine Chloride Hydrochloride
1.0
0.75
0.25 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 100,000)
JP XV
Ultraviolet-visible Reference Spectra 1645
Thiamylal Sodium
1.0
0.75-
0.25-
~i — r t T i "i" i i — r | " i — i — i — i — i — r
200.0 250.0
A solution in ethanol (95) (7 in 1,000,000)
-i — i — j — i — i — i — i — i — I — i — i — I — |~~i — I — i — i — i — i — i — i — r
300.0 350.0 400.0
Timepidium Bromide Hydrate
1.0
0.75
0.25
0.0
i , i i j i i i "T i 1 . 1 1 j , r r- i j— i v i -T-] 1 1 1 1 i 1 1 1 , j 1 r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Timolol Maleate
1.0
0.75-
0.5-
0.25 — -
0.0 — I i - r • i — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i — i — j — i — i — i — i — i — i — i — i i | i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
1646 Ultraviolet-visible Reference Spectra
JP XV
Tinidazole
1.0
0.75 -
0.25
— i r 1 t — l — t 1 "i t — t™| r™n 1 1 1 1 1 r
200.0 225.0 250.0 275.0
A solution in methanol (1 in 50,000)
■[■■'" v ' ■ r
350.0 375.0
4000
Tipepidine Hibenzate
1.0
0.75
0.5
0.25 -
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 100,000)
Tizanidine Hydrochloride
1.0
0.75
0.5-
0.25-
0.0
i — i — i — i — i — i — i — i — i — i — r
200.0 300.0
A solution in diluted 1 mol/L ammonia TS (1 in 10) (1 in 125,000)
t — i — r ■ ■ i ~ | — r~i — i — t^ i — r~n — v
400.0 500.0
JP XV
Ultraviolet-visible Reference Spectra 1647
Tocopherol Nicotinate
1.0
0.75 -
0.5 -
0.25 -
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (99.5) (1 in 20,000)
Todralazine Hydrochloride Hydrate
1.0
0.75
0.5
0.25
0.0
_
—
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (3 in 100,000)
Tofisopam
1.0 -
0.5 -
0.0-
■■r r- r ■(■,,,, j ,,, ,
r < ' r j i i i i r i i i t i i i i i r i i r i j i i i i i i i i 1
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0 700.0
A solution in ethanol (95) (1 in 100,000)
1648 Ultraviolet-visible Reference Spectra
Tolazamide
1.0
JP XV
0.75
0.5
0.25
0.0
-
i j
!
-
1
i
1
i
i l i i t ■ r • -r "l — l
i.ii
fs-r-i — i i | , i i ,
1 — r —•"- '■""'' "
"' ' ' '
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 100,000)
Tolnaftate
l.U
0.75 ~
1 , ;
-
0.5 -_
0.25 -
0.0
I
1
1 r 1 1 r 1 r — t 1
" T 1""— 1 1
-.,.-,.,..,. j r -, , , ■
'"-Ill"
. . . ,
ill.'
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in methanol (1 in 100,000)
0.75
0.25 "
-1 1 1 ] 1 r— , , ■ [ ■-, 1 . . | 1 . 1 1 1 r 1 1 1 "J '^1 1 1 1 1 1 1 1 [ - ■'■ - ' ■ r-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 125,000)
JP XV
Trepibutone
1.0
0.75
Ultraviolet-visible Reference Spectra 1649
0.5
0.0
- III!
I \ 1 j
— ■ — i — "r — i 1 [ 1 1 1 j i 1 1 t r 1 1 ■ t — t~ — i ■ ■ r 1 1 t ■ n i r 1 r
200.0 225.0 250.0 275.0 300.0 325.0
A solution in diluted dilute sodium hydroxide TS (1 in 10) (1 in 100,000)
400.0
Triamcinolone Acetonide
0.75
0.25
-i 1 1 1 [ 1 1 1 1 1 1 1 c 1 1 i 1— =t="^ — r^r — i 1 1 1 — i 1 -
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in ethanol (95) (1 in 100,000)
Triamterene
1.0
0.75
0.5
0.25
0.0
-I | \ \ ! I
■— i — i — j— ■—>—■— i— j— ■ — ' ■ ■ i — <— •—> — ' j ' ■ ■ ' j ' ■ ■ ' j ■■'■■■' — <— ^ r =t= ~ l r i j i . i i
250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0
A solution prepared as follows: Dissolve 0.01 g in 100 mL of acetic acid (100). To 10 mL of this solution add water
to make 100 mL.
1650 Ultraviolet-visible Reference Spectra
JP XV
Trichlormethiazide
1.0
0.75 —
0.25
i — i — i — i — i - ~i — r • " i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — rT — i — | — i — i — i — i — r
200.0 250.0 300.0 350.0
A solution in ethanol (95) (3 in 250,000)
400.0
Trimebutine Maleate
1.0
0.75-
0.25 —
0.0
1 — i — i — i — i — i — i — i — i — | — i — i — i — i — i — i — i — i — i — | — i — i — i — i — n — i — i — i — | — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 50,000)
400.0
Trimetazidine Hydrochloride
1.0
0.75
0.5
0.25
0.0
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.1 mol/L hydrochloric acid TS (1 in 6250)
JP XV
Ultraviolet-visible Reference Spectra 1651
Trimetoquinol Hydrochloride Hydrate
1.0
0.75
0.5
0.25
0.0
. . . r I , i ' ■— T- 1 ' ' i i >-■--!■ r i r--T— I T"\
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 20,000)
Tubocurarine Chloride Hydrochloride Hydrate
1.0
0.75
0.5
0.25
0.0
1 1 1 1 i ] 1 r 1 i 1 1 1 r — r 1 1 r ■■ "r^^ — r 1 r ■■ r r r r 1 r r r — i 1 f—^\ T 1 T
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (3 in 100,000)
Tulobuterol Hydrochloride
-| 1 1 1 1 , 1 r , , ,-
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 2500)
1652 Ultraviolet-visible Reference Spectra
Ulinastatin
1.0
0.75 -
JP XV
0.5
0.25
0.0
200.0 225.0 250.0 275.0
An aqueous solution 2000 units/mL
p=r=i i i | i i ——- *■
350.0 375.0
400.0
Urapidil
1.0
0.75 -
0.25
200.0 250.0
A solution in ethanol (95) (1 in 100,000)
Vancomycin Hydrochloride
1.0
0.75 —
0.5-
0.25 —
0.0
i — i — i — i — i — i — i — i — i — ] — i — i — i — i — i — i — i — i — i — i — i I — i — i — ] — i — i — i — i — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0 400.0
An aqueous solution (1 in 10,000)
JP XV
Ultraviolet-visible Reference Spectra 1653
Verapamil Hydrochloride
1.5
n — ' — ' — ■ — ■ — i — ■ — < — ■ — > — i — ' — ■ — ■ — ' — i — i — i — ' — i — i — ■ — ' — i — ■ — i — ' — ■ — i — ' — r
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.01 mol/L hydrochloric acid TS (1 in 50,000)
Vinblastine Sulfate
1.0
0.75
0.5
0.25
0.0
;7Y
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
An aqueous solution (1 in 50,000)
Vincristine Sulfate
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An aqueous solution (1 in 50,000)
1654 Ultraviolet-visible Reference Spectra
Warfarin Potassium
1.0
0.75
0.5
JP XV
0.25
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200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0
A solution in 0.02 mol/L potassium hydroxide TS (1 in 100,000)
Zaltoprofen
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0.5-
0.25
0.0
i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r
200.0 250.0 300.0 350.0
A solution in ethanol (99.5) (1 in 100,000)
400.0
GENERAL INFORMATION
1. Amino Acid Analysis
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia.
Amino acid analysis refers to the methodology used to de-
termine the amino acid composition or content of proteins,
peptides, and other pharmaceutical preparations. Proteins
and peptides are macromolecules consisting of covalently
bonded amino acid residues organized as a linear polymer.
The sequence of the amino acids in a protein or peptide
determines the properties of the molecule. Proteins are
considered large molecules that commonly exist as folded
structures with a specific conformation, while peptides are
smaller and may consist of only a few amino acids. Amino
acid analysis can be used to quantify protein and peptides, to
determine the identity of proteins or peptides based on their
amino acid composition, to support protein and peptide
structure analysis, to evaluate fragmentation strategies for
peptide mapping, and to detect atypical amino acids that
might be present in a protein or peptide. It is necessary to
hydrolyze a protein/peptide to its individual amino acid
constituents before amino acid analysis. Following pro-
tein/peptide hydrolysis, the amino acid analysis procedure
can be the same as that practiced for free amino acids in other
pharmaceutical preparations. The amino acid constituents of
the test sample are typically derivatized for analysis.
Apparatus
Methods used for amino acid analysis are usually based on
a chromatographic separation of the amino acids present in
the test sample. Current techniques take advantage of the au-
tomated chromatographic instrumentation designed for ana-
lytical methodologies. An amino acid analysis instrument will
typically be a low-pressure or high-pressure liquid chromato-
graph capable of generating mobile phase gradients that
separate the amino acid analytes on a chromatographic
column. The instrument must have postcolumn derivatiza-
tion capability, unless the sample is analyzed using
precolumn derivatization. The detector is usually an ultrav-
iolet-visible or fluorescence detector depending on the
derivatization method used. A recording device (e.g.,
integrator) is used for transforming the analog signal from
the detector and for quantitation. It is preferred that
instrumentation be dedicated particularly for amino acid
analysis.
General Precautions
Background contamination is always a concern for the
analyst in performing amino acid analysis. High purity
reagents are necessary (e.g., low purity hydrochloric acid can
contribute to glycine contamination). Analytical reagents are
changed routinely every few weeks using only high-pressure
liquid chromatography (HPLC) grade solvents. Potential
microbial contamination and foreign material that might be
present in the solvents are reduced by filtering solvents before
use, keeping solvent reservoirs covered, and not placing ami-
no acid analysis instrumentation in direct sunlight.
Laboratory practices can determine the quality of the
amino acid analysis. Place the instrumentation in a low traffic
area of the laboratory. Keep the laboratory clean. Clean and
calibrate pipets according to a maintenance schedule. Keep
pipet tips in a covered box; the analysts may not handle pipet
tips with their hands. The analysts may wear powder-free la-
tex or equivalent gloves. Limit the number of times a test
sample vial is opened and closed because dust can contribute
to elevated levels of glycine, serine, and alanine.
A well-maintained instrument is necessary for acceptable
amino acid analysis results. If the instrument is used on a
routine basis, it is to be checked daily for leaks, detector and
lamp stability, and the ability of the column to maintain reso-
lution of the individual amino acids. Clean or replace all
instrument filters and other maintenance items on a routine
schedule.
Reference Standard Material
Acceptable amino acid standards are commercially
available for amino acid analysis and typically consist of an
aqueous mixture of amino acids. When determining amino
acid composition, protein or peptide standards are analyzed
with the test material as a control to demonstrate the integrity
of the entire procedure. Highly purified bovine serum
albumin has been used as a protein standard for this purpose.
Calibration of Instrumentation
Calibration of amino acid analysis instrumentation
typically involves analyzing the amino acid standard, which
consists of a mixture of amino acids at a number of concen-
trations, to determine the response factor and range of
analysis for each amino acid. The concentration of each ami-
no acid in the standard is known. In the calibration proce-
dure, the analyst dilutes the amino acid standard to several
different analyte levels within the expected linear range of the
amino acid analysis technique. Then, replicates at each of the
different analyte levels can be analyzed. Peak areas obtained
for each amino acid are plotted versus the known concentra-
tion for each of the amino acids in the standard dilution.
These results will allow the analyst to determine the range of
amino acid concentrations where the peak area of a given
amino acid is an approximately linear function of the amino
acid concentration. It is important that the analyst prepare
the samples for amino acid analysis so that they are within
the analytical limits (e.g., linear working range) of the tech-
nique employed in order to obtain accurate and repeatable
results.
Four to six amino acid standard levels are analyzed to
determine a response factor for each amino acid. The
response factor is calculated as the average peak area or peak
height per nmol of amino acid present in the standard. A
calibration file consisting of the response factor for each ami-
no acid is prepared and used to calculate the concentration of
each amino acid present in the test sample. This calculation
involves dividing the peak area corresponding to a given ami-
1655
1656 Amino Acid Analysis / General Information
JP XV
no acid by the response factor for that amino acid to give the
nmol of the amino acid. For routine analysis, a single-point
calibration may be sufficient; however, the calibration file is
updated frequently and tested by the analysis of analytical
controls to ensure its integrity.
Repeatability
Consistent high quality amino acid analysis results from an
analytical laboratory require attention to the repeatability of
the assay. During analysis of the chromatographic separation
of the amino acids or their derivatives, numerous peaks can
be observed on the chromatogram that correspond to the
amino acids. The large number of peaks makes it necessary to
have an amino acid analysis system that can repeatedly iden-
tify the peaks based on retention time and integrate the peak
areas for quantitation. A typical repeatability evaluation in-
volves preparing a standard amino acid solution and analyz-
ing many replicates (i.e., six analyses or more) of the same
standard solution. The relative standard deviation (RSD) is
determined for the retention time and integrated peak area of
each amino acid. An evaluation of the repeatability is ex-
panded to include multiple assays conducted over several
days by different analysts. Multiple assays include the prepa-
ration of standard dilutions from starting materials to deter-
mine the variation due to sample handling. Often the amino
acid composition of a standard protein (e.g., bovine serum
albumin) is analyzed as part of the repeatability evaluation.
By evaluating the replicate variation (i.e., RSD), the labora-
tory can establish analytical limits to ensure that the analyses
from the laboratory are under control. It is desirable to estab-
lish the lowest practical variation limits to ensure the best
results. Areas to focus on to lower the variability of the ami-
no acid analysis include sample preparation, high back-
ground spectral interference due to quality of reagents
and/or laboratory practices, instrument performance and
maintenance, data analysis and interpretation, and analyst
performance and habits. All parameters involved are fully in-
vestigated in the scope of the validation work.
Sample Preparation
Accurate results from amino acid analysis require purified
protein and peptide samples. Buffer components (e.g., salts,
urea, detergents) can interfere with the amino acid analysis
and are removed from the sample before analysis. Methods
that utilize postcolumn derivatization of the amino acids are
generally not affected by buffer components to the extent seen
with precolumn derivatization methods. It is desirable to
limit the number of sample manipulations to reduce potential
background contamination, to improve analyte recovery,
and to reduce labor. Common techniques used to remove
buffer components from protein samples include the follow-
ing methods: (1) injecting the protein sample onto a reversed-
phase HPLC system, eluting the protein with a volatile sol-
vent containing a sufficient organic component, and drying
the sample in a vacuum centrifuge; (2) dialysis against a vola-
tile buffer or water; (3) centrifugal ultrafiltration for buffer
replacement with a volatile buffer or water; (4) precipitating
the protein from the buffer using an organic solvent (e.g.,
acetone); and (5) gel filtration.
Internal Standards
It is recommended that an internal standard be used to mo-
nitor physical and chemical losses and variations during ami-
no acid analysis. An accurately known amount of internal
standard can be added to a protein solution prior to hydroly-
sis. The recovery of the internal standard gives the general
recovery of the amino acids of the protein solution. Free ami-
no acids, however, do not behave in the same way as protein-
bound amino acids during hydrolysis because their rates of
release or destruction are variable. Therefore, the use of an
internal standard to correct for losses during hydrolysis may
give unreliable results. It will be necessary to take this point
under consideration when interpreting the results. Internal
standards can also be added to the mixture of amino acids af-
ter hydrolysis to correct for differences in sample application
and changes in reagent stability and flow rates. Ideally, an in-
ternal standard is an unnaturally occurring primary amino
acid that is commercially available and inexpensive. It should
also be stable during hydrolysis, its response factor should be
linear with concentration, and it needs to elute with a unique
retention time without overlapping other amino acids. Com-
monly used amino acid standards include norleucine,
nitrotyrosine, and a-aminobutyric acid.
Protein Hydrolysis
Hydrolysis of protein and peptide samples is necessary for
amino acid analysis of these molecules. The glassware used
for hydrolysis must be very clean to avoid erroneous results.
Glove powders and fingerprints on hydrolysis tubes may
cause contamination. To clean glass hydrolysis tubes, boil
tubes for 1 hour in 1 mol/L hydrochloric acid or soak tubes
in concentrated nitric acid or in a mixture of concentrated
hydrochloric acid and concentrated nitric acid (1:1). Clean
hydrolysis tubes are rinsed with high-purity water followed
by a rinse with HPLC grade methanol, dried overnight in an
oven, and stored covered until use. Alternatively, pyrolysis of
clean glassware at 500°C for 4 hours may also be used to
eliminate contamination from hydrolysis tubes. Adequate
disposable laboratory material can also be used.
Acid hydrolysis is the most common method for hydrolyz-
ing a protein sample before amino acid analysis. The acid
hydrolysis technique can contribute to the variation of the
analysis due to complete or partial destruction of several ami-
no acids. Tryptophan is destroyed; serine and threonine are
partially destroyed; methionine might undergo oxidation;
and cysteine is typically recovered as cystine (but cystine
recovery is usually poor because of partial destruction or
reduction to cysteine). Application of adequate vacuum
( ^ less than 200 /xm of mercury or 26.7 Pa) or introduction of
an inert gas (argon) in the headspace of the reaction vessel
can reduce the level of oxidative destruction. In peptide
bonds involving isoleucine and valine the amido bonds of Ile-
Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved; and
asparagine and glutamine are deamidated, resulting in aspar-
tic acid and glutamic acid, respectively. The loss of trypto-
phan, asparagine, and glutamine during an acid hydrolysis
limits quantitation to 17 amino acids. Some of the hydrolysis
techniques described are used to address these concerns.
Some of the hydrolysis techniques described (i.e., Methods
4-11) may cause modifications to other amino acids. There-
fore, the benefits of using a given hydrolysis technique are
weighed against the concerns with the technique and are test-
ed adequately before employing a method other than acid
hydrolysis.
A time-course study (i.e., amino acid analysis at acid
hydrolysis times of 24, 48, and 72 hours) is often employed to
analyze the starting concentration of amino acids that are
JPXV
General Information / Amino Acid Analysis 1657
partially destroyed or slow to cleave. By plotting the observed
concentration of labile amino acids (i.e., serine and threo-
nine) versus hydrolysis time, the line can be extrapolated to
the origin to determine the starting concentration of these
amino acids. Time-course hydrolysis studies are also used
with amino acids that are slow to cleave (e.g., isoleucine and
valine). During the hydrolysis time course, the analyst will
observe a plateau in these residues. The level of this plateau is
taken as the residue concentration. If the hydrolysis time is
too long, the residue concentration of the sample will begin
to decrease, indicating destruction by the hydrolysis condi-
tions.
An acceptable alternative to the time-course study is to
subject an amino acid calibration standard to the same
hydrolysis conditions as the test sample. The amino acid in
free form may not completely represent the rate of destruc-
tion of labile amino acids within a peptide or protein during
the hydrolysis. This is especially true for peptide bonds that
are slow to cleave (e.g., Ile-Val bonds). However, this
technique will allow the analyst to account for some residue
destruction. Microwave acid hydrolysis has been used and is
rapid but requires special equipment as well as special precau-
tions. The optimal conditions for microwave hydrolysis must
be investigated for each individual protein/peptide sample.
The microwave hydrolysis technique typically requires only a
few minutes, but even a deviation of one minute may give in-
adequate results (e.g., incomplete hydrolysis or destruction
of labile amino acids). Complete proteolysis, using a mixture
of proteases, has been used but can be complicated, requires
the proper controls, and is typically more applicable to pep-
tides than proteins.
Note: During initial analyses of an unknown protein,
experiments with various hydrolysis time and temperature
conditions are conducted to determine the optimal condi-
tions.
Method 1
Acid hydrolysis using hydrochloric acid containing phenol
is the most common procedure used for protein/peptide
hydrolysis preceding amino acid analysis. The addition of
phenol to the reaction prevents the halogenation of tyrosine.
Hydrolysis Solution 6 mol/L hydrochloric acid contain-
ing 0.1% to 1.0% of phenol.
Procedure —
Liquid Phase Hydrolysis Place the protein or peptide
sample in a hydrolysis tube, and dry. [Note: The sample is d-
ried so that water in the sample will not dilute the acid used
for the hydrolysis.] Add 200 /uL of Hydrolysis Solution per
500 /ug of lyophilized protein. Freeze the sample tube in a dry
ice-acetone bath, and flame seal in vacuum. Samples are typi-
cally hydrolyzed at 110°C for 24 hours in vacuum or inert at-
mosphere to prevent oxidation. Longer hydrolysis times
(e.g., 48 and 72 hours) are investigated if there is a concern
that the protein is not completely hydrolyzed.
Vapor Phase Hydrolysis This is one of the most common
acid hydrolysis procedures, and it is preferred for microanal-
ysis when only small amounts of the sample are available.
Contamination of the sample from the acid reagent is also
minimized by using vapor phase hydrolysis. Place vials con-
taining the dried samples in a vessel that contains an ap-
propriate amount of Hydrolysis Solution. The Hydrolysis
Solution does not come in contact with the test sample. App-
ly an inert atmosphere or vacuum (^ less than 200 //m of
mercury or 26.7 Pa) to the headspace of the vessel, and heat
to about 110°C for a 24-hour hydrolysis time. Acid vapor
hydrolyzes the dried sample. Any condensation of the acid in
the sample vials is minimized. After hydrolysis, dry the test
sample in vacuum to remove any residual acid.
Method 2
Tryptophan oxidation during hydrolysis is decreased by us-
ing mercaptoethanesulfonic acid (MESA) as the reducing
acid.
Hydrolysis Solution 2.5 mol/L MESA solution.
Vapor Phase Hydrolysis About 1 to 100 /xg of the
protein/peptide under test is dried in a hydrolysis tube. The
hydrolysis tube is placed in a larger tube with about 200 /xL of
the Hydrolysis Solution. The larger tube is sealed in vacuum
(about 50 /xm of mercury or 6.7 Pa) to vaporize the Hydroly-
sis Solution. The hydrolysis tube is heated to 170°C to 185 C C
for about 12.5 minutes. After hydrolysis, the hydrolysis tube
is dried in vacuum for 15 minutes to remove the residual acid.
Method 3
Tryptophan oxidation during hydrolysis is prevented by
using thioglycolic acid (TGA) as the reducing acid.
Hydrolysis Solution A solution containing 7 mol/L
hydrochloric acid, 10% of trifluoroacetic acid, 20% of
thioglycolic acid, and 1% of phenol.
Vapor Phase Hydrolysis About 10 to 50 /xg of the
protein/peptide under test is dried in a sample tube. The sam-
ple tube is placed in a larger tube with about 200 /xL of the
Hydrolysis Solution. The larger tube is sealed in vacuum
(about 50 /xm of mercury or 6.7 Pa) to vaporize the TGA.
The sample tube is heated to 166°C for about 15 to 30
minutes. After hydrolysis, the sample tube is dried in vacuum
for 5 minutes to remove the residual acid. Recovery of tryp-
tophan by this method may be dependent on the amount of
sample present.
Method 4
Cysteine-cystine and methionine oxidation is performed
with performic acid before the protein hydrolysis.
Oxidation Solution The performic acid is prepared fresh
by mixing formic acid and 30 percent hydrogen peroxide
(9:1), and incubated at room temperature for 1 hour.
Procedure The protein/peptide sample is dissolved in
20 /xL of formic acid, and heated at 50°C for 5 minutes; then
100 //L of the Oxidation Solution is added. The oxidation is
allowed to proceed for 10 to 30 minutes. In this reaction, cys-
teine is converted to cysteic acid and methionine is converted
to methionine sulfone. The excess reagent is removed from
the sample in a vacuum centrifuge. This technique may cause
modifications to tyrosine residues in the presence of halides.
The oxidized protein can then be acid hydrolyzed using
Method 1 or Method 2.
Method 5
Cysteine-cystine oxidation is accomplished during the
liquid phase hydrolysis with sodium azide.
Hydrolysis Solution 6 mol/L hydrochloric acid contain-
ing 0.2% of phenol, to which is added sodium azide to obtain
a final concentration of 0.2% (w/v). The added phenol pre-
vents halogenation of tyrosine.
Liquid Phase Hydrolysis The protein/peptide hydrolysis
is conducted at about 110°C for 24 hours. During the hydrol-
ysis, the cysteine-cystine present in the sample is converted to
cysteic acid by the sodium azide present in the Hydrolysis So-
1658 Amino Acid Analysis / General Information
JP XV
lution. This technique allows better tyrosine recovery than
Method 4, but it is not quantitative for methionine. Methio-
nine is converted to a mixture of the parent methionine and
its two oxidative products, methionine sulfoxide and methio-
nine sulfone.
Method 6
Cysteine-cystine oxidation is accomplished with dimethyl
sulfoxide (DMSO).
Hydrolysis Solution 6 mol/L hydrochloric acid contain-
ing 0.1% to 1.0% of phenol, to which DMSO is added to
obtain a final concentration of 2% (v/v).
Vapor Phase Hydrolysis The protein/peptide hydrolysis
is conducted at about 1 10°C for 24 hours. During the hydrol-
ysis, the cysteine-cystine present in the sample is converted to
cysteic acid by the DMSO present in the Hydrolysis Solution.
As an approach to limit variability and compensate for par-
tial destruction, it is recommended to evaluate the cysteic
acid recovery from oxidative hydrolyses of standard proteins
containing 1 to 8 mol of cysteine. The response factors from
protein/peptide hydrolysates are typically about 30% lower
than those for nonhydrolyzed cysteic acid standards. Because
histidine, methionine, tyrosine, and tryptophan are also mo-
dified, a complete compositional analysis is not obtained with
this technique.
Method 7
Cysteine-cystine reduction and alkylation is accomplished
by a vapor phase pyridylethylation reaction.
Reducing Solution Transfer 83.3 fiL of pyridine, 16.7 /uL
of 4-vinylpyridine, 16.7 /xL of tributylphosphine, and 83.3
/uL of water to a suitable container, and mix.
Procedure Add the protein/peptide (between 1 and 100
fig) to a hydrolysis tube, and place in a larger tube. Transfer
the Reducing Solution to the large tube, seal in vacuum
(about 50 /um of mercury or 6.7 Pa), and incubate at about
100°C for 5 minutes. Then remove the inner hydrolysis tube,
and dry it in a vacuum desiccator for 15 minutes to remove
residual reagents. The pyridylethylated protein/peptide can
then be acid hydrolyzed using previously described
procedures. The pyridylethylation reaction is performed
simultaneously with a protein standard sample containing 1
to 8 mol of cysteine to improve accuracy in the pyridylethyl-
cysteine recovery. Longer incubation times for the
pyridylethylation reaction can cause modifications to the
a-amino terminal group and the £-amino group of lysine in
the protein.
Method 8
Cysteine-cystine reduction and alkylation is accomplished
by a liquid phase pyridylethylation reaction.
Stock Solutions Prepare and filter three solutions:
1 mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/L
disodium dihydrogen ethylendiamine tetraacetate (Stock So-
lution A), 8 mol/L guanidine hydrochloride (Stock Solution
B), and 10% of 2-mercaptoethanol in water (Stock Solution
Q.
Reducing Solution Prepare a mixture of Stock Solution B
and Stock Solution A (3:1) to obtain a buffered solution
of 6 mol/L guanidine hydrochloride in 0.25 mol/L Tris
hydrochloride.
Procedure Dissolve about 10 /ug of the test sample in 50
fxL of the Reducing Solution, and add about 2.5 juh of Stock
Solution C. Store under nitrogen or argon for 2 hours at
room temperature in the dark. To achieve the pyridylethyla-
tion reaction, add about 2//L of 4-vinylpyridine to the
protein solution, and incubate for an additional 2 hours at
room temperature in the dark. The protein/peptide is
desalted by collecting the protein/peptide fraction from a
reversed-phase HPLC separation. The collected sample can
be dried in a vacuum centrifuge before acid hydrolysis.
Method 9
Cysteine-cystine reduction and alkylation is accomplished
by a liquid phase carboxymethylation reaction.
Stock Solutions Prepare as directed for Method 8.
Carboxymethylation Solution Prepare a solution con-
taining 100 mg of iodoacetamide per mL of ethanol (95).
Buffer Solution Use the Reducing Solution, prepared as
directed for Method 8.
Procedure Dissolve the test sample in 50 fiL of the Buffer
Solution, and add about 2.5 [iL of Stock Solution C. Store
under nitrogen or argon for 2 hours at room temperature in
the dark. Add the Carboxymethylation Solution in a ratio 1.5
fold per total theoretical content of thiols, and incubate for
an additional 30 minutes at room temperature in the dark.
[Note: If the thiol content of the protein is unknown, then
add 5 fiL of 100 mmol/L iodoacetamide for every 20 nmol of
protein present.] The reaction is stopped by adding excess of
2-mercaptoethanol. The protein/peptide is desalted by col-
lecting the protein/peptide fraction from a reversed-phase
HPLC separation. The collected sample can be dried in a
vacuum centrifuge before acid hydrolysis. The
S-carboxyamidomethyl-cysteine formed will be converted to
S-carboxymethylcysteine during acid hydrolysis.
Method 10
Cysteine-cystine is reacted with dithiodiglycolic acid or
dithiodipropionic acid to produce a mixed disulfide. [Note:
The choice of dithiodiglycolic acid or dithiodipropionic acid
depends on the required resolution of the amino acid analysis
method.]
Reducing Solution A solution containing 10 mg of
dithiodiglycolic acid (or dithiodipropionic acid) per mL of
0.2 mol/L sodium hydroxide.
Procedure Transfer about 20 /ug of the test sample to a
hydrolysis tube, and add 5 /uL of the Reducing Solution. Add
10 /uL of isopropyl alcohol, and then remove all of the sample
liquid by vacuum centrifugation. The sample is then hydro-
lyzed using Method 1. This method has the advantage that
other amino acid residues are not derivatized by side reac-
tions, and the sample does not need to be desalted prior to
hydrolysis.
Method 11
Asparagine and glutamine are converted to aspartic acid
and glutamic acid, respectively, during acid hydrolysis.
Asparagine and aspartic acid residues are added and
represented by Asx, while glutamine and glutamic acid
residues are added and represented by Glx. Proteins/peptides
can be reacted with bis(l,l-trifluoroacetoxy)iodobenzene
(BTI) to convert the asparagine and glutamine residues to
diaminopropionic acid and diaminobutyric acid residues,
respectively, upon acid hydrolysis. These conversions allow
the analyst to determine the asparagine and glutamine
content of a protein/peptide in the presence of aspartic acid
and glutamic acid residues.
Reducing Solutions Prepare and filter three solutions: a
JPXV
General Information / Amino Acid Analysis 1659
solution of 10 mmol/L trifluoroacetic acid (Solution A), a
solution of 5 mol/L guanidine hydrochloride and 10 mmol/L
trifluoroacetic acid (Solution B), and a freshly prepared
solution of A^Af-dimethylformamide containing 36 mg of
BTI per mL (Solution C).
Procedure In a clean hydrolysis tube, transfer about
200 pig of the test sample, and add 2 mL of Solution A or
Solution B and 2 mL of Solution C. Seal the hydrolysis tube
in vacuum. Heat the sample at 60°C for 4 hours in the dark.
The sample is then dialyzed with water to remove the excess
reagents. Extract the dialyzed sample three times with equal
volumes of w-butyl acetate, and then lyophilize. The protein
can then be acid hydrolyzed using previously described proce-
dures. The a,/?-diaminopropionic and a,y-diaminobutyric
acid residues do not typically resolve from the lysine residues
upon ion-exchange chromatography based on amino acid
analysis. Therefore, when using ion-exchange as the mode of
amino acid separation, the asparagine and glutamine con-
tents are the quantitative difference in the aspartic acid and
glutamic acid content assayed with underivatized and BTI-
derivatized acid hydrolysis. [Note: The threonine, methio-
nine, cysteine, tyrosine, and histidine assayed content can be
altered by BTI derivatization; a hydrolysis without BTI will
have to be performed if the analyst is interested in the compo-
sition of these other amino acid residues of the protein/pep-
tide.]
Methodologies of Amino Acid Analysis General Principles
Many amino acid analysis techniques exist, and the choice
of any one technique often depends on the sensitivity
required from the assay. In general, about one-half of the
amino acid analysis techniques employed rely on the
separation of the free amino acids by ion-exchange
chromatography followed by postcolumn derivatization
(e.g., with ninhydrin or o-phthalaldehyde). Postcolumn
detection techniques can be used with samples that contain
small amounts of buffer components, such as salts and
urea, and generally require between 5 and 10 /xg of protein
sample per analysis. The remaining amino acid techniques
typically involve precolumn derivatization of the free amino
acids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-iV-
hydroxysuccinimidyl carbamate or o-phthalaldehyde;
(dimethylamino)azobenzenesulfonyl chloride; 9-
fiuorenylmethylchloroformate; and, 7-fluoro-4-nitrobenzo-2-
oxa-l,3-diazole) followed by reversed-phase HPLC.
Precolumn derivatization techniques are very sensitive and
usually require between 0.5 and 1.0 /xg of protein sample per
analysis but may be influenced by buffer salts in the samples.
Precolumn derivatization techniques may also result in
multiple derivatives of a given amino acid, which complicates
the result interpretation. Postcolumn derivatization tech-
niques are generally influenced less by performance variation
of the assay than precolumn derivatization techniques.
The following Methods may be used for quantitative
amino acid analysis. Instruments and reagents for these
procedures are available commercially. Furthermore, many
modifications of these methodologies exist with different
reagent preparations, reaction procedures, chromatographic
systems, etc. Specific parameters may vary according to the
exact equipment and procedure used. Many laboratories will
utilize more than one amino acid analysis technique to exploit
the advantages offered by each. In each of these Methods, the
analog signal is visualized by means of a data acquisition sys-
tem, and the peak areas are integrated for quantification pur-
poses.
Method 1 — Postcolumn Ninhydrin Detection General
Principle
Ion-exchange chromatography with postcolumn ninhydrin
detection is one of the most common methods employed for
quantitative amino acid analysis. As a rule, a Li-based
cation-exchange system is employed for the analysis of the
more complex physiological samples, and the faster Na-based
cation-exchange system is used for the more simplistic amino
acid mixtures obtained with protein hydrolysates (typically
containing 17 amino acid components). Separation of the
amino acids on an ion-exchange column is accomplished
through a combination of changes in pH and cation strength.
A temperature gradient is often employed to enhance separa-
tion.
When the amino acid reacts with ninhydrin, the reactant
has characteristic purple or yellow color. Amino acids, except
imino acid, give a purple color, and show the maximum ab-
sorption at 570 nm. The imino acids such as proline give a
yellow color, and show the maximum absorption at 440 nm.
The postcolumn reaction between ninhydrin and amino acid
eluted from column is monitored at 440 and 570 nm, and the
chromatogram obtained is used for the determination of ami-
no acid composition.
Detection limit is considered to be 10 pmol for most of the
amino acid derivatives, but 50 pmol for proline. Response
linearity is obtained in the range of 20 to 500 pmol with
correlation coefficients exceeding 0.999. To obtain good com-
position data, samples larger than 1 \xg before hydrolysis are
best suited for this amino acid analysis of protein/peptide.
Method 2 — Postcolumn OPA Fluorometric Detection
General Principle
o-Phthalaldehyde (OPA) reacts with primary amines in the
presence of thiol compound, to form highly fluorescent isoin-
dole products. This reaction is utilized for the postcolumn
derivatization in analysis of amino acids by ion-exchange
chromatography. The rule of the separation is the same as
Method 1. Instruments and reagents for this form of amino
acid analysis are available commercially. Many modifications
of this methodology exist.
Although OPA does not react with secondary amines
(imino acids such as proline) to form fluorescent substances,
the oxidation with sodium hypochlorite allows secondary
amines to react with OPA. The procedure employs a strongly
acidic cation-exchange column for separation of free amino
acids followed by postcolumn oxidation with sodium
hypochlorite and postcolumn derivatization using OPA and
thiol compound such as A^-acetyl-L-cysteine and 2-mercap-
toethanol. The derivatization of primary amino acids are not
noticeably affected by the continuous supply of sodium
hypochlorite.
Separation of the amino acids on an ion-exchange column
is accomplished through a combination of changes in pH and
cation strength. After postcolumn derivatization of eluted
amino acids with OPA, the reactant passes through the
fluorometric detector. Fluorescence intensity of OPA-deriva-
tized amino acids are monitored with an excitation
wavelength of 348 nm and an emission wavelength of
450 nm.
Detection limit is considered to be a few tens of picomole
level for most of the amino acid derivatives. Response lineari-
1660 Amino Acid Analysis / General Information
JP XV
ty is obtained in the range of a few picomole level to a few
tens of nanomole level. To obtain good compositional data,
the starting with greater than 500 ng of sample before hydrol-
ysis is best suited for the amino acid analysis of
protein/peptide.
Method 3 — Precolumn PITC Derivatization General
Principle
Phenylisothiocyanate (PITC) reacts with amino acids to
form phenylthiocarbamyl (PTC) derivatives which can be de-
tected with high sensitivity at 245 nm. Therefore, precolumn
derivatization of amino acids with PITC followed by a rever-
sed-phase HPLC separation with UV detection is used to
analyze the amino acid composition.
After the reagent is removed under vacuum, the deriva-
tized amino acids can be stored dry and frozen for several
weeks with no significant degradation. If the solution for in-
jection is kept cold, no noticeable loss in chromatographic
response occurs after three days.
Separation of the PTC-amino acids on a reversed-phase
HPLC with ODS column is accomplished through a combi-
nation of changes in concentrations of acetonitrile and buffer
ionic strength. PTC-amino acids eluted from column are
monitored at 254 nm.
Detection limit is considered to be 1 pmol for most of the
amino acid derivatives. Response linearity is obtained in the
range of 20 to 500 pmol with correlation coefficients exceed-
ing 0.999. To obtain good compositional data, samples larger
than 500 ng of protein/peptide before hydrolysis is best suit-
ed for this amino acid analysis of proteins/peptides.
Method 4 — Precolumn AQC Derivatization General
Principle
Precolumn derivatization of amino acids with 6-amino-
quinolyk/V-hydroxysuccinimidyl carbamate (AQC) followed
by reversed-phase HPLC separation with fluorometric
detection is used.
6-Aminoquinolyl-/V-hydroxysuccinimidyl carbamate
(AQC) reacts with amino acids to form stable, fluorescent un-
symmetric urea derivatives (AQC-amino acids) which are rea-
dily amenable to analysis by reversed-phase HPLC. There-
fore, precolumn derivatization of amino acids with AQC fol-
lowed by reversed-phase HPLC separation is used to analyze
the amino acid composition.
Separation of the AQC-amino acids on ODS column is
accomplished through a combination of changes in concen-
trations of acetonitrile and salt. Selective fluorescence detec-
tion of the derivatives with excitation wavelength at 250 nm
and emission wavelength at 395 nm allows for the direct
injection of the reaction mixture with no significant interfer-
ence from the only major fluorescent reagent by-product,
6-aminoquinoline. Excess reagent is rapidly hydrolyzed (t 1/2
<15 seconds) to yield 6-aminoquinoline, AMiydroxysuc-
cinimide and carbon dioxide, and after 1 minute no further
derivatization can take place.
Peak areas for AQC-amino acids are essentially unchanged
for at least 1 week at room temperature, and the derivatives
have more than sufficient stability to allow for overnight au-
tomated chromatographic analysis.
Detection limit is considered to be ranging from ca. 40 to
320 fmol for each amino acid, except for Cys. Detection limit
for Cys is approximately 800 fmol. Response linearity is ob-
tained in the range of 2.5 to 200,Mmol/L with correlation
coefficients exceeding 0.999. Good compositional data could
be obtained from the analysis of derivatized protein hydroly-
sates containing as little as 30 ng of protein/peptide.
Method 5 — Precolumn OPA Derivatization General
Principle
Precolumn derivatization of amino acids with o-phthalal-
dehyde (OPA) followed by reversed-phase HPLC separation
with fluorometric detection is used. This technique does not
detect amino acids that exist as secondary amines (e.g.,
proline).
o-Phthalaldehyde (OPA) in conjunction with a thiol
reagent reacts with primary amine groups to form highly
fluorescent isoindole products. 2-Mercaptoethanol or 3-mer-
captopropionic acid can be used as the thiol. OPA itself does
not fluoresce and consequently produces no interfering
peaks. In addition, its solubility and stability in aqueous
solution, along with the rapid kinetics for the reaction, make
it amenable to automated derivatization and analysis using
an autosampler to mix the sample with the reagent. However,
lack of reactivity with secondary amino acids has been
predominant drawback. This method does not detect amino
acids that exist as secondary amines (e.g., proline). To com-
pensate for this drawback, this technique may be combined
with another technique described in Method 7 or Method 8.
Precolumn derivatization of amino acids with OPA is
followed by a reversed-phase HPLC separation. Because of
the instability of the OPA-amino acid derivative, HPLC
separation and analysis are performed immediately following
derivatization. The liquid chromatograph is equipped with a
fluorometric detector for the detection of derivatized amino
acids. Fluorescence intensity of OPA-derivatized amino acids
is monitored with an excitation wavelength of 348 nm and an
emission wavelength of 450 nm.
Detection limits as low as 50 fmol via fluorescence have
been reported, although the practical limit of analysis
remains at 1 pmol.
Method 6 — Precolumn DABS-C1 Derivatization General
Principle
Precolumn derivatization of amino acids with
(dimethylamino)azobenzenesulfonyl chloride (DABS-C1) fol-
lowed by reversed-phase HPLC separation with visible light
detection is used.
(Dimethylamino)azobenzenesulfonyl chloride (DABS-C1)
is a chromophoric reagent employed for the labeling of
amino acids. Amino acids labeled with DABS-C1 (DABS-
amino acids) are highly stable and show the maximum
absorption at 436 nm.
DABS-amino acids, all 19 naturally occurring amino acids
derivatives, can be separated on an ODS column of a
reversed-phase HPLC by employing gradient systems consist-
ing of acetonitrile and aqueous buffer mixture. Separated
DABS-amino acids eluted from column are detected at
436 nm in the visible region.
This Method can analyze the imino acids such as proline
together with the amino acids at the same degree of sensitivi-
ty, DABS-C1 derivatization method permits the simultaneous
quantification of tryptophan residues by previous hydrolysis
of the protein/peptide with sulfonic acids such as mercap-
toethanesulfonic acid, /?-toluenesulfonic acid or methanesul-
fonic acid described under Method 2 in "Protein Hydroly-
sis". The other acid-labile residues, asparagine and gluta-
mine, can also be analysed by previous conversion into di-
aminopropionic acid and diaminobutyric acid, respectively,
JPXV
General Information / Amino Acid Analysis 1661
by treatment of protein/peptide with BTI described under
Method 11 in "Protein Hydrolysis".
The non-proteinogenic amino acid, norleucine cannot be
used as internal standard in this method, as this compound is
eluted in a chromatographic region crowded with peaks of
primary amino acids. Nitrotyrosine can be used as an internal
standard, because it is eluted in a clean region.
Detection limit of DABS-amino acid is about 1 pmol. As
little as 2 to 5 pmol of an individual DABS-amino acid can be
quantitatively analysed with reliability, and only 10 to 30 ng
of the dabsylated protein hydrolysate is required for each
analysis.
Method 7 — Precolumn FMOC-CI Derivatization General
Principle
Precolumn derivatization of amino acids with 9-
fluorenylmethyl chloroformate (FMOC-CI) followed by
reversed-phase HPLC separation with fluorometric detection
is used.
9-Fluorenylmethyl chloroformate (FMOC-CI) reacts with
both primary and secondary amino acids to form highly
fluorescent products. The reaction of FMOC-CI with amino
acid proceeds under mild conditions in aqueous solution and
is completed in 30 seconds. The derivatives are stable, only
the histidine derivative showing any breakdown. Although
FMOC-CI is fluorescent itself, the reagent excess and
fluorescent side-products can be eliminated without loss of
FMOC-amino acids.
FMOC-amino acids are separated by a reversed-phase
HPLC using ODS column. The separation is carried out by
gradient elution varied linearly from a mixture of acetonitrile
methanol and acetic acid buffer (10:40:50) to a mixture of
acetonitrile and acetic acid buffer (50:50), and 20 amino acid
derivatives are separated in 20 minutes. Each derivative elut-
ed from column is monitored by a fluorometric detector set at
an excitation wavelength of 260 nm and an emission
wavelength of 313 nm.
The detection limit is in the low fmol range. A linearity
range of 0.1 to 50//mol/L is obtained for most of the amino
acids.
Method 8 — Precolumn NBD-F Derivatization General
Principle
Precolumn derivatization of amino acids with 7-fiuoro-4-
nitrobenzo-2-oxa-1.3-diazole (NBD-F) followed by reversed-
phase HPLC separation with fluorometric detection is used.
7-fluoro-4-nitrobenzo-2-oxa-1.3-diazole (NBD-F) reacts
with both primary and secondary amino acids to form highly
fluorescent products. Amino acids are derivatized with NBD-
F by heating to 60°C for 5 minutes.
NBD-amino acid derivatives are separated on an ODS
column of a reversed-phase HPLC by employing gradient
elution system consisting of acetonitrile and aqueous buffer
mixture, and 17 amino acid derivatives are separated in 35
minutes. e-Aminocaproic acid can be used as an internal
standard, because it is eluted in a clean chromatographic
region. Each derivative eluted from column is monitored by a
fluorometric detector set at an excitation wavelength of
480 nm and an emission wavelength of 530 nm.
The sensitivity of this method is almost the same as
for precolumn OPA derivatization method (Method 5),
excluding proline to which OPA is not reactive, and might be
advantageous for NBD-F against OPA. The detection limit
for each amino acid is about 10 fmol. Profile analysis was
achieved for about 1 .5 /^g of protein hydrolysates in the final
precolumn labeling reaction mixture for HPLC.
Data Calculation and Analysis
When determining the amino acid content of a pro-
tein/peptide hydrolysate, it should be noted that the acid
hydrolysis step destroys tryptophan and cysteine. Serine and
threonine are partially destroyed by acid hydrolysis, while
isoleucine and valine residues may be only partially cleaved.
Methionine can undergo oxidation during acid hydrolysis,
and some amino acids (e.g., glycine and serine) are common
contaminants. Application of adequate vacuum (^0.0267
kPa) or introduction of inert gas (argon) in the headspace of
the reaction vessel during vapor phase hydrolysis can reduce
the level of oxidative destruction. Therefore, the quantitative
results obtained for cysteine, tryptophan, threonine, isoleu-
cine, valine, methionine, glycine, and serine from a pro-
tein/peptide hydrolysate may be variable and may warrant
further investigation and consideration.
Calculations
Amino Acid Mole Percent This is the number of specific
amino acid residues per 100 residues in a protein. This result
may be useful for evaluating amino acid analysis data when
the molecular weight of the protein under investigation is
unknown. This information can be used to corroborate the
identity of a protein/peptide and has other applications.
Carefully identify and integrate the peaks obtained as direct-
ed for each Procedure. Calculate the mole percent for each
amino acid present in the test sample by the formula:
100/u/r,
in which r v is the peak response, in nmol, of the amino acid
under test; and r is the sum of peak responses, in nmol, for all
amino acids present in the test sample. Comparison of the
mole percent of the amino acids under test to data from
known proteins can help establish or corroborate the identity
of the sample protein.
Unknown Protein Samples This data analysis technique
can be used to estimate the protein concentration of an
unknown protein sample using the amino acid analysis data.
Calculate the mass, in /ug, of each recovered amino acid by
the formula:
»jM w /1000,
in which m is the recovered quantity, in nmol, of the amino
acid under test; and M w is the average molecular weight for
that amino acid, corrected for the weight of the water
molecule that was eliminated during peptide bond formation.
The sum of the masses of the recovered amino acids will give
an estimate of the total mass of the protein analyzed after ap-
propriate correction for partially and completely destroyed
amino acids. If the molecular weight of the unknown protein
is available (i.e., by SDS-PAGE analysis or mass spec-
troscopy), the amino acid composition of the unknown pro-
tein can be predicted. Calculate the number of residues of
each amino acid by the formula:
m/(1000M/M WT ),
in which m is the recovered quantity, in nmol, of the amino
acid under test; M is the total mass, in fig, of the protein; and
M WT is the molecular weight of the unknown protein.
Known Protein Samples This data analysis technique can
be used to investigate the amino acid composition and pro-
tein concentration of a protein sample of known molecular
weight and amino acid composition using the amino acid
analysis data. When the composition of the protein being
1662 Aristolochic Acid / General Information
JP XV
analyzed is known, one can exploit the fact that some amino
acids are recovered well, while other amino acid recoveries
may be compromised because of complete or partial destruc-
tion (e.g., tryptophan, cysteine, threonine, serine, methio-
nine), incomplete bond cleavage (i.e., for isoleucine and
valine) and free amino acid contamination (i.e., by glycine
and serine).
Because those amino acids that are recovered best
represent the protein, these amino acids are chosen to
quantify the amount of protein. Well-recovered amino acids
are, typically, aspartate-asparagine, glutamate-glutamine,
alanine, leucine, phenylalanine, lysine, and arginine. This list
can be modified based on experience with one's own analysis
system. Divide the quantity, in nmol, of each of the well-
recovered amino acids by the expected number of residues for
that amino acid to obtain the protein content based on each
well-recovered amino acid. Average the protein content
results calculated. The protein content determined for each
of the well-recovered amino acids should be evenly distribut-
ed about the mean. Discard protein content values for those
amino acids that have an unacceptable deviation from the
mean. Typically a greater than 5% variation from the mean
is considered unacceptable. Recalculate the mean protein
content from the remaining values to obtain the protein con-
tent of the sample. Divide the content of each amino acid by
the calculated mean protein content to determine the amino
acid composition of the sample by analysis.
Calculate the relative compositional error, in percentage,
by the formula:
100m/m s ,
in which m is the experimentally determined quantity, in
nmol per amino acid residue, of the amino acid under test;
and m s is the known residue value for that amino acid. The
average relative compositional error is the average of the
absolute values of the relative compositional errors of the
individual amino acids, typically excluding tryptophan and
cysteine from this calculation. The average relative composi-
tional error can provide important information on the stabil-
ity of analysis run over time. The agreement in the amino
acid composition between the protein sample and the known
composition can be used to corroborate the identity and puri-
ty of the protein in the sample.
2. Aristolochic Acid
Aristolochic acid, which occurs in plants of Aristolochia-
ceae, is suspected to cause renal damage. It is also reported to
be oncogenic (see References).
Aristolochic acid toxicity will not be a problem if crude
drugs of the origin and parts designated in the JP are used,
but there may be differences in crude drug nomenclature be-
tween different countries, and it is known that crude drug
preparations not meeting the specifications of the JP are cir-
culating in some countries. Consequently, when crude drugs
or their preparations are used, it is important that the materi-
als should not include any plant containing aristolochic acid.
Since Supplement I to JP14, the test for aristolochic acid I
was added to the Purity under Asiasarum Root, which con-
sists of the rhizome and root. Because the aerial part of the
plant may contain aristolochic acid and may have been
improperly contaminated in Asiasarum Root.
It is considered that Akebia Stem, Sinomenium Stem and
Saussurea Root do not contain aristolochic acid, unless
plants of origin other than that designated in the JP are used.
However, contamination of aristolochic acid might occur, as
mentioned above. In this case, the test described in the Purity
under Asiasarum Root is useful for checking the presence of
aristolochic acid.
References:
Drug & Medical Device Safety Information (No. 161) (July,
2000).
New England Journal of Medicine (June 8, 2000).
Mutation Research 515, 63-72 (2002).
3. Basic Requirements for Viral
Safety of Biotechnological/
Biological Products listed in
Japanese Pharmacopoeia
Introduction
The primary role of specification of biotechnological/bio-
logical products listed in Japanese Pharmacopoeia (JP) is not
only for securing quality control or consistency of the quality
but also for assuring their efficacy and safety. In the mean-
time, the requirements to assure quality and safety of drugs
have come to be quite strict recently, and a rigid attitude
addressing safety assurance is expected for biotechnological/
biological products. The key points for quality and safety as-
surance of biotechnological /biological products are selection
and appropriate evaluation of source material, appropriate
evaluation of manufacturing process and maintenance of
manufacturing consistency, and control of specific physical
properties of the products. Now, how to assure quality and
safety of such drugs within a scope of JP has come to be
questioned. This General Information describes what sorts of
approaches are available to overcome these issues.
It is desired that quality and safety assurance of JP listed
products are achieved by state-of-the-art methods and con-
cepts which reflect progress of science and accumulation of
experiences. This General Information challenges to show the
highest level of current scientific speculation. It is expected
that this information will contribute to promotion of scien-
tific understanding of quality and safety assurance of not
only JP listed products but also the other biotechnological/
biological products and to promotion of active discussion of
each Official Monograph in JP.
1. Fundamental measures to ensure viral safety of JP listed
biotechnological /biological products
The biotechnological/biological product JP includes the
products derived from living tissue and body fluid (urine,
blood, etc.) of mammals, etc. Protein drugs derived from cell
lines of human or animal origin (e.g., recombinant DNA
drug, cell culture drug) are also included. The fundamental
measures required for comprehensive viral safety of JP listed
biotechnological/biological products are as follows: 1) ac-
quaintance of possible virus contamination (source of con-
tamination); 2) careful examination of eligibility of raw
materials and their sources, e.g. human/animal, and
thorough analysis and screening of the sample chosen as a
substrate for drug production (e.g., pooled body fluid, cell
JPXV
General Information / Basic Requirements for Viral 1663
bank, etc.) to determine any virus contamination and deter-
mination of type and nature of the virus, if contaminated; 3)
evaluation to determine virus titer and virus-like particles
hazardous to human, if exists; 4) selection of production
related material (e.g., reagent, immune antibody column)
free from infectious or pathogenic virus; 5) performance of
virus free test at an appropriate stage of manufacturing in-
cluding the final product, if necessary; 6) adoption of effec-
tive viral clearance method in the manufacturing process to
remove /inactivate virus. Combined method sometimes
achieves higher level of clearance; 7) development of a
deliberate viral clearance scheme; 8) performance of the test
to evaluate viral removal and inactivation. It is considered
that the stepwise and supplemental adoption of the said
measures will contribute to ensure viral safety and its im-
provement.
2. Safety assurance measures described in the Official
Monograph and this General Information
As mentioned in above 1, this General Information
describes, in package, points to be concerned with and con-
crete information on the measures taken for viral safety of JP
listed products. Except where any specific caution is provided
in Official Monograph of a product in question, Official
Monograph provides in general that "Any raw material, sub-
strate for drug production and production related material
used for production of drug should be derived from healthy
animals and should be shown to be free of latent virus which
is infectious or pathogenic to human", "Cell line and culture
method well evaluated in aspects of appropriateness and ra-
tionality on viral safety are used for production, and the
presence of infectious or pathogenic latent virus to human in
process related materials derived from living organisms
should be denied", and "biotechnological/biological drug
should be produced through a manufacturing process which
is capable of removing infectious or pathogenic virus", etc.,
to raise awareness on viral safety and on necessity to conduct
test and process evaluation for viral safety.
3. Items and contents described in this General Information
As for viral safety of protein drug derived from cell line of
human or animal origin, there is a Notice in Japan entitled
"Viral safety evaluation of biotechnology products derived
from cell lines of human or animal origin" (Iyakushin No.
329 issued on February 22, 2000 by Director, Evaluation and
Licensing Division, Pharmaceutical and Medical Safety
Bureau, Ministry of Health and Welfare) to reflect the inter-
nationally harmonized ICH Guideline, and as for blood plas-
ma protein fraction preparations, there is a document enti-
tled "Guideline for ensuring viral safety of blood plasma
protein fraction preparations". This General Information
for ensuring viral safety of JP listed biotechnological/biolog-
ical products has been written, referencing the contents of
those guidelines, to cover general points and their details to
be concerned for ensuring viral safety of not only JP listed
biotechnological/biological products but also all products
which would be listed in JP in future, i.e., biological
products derived from living tissue and body fluid, such as
urine, and protein drugs derived from cell line of human or
animal origin (Table 1).
3.1 Purpose
The purpose of this document is to propose the compre-
hensive concepts of the measures to be taken for ensuring
viral safety of biotechnological/biological products derived
Table 1. Items described in General Information
for Viral Safety Assurance of JP listed
Biotechnological/Biological Product
I. Introduction
1 . Fundamental measures to ensure viral safety of JP listed
biotechnological/biological products
2. Safety assurance measures described in the Official
Monograph and this General Information
3. Items and contents described in this General Informa-
tion
II. General Matters
1. Purpose
2. Background
3. Unknown risk on the measures taken for ensuring viral
safety
4. Applicable range
5. Possible viral contamination to a JP listed biotechno-
logical/biological product (source of virus contamina-
tion)
6. Basis for ensuring viral safety
7. Limit of virus test
8. Roles of viral clearance studies
III. Raw material /substrate for drug production
1. Issues relating to animal species and its region as a
source of raw material/substrate for drug production
and countermeasures to be taken thereto
2. Qualification evaluation test on human or animal as a
source of raw material/substrate for drug production
IV. Points of concern with respect to manufacturing and vi-
rus testing
1. Virus test conducted in advance of purification process
2. Virus test as an acceptance test of an intermediate
material, etc.
3. Virus test on a final product
V. Process evaluation on viral clearance
1. Rationale, objective and general items to be concerned
with respect to viral clearance process evaluation
2. Selection of virus
3. Design of viral clearance studies
4. Interpretation of viral clearance studies
1) Evaluation of viral clearance factor
2) Calculation of viral clearance index
3) Interpretation of results and items to be concerned at
evaluation
VI. Statistics
1. Statistical considerations for assessing virus assays
2. Reproducibility and confidence limit of viral clearance
studies
VII. Re-evaluation of viral clearance
VIII. Measurement for viral clearance studies
1. Measurement of virus infective titer
2. Testing by nucleic-acid amplification test (NAT)
IX. Reporting and preservation
X. Others
from living tissue or body fluid of mammals, etc. and of pro-
tein drugs derived from cell lines of human or animal origin.
That is to say, this document describes the measures and the
points of concern on the items, such as ® consideration of
the source of virus contamination; (2) appropriate evaluation
on eligibility at selecting the raw material and on qualification
1664 Basic Requirements for Viral / General Information
JP XV
of its source, e.g. human or animal; © virus test, and its
analysis and evaluation at a stage of cell substrate for drug
production; © appropriate evaluation to choose product
related materials derived from living organisms (e.g. reagent,
immune antibody column, etc.); © conduct of necessary vi-
rus test on the product at an appropriate stage of manufac-
turing; © development of viral clearance test scheme; © per-
formance and evaluation of viral clearance test. This docu-
ment is also purposed to comprehensively describe in details
that supplemental and combining adoption of the said meas-
ures will contribute to secure viral safety and its improve-
ment.
3.2 Background
One of the most important issues to be cautioned for safety
of a biological product, which is directly derived from human
or animal, or of a protein drug, which is derived from cell
line of human or animal origin (recombinant DNA derived
product, cell culture derived product, etc.), is risk of virus
contamination. Virus contamination may cause serious situa-
tion at clinical use once it occurs. Virus contamination may
be from a raw material or from a cell substrate for drug
production, or may be from an adventitious factor in-
troduced to the manufacturing process.
JP listed biological drugs or protein drugs derived from
cell line have achieved drastic contribution to the medical
society, and to date, there has not been any evidence of any
safety problem on them caused by virus. But, social require-
ment of health hazard prevention is strong, and it is now very
important to prevent accidental incidence, taking security
measures carefully supported by scientific rationality. It is
always great concern among the persons involved that under
what sort of viewpoint and to what extent we have to pursue
for ensuring viral safety of a biotechnological /biological
product. Before discussing these issues, two fundamental
points have to be reconfirmed. One is that; we have to con-
sider scientific, medical, and social profiles a drug has. In
other words, "Medicine is a social asset which is utilized in
medical practice paying attention to the risk and benefit from
the standpoints of science and society". It is the destine and
the mission of the medical/pharmaceutical society to realize
prompt and stable supply of such a social asset, drug, among
the medical work front to bring gospel to the patients.
The other is that; issue of viral safety is independent from
safety of the components of a drug per se (narrow sense of
safety). It is important to consider that this is the matter of
general safety of drug (broad sense of safety). In case of a
drug which has been used for a long time in the medical
front, such as a JP listed product, its broad sense of safety is
considered to have been established epidemiologically, and
its usage past records have a great meaning. However, differ-
ent from safety of drug per se (its components), taking into
account any possibility of virus contamination, we have to
say that only the results accumulated can not always assure
viral safety of a drug used in future. Accordingly, the basis
for securing broad sense of viral safety of JP listed
biotechnological /biological products is to pay every attention
to the measures to take for prevention, while evaluating the
accumulated results.
Adopting strict regulations and conducting tests at maxi-
mum level to the extent theoretically considered may be the
ways off assuring safety, but applying such way generally,
without sufficient scientific review of the ways and evaluation
of usage results, causes excessive requirement of regulation
and test not having scientific rationality. As the results, effec-
tive and prompt supply of an important drug, already having
enough accumulation of experiences, to the medical work
front will be hampered, and the drug, a social asset, may not
to be utilized effectively. Medicine is a sword used in medical
field having double-edge named effectiveness and safety.
Effectiveness and safety factors have to be derived as the
fruits of leading edge of science, and relatively evaluated on a
balance sheet of usefulness. Usefulness evaluation should not
be unbalanced in a way that too much emphasis is placed on
safety concern without back-up of appropriate scientific ra-
tionality. A drug can play an important role as a social asset
only when well balanced appropriate scientific usefulness
evaluation in addition to social concern of the age are given.
In other words, drug is a common asset utilized by society for
medication as a fruit of science of the age, and the key point
of its utilization lies on a balance of risk and benefit produced
from scientific and social evaluation. So, those factors have
to be taken into account when target and pursuance levels for
ensuring viral safety of a JP listed biotechnological /biologi-
cal product are reviewed.
And, in general, the risk and benefit of drugs should be
considered with the relative comparison to alternative drugs
or medical treatment. The usefulness of certain drug should
be reviewed finally after the competitive assessment on the
risk and benefit on the alternative drugs, relevant drugs and/
or alternative medical treatment.
Under such background, the purpose of this article is to
describe the scientific and rational measures to be taken for
ensuring viral safety of JP listed biotechnological /biological
products. Giving scientific and rational measures mean that;
appropriate and effective measures, elaborated from the cur-
rent scientific level, are given to the issues assumable under
the current scientific knowledge. In other words, possible
contaminant virus is assumed to have the natures of genus,
morph, particle size, physical /chemical properties, etc. which
are within the range of knowledge of existing virology, and is
those assumed to exist in human and animal, tissue and body
fluid, which are the source of biotechnological/biological
product, reagent, material, additives, etc. Accordingly, viral
clearance studies using a detection method which target those
viruses have to be designed.
3.3 Unknown risk on the measures taken for ensuring viral
safety
There are known and unknown risks.
It is easy to determine a test method and an evaluation
standard on the known risk, which exists in the drug per se
(pharmaceutical component) or inevitably exists due to a
quality threshold, and quantification of such risk is possible.
In other words, it is easy to evaluate the known risk on a
balance sheet in relation to the benefit, and we can say that
valuation even in this respect has been established to some ex-
tent.
On the other hand, as for the unknown risk which is inevit-
able for ensuring viral safety, the subject of the risk can not
be defined and quantitative concept is hard to introduce, and,
therefore, taking a counter measure and evaluating its effect
are not so easy. Therefore, this is the subject to be challenged
calling upon wisdom of the related parties among the society
of drug.
Talking about the unknown risk, there are view points that
say "It is risky because it is unknown." and "What are the
unknowns, and how do we cope with them in ensuring safe-
JP XV
General Information / Basic Requirements for Viral 1665
ty?".
The view of "It is risky because it is unknown." is already
nothing but a sort of evaluation result, and directly connects
to a final decision if it can be used as a drug. Such evaluation/
decision has to be made based upon a rational, scientific or
social judgment.
For example, in the case that "In a manufacturing process
of drug, virus, virus-like particle or retrovirus was detected,
but its identification could not be confirmed, and, therefore,
its risk can not be denied.", the evaluation of "It is risky be-
cause it is unknown." is scientifically rational and reasona-
ble. On the other hand, however, if we reach a decision of "It
is risky because it is unknown." due to the reason that "In a
manufacturing process of drug, virus, virus-like particle or
retrovirus was not detected, but there is a 'concern' that
something unknown may exist.", it can not be said that such
evaluation is based upon a rational, scientific or social judg-
ment. It goes without saying that the utmost care has to be
taken for viral safety, but the substance of 'concern' has to
be at least clearly explainable. Otherwise, the 'concern' may
result in causing contradiction in the meaningful mission to
utilize a social asset, drug, in medical practice.
From scientific view point, we should not be narrow mind-
ed by saying "it is risky" because "there is a 'concern' that
something unknown may exists", but challenge to clarify the
subject of "What is unknown, and how to cope with it for
ensuring safety" using wisdom. What is important at the
time is to define "what is unknown" based upon current
scientific knowledge. Only through this way, is it possible for
us to elaborate the measures for ensuring safety.
Once we chase up the substance of unknown risk for viral
safety without premises of "what is unknown", "unknown"
will be an endless question because it theoretically remains
unresolved forever. If this kind of approach is taken, the is-
sue and the measure can not be scientifically connected to
each other, which will result in the excessive requirement of
regulation and of test to be conducted. Yet, it is unlikely that
the measure which has no relation with science will be effec-
tive to the subject of "What is unknown is unknown."
For example, "what is unknown" at the "evaluation of a
purification process which can completely clear up every virus
that contaminated in a manufacturing process" should be the
subject of "what sort of existing virus that contaminated is
unknown", not on the subject of "what sort of virus that ex-
ist in the world is unknown. In the former subject, the
premise of the study is based on all the knowledge on viruses
including DNA/RNA-virus, virus with/without envelope,
particle size, physical/chemical properties, etc. The premise
is that the virus contaminated should be within range of exist-
ing wisdom and knowledge of virus such as species, type, na-
ture, etc., even though the virus that contaminated is
unknown. Under such premise, when evaluation is made on a
purification process to decide its capability of clearing a der-
ived virus, which is within the range of existing wisdom and
learning, specific viral clearance studies designed to combine
a few model viruses with different natures, such as type of
nucleic acid, with/without envelope, particle size, physical/
chemical properties, etc., would be enough to simulate every
sort of the virus already known, and will be a "good measure
for ensuring safety".
The issue of "the sort of viruses that exist in the world is
unknown" may be a future study item, but it is not an ap-
propriate subject for the viral clearance test. Further, even if
the subject of "unknown viruses, which have a particle size
smaller than that of currently known viruses, may exists" or
"unknown viruses, which have special physical/chemical
properties that can not be matched to any of the currently
known viruses, may exists" is set up as an armchair theory,
any experimental work can not be pursued under the current
scientific level, since such virus model is not available. Fur-
ther, any viral clearance test performed by using the currently
available methods and technologies will be meaningless "for
ensuring safety", since particle size or natures of such specu-
lated virus are unknown. Likewise, any counter measures can
not be taken on the subject of "unknown virus, which can
not be detected by currently available screening method, may
exist", and conducting any virus detection test at any stage
will be useless "for ensuring safety".
The requirement of regulations or tests excessively over
scientific rationality will raise human, economical and tem-
poral burden to the pharmaceutical companies, and will ad-
versely affect prompt, effective and economical supply of a
drug to the medical front. As drug is a sort of social asset,
which has to be scientifically evaluated, how to assure max-
imization of its safety by means of scientifically rational ap-
proach at minimum human, economical and time resources is
important.
It is also important to reconfirm that achievement of those
issues is on the premise that appropriate measures are taken
on the supply source of drugs. For example, in a case of "In a
manufacturing process of drug, virus, virus-like particle or
retrovirus was not detected, but there is a 'concern' that
something unknown may exist.", appropriateness of the test,
which resulted in the judgment that "virus, virus-like particle
or retrovirus was not detected in a process of drug produc-
tion", should be a prerequisite premise when judged by
science standard ate the time. If there is any question on the
premise, it is quite natural that the question of "there is a
'concern' that unknown something may exist." will be effec-
tive.
3.4 Applicable range
This General Information is on JP listed biological
products, derived from living tissue or body fluid, and pro-
tein drugs, derived from human or animal cell line, that in
Japan. In the case of protein drugs derived from human or
animal cell line, the products developed and approved before
enforcement of the Notice Iyakushin No. 329 entitled "Viral
safety evaluation of biotechnology products derived from cell
lines of human or animal origin" should have been treated
under the Notice had there been one, and it is inevitable that
some products approved after the Notice might not have been
sufficiently treated. It is expected that such biodrug will be
sufficiently examined to meet such General Information be-
fore being listed in JP. On the other hand, blood prepara-
tions listed in the biological products standard and covered
by "Guideline for securing safety of blood plasma protein
fraction preparations against virus", are out of the scope of
this General Information. Further, in case of a relatively low-
er molecular biogenous substance, such as amino acid, sac-
charide and glycerin, and of gelatin, which is even classified
as infectious or pathogenic polymer, there are cases that viral
contamination can not be considered due to its manufactur-
ing or purification process, and that potent viral inactivation/
removal procedure that can not be applied to protein, can be
used, and, therefore, it is considered reasonable to omit such
substances from the subject for application. However, some
1666 Basic Requirements for Viral / General Information
JP XV
part of this General Information may be used as reference.
Further, a comprehensive assurance measure for viral safety
is recommendable on a biotechnological/biological product
not listed in JP using this document as a reference so long as
it is similar to the biotechnological/biological product JP.
3.5 Possible viral contamination to a JP listed biotechno-
logical/biological product (source of virus contamination)
Promoting awareness of virus contamination to a JP listed
biotechnological/biological product (source of virus con-
tamination) and citing countermeasure thereof are important
for eradicating any possible virus contamination and raising
probability of safety assurance. Many biotechnological/bio-
logical products are produced from a "substrate" which is
derived from human or animal tissue, body fluid, etc. as an
origin /raw material, and in purification or pharmaceutical
processing of such products column materials or additives,
which are living organism origin, are occasionally used. Ac-
cordingly, enough safety measures should be taken against
diffusion of the contaminant virus. Further, as mentioned in
Notice Iyakushin No. 329, any protein drug derived from cell
lines of human or animal origin should be carefully examined
with respect to the risk of virus contamination through the
cell line, the cell substrate for drug production, and through
the manufacturing process applied thereafter.
"Substrate for drug production" is defined as a starting
material which is at a stage where it is deemed to be in a posi-
tion to ensure quality /safety of an active substance. The
"substrate for drug production" is sometimes tissue, body
fluid, etc. of human or animal per se and pooled material
such as urine, and sometimes a material after some treat-
ment. In many cases, it is considered rational that starting
point of full-scale test, evaluation and control should be at
the stage of "substrate for drug production". The more strict
levels of test, evaluation and control achieved at the stage of
"substrate for drug production" can more rationalize evalu-
ation and control of the raw material or individual level of
upper stream. On the contrary, strict evaluation and control
of the raw material or individual level at an upper stream
stage can rationalize tests, evaluation or quality control at the
stage of "substrate for drug production".
The measures taken for ensuring viral safety on a
biotechnological/biological product currently listed in JP can
be assumed from the provisions of manufacturing method,
specification and test methods of each preparation. However,
unitary principles or information with respect to the meas-
ures to be taken for ensuring viral safety, totally reviewing
the entire process up to the final product rationally and com-
prehensively, including source /raw material /substrate,
purification process, etc. have not been clarified. The most
important thing for ensuring viral safety is to take thorough
measures to eliminate the risk of virus contamination at any
stage of source animal, raw material and substrate. Although
not the cases of a biotechnological/biological product,
known examples of a virus contamination from a raw materi-
al/substrate for drug production in old times are Hepatitis A
Virus (HAV) or Hepatitis C Virus (HCV) contamination in
blood protein fraction preparations. It is also well known
that Human Immunodeficiency Virus (HIV) infection caused
by blood plasma protein fraction preparations occurred in
1980s. The aim of this General Information is to show con-
crete guidelines for comprehensive viral safety assurance of
the JP listed biotechnological/biological products. The
pathogenic infectious viruses, currently known to con-
taminate to raw materials, etc. of drug and have to be cau-
tioned, are HIV, HAV, Hepatitis B Virus (HBV), HCV, Hu-
man T-Lymphotropic Virus (HTLV-I/II), Human Parvovi-
rus B19, Cytomegalovirus (CMV), etc. Biotechnological/bio-
logical products produced from raw material/cell substrate
derived from tissue or body fluid of human or animal origin
always have a risk of contamination of pathogenic or other
latent virus. Therefore, safety measures should be thoroughly
taken. There is also the case that a material, other than the
biological component such as raw material/substrate, causes
virus contamination. Using enzymatic or monoclonal an-
tibody column or using albumin etc. as a stabilizer, is the ex-
ample of the case, in which caution has to be taken on risk of
virus contamination from the source animal or cell. Further,
there is a possibility of contamination from environment or
personnel in charge of production or at handling of the
product. So, caution has to be taken on these respects as well.
In case of protein drugs derived from cell line of human or
animal origin, there may be cases where latent or persistent
infectious viruses (e.g., herpesvirus) or endogenous
retroviruses exist in the cell. Further, adventitious viruses
may be introduced through the routes such as: 1) derivation
of a cell line from an infected animal; 2) use of a virus to
drive a cell line; 3) use of a contaminated biological reagent
(e.g., animal serum components); 4) contamination during
cell handling. In the manufacturing process of drug, an
adventitious virus may contaminate to the final product
through the routes, such as 1) contamination through a rea-
gent of living being origin, such as serum component, which
is used for culturing, etc.; 2) use of a virus for introduction of
a specific gene expression to code an objective protein; 3)
contamination through a reagent used for purification such
as monoclonal antibody affinity column; 4) contamination
through an additive used for formulation production; 5) con-
tamination at handling of cell and culture media, etc. It is
reported that monitoring of cell culture parameter may be
helpful for early detection of an adventitious viral contami-
nation.
3.6 Basis for ensuring viral safety
Viral safety of a biotechnological/biological product pro-
duced from a raw material/substrate, which derived from tis-
sue, body fluid, cell line, etc. of human or animal origin, can
be achieved by supplemental and appropriate adoption of the
following plural methods.
(1) Acquaintance of possible virus contamination (source
of contamination).
(2) Careful examination of eligibility of the raw material
and its source, i.e., human or animal, thorough analysis
and screening of the sample chosen as the substrate for
drug production to determine virus contamination and
through examination of the type of virus and its nature,
if contaminated.
(3) Evaluation to determine hazardous properties of the vi-
rus or virus-like particle to human, if exists.
(4) Choosing a product related material of living organism
origin (e.g., reagent, immune anti-body column, etc.)
which is free from infectious or pathogenic virus.
(5) Conduct virus free test at an appropriate stage of
manufacturing including the final product, if necessary.
(6) Adoption of an effective method to remove /inactivate
the virus in the manufacturing process for viral clear-
ance. Combined processes sometimes achieve higher lev-
el of viral clearance.
JPXV
General Information / Basic Requirements for Viral 1667
(7) Develop a deliberate viral clearance scheme.
(8) Conduct test and evaluation to confirm removal /inacti-
vation of the virus.
Manufacturer is responsible for explaining rationality of
the way of approach adopted among the comprehensive
strategy for viral safety on each product and its manufactur-
ing process. At the time, the approach described in this
General Information shall be applicable as far as possible.
3.7 Limit of virus test
Virus test has to be conducted to define existence of virus,
but it should be noted that virus test alone can not reach a
conclusion of inexistence of virus nor sufficient to secure safe-
ty of the product. Examples of a virus not being detected are
as follows: 1) Due to statistical reason, there is an inherent
quantitative limit, such as detection sensitivity at lower con-
centration depends upon the sample size. 2) Generally, every
virus test has a detection limit, and any negative result of a vi-
rus test can not completely deny existence of a virus. 3) A vi-
rus test applied is not always appropriate in terms of specifici-
ty or sensitivity for detection of a virus which exists in the tis-
sue or body fluid of human or animal origin.
Virus testing method is improved as science and technology
progress, and it is important to apply scientifically the most
advanced technology at the time of testing so that it can be
possible to raise the assurance level of virus detection. It
should be noted, however, that the limit as mentioned above
can not always be completely overcome. Further, risk of vi-
rus contamination in a manufacturing process can not be
completely denied, and, therefore, it is necessary to elaborate
the countermeasure taken these effects into account.
Reliable assurance of viral free final product can not be ob-
tained only by negative test results on the raw material/sub-
strate for drug production or on the product in general, it is
also necessary to demonstrate inactivation/removal capabil-
ity of the purification process.
3.8 Roles of viral clearance studies
Under the premises as mentioned in the preceding clause
that there is a limit of a virus test, that there is a possibility of
existence of latent virus in a raw material /substrate for drug
production and that there is a risk of entry of a non-en-
dogenous virus in a manufacturing process, one of the im-
portant measures for viral safety is how to remove or inacti-
vate the virus, which exists in a raw material, etc. and can not
be detected, or the virus, which is contingently contaminated
in a manufacturing process. The purpose of viral clearance
study is to experimentally evaluate the viral removal /inacti-
vation capability of a step that mounted in a manufacturing
process. So, it is necessary to conduct an experimental scale
spike test using an appropriate virus that is selected by taking
account the properties, such as particle size, shape, with or
without envelope, type of nucleic acid (DNA type, RNA
type), heat and chemical treatment tolerance, etc., with an
aim to determine removal /inactivation capability of the virus
that can not be detected in a raw material or contingently
contaminated.
As mentioned above, the role of viral clearance study is to
speculate removal /inactivation capability of a process
through a model test, and it contributes to give scientific basis
to assure that a biotechnological/biological product of hu-
man or animal origin has reached an acceptable level in
aspect of viral safety.
At a viral clearance study, it is necessary to adopt an ap-
propriate approach method which is definitive and rational
and can assure viral safety of a final product, taking into con-
sideration the source and the properties of the raw material/
substrate as well as the manufacturing process.
4. Raw material /substrate for drug production
4.1 Issues relating to animal species and its region as a
source of raw material/substrate for drug production and
countermeasures to be taken thereto
For manufacturing JP listed biotechnological/biological
products, which require measures for viral safety, a raw
material/substrate derived mainly from human, bovine,
swine or equine is used, and it is obvious that such human
and animal has to be healthy nature. A wild animal should be
avoided, and it is recommended to use animals derived from
a colony controlled by an appropriate SPF (Specific
Pathogen-Free) condition and bred under a well deigned
hygienic control, including appropriate control for preven-
tion of microbial contamination and contamination monitor-
ing system. If a meat standard for food is available, an
animal meeting this standard has to be used. The type of vi-
rus to be concerned about depend on animal species, but it
may be possible to narrow down the virus for investigation by
means of examining the hygiene control, applicability of a
meat standard for food, etc. On the other hand, even with the
animals of the same species, a different approach may be
necessary depending upon the region where the specimen for
a raw material /substrate is taken. For example, in case of ob-
taining raw material/substrate from blood or other specific
region, it is necessary to be aware of the risk level, virus mul-
tiplication risk, etc. which may specifically exists depending
upon its region. Such approach may be different from those
applied to body waste such as urine, milk, etc. as a source of
raw material/substrate. Further, caution has to be taken on
transmissible spongiform encephalopathy (TSE) when pitui-
tary gland, etc. is used as a raw material. This report does not
include detailed explanation on TSE, but recommendations
are to use raw material derived from 1) animals originated in
the countries (area) where incidence of TSE has not been
reported; 2) animals not infected by TSE; or 3) species of
animal which has not been reported on TSE. It is recom-
mended to discuss the matters concerned with TSE with the
regulatory authority if there is any unclear point.
Followings are the raw material/substrate used for
manufacturing biotechnological/biological products in
Japan.
(1) Biological products derived from human
Blood plasma, placenta, urine, etc. derived from human
are used as the sources of raw material of biotechnological/
biological product. As for these raw materials, there are 2
cases: 1) Appropriateness can be confirmed by interview or
by examination of the individual who supplies each raw
material, and 2) Such sufficient interview or examination of
the individual can not be made due to type of raw material.
In case that sufficient examination of individual level is not
possible, it is necessary to perform test to deny virus contami-
nation at an appropriate manufacturing stage, for example,
the stage to decide it as a substrate for drug production.
(2) Biological products derived from animal besides human
Insulin, gonadotropin, etc. are manufactured from blood
plasma or from various organs of bovine, swine and equine.
(3) Protein drug derived from cell line of human or animal
origin
In the case of protein drugs derived from cell line of human
1668 Basic Requirements for Viral / General Information
JP XV
or animal origin, a cell line of human or animal is the raw
material per se, and the substrate for drug production is a cell
bank prepared from cloned cell line (master cell bank or
working cell bank). Examination at cell bank level is consi-
dered enough for viral safety qualification, but it goes
without saying that the more appropriate and rational qualifi-
cation evaluation test of cell bank can be realized when more
information is available on the virus of the source animal or
on prehistory of driving the cell line, the base of cell bank.
4.2 Qualification evaluation test on human or animal as a
source of raw material/substrate for drug production
(1) Biological products derived from human
Body fluid etc. obtained from healthy human must be used
for biological products production. Further, in case that in-
terview or examination of the individual, who supplies the
raw material, can be possible and is necessary, interview un-
der an appropriate protocol and a serologic test well evaluat-
ed in aspects of specificity, sensitivity and accuracy have to be
performed, so that only the raw material, which is denied la-
tent HBV, HCV and HIV, will be used. In addition to the
above, it is necessary to test for gene of HBV, HCV and HIV
by a nucleic amplification test (NAT) well evaluated in
aspects of specificity, sensitivity and accuracy.
In case of the raw material (e.g., urine), which can not be
tested over the general medical examination of the individual
who supplies the material, or of the raw material which is ir-
rational to conduct individual test, the pooled raw material,
as the substrate for drug production, has to be conducted at
least to deny existence of HBV, HCV and HIV, using a
method well evaluated in aspects of specificity, sensitivity and
accuracy, such as the antigen test or NAT.
(2) Biological products derived from animal besides human
The animal used for manufacturing biological product has
to be under appropriate health control, and has to be con-
firmed of its health by various tests. Further, it is necessary
that the population, to which the animal belongs, has been
under an appropriate breeding condition, and that no abnor-
mal individual has been observed in the population. Further,
it is necessary to demonstrate information or scientific basis
which can deny known causes infection or disease to human,
or to deny such animal inherent latent virus by serologic test
or by nucleic amplification test (NAT). The infectious virus
that is known to be common between human and animal, and
known to cause infection in each animal are tentatively listed
in Table 2. It is necessary that the table is completed under
careful examination, and denial of all of them, by means of
tests on individual animal, tissue, body fluid, etc. as a raw
material, or on pooled raw material (as a direct substrate for
drug production), is not always necessary. Table 2 can be
used as reference information, in addition to the other infor-
mation, such as; source of animal, health condition, health
and breeding control, conformity to the meat standard for
food, etc., to elaborate to which virus what kind of test has to
be performed, and for which virus it is not always necessary
to test for, etc. It is important to clarify and record the basis
of choosing the virus and the test conducted thereof.
Table 2. Infectious viruses known to be common
between human and animal and known to
cause infection to each animal
bovine
Cowpox virus ©
Paravaccinia virus ©
Murray valley encephalitis
virus
©
Louping ill virus
Wesselsbron virus
Foot-and-mouth disease
©
©
Japanese encephalitis
virus
Vesicular stomatitis virus
Bovine papular stomatitis
virus
©
Orf virus
Borna disease virus
Rabies virus
©
Influenza virus
Hepatitis E virus
Encephalomyocarditis
virus
©
Rotavirus
Eastern equine
encephalitis virus
Western equine
encephalitis virus
Venezuelan equine
encephalitis virus
Morbillivirus
Hendra virus
©
Nipah virus
Transmissible gastroente-
ritis virus
Porcine respiratory
coronavirus
Porcine epidemic
diarrhea virus
Hemagglutinating
encephalomyelitis virus
Porcine respiratory and
reproductive syndrome virus
Hog cholera virus
swine
-
: ;
©
©
©
©
: '
©
©
©
©
©
©
©
©
©
sheep
©
©
©
©
©
©
goat
equine
©
©
©
©
©
©
©
©
©
JPXV
General Information / Basic Requirements for Viral 1669
Parainfluenza virus Type 3
©
Telfan/Teschen disease
virus
©
Reovirus
©
Endogenous retrovirus
©
Porcine adenovirus
©
Porcine circovirus
©
Porcine parvovirus
©
Porcine poxvirus
©
Porcine cytomegalovirus
©
Pseudorabies virus
©
Russian spring-summer
encephalitis virus
©
©
Rift Valley fever virus
©
©
Crimean-Congo hemor-
rhagic fever virus
(Nairovirus)
©
©
©
Torovirus
©
(3) Protein drug derived from cell line of human or animal
origin
It is important to conduct thorough investigation on latent
endogenous and non-endogenous virus contamination in a
master cell bank (MCB), which is the cell substrate for drug
production, in accordance with the Notice Iyakushin No. 329
entitled "Viral safety evaluation of biotechnology products
derived from cell lines of human or animal origin". Further,
it is necessary to conduct an appropriate adventitious virus
test (e.g., in vitro and in vivo test) and a latent endogenous vi-
rus test on the cell at the limit of in vitro cell age (CAL) for
drug production. Each WCB as a starting cell substrate for
drug production should be tested for adventitious virus either
by direct testing or by analysis of cells at the CAL, initiated
from the WCB. When appropriate non-endogenous virus
tests have been performed on the MCB and cells cultured up
to or beyond the CAL have been derived from the WCB and
used for testing for the presence of adventitious viruses, simi-
lar tests need not be performed on the initial WCB.
5. Points of concern with respect to manufacturing and
virus testing
To ensure viral safety of a biological product derived from
tissue, body fluid etc. of human or animal origin, it is neces-
sary to exclude any possibility of virus contamination from a
raw material, such as tissue and body fluid, or a substrate,
paying attention to the source of virus contamination as men-
tioned in above 3.5, and to adopt appropriate manufacturing
conditions and technologies in addition to enhancement of
manufacturing environment, so that virus contamination in
the course of process and handling and from operators, facil-
ities and environment can be minimized.
In addition to the above, effective virus test and viral inac-
tivation /removal technology, which are reflected by rapid
progress of science, have to be introduced. Adoption of two
or more steps with different principles is recommended for vi-
rus inactivation/removal process. Further, it is important to
minimize any possible virus derivation by using a reagent,
which quality is equivalent to that of a drug. Examples of vi-
rus inactivation/removal processes are © heating (It is
reported that almost viruses are inactivated by heating at 55 -
60°C for 30 minutes with exceptions of hepatitis virus, etc.
and that dry heating at 60°C for 10 - 24 hours is effective in
case of the products of blood or urine origin.), (2) treatment
with organic solvent /surfactant (S/D treatment), © mem-
brane filtration (15 - 50 nm), @ acid treatment, © irradia-
tion (y-irradiation, etc.), © treatment with column chro-
matograph (e.g. affinity chromatography, ion-exchange chro-
matography), © fractionation (e.g. organic solvent or am-
monium sulfate fractionation), ® extraction.
5.1 Virus test conducted in advance of purification process
(1) Biological products derived from human
In many cases, samples for virus test before purification
process are body fluid or tissue of individual collected as a
raw material, or its pooled material or extraction as a sub-
strate. As mentioned in 4.2 (1), it is necessary to deny latent
HBV, HCV and HIV by the test evaluated enough in aspects
of specificity, sensitivity and accuracy. Even in a case that a
non-purified bulk before purification process is produced
from a substrate, it is not always necessary to conduct virus
test again at the stage before purification, so long as the
presence of any latent virus can be denied at the stage of sub-
strate by an appropriate virus test, with cases where the non-
purified bulk is made from the substrate by adding any rea-
gent etc. of living organisms origin are an exception.
(2) Biological products derived from animal besides human
Similar to (1) above, samples for virus test before purifica-
tion process are, in many cases, body fluid or tissue of in-
dividual collected as a raw material, or its pooled material or
extraction as a substrate. In these cases, it is necessary to have
a data, which can deny latent virus of probable cause of hu-
man infection or disease as mentioned in the above 4.2 (2), or
to have a result of serologic test or nucleic amplification test
(NAT) evaluated enough in aspects of specificity, sensitivity
and accuracy. The concept, which is applied to a case that
non-purified bulk before purification process is produced
from substrate, is the same as those provided in the above 4.2
(!)■
(3) Protein drug derived from cell line of human or animal
origin
Generally, substrate in this case is cell bank, and the sam-
ple for testing before purification process is a harvested cell
after cell culturing or unprocessed bulk which consists of sin-
gle or pooled complex culture broth. The unprocessed bulk
may be sometimes culture broth without cell. Denial of latent
virus, which is determined by virus test at a MCB or WCB
level, does not always deny latent virus in unprocessed bulk
after culturing. Further, it is noted that the viral test at the
CAL is meaningful as a validation but can not guarantee
definite assurance of latent virus denial, since the test is
generally performed only once. In case of using a serum or a
component of blood origin in a culture medium, definite
denial of latent virus at the level of unprocessed bulk can not
be assured so long as the viral test has not been conducted on
each lot at the CAL, since lot renewal can be a variable factor
on viral contamination.
A representative sample of the unprocessed bulk, removed
from the production reactor prior to further processing,
1670 Basic Requirements for Viral / General Information
JP XV
represents one of the most suitable levels at which the pos-
sibility of adventitious virus contamination can be deter-
mined with a high probability of detection. Appropriate test-
ing for viruses should be performed at the unprocessed bulk
level unless virus testing is made more sensitive by initial par-
tial processing (e.g., unprocessed bulk may be toxic in test
cell cultures, whereas partially processed bulk may not be
toxic). In certain instances it may be more appropriate to test
a mixture consisting of both intact and disrupted cells and
their cell culture supernatants removed from the production
reactor prior to further processing.
In case of unprocessed bulk, it is required to conduct virus
test on at least 3 lots obtained from pilot scale or commercial
scale production. It is recommended that manufacturers
develop programs for the ongoing assessment of adventitious
viruses in production batches. The scope, extent and fre-
quency of virus testing on the unprocessed bulk should be de-
termined by taking several points into consideration includ-
ing the nature of the cell lines used to produce the desired
products, the results and extent of virus tests performed dur-
ing the qualification of the cell lines, the cultivation method,
raw material sources and results of viral clearance studies.
Screening in vitro tests, using one or several cell lines, are
generally employed to test unprocessed bulk. If appropriate,
a NAT test or other suitable methods may be used.
Generally, harvest material in which adventitious virus has
been detected should not be used to manufacture the
product. If any adventitious viruses are detected at this level,
the process should be carefully checked to determine the
cause of the contamination, and appropriate actions taken.
5.2 Virus test as an acceptance test of an intermediate
material, etc.
When a biological product is manufactured from tissue,
body fluid etc. of human or animal origin, there are cases that
an intermediate material, partially processed as a raw materi-
al or substrate by outside manufacturer, is purchased and
used for manufacturing. In such case, if any test to meet this
General Information has been conducted by such outside
manufacturer, it is necessary for the manufacturer of the bio-
logical product, who purchased the intermediate material, to
examine what sort of virus test has to be conducted as accep-
tance tests, and to keep record on the basis of rationality in-
cluding the details of the test conducted.
On the other hand, if no test to meet this General Informa-
tion has been conducted by such outside manufacturer of the
raw material, all necessary virus free test has to be conducted
to meet this General Information on the intermediate materi-
al regarding it as the direct substrate for drug production.
5.3 Virus test on a final product
Virus tests to be conducted on a final product (or on a
product to reach the final product) has to be defined under
comprehensive consideration of the type of raw material or
substrate, the result of virus test conducted on raw material/
substrate, the result of evaluation on viral removal /inactiva-
tion process, any possibility of virus contamination in the
manufacturing process, etc. Comprehensive viral safety as-
surance can only be achieved by appropriate selection of the
raw material/substrate, an appropriate virus test conducted
on the raw material /substrate /intermediate material, the
virus test conducted at an appropriate stage of manufactur-
ing, an appropriate viral clearance test, etc. However, there
are cases of having specific backgrounds, such as 1) use of the
raw material derived from unspecified individual human, 2)
possible existence of virus at window period, 3) specific detec-
tion limit of virus test, etc. and in these cases, virus contami-
nation to the final product may occur if there is any deficiency
on the manufacturing process (e.g., damage of membrane
filter) or any mix-up of the raw materials, etc. To avoid such
accidental virus contamination, it may be recommended to
conduct nucleic amplification test (NAT) on the final product
focusing on the most risky virus among those that may possi-
bly to exist in the raw material.
6. Process evaluation on viral clearance
6.1 Rationale, objective and general items to be concerned
with respect to viral clearance process evaluation
Evaluation of a viral inactivation /removal process is im-
portant for ensuring safety of a biological product derived
from tissue or body fluid of human or animal origin. Con-
ducting evaluation on viral clearance is to assure, even to
some extent, elimination of the virus, which may exist in a
raw material, etc. or may be derived to the process due to un-
expected situation. Viral clearance studies should be made by
a carefully designed appropriate method, and has to be ra-
tionally evaluated.
The objective of viral clearance studies is to assess process
step(s) that can be considered to be effective in inactivating/
removing viruses and to estimate quantitatively the overall
level of virus reduction obtained by the process. This should
be achieved by the deliberate addition ("spiking") of sig-
nificant amounts of a virus at different manufacturing /purifi-
cation steps and demonstrating its removal or inactivation
during the subsequent steps. It is not necessary to evaluate or
characterize every step of a manufacturing process if ade-
quate clearance is demonstrated by the use of fewer steps. It
should be borne in mind that other steps in the process may
have an indirect effect on the viral inactivation /removal
achieved. Manufacturers should explain and justify the ap-
proach used in studies for evaluating viral clearance.
The reduction of virus infectivity may be achieved by
removal of virus particles or by inactivation of viral infectivi-
ty. For each production step assessed, the possible mechan-
ism of loss of viral infectivity should be described with regard
to whether it is due to inactivation or removal. For
inactivation steps, the study should be planned in such a way
that samples are taken at different times and an inactivation
curve constructed.
6.2 Selection of virus
To obtain broad range of information of viral inactivation
/removal, it is desirable that a model virus used for viral
clearance studies should be chosen from the viruses with
broad range of characteristics in aspects of DNA/RNA, with
or without envelope, particle size, significant resistance to
physical/chemical treatment, etc. and it is necessary to com-
bine about 3 model viruses to cover these characteristics.
At choice of a model virus, there are also the ways to
choose a virus closely related to or having the same character-
istics of the virus known to exist in the raw material. In such
case, it is in principle recommendable to choose a virus which
demonstrates a higher resistance to inactivation/removal
treatment if two or more candidate viruses are available for
choice. Further, a virus which can grow at a high titer is
desirable for choice, although this may not always be possi-
ble. In addition to the above, choosing a virus, which will
provide effective and reliable assay result at each step, is
necessary, since sample condition to be tested at each step of
JP XV
General Information / Basic Requirements for Viral 1671
Table 3. Example of viruses which have been used for viral clearance studies
Virus
Family
Genus
Natural host
Genome
Env
Size (nm)
Shape
Resistance
Vesicular Stomatitis Virus
Rhabdo
Vesiculovirus
Equine
Bovine
RNA
yes
70 x 150
Bullet
Low
Parainfluenza Virus
Paramyxo
Type 1,3
Respirovirus
Type 2,4
Rubulavirus
Various
RNA
yes
100-200 +
Pleo-Spher
Low
MuLV
Retro
Type C
oncovirus
Mouse
RNA
yes
80-110
Spherical
Low
Sindbis Virus
Toga
Alphavirus
Human
RNA
yes
60-70
Spherical
Low
BVDV
Flavi
Pestivirus
Bovine
RNA
yes
50-70
Pleo-Spher
Low
Pseudorabies Virus
Herpes
Varicellovirus
Swine
DNA
yes
120 - 200
Spherical
Med
Poliovirus Sabin Type 1
Picorna
Enterovirus
Human
RNA
no
25-30
Icosahedral
Med
Encephalomyocardititis
Picorna
Cardiovirus
Mouse
RNA
no
25-30
Icosahedral
Med
Virus
Reovirus 3
Reo
Orthoreovirus
Various kind
RNA
no
60-80
Spherical
Med
SV 40
Papova
Polyomavirus
Monkey
DNA
no
40-50
Icosahedral
Very high
Parvovirus: canine, por-
Parvo
Parvovirus
Canine
DNA
no
18-24
Icosahedral
Very high
cine
Porcine
a production process may influence the detection sensitivity.
Consideration should also be given to health hazard which
may pose to the personnel performing the clearance studies.
For the other items taken for consideration at choice of
virus, the Notice, Iyakushin No. 329 can be used as a refer-
ence. Examples of the virus which have been used for viral
clearance studies are shown in Table 3 which was derived
from Iyakushin No. 329. However, the Notice, Iyakushin
No. 329, is on viral safety of a product derived cell line of hu-
man or animal origin, and a more appropriate model virus
has to be chosen taking into account the origin/raw material
of biological products.
6.3 Design of viral clearance studies
The purpose of viral clearance studies is to quantitatively
evaluate removal or inactivation capability of a process, in
which a virus is intentionally spiked to a specific step of a
manufacturing process.
Following are the precautions to be taken at planning viral
clearance studies.
(1) Care should be taken in preparing the high-titer virus
to avoid aggregation which may enhance physical removal
and decrease inactivation thus distorting the correlation with
actual production.
(2) Virus detection method gives great influence to viral
clearance factor. Accordingly, it is advisable to gain detec-
tion sensitivity of the methods available in advance, and use a
method with a detection sensitivity as high as possible. Quan-
titative infectivity assays should have adequate sensitivity and
reproducibility in each manufacturing process, and should be
performed with sufficient replicates to ensure adequate
statistical validity of the result. Quantitative assays not asso-
ciated with infectivity may be used if justified. Appropriate
virus controls should be included in all infectivity assays to
ensure the sensitivity of the method. Also, the statistics of
sampling virus when at low concentrations (for example,
number of virus is 1-1000/L) should be considered.
(3) Viral clearance studies are performed in a miniature
size system that simulates the actual production process of
the biotechnological /biological product used by the
manufacturer. It is inappropriate to introduce any virus into
a production facility because of GMP constraints. Therefore,
viral clearance studies should be conducted in a separate
laboratory equipped for virological work and performed by
staff with virological expertise in conjunction with produc-
tion personnel involved in designing and preparing a scaled-
down version of the purification process. The viral clearance
studies should be performed under the basic concept of GLP.
(4) Each factor on a viral clearance study of a process,
which is performed in miniature size, should reflect that of
actual manufacturing as far as possible, and its rationality
should be clarified. In case of chromatograph process, length
of column bed, linear velocity, ratio of bed volume per veloc-
ity (in other words, contact time), buffer, type of column
packing, pH, temperature, protein concentration, salt con-
centration and concentration of the objective product are all
correspondent to those of the actual production. Further,
similarity of elution profile should be achieved. For the other
process, similar concept should be applied. If there is any fac-
tor which can not reflect the actual production, its effect to
the result should be examined.
(5) It is desirable that two or more inactivation /removal
processes of different principles are selected and examined.
(6) As for the process which is expected to inactivate /re-
move virus, each step should be evaluated in aspect of clear-
ance capability, and carefully determined if it is the stage of
inactivation, removal or their combination for designing the
1672 Basic Requirements for Viral / General Information
JP XV
test. Generally, in viral clearance test, a virus is spiked in each
step which is the object of the test, and after passing through
the process in question, the reduction level of infectivity is
evaluated. But, in some case, it is accepted that a high poten-
tial virus is spiked at a step of the process, and virus concen-
tration of each succeeding step is carefully monitored. When
removal of virus is made by separation or fractionation, it is
desirable to investigate how the virus is separated or fractio-
nated (mass balance).
(7) For assessment of viral inactivation, unprocessed
crude material or intermediate material should be spiked with
infectious virus and the reduction factor calculated. It should
be recognized that virus inactivation is not a simple, first ord-
er reaction and is usually more complex, with a fast "phase
1" and a slow "phase 2". The study should, therefore, be
planned in such a way that samples are taken at different
times and an inactivation curve constructed. It is recom-
mended that studies for inactivation include at least one time
point less than the minimum exposure time and greater than
zero, in addition to the minimum exposure time. The
reproducible clearance should be demonstrated in at least two
independent studies. When there is a possibility that the virus
is a human pathogen, it is very important that the effective in-
activation process is designed and additional data are ob-
tained. The initial virus load should be determined from the
virus which can be detected in the spiked starting material. If
this is not possible, the initial virus load may be calculated
from the titer of the spiking virus preparation. Where inacti-
vation is too rapid to plot an inactivation curve using process
conditions, appropriate controls should be performed to
demonstrate that infectivity is indeed lost by inactivation.
(8) If antibody against virus exists in an unprocessed
material, caution should be taken at clearance studies, since it
may affect the behavior of virus at viral removal or inactiva-
tion process.
(9) Virus spiked in unprocessed material should be
sufficient enough to evaluate viral removal or inactivation
capability of the process. However, the virus "spike" to be
added to the unprocessed material should be as small as pos-
sible in comparison with the sample volume of the unproc-
essed material so as not to cause characteristic change of the
material by addition of the virus nor to cause behavioral
change of the protein in the material.
(10) It is desirable that the virus in the sample is subject
for quantitative determination without applying ultracen-
trifuge, dialysis, storage, etc. as far as possible. However,
there may be a case that any handling before quantitative
test, such as remove procedure of inhibitor or toxic sub-
stance, storage for a period to realize test at a time, etc., is in-
evitable. If any manipulation, such as dilution, concentra-
tion, filtration, dialysis, storage, etc., is applied for prepara-
tion of the sample for testing, a parallel control test, which
passes through a similar manipulation, should be conducted
to assess infectivity variance at the manipulation.
(11) Buffers and product (desired protein or other com-
ponent contained therein) should be evaluated independently
for toxicity or interference in assays used to determine the vi-
rus titer, as these components may adversely affect the indica-
tor cells. If the solutions are toxic to the indicator cells, dilu-
tion, adjustment of the pH, or dialysis of the buffer contain-
ing spiked virus might be necessary. If the product itself has
anti-viral activity, the clearance study may need to be per-
formed without the product in a "mock" run, although
omitting the product or substituting a similar protein that
does not have anti-viral activity could affect the behaviour of
the virus in some production steps.
(12) Many purification schemes use the same or similar
buffers or columns, repetitively. The effects of this approach
should be taken into account when analyzing the data. The
effectiveness of virus elimination by a particular process may
vary with the stage in manufacture at which it is used.
(13) Overall reduction factors may be underestimated
where production conditions or buffers are too cytotoxic or
virucidal and should be discussed on a case-by-case basis.
Overall reduction factors may also be overestimated due to
inherent limitations or inadequate design of viral clearance
studies.
(14) It has to be noted that clearance capability of viral
removal/inactivation process may vary depending upon the
type of virus. The viral removal/inactivation process, which
displays viral clearance by specific principle or mechanism,
may be quite effective to the virus, which meets such mechan-
ism of action, but not effective to the other type of viruses.
For example, S/D treatment is generally effective to the virus
with lipid membrane, but not effective to the virus not having
such membrane. Further, some virus is resistant to the gener-
al heating process (55 - 60°C, 30 minutes). When clearance is
expected for such virus, introduction of a further severe con-
dition or process, which has different principle or mechan-
ism, is necessary. Virus removal membrane filtration, which
is different from S/D or heat treatment in aspect of principle,
is effective to a broad range of virus that can not pass through
the membrane. Affinity chromatography process, which spe-
cifically absorbs the objective protein, can thoroughly wash
out the materials other than the objective protein including
virus etc. and is generally effective for viral removal. Separa-
tion /fractionation of a virus from an objective protein is
sometimes very difficult, but there are not so rare that ion ex-
change chromatography, ethanol fractionation, etc. is effec-
tive for clearance of a virus which can not be sufficiently inac-
tivated or removed by the other process.
(15) Effective clearance may be achieved by any of the
following: multiple inactivation steps, multiple complemen-
tary separation steps, or combinations of inactivation and
separation steps. Separation methods may be dependent on
the extremely specific physico-chemical properties of a virus
which influence its interaction with gel matrices and precipi-
tation properties. However, despite these potential variables,
effective removal can be obtained by a combination of com-
plementary separation steps or combinations of inactivation
and separation steps. Well designed separation steps, such as
chromatographic procedures, filtration steps and extractions,
can be also effective virus removal steps provided that they
are performed under appropriately controlled conditions.
(16) An effective virus removal step should give
reproducible reduction of virus load shown by at least two in-
dependent studies.
(17) Over time and after repeated use, the ability of chro-
matography columns and other devices used in the purifica-
tion scheme to clear virus may vary. Some estimate of the sta-
bility of the viral clearance after several uses may provide
support for repeated use of such columns.
(18) The Notice, Iyakushin No. 329, would be used as a
reference when viral clearance studies on biological products
are designed.
6.4 Interpretation of viral clearance studies
JPXV
General Information / Basic Requirements for Viral 1673
6.4.1 Evaluation on viral clearance factor
Viral clearance factor is a logarithm of reduction ratio of
viral amount (infectious titer) between each step applied for
viral clearance of a manufacturing process. Total viral clear-
ance factor throughout the process is sum of the viral clear-
ance factor of each step appropriately evaluated.
Whether each and total viral clearance factor obtained are
acceptable or should not be evaluated in aspects of every
virus that can be realistically anticipated to derive into the
raw material or the manufacturing process, and its rationality
should be recorded.
In case that existence of any viral particle is recognized in a
substrate for drug production, e.g., a substrate of rodent ori-
gin for biodrug production, it is important not only to
demonstrate removal or inactivation of such virus, but also
to demonstrate that the purification process has enough
capability over the required level to assure safety of the final
product at an appropriate level. The virus amount removed
or inactivated in a manufacturing process should be com-
pared with the virus amount assumed to exist in the substrate
etc. used for manufacturing drug, and for this purpose, it is
necessary to obtain the virus amount in the raw materials/
substrate, etc. Such figure can be obtained by measuring in-
fectious titer or by the other method such as transmission
electron microscope (TEM). For evaluation of overall proc-
ess, a virus amount, far larger than that assumed to exist in
the amount of the raw materials /substrate which is equiva-
lent to single administration of the final product, has to be re-
moved. It is quite rare that existence of virus can be assumed
in a substrate for drug production, with the exception of the
substrate of rodent origin, and such suspicious raw material/
substrate should not be used for manufacturing drug with a
special exceptional case that the drug in question is not avail-
able from the other process and is clinically indispensable,
and that the information including infectious properties of
the virus particle assumed to exist has been clarified.
6.4.2 Calculation of viral clearance index
Viral clearance factor, "R", for viral removal /inactivation
process can be calculated by the following formula.
U = log[(K,xr,)/(K 2 x7' J )]
In which
R: Logarithm of reduction ratio
V\. Sample volume of the unprocessed material
7\: Virus amount (titer) of the unprocessed material
V 2 : Sample volume of the processed material
T 2 : Virus amount (titer) of the processed material
At the calculation of viral clearance factor, it is recom-
mendable to use the virus titer detected in the sample prepa-
ration of the unprocessed material after addition of virus, not
the viral titer added to the sample preparation wherever pos-
sible. If this is not possible, loaded virus amount is calculated
from virus titer of the solution used for spike.
6.4.3 Interpretation of results and items to be concerned at
evaluation
At the interpretation and the evaluation of the data on
effectiveness of viral inactivation /removal process, there are
various factors to be comprehensively taken into account,
such as ® appropriateness of the virus used for the test, ©
design of the viral clearance studies, © virus reduction ratio
shown in logarithm, @ time dependence of inactivation, ©
factors /items which give influence to the inactivation/
removal process, © sensitivity limit of virus assay method, ©
possible effect of the inactivation /removal process which is
specific to certain class of viruses.
Additional items to be concerned at appropriate interpreta-
tion and evaluation of the viral clearance data are as follows:
(1) Behavior of virus used to the test
At interpretation of the vial clearance results, it is necessa-
ry to recognize that clearance mechanism may differ depend-
ing upon the virus used for the test. Virus used for a test is
generally produced in tissue culture, but behavior of the virus
prepared in the tissue culture may be different from that of
the native virus. Examples are possible differences of purity
and degree of aggregation between the native and the cul-
tured viruses. Further, change of surface properties of a vi-
rus, e.g., addition of a sucrose chain which is ascribed to
specific nature of a separation process, may give effect to the
separation. These matters should be also considered at inter-
pretation of the results.
(2) Design of test
Viral clearance test should have been designed taking into
account variation factors of the manufacturing process and
scaling down, but there still remain some variance from
actual production scale. It is necessary to consider such vari-
ance at the interpretation of the data and limitation of the
test.
(3) Acceptability of viral reduction data
Total viral clearance factor is expressed as a sum of
logarithm of reduction ratio obtained at each step. The sum-
mation of the reduction factor of multiple steps, particularly
of steps with little reduction (e.g., below 1 log 10 ), may overes-
timate viral removal /inactivation capability of the overall
process. Therefore, virus titer of the order of 1 logio or less
has to be ignored unless justified. Further, viral clearance fac-
tor achieved by repeated use of the same or similar method
should be ignored for calculation unless justified.
(4) Time dependence of inactivation
Inactivation of virus infectivity frequently shows biphasic
curve, which consists of a rapid initial phase and subsequent
slow phase. It is possible that a virus not inactivated in a step
may be more resistant to the subsequent step. For example, if
an inactivated virus forms coagulation, it may be resistant to
any chemical treatment and heating.
(5) Evaluation of viral reduction ratio shown logarithm
Viral clearance factor shown in logarithm of reduction
ratio of virus titer can demonstrate drastic reduction of resid-
ual infectious virus, but there is a limit that infectious titer
can never be reduced to zero. For example, reduction in in-
fectivity of a preparation containing 8 log 10 infectious unit
per mL by a factor of 8 log 10 leaves zero log 10 per mL or one
infectious unit per mL, taking into account the detection
limit of assay.
(6) Variable factor of manufacturing process
Minor variance of a variation factor of a manufacturing
process, e.g., contact time of a spiked sample to a buffer or a
column, will sometimes give influence to viral removal or in-
activation effect. In such case, it may be necessary to inves-
tigate to what extent such variance of the factor has given in-
fluence to the process concerned in aspect of viral inactiva-
tion.
(7) Existence of anti-viral antiserum
Anti-viral antiserum that exists in the sample preparation
used for a test may affect sensitivity of distribution or inacti-
vation of a virus, which may result in not only defusing the
1674 Basic Requirements for Viral / General Information
JP XV
virus titer but complicating interpretation of the test result.
So, existence of anti-viral antiserum is one of the important
variable factors.
(8) Introduction of a new process for removal/inactivation
Viral clearance is an important factor for securing safety of
drug. In case that an achievement level of infective clearance
of a process is considered insufficient, a process which is
characterized by inactivation /removal mechanism to meet
the purpose or an inactivation /removal process which can
mutually complement to the existence process has to be in-
troduced.
(9) Limit of viral clearance studies
Viral clearance studies are useful for contributing to the as-
surance that an acceptable level of safety in the final product
is achieved but do not by themselves establish safety.
However, a number of factors in the design and execution of
viral clearance studies may lead to an incorrect estimate of
the ability of the process to remove virus infectivity, as de-
scribed above.
7. Statistics
The viral clearance studies should include the use of
statistical analysis of the data to evaluate the results. The
study results should be statistically valid to support the con-
clusions reached.
7.1 Statistical considerations for assessing virus assays
Virus titrations suffer the problems of variation common
to all biological assay systems. Assessment of the accuracy of
the virus titrations and reduction factors derived from them
and the validity of the assays should be performed to define
the reliability of a study. The objective of statistical evalua-
tion is to establish that the study has been carried out to an
acceptable level of virological competence.
Assay
1. Assay methods may be either quantal or quantitative.
Both quantal and quantitative assays are amenable to statisti-
cal evaluation.
2. Variation can arise within an assay as a result of dilu-
tion errors, statistical effects and differences within the assay
system which are either unknown or difficult to control.
These effects are likely to be greater when different assay runs
are compared (between-assay variation) than when results wi-
thin a single assay run are compared (within-assay variation).
3. The 95% confidence limits for results of within-assay
variation normally should be on the order of ±0.5 log 10 of
the mean. Within-assay variation can be assessed by standard
textbook methods. Between-assay variation can be moni-
tored by the inclusion of a reference preparation, the estimate
of whose potency should be within approximately 0.5 log 10 of
the mean estimate established in the laboratory for the assay
to be acceptable. Assays with lower precision may be accepta-
ble with appropriate justification.
7.2 Reproducibility and confidence limit of viral clearance
studies
An effective virus inactivation /removal step should give
reproducible reduction of virus load shown by at least two in-
dependent studies. The 95% confidence limits for the reduc-
tion factor observed should be calculated wherever possible
in studies of viral clearance. If the 95% confidence limits for
the viral assays of the starting material are ± s, and for the
viral assays of the material after the step are ±a, the 95%
confidence limits for the reduction factor are ± A /s 2 + a 2 .
8. Re-evaluation of viral clearance
Whenever significant changes in the production or purifica-
tion process are made, the effect of that change, both direct
and indirect, on viral clearance should be considered and the
system re-evaluated as needed. Changes in process steps may
also change the extent of viral clearance.
9. Measurement for viral clearance studies
9.1 Measurement of virus infective titer
Assay methods may be either quantal or quantitative.
Quantal methods include infectivity assays in animals or in
cultured cell infections dose (CCID) assays, in which the
animal or cell culture is scored as either infected or not. In-
fectivity titers are then measured by the proportion of
animals or culture infected. In quantitative methods, the in-
fectivity measured varies continuously with the virus input.
Quantitative methods include plaque assays where each
plaque counted corresponds to a single infectious unit. Both
quantal and quantitative assays are amenable to statistical
evaluation.
9.2 Testing by nucleic-acid-amplification test (NAT)
NAT can detect individual or pooled raw material/cell sub-
strate or virus genome at a high sensitivity even in a stage that
serum test on each virus is negative. Further, it can detect
HBV or HCV gene, which can not be measured in culture sys-
tem. Window period can be drastically shortened at the test
on HBV, HCV and HIV, and the method is expected to con-
tribute as an effective measure for ensuring viral safety.
However, depending upon a choice of primer, there may be a
case that not all the subtype of objective virus can be detected
by this method, and, therefore, it is recommendable to evalu-
ate, in advance, if subtype of a broad range can be detected.
NAT will be an effective evaluation method for virus
removal capability at viral clearance studies. However, in
case of viral inactivation process, viral inactivation obtained
by this method may be underrated, since there is a case that
inactivated virus still shows positive on nucleic acid. Further,
at introduction of NAT, cautions should be taken on ration-
ality of detection sensitivity, choice of a standard which is
used as run-control, quality assurance and maintenance of a
reagent used for primer, interpretation of positive and nega-
tive results, etc.
10. Reporting and preservation
All the items relating to virus test and viral clearance stu-
dies should be reported and preserved.
11. Others
The Notice, Iyakushin No. 329, should be used as a refer-
ence at virus test and viral clearance studies.
Conclusion
As mentioned at the Introduction, assurance of quality/
safety etc. of JP listed drugs should be achieved by state-of-
the-art methods and concepts reflecting the progress of
science and accumulation of experiences.
The basis for ensuring viral safety of JP listed biotechno-
logical/biological products is detailed in this General Infor-
mation. What is discussed here is that an almost equal level
of measures are required for both development of new drugs
and for existing products as well, which means that similar
level of concerns should be paid on both existing and new
products in aspect of viral safety. This document is intended
to introduce a basic concept that quality and safety assurance
of JP listed product should be based upon the most advanced
JPXV
General Information / Capillary Electrophoresis 1675
methods and concepts. This document has been written to
cover all conceivable cases, which can be applied to all
biotechnological/biological products. Therefore, there may
be cases that it is not so rational to pursue virus tests and viral
clearance studies in accordance with this document on each
product, which has been used for a long time without any
safety issue. So, it will be necessary to elaborate the most ra-
tional ways under a case-by-case principle taking into due
consideration source, origin, type, manufacturing process,
characteristics, usages at clinical stage, accumulation of the
past usage record, etc. relating to such biotechnological/bio-
logical products.
4. Capillary Electrophoresis
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia.
General Principles
Capillary electrophoresis is a physical method of analysis
based on the migration, inside a capillary, of charged
analytes dissolved in an electrolyte solution, under the
influence of a direct-current electric field.
The migration velocity of an analyte under an electric field
of intensity E, is determined by the electrophoretic mobility
of the analyte and the electro-osmotic mobility of the buffer
inside the capillary. The electrophoretic mobility of a solute
(ju ep ) depends on the characteristics of the solute (electric
charge, molecular size and shape) and those of the buffer in
which the migration takes place (type and ionic strength of
the electrolyte, pH, viscosity and additives). The elec-
trophoretic velocity (v ep ) of a solute, assuming a spherical
shape, is given by the equation:
Vep=/VE =
(mrjr
q: effective charge of the solute,
rj: viscosity of the electrolyte solution,
r: Stoke's radius of the solute,
V: applied voltage,
L: total length of the capillary.
When an electric field is applied through the capillary filled
with buffer, a flow of solvent is generated inside the capillary,
called electro-osmotic flow. The velocity of the electro-os-
motic flow depends on the electro-osmotic mobility (w eo )
which in turn depends on the charge density on the capillary
internal wall and the buffer characteristics. The electro-os-
motic velocity (v eo ) is given by the equation:
"M,oE =
n
e: dielectric constant of the buffer,
f: zeta potential of the capillary surface.
The velocity of the solute (v) is given by:
v = v ep + v eo
The electrophoretic mobility of the analyte and the electro-
osmotic mobility may act in the same direction or in opposite
directions, depending on the charge of the solute. In normal
capillary electrophoresis, anions will migrate in the opposite
direction to the electro-osmotic flow and their velocities will
be smaller than the electro-osmotic velocity. Cations will mi-
grate in the same direction as the electro-osmotic flow and
their velocities will be greater than the electro-osmotic veloc-
ity. Under conditions in which there is a fast electro-osmotic
velocity with respect to the electrophoretic velocity of the so-
lutes, both cations and anions can be separated in the same
run.
The time (t) taken by the solute to migrate the distance (0
from the injection end of the capillary to the detection point
(capillary effective length) is given by the expression:
t =
l
IxL
, + v.,
(M cp +Mco)V
In general, uncoated fused-silica capillaries above pH 3
have negative charge due to ionized silanol groups in the
inner wall. Consequently, the electro-osmotic flow is from
anode to cathode. The electro-osmotic flow must remain con-
stant from run to run if good reproducibility is to be obtained
in the migration velocity of the solutes. For some applica-
tions, it may be necessary to reduce or suppress the electro-
osmotic flow by modifying the inner wall of the capillary or
by changing the concentration, composition and/or pH of
the buffer solution.
After the introduction of the sample into the capillary,
each analyte ion of the sample migrates within the back-
ground electrolyte as an independent zone, according to its
electrophoretic mobility. Zone dispersion, that is the spread-
ing of each solute band, results from different phenomena.
Under ideal conditions the sole contribution to the solute-
zone broadening is molecular diffusion of the solute along the
capillary (longitudinal diffusion). In this ideal case the
efficiency of the zone, expressed as the number of theoretical
plates (N), is given by:
N=
C"ep+/QX^X£
2XDXL
D:
molecular diffusion coefficient of the solute in the
buffer.
In practice, other phenomena such as heat dissipation,
sample adsorption onto the capillary wall, mismatched
conductivity between sample and buffer, length of the injec-
tion plug, detector cell size and unlevelled buffer reservoirs
can also significantly contribute to band dispersion.
Separation between two bands (expressed as the resolution,
R s ) can be obtained by modifying the electrophoretic mobili-
ty of the analytes, the electro-osmotic mobility induced in the
capillary and by increasing the efficiency for the band of each
analyte, according to the equation:
* s
4(«ep+/0
jU epa and fi cph : electrophoretic mobilities of the two analytes
separated,
/i e p: mean electrophoretic mobility of the two analytes
,"ep= y t"epb+/^epa).
Apparatus
An apparatus for capillary electrophoresis is composed of:
— a high-voltage, controllable direct-current power supply,
— two buffer reservoirs, held at the same level, containing
the prescribed anodic and cathodic solutions,
1676 Capillary Electrophoresis / General Information
JP XV
— two electrode assemblies (the cathode and the anode),
immersed in the buffer reservoirs and connected to the
power supply,
— a separation capillary (usually made of fused-silica)
which, when used with some specific types of detectors,
has an optical viewing window aligned with the detector.
The ends of the capillary are placed in the buffer reser-
voirs. The capillary is filled with the solution prescribed
in the monograph,
— a suitable injection system,
— a detector able to monitor the amount of substances of
interest passing through a segment of the separation
capillary at a given time. It is usually based on absorp-
tion spectrophotometry (UV and visible) or fluorometry,
but conductimetric, amperometric or mass spectrometric
detection can be useful for specific applications. Indirect
detection is an alternative method used to detect non-
UV-absorbing and non-fluorescent compounds,
— a thermostatic system able to maintain a constant tem-
perature inside the capillary is recommended to obtain a
good separation reproducibility,
— a recorder and a suitable integrator or a computer.
The definition of the injection process and its automation
are critical for precise quantitative analysis. Modes of
injection include gravity, pressure or vacuum injection and
electrokinetic injection. The amount of each sample
component introduced electrokinetically depends on its
electrophoretic mobility, leading to possible discrimination
using this injection mode.
Use the capillary, the buffer solutions, the preconditioning
method, the sample solution and the migration conditions
prescribed in the monograph of the considered substance.
The employed electrolytic solution is filtered to remove
particles and degassed to avoid bubble formation that could
interfere with the detection system or interrupt the electrical
contact in the capillary during the separation run. A rigorous
rinsing procedure should be developed for each analytical
method to achieve reproducible migration times of the so-
lutes.
1. Capillary Zone Electrophoresis
Principle
In capillary zone electrophoresis, analytes are separated in
a capillary containing only buffer without any anticonvective
medium. With this technique, separation takes place because
the different components of the sample migrate as discrete
bands with different velocities. The velocity of each band
depends on the electrophoretic mobility of the solute and the
electro-osmotic flow in the capillary (see General Principles).
Coated capillaries can be used to increase the separation
capacity of those substances adsorbing on fused-silica
surfaces.
Using this mode of capillary electrophoresis, the analysis
of both small (M r <2000) and large molecules (2000 <M r
< 100,000) can be accomplished. Due to the high efficiency
achieved in free solution capillary electrophoresis, separation
of molecules having only minute differences in their charge-
to-mass ratio can be effected. This separation mode also al-
lows the separation of chiral compounds by addition of chiral
selectors to the separation buffer.
Optimization
Optimization of the separation is a complex process where
several separation parameters can play a major role. The
main factors to be considered in the development of separa-
tions are instrumental and electrolytic solution parameters.
Instrumental parameters
Voltage: A Joule heating plot is useful in optimizing the
applied voltage and column temperature. Separation time is
inversely proportional to applied voltage. However, an
increase in the voltage used can cause excessive heat produc-
tion, giving rise to temperature and, as a result thereof,
viscosity gradients in the buffer inside the capillary. This
effect causes band broadening and decreases resolution.
Polarity: Electrode polarity can be normal (anode at the
inlet and cathode at the outlet) and the electro-osmotic flow
will move toward the cathode. If the electrode polarity is
reversed, the electro-osmotic flow is away from the outlet and
only charged analytes with electro-osmotic mobilities greater
than the electro-osmotic flow will pass to the outlet.
Temperature: The main effect of temperature is observed
on buffer viscosity and electrical conductivity, and therefore
on migration velocity. In some cases, an increase in capillary
temperature can cause a conformational change in proteins,
modifying their migration time and the efficiency of the sepa-
ration.
Capillary: The dimensions of the capillary (length and in-
ternal diameter) contribute to analysis time, efficiency of
separations and load capacity. Increasing both effective
length and total length can decrease the electric fields
(working at constant voltage) which increases migration time.
For a given buffer and electric field, heat dissipation, and
hence sample band-broadening, depend on the internal di-
ameter of the capillary. The latter also affects the detection
limit, depending on the sample volume injected and the
detection system employed.
Since the adsorption of the sample components on the
capillary wall limits efficiency, methods to avoid these
interactions should be considered in the development of a
separation method. In the specific case of proteins, several
strategies have been devised to avoid adsorption on the capil-
lary wall. Some of these strategies (use of extreme pH and ad-
sorption of positively charged buffer additives) only require
modification of the buffer composition to prevent protein ad-
sorption. In other strategies, the internal wall of the capillary
is coated with a polymer, covalently bonded to the silica, that
prevents interaction between the proteins and the negatively
charged silica surface. For this purpose, ready-to-use capilla-
ries with coatings consisting of neutral-hydrophilic, cationic
and anionic polymers are available.
Electrolytic solution parameters
Buffer type and concentration: Suitable buffers for
capillary electrophoresis have an appropriate buffer capacity
in the pH range of choice and low mobility to minimize
current generation.
Matching buffer-ion mobility to solute mobility, whenever
possible, is important for minimizing band distortion. The
type of sample solvent used is also important to achieve
on-column sample focusing, which increases separation
efficiency and improves detection.
An increase in buffer concentration (for a given pH)
decreases electro-osmotic flow and solute velocity.
Buffer pH: The pH of the buffer can affect separation by
modifying the charge of the analyte or additives, and by
changing the electro-osmotic flow. In protein and peptide
separation, changing the pH of the buffer from above to
below the isoelectric point (pi) changes the net charge of the
JPXV
General Information / Capillary Electrophoresis 1677
solute from negative to positive. An increase in the buffer pH
generally increases the electro-osmotic flow.
Organic solvents: Organic modifiers (methanol, acetoni-
trile, etc.) may be added to the aqueous buffer to increase the
solubility of the solute or other additives and/or to affect the
degree of ionization of the sample components. The addition
of these organic modifiers to the buffer generally causes a
decrease in the electro-osmotic flow.
Additives for chiral separations: For the separation of
optical isomers, a chiral selector is added to the separation
buffer. The most commonly used chiral selectors are
cyclodextrins, but crown ethers, polysaccharides and proteins
may also be used. Since chiral recognition is governed by the
different interactions between the chiral selector and each of
the enantiomers, the resolution achieved for the chiral com-
pounds depends largely on the type of chiral selector used. In
this regard, for the development of a given separation it may
be useful to test cyclodextrins having a different cavity size
(a-, /?-, or y-cyclodextrin) or modified cyclodextrins with neu-
tral (methyl, ethyl, hydroxyalkyl, etc.) or ionizable
(aminomethyl, carboxymethyl, sulfobutyl ether, etc.) groups.
When using modified cyclodextrins, batch-to-batch varia-
tions in the degree of substitution of the cyclodextrins must
be taken into account since it will influence the selectivity.
Other factors controlling the resolution in chiral separations
are concentration of chiral selector, composition and pH of
the buffer and temperature. The use of organic additives,
such as methanol or urea can also modify the resolution
achieved.
2. Capillary Gel Electrophoresis
Principle
In capillary gel electrophoresis, separation takes place
inside a capillary filled with a gel that acts as a molecular
sieve. Molecules with similar charge-to-mass ratios are
separated according to molecular size since smaller molecules
move more freely through the network of the gel and there-
fore migrate faster than larger molecules. Different biological
macromolecules (for example, proteins and DNA frag-
ments), which often have similar charge-to-mass ratios, can
thus be separated according to their molecular mass by capil-
lary gel electrophoresis.
Characteristics of Gels
Two types of gels are used in capillary electrophoresis: per-
manently coated gels and dynamically coated gels. Perma-
nently coated gels, such as cross-linked polyacrylamide, are
prepared inside the capillary by polymerization of the
monomers. They are usually bonded to the fused-silica wall
and cannot be removed without destroying the capillary. If
the gels are used for protein analysis under reducing condi-
tions, the separation buffer usually contains sodium dodecyl
sulfate and the samples are denatured by heating a mixture of
sodium dodecyl sulfate and 2-mercaptoethanol or
dithiothreitol before injection. When non-reducing condi-
tions are used (for example, analysis of an intact antibody),
2-mercaptoethanol and dithiothreitol are not used. Separa-
tion in cross-linked gels can be optimized by modifying the
separation buffer (as indicated in the free solution capillary
electrophoresis section) and controlling the gel porosity
during the gel preparation. For cross-linked polyacrylamide
gels, the porosity can be modified by changing the concentra-
tion of acrylamide and/or the proportion of cross-linker. As
a rule, a decrease in the porosity of the gel leads to a decrease
in the mobility of the solutes. Due to the rigidity of these gels,
only electrokinetic injection can be used.
Dynamically coated gels are hydrophilic polymers, such as
linear polyacrylamide, cellulose derivatives, dextran, etc.,
which can be dissolved in aqueous separation buffers giving
rise to a separation medium that also acts as a molecular
sieve. These separation media are easier to prepare than
cross-linked polymers. They can be prepared in a vial and
filled by pressure in a wall-coated capillary (with no electro-
osmotic flow). Replacing the gel before every injection gener-
ally improves the separation reproducibility. The porosity of
the gels can be increased by using polymers of higher molecu-
lar mass (at a given polymer concentration) or by decreasing
the polymer concentration (for a given polymer molecular
mass). A reduction in the gel porosity leads to a decrease in
the mobility of the solute for the same buffer. Since the disso-
lution of these polymers in the buffer gives low viscosity solu-
tions, both hydrodynamic and electrokinetic injection tech-
niques can be used.
3. Capillary Isoelectric Focusing
Principle
In isoelectric focusing, the molecules migrate under the
influence of the electric field, so long as they are charged, in a
pH gradient generated by ampholytes having pi values in a
wide range (poly-aminocarboxylic acids), dissolved in the
separation buffer.
The three basic steps of isoelectric focusing are loading,
focusing and mobilization.
Loading step: Two methods may be employed:
— loading in one step: the sample is mixed with ampholytes
and introduced into the capillary either by pressure or
vacuum;
— sequential loading: a leading buffer, then the ampho-
lytes, then the sample mixed with ampholytes, again am-
pholytes alone and finally the terminating buffer are in-
troduced into the capillary. The volume of the sample
must be small enough not to modify the pH gradient.
Focusing step: When the voltage is applied, ampholytes
migrate toward the cathode or the anode, according to their
net charge, thus creating a pH gradient from anode (lower
pH) to cathode (higher pH). During this step the components
to be separated migrate until they reach a pH corresponding
to their isoelectric point (pi) and the current drops to very
low values.
Mobilization step: If mobilization is required for detec-
tion, use one of the following methods. Three methods are
available:
— in the first method, mobilization is accomplished during
the focusing step under the effect of the electro-osmotic
flow; the electro-osmotic flow must be small enough to
allow the focusing of the components;
— in the second method, mobilization is accomplished by
applying positive pressure after the focusing step;
— in the third method, mobilization is achieved after the
focusing step by adding salts to the cathode reservoir or
the anode reservoir (depending on the direction chosen
for mobilization) in order to alter the pH in the capillary
when the voltage is applied. As the pH is changed, the
proteins and ampholytes are mobilized in the direction
of the reservoir which contains the added salts and pass
the detector.
The separation achieved, expressed as Apl, depends on the
1678 Capillary Electrophoresis / General Information
JP XV
pH gradient (dpH/dx), the number of ampholytes having
different pi values, the molecular diffusion coefficient (D), the
intensity of the electric field (E) and the variation of the elec-
trophoretic mobility of the analyte with the pH ( — dfi/dpH):
API=3
D(dpH/dx)
E(-dn/dpH)
Optimization
The main parameters to be considered in the development
of separations are:
Voltage: Capillary isoelectric focusing utilises very high
electric fields, 300 V/cm to 1000 V/cm in the focusing step.
Capillary: The electro-osmotic flow must be reduced or
suppressed depending on the mobilization strategy (see
above). Coated capillaries tend to reduce the electro-osmotic
flow.
Solutions: The anode buffer reservoir is filled with a
solution with a pH lower than the pi of the most acidic
ampholyte and the cathode reservoir is filled with a solution
with a pH higher than the pi of the most basic ampholyte.
Phosphoric acid for the anode and sodium hydroxide for the
cathode are frequently used.
Addition of a polymer, such as methylcellulose, in the
ampholyte solution tends to suppress convective forces (if
any) and electro-osmotic flow by increasing the viscosity.
Commercial ampholytes are available covering many pH
ranges and may be mixed if necessary to obtain an expanded
pH range. Broad pH ranges are used to estimate the isoelec-
tric point whereas narrower ranges are employed to improve
accuracy. Calibration can be done by correlating migration
time with isoelectric point for a series of protein markers.
During the focusing step precipitation of proteins at their
isoelectric point can be prevented, if necessary, using buffer
additives such as glycerol, surfactants, urea or zwitterionic
buffers. However, depending on the concentration, urea
denatures proteins.
4. Micellar Electrokinetic Chromatography (MEKC)
Principle
In micellar electrokinetic chromatography, separation
takes place in an electrolyte solution which contains a sur-
factant at a concentration above the critical micellar concen-
tration (cmc). The solute molecules are distributed between
the aqueous buffer and the pseudo-stationary phase com-
posed of micelles, according to the partition coefficient of the
solute. The technique can therefore be considered as a hybrid
of electrophoresis and chromatography. It is a technique that
can be used for the separation of both neutral and charged
solutes, maintaining the efficiency, speed and instrumental
suitability of capillary electrophoresis. One of the most wide-
ly used surfactants in MEKC is the anionic surfactant sodium
dodecyl sulfate, although other surfactants, for example
cationic surfactants such as cetyltrimethylammonium salts,
are also used.
The separation mechanism is as follows. At neutral and
alkaline pH, a strong electro-osmotic flow is generated and
moves the separation buffer ions in the direction of the
cathode. If sodium dodecyl sulfate is employed as the sur-
factant, the electrophoretic migration of the anionic micelle
is in the opposite direction, towards the anode. As a result,
the overall micelle migration velocity is slowed down com-
pared to the bulk flow of the electrolytic solution. In the case
of neutral solutes, since the analyte can partition between the
micelle and the aqueous buffer, and has no electrophoretic
mobility, the analyte migration velocity will depend only on
the partition coefficient between the micelle and the aqueous
buffer. In the electropherogram, the peaks corresponding to
each uncharged solute are always between that of the electro-
osmotic flow marker and that of the micelle (the time elapsed
between these two peaks is called the separation window).
For electrically charged solutes, the migration velocity de-
pends on both the partition coefficient of the solute between
the micelle and the aqueous buffer, and on the electrophoretic
mobility of the solute in the absence of micelle.
Since the mechanism in MEKC of neutral and weakly
ionized solutes is essentially chromatographic, migration of
the solute and resolution can be rationalized in terms of the
retention factor of the solute (k'), also referred to as mass
distribution ratio (D m ), which is the ratio of the number of
moles of solute in the micelle to those in the mobile phase.
For a neutral compound, k' is given by:
^R tn
a i
M11C
t R : migration time of the solute,
t : analysis time of an unretained solute (determined by in-
jecting an electro-osmotic flow marker which does not
enter the micelle, for instance methanol),
t mc : micelle migration time (measured by injecting a micelle
marker, such as Sudan III, which migrates while con-
tinuously associated in the micelle),
K: partition coefficient of the solute,
V s : volume of the micellar phase,
V M : volume of the mobile phase.
Likewise, the resolution between two closely-migrating
solutes (R s ) is given by:
Rs
a-1
a
1-
ta
k\+\
1 +
TV: number of theoretical plates for one of the solutes,
a: selectivity,
k\ and k\: retention factors for both solutes, respectively
(k\>k\).
Similar, but not identical, equations give k' and R s values
for electrically charged solutes.
Optimization
The main parameters to be considered in the development
of separations by MEKC are instrumental and electrolytic so-
lution parameters.
Instrumental parameters
Voltage: Separation time is inversely proportional to
applied voltage. However, an increase in voltage can cause
excessive heat production that gives rise to temperature
gradients and viscosity gradients of the buffer in the cross-
section of the capillary. This effect can be significant with
high conductivity buffers such as those containing micelles.
Poor heat dissipation causes band broadening and decreases
resolution.
Temperature: Variations in capillary temperature affect
the partition coefficient of the solute between the buffer and
the micelles, the critical micellar concentration and the
JPXV
General Information / Capillary Electrophoresis 1679
viscosity of the buffer. These parameters contribute to the
migration time of the solutes. The use of a good cooling sys-
tem improves the reproducibility of the migration time for
the solutes.
Capillary: As in free solution capillary electrophoresis,
the dimensions of the capillary (length and internal diameter)
contribute to analysis time and efficiency of separations. In-
creasing both effective length and total length can decrease
the electric fields (working at constant voltage), increase
migration time and improve the separation efficiency. The in-
ternal diameter controls heat dissipation (for a given buffer
and electric field) and consequently the sample band broaden-
ing.
Electrolytic solution parameters
Surfactant type and concentration: The type of sur-
factant, in the same way as the stationary phase in chro-
matography, affects the resolution since it modifies separa-
tion selectivity. Also, the log k' of a neutral compound
increases linearly with the concentration of surfactant in the
mobile phase. Since resolution in MEKC reaches a maximum
when k' approaches the value of *jt m /t Q , modifying the con-
centration of surfactant in the mobile phase changes the reso-
lution obtained.
Buffer pH: Although pH does not modify the partition
coefficient of non-ionized solutes, it can modify the electro-
osmotic flow in uncoated capillaries. A decrease in the buffer
pH decreases the electro-osmotic flow and therefore increases
the resolution of the neutral solutes in MEKC, resulting in a
longer analysis time.
Organic solvents: To improve MEKC separation of
hydrophobic compounds, organic modifiers (methanol,
propanol, acetonitrile, etc.) can be added to the electrolytic
solution. The addition of these modifiers usually decreases
migration time and the selectivity of the separation. Since the
addition of organic modifiers affects the critical micellar con-
centration, a given surfactant concentration can be used only
within a certain percentage of organic modifier before the
micellization is inhibited or adversely affected, resulting in
the absence of micelles and, therefore, in the absence of par-
tition. The dissociation of micelles in the presence of a high
content of organic solvent does not always mean that the
separation will no longer be possible; in some cases the
hydrophobic interaction between the ionic surfactant
monomer and the neutral solutes forms solvophobic
complexes that can be separated electrophoretically.
Additives for chiral separations: For the separation of
enantiomers using MEKC, a chiral selector is included in the
micellar system, either covalently bound to the surfactant or
added to the micellar separation electrolyte. Micelles that
have a moiety with chiral discrimination properties include
salts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiral
resolution can also be achieved using chiral discriminators,
such as cyclodextrins, added to the electrolytic solutions
which contain micellized achiral surfactants.
Other additives: Several strategies can be carried out to
modify selectivity, by adding chemicals to the buffer. The ad-
dition of several types of cyclodextrins to the buffer can also
be used to reduce the interaction of hydrophobic solutes with
the micelle, thus increasing the selectivity for this type of
compound.
The addition of substances able to modify solute-micelle
interactions by adsorption on the latter, is used to improve
the selectivity of the separations in MEKC. These additives
may be a second surfactant (ionic or non-ionic) which gives
rise to mixed micelles or metallic cations which dissolve in the
micelle and form co-ordination complexes with the solutes.
Quantification
Peak areas must be divided by the corresponding migration
time to give the corrected area in order to:
— compensate for the shift in migration time from run to
run, thus reducing the variation of the response,
— compensate for the different responses of sample
constituents with different migration times.
Where an internal standard is used, verify that no peak of
the substance to be examined is masked by that of the
internal standard.
Calculations
From the values obtained, calculate the content of the
component or components being examined. When
prescribed, the percentage content of one or more compo-
nents of the sample to be examined is calculated by determin-
ing the corrected area(s) of the peak(s) as a percentage of the
total of the corrected areas of all peaks, excluding those due
to solvents or any added reagents (normalization procedure).
The use of an automatic integration system (integrator or
data acquisition and processing system) is recommended.
System Suitability
In order to check the behavior of the capillary electropho-
resis system, system suitability parameters are used. The
choice of these parameters depends on the mode of capillary
electrophoresis used. They are: retention factor (k') (only for
micellar electrokinetic chromatography), apparent number
of theoretical plates (N), symmetry factor (A s ) and resolution
(i? s ). In previous sections, the theoretical expressions for N
and R s have been described, but more practical equations
that allow these parameters to be calculated from the elec-
tropherograms are given below.
Apparent Number of Theoretical Plates
The apparent number of theoretical plates (N) may be
calculated using the expression:
Ar =5 . 54 / J^ W
\ Wh
t R : migration time or distance along the baseline from the
point of injection to the perpendicular dropped from
the maximum of the peak corresponding to the compo-
nent,
vf h : width of the peak at half-height.
Resolution
The resolution (R s ) between peaks of similar height of two
components may be calculated using the expression:
Rs
1.18(r R2 -f R1 )
Whi + w h2
tR2> tRl
t K1 and t R2 : migration times or distances along the baseline
from the point of injection to the perpendicu-
lars dropped from the maxima of two adjacent
peaks,
w h i and w h 2: peak widths at half-height.
When appropriate, the resolution may be calculated by
measuring the height of the valley (H v ) between two partly
resolved peaks in a standard preparation and the height of
the smaller peak (H v ) and calculating the peak-to-valley ratio:
1680 Decision of Limit for Bacterial / General Information
JP XV
p/v-
Ik
Symmetry Factor
The symmetry factor (^4 S ) of a peak may be calculated
using the expression:
A s =
Wp.QS
Id
W0.05: width of the peak at one-twentieth of the peak
height,
d: distance between the perpendicular dropped from the
peak maximum and the leading edge of the peak at one-
twentieth of the peak height.
Tests for area repeatability (standard deviation of areas or
of the area/migration-time ratio) and for migration time
repeatability (standard deviation of migration time) are
introduced as suitability parameters. Migration time
repeatability provides a test for the suitability of the capillary
washing procedures. An alternative practice to avoid the lack
of repeatability of the migration time is to use migration time
relative to an internal standard.
A test for the verification of the signal-to-noise ratio for a
standard preparation (or the determination of the limit of
quantification) may also be useful for the determination of
related substances.
Signal-to-noise Ratio
The detection limit and quantification limit correspond to
signal-to-noise ratios of 3 and 10 respectively. The signal-to-
noise ratio (S/N) is calculated using the expression:
S/N =
2H
h
H: height of the peak corresponding to the component
concerned, in the electropherogram obtained with the
prescribed reference solution, measured from the maximum
of the peak to the extrapolated baseline of the signal observed
over a distance equal to twenty times the width at half-height,
h: range of the background in an electropherogram ob-
tained after injection of a blank, observed over a distance
equal to twenty times the width at the half-height of the peak
in the electropherogram obtained with the prescribed refer-
ence solution and, if possible, situated equally around the
place where this peak would be found.
5. Decision of Limit for Bacterial
Endotoxins
The endotoxin limit for injections is to be decided as fol-
lows:
Endotoxin limit =K/M
where K is a minimum pyrogenic dose of endotoxin per kg
body mass (EU/kg), and Mis equal to the maximum dose of
product per kg per hour.
Mis expressed in mL/kg for products to be administered
by volume, in mg/kg or mEq/kg for products to be
administered by mass, and in Unit/kg for products to be
administered by biological units. Depending on the adminis-
tration route, values for K are set as in the following table.
Intended route of administration
K (EU/kg)
Intravenous
5.0
Intravenous, for
radiopharmaceuticals
2.5
Intraspinal
0.2
Notes:
1) For products to be administered by mass or by units,
the endotoxin limit should be decided based on the labeled
amount of the principal drug.
2) Sixty kg should be used as the average body mass of an
adult when calculating the maximum adult dose per kg.
3) The pediatric dose per kg body mass should be used
when this is higher than the adult dose.
4) The K values for the intravenous route are applicable
to drugs to be administered by any route other than those
shown in the table.
6. Disinfection and Sterilization
Methods
Disinfection and Sterilization Methods are applied to kill
microorganisms in processing equipment /utensils and areas
used for drug manufacturing, as well as to perform microbio-
logical tests specified in the monographs, and so differ from
"Terminal Sterilization" and "Filtration Method" described
in "Terminal Sterilization and Sterilization Indicators". The
killing effect on microorganisms or the estimated level of
sterility assurance is greatly variable, so the conditions for
disinfection and sterilization treatment must be chosen
appropriately for each application. Generally, the following
methods are to be used singly or in combination after
appropriate optimization of operation procedures and condi-
tions, in accordance with the kind and the degree of the
contaminating microorganisms and the nature of the item to
which the methods are applied.
The validation of sterilization in accordance with Terminal
Sterilization and Sterilization Indicators is required when the
methods are applied to the manufacturing processes of drug
products.
1. Disinfection methods
These methods are used to reduce the number of living
microorganisms, but do not always remove or kill all
microorganisms present. Generally, disinfection is classified
into chemical disinfection with the use of chemical drugs (dis-
infectants) and physical disinfection with the use of moist
heat, ultraviolet light, and other agents.
1-1. Chemical disinfection
Microorganisms are killed with chemical drugs. The killing
effect and mechanisms of a chemical drug differ depending on
the type, applied concentration, action temperature, and
action time of the chemical drug used, the degree of contami-
nation on the object to be disinfected, and the species and
state (e.g., vegetative bacteria or spore bacteria) of microor-
ganisms.
JP XV
General Information / Disinfection and Sterilization 1681
Therefore, in applying the method, full consideration is
required of the sterility and permissible storage period of the
prepared chemical drug, the possibility of resistance of
microorganisms at the site of application, and the effect of
residual chemical drug on the product. In selecting a suitable
chemical drug, the following items should be considered in
relation to the intended use.
1) The antimicrobial spectrum
2) Action time for killing microorganisms
3) Action durability
4) Effect of the presence of proteins
5) Influence on the human body
6) Solubility in water
7) Influence on the object to be disinfected
8) Odor
9) Convenience of use
10) Easy disposability
11) Influence on the environment at disposal
12) Frequency of occurrence of resistance
1-2. Physical disinfection
Microorganisms are killed without a chemical drug.
(i) Steam flow method
Microorganisms are killed by direct application of steam.
This method is used for a product which may be denatured by
the moist heat method. As a rule, the product is kept in
flowing steam at 100°C for 30 - 60 minutes.
(ii) Boiling method
Microorganisms are killed by putting the object in boiling
water. This method is used for a product which may be
denatured by the moist heat method. As a rule, the product is
put in boiling water for 15 minutes or more.
(iii) Intermittent method
Microorganisms are killed by heating for 30 - 60 minutes
repeatedly, three to five times, once a day in water at 80 -
100°C or in steam. This method is used for a product which
may be denatured by the moist heat method. There is another
method called the low temperature intermittent method with
repeated heating at 60-80°C. During the intermission
periods between heating or warming, a suitable temperature
for the growth of microorganisms of 20°C or higher, must be
maintained.
(iv) Ultraviolet method
As a rule, microorganisms are killed by irradiation with
ultraviolet rays at a wavelength of around 254 nm. This
method is used for products which are resistant to ultraviolet
rays, such as smooth-surfaced articles, facilities, and equip-
ment, or water and air. This method does not suffer from the
occurrence of resistance, which is observed in chemical disin-
fection, and shows a killing effect on bacteria, fungi, and
viruses. It must be taken into consideration that direct ultrav-
iolet irradiation of the human body can injure the eyes and
skin.
2. Sterilization methods
2-1. Heating methods
In these methods, the heating time before the temperature
or pressure reaches the prescribed value differs according to
the properties of the product, the size of the container, and
the conditions. The duration of heating in conducting these
methods is counted from the time when all the parts of the
product have reached the prescribed temperature.
(i) Moist heat method
Microorganisms are killed in saturated steam at a suitable
temperature and pressure. This method is generally used for
heat-stable substances, such as glass, porcelain, metal, rub-
ber, plastics, paper, and fiber, as well as heat-stable liquids,
such as water, culture media, reagents, test solutions, liquid
samples, etc. As a rule, one of the following conditions is
used.
115- 118°C for 30 minutes
121 - 124°C for 15 minutes
126- 129°C for 10 minutes
(ii) Dry-heat method
Microorganisms are killed in dry-heated air. This method
is generally used for heat-stable substances, such as glass,
porcelain, and metal, as well as heat-stable products, such as
mineral oils, fats and oils, powder samples, etc. This method
is generally conducted in the way of direct heating by gas or
electricity or circulating heated air. As a rule, one of the fol-
lowing conditions is used.
160 - 170°C for 120 minutes
170-180°Cfor 60 minutes
180-190°Cfor 30 minutes
2-2. Irradiation methods
(i) Radiation method
Microorganisms are killed by gamma-rays emitted from a
radioisotope or electron beam and bremsstrahlung (X-ray)
generated from an electron accelerator. This method is gener-
ally used for radiation-resistant substances such as glass, por-
celain, metal, rubber, plastics, fiber, etc. The dose is decided
according to the material properties, and the degree of con-
tamination of the product to be sterilized. Special considera-
tion is necessary of the possibility of qualitative change of the
product after the application of the method.
(ii) Microwave method
Microorganisms are killed by the heat generated by direct
microwave irradiation. This method is generally used for
microwave-resistant products such as water, culture media,
test solutions, etc. As a rule, microwave radiation with a
wavelength of around 2450 ±50 MHz is used.
2-3. Gas methods
Microorganisms are killed by a sterilizing gas. Suitable
gases for killing microorganisms include ethylene oxide gas,
formaldehyde gas, hydrogen peroxide gas, chlorine dioxide
gas, etc. Temperature, humidity, the concentration of gas,
and the exposure time differ in accordance with the species of
gas used. As sterilizing gases are generally toxic to humans,
full consideration is required of the environmental control
for the use of gases and the concentration of residual gas. In
some of the gas methods, it may be difficult to measure or
estimate quantitatively the killing of microorganisms.
2-4. Filtration method
Microorganisms are removed by filtration with a suitable
filtering device. This method is generally used for gas, water,
or culture media and test solutions containing a substance
that is water-soluble and unstable to heat. As a rule, a filter
having a pore size of 0.22 /an or smaller is used for the sterili-
zation. However, in this method, a filter with a pore size of
0.45 /um or smaller is permitted to be used.
1682 Guideline for Residual Solvents, / General Information
JP XV
7. Guideline for Residual Solvents,
Residual Solvents Test, and Models
for the Test in Monographs
1. Guideline for Residual Solvents
Refer to the Guideline for Residual Solvents in Phar-
maceuticals (PAB/ELD Notification No. 307; dated March
30, 1998).
The acceptable limits of residual solvents recommended in
the Guideline were estimated to keep the safety of patients.
The levels of residual solvents in pharmaceuticals should not
exceed the limits, except for in a special case. Accordingly,
pharmaceutical manufacturers should assure the quality of
their products by performing the test with the products
according to the Residual Solvents Test, to keep the limits
recommended in the Guideline.
2. Residual Solvents Test
Method — Perform the test as directed in the Residual Sol-
vents Test under the General Tests, Processes and Apparatus.
International harmonization — The test may also be per-
formed according to the Residual solvents in the EP or the
Organic volatile impurities in the USP. Even in this case,
Monographs should be described in the JP style.
3. Models for the Test in Monographs
The following are typical examples for the test in Mono-
graphs, but these do not necessarily imply that other suitable
operating conditions can not be used. It is important to pre-
pare (draft) monographs according to the Guideline for the
Preparation of the Japanese Pharmacopoeia.
1) A model for test item, amounts of test sample and refer-
ence standard (reference substance), preparation of the sam-
ple solution and the standard solution, injection amount for
gas chromatography, calculation formula, and preparation
of the internal standard solution
Residual solvents (or name of the solvent) — Weigh ac-
curately about 0.200 g of AAA (name of the substance to be
tested), add exactly 5 mL of the internal standard solution to
dissolve, add water to make exactly 20 mL, and use this solu-
tion as the sample solution. If necessary filter or centrifuge.
Separately, weigh exactly 0.10 g of OOO reference sub-
stance (name of the solvent), put in a vessel containing 50 mL
of water, and add water to make exactly 100 mL. Pipet 5 mL
of this solution, and add water to make exactly 100 mL. Pipet
2 mL of this solution, add exactly 5 mL of the internal stan-
dard solution and 20 mL of water, and use this solution as the
standard solution. Perform the test with 1 /uL each of the
sample solution and standard solution as directed (in the
head-space method) under Gas Chromatography according
to the following conditions, and calculate the ratios, Qt and
Qs, of the peak area of AAA (name of the substance to be
tested) to that of the internal standard of each solution,
respectively. The amount of AAA should be not more than
x x ppm.
Amount of OOO (MS) q
= amount of OOO reference substance (jug) x -*p^
Lis
Internal standard solution — A solution of AAA in V VV
(name of solvent) (1 in 1000).
2) Models for operating conditions for a head-space sample
injection device
Operating conditions (1) for the head-space sample injection
device—
Equilibration temperature for
inside vial
Equilibration time for inside vial
Transfer-line temperature
Carrier gas
Pressurisation time
Injection volume of sample
Operating conditions (2) for the head-
device—
Equilibration temperature for
inside vial
Equilibration time for inside vial
Transfer-line temperature
Carrier gas
Pressurisation time
Injection volume of sample
Operating conditions (3) for the head-
device —
Equilibration temperature for
inside vial
Equilibration time for inside vial
Transfer-line temperature
Carrier gas
Pressurisation time
Injection volume of sample
A constant tempera-
ture of about 80°C
60 minutes
A constant tempera-
ture of about 85 °C
Nitrogen
30 seconds
l.OmL
■space sample injection
A constant tempera-
ture of about 105°C
45 minutes
A constant tempera-
ture of about 110°C
Nitrogen
30 seconds
l.OmL
■space sample injection
A constant tempera-
ture of about 80°C
45 minutes
A constant tempera-
ture of about 105°C
Nitrogen
30 seconds
l.OmL
3) Models for operating conditions and system suitability
In the operating conditions, generally, items required for
the test such as detector, column, column temperature, carri-
er gas, flow rate, and time span of measurement should be
specified, and in the system suitability, items such as test for
required detectability, system performance, and system
repeatability should be specified.
The following are several models for the operating condi-
tions and the system suitability:
Operating conditions —
Detector: Specify in the following manner: "Hydrogen
flame-ionization detector".
Column: Specify the inside diameter, length, material of
column, name of stationary phase liquid and thickness of sta-
tionary phase in the following manner: "Coat the inside wall
of a fused silica tube, 0.3 mm in inside diameter and 30 m in
length, to 0.25 /xm thickness with polyethylene glycol 20M."
Describe the inside diameter and length of the column, and
thickness or particle size of the stationary phase based on the
data obtained for validation of the test method.
Column temperature: Specify in the following manner: "A
constant temperature of about x °C." or "Maintain at 40°C
for 20 minutes, then increase to 240°C at 10°C per minute,
and keep at 240°C for 20 minutes."
Carrier gas: Specify in the following manner: "Helium".
Flow rate: Specify in the following manner: "Adjust the
flow rate so that the retention time of AAA is about x
minutes." or "35 cm/second". Describe the flow rate based
on the data obtained for validation of the test method.
Time span of measurement: About x times as long as the
JPXV
General Information / Guideline for Residual Solvents, 1683
retention time of AAA beginning after the air peak.
System suitability —
Test for required detectability: Specify in the following
manner: "Measure exactly x mL of the standard solution,
and add DDD to make exactly x mL. Confirm that the
peak area of DDD obtained from x /xL of this solution is
equivalent to x to X x % of that of DDD obtained from
the standard solution". This item should be specified when,
in Purity, the amount of an impurity is controlled by compar-
ing the area of a specific peak from the sample solution with
that of the peak of x x x x from the standard solution and
the system repeatability alone is not sufficient to check the
system suitability.
System performance: Specify in the following manner:
"Dissolve x g of DDD and x g of AAA in x mLofOO
O- When the procedure is run with x /xh of this solution un-
der the above operating conditions, DDD and AAA are
eluted in this order with the resolution between these peaks
being not less than x , and the number of theoretical plates
and the symmetry factor of the peak of DDD are not less
than x x steps and not more than x x x, respectively."
This item should be specified in all of the test methods.
Generally, the order of elution and the resolution, and in case
of need (such as when the peak is asymmetrical) the symmet-
ry factor, should be specified. The order of elution and the
resolution may be replaced by the resolution and the number
of theoretical plates. When no suitable reference substance is
available to check separation, the number of theoretical
plates and the symmetry factor of the peak of the substance
to be tested may be specified.
System repeatability: Specify in the following manner:
"When the test is repeated x times with x /uL of the solution
for the system suitability test under the above operating con-
ditions, the relative standard deviation of the peak areas of
AAA is not more than x%." The system repeatability
should be specified in any case, except in a qualitative test.
Examples of operating conditions and system suitability
Test conditions (1)
Operating conditions —
Detector: Hydrogen flame-ionization detector
Column: Coat the inside wall of a fused silica tube, 0.53
mm in inside diameter and 30 m in length, to 3 //m thickness
with 6% cyanopropylphenyl-methyl silicon polymer for gas
chromatography. Use a guard column if necessary.
Column temperature: Maintain at 40°C for 20 minutes,
then increase to 240°C at 10°C per minute if necessary, and
keep at 240 C C for 20 minutes.
Injection port temperature: A constant temperature of
about 140°C
Detector temperature: A constant temperature of about
250°C
Carrier gas: Helium
Flow rate: 35 cm/second
Split ratio: 1:5
System suitability —
System performance: When the procedure is run with the
standard solution under the above operating conditions, the
resolution between the peaks is not less than 1.0. (Note: In
the case that the number of substances to be tested is two or
more.)
System repeatability: When the test is repeated 3 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak areas of the sub-
stance to be tested is not more than 15%.
Test conditions (2)
Operating conditions —
Detector: Hydrogen flame-ionization detector
Column: Coat the inside wall of a fused silica tube, 0.53
mm in inside diameter and 30 m in length, to 5 /xm thickness
with 5% phenyl-methyl silicon polymer for gas chro-
matography. If necessary use a guard column prepared by
coating the inside wall of a fused silica tube, 0.53 mm in in-
side diameter and 5 m in length, to 5 /urn thickness with 5%
phenyl-methyl silicon polymer for gas chromatography.
Column temperature: Maintain at 35 °C for 5 minutes,
then increase to 175°C at 8°C per minute, further increase to
260°C at 35°C per minute if necessary, and keep at 260°C for
16 minutes.
Injection port temperature: A constant temperature of
about 70°C
Detector temperature: A constant temperature of about
260°C
Carrier gas: Helium
Flow rate: 35 cm/second
Split ratio: Splitless
System suitability —
System performance: When the procedure is run with the
standard solution under the above operating conditions, the
resolution between the peaks is not less than 1.0. (Note: In
the case that the number of substances to be tested is two or
more.)
System repeatability: When the test is repeated 3 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak areas of the sub-
stance to be tested is not more than 15%.
Test conditions (3)
Operating conditions —
Detector: Hydrogen flame-ionization detector
Column: Coat the inside wall of a fused silica tube, 0.32
mm in inside diameter and 30 m in length, to 0.25 //m thick-
ness with polyethylene glycol 20M for gas chromatography.
Use a guard column if necessary.
Column temperature: Maintain at 50°C for 20 minutes,
then increase to 165 °C at 6°C per minute if necessary, and
keep at 165°C for 20 minutes.
Injection port temperature: A constant temperature of
about 140°C
Detector temperature: A constant temperature of about
250°C
Carrier gas: Helium
Flow rate: 35 cm/second
Split ratio: 1:5
System suitability —
System performance: When the procedure is run with the
standard solution under the above operating conditions, the
resolution between the peaks is not less than 1.0. (Note: In
the case that the number of substances to be tested is two or
more.)
System repeatability: When the test is repeated 3 times with
the standard solution under the above operating conditions,
the relative standard deviation of the peak areas of the sub-
stance to be tested is not more than 15%.
1684 International Harmonization / General Information
JP XV
8. International Harmonization
Implemented in the Japanese
Pharmacopoeia Fifteenth Edition
Items for which harmonization has been agreed among the
European Pharmacopoeia, the United States Pharmacopeia
and the Japanese Pharmacopoeia are implemented in the
Japanese Pharmacopoeia Fifteenth Edition (JP 15). They are
shown in the tables below.
The column headed Harmonized items shows the har-
monized items written in the Pharmacopoeial Harmonization
Agreement Document, and the column headed JP 15 shows
the items as they appear in JP 15. In the Remarks column,
notes on any differences between JP 15 and the agreement are
shown as occasion demands.
The date on which the agreement has been signed is shown
on the top pf each table. In the case where the harmonized i-
tems have been revised, this is indicated in parenthesis.
Oct. 2004 (Rev. 2)
Harmonized items
Residue on Ignition/Sulphated Ash
Test
(Introduction)
Procedure
JP 15
2.44 Residue on Ignition Test
(Introduction)
Procedure
Remarks
JP's particular description: Explana-
tion of the description in mongraph,
etc.
Nov. 2003
Harmonized items
JP 15
Remarks
Specific Surface Area
(Introduction)
Multi-point measurement
Single-point measurement
Sample preparation
Outgassing
Adsorbate
Quantity of sample
Method 1: The dynamic flow method
Method 2: The volumetric method
Reference materilals
Figure 1 Schematic diagram of the
dynamic flow method apparatus
Figure 2 Schematic diagram of the
volumetric method apparatus
3.02 Specific Surface Area
(Introduction)
Multi-point measurement
Single-point measurement
Sample preparation
Method 1: The dynamic flow method
Method 2: The volumetric method
Reference materilals
Fig. 3.02-1 Schematic diagram of the
dynamic flow method apparatus
Fig. 3.02-2 Schematic diagram of the
volumetric method apparatus
JP's particular description: Explana-
tion on this test.
JPXV
June 2004
General Information / Isoelectric Focusing 1685
Harmonized items
JP 15
Remarks
(Introduction)
3.04 Particle Size Determination
(Introduction)
JP's particular description: Explana-
tion on this test.
Optical microscopy
Apparatus
Adjustment
Illumination
Visual characterization
Photographic characterization
Preparation of the mount
Crystallinity characterization
Limit test of particle size by
microscopy
Particle size characterization
Particle shape characterization
General observations
Figure 1 Commonly used measure-
ments of particle size
Figure 2 Commonly used descriptions
of particle shape
Analytical sieving
Principles of analytical sieving
Test sieves
Test specimen
Agitation methods
Endpoint determination
Sieving methods
1) Methanical agitation
dry sieving method
2) Air entrainment methods
air jet and sonic sifter sieving
Table 1 Size of standard sieve series in
range of interest
Interpretation
Method 1. Optical microscopy
Apparatus
Adjustment
Illumination
Visual characterization
Photographic characterization
Preparation of the mount
Crystallinity characterization
Limit test of particle size by
microscopy
Particle size characterization
Particle shape characterization
General observations
Fig. 3.04-1 Commonly used measure-
ments of particle size
Fig. 3.04-2 Commonly used descrip-
tions of particle shape
Method 2. Analytical sieving method
Principles of analytical sieving
Test sieves
Test specimen
Agitation methods
Endpoint determination
Sieving methods
Methanical agitation
Dry sieving method
Air entrainment methods
Air jet and sonic shifter sieving
Table 3.04-1 Size of standard sieve
series in range of interest
Interpretation
JP's particular description: Explana-
tion on this test.
JP's particular description: Explana-
tion on the method of particle size
measurement.
JP's particular description: Explana-
tion on this method.
JP's particular description: How to
treat the fine powder adherent on back
surface of the sieve.
1686 Isoelectric Focusing / General Information
Jan. 2000
JP XV
Harmonized items
JP 15
Remarks
Bacterial Endotoxins Test
(Introduction)
Apparatus
Preparation of standard endotoxin
stock solution
4.01 Bacterial Endotoxins Test
(Introduction)
Apparatus
Preparation of standard endotoxin
stock solution
Preparation of standard endotoxin so- Preparation of standard endotoxin so-
lution lution
Preparation of sample solutions
Determination of maximum valid di-
lution
Gel-clot technique
(1) Preparatory testing
(i) Test for confirmation of labeled
lysate sensitivity
(ii) Test for interfering factors
(2) Limit test
(i) Procedure
(ii) Interpretation
(3) Assay
(i) Procedure
(ii) Calculation and interpretation
Photometric techniques
(1) Turbidimetric techniques
(2) Chromogenic technique
(3) Preparatory testing
(i) Assurance of criteria for the
standard curve
(ii) Test for interfering factors
(4) Assay
(i) Procedure
(ii) Calculation
Preparation of sample solutions
Determination of maximum valid di-
lution
Gel-clot technique
(1) Preparatory testing
(i) Test for confirmation of labeled
lysate sensitivity
(ii) Test for interfering factors
(2) Limit test
(i) Procedure
(ii) Interpretation
(3) Assay
(i) Procedure
(ii) Calculation and interpretation
Photometric techniques
(1) Turbidimetric techniques
(2) Chromogenic technique
(3) Preparatory testing
(i) Assurance of criteria for the
standard curve
(ii) Test for interfering factors
(4) Assay
(i) Procedure
(ii) Calculation
JP's particular description: Endotoxin
10000 Reference Standard and En-
dotoxin 100 Reference Standard.
JP's particular description: Addition
of the preparation of sample solutions
for containers, and deletion of that for
medical devices.
JP's particular description: Addition
of the concentration unit of sample so-
lutions in the case where the endotoxin
limit per equivalent is specified.
JP's particular description: The test for
assurance of criteria for the standard
curve must be carried out for each lot
of lysate reagent.
JP's particular description:
Two conditions which the test must
meet are specified.
Explanation of the test method
where the interfering action is found.
Explanation of the usual methods
for eliminating the interference.
JP's particular description: Addition
of the alternative requirement to which
solution D must meet for valid test.
(hi) Interpretation
(iii) Interpretation
JPXV
General Information / Isoelectric Focusing 1687
Regents, test solutions
Amebocyte lysate
Lysate TS
Water for bacterial endotoxins test
(BET)
Deletion
Deletion
Deletion
Deletion
Oct. 2002
Harmonized items
Sterility
(Introduction)
Precautions against microbial con-
tamination
Culture media and incubation temper-
atures
Fluid thioglycollate medium
Soya-bean casein digest medium
Sterility
Growth promotion test of aerobes,
anaerobes and fungi
Validation test
Membrane filtration
Direct inoculation
Test for sterility of the product to be
examined
Membrane filtration
Aqueous solutions
Soluble solids
Oils and oily solutions
Ointments and creams
Direct inoculation of the culture medi-
um
Oily liquids
Ointments and creams
Catgut and other surgical sutures for
veterinary use
Observation and interpretation of
results
Application of the test to parenteral
preparations, ophthalmic and other
non-injectable preparations required
to comply with the test for sterility
Table 2.6.1.-1. Strains of the test
micro-organisms suitable for use in
the Growth Promotion Test and the
Validation Test
Table 2.6.1.-2. Minimum quantity
to be used for each medium
JP 15
4.06 Sterility Test
(Introduction)
Media and rinsing fluids
Fluid thioglycolate medium
Soybean-casein digest medium
Sterility of media
Growth promotion test
Validation test
Membrane filtration
Direct inoculation
Test for sterility of the products to be
examined
Membrane filtration
a) Liquid medicines
b) Solid medicines
c) Oils and oily solutions
d) Ointments and creams
Direct inoculation of the culture medi-
um
a) Oily liquids
b) Ointments and creams
Cultivation and observation, Observa-
tion and interpretation of results
Remarks
The same statement appears in the in-
troduction.
Note 1 - 5
Note 6
Note 7
The items not included in JP.
Note 8-9
A part of this is directed in the General
Test.
Table 4.06-1. Microorganisms for
growth promotion test and the vali-
dation test
Table 4.06-3. Minimum quantity to
be used for each medium
1688 Isoelectric Focusing / General Information
JP XV
Table 2.6.1.-3. Minimum number of
items to be tested
Table 4.06-2. Number of items to be
taken from the lot
Note 10-11
Note: 1
2
3
4
5
Non-pharmaceutical media: Not to be used.
Water content of agar: Not being specified.
Effective period of media: Unnecessary to be validated.
Effective period of media stored in hermetic containers: Usable for maximum one year.
Medium for sterility test of the products containing a mercurial preservative: Specified.
Periodic growth promotion test for a ready-made powder medium: Specified.
Amount of the rinsing fluid each time in the membrane-filtration method: 100 mL per filter.
Transferring amount from turbid medium to fresh medium: A suitable amount.
Requirements for the retesting in the case when the evidence of microbial growth is found: Specified.
The table of 'Number of items to be taken from the lot': Specified as a part of the General Test.
Number of large-volume products (more than 100 mL) to be taken from the lot: Maximum 10 containers.
Feb. 2004
Harmonized items
JP 15
Remarks
Uniformity of Dosage Units
(Introduction)
Content uniformity
Solid dosage forms
Liquid dosage forms
Calculation of acceptance value
Mass variation
Uncoated or film-coated tablets
Hard capsules
Soft capsules
Solid dosage forms other than tablets
and capsules
Liquid dosage forms
Calculation of acceptance value
Criteria
Solid and liquid dosage forms
Table 1
Application of content uniformity
(CU) and mas variation (MV) test for
dosage forms
Table 2
6.02 Uniformity of Dosage Units
(Introduction)
Content uniformity
Solid dosage forms
Liquid dosage forms
Calculation of acceptance value
Mass variation
Uncoated or film-coated tablets
Hard capsules
Soft capsules
Solid dosage forms other than tablets
and capsules
Liquid dosage forms
Calculation of acceptance value
Criteria
Solid and liquid dosage forms
Table 6.02-1
Application of content uniformity
(CU) and mas variation (MV) test for
dosage forms
Table 6.02-2
JP's particular description:
Additional explanation on Liquids.
Additional explanation for the part
not containing drug substance.
JP's particular description: Assuming
that the concentration of drug sub-
stance is uniform in each lot.
The phrase "in conditions of normal
use. If necessary, compute the equiva-
lent volume after determining the den-
sity." is deleted.
JP's particular description:
Addition of "(divided forms, lyophi-
lized forms)" and "(true solution)".
The phrases "at time of manufacture"
and "For purposes of this Phar-
macopoeia" are deleted
JP XV
June 2004 (Rev.l)
General Information / Isoelectric Focusing 1689
Harmonized items
JP 15
Test for Extractable Volume of Paren- 6.05 Test for Extractable Volume of
teral Preparations
(Introduction)
Single-dose containers
Multi-dose containers
Cartridges and prefilled syringes
Parenteral infusions
Parenteral Preparations
(Introduction)
(1) Single-dose containers
(2) Multi-dose containers
(3) Cartridges and prefilled syringes
(4) Parenteral infusions
Remarks
JP's particular description: Explana-
tion of this Test
June 2004 (Rev.l)
Harmonized items
Particulate Matter in Injectables
(Introduction)
Method 1.
Light obscuration particle count test
General precautions
Method
Evaluation
Method 2.
Microscopic particle count test
General precautions
Method
Evaluation
1 . Circular diameter graticule
JP 15
6.07 Insoluble Particulate Matter Test
for Injections
(Introduction)
Method 1.
Light obscuration particle count test
Apparatus
Calibration
Manual methods
Electronic methods
Automated methods
Sample volume accuracy
Sample flow rate
Sensor
Particle resolution
Particle counting accuracy
Threshold accuracy
Reagents
General precautions
Method
Evaluation
Method 2.
Microscopic particle count test
Apparatus
General precautions
Method
Evaluation
Fig. 6.07-1 Circular diameter graticule
Remarks
The phrase "other than gas bubbles" is
deleted.
JP's particular description: Description
of evaluation frequency.
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description
JP's particular description: "equal to
or more than 100 mL".
JP's particular description: "equal to
or more than 100 mL".
1690 Isoelectric Focusing / General Information
June 2004
JP XV
Harmonized items
Disintegration
Apparatus
Basket-rack assembly
Disk
Procedure
JP 15
6.09 Disintegration
Apparatus
Basket-rack assembly
Disk
Auxiliary tube
Procedure
(1) Immediate-release preparation
Remarks
JP's particular description: This test is
applied also to Granules and Pills.
JP's particular description: The ap-
paratus may be varied somewhat
provided the specifications.
JP's particular description
JP's particular description: Making use
of water also as the test fluid.
Setting each intervals of the immersion.
Making a definition of complete disin-
tegration.
Setting a procedure for Granules.
JP's particular description:
Setting a test for Granules and Pills.
Figure 1 Disintegration apparatus
(2) Enteric coated preparations
Fig. 6.09-1 Disintegration apparatus
Fig. 6.09-2 Auxiliary tube
JP's particular description:
Setting a test for enteric coated prepa-
rations.
JP's particular description.
June 2004
Harmonized items
Dissolution
Apparatus
Apparatus 1 (Basket apparatus)
Apparatus 2 (Paddle apparatus)
Apparatus 3 (Reciprocating cylinder)
Apparatus 4 (Flow-through cell)
Procedure
Apparatus 1 or 2
Immediate-release dosage forms
Procedure
Dissolution medium
Time
Extended-release dosage forms
Procedure
Dissolution medium
Time
JP 15
6.10 Dissolution Test
Apparatus
Apparatus for Basket Method
(Apparatus 1)
Apparatus for Paddle Method
(Apparatus 2)
Apparatus for Flow-Through Cell
Method (Apparatus 3)
Procedure
Basket Method or Paddle Method
Immediate-release dosage forms
Procedure
Dissolution medium
Time
Extended-release dosage forms
Procedure
Dissolution medium
Time
Remarks
JP's particular description: The test
also aims at preventing significant
bioinequivalence.
JP's particular description: The sinker
is allowed to use in case only when spe-
cified in the monograph.
JP's particular description: A pump
without the pulsation may also be used.
JPXV
General Information / Isoelectric Focusing 1691
Delayed-release dosage forms
Procedure
Method A
Method B
Time
Apparatus 3
Immediate-release dosage forms
Procedure
Dissolution medium
Time
Extended-release dosage forms
Procedure
Dissolution medium
Time
Delayed-release dosage forms
Procedure
Time
Apparatus 4
Immediate-release dosage forms
Procedure
Delayed-release dosage forms
Procedure
Dissolution medium
Time
not specified
Flow-Through Cell Method
Immediate-release dosage forms
Procedure
In the Harmonized text: Alternative
usage of Method A or B.
JP's particular description.
JP's particular description: Time is
specified each for the 1st and 2nd
fluids.
Dissolution medium
Time
Extended-release dosage forms
Procedure
Dissolution medium
Time
Delayed-release dosage forms
Procedure
Time
Interpretation
Immediate-release dosage forms
Extended-release dosage forms
Delayed-release dosage forms
Dissolution medium
Time
Extended-release dosage forms
Procedure
Dissolution medium
Time
Interpretation
Immediate-release dosage forms
Interpretation 1
Interpretation 2
Extended-release dosage forms
Interpretation 1
Interpretation 2
Delayed-release dosage forms
JP's particular description: Follow In-
terpretation 1 when the value Q is spe-
cified in the individual monograph,
otherwise follow Interpretation 2.
JP's particular description:
Setting Interpretation 2.
JP's particular description:
Setting Interpretation 2.
Non-harmonized items: Different dis-
solution medium. Deletion of dishar-
monized part on the value Q.
1692 Isoelectric Focusing / General Information
JP XV
Acceptance Table 1
Acceptance Table 2
Acceptance Table 3
Acceptance Table 4
Figure 1 Apparatus 1. Basket stirring
element
Figure 2 Paddle stirring element
Figure 2 a Alternative sinker
Figure 3 Apparatus
Figure 4 Apparatus 4
(top) large cell tablets and capsules
(bottom) tablet holder for the large
cell
Figure 5 Apparatus 4
(top) small cell tablets and capsules
(bottom) tablet holder for the small
cell
Interpretation 1
Interpretation 2
Acceptance Table 6.10-1
Acceptance Table 6.10-2
Acceptance Table 6.10-3
Acceptance Table 6.10-4
Fig. 6.10-1 Apparatus 1. Basket stir-
ring element
Fig. 6.10-2 Paddle stirring element
Fig. 6.10-2 a Alternative sinker
not specified
Fig. 6.10-3 Apparatus 3
(top) large cell tablets and capsules
(bottom) tablet holder for the large
cell
Fig. 6.10-4 Apparatus 3
(top) small cell tablets and capsules
(bottom) tablet holder for the small
cell
JP's particular description: Setting In-
terpretation 2.
The value Q is specified in the
individual monograph.
Sep. 2002 (Rev. 1)
Harmonized items
JP 15
Remarks
Ethanol
Identification A
Identification B
Relative density
Appearance
Acidity or alkalinity
Volatile impurities
Absorbance
Residue on
evaporation
Storage
Ethanol
not specified as Identification
Identification
Specific gravity
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid or alkali
Purity (3) Volatile impurities
Purity (4) Other impurities
Purity (5) Residue on evaporation
Containers and storage
Setting Specific gravity as specification.
Setting Specific gravity at 15°C.
Sep. 2002 (Rev. 1)
Harmonized items
JP 15
Remarks
Ethanol, Anhydrous
Definition
Anhydrous Ethanol
limits of content
Identification A
Identification B
Relative density
not specified as Identification
Identification
Specific gravity
Setting Specific gravity as specification.
Setting Specific gravity at 15°C.
JPXV
General Information / Isoelectric Focusing 1693
Appearance
Acidity or alkalinity
Volatile impurities
Absorbance
Residue on
evaporation
Storage
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid or alkali
Purity (3) Volatile impurities
Purity (4) Other impurities
Purity (5) Residue on evaporation
Containers and storage
Nov. 2003 (Rev. 2)
Harmonized items
Sodium Chloride
Definition
Identification A
Identification B
Acidity or alkalinity
Sulphates
Phosphates
Bromides
Iodides
Ferrocyanides
Iron
Barium
Magnesium and alkaline-earth metals
Aluminium
Nitrites
Potassium
Loss on drying
Assay
JP 15
Sodium Chloride
limits of the content
Identification (1)
Identification (2)
Purity (2) Acidity or alkalinity
Purity (3) Sulfates
Purity (4) Phosphates
Purity (5) Bromides
Purity (6) Iodides
Purity (7) Ferrocyanides
Purity (9) Iron
Purity (10) Barium
Purity (11) Magnesium and alkaline-
earth materials
not specified
not specified
not specified
Loss on drying
Assay
Remarks
July 2003 (Rev. 1)
Harmonized items
Carboxymethylcellulose Calcium
Definition
Identification A
Identification B
Identification C
Identification D
Alkalinity
Loss on drying
Residue on ignition
Limit of chloride
Limit of sulfate
JP 15
Carmellose Calcium
origin
Identification (1)
Identification (2)
Identification (3)
Identification (4)
Purity (1) Alkali
Loss on drying
Residue on ignition
Purity (2) Chloride
Purity (3) Sulfate
Remarks
1694 Isoelectric Focusing / General Information
JP XV
Nov. 2003 (Rev. 1)
Harmonized items
JP 15
Remarks
Citric Acid, Anhydrous
Definition
Appearance of solution
Sulphates
Oxalic acid
Readily carbonisable substances
Aluminium
Water
Sulphated ash
Assay
Anhydrous Citric Acid
limits of the content
Purity (1) Clarity and color of solu-
tion
Purity (2) Sulfates
Purity (3) Oxalate
Purity (5) Readily carbonizable sub-
stances
not specified
Water
Residue on ignition
Assay
Nov. 2003 (Rev. 1)
Harmonized items
Citric Acid Monohydrate
Definition
Appearance of solution
Sulphates
Oxalic acid
Readily carbonisable substances
Aluminium
Water
Sulphated ash
Assay
JP 15
Citric Acid Hydrate
limits of the content
Purity (1) Clarity and color of solu-
tion
Purity (2) Sulfates
Purity (3) Oxalate
Purity (5) Readily carbonizable sub-
stances
not specified
Water
Residue on ignition
Assay
Remarks
Oct. 2001
Harmonized items
JP 15
Remarks
Croscarmellose Sodium
Croscarmellose Sodium
Identification A
B
C
PH
Settling volume
Degree of substitution
Loss on drying
Loss on drying
Packaging and storage
Definition
origin
Identification (1)
Identification (2)
Identification (3)
pH
Precipitation test
Degree of substitution
Residue on ignition
Residue on ignition
Containers and storate
JPXV
General Information / Isoelectric Focusing 1695
Oct. 2001
Harmonized items
JP 15
Remarks
Wheat Starch
Definition
Identification A
Identification B
Identification C
pH
Iron
Total protein
Oxidising substances
Sulphur dioxide
Loss on drying
Sulphated ash
Wheat Starch
origin
Identification (1)
Identification (2)
Identification (3)
pH
Purity (1) Iron
not specified
Purity (2) Oxidizing substances
Purity (3) Sulfur dioxide
Loss on drying
Residue on ignition
Feb. 2003
Harmonized items
JP 15
Remarks
Saccharin
Definition
Limit of benzoate and salicylate
Readily carbonisable substances
Loss on drying
Residue on ignition
Assay
Packaging and storage
Saccharin
limits of content
Purity (3) Benzoate and salicylate
Purity (5) Readily carbonizable sub-
stances
Loss on drying
Residue on ignition
Assay
Containers and storage
Feb. 2004 (Rev. 1)
Harmonized items
Saccharin Sodium
Definition
Identification A, B
Acidity or alkalinity
Limit of benzoate and salicylate
Readily carbonisable substances
Water
Assay
JP 15
Saccharin Sodium Hydrate
limits of content
Identification (2)
Purity (2) Acid or alkali
Purity (4) Benzoate and salicylate
Purity (6) Readily carbonizable sub-
stances
Water
Assay
Remarks
1696 Isoelectric Focusing / General Information
JP XV
Oct. 2001
Harmonized items
JP 15
Remarks
Cellacefate
Definition
Identification
Viscosity
Limit of free acid
Water
Residue on ignition
Phthalyl content
Content of acetyl
Packaging and storage
Cellulose Acetate Phthalate
content of the acetyl and carboxyben-
zoyl groups
Identification
Viscosity
Purity (2) Free acids
Water
Residue on ignition
Assay (1) Carboxybenzoyl group
Assay (2) Acetyl group
Containers and storage
May 2005 (Rev. 1)
Harmonized items
Microcrystalline Cellulose
Definition
Identification A
Identification B
pH
Water-soluble substances
Ether-soluble substances
Conductivity
Loss on drying
Residue on ignition
Bulk density
JP 15
Microcrystalline Cellulose
origin
Identification (1)
Identification (3)
pH
Purity (2) Water- soluble substances
Purity(3) Diethyl ether-soluble sub-
stances
Conductivity
Loss on drying
Residue on ignition
Bulk density
Remarks
May 2005 (Rev. 1)
Harmonized items
Powdered Cellulose
Definition
Identification A
Identification B
pH
Water-soluble substances
Ether-soluble substances
Loss on drying
Residue on ignition
JP 15
Powdered Cellulose
origin
Identification (1)
Identification (3)
pH
Purity (2) Water-soluble substances
Purity (3) Diethyl ether- soluble sub-
stances
Loss on drying
Residue on ignition
Remarks
JPXV
General Information / Isoelectric Focusing 1697
Feb. 2004 (Rev. 1)
Harmonized items
JP 15
Remarks
Corn Starch
Definition
Identification A
Identification B
Identification C
pH
Loss on drying
Residue on ignition
Limit of iron
Limit of oxidizing substances
Sulfur dioxide determination
Corn Starch
origin
Identification (1)
Identification (2)
Identification (3)
pH
Loss on drying
Residue on ignition
Purity (1) Iron
Purity (2) Oxidizing substances
Purity (3) Sulfur dioxide
Feb. 2003 (Rev. 1)
Harmonized items
JP 15
Remarks
Anhydrous Lactose
Definition
Labeling
Identification
Specific rotation
Clarity and color of solution
Acidity or alkalinity
Protein and light-absorbing impurities
Loss on drying
Water
Residue on ignition
Content of alpha and beta anomers
Anhydrous Lactose
origin
labeling of relative quantities of a and
fS lactose
Identification
Optical rotation
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid and alkali
Purity (4) Proteins and light absorb-
ing substances
Loss on drying
Water
Residue on ignition
Isomer ratio
Sep. 2002
Harmonized items
Lactose Monohydrate
Definition
Identification
Specific rotation
Clarity and color of solution
Acidity or alkalinity
Protein and light-absorbing impurities
Water
Residue on ignition
JP 15
Lactose Hydrate
origin
Identification
Specific rotation
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid and alkali
Purity (4) Protein and light absorbing
substances
Water
Residue on ignition
Remarks
1698 Isoelectric Focusing / General Information
Feb. 2004
JP XV
Harmonized items
Ethyl Parahydroxybenzoate
Definition
Identification A
Appearance of solution
Acidity
Related substances
Sulphated ash
Assay
JP 15
Ethyl Parahydroxybenzoate
limits of content
Identification (1)
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid
Purity (4) Related substances
Residue on ignition
Assay
Remarks
System suitability is not specified.
Feb. 2004
Harmonized items
JP 15
Remarks
Butyl Parahydroxybenzoate
Definition
Identification A
Appearance of solution
Acidity
Related substances
Sulphated ash
Assay
Butyl Parahydroxybenzoate
limits of content
Identification
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid
Purity (4) Related substances
Residue on ignition
Assay
System suitability is not specified.
Feb. 2004
Harmonized items
Propyl Parahydroxybenzoate
Definition
Identification A
Appearance of solution
Acidity
Related substances
Sulphated ash
Assay
JP 15
Propyl Parahydroxybenzoate
limits of content
Identification (1)
Purity (1) Clarity and color of solu-
tion
Purity (2) Acid
Purity (4) Related substances
Residue on ignition
Assay
Remarks
System suitability is not specified.
Feb. 2004
Harmonized items
Methyl Parahydroxybenzoate
Definition
Identification A
Appearance of solution
JP 15
Methyl Parahydroxybenzoate
limits of content
Identification (1)
Purity (1) Clarity and color of solu-
tion
Remarks
JPXV
General Information / Isoelectric Focusing 1699
Acidity
Related substances
Sulphated ash
Assay
Purity (2) Acid
Purity (4) Related substances
Residue on ignition
Assay
System suitability is not specified.
Oct. 2001
Harmonized items
Potato Starch
Definition
Identification A
Identification B
Identification C
pH
Iron
Oxidising substances
Sulphur dioxide
Loss on drying
Sulphated ash
JP 15
Potato Starch
origin
Identification (1)
Identification (2)
Identification (3)
pH
Purity (1) Iron
Purity (2) Oxidizing substances
Purity (3) Sulfur dioxide
Loss on drying
Residue on ignition
Remarks
Nov. 2003
Harmonized items
Hydroxypropyl Methylcellulose
Definition
Labeling
Identification (1)
Identification (2)
Identification (3)
Identification (4)
Identification (5)
Viscosity
Method 1
Method 2
pH
Heavy metals
Loss on drying
Residue on ignition
Assay
JP 15
Hypromellose
limits of content of methoxy group
and hydroxypropoxy group
labeling of viscosity
Identification (1)
Identification (2)
Identification (3)
Identification (4)
Identification (5)
Viscosity
Method I
Method II
pH
Purity Heavy metals
Loss on drying
Residue on ignition
Assay
Remarks
1700 Isoelectric Focusing / General Information
JP XV
July, 2000
Harmonized items
JP 15
Remarks
Benzyl Alcohol
Definition
Identification
Appearance of solution
Refractive index
Acidity
Benzaldehyde and other related
substances
Peroxide value
Residue on evaporation
Assay
Benzyl Alcohol
limits of the content
Identification
Purity (1) Clarity and color of solu-
tion
Refractive index
Purity (2) Acid
Purity (3) Benzaldehyde and other
related substances
Purity (4) Peroxide value
Purity (5) Residue on evaporation
Assay
Nov. 2003
Harmonized items
Methylcellulose
Definition
Labeling
Identification (1)
Identification (2)
Identification (3)
Identification (4)
Identification (5)
Viscosity
Method 1
Method 2
PH
Heavy metals
Loss on drying
Residue on ignition
Assay
JP 15
Methylcellulose
limits of content of methoxy group
labeling of viscosity
Identification (1)
Identification (2)
Identification (3)
Identification (4)
Identification (5)
Viscosity
Method I
Method II
PH
Purity Heavy metals
Loss on drying
Residue on ignition
Assay
Remarks
Sep. 2002
Harmonized items
Amino Acid Analysis
Apparatus
General Precautions
Reference Standard Material
Calibration of Instrumentation
Repeatability
Sample Preparation
Internal Standards
JP 15
General Information
Amino Acid Analysis
Apparatus
General Precautions
Reference Standard Material
Calibration of Instrumentation
Repeatability
Sample Preparation
Internal Standards
Remarks
JPXV
General Information / Isoelectric Focusing 1701
Protein Hydrolysis
Method 1
Hydrolysis Solution
Procedure
Method 2
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 3
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 4
Oxidation Solution
Procedure
Method 5
Hydrolysis Solution
Liquid Phase Hydrolysis
Method 6
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 7
Reducing Solution
Procedure
Method 8
Stock Solutions
Reducing Solution
Procedure
Method 9
Stock Solutions
Carboxymethylation Solution
Buffer Solution
Procedure
Method 10
Reducing Solution
Procedure
Method 11
Reducing Solutions
Procedure
Methodologies of amino acid analysis
general principles
Method 1-Postcolumn ninhydrin
detection general principle
Method 2-Postcolumn OPA fluoro-
metric detection general principle
Method 3-Precolumn PITC derivati-
zation general principle
Method 4-Precolumn AQC derivati-
zation general principle
Protein Hydrolysis
Method 1
Hydrolysis Solution
Procedure
Method 2
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 3
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 4
Oxidation Solution
Procedure
Method 5
Hydrolysis Solution
Liquid Phase Hydrolysis
Method 6
Hydrolysis Solution
Vapor Phase Hydrolysis
Method 7
Reducing Solution
Procedure
Method 8
Stock Solutions
Reducing Solution
Procedure
Method 9
Stock Solutions
Carboxymethylation Solution
Buffer Solution
Procedure
Method 10
Reducing Solution
Procedure
Method 11
Reducing Solutions
Procedure
Methodologies of Amino Acid Analy-
sis General Principles
Method 1-Postcolumn Ninhydrin
Detection General Principle
Method 2-Postcolumn OPA Fluoro-
metric Detection General Principle
Method 3-Precolumn PITC Derivati-
zation General Principle
Method 4-Precolumn AQC Dereali-
zation General Principle
1702 Isoelectric Focusing / General Information
JP XV
Method 5-Precolumn OPA derivati-
zation general principle
Method 6-Precolumn DABS-C1
derivatization general principle
Method 7-Precolumn FMOC-C1
derivatization general principle
Method 8-Precolumn NBD-F
derivatization general principle
Data calculation and analysis
Calculations
Amino acid mole percent
Unknown protein samples
Known protein samples
Method 5-Precolumn OPA Derivati-
zation General Principle
Method 6-Precolumn DABS-C1
Derivatization General Principle
Method 7-Precolumn FMOC-C1
Derivatization General Principle
Method 8-Precolumn NBD-F
Derivatization General Principle
Data Calculation and Analysis
Calculations
Amino Acid Mole Percent
Unknown Protein Samples
Known Protein Samples
Oct. 1999
Harmonized items
Sodium Dodecyl Sulphate Poly-
acrylamide Gel Electrophoresis
(SDS-PAGE)
Characteristics of polyacrylamide gels
Denaturing polyacrylamide gel elec-
trophoresis
Reducing conditions
Non-reducing conditions
Characteristics of discontinuous
buffer system gel electrophoresis
Preparing vertical discontinuous
buffer SDS polyacrylamide gels
Assembling of the gel moulding cas-
sette
Preparation of the gel
Mounting the gel in the electrophore-
sis apparatus and electrophoretic
separation
Detection of protein in gels
Coomassie staining
Silver staining
Drying of stained SDS polyacrylamide
gels
Molecular-mass determination
Validation of the test
Quantification of impurities
Reagents, test solutions
Blocking solution
Coomassie staining solution
Destaining solution
Developer solution
JP 15
General Information
SDS-Polyacrylamide Gel Electrophore-
1. Characteristics of Polyacrylamide
Gels
2. Polyacrylamide Gel Electrophore-
sis under Denaturing Conditions
1) Reducing conditions
2) Non-reducing conditions
3. Characteristics of Discontinuous
Buffer System Gel Electrophoresis
4. Preparing Vertical Discontinuous
Buffer SDS-Polyacrylamide Gels
1) Assembling of the gel moulding
cassette
2) Preparation of the gel
3) Mounting the gel in the electro-
phoresis apparatus and electro-
phoretic separation
5. Detection of Proteins in Gels
1) Coomassie staining
2) Silver staining
6. Drying of Stained SDS-
Polyacrylamide Gels
7. Molecular-Mass Determination
8. Suitability of the Test
9. Quantification of Impurities
Test Solutions
Blocking TS
Coomassie staining TS
Destaining TS
Developer TS
Remarks
JPXV
General Information / Isoelectric Focusing 1703
Fixing solution
Silver nitrate reagent
Trichloroacetic acid reagent
Table 1 - Preparation of resolving
gel
Table 2 - Preparation of stacking gel
Fixing TS
Silver nitrate TS for silver staining
Trichloroacetic acid TS for fixing
Table 1. Preparation of resolving gel
Table 2. Preparation of stacking gel
Sep. 2002
Harmonized items
Capillary Electrophoresis
Apparatus
Capillary zone electrophoresis
Optimisation
Instrumental parameters
Voltage
Polarity
Temperature
Capillary
Electrolytic solution parameters
Buffer type and concentration
Buffer pH
Organic solvents
Additives for chiral separations
Capillary gel electrophoresis
Characteristics of gels
Capillary isoelectric focusing
Loading step
loading in one step
sequential loading
Focusing step
Mobilisation step
Optimisation
Voltage
Capillary
Solutions
Micellar electrokinetic chro-
matography (MEKC)
Optimisation
Instrumental parameters
Voltage
Temperature
Capillary
Electrolytic solution parameters
Surfactant type and concentration
Buffer pH
JP 15
General Information
Capillary Electrophoresis
Apparatus
1 . Capillary Zone Electrophoresis
Optimization
Instrumental parameters
Voltage
Polarity
Temperature
Capillary
Electrolytic solution parameters
Buffer type and concentration
Buffer pH
Organic solvents
Additives for chiral separations
2. Capillary Gel Electrophoresis
Characteristics of Gels
3. Capillary Isoelectric Focusing
Loading step
loading in one step
sequential loading
Focusing step
Mobilization step
Optimization
Voltage
Capillary
Solutions
4. Micellar Electrokinetic Chro-
matography (MEKC)
Optimization
Instrumental parameters
Voltage
Temperature
Capillary
Electrolytic solution parameters
Surfactant type and concentration
Buffer pH
Remarks
1704 Isoelectric Focusing / General Information
JP XV
Organic solvents
Additives for chiral separations
Other additives
Quantification
Calculations
System Suitability
Apparent number of theoretical
plates
Resolution
Symmetry factor
Signal-to-noise ratio
Organic solvents
Additives for chiral separations
Other additives
Quantification
Calculations
System Suitability
Apparent Number of Theoretical
Plates
Resolution
Symmetry Factor
Signal-to-noise Ratio
Feb. 2004
Harmonized items
Tablet Friability
JP 15
General Information
Tablet Friability Test
Remarks
Sep. 2002
Harmonized items
Total Protein Assay
Method 1
Standard Solution
Test Solution
Procedure
Light-scattering
Calculations
Method 2
Standard solutions
Test solution
Blank
Reagents and solutions
Copper sulfate reagent
SDS Solution
Sodium Hydroxide Solution
Alkaline copper reagent
Diluted Folin-Ciocalteu's Phenol
Ragent
Procedure
Calculations
Interfering substances
Sodium deoxycholate reagent
Trichloroacetic acid reagent
Procedure
Method 3
Standard solutions
JP 15
General Information
Total Protein Assay
Method 1
Standard Solution
Test Solution
Procedure
Light-Scattering
Calculations
Method 2
Standard Solutions
Test Solution
Blank
Reagents and Solutions
Copper Sulfate Reagent
5% SDS TS
Sodium Hydroxide Solution
Alkaline Copper Reagent
Diluted Folin's TS
Procedure
Calculations
Interfering Substances
Sodium Deoxycholate Reagent
Trichloroacetic Acid Reagent
Procedure
Method 3
Standard Solutions
Remarks
Explanatory footnote "Example: the
Minimum Requirements for Biological
Products and individual monograph of
JP" is added.
JPXV
General Information / Isoelectric Focusing 1705
Test solution
Blank
Coomassie reagent
Procedure
Calculations
Method 4
Standard solutions
Test solution
Blank
Reagents
BCA Reagent
Copper sulfate reagent
Copper-BCA reagent
Procedure
Calculations
Method 5
Standard solutions
Test solution
Blank
Biuret reagent
Procedure
Calculations
Interfering substances
Comments
Method 6
Standard solutions
Test solution
Blank
Reagents
Borate buffer
Stock OPA reagent
OPA reagent
Procedure
Calculations
Method 7
Procedure A
Procedure B
Calculations
Test Solution
Blank
Coomassie Reagent
Procedure
Calculations
Method 4
Standard Solutions
Test Solution
Blank
Reagents and Solutions
BCA Reagent
Copper Sulfate Reagent
Copper-BCA Reagent
Procedure
Calculations
Method 5
Standard Solutions
Test Solution
Blank
Biuret Reagent
Procedure
Calculations
Interfering Substances
Comments
Method 6
Standard Solutions
Test Solution
Blank
Reagents and Solutions
Borate Buffer
Stock OPA Reagent
OPA Reagent
Procedure
Calculations
Method 7
Procedure A
Procedure B
Calculations
Sep. 2002
Harmonized items
Isoelectric Focusing
Theoretical aspects
Practical aspects
JP 15
General Information
Isoelectric Focusing
Theoretical Aspects
Practical Aspects
Remarks
1706 Isoelectric Focusing / General Information
JP XV
Apparatus
Isoelectric Focusing in polyacrylamide
gels: detailed procedure
Preparation of the gels
1) 7.5 per cent polyacrylamide gel
2) Preparation of the mould
Method
Variations to the detailed procedure
(subject to validation)
Validation of iso-electric focusing
procedures
Specified variation to the general
method
Point to consider
Figure-Mould
Reagents
Fixing solution for isoelectric focus-
ing in polyacrylamide gel
Apparatus
Isoelectric Focusing in Poly-
acrylamide Gels: Detailed Procedure
Preparation of the Gels
7.5% Polyacrylamide gel
Preparation of the mould
Method
Variations to the Detailed Procedure
(Subject to Validation)
Validation of Iso-Electric Focusing
Procedures
Specified Variations to the General
Method
Points to Consider
Figure. Mould
Reagents and Solutions
Fixing solution for isoelectric focus-
ing in polyacrylamide gel
Coomassie staining TS
Destaining solution
Coomassie staining TS and destaining
solution are specified.
June 2004
Harmonized items
JP 15
Remarks
Powder Flow
Angel of repose
Basic methods for angel of repose
Variations in angel of repose
methods
Angel of repose general scale of
flowability
Experimental considerations for an-
gle of repose
Recommended procedure for angle
of repose
Compressibility index and Hausner
ratio
Basic methods for compressibility in-
dex and Hausner ratio
Experimental considerations for the
compressibility index and Hausner
ratio
Recommended procedure for com-
pressibility index and Hausner ra-
tio
Flow through an orifice
Basic methods for flow through an
orifice
General Information
Powder Flow
1 . Angel of repose
1.1 Basic methods for angel of re-
pose
1.2 Variations in angel of repose
methods
1.3 Angel of repose general scale of
flowability
1.4 Experimental considerations for
angle of repose
1.5 Recommended procedure for an-
gle of repose
2. Compressibility index and Hausner
ratio
2.1 Basic methods for compressibili-
ty index and Hausner ratio
2.2 Experimental considerations for
the compressibility index and
Hausner ratio
2.3 Recommended procedure for
compressibility index and Hausner
ratio
3. Flow through an orifice
3.1 Basic methods for flow through
an orifice
JPXV
General Information / Isoelectric Focusing 1707
Variations in methods for flow
through an orifice
General scale of flowability for flow
through an orifice
Experimental considerations for
flow through an orifice
Recommended procedure for flow
through an orifice
Shear cell methods
Basic methods for shear cell
Recommendation for shear cell
Table 1 Flow properties and cor-
responding angle repose
Table 2 Scale of flowability
3.2 Variations in methods for flow
through an orifice
3.3 General scale of flowability for
flow through an orifice
3.4 Experimental considerations for
flow through an orifice
3.5 Recommended procedure for
flow through an orifice
4. Shear cell methods
4.1 Basic methods for shear cell
4.2 Recommendation for shear cell
Table 1 Flow properties and cor-
responding angle repose
Table 2 Scale of flowability
Sep. 2002
Harmonized items
JP 15
Remarks
Peptide Mapping
Purpose and scope
The peptide map
Isolation and purification
Selective cleavage of peptide bonds
Pretreatment of sample
Pretreatment of the cleavage agent
Pretreatment of the protein
Establishment of optimal digestion
conditions
PH
Temperature
Time
Amount of cleavage agent
Chromatographic separation
Chromatographic column
solvent
Mobile phase
Gradient selection
Isocratic selection
Other parameters
Validation
Analysis and identification of peptides
Table 1. Examples of cleavage agents.
Table 2. Techniques used for the sepa-
ration of peptides.
General Information
Peptide Mapping
Purpose and Scope
The Peptide Map
Isolation and Purification
Selective Cleavage of Peptide Bonds
Pretreatment of Sample
Pretreatment of the Cleavage Agent
Pretreatment of the Protein
Establishment of Optimal Digestion
Conditions
pH
Temperature
Time
Amount of Cleavage Agent
Chromatographic Separation
Chromatographic Column
Solvent
Mobile Phase
Gradient Selection
Isocratic Selection
Other Parameters
Validation
Analysis and Identification of Pep-
tides
Table 1 . Examples of Cleavage Agents
Table 2. Techniques Used for the
Separation of Peptides
1708 Isoelectric Focusing / General Information
JP XV
9. Isoelectric Focusing
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia. The parts of the text that are not
harmonized are marked with symbols (* ♦).
General Principles
Isoelectric focusing (IEF) is a method of electrophoresis
that separates proteins according to their isoelectric point.
Separation is carried out in a slab of polyacrylamide or
agarose gel that contains a mixture of amphoteric electrolytes
(ampholytes). When subjected to an electric field, the ampho-
lytes migrate in the gel to create a pH gradient. In some cases
gels containing an immobilized pH gradient, prepared by in-
corporating weak acids and bases to specific regions of the gel
network during the preparation of the gel, are used. When
the applied proteins reach the gel fraction that has a pH that
is the same as their isoelectric point (pi), their charge is neu-
tralized and migration ceases. Gradients can be made over
various ranges of pH, according to the mixture of ampho-
lytes chosen.
Theoretical Aspects
When a protein is at the position of its isoelectric point, it
has no net charge and cannot be moved in a gel matrix by the
electric field. It may, however, move from that position by
diffusion. The pH gradient forces a protein to remain in its
isoelectric point position, thus concentrating it; this concen-
trating effect is called "focusing". Increasing the applied
voltage or reducing the sample load result in improved
separation of bands. The applied voltage is limited by the
heat generated, which must be dissipated. The use of thin gels
and an efficient cooling plate controlled by a thermostatic cir-
culator prevents the burning of the gel whilst allowing sharp
focusing. The separation is estimated by determining the
minimum pi difference (ApI), which is necessary to separate 2
neighboring bands:
ApI =3
D(dpH/dx)
E( - dfi/dpH)
D: Diffusion coefficient of the protein
dpH/dx: pH gradient
E: Intensity of the electric field, in volts per centimeter
- d/x/dpH: Variation of the solute mobility with the pH in
the region close to the pi
Since D and — d/u/dpH for a given protein cannot be
altered, the separation can be improved by using a narrower
pH range and by increasing the intensity of the electric field.
Resolution between protein bands on an IEF gel prepared
with carrier ampholytes can be quite good. Improvements in
resolution may be achieved by using immobilized pH
gradients where the buffering species, which are analogous to
carrier ampholytes, are copolymerized within the gel matrix.
Proteins exhibiting pis differing by as little as 0.02 pH units
may be resolved using a gel prepared with carrier ampholytes
while immobilized pH gradients can resolve proteins differing
by approximately 0.001 pH units.
Practical Aspects
Special attention must be paid to sample characteristics
and/or preparation. Having salt in the sample can be prob-
lematic and it is best to prepare the sample, if possible, in
deionized water or 2 per cent ampholytes, using dialysis or gel
filtration if necessary.
The time required for completion of focusing in thin-layer
polyacrylamide gels is determined by placing a colored
protein (e.g. hemoglobin) at different positions on the gel sur-
face and by applying the electric field: the steady state is
reached when all applications give an identical band pattern.
In some protocols the completion of the focusing is indicated
by the time elapsed after the sample application.
The IEF gel can be used as an identity test when the
migration pattern on the gel is compared to a suitable
standard preparation and IEF calibration proteins, the IEF
gel can be used as a limit test when the density of a band on
IEF is compared subjectively with the density of bands
appearing in a standard preparation, or it can be used as a
quantitative test when the density is measured using a
densitometer or similar instrumentation to determine the
relative concentration of protein in the bands subject to
validation.
Apparatus
An apparatus for IEF consists of:
— a controllable generator for constant potential, current
and power. Potentials of 2500 V have been used and are
considered optimal under a given set of operating
conditions. Supply of up to 30 W of constant power is
recommended,
— a rigid plastic IEF chamber that contains a cooled plate,
of suitable material, to support the gel,
— a plastic cover with platinum electrodes that are connect-
ed to the gel by means of paper wicks of suitable width,
length and thickness, impregnated with solutions of a-
nodic and cathodic electrolytes.
Isoelectric Focusing in Polyacrylamide Gels: Detailed
Procedure
The following method is a detailed description of an IEF
procedure in thick polyacrylamide slab gels, which is used un-
less otherwise stated in the monograph.
Preparation of the Gels
Mould The mould (see Figure) is composed of a glass
plate (A) on which a polyester film (B) is placed to facilitate
handling of the gel, one or more spacers (C), a second glass
plate (D) and clamps to hold the structure together.
7.5% Polyacrylamide gel Dissolve 29.1 g of acrylamide
and 0.9 g of A f ,A r '-methylenebisacrylamide in 100 mL of
water. To 2.5 volumes of this solution, add the mixture of
ampholytes specified in the monograph and dilute to 10
Figure. Mould
JPXV
General Information / Isoelectric Focusing 1709
volumes with water. Mix carefully and degas the solution.
Preparation of the mould Place the polyester film on the
lower glass plate, apply the spacer, place the second glass
plate and fit the clamps. Place 7.5% polyacrylamide gel
prepared before use on a magnetic stirrer, and add 0.25
volumes of a solution of ammonium persulfate (1 in 10) and
0.25 volumes of Af./V./V'./V'-tetramethylethylenediamine.
Immediately fill the space between the glass plates of the
mould with the solution.
Method
Dismantle the mould and, making use of the polyester film,
transfer the gel onto the cooled support, wetted with a few
millilitres of a suitable liquid, taking care to avoid forming
air bubbles. Prepare the test solutions and reference solutions
as specified in the monograph. Place strips of paper for sam-
ple application, about 10 mm x 5 mm in size, on the gel and
impregnate each with the prescribed amount of the test and
reference solutions. Also apply the prescribed quantity of a
solution of proteins with known isoelectric points as pH mar-
kers to calibrate the gel. In some protocols the gel has pre-
cast slots where a solution of the sample is applied instead of
using impregnated paper strips. Cut 2 strips of paper to the
length of the gel and impregnate them with the electrolyte so-
lutions: acid for the anode and alkaline for the cathode. The
compositions of the anode and cathode solutions are given in
the monograph. Apply these paper wicks to each side of the
gel several millimetres from the edge. Fit the cover so that the
electrodes are in contact with the wicks (respecting the anodic
and cathodic poles). Proceed with the isoelectric focusing by
applying the electrical parameters described in the mono-
graph. Switch off the current when the migration of the mix-
ture of standard proteins has stabilized. Using forceps, re-
move the sample application strips and the 2 electrode wicks.
Immerse the gel in fixing solution for isoelectric focusing in
polyacrylamide gel. Incubate with gentle shaking at room
temperature for 30 minutes. Drain off the solution and add
200 mL of destaining solution. Incubate with shaking for 1
hour. Drain the gel, add coomassie staining TS. Incubate for
30 minutes. Destain the gel by passive diffusion with destain-
ing solution until the bands are well visualized against a clear
background. Locate the position and intensity of the bands in
the electropherogram as prescribed in the monograph.
Variations to the Detailed Procedure (Subject to Validation)
Where reference to the general method on isoelectric focus-
ing is made, variations in methodology or procedure may be
made subject to validation. These include:
— the use of commercially available pre-cast gels and of com-
mercial staining and destaining kits,
— the use of immobilized pH gradients,
— the use of rod gels,
— the use of gel cassettes of different dimensions, including
ultra-thin (0.2 mm) gels,
— variations in the sample application procedure, including
different sample volumes or the use of sample application
masks or wicks other than paper,
— the use of alternate running conditions, including varia-
tions in the electric field depending on gel dimensions and
equipment, and the use of fixed migration times rather
than subjective interpretation of band stability,
— the inclusion of a pre-focusing step,
— the use of automated instrumentation,
— the use of agarose gels.
Validation of lso-Electric Focusing Procedures
Where alternative methods to the detailed procedure are
employed they must be validated. The following criteria may
be used to validate the separation:
— formation of a stable pH gradient of desired characteris-
tics, assessed for example using colored pH markers of
known isoelectric points,
— comparison with the electropherogram provided with the
chemical reference substance for the preparation to be
examined,
— any other validation criteria as prescribed in the mono-
graph.
Specified Variations to the General Method
Variations to the general method required for the analysis
of specific substances may be specified in detail in mono-
graphs. These include:
— the addition of urea in the gel (3 mol/L concentration is
often satisfactory to keep protein in solution but up to 8
mol/L can be used): some proteins precipitate at their isoe-
lectric point. In this case, urea is included in the gel formu-
lation to keep the protein in solution. If urea is used, only
fresh solutions should be used to prevent carbamylation of
the protein,
— the use of alternative staining methods,
— the use of gel additives such as non-ionic detergents (e.g.
octylglucoside) or zwitterionic detergents (e.g., CHAPS or
CHAPSO), and the addition of ampholyte to the sample,
to prevent proteins from aggregating or precipitating.
Points to Consider
Samples can be applied to any area on the gel, but to pro-
tect the proteins from extreme pH environments samples
should not be applied close to either electrode. During
method development the analyst can try applying the protein
in 3 positions on the gel (i.e. middle and both ends); the
pattern of a protein applied at opposite ends of the gel may
not be identical.
A phenomenon known as cathodic drift, where the pH
gradient decays over time, may occur if a gel is focused too
long. Although not well understood, electroendoosmosis and
absorption of carbon dioxide may be factors that lead to
cathodic drift. Cathodic drift is observed as focused protein
migrating off the cathode end of the gel. Immobilized pH
gradients may be used to address this problem.
Efficient cooling (approximately 4°C) of the bed that the
gel lies on during focusing is important. High field strengths
used during isoelectric focusing can lead to overheating and
affect the quality of the focused gel.
Reagents and Solutions —
Fixing solution for isoelectric focusing in polyacrylamide
gel Dissolve 35 g of 5-sulfosalicylic acid dihydrate and 100 g
of trichloroacetic acid in water to make 1000 mL.
*Coomassie staining TS Dissolve 125 mg of coomassie
brilliant blue R-250 in 100 mL of a mixture of water,
methanol and acetic acid (100) (5:4:1), and filter.
Destaining solution A mixture of water, methanol and
acetic acid (100) (5:4:1). »
1710 Laser Diffraction Measurement / General Information
JP XV
10. Laser Diffraction Measurement
of Particle Size
Particle size is one of the important factors concerning the
powder characteristics of solid-state drugs and pharmaceuti-
cal excipients, and therefore prompt and highly accurate de-
termination method is necessary. The laser diffraction
method is considered as a potent candidate, but due to the
limit of kind of particles and the range of particle diameter
that can be measured, the optical microscopy method should
be sometimes applied in parallel. In the pharmaceutical field,
the reliable and reproducible particle size determination
method is required for quality control of the composition of
the formulation, especially, of the pharmaceutical excipients
that are difficult to apply the analytical sieving method due to
the particle diameter of not larger than 100 /um.
In the Japanese Pharmacopoeia, the optical microscopy
(Method 1) for the particles ranging from approximately 1
/um to 100 /um and the analytical sieving method (Method 2)
for the particles of not smaller than 75 /um are listed as the
powder particle size determination.
This document describes the laser diffraction technique for
particle size determination as the method for analyzing the
particles having the diameter ranging from approximately 1
fim to 1000 /um by utilizing the diffraction phenomenon
against the laser beam (Fraunhofer diffraction). There are
methods utilizing the principles such as the Mie scattering
other than the Fraunhofer diffraction, which can be also used
for particle size determination of the particles in sub-micron
range (not larger than 1 /um).
1. Principle of laser diffraction technique
The laser diffraction technique is based upon the beam
diffraction phenomenon (Fraunhofer diffraction), in which
particle size-dependent intensity patterns exhibit when the
powder particles are exposed to the parallel rays, and ac-
curate and reproducible particle size distribution image can
be obtained by mathematical analysis of the diffraction pat-
tern. In other words, this apparatus is designed to detect the
beams diffracted to each angle by the particles when the laser
beam is radiated to the powder sample dispersed in a liquid or
gas, in a way that the beam passes over the specimen and, the
diffraction pattern is obtained and recorded for data analysis.
For the analysis of data, the particles are qualified as spheri-
cal form. In this technique, at every particle diameter interval
that is provided in the apparatus, distribution of the spherical
equivalent diameter in the standard volume is calculated by
inverse operation from the optical model to attain the highest
coherence to the diffraction intensity pattern.
For the particles in sub-micron region, it is not easy to ob-
tain an accurate particle size distribution due to difficulty in
distinction of the particle diameter-dependent scattering pat-
tern because of weakness of the forward light scattering in-
tensity, and it is generally considered that the Fraunhofer
diffraction can not be applied for the evaluation of particle
diameter in the sub-micron range. Therefore, for the particles
in sub-micron region, the method that applies the Mie scat-
tering theory is introduced here. The particle diameter is
measured by the analysis of the scattered lights toward back
and side that are easy to catch the signals of transmitted and
refracted light. However, since the scattering patterns of the
non-spherical particles vary depending on the particle shape,
the correct particle diameter can not be obtained even if the
accurate physical properties (refraction index or transmit-
tance) of the dispersing medium are given. Therefore, the
Mie scattering theory can be effectively applied if the particles
are nearly spherical, and for the other cases, evaluation of the
data is in general difficult due to strongly be affected by the
physical properties or the particle concentration.
2. Apparatus
A typical set-up for a laser diffraction instrument is given
in Figure 1. As the light source, the laser beam of a single
wavelength is generally used. A beam expander is equipped as
the laser beam processing unit H for irradiation to the parti-
cle ensemble dispersed. When the sample (particle ensemble
F) passes through the laser beam, a part of the laser beam is
scattered by the particles, and the diffraction images given by
the scattered and transmitted beams are transcribed by the
detector J placed at the focal length of the Fourier lens D.
A representative sample is dispersed in a liquid or gas, and
the recirculating system consisting of the optical measure-
ment cell, a dispersion bath (usually equipped with the stirrer
and ultrasonic elements), a pump and tubing are commonly
most used.
(1) Calibration of apparatus
According to the description in ISO standard 13320-1
(1999): Particle size analysis-Laser diffraction methods-Part
1: General principles, apparatus should be calibrated by us-
ing the plural standard particles for particle size measurement
having a known particle size distribution and over at least one
decade of size.
(2) Particle size determination in sub-micron region
In order to extend the measuring range of particle diameter
to sub-micron region, apparatuses based on the Mie scatter-
ing theory have been developed and used. This type of instru-
ment is designed so that the scattered lights toward back and
side are detected and analyzed, but there are large variations
in the analytical results depending on the type of instrument.
However, this type of apparatus can produce reproducible
data even in a sub-micron region, if the apparatus used and
the sample tested are restricted under the specific conditions
in which the input number for data analysis, e.g., physical
properties, are fixed for the apparatus.
(3) Particle size determination of emulsion
In case of particle size determination of emulsion, the
method utilizing dynamic light scattering or static light scat-
tering is generally used. Since the emulsion particles transmit
the light, the laser diffraction method is not very appropriate
to apply. However, it is possible to evaluate emulsion particle
diameter when the apparatus has been appropriately calibrat-
ed using the standard particles for particle size measurement.
3. Measurement
In case of the laser diffraction method, the particle concen-
tration affects the accuracy of measurement. Therefore, for
the sample having the size near the upper measuring limit of
particle diameter, the particle concentration should be high
enough to attain the static analysis. On the contrary, for the
sample having smaller particle diameter, it is necessary for
accurate measurement to adjust the particle concentration
lower to some extent to prevent the multiple scattering.
(1) Conditions of measurement
•Location of the apparatus: It is important that the appara-
JPXV
General Information / Laser Diffraction Measurement
I K
1711
A: Absoiplion (obscuration) detector
B: Scattered light
C: Direct rays of light
D: Fourier lens
K: Scattered light not collected with lens D
V: Particle assembly
G: I.aserbeani
II: Ream adjustment parts
I: Measurement point
J: Detector with plural dements
K: Focal length of lens D
Fig. 1 Construction of the laser diffraction apparatus
tus is not affected by electrical noise and mechanical vibra-
tions. It is also desirable that the apparatus is located under
the environment where there is no variation of temperature
and humidity to the extent that affects the measurement and
is no exposure of direct sun light.
•Blank measurement: After selection of proper alignment of
the optical part of the instrument, a blank measurement per-
formed in a pure dispersing medium using the same method
as that for measurement of the sample.
•Preliminary observation of the sample: Prior to the meas-
urement, the powder sample should be observed in advance
by visual/microscopic inspection to confirm the approxi-
mate particle diameter range and particle shape.
•Establishment of the measurement conditions: The time for
measurement, the reading time of detector and the number
of repetition should be experimentally decided in accor-
dance with required measurement accuracy.
•Measurement of the sample: The sample is measured under
the established measurement conditions. Data analysis is
performed based on the pulse of dispersed particles from the
difference of diffraction intensity between the sample and
the blank.
(2) Preparation of the sample
The dry powder sample can be dispersed in a liquid or gas,
and the appropriate method is selected depending on the pur-
pose of measurement. There are two types of dispersion
method, wet type using a liquid as the dispersion medium and
dry type using a gas as the dispersion medium. It is advisable
that the powder sample for measurement is divided to an ap-
propriate apparent volume using a sample splitting tech-
nique.
The result of the particle size distribution much varies de-
pending on the conditions of preparation of the sample.
Therefore, the procedures and conditions of sample prepara-
tion should be defined in advance.
(3) Sampling
The amount of the sample necessary for the laser diffrac-
tion for powder particle size measurement has deceased due
to technical advancement of the apparatus, but the sample
for the measurement should represent the whole sample.
Therefore, how to sample a small amount for actual meas-
urement from a sample of large volume is an important prob-
lem. It is general that a large amount of the sample is
divided/reduced to reach the amount necessary for measure-
ment, and the divider or the cone and quartering method, etc.
are available for this purpose. The cone and quartering
method is used for reduction of a small amount of sample
with poor flowability, and is a simple method without using
any divider.
For sampling of the powder particles dispersed in a liquid,
the liquid is first stirred uniformly and mixed, and it should
be confirmed that no precipitate remains at the bottom of the
vessel. Then, the amount necessary for measurement is
quickly sampled using a pipette, etc. In case of the sample
that contains lager particles, it is easy to cause precipitates,
and they are precipitated at the bottom after once stopping
agitation. Therefore, the sampling of the suspension should
be made under agitation.
(4) Pretreatment of the sample for measurement
If the sample contains the particles or agglomerates over
the upper limit of measurable particle diameter range of the
laser diffraction instrument, they should be removed by siev-
ing in advance, and the mass and percent of such particles or
agglomerates removed should be recorded.
(5) Liquids used as the dispersion medium
A variety of liquids is available for the dispersion of the
powder samples, and at selection of the dispersion liquids,
the followings should be taken into account:
— be compatible with the materials used in the instrument
(O-ring, tubing, etc.)
— not dissolve or alter the size of particular materials,
— favour easy and stable dispersion of the particulate materi-
als,
— have suitable viscosity in order to enable recirculation,
— not be hazardous to health and should meet safety require-
ments.
A low-foaming surfactant and dispersant may be used to
facilitate the wetting of the particles, and to stabilize the dis-
persion. A preliminary check on the dispersion quality can be
made by visual/microscopic inspection of the suspension.
(6) Dispersion of the agglomerated particles by ultrasonica-
tion
For the powder sample containing fine particles, intense
physical power should be given to disperse the agglomerated
particles. The ultrasonication is generally used for this pur-
pose. In this case, since the ultrasonic output, irradiation
time and the volumes of both the suspension and the vessel
1712 Media Fill Test / General Information
JP XV
used significantly affect the intensified dispersion effect, the
optimum irradiation conditions should be established taking
account of these factors. In case that an ultrasonic bath is
used for dispersion, kinetic condition of the dispersant and
dispersion effect may change depending on the altitude of its
surface. Therefore, it is necessary to confirm the resonance
point where the ultrasonic wave is intensely irradiated. Fur-
ther, in case of stronger ultrasonic output or longer irradia-
tion time, the particles for measurement may be destructed.
Therefore, the change of the particle size distribution de-
pending on the variation of the irradiation conditions should
be checked to determine the appropriate ultrasonic output
and irradiation time.
(7) Dispersion gases as the dispersion medium
An appropriate dry type dispersion apparatus using com-
pressed gas is used for dispersion of the powder sample in a
gas. Any substance that may affect the measurement, such as
oil, water and particulate matter, should not be contaminated
in the dispersion medium gas, and such substances should be
removed, if necessary, by a filter, etc. In case of the dry dis-
persion method, it is advisable to measure all the samples
reduced. A larger amount of sample diminishes the statistical
error at a rough and large particle size area even if the particle
size distribution is broad. It is also important that the parti-
cles are dispersed uniformly, It can be confirmed by compar-
ing the results obtained by dry and wet dispersion methods. It
is desirable that the equivalent results are obtained by dry and
wet measurements.
4. Data analysis
Various softwares have been developed to analyze the data
utilizing some mathematical methods, and the particle di-
ameter and particle size distribution can be calculated from
the data to meet its object. As for the accuracy of measure-
ment, the reproducibility required for each apparatus is at-
tained when measured on the independent triplicate sam-
plings of the same batch.
(1) Calculation of the particle size distribution
The instrument manufacturers may use different al-
gorithms for calculation of the particle size distribution from
the intensity of diffraction (or scattering) beams obtained by
the laser diffraction instrument, but the principle of estimat-
ing particle diameter from the intensity of light diffraction
obtained by the detector under the diffraction (or scattering)
theory is fundamentally the same. Since this method is based
upon the optical model that assumes spherical particle, the
sphere equivalent diameter is obtained for the non-spherical
particles. The laser diffraction method can not distinguish be-
tween the diffraction of a single particle and that of the ag-
glomerate or of the primary particle cluster that constitutes
an aggregates or agglomerates.
(2) Weaving of the results
For the samples having particle size distributions, the me-
dian diameter or the modal diameter are used as the charac-
teristic diameters, and the particle size distribution is ex-
pressed either as the cumulative size distribution or the fre-
quency size distribution. The cumulative size distribution is
expressed as a quantitative proportion of the particles smaller
than the specified particle diameter among the whole sample.
The analytical sieving method is generally used for the parti-
cle size distribution determination, and this cumulative distri-
bution is represented as the cumulative undersize distribu-
tion. On the other hand, the frequency size distribution is ex-
pressed as the frequency that is shared by the particles that
belong to the specified particle diameter fraction among the
whole sample.
•Mathematical expression of the particle size distribution
The particle size distribution is expressed by a function of
either the normal distribution or the lognormal distribution.
The lognormal distribution is characterized by distribution
pattern that splays out toward large particle size range, and is
generally applied for the samples having a large amount of
larger particles and a small amount of smaller particles.
5. Experimental considerations on the measurement
Accurate particle diameter measurement can not be expect-
ed for the laser diffraction method in case of the samples hav-
ing wide range of particle size distribution. Therefore, the
setting the measurement range is essential for the measure-
ment of the particle diameter by the laser diffraction method,
because the scattering intensity is much influenced by the low-
er limit diameter (1 //m) and upper limit diameter (1000 //m)
of sample particles. It is desirable in this method that the
measurement diameter range is confirmed by interpolation
using the standard sample having a known particle size distri-
bution considering the accuracy of measurement and the sen-
sitivity of instrument used. For the particles of not larger
than 10 /xm, it is difficult to obtain accurate particle diameter,
because the difference of diffraction pulse detected by the de-
tector is small. Particularly, for the particles in sub-micron
region, there is almost no difference of the scattered light for-
ward, and it is hard to distinguish them. Further, in addition
to the error caused by recognizing the non-spherical particles
as spherical particles, decrease of the scattering intensity,
which is much bigger than that estimated from the reduction
of geometrical particle diameter, occurs as the particle di-
ameter decreases, and securing the accuracy of measurement
comes to be difficult. Therefore, the lower limit for measure-
ment of the particle diameter by the laser diffraction tech-
nique is approximately 1 /urn, similar to that by the optical
microscopic method. In case of the laser diffraction tech-
nique, since the scattering intensity is related to the volume
concentration of the particles, and the maximum measure-
ment range of particle diameter should be decided taking ac-
count of the relative variation of the scattering intensity. A
particle of 10 times larger in diameter has a volume of 1000
times larger. This means that in case of 1 ^m for lower limit
measurement of particle diameter, the particles of 1000 /um
have a volume of 1 billion times larger, and consequently the
sample necessary for measurement is 10 billion times larger,
similar to the case of volume. Therefore, approximately 1000
/um is appropriate for the maximum particle diameter for
measurement.
Example of the standard reference particles
Use the standard reference particles for SAP 10-03, the
Specification of Association of Powder Process Industry and
Engineering, Japan. Either of MBP1-10 or MBP10-100 is
used to meet the particle size measurement range (1 - lO^m
or 10- 100 /um).
11. Media Fill Test
The media fill test (MFT) is one of the processing valida-
tions employed to evaluate the propriety of the aseptic
JP XV
General Information / Media Fill Test 1713
Table 1. Alert and action levels for large numbers of
media filled units
Table 3. Initial performance qualification: Media fills
Number of
Number of contaminated units
units* 1
Acceptance levels
Alert levels
Action levels
3,000
not applicable
§ 1
4,750
1
g 2
6,300
1 -2
§ 3
7,760
1 -3
§ 4
9,160
1 -4
§ 5
10,520
1
2-5
S 6
11,850
1
2-6
g 7
13,150
1 -2
3-7
S 8
14,440
1 -2
3-8
S 9
15,710
1 -3
4-9
2=10
16,970
1 -3
4-10
§11
*1 It is not necessary to relate the number of units with lot size of
actual products.
Table 2. Upper 95% confidence limit of a Poisson variable
for numbers of contaminated units
Observed numbers of contaminated
Upper 95% confidence
units (k)
limit (U)
2.9957
1
4.7439
2
6.2958
3
7.7537
4
9.1537
5
10.5130
6
11.8424
7
13.1481
8
14.4346
9
15.7052
10
16.9622
Using the 95% confidence limit (U) in Table 2, the contamination
rate (P) of observed numbers of contaminated units (k) per filled
units (n) can be calculated as P = U/n (equation 1). For example:
If 5,000 units were filled and two contaminated units were ob-
served, n = 5,000 and U = 6.30 (k = 2) are substituted in the equa-
tion 1; P = 6.30/5,000 = 0.0013. The upper 95% confidence limit
for the contamination rate would be 0.13%.
processing of pharmaceutical products using sterile media,
etc. instead of actual products. Therefore, media fill tests
should be conducted with the manipulations normally per-
formed in actual processing, e.g. filling and closing opera-
tion, operating environment, processing operation, number
of personnel involved, etc., and conducted under processing
conditions that include "worst case" conditions. Refer to
GMP (1), WHO/GMP for pharmaceutical products (2), and
ISO 13408 (3), etc. for necessary information to conduct this
test.
1. Frequency of media fills
1 . 1 Initial performance qualification
Initial performance qualification should be conducted for
each new facility, item of equipment, filling line, and contain-
er design (except for multiple sizes of the same container de-
sign), etc. For production batch sizes exceeding 3,000 units, a
minimum of three media fill runs should be conducted on
separate days. For production batch sizes of less than 3,000
units, see Table 3.
Production
lot size
Numbers of
media fill runs
Alert level and
action required
Action level and
action required
A minimum of One contaminat- Two contaminat-
< 500
500 - 2,9
10 media fill
runs using the
maximum lot
size of the
product
A minimum of 3
media fill runs
using the maxi-
mum lot size of
the product
ed unit in any
run. Investigate
cause.
The same as
above
ed units in single
run, or one each
in two runs.
Investigate
cause and repeat
initial qualifica-
tion media fills.
The same as
above
A minimum of 3 When any of the When any of the
media fill runs
using at least
3,000 3,000 units
media fill runs
exceeds the alert
level shown in
Table 1, take
the action set
out in 2.1.1.
media fill runs
exceeds the ac-
tion level shown
in Table 1, take
the action set
out in 2.1.1
Table 4. Periodic performance requalification: Media fills
Production
Numbers of
Alert level and
Action level and
lot size
media fill runs
action required
action required
A minimum of 3
One contaminat-
media fill runs
ed unit in any
< 500
using the maxi-
run. Investigate
mum lot size of
cause and repeat
the product
initial qualifica-
tion media fill
runs.
One media fill
One contaminat-
run using the
ed unit. Inves-
500 - 2,999
maximum lot
tigate cause and
size of the
repeat initial
product
qualification me-
dia fill runs.
One media fill
When the media
When the media
run using
fill run exceeds
fill run exceeds
at least 3,000
the alert level
the action level
g 3,000
units
shown in Table
shown in Table
1 , take the ac-
1, take the ac-
tion set out in 2.
tion set out in 2.
1. 1.
1. 1
1.2 Periodic performance requalification
1) Conduct media fill requalifications periodically on
each working shift for the filling line. Employees working in
the aseptic processing area should be trained for aseptic
processing operations and take part in media fills.
2) When filling lines have not been used for over six
months, conduct appropriate numbers of media fill runs in
the same way as for the initial performance qualification
prior to resumption of use of the filling lines.
3) In cases of facility and equipment modification (inter-
changing parts may not require requalification), changes in
personnel working in critical aseptic processing (e.g. new
crews), anomalies in environmental testing results, or a
1714 Media Fill Test / General Information
JP XV
product sterility test showing contaminated products, con-
duct appropriate numbers of media fill runs in the same way
as for the initial performance qualification prior to the sched-
uled media fills.
2. Acceptance criteria of media fills
A large number of media filled units is required to detect
0.1% contamination rate. The alert level is more than 0.05%
but less than 0.1%, and the action level is more than 0.1%
contamination rate at the upper 95% confidence level.
Table 1 shows alert and action levels for media filled units.
Table 2 is to be used for the calculation of contamination rate
of contaminated units found in media filled units. The con-
tamination rate of 0.05% in media fills is the minimum ac-
ceptable level, and manufacturers should make efforts to
achieve lower contamination rate than this. Table 3 shows the
alert and action levels for initial performance qualification of
an aseptic processing line, and actions required for each level.
Table 4 shows the alert and action levels for requalification of
an aseptic processing line, and actions required for each level.
The alert and action levels for production batch sizes of less
than 3,000 units take semiautomatic or manual operation
into consideration.
2.1 Actions required for each level
2.1.1 Initial performance qualification
1) When the result of the media fill run is less than the
alert level, the media fill run meets the requirement of the
MFT.
2) When the results of any media fill run done with at
least three replicate runs reach the alert or the action level, an
investigation regarding the cause is required, and initial
qualification media fills are to be repeated. When the result of
each media fill run is less than the alert level, the initial
qualification media fills meet the requirement of the MFT.
2.1.2 Requalification
1) When the result of the media fill run is less than the
alert level, the media fill run meets the requirement of the
MFT.
2) When the result of the media fill run exceeds the alert
level, an investigation regarding the cause is required, and
one more media fill run is to be done. If the result is less than
the alert level, the media fill run meets the requirement of the
MFT.
3) When the result of the media fill run exceeds the action
level, a prompt review of all appropriate records relating to
aseptic production between the current media fill and the last
successful one, and an investigation regarding the cause must
be conducted simultaneously. If necessary, appropriate ac-
tion to sequester stored and/or distributed products should
be taken. After investigation regarding the cause, repeat
three serial media fill runs. If the results are less than the alert
level, the media fill runs meet the requirement of the MFT.
2.2 Parameters which affect sterility
When media fill alert and action levels are exceeded, an in-
vestigation should be conducted regarding the cause, taking
into consideration the following points:
1) Microbial environmental monitoring data
2) Particulate monitoring data
3) Personnel monitoring data (microbial monitoring data
on gloves, gowns, etc. at the end of work)
4) Sterilization cycles for media, commodities, equip-
ment, etc.
5) Calibration of sterilization equipment
6) Storage conditions of sterile commodities
7) HEP A filter evaluation (airborne particulate levels,
DOP test, velocity measurements, etc.)
8) Pre and post filter integrity test data (including filter
housing assembly)
9) Room air flow patterns and pressures
10) Unusual events that occurred during the media fill run
11) Characterization of contaminants
12) Hygienic control and training programs
13) Gowning procedures and training programs
14) Aseptic processing technique and training programs
15) Operator's health status (especially coughing, sneez-
ing, etc., due to respiratory diseases)
16) Other factors that affect sterility
3. Data guidance for media fills
Each media fill run should be fully documented and the
following information recorded:
1) Data and time of media fill
2) Identification of filling room and filling line used
3) Container/closure type and size
4) Volume filled per container
5) Filling speed
6) Filter lot and catalogue number
7) Type of media filled
8) Number of units filled
9) Number of units not incubated and reason
10) Number of units incubated
11) Number of units positive
12) Incubation time and temperature
13) Procedures used to simulate any step of a normal
production fill (e.g., mock lyophilization or substitution of
vial headspace gas)
14) Microbiological monitoring data obtained during the
media fill set-up and run
15) List of personnel who took part in the media fill
16) Growth promotion results of the media (in case of
powder fill, an antimicrobial activity test for the powder is
necessary)
17) Characterization of the microorganisms from any
positive units
18) Review
4. Media fill procedures
Methods to validate aseptic processing of liquid, powder
and freeze-dried products are described. Basically, it is possi-
ble to apply media fill procedures for liquid products to other
dosage forms and container configurations.
4.1 Media selection and growth promotion
Soybean-casein digest medium or other suitable media are
used. As growth promotion testing microorganisms, strains
listed in the Sterility Test and, if necessary, one to two
representative microorganisms which are frequently isolated
in environmental monitoring should be used. The media in-
oculated with 10 to 100 viable microorganisms of each strain
should show obvious growth when incubated at the predeter-
mined temperature for 5 days.
4.2 Sterile medium preparation
The medium is sterilized according to the pre-validated
method.
4.3 Incubation and inspection of media filled units
Leaking or damaged media fill evaluation units should be
removed and recorded prior to incubation of media filled
units. Incubate at 20-25°C for 1 week, and then at 30-
JPXV
General Information / Microbial Attributes 1715
35°C for 1 week (or at 30 - 35°C for 1 week, and then at 20 -
25°C for 1 week), or at 30 - 35°C for 2 weeks. Observe the
media filled units for growth of microorganisms at least once
between the third day and seventh day and on the last day of
the test period, twice in total. Microorganisms present in con-
taminated units should be characterized.
A. Liquid products
Media fill procedure
Media fill should include normal facility/equipment opera-
tions and clean-up routines. Containers, closures, parts of
the filling machine, trays, etc. are washed and sterilized ac-
cording to the standard operating procedures. Media fills
should be conducted under processing conditions that include
"worst case" conditions, e.g., correction of line stoppage,
repair or replacement of filling needles/tubes, replacement of
on-line filters, permitted interventions, duration and size of
run, number of personnel involved, etc.
A predetermined volume of medium is filled into sterilized
containers at a predetermined filling speed and the containers
are sealed. The media are contacted with all product contact
surfaces in the containers by an appropriate method, and
then incubated at the predetermined temperature.
B. Powder products
B.l Powder selection and antimicrobial activity test
Actual products or placebo powder are used. In general,
lactose monahydrate, D-mannitol, polyethylene glycol 6,000,
carboxymethyl cellulose salts or media powder, etc. are used
as placebo powders. Prior to employing any of the powders,
evaluate whether the powder has antimicrobial activity. Me-
dia powders are dissolved in water and other powders in liq-
uid medium, and the solutions are inoculated with 10 to 100
viable microorganisms of each kind, shown in 4.1, for the
growth promotion test. If obvious growth appears in the
medium incubated at the predetermined temperature for 5
days, the powder has no antimicrobial activity and is availa-
ble for the media fill test.
B.2 Sterilization of powders
Dry powders are bagged in suitable containers (e.g. double
heat-sealed polyethylene bags), and are subjected to radiation
sterilization.
B.3 Sterility of filling powders
The powders must pass the Sterility Test. However, if the
sterilization is fully validated, sterility testing of the powders
can be omitted.
B.4 Media fill procedures
Chose a suitable procedure from among the following
procedures.
1) Fill sterilized liquid media into containers by suitable
methods, and then fill actual products or sterilized placebo
powder with the powder filling machine. If sterilized powder
media are used as a placebo powder, fill sterilized water in-
stead of sterilized liquid media.
2) Distribute liquid media into containers, and then steri-
lize them in an autoclave. Remove the containers to the filling
area, and then fill actual products or sterilized placebo pow-
der into the containers with the powder filling machine.
3) Fill actual products or sterilized placebo powder into
containers with the powder filling machine, and then fill steri-
lized liquid media into the containers by appropriate
methods. If sterilized powder media are used as a placebo
powder, fill sterilized water instead of sterilized liquid media.
C. Lyophilized products
In the case of lyophilized products, it may be impossible to
conduct a media fill run in the same way as used for actual
processing of lyophilized products. The process of freezing
and lyophilization of the solution may kill contaminant or-
ganisms and change the characteristics of the media too. The
use of inert gas as a blanket gas may inhibit the growth of
aerobic bacteria and fungi. Therefore, in general, the actual
freezing and lyophilization process should be avoided and air
used as the blanket gas.
Media fill procedures
Use the following method or other methods considered to
be equivalent to these methods.
1) After filling of the media into containers by the filling
machine, cap the containers loosely and collect them in pre-
sterilized trays.
2) After placing the trays in the lyophilizer, close the
chamber door, and conduct lyophilization according to the
procedures for production operation. Hold them without
freezing under weak vacuum for the predetermined time.
3) After the vacuum process, break the vacuum, and seal
the stoppers.
4) Contact the media with all product contact surfaces in
the containers by appropriate methods, and then cultivate
them at the predetermined temperature.
References
1) Good manufacturing practices for pharmaceutical
products (WHO-GMP, 1992)
2) ISO 13408-1 (Aseptic processing of health care
products: Generals)
12. Microbial Attributes of
Nonsterile Pharmaceutical
Products
The presence of microbial contaminants in nonsterile phar-
maceutical products can reduce or even inactivate the ther-
apeutic activity of the product and has the potential to affect
adversely the health of patients. Manufacturers, therefore,
should ensure as low as possible a contamination level for fin-
ished dosage forms, raw materials and packaging compo-
nents to maintain appropriate quality, safety and efficacy of
nonsterile pharmaceutical products. This chapter provides
guidelines for acceptable limits of viable microorganisms
(bacteria and fungi) existing in raw materials and nonsterile
pharmaceutical products. Testing methods for the counting
of total viable microorganisms and methods for the detection
and identification of specified microorganisms {Escherichia
coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus
aureus, etc.) are given under the "Microbial Limit Test".
When these tests are carried out, a microbial control program
must be established as an important part of the quality
management system of the product. Personnel responsible
for conducting the tests should have specialized training in
microbiology and in the interpretation of the testing results.
1. Definitions
1.1 Nonsterile pharmaceutical products: Nonsterile drugs
shown in monographs of the JP and nonsterile products
including intermediate products and finished dosage
1716 Microbial Attributes / General Information
JP XV
forms.
1.2 Raw materials: All materials, including raw ingredients
and excipients, used for the preparation of drugs, except
for water and gases.
1.3 Bioburden: Number and type of viable microorganisms
existing in nonsterile pharmaceutical products.
1.4 Action levels: Established bioburden levels that require
immediate follow-up and corrective action if they are ex-
ceeded.
1.5 Alert levels: Established bioburden levels that give early
warning of a potential drift from normal bioburden level,
but which are not necessary grounds for definitive correc-
tive action, though they may require follow-up investiga-
tion.
1.6 Quality management system: The procedures, opera-
tion methods and organizational structure of a manufac-
turer (including responsibilities, authorities and relation-
ships between these) needed to implement quality manage-
ment.
2. Scope
In general, the tests for total viable aerobic count and for
the detection of specified microorganisms are not applied to
antibiotic or bacteriostatic drugs. However, the tests should
be done for microorganisms that are not affected by the drug.
The test for total viable aerobic count is not applied to drugs
containing viable microorganisms as an active ingredient.
3. Sampling plan and frequency of testing
3.1 Sampling methods
Microbial contaminants are usually not uniformly dis-
tributed throughout the batches of non-sterile pharmaceuti-
cal products or raw materials. A biased sampling plan, there-
fore, cannot be used to estimate the real bioburden in the
product. A sampling plan which can properly reflect the sta-
tus of the product batch should be established on the basis of
the bioburden data obtained by retrospective validation and/
or concurrent validation. In general, a mixture of samples
randomly taken from at least different three portions, almost
the same amount for each portion, is used for the tests of the
product. When the sampling is difficult in a contamination-
controlled environment, special care is required during sam-
pling to avoid introducing microbial contamination into the
product or affecting the nature of the product bioburden. If it
is confirmed that the product bioburden is stable for a certain
period, as in the case of nonaqueous or dried products, it is
not necessary to do the tests for total viable aerobic counts
and for the detection of specified microorganisms, immedi-
ately after the sampling.
3.2 Testing frequency
The frequency of the tests should be established on the ba-
sis of a variety of factors unless otherwise specified. These
factors include:
a) Dosage forms of non-sterile pharmaceutical products
(dosage directions);
b) Manufacturing processes;
c) Manufacturing frequency;
d) Characteristics of raw materials (natural raw material,
synthetic compound, etc.);
e) Batch sizes;
f) Variations in bioburden estimates (changes in batches,
seasonal variations, etc.);
g) Changes affecting the product bioburden (changes in
manufacturing process, supplier of raw materials, lot
number of raw materials, etc.);
h) Others.
In general, the tests may be performed at a high frequency
during the initial production of a drug to get information on
the microbiological attributes of the product or raw materials
used for the production. However, this frequency may be
reduced as bioburden data are accumulated through
retrospective validation and/or concurrent validation. For
example, the tests may be performed at a frequency based on
time (e.g., weekly, monthly or seasonally), or on alternate
batches.
4. Microbial control program
When the "Microbial Limit Test" is applied to a nonsterile
pharmaceutical product, the methods for the recovery, culti-
vation and estimation of the bioburden from the product
must be validated and a "Microbial control program" cover-
ing the items listed below must be prepared.
a) Subject pharmaceutical name (product name);
b) Frequency of sampling and testing;
c) Sampling methods (including responsible person, quanti-
ty, environment, etc. for sampling);
d) Transfer methods of the samples to the testing area (in-
cluding storage condition until the tests);
e) Treatment of the samples (recovery methods of microbi-
al contaminants);
f) Enumeration of viable microorganisms (including testing
quantity, culture media, growth-supporting test of the
media, culturing methods, etc.);
g) Detection of specified microorganisms (including testing
quantity, culture media, growth-supporting test of the
media, culturing methods, etc.);
h) Estimation of the number of and characterization of
microbial contaminants;
i) Establishment of "Microbial contamination limits" (in-
cluding alert level and action level);
j) Actions to be taken when the levels exceed "Microbial
contamination limits";
k) Persons responsible for the testing and evaluation, etc.;
1) Other necessary items
5. Microbial contamination limits for nonsterile phar-
maceutical products
By establishing "Microbial contamination limits", it is
possible to evaluate at the initial processing stage of the
product whether the microbiological quality of the raw
materials is adequate or not. Furthermore, it is then possible
to implement appropriate corrective action as needed to
maintain or improve the microbiological quality of the
product. The target limits of microbial levels for raw materi-
als (systhetic compounds and minerals) are shown in Table 1 .
In general, synthetic compounds have low bioburden levels
due to the high temperatures, organic solvents, etc., used in
their manufacturing processes. Raw materials originated
from plants and animals in general have higher bioburdens
than synthetic compounds.
The microbial quality of the city water or purified water
used in the processing of active ingredients or nonsterile
pharmaceuticals may have a direct effect on the quality of the
finished dosage form. This means it is necessary to keep the
level of microbial contaminants in the water as low as possi-
ble.
The microbial contamination limits for nonsterile finished
dosage forms are shown in Table 2. These microbial limits
JPXV
General Information / Microbiological Evaluation 1717
are based primarily on the type of dosage form, water activi-
ty, and so on. For oral liquids and pharmaceutical products
having a high water activity, in general, low microbial con-
tamination limits are given. In this guideline, Escherichia
coli, Pseudomonas aeruginosa, Staphylococcus aureus, and
Candida albicans are shown as specified microorganisms, but
it is also necessary to test certain pharmaceutical products for
other microorganisms (for example, certain species of Clos-
tridia, Pseudomonas, Burkholderia, Aspergillus, and En-
terobacter species) that may have the potential to present a
microbial risk to patients. The selection of the specified
microorganisms was based on following criteria; indicator
for poor hygienic practices, pathogenic potential for route of
administration, and survival profile of the microorganism
and recoverability in the product. Due to the inherent preci-
sion limitations of the enumeration methods, a value exceed-
ing the target limit by not more than 2 times.
6. Microbial contamination limits for herbal drugs
Target limits of microbial contamination for herbal drugs
and herbal drug containing preparations are shown in Table
3. Category 1 indicates herbal drugs and their preparations to
which boiling water is added before use, and category 2 indi-
cates other herbal drugs and their preparations. In this guide-
line, enterobacteria and other gram-negative bacteria, Es-
cherichia coli, Salmonella, and Staphylococcus aureus are
mentioned as specidied microorganisms, but other microor-
ganisms such as certain species of Bacillus cereus, Clostridia,
Pseudomonas, Burkholderia, Asperigillus and Enterobacter
species are also necessary to be tested depending on the origin
of the herbal drug raw materials or the preparation method
of the preparations.
Table 1. Microbial enumeration limits for raw materials
Target limit
Microorganisms (cfu/g or
cfu/mL)
Total aerobic microbial count (TAMC) ^ 1000
Total combined yeasts /molds count
(TYMC)
100
Table 2. Microbial enumeration limits for
nonsterile finished dosage forms
Route of
administration
Inhalation
(liquid)
TAMC TYMC Examples of
(cfu/g or (cfu/g or objectionable
cfu/mL) cfu/mL) microorganisms
=£20
=£20
Staphylococcus
aureus
Pseudomonas
aeruginosa
Inhalation
(powder)
=£100
=g50
Staphylococcus
aureus
Pseudomonas
aeruginosa
Nasal
;ioo
;50
Staphylococcus
Varginal
Pseudomonas
aeruginosa
1100 =£50 Escherichia
coli
Staphylococcus
aureus
Candida albicans
Otic or Topical
(including
transdermal
patches)
=£100*
=£50*
Pseudomonas
aeruginosa
Staphylococcus
aureus
Rectal
^1000
^100
Not specified
Oral (solid)
^1000
=£100
Escherichia coli
Oral (liquid)
^100
=£50
Escherichia coli
For transdermal patches, the limits are expressed as cfu per
transdermal patch.
Table 3. Microbial enumeration limits for
herbal drugs and their preparations
Microorganisms
Category 1
Category 2
(cfu/g or cfu/mL) (cfu/g or cfu/mL)
Aerobic bacteria
10 7
10 5
Molds and yeasts
10 4
10 3
Enterobacteria
and other gram-
*
10 3
negative bacteria
Escherichia coli
10 2
not detected
Salmonella
not detected
not detected
Staphylococcus
aureus
* The limits are not specified.
13. Microbiological Evaluation of
Processing Areas for
Sterile Pharmaceutical Products
This chapter describes the methods for the control and
evaluation of microbial contamination in areas used for the
processing of sterile pharmaceutical products. Such process-
ing areas are classified into critical areas and clean areas
according to the required levels of air-cleanliness. A critical
area is a defined space in which the airborne particulate and
microorganism levels are controlled to meet grade A. The
cleanliness requirements for such a space extend to the sur-
faces of the facilities and equipment which form or are locat-
ed within the space, as well as to the supplied raw materials,
chemicals, water, etc. Environmental conditions, such as
temperature, humidity, and air pressure, are also controlled
in this space when required. A clean area is a controlled space
such that the levels of contaminants (particulates and
microorganisms) in air, gases and liquids are maintained
within specified limits, which are less stringent than those of
grade A. When sterile pharmaceutical products are manufac-
1718 Microbiological Evaluation / General Information JP XV
Table 1. Air-cleanliness requirements for processing of sterile pharmaceutical products
Air cleanliness
Maximum number of airborne particulates per m 3
Grade*
at rest
in operation
^0.5 /urn.
A (Laminar-airflow zone)
B (Non laminar-airflow zone)
C
D
3,530
3,530
353,000
3,530,000
3,530
353,000
3,530,000
*2
*1 The maximum permitted number of particles in the "in operation" condition corresponds to the standards described under USP <1116> as
follows.
Grade A: Class 100 (M3.5); Grade B: Class 10,000 (M5.5); Grade C: Class 100,000 (M6.5);
Grade D: no corresponding standard.
*2 The limit for this area will depend on the nature of the operation carried out there.
Table 2. Suggested frequency of environmental monitoring
Processing area
Frequency of monitoring
Critical area (Grade A)
Clean area adjacent to critical area (Grade B)
Other clean areas (Grade C, D)
Potential product/container contact areas
Non-product/container contact areas
Each shift
Each shift
Twice a week
Once a week
Table 3. Media and culture conditions
Microorganisms
to be detected
Media*
Culture conditions
Aerobes
Soybean-casein digest agar (or fluid) medium
Brain-heart infusion agar (or fluid) medium
Nutrient agar (or fluid) medium
30-35°C* 2
More than 5 days* 3
Yeast and fungi
Anaerobes* 4
Soybean-casein digest agar (or fluid) medium
Sabouraud dextrose agar (or fluid) medium
Potato-dextrose agar (or fluid) medium
Glucose peptone agar (or fluid) medium
Soybean-casein digest agar medium
Fluid cooked meat medium
Reinforced clostridial agar (or fluid) medium
Thioglycolate medium I (or thioglycolate agar
medium) for sterility test
20-25°C* 2
More than 5 days* 3
30-35°C
More than 5 days* 3
*1 If necessary, antibiotics may be added to media in an appropriate concentration (see Microbial Limit Test). If the existence of disinfectants
that may interfere with the test on the surface of the specimen is suspected, add a substance to inactivate them.
*2 When soybean-casein digest agar medium is used for the detection of aerobes, yeast and fungi, incubation at 25 to 30°C for more than 5
days is acceptable.
*3 If a reliable count is obtained in a shorter incubation time than 5 days, this may be adopted.
*4 Generally, anaerobes are not targets for the monitoring. For the detection of anaerobes, agar medium is incubated in an appropriate
anaerobic jar.
Table 4. Recommended limits for environmental microorganisms* 1
Grade
Airborne
microorganisms*
(CFU/m 3 )
CFU on a surface
Minimum air sample
(m 3 )
instruments/ facilities
gloves
(CFU/24-30 cm 2 )*
A
B
C
D
<1
10
100
200
0.5
0.5
0.2
0.2
<1
5
25
50
<1
5
*1 Maximum acceptable average numbers of microorganisms under each condition.
*2 These values are by using a slit sampler or equivalent.
*3 Viable microbe cell number per contact plate (5.4-6.2 cm in diameter). When swabbing is used in sampling, the number of microorganisms
is calculated per 25 cm 2 . For gloves, usually, put their all fingers on the plate.
JPXV
General Information / Microbiological Evaluation 1719
tured, the environment, facilities/equipment, and personnel
should be routinely monitored to ensure appropriate
microbiological control in the processing areas. The detection
of microorganisms should be performed under normal opera-
tional conditions, using an appropriate sampling device,
according to an environmental control program established
previously. The sampling, cultivation, counting, and evalua-
tion methods for airborne microorganisms, as well as those
found on surfaces, should also be chosen appropriately,
depending on the monitoring purpose, monitoring items, and
microorganisms being detected. Sampling devices, measure-
ment methods, media, culture conditions, frequency of
monitoring, and recommended limits for environmental
microorganisms shown in this chapter are for information
only, and are not requirements.
1. Definitions
For the purposes of this chapter, the following definitions
apply.
1) Processing areas: Areas in which actions such as culti-
vation, extraction/purification, weighing of raw materials,
washing and drying of containers and stoppers, preparation
of solutions, filling, sealing and packaging are performed, in-
cluding the gowning area.
2) Action levels: Established microbial levels (and type of
microorganisms, if appropriate) that require immediate fol-
low-up and corrective action if they are exceeded.
3) Alert levels: Established microbial levels (and type of
microorganisms if appropriate) that give early warning of a
potential drift from normal operating conditions, but which
are not necessarily grounds for definitive corrective action,
though they may require follow-up investigation.
4) Contaminants: Particulates and microorganisms caus-
ing contamination by adhering to surfaces or by being incor-
porated into materials.
5) Cleanliness: A quantity which indicates the condition
of cleanliness of a monitored item, expressed as mass or num-
ber of contaminants contained in a certain volume or area.
6) Contamination control: The planning, establishment
of systems and implementation activities performed in order
to maintain the required cleanliness of a specified space or
surface.
7) Shift: Scheduled period of work or production, usual-
ly less than 12 hours in length, during which operations are
conducted by a single defined group of workers.
8) Characterization of contaminants: Procedures for
classifying contaminants so that they can be differentiated. In
routine control, classification to the genus level is sufficient;
as required, identification to the species level is performed.
2. Air-cleanliness of processing areas for sterile pharmaceu-
tical products
Airborne particulates in areas used for the processing of
pharmaceutical products may act physically as a source of in-
soluble particles in the products, and biologically as a carrier
of microorganisms. So, it is necessary to control strictly the
number of particles in the air. The air-cleanliness criteria are
shown in Table 1.
2.1 Terminally sterilized products
Solutions should generally be prepared in a grade C en-
vironment. Solution preparation may be permitted in a grade
D environment if additional measures are taken to minimize
contamination. For parenterals, filling should be done in a
grade A workstation in a grade B or C environment. The re-
quirements during the preparation and filling of other sterile
products are generally similar to those for parenterals.
2.2 Sterile products prepared aseptically after filtration
The handling of starting materials and the preparation of
solutions should be done in a grade C environment. These ac-
tivities may be permitted in a grade D environment if addi-
tional measures are taken to minimize contamination, such as
the use of closed vessels prior to filtration. After sterile filtra-
tion, the product must be handled and filled into containers
under grade A aseptic conditions.
2.3 Sterile products prepared aseptically from sterile start-
ing materials
The handling of starting materials and all further process-
ing should be done under grade A conditions.
3. Microbiological environmental monitoring program
Environmental monitoring is especially important in steril-
ity assurance for sterile pharmaceutical products that are
manufactured by aseptic processing. The major purpose of
environmental monitoring is to predict potential deteriora-
tion of the processing environment before it occurs, and to
produce high-quality, sterile pharmaceutical products under
appropriate contamination control.
3.1 Monitoring of environmental microorganisms
a) An environmental control program document is pre-
pared for each area used for the processing of sterile phar-
maceutical products. The procedures in the document in-
clude: 1) items to be monitored, 2) type of microorganisms to
be monitored, 3) frequency of monitoring, 4) methods of
monitoring, 5) alert and action levels, and 6) actions to be
taken when specified levels are exceeded.
b) The aseptic processing areas and other processing
areas maintained under controlled conditions are monitored
on a routine basis. The critical processing areas where sterile
products are in contact with environmental air are monitored
during every operational shift. The items to be monitored in-
clude the air, floor, walls, equipment surfaces, and the gowns
and gloves of the personnel. Table 2 shows suggested fre-
quencies of the environmental monitoring.
c) The sampling devices used for monitoring environ-
mental microorganisms, as well as the methods and culture
media, should be suitable to detect microorganisms that may
be present (aerobic bacteria, anaerobic bacteria, molds,
yeast, etc.). The cultivation conditions, such as incubation
temperature and time, are selected to be appropriate for the
specific growth requirements of microorganisms to be detect-
ed. Table 3 shows the culture media and cultivation condi-
tions that are generally used in testing for environmental
microorganisms.
d) The number of microorganisms in the samples is esti-
mated by using the Membrane Filtration, Pour Plating,
Spread Plating, or Serial Dilution (Most Probable Number)
Methods described in the Microbial Limit Test.
e) Table 4 shows recommended limits for environmental
microorganisms. The alert and action levels may be adjusted
if necessary after sufficient data have been accumulated. The
most important point in environmental monitoring is to con-
firm that an acceptable value of each monitoring item is
maintained consistently.
f ) Microorganisms isolated are characterized if necessa-
ry. In addition, analysis of hourly or daily variation of air-
borne particulate numbers will provide data to assist in the
control of the cleanliness of processing areas.
1720 Microbiological Evaluation / General Information
JP XV
3.2. Evaluation of environmental monitoring data
a) The data from the environmental monitoring are eval-
uated on a routine basis for each area and location. The
source of any discrepancy should be investigated immediately
and the investigation should be documented in a report. Af-
ter corrective action has been taken, follow-up monitoring
should be done to demonstrate that the affected area is once
again within specification.
b) The report is reviewed and approved by personnel
responsible for quality control and distributed to all key per-
sonnel associated with the aseptic processing operation.
4. Sampling devices and measuring methodology
Various types of sampling devices and measurement
methods are available for the sampling and measurement of
microorganisms in the air and on surfaces, and appropriate
samplers and measuring methodology are selected according
to the purpose of monitoring and the items to be monitored.
4.1 Evaluation of airborne microorganisms
a) Settle plates
Petri dishes of a specified diameter containing a suitable
culture medium are placed at the measurement location and
the cover is removed there. The plates are exposed for a given
time and the microorganisms deposited from the air onto the
agar surface are enumerated after incubation. This method is
not effective for quantitative monitoring of total airborne
microorganisms because it does not detect microorganisms
that do not settle onto the surface of the culture media, and
the settling velocity of aggregates of microorganisms is affect-
ed by air currents and disturbances in airflow. Although the
results obtained by the settle plate method are only qualita-
tive or semi-quantitative, this method is suitable for long-
term evaluation of possible contamination of products or
devices by airborne microorganisms.
b) Active microbial sampling methods
1) Measuring methods
Methods in which a fixed volume of air is aspirated include
filtration-type sampling devices and impact-type sampling
devices. With the filter-type sampling devices the desired
volume of air can be collected by appropriately changing the
air intake rate or the filter size. However, care must be taken
to ensure that sterility is maintained while the filter is placed
in and removed from the holder. When air sampling devices
are used in critical areas, care must be taken to avoid distur-
bance of the airflow around the products. There are two types
of filters; wet-type used gelatin filters and dry-type used mem-
brane filters. With the dry-type filters, static electricity effects
can make it impossible to collect quantitatively microorgan-
isms on the filter. When an impact-type sampling device is
used, the following points are important: 1) The speed at
which the collected air strikes the culture medium surface
must be sufficient to capture the microorganisms, but must
not have an adverse effect on the collected microorganisms.
2) A sufficient volume of air must be sampled so that even ex-
tremely low levels of microbiological contaminants are de-
tected, but the procedure must not cause a significant change
in the physical or chemical properties of the culture medium.
3) When the device is used in critical areas, care must be exer-
cised to ensure that the processing of the sterile pharmaceuti-
cal products is not adversely affected by the air disturbance.
2) Sampling devices
The most commonly used samplers are as follows: Slit
sampler, Andersen sampler, pinhole sampler, centrifugal
sampler and filtration-type sampler. Each sampler has
specific characteristics. The slit sampler is a device to trap
microorganisms in a known volume of air passed through a
standardized slit. The air is impacted on a slowly revolving
Petri dish containing a nutrient agar. The rotation rate of the
Petri dish and the distance from the slit to the agar surface
are adjustable and it is possible to estimate the number of
microorganisms in the air passed through the device for a
period of up to 1 hr. The Andersen sampler consists of a per-
forated cover and several pieces of Petri dishes containing a
nutrient agar, and a known volume of air passed through the
perforated cover impacts on the agar medium in the Petri dis-
hes. The sampler is suitable for the determination of the dis-
tribution of size ranges of microorganism particulates in the
air. The pinhole sampler resembles the slit of the slit sampler,
but has pinholes in place of the slit. A known volume of air
passed through several pinholes impacts on agar medium in a
slowly revolving Petri dish. The centrifugal sampler consists
of a propeller that pulls a known volume of air into the device
and then propels the air outward to impact on a tangentially
placed nutrient agar strip. The sampler is portable and can be
used anywhere, but the sampling volume of air is limited.
See above 1) on the characteristics of the filtration-type
sampler.
4.2 Measurement methods for microorganisms on surfaces
a) Contact plates
Use a contact plate with an appropriate contact surface.
The culture medium surface should be brought into contact
with the sampling site for several seconds by applying
uniform pressure without circular or linear movement. After
contact and removal, the plates are covered and, as soon as
possible, incubated using appropriate culture conditions. Af-
ter a contact plate has been used, the site to which the plate
was applied must be wiped aseptically to remove any adher-
ent culture medium.
b) Swabs
A piece of sterilized gauze, absorbent cotton, cotton swab,
or other suitable material premoistened with an appropriate
rinse fluid is stroked in closely parallel sweeps or slowly rotat-
ed over the defined sampling area. After sampling, the swab
is agitated in a specified amount of an appropriate sterilized
rinse fluid, and the rinse fluid is assayed for viable organisms.
5. Test methods for collection performance of a sampling
device for airborne microorganisms
The testing of the collection performance of sampling
devices for airborne microorganisms is performed in accor-
dance with JIS K 3836 (Testing methods for collection
efficiency of airborne microbe samplers) or ISO 14698 - 1
(Cleanrooms and associated controlled environments.
Biocontamination control. General principles).
6. Growth-promotion test of media and confirmation of an-
timicrobial substances
This test and confirmation are performed according to
"Effectiveness of culture media and confirmation of an-
timicrobial substances" in the Microbial Limit Test.
Media
Brain-heart infusion agar medium or Fluid brain-heart infu-
sion medium
Bovine brain extract powder* 1
JPXV
General Information / Mycoplasma Testing 1721
An amount equivalent to 200 g of calf brain
Bovine heart extract powder* 2
An amount equivalent to 250 g of the material
Peptone 10.0 g
Glucose 2.0 g
Sodium chloride 5.0 g
Disodium hydrogenphosphate
dodecahydrate 2.5 g
Agar 15.0 g
Water 1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 7.2 - 7.6.
Fluid cooked meat medium
Bovine heart extract powder* 2
An amount equivalent to 450 g of the material
Peptone 20.0 g
Glucose 2.0 g
Sodium chloride 5.0 g
Water 1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 7.2 - 7.6.
Glucose peptone agar medium or Fluid glucose peptone
medium
See Microbial Limit Test. Antibiotic is added if necessary.
Nutrient agar medium or Fluid nutrient medium
Meat extract
Peptone
Agar
Water
3.0 g
5.0 g
15.0 g
1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 6.6 - 7.0.
Potato-dextrose agar medium or Fluid potato-dextrose medi-
um
See Microbial Limit Test. Antibiotic is added if necessary.
Thioglycolate agar medium or thioglycolate medium I for
sterility test
See Sterility Test. The agar concentration of Thioglycolate
agar medium is about 1.5%.
*1 Bovine brain extract powder Dried extract of bovine
fresh brain. A yellow-brown powder having a characteristic
odor.
Loss on drying: not more than 5%.
*2 Bovine heart extract powder Dried extract of bovine
fresh heart. A yellow-brown powder having a characteristic
odor.
Loss on drying: not more than 5%.
Rinsing Liquids
Buffered sodium chloride-peptone solution (pH 7.0)
See Microbial Limit Test.
LP liquid
Casein peptone
Soybean lecithin
Polysorbate 80
Water
1.0 g
0.7 g
1.0-20.0 g
1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 7.2.
Phosphate buffered solution (pH 7.2)
See Microbial Limit Test.
Ringer's solution, 1/4 concentration
Sodium chloride 2.25 g
Potassium chloride 0.105 g
Calcium chloride dihydrate 0.16 g
Water 1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 7.0.
Reinforced clostridial agar medium or Fluid reinforced clos-
tridial medium
Meat extract 10.0 g
Peptone 10.0 g
Yeast extract 3.0 g
Soluble starch 1.0 g
Glucose 5.0 g
L-Cystein hydrochloride monohydrate 0.5 g
Sodium chloride 5.0 g
Sodium acetate trihydrate 3.0 g
Agar 15.0 g
Water 1,000 mL
For fluid medium, add 0.5 g of agar. Sterilize by heating
in an autoclave at 121°C for 15 to 20 min. pH after
sterilization: 6.7 - 6.9.
Sabouraud dextrose agar medium or Fluid sabouraud dex-
trose medium
See Microbial Limit Test. Antibiotic is added if necessary.
Soybean-casein digest agar medium or Fluid soybean-casein
digest medium
See Microbial Limit Test.
Thiosulfate-Ringer's solution
Sodium thiosulfate pentahydrate 0.8 g
Ringer's solution, 1/4 concentration 1,000 mL
Sterilize by heating in an autoclave at 121 °C for 15 to 20
min. pH after sterilization: 6.6.
14. Mycoplasma Testing for
Cell Substrates used for the
Production of Biotechnological/
Biological Products
This document describes the currently available methods
of mycoplasma testing that should be performed for cell sub-
strates that are used in the manufacture of biotechnological/
biological products.
Methods suggested for detection of mycoplasma are, A.
culture method, B. indicator cell culture method, and C.
polymerase chain reaction (PCR) method.
Mycoplasma testing should be performed on the master
cell bank (MCB) and the working cell bank (WCB), as well as
on the cell cultures used during the manufacturing process of
1722 Mycoplasma Testing / General Information
JP XV
the product. For the assessment of these cells, mycoplasma
testing should be performed using both methods A and B.
Method B, however, does not detect only DNA derived from
mycoplasma. Therefore, if a positive result is obtained only
from method B, method C can be used to determine whether
mycoplasma is actually present. When method C is used, it is
necessary to demonstrate the rationale for determining a
negative result. In such a case, the sensitivity and specificity
of the method, the appropriateness of the sample prepara-
tion, and the suitability of the selection of the test method,
including selection of reagents, reaction conditions and
primers should be taken into account.
Prior to mycoplasma testing, the sample should be tested
to detect the presence of any factors inhibiting the growth of
mycoplasma. If such growth-inhibiting factors are detected
they should be neutralized or eliminated by an appropriate
method, such as centrifugation or cell passage.
If the test will be performed within 24 hours of obtaining
the sample, the sample should be stored at a temperature be-
tween 2°C and 8°C. If more than 24 hours will elapse before
the test is performed, the sample should be stored at -60°C
or lower.
If mycoplasma is detected, additional testing to identify
the species may be helpful in determining the source of con-
tamination.
A. Culture Method
1. Culture Medium
Both agar plates and broth are used. Each lot of agar and
broth medium should be free of antibiotics except for penicil-
lin. Refer to the Minimum Requirements for Biological
Products regarding selection of the culture media. Other cul-
ture media may be used if they fulfill the requirements
described in the following section 2.
2. Suitability of Culture Medium
Each lot of medium should be examined for mycoplasma
growth-promoting properties. To demonstrate the capacity
of the media to detect known mycoplasma, each test should
include control cultures of at least two known species or
strains of mycoplasma, one of which should be a dextrose
fermenter (i.e., M. pneumoniae strain FH or equivalent spe-
cies or strains) and one of which should be an arginine
hydrolyser (i.e., M. orale CH 19299 or equivalent species or
strains). The mycoplasma strains used for the positive control
tests should be obtained from an official or suitably accredit-
ed agency, and handled appropriately. Inoculate the culture
medium with no more than 100 colony-forming units (CFU).
3. Culture and Observation
1) Inoculate no less than 0.2 mL of test sample (cell sus-
pension) in evenly distributed amounts over the surface of
each of four or more agar plates. After the surfaces of the in-
oculated plates are dried, one half of the plates should be in-
cubated under aerobic conditions in an atmosphere of air
containing 5 to 10 percent carbon dioxide and adequate hu-
midity, and the other half under anaerobic conditions in an
atmosphere of nitrogen containing 5 to 10 percent carbon di-
oxide and adequate humidity at 36 ± 1 °C for no less than 14
days.
2) Inoculate no less than 10 mL of the test sample (cell
suspension) into each of two vessels containing 100 mL of
broth medium. One vessel should be incubated under aerobic
conditions, and the other under anaerobic conditions.
If the culture medium for the sample cells contains any
growth-inhibiting factors, such as antibiotics, these factors
must be removed. A method such as centrifugation is recom-
mended for this purpose.
3) Subculture 0.2 mL of broth culture from each vessel
on the 3 rd , 7 th , and 14 th days of incubation onto two or more
agar plates. The plates inoculated with aerobic or anaerobic
broth cultures should be incubated aerobically or anaerobi-
cally, respectively at 36 ± 1°C for no less than 14 days.
4) Examination of all plates for mycoplasma colonies
should be done microscopically on the 7 th and 14 th day at 100
times magnification or greater.
B. Indicator Cell Culture Method
Using Vero cell culture substrate, pretest the suitability of
the method using an inoculum of no more than 100 CFU of
M. hyorhinis DBS 1050 or M. orale CH 19299.
An equivalent indicator cell substrate and suitable
mycoplasma strains may be acceptable if data demonstrate at
least equal sensitivity for the detection of known mycoplasma
contaminants. The mycoplasma strains should be obtained
from an official or suitably accredited agency, and handled
appropriately. The cell substrate used should be obtained
from a qualified cell bank and certified to be mycoplasma
free. The acquired cells should be carefully cultured and
propagated, and sufficient volumes of seed stock should be
prepared with the proper precautions to avoid mycoplasma
contamination. The stock should be tested for mycoplasma
contamination using at least one of the methods described in
this document, then frozen for storage. For each test a new
container from the stock should be thawed and used within 6
passages.
Indicator cell cultures should be grown on cover slips sub-
merged in culture dishes or equivalent containers for one day.
Inoculate no less than 1 mL of the test sample (cell culture su-
pernatant) into two or more of the culture dishes.
The test should include a negative (non-infected) control
and two positive mycoplasma controls, such as M. hyorhinis
DBS 1050 or M. orale CH 19299. Use an inoculum of no
more than 100 CFU for the positive controls.
Incubate the cell cultures for 3 to 6 days at 36 ± 1°C in an
atmosphere of air containing 5 percent carbon dioxide.
Examine the cell cultures after fixation for the presence of
mycoplasma by epifluorescence microscopy (400 to 600 times
magnification or greater) using a DNA-binding
fluorochrome, such as bisbenzimidazole or an equivalent
stain. Compare the microscopical appearance of the test cul-
tures with that of the negative and positive controls.
Procedure
1) Aseptically place a sterilized glass cover slip into each
cell culture dish (35 mm diameter).
2) Prepare Vero cell suspension in Eagle's minimum es-
sential medium containing 10 percent bovine calf serum at a
concentration of 1 x 10 4 cells per 1 mL. The bovine calf
serum should be tested and confirmed to be free from
mycoplasma prior to use.
3) Inoculate aliquots of 2 mL of the Vero cell suspension
into each culture dish. Ensure that the cover slips are com-
pletely submerged, and not floating on the surface of the cul-
ture medium. Incubate the cultures at 36±1°C in an at-
mosphere of air containing 5 percent carbon dioxide for one
day, so that the cells are attached to the glass cover slip.
4) Replace 2 mL of the culture medium with fresh medi-
um, then add 0.5 mL of the test sample (cell culture super-
JPXV
General Information / Mycoplasma Testing 1723
natant) to each of two or more culture dishes. Perform the
same procedure for the positive (2 types of mycoplasma, such
as M. hyorhinis and M. orale) and negative controls.
5) Incubate the cultures for 3 to 6 days at 36 ± 1 °C in an
atmosphere of air containing 5 percent carbon dioxide.
6) Remove the culture medium from the culture dishes,
and add 2 mL of a mixture of acetic acid (100) and methanol
(1:3) (fixative) to each dish; then, allow them to stand for 5
minutes.
7) Remove the fixative from each dish, then add the same
amount of fixative again, and leave the dishes to stand for 10
minutes.
8) Remove the fixative and then completely air-dry all the
dishes.
9) Add 2 mL of bisbenzamide fluorochrome staining so-
lution to each culture dish. Cover the dishes and let them
stand at room temperature for 30 minutes.
10) Aspirate the staining solution and rinse each dish
with 2 mL of distilled water 3 times. Take out the glass cover
slips and dry them.
11) Mount each cover slip with a drop of a mounting
fluid. Blot off surplus mounting fluid from the edges of the
cover slips.
12) Examine by epifluorescence microscopy at 400 to 600
times magnification or greater.
13) Compare the microscopic appearance of the test sam-
ple with that of the negative and positive controls.
14) The test result is judged to be positive if there are
more than 5 cells per 1000 (0.5%) that have minute fluores-
cent spots that appear to surround, but are outside, the cell
nucleus.
C. Polymerase Chain Reaction (PCR) Detection Method
The PCR method is a highly specific method that enables
the detection of trace amounts of mycoplasma DNA, and has
come to be widely used in recent years as a means of detecting
mycoplasma contamination. However, the sensitivity and
specificity depend on the procedure employed, and a positive
result from PCR does not always indicate the presence of via-
ble mycoplasma.
The PCR method is based on amplifying DNA extracted
from the cell culture with specific primers so that the presence
of the target DNA is detected. A two-step PCR (nested PCR)
is recommended in order to increase sensitivity and specifici-
ty. The tests should include both a positive control (such as
M. hyorhinis of 100 CFU or less) and a negative control.
Mycoplasma DNA from the sample of cells or cell cultures
is amplified using primers which should be able to amplify
some common conserved mycoplasma DNA sequence. The
amplification should be performed using an appropriate heat-
resistant DNA polymerase, and suitable conditions. The am-
plified DNA can be identified after agarose gel electrophore-
sis, followed by ethidium bromide staining and UV irradia-
tion of the gel.
For this method, it is important to use primers that are
specific to mycoplasma by choosing base sequences that are
well-conserved for a wide range of mycoplasma species, for
example, the spacer region between the 16S-23S ribosome
genes.
It is recommended that a two-step PCR using nested
primers should be performed to increase the sensitivity and
specificity, if the one-step PCR is negative.
The primers to be selected for the second stage of a two-
step PCR are nested primers from the inner portion of the
sequence. The outer and inner primers should have proven
effectiveness and specificity as described in publications or be
validated experimentally.
It is possible to increase the accuracy of the detection of
mycoplasma DNA by performing PCR tests after cultivation
of mycoplasma that may be present in samples using Vero
cells.
The following is an example of a two-step PCR procedure.
The reagents and reaction conditions in this example are not
exclusive. If the suitability of other reagents and conditions is
verified, they may be used. If another procedure is used, the
procedure should be justified and documented in detail, and
the information provided should include the sensitivity and
specificity of the method.
Example Procedure
1. Preparation of template
1) Place 600 /xL of the test cell suspension (if necessary,
subcultured with Vero cells) in a tube and dissolve the cells
with 0.1% SDS or an equivalent. Add an equal volume (600
,uL) of TE (lOmmol/L tris-hydrochloric acid (pH 8.0), 1
mmol/L EDTA) buffer-saturated phenol, and mix.
2) Centrifuge at 15,000 min~* for 5 minutes at room tem-
perature.
3) Transfer 400 /uL of the supernatant to another tube,
and add 10 /uL of 3 mol/L sodium acetate.
4) Add 1 mL (2.5 volumes) of ethanol (95) and stir
thoroughly. Ice the mixture for 15 minutes, then centrifuge at
15,000 min-' for 10 minutes at 4°C.
5) Discard the supernatant and rinse the precipitate once
or twice with 200 to 300 fiL of 80% ethanol. Remove the rinse
solution using a pipette. Centrifuge at 15,000 min -1 for 10
minutes at 4°C, then remove the supernatant thoroughly and
dry up the precipitate.
6) Dissolve the precipitate in 40 fiL of distilled water.
2. Perform the same procedure for the positive and nega-
tive controls
3. First stage of a two-step PCR
1) Make a mixture of the heat-resistant DNA poly-
merase, dNTP solution, outer primer, and reaction buffer so-
lution (including Mg ions), and place 90 /xL in each tube.
2) Add 10 /xL of the template prepared as above to each
tube containing the first stage PCR solution (90 /xL).
3) Perform the DNA amplification by repeating 30 cycles
of denaturation at 94 °C for 30 seconds, annealing at an ap-
propriate temperature for the primer (55 °C for the primer in
this example), and elongation at 72°C for 2 minutes. Add
drops of mineral oil or suitable equivalent as needed during
the reaction to prevent evaporation.
4. Second stage of a two-step PCR
1) Make a mixture of the heat-resistant DNA poly-
merase, dNTP solution, inner primer, and reaction buffer
solution (including Mg ions), and place 99 /uL in each tube.
2) Add 1 /xL of the first stage PCR product from each
tube to a tube containing the second stage PCR solution (99
UL).
3) Perform the DNA amplification by repeating 30 cycles
of denaturation at 94 °C for 30 seconds, annealing at an ap-
propriate temperature for the primer (55 °C for the primer in
this example), and elongation at 72°C for 2 minutes. Add
drops of mineral oil or suitable equivalent as needed during
the reaction to prevent evaporation.
5. Agarose gel electrophoresis
1724 Peptide Mapping / General Information
JP XV
1) Mix 10 ,«L of each of the first stage and second stage
PCR products with 2 /uL of an appropriate dye as a migration
marker, and perform 1% agarose gel electrophoresis.
2) Stain the gel with ethidium bromide and take a photo-
graph under UV irradiation.
3) The test is judged to be positive if a DNA band is de-
tected.
[An Example of Primer]
For mycoplasma detection
Outer primer
F 1 : 5 ' -AC ACC ATGGGAG(C/T)TGGT AAT-3 '
R 1 : 5 ' -CTTC(A/T)TCG ACTT(C/T)C AGACCC AAGG-
CAT-3'
Inner primer
F2:5'-GTG(G/C)GG(A/C)TGGATCACCTCCT-3'
R2 : 5 ' -GC ATCCACC A(A/T)A(A/T)AC(C/T)CTT-3 '
( ) indicates a mixture.
[PCR reaction solution]
[First stage] [Second stage]
dNTP solution (each 1 .25 mol) 16 /uL 16 ixL
Primer (10 pmol/uL) Fl 2,wL F2 2//L
Primer (10 pmol/uL) Rl 2,mL R2 2//L
Heat-resistant DNA poly-
merase (1 U/mL)
Reaction buffer solution 68 /uL 77 /xL
25 mmol/L magnesium chlo-
ride hexahydrate
10-fold buffer solution* 10,mL 10 fiL
Sterile distilled water 50 /uL 59 /xL
2 /ih
8/uL
2//L
8//L
"Composition of 10-fold buffer solution
2-amino-2-hydroxymethyl-l ,3-
propanediol-hydrochloric acid
(pH 8.4) 100 mmol/L
Potassium chloride 500 mmol/L
Magnesium chloride hexahydrate 20 mmol/L
Gelatin 0.1 g/L
[Method of cultivating mycoplasma within Vero cells]
1) Use at least two cell culture dishes for each of the test
sample, positive control and negative control.
2) Into each cell culture dish (diameter 35 mm), inoculate
2 mL of the Vero cell suspension (1 X 10 4 cells per 1 mL) in
Eagle's minimum essential medium containing 10 percent bo-
vine calf serum (tested in advance using the PCR method to
verify that it does not contain any detectable mycoplasma
DNA). Incubate the cultures at 36 ± 1 °C in an atmosphere of
air containing 5 percent carbon dioxide for one day.
3) Replace the culture media with fresh media, and add
0.5 mL of the test sample (cell culture supernatant) to each of
two or more Vero cell culture dishes. Perform the same
procedure for the positive (such as 100 CFU or less M.
hyorhinis) and negative controls.
4) Incubate the Vero cell culture dishes for the test sam-
ple, positive and negative controls for 3 to 6 days at 36 ± 1°C
in an atmosphere of air containing 5 percent carbon dioxide.
15. Peptide Mapping
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia.
Purpose and Scope
Peptide mapping is an identity test for proteins, especially
those obtained by r-DNA technology. It involves the chemi-
cal or enzymatic treatment of a protein resulting in the for-
mation of peptide fragments followed by separation and
identification of the fragments in a reproducible manner. It is
a powerful test that is capable of identifying single amino
acid changes resulting from events such as errors in the
reading of complementary DNA (cDNA) sequences or point
mutations. Peptide mapping is a comparative procedure
because the information obtained, compared to a reference
standard or reference material similarly treated, confirms the
primary structure of the protein, is capable of detecting
whether alterations in structure have occurred, and demon-
strates process consistency and genetic stability. Each protein
presents unique characteristics which must be well
understood so that the scientific and analytical approaches
permit validated development of a peptide map that provides
sufficient specificity.
This chapter provides detailed assistance in the application
of peptide mapping and its validation to characterize the
desired protein product, to evaluate the stability of the ex-
pression construct of cells used for recombinant DNA
products and to evaluate the consistency of the overall proc-
ess, to assess product stability as well as to ensure the identity
of the protein product, or to detect the presence of protein
variant.
The Peptide Map
Peptide mapping is not a general method, but involves
developing specific maps for each unique protein. Although
the technology is evolving rapidly, there are certain methods
that are generally accepted. Variations of these methods will
be indicated, when appropriate, in specific monographs.
A peptide map may be viewed as a fingerprint of a protein
and is the end product of several chemical processes that pro-
vide a comprehensive understanding of the protein being ana-
lyzed. Four major steps are necessary for the development of
the procedure: isolation and purification of the protein, if the
protein is part of a formulation; selective cleavage of the pep-
tide bonds; chromatographic separation of the peptides; and
analysis and identification of the peptides. A test sample is
digested and assayed in parallel with a reference standard or a
reference material. Complete cleavage of peptide bonds is
more likely to occur when enzymes such as endoproteases
(e.g., trypsin) are used, instead of chemical cleavage rea-
gents. A map should contain enough peptides to be meaning-
ful. On the other hand, if there are too many fragments, the
map might lose its specificity because many proteins will then
have the same profiles.
Isolation and Purification
Isolation and purification are necessary for analysis of bulk
drugs or dosage forms containing interfering excipients and
carrier proteins and, when required, will be specified in the
monograph. Quantitative recovery of protein from the
dosage form should be validated.
Selective Cleavage of Peptide Bonds
The selection of the approach used for the cleavage of pep-
tide bonds will depend on the protein under test. This selec-
tion process involves determination of the type of cleavage to
be employed — enzymatic or chemical — and the type of
JPXV
General Information / Peptide Mapping 1725
cleavage agent within the chosen category. Several cleavage
agents and their specificity are shown in Table 1. This list is
not all-inclusive and will be expanded as other cleavage
agents are identified.
Table 1. Examples of Cleavage Agents
Type
Agent
Specificity
Enzymatic Trypsin (EC 3.4.21.4)
Chymotrypsin
(EC 3.4.21.1)
Pepsin (EC 3.4.23.1 & 2)
Lysyl endopeptidase
(Lys-C Endopeptidase)
(EC 3.4.21.50)
Glutamyl endopeptidase
(from S. aureus strain V8)
(EC 3.4.21.19)
Peptidyl-Asp metallo
endopeptidase
(Endoproteinase Asp-N)
(EC 3.24.33)
Clostripain (EC 3.4.22.8)
C-terminal side of
Arg and Lys
C-terminal side of
hydrophobic
residues (e.g.,
Leu, Met, Ala,
aromatics)
Nonspecific digest
C-terminal side of
Lys
C-terminal side of
Glu and Asp
N-terminal side of
Asp
C-terminal side of
Arg
Chemical Cyanogen bromide
2-Nitro-5-thio-cyanobenzoic
acid
o-Iodosobenzoic acid
Dilute acid
BNPS-skatole
C-terminal side of
Met
N-terminal side of
Cys
C-terminal side of
Trp and Tyr
Asp and Pro
Trp
Pretreatment of Sample Depending on the size or the
configuration of the protein, different approaches in the
pretreatment of samples can be used. For monoclonal
antibodies the heavy and light chains will need to be separat-
ed before mapping. If trypsin is used as a cleavage agent for
proteins with a molecular mass greater than 100,000 Da,
lysine residues must be protected by citraconylation or maley-
lation; otherwise, many peptides will be generated.
Pretreatment of the Cleavage Agent Pretreatment of
cleavage agents — especially enzymatic agents — might be
necessary for purification purposes to ensure reproducibility
of the map. For example, trypsin used as a cleavage agent will
have to be treated with tosyl-L-phenylalanine chloromethyl
ketone to inactivate chymotrypsin. Other methods, such as
purification of trypsin by HPLC or immobilization of en-
zyme on a gel support, have been successfully used when only
a small amount of protein is available.
Pretreatment of the Protein Under certain conditions, it
might be necessary to concentrate the sample or to separate
the protein from added substances and stabilizers used in for-
mulation of the product, if these interfere with the mapping
procedure. Physical procedures used for pretreatment can in-
clude ultrafiltration, column chromatography, and lyophili-
zation. Other pretreatments, such as the addition of
chaotropic agents (e.g., urea) can be used to unfold the pro-
tein prior to mapping. To allow the enzyme to have full ac-
cess to cleavage sites and permit some unfolding of the pro-
tein, it is often necessary to reduce and alkylate the disulfide
bonds prior to digestion.
Digestion with trypsin can introduce ambiguities in the
tryptic map due to side reactions occurring during the
digestion reaction, such as nonspecific cleavage, deamida-
tion, disulfide isomerization, oxidation of methionine
residues, or formation of pyroglutamic groups created from
the deamidation of glutamine at the N-terminal side of a pep-
tide. Furthermore, peaks may be produced by autohydrolysis
of trypsin. Their intensities depend on the ratio of trypsin to
protein. To avoid autohydrolysis, solutions of proteases may
be prepared at a pH that is not optimal (e.g., at pH 5 for
trypsin), which would mean that the enzyme would not
become active until diluted with the digest buffer.
Establishment of Optimal Digestion Conditions Factors
that affect the completeness and effectiveness of digestion of
proteins are those that could affect any chemical or enzymatic
reactions.
pH: The pH of the digestion mixture is empirically deter-
mined to ensure the optimization of the performance of the
given cleavage agent. For example, when using cyanogen
bromide as a cleavage agent, a highly acidic environment
(e.g., pH 2, formic acid) is necessary; however, when using
trypsin as a cleavage agent, a slightly alkaline environment
(pH 8) is optimal. As a general rule, the pH of the reaction
milieu should not alter the chemical integrity of the protein
during the digestion and should not change during the course
of the fragmentation reaction.
Temperature: A temperature between 25 °C and 37°C is
adequate for most digestions. The temperature used is
intended to minimize chemical side reactions. The type of
protein under test will dictate the temperature of the reaction
milieu, because some proteins are more susceptible to denatu-
ration as the temperature of the reaction increases. For exam-
ple, digestion of recombinant bovine somatropin is conduct-
ed at 4°C, because at higher temperatures it will precipitate
during digestion.
Time: If sufficient sample is available, a time course
study is considered in order to determine the optimum time to
obtain a reproducible map and avoid incomplete digestion.
Time of digestion varies from 2 to 30 hours. The reaction is
stopped by the addition of an acid which does not interfere in
the tryptic map or by freezing.
Amount of Cleavage Agent: Although excessive amounts
of cleavage agent are used to accomplish a reasonably rapid
digestion time (i.e., 6 to 20 hours), the amount of cleavage a-
gent is minimized to avoid its contribution to the chromato-
graphic map pattern. A protein to protease ratio between
20:1 and 200:1 is generally used. It is recommended that the
cleavage agent can be added in two or more stages to op-
timize cleavage. Nonetheless, the final reaction volume
remains small enough to facilitate the next step in peptide
mapping — the separation step. To sort out digestion artifacts
that might be interfering with the subsequent analysis, a
blank determination is performed, using a digestion control
with all the reagents, except the test protein.
Chromatographic Separation
Many techniques are used to separate peptides for
1726 Peptide Mapping / General Information
JP XV
mapping. The selection of a technique depends on the protein
being mapped. Techniques that have been successfully used
for separation of peptides are shown in Table 2. In this sec-
tion, a most widely used reverse-phase High Performance
Liquid Chromatographic (RP-HPLC) method is described as
one of the procedures of chromatographic separation.
Table 2. Techniques Used for the Separation of Peptides
Reverse-Phase High Performance Liquid Chromatography
(RP-HPLC)
Ion-Exchange Chromatography (IEC)
Hydrophobic Interaction Chromatography (HIC)
Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat-
ing
SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Capillary Electrophoresis (CE)
Paper Chromatography-High Voltage (PCHV)
High-Voltage Paper Electrophoresis (HVPE)
The purity of solvents and mobile phases is a critical factor
in HPLC separation. HPLC-grade solvents and water that
are commercially available, are recommended for RP-
HPLC. Dissolved gases present a problem in gradient
systems where the solubility of the gas in a solvent may be less
in a mixture than in a single solvent. Vacuum degassing and
agitation by sonication are often used as useful degassing
procedures. When solid particles in the solvents are drawn
into the HPLC system, they can damage the sealing of pump
valves or clog the top of the chromatographic column. Both
pre- and post-pump filtration is also recommended.
Chromatographic Column The selection of a chromato-
graphic column is empirically determined for each protein.
Columns with 100 A or 300 A pore size with silica support
can give optimal separation. For smaller peptides, octylsilane
chemically bonded to totally porous silica articles, 3 to 10 /xm
in diameter (L7) and octadecylsilane chemically bonded to
porous silica or ceramic micro-particles, 3 to 10 /xm in di-
ameter (LI) column packings are more efficient than the butyl
silane chemically bonded to totally porous silica particles, 5
to 10 ^m in diameter (L26) packing.
Solvent The most commonly used solvent is water with
acetonitrile as the organic modifier to which less than 0.1%
trifluoroacetic acid is added. If necessary, add isopropyl
alcohol or n-propyl alcohol to solubilize the digest compo-
nents, provided that the addition does not unduly increase
the viscosity of the components.
Mobile Phase Buffered mobile phases containing phos-
phate are used to provide some flexibility in the selection of
pH conditions, since shifts of pH in the 3.0 to 5.0 range
enhance the separation of peptides containing acidic residues
(e.g., glutamic and aspartic acids). Sodium or potassium
phosphates, ammonium acetate, phosphoric acid, and a pH
between 2 and 7 (or higher for polymer-based supports) have
also been used with acetonitrile gradients. Acetonitrile con-
taining trifluoroacetic acid is used quite often.
Gradient Selection Gradients can be linear, nonlinear, or
include step functions. A shallow gradient is recommended in
order to separate complex mixtures. Gradients are optimized
to provide clear resolution of one or two peaks that will
become "marker" peaks for the test.
Isocratic Selection Isocratic HPLC systems using a single
mobile phase are used on the basis of their convenience of use
and improved detector responses. Optimal composition of a
mobile phase to obtain clear resolution of each peak is some-
times difficult to establish. Mobile phases for which slight
changes in component ratios or in pH significantly affect
retention times of peaks in peptide maps should not be used
in isocratic HPLC systems.
Other Parameters Temperature control of the column is
usually necessary to achieve good reproducibility. The flow
rates for the mobile phases range from 0.1 to 2.0 mL per
minute, and the detection of peptides is performed with a UV
detector at 200 to 230 nm. Other methods of detection have
been used (e.g., postcolumn derivatization), but they are not
as robust or versatile as UV detection.
Validation This section provides an experimental
means for measuring the overall performance of the test
method. The acceptance criteria for system suitability depend
on the identification of critical test parameters that affect data
interpretation and acceptance. These critical parameters are
also criteria that monitor peptide digestion and peptide anal-
ysis. An indicator that the desired digestion endpoint was
achieved is by the comparison with a Reference Standard,
which is treated exactly as the article under test. The use of a
reference standard or reference material in parallel with the
protein under test is critical in the development and establish-
ment of system suitability limits. In addition a specimen
chromatogram should be included with the Reference Stan-
dard or Reference Material for additional comparison pur-
poses. Other indicators may include visual inspection of pro-
tein or peptide solubility, the absence of intact protein, or
measurement of responses of a digestion-dependent peptide.
The critical system suitability parameters for peptide analysis
will depend on the particular mode of peptide separation and
detection and on the data analysis requirements.
When peptide mapping is used as an identification test, the
system suitability requirements for the identified peptides
covers selectivity and precision. In this case, as well as when
identification of variant protein is done, the identification of
the primary structure of the peptide fragments in the peptide
map provides both a verification of the known primary struc-
ture and the identification of protein variants by comparison
with the peptide map of the reference standard/reference
material for the specified protein. The use of a digested refer-
ence standard or reference material for a given protein in the
determination of peptide resolution is the method of choice.
For an analysis of a variant protein, a characterized mixture
of a variant and a reference standard or reference material
can be used, especially if the variant peptide is located in a
less-resolved region of the map. The index of pattern con-
sistency can be simply the number of major peptides detect-
ed. Peptide pattern consistency can be best defined by the
resolution of peptide peaks. Chromatographic parameters
— such as peak-to-peak resolution, maximum peak width,
peak area, peak tailing factors, and column efficiency — may
be used to define peptide resolution. Depending on the pro-
tein under test and the method of separation used, single pep-
tide or multiple peptide resolution requirements may be
necessary.
The replicate analysis of the digest of the reference
standard or reference material for the protein under test
yields measures of precision and quantitative recovery.
Recovery of the identified peptides is generally ascertained by
the use of internal or external peptide standards. The
JPXV
General Information / pH Test for Gastrointestinal 1727
precision is expressed as the relative standard deviation
(RSD). Differences in the recovery and precision of the
identified peptides are expected; therefore, the system
suitability limits will have to be established for both the
recovery and the precision of the identified peptides. These
limits are unique for a given protein and will be specified in
the individual monograph.
Visual comparison of the relative retention times, the peak
responses (the peak area or the peak height), the number of
peaks, and the overall elution pattern is completed initially. It
is then complemented and supported by mathematical analy-
sis of the peak response ratios and by the chromatographic
profile of a 1:1 (v/v) mixture of sample and reference stan-
dard or reference material digest. If all peaks in the sample
digest and in the reference standard or reference material
digest have the same relative retention times and peaks
response ratios, then the identity of the sample under test is
confirmed.
If peaks that initially eluted with significantly different rela-
tive retention times are then observed as single peaks in the
1:1 mixture, the initial difference would be an indication of
system variability. However, if separate peaks are observed
in the 1:1 mixture, this would be evidence of the nonequiva-
lence of the peptides in each peak. If a peak in the 1 : 1 mixture
is significantly broader than the corresponding peak in the
sample and reference standard or reference material digest, it
may indicate the presence of different peptides. The use of
computer-aided pattern recognition software for the analysis
of peptide mapping data has been proposed and applied, but
issues related to the validation of the computer software
preclude its use in a compendial test in the near future. Other
automated approaches have been used that employ mathe-
matical formulas, models, and pattern recognition. Such ap-
proaches are, for example, the automated identification of
compounds by IR spectroscopy and the application of diode-
array UV spectral analysis for identification of peptides.
These methods have limitations due to inadequate resolu-
tions, co-elution of fragments, or absolute peak response
differences between reference standard or reference material
and sample fragments.
The numerical comparison of the retention times and peak
areas or peak heights can be done for a selected group of
relevant peaks that have been correctly identified in the
peptide maps. Peak areas can be calculated using one peak
showing relatively small variation as an internal reference,
keeping in mind that peak area integration is sensitive to
baseline variation and likely to introduce error in the
analysis. Alternatively, the percentage of each peptide peak
height relative to the sum of all peak heights can be calculated
for the sample under test. The percentage is then compared to
that of the corresponding peak of the reference stan-
dard/reference material. The possibility of auto-hydrolysis
of trypsin is monitored by producing a blank peptide map,
that is, the peptide map obtained when a blank solution is
treated with trypsin.
The minimum requirement for the qualification of peptide
mapping is an approved test procedure that includes system
suitability as a test control. In general, early in the regulatory
process, qualification of peptide mapping for a protein is
sufficient. As the regulatory approval process for the protein
progresses, additional qualifications of the test can include a
partial validation of the analytical procedure to provide
assurance that the method will perform as intended in the de-
velopment of a peptide map for the specified protein.
Analysis and Identification of Peptides
This section gives guidance on the use of peptide mapping
during development in support of regulatory applications.
The use of a peptide map as a qualitative tool does not
require the complete characterization of the individual
peptide peaks. However, validation of peptide mapping in
support of regulatory applications requires rigorous cha-
racterization of each of the individual peaks in the peptide
map. Methods to characterize peaks range from N-terminal
sequencing of each peak followed by amino acid analysis to
the use of mass spectroscopy (MS).
For characterization purposes, when N-terminal sequenc-
ing and amino acids analysis are used, the analytical separa-
tion is scaled up. Since scale-up might affect the resolution of
peptide peaks, it is necessary, using empirical data, to assure
that there is no loss of resolution due to scale-up. Eluates cor-
responding to specific peptide peaks are collected, vacuum-
concentrated, and chromatographed again, if necessary.
Amino acid analysis of fragments may be limited by the pep-
tide size. If the N-terminus is blocked, it may need to be
cleared before sequencing. C-terminal sequencing of proteins
in combination with carboxypeptidase and MALDITOF-MS
can also be used for characterization purposes.
The use of MS for characterization of peptide fragments is
by direct infusion of isolated peptides or by the use of on-line
LC-MS for structure analysis. In general, it includes elec-
trospray and matrix-assisted laser desorption ionization cou-
pled to time-of-flight analyzer (MALDITOF) as well as fast
atom bombardment (FAB). Tandem MS has also been used
to sequence a modified protein and to determine the type of
amino acid modification that has occurred. The comparison
of mass spectra of the digests before and after reduction pro-
vides a method to assign the disulfide bonds to the various
sulfhydryl-containing peptides.
If regions of the primary structure are not clearly demon-
strated by the peptide map, it might be necessary to develop a
secondary peptide map. The goal of a validated method of
characterization of a protein through peptide mapping is to
reconcile and account for at least 95% of the theoretical com-
position of the protein structure.
16. pH Test for Gastrointestinal
Medicine
In this test, medicine for the stomach and bowels, which is
said to control stomach acid, is stirred in a fixed amount of
the 0.1 mol/L hydrochloric acid for a fixed duration, and the
pH value of this solution is obtained. The pH value of a
stomach medicine will be based on the dose and the dosage of
the medicine (when the dosage varies, a minimum dosage is
used) and expressed in the pH value obtained from the test
performed by the following procedure.
Preparation of Sample
Solid medicine which conforms to the general regulations
for medicine (the powdered medicine section) can be used as
a sample. When the medicine is in separate packages, the
content of 20 or more packages is accurately weighed to cal-
culate the average mass for one dose and mixed evenly to
1728 Plastic Containers / General Information
JP XV
make a sample. For granules and similar types in separate
packages, among the solid medicine which does not conform
to the general regulations for medicine (the powdered medi-
cine section), the content of 20 or more packages is accurately
weighed to calculate the average mass for one dose and is
then powdered to make sample. For granules and similar
types not in separate packages, among solid medicine which
does not conform to the general regulations for medicine (the
powdered medicine section), 20 doses or more are powdered
to make a sample. For capsules and tablets, 20 doses or more
are weighed accurately to calculate the average mass for one
dose or average mass and then powdered to make a sample.
Liquid medicine is generously mixed to make a sample.
Procedure
Put 50 mL of the 0.1 mol/L hydrochloric acid with the
molarity coefficient adjusted to 1.000, or equivalent 0.1 mol/
L hydrochloric acid with its volume accurately measured in a
100-mL beaker. Stir this solution with a magnetic stirrer and
a magnetic stirrer rotator (35 mm length, 8 mm diameter) at
the speed of about 300 revolutions per minute. While stirring,
add the accurately weighed one-dose sample. After 10
minutes, measure the pH value of the solution using the pH
Determination. The solution temperature should be main-
tained at 37±2°C throughout this operation.
17. Plastic Containers for
Pharmaceutical Products
Various kinds of plastics are used in the manufacture of
containers for pharmaceutical products. Such plastics should
not alter the efficacy, safety or stability of the pharmaceutical
products. In selecting a suitable plastic container, it is desira-
ble to have full information on the manufacturing processes
of the plastic container including the substances added. Since
each plastic has specific properties and a wide variety of phar-
maceutical products may be stored in containers made from
it, the compatibility of plastic containers with pharmaceutical
products should be judged for each combination of container
and the specific pharmaceutical product to be contained
therein. This judgement should be carried out by verifying
that a type sample of the container for the pharmaceutical
preparation fulfills the essential requirements, i.e., the design
specifications, according to experiments and/or scientific
documentation, etc. In addition, the compatibility must be
ensured based upon an appropriate quality assurance system.
Furthermore, in introducing a plastic container, it is
desirable that proper disposal after use is taken into consider-
ation.
Essential Requirements in Designing Plastic Containers for
Pharmaceutical Products
The plastic material for the container should be of high
quality. Therefore, recycled plastic materials, which are of
unknown constitution, must not be used.
The leachables or migrants from the container should not
alter the efficacy or stability of the pharmaceutical products
contained therein. In addition, the possible toxic hazards of
the leachables or migrants should not exceed a given level.
Furthermore, the amounts of leachable or migratable chemi-
cal substances, such as monomers and additives, from the
containers to the pharmaceutical products contained therein
must be sufficiently small from the viewpoint of safety.
The container should have a certain level of physical
properties such as hardness, flexibility, shock resistance, ten-
sile strength, tear strength, bending strength, heat resistance
and the like, in accordance with the intended usage.
The quality of the pharmaceutical products contained in
the container must not deteriorate during storage. For exam-
ple, in the case of pharmaceutical products which are unsta-
ble to light, the container should provide a sufficient level of
light shielding. In the case of pharmaceutical products which
are easily oxidized, the container material should not allow
the permeation of oxygen. In the case of aqueous pharmaceu-
tical products and pharmaceutical products that must be kept
dry, the container material should not allow the permeation
of water vapor. In addition, care should be taken that the
container is impermeable to the solvent in the case of solvents
other than water. The concentration of the pharmaceuticals
must not be decreased by more than a certain level due to the
absorption of the pharmaceuticals on the surface of the con-
tainer, the migration of the pharmaceuticals into the inside of
the material of the container, or the loss of pharmaceuticals
through the container. Also, the pharmaceutical products
contained therein must not be degraded by an interaction
with the material of the container.
The container should not be deformed, should not de-
teriorate and should not be degraded by the pharmaceutical
products contained therein. Unacceptable loss of function of
the container should not result from possible high tempera-
ture or low temperature or cycles thereof encountered during
storage or transportation.
The container should be of a required level of transparen-
cy, when it is necessary to examine foreign insoluble matter
and/or turbidity of the pharmaceutical products by visual ob-
servation.
In the case of pharmaceutical products which must be steri-
lized, it is required to satisfy the above-mentioned essential
requirements of the container after the sterilization if there is
a possibility that the quality of the container may change
after the sterilization. There should not be any residue or
generation of new toxic substances of more than certain risk
level after the sterilization. In addition, the container should
not have any inappropriate structure and/or material that
might result in any bacterial contamination of the phar-
maceutical products contained therein during storage and
transportation after sterilization.
Toxicity Evaluation of Container at Design Phase
For design verification, the toxicity of the container should
be evaluated. For the toxicity evaluation, it is desirable to
select appropriate test methods and criteria for the evalua-
tion, and to clarify the rationales for the selection. The tests
should be conducted using samples of the whole or a part of
the prototype container. If the container consists of plural
parts of different materials, each part should be tested
separately. Such materials as laminates, composites, and like
are regarded as a single material. To test containers made of
such materials, it is recommended to expose the inner surface
of the container, which contacts the pharmaceutical products
contained therein, to the extraction media used in the tests as
far as possible.
The tests required for the toxicity evaluation of the con-
tainer are different depending upon the tissue to which the
pharmaceutical products contained therein are to be applied.
JPXV
General Information / Powder Flow 1729
The following tests are required for containers for
1) preparations contacting blood:
Acute toxicity test, cytotoxicity test, sensitization test
and hemolysis test
2) preparations contacting skin or mucous membranes:
Cytotoxicity test and sensitization test
3) liquid orally administered preparations:
Cytotoxicity test
It is recommended to conduct the tests in accordance with
the latest versions of the standard test methods on medical
devices and materials published in Japan and other countries.
Those standard test methods are listed for information:
(A) Selection of Tests
•Guidelines for Basic Biological Tests of Medical Devices
and Materials (PAB Notification, YAKU-KI NO. 99, June 27,
1995), Principles and selection of tests
• ISO 10993-1: Biological evaluation of medical
devices — Evaluation and testing
(B) Acute Toxicity Test
• ASTM F750-82: Standard practice for evaluating material
extracts by systemic injection in the mice
•BS5736: Part 3 Method of test for systemic toxicity; as-
sessment of acute toxicity of extracts from medical devices
•USP 24 <88> Biological reactivity tests, in vivo
(C) Cytotoxicity Test
•Guidelines for Basic Biological Tests of Medical Devices
and Materials, I. Cytotoxicity Test 10. Cytotoxicity test using
extract of medical device or material
• ISO 10993-5: Biological evaluation of medical
devices — Tests for cytotoxicity : in vitro methods
•USP 24 <87> Biological reactivity tests, in vitro
(D) Hemolysis Test
•Guidelines for Basic Biological Tests of Medical Devices
and Materials, VII. Hemolysis Test
• ISO 10993-4: Biological evaluation of medical
devices — Selection of tests for interaction with blood. Annex
D
•ASTM F756-82: Standard practice for assessment of
hemolytic properties of materials
(E) Sensitization Test
•Guidelines for Basic Biological Tests of Medical Devices
and Materials, II. Sensitization Test
•ISO 10993-10: Biological evaluation of medical
devices — Tests for irritation and sensitization
Test Results to be Recorded per Production Unit
At the line production phase, it is required to establish the
values of acceptable limits on at least the test items men-
tioned below and to record the test results of each production
unit of plastic containers for pharmaceutical products. In ad-
dition, it is desirable to clarify the rationales for setting the
values of limits. However, these requirements should not be
applied to orally administered preparations except liquid
ones.
1) Combustion Tests: Residue of ignition, heavy metals.
If necessary, the amounts of the specified metals (lead, cad-
mium, etc.)
2) Extraction Tests: pH, ultraviolet absorption spectra,
potassium permanganate-reducing substances, foaming,
non-volatile residue
3) Cytotoxicity Test
4) Any other necessary tests for the specific container for
aqueous infusions.
18. Powder Flow
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia. The parts of the text that are not
harmonized are marked with symbols (* ♦).
Four commonly reported methods for testing powder flow
are (1) angle of repose, (2) compressibility index of Hausner
ratio, (3) flow rate through an orifice, and (4) shear cell. In
addition, numerous variations of each of these basic methods
are available.
In general, any method of measuring powder flow should
be practical, useful, reproducible, sensitive, and yield
meaningful results. It bears repeating that no one simple
powder flow method will adequately or completely character-
ize the wide range of flow properties. An appropriate strategy
may well be the use of multiple standardized test methods to
characterize the various aspects of powder flow as needed by
the pharmaceutical scientist.
1. Angle of Repose
The angle of repose is the constant, three-dimensional an-
gle (relative to the horizontal base) assumed by a cone-like
pile of material formed by any of several different methods.
The angle of repose has been used in several branches of
science to characterize the flow properties of solids. Angle of
repose is a characteristic related to interparticulate friction,
or resistance to movement between particles. Angle of repose
test results are reported to be very dependent upon the
method used. Experimental difficulties arise due to segrega-
tion of material and consolidation or aeration of the powder
as the cone is formed.
1.1 Basic Methods for Angle of Repose
A variety of angle of repose test methods are reported in
the literature. The most common methods for determining
the static angle of repose can be classified based on two im-
portant experimental variables:
(1) The height of the "funnel" through which the powder
passes may be fixed relative to the base, or the height may be
varied as the pile forms.
(2) The base upon which the pile forms may be of fixed
diameter or the diameter of the powder cone may be allowed
to vary as the pile forms.
1.2 Variations in Angle of Repose Methods
In addition to the above methods, variations of them have
been used to some extent.
•Drained angle of repose. This is determined by allowing an
excess quantity of material positioned above a fixed di-
ameter base to "drain" from the container. Formation of a
cone of powder on the fixed diameter base allows determina-
tion of the drained angle of repose.
•Dynamic angle of repose. This is determined by filling a
cylinder (with a clear, flat cover on one end) and rotating it
at a specified speed. The dynamic angle of repose is the an-
gle (relative to the horizontal) formed by the flowing pow-
der. The internal angle of kinetic friction is defined by the
plane separating those particles sliding down the top layer of
the powder and those particles that are rotating with the
drum (with roughened surface).
1.3 Angle of Repose General Scale of Flowability
1730 Powder Flow / General Information
JP XV
While there is some variation in the qualitative description
of powder flow using the angle of repose, much of the phar-
maceutical literature appears to be consistent with the classifi-
cation by Carr 1 , which is shown in Table 1. There are exam-
ples of formulations with an angle of repose in the range of
40 to 50 degrees that manufactured satisfactorily. When the
angle of repose exceeds 50 degrees, the flow is rarely accepta-
ble for manufacturing purposes.
Table 1 Flow Properties and Corresponding
Angles of Repose 1 '
Flow Property
Excellent
Good
Fair
Passable
Poor
Very poor
Very, very poor
Angle of Repose (degrees)
25-30
31 -35
36-40
41 -45
46-55
56-65
>66
1.4 Experimental Considerations for Angle of Repose
Angle of repose is not an intrinsic property of the powder,
that is to say, it is very much dependent upon the method
used to form the cone of powder. On this subject, the existing
literature raises these important considerations:
•The peak of the cone of powder can be distorted by the im-
pact of powder from above. By carefully building the pow-
der cone, the distortion caused by impact can be minimized.
•The nature of the base upon which the powder cone is
formed influences the angle of repose. It is recommended
that the powder cone be formed on a "common base",
which can be achieved by forming the cone of powder on a
layer of powder. This can be done by using a base of fixed
diameter with a protruding outer edge to retain a layer of
powder upon which the cone is formed.
1.5 Recommended Procedure for Angle of Repose
Form the angle of repose on a fixed base with a retaining
lip to retain a layer of powder on the base. The base should
be free of vibration. Vary the height of the funnel to carefully
build up a symmetrical cone of powder. Care should be taken
to prevent vibration as the funnel is moved. The funnel
height should be maintained approximately 2-4 cm from the
top of the powder pile as it is being formed in order to
minimize the impact of falling powder on the tip of the cone.
If a symmetrical cone of powder cannot be successfully or
reproducibly prepared, this method is not appropriate. De-
termine the angle of repose by measuring the height of the
cone of powder and calculating the angle of repose, a, from
the following equation:
tan a = height/0.5 base
2. Compressibility Index and Hausner Ratio
In recent years the compressibility index and the closely
related Hausner ratio have become the simple, fast and popu-
lar methods of predicting powder flow characteristics. The
compressibility index bas been proposed as an indirect meas-
ure of bulk density, size and shape surface area, moisture
content, and cohesiveness of materials because all of these
can influence the observed compressibility index. The com-
pressibility index and the Hausner ratio are determined by
measuring both the bulk volume and tapped volume of a
powder.
2.1 Basic Methods for Compressibility Index and Haus-
ner Ratio
While there are some variations in the method of determin-
ing the compressibility index and Hausner ratio, the basic
procedure is to measure (1) the unsettled apparent volume,
V ot and (2) the final tapped volume, V f , of the powder after
tapping the material until no further volume changes occur
(refer to the General Test Method, 3.01 Determination of
Bulk and Tapped Densities). The compressibility index and
the Hausner ratio are calculated as follows:
Compressibility Index = 100 x {{V - V t )/V }
Hausner Ratio = VJ V t
Alternatively, the compressibility index and Hausner ratio
may be calculated using measured values for bulk density
(p B ) and tapped density (p T ) as follows:
Compressibility Index = 100 x {(p T — p B ) /p T }
Hausner Ratio =p T //? B
In a variation of these methods, the rate of consolidation is
sometimes measured rather than, or in addition to, the
change in volume that occurs on tapping. For the compres-
sibility index and the Hausner ratio, the generally accepted
scale of flowability is given in Table 2.
Table 2. Scale of Flowability 1 *
ndex (%)
Flow Character
Hausner Ratio
=£10
Excellent
1.00- 1.11
11 -15
Good
1.12- 1.18
16-20
Fair
1.19- 1.25
21 -25
Passable
1.26- 1.34
26-31
Poor
1.35- 1.45
32-37
Very poor
1.46- 1.59
♦a38»
Very, very poor
> 1.60
2.2 Experimental Considerations for the Compressibility
Index and Hausner Ratio
Compressibility index and Hausner ratio are not intrinsic
properties of the powder, that is to say, they are dependent
upon the methodology used. The existing literature points
out several important considerations affecting the determina-
tion of the (1) unsettled apparent volume, V , (2) the final
tapped volume, V t , (3) the bulk density, p B , and (4) the
tapped density, p T :
•The diameter of the cylinder used
•The number of times the powder is tapped to achieve the
tapped density
•The mass of material used in the test
•Rotation of the sample during tapping
2.3 Recommended Procedure for Compressibility Index
and Hausner Ratio
Use a 250-mL volumetric cylinder with a test sample weight
of 100 grams. Smaller weights and volumes may be used, but
weight of the test sample and volume of the cylinder used
should be described with the results. An average of three de-
terminations is recommended.
JPXV
General Information / Powder Flow 1731
3. Flow through an Orifice
The flow rate of a material depends upon many factors,
some of which are particle-related and some related to the
process. Monitoring the rate of flow of material through an
orifice has been proposed as a better measure of powder
flowability. Of particular significance is the utility of
monitoring flow continuously since pulsating flow patterns
have been observed even for free flowing materials. Changes
in flow rate as the container empties can also be observed.
Empirical equations relating flow rate to the diameter of the
opening, particle size, and particle density have been deter-
mined. However, determining the flow rate through an orifice
is useful only with free-flowing materials.
The flow rate through an orifice is generally measured as
the mass per time flowing from any of a number of types of
containers (cylinders, funnels, hoppers). Measurement of the
flow rate can be in discrete increments or continuous.
3.1 Basic Methods for Flow through an Orifice
There are a variety of methods described in the literature.
The most common for determining the flow rate through an
orifice can be classified based on three important experimen-
tal variables:
(1) The type of container used to contain the powder.
Common containers are cylinders, funnels and hoppers from
production equipment.
(2) The size and shape of the orifice used. The orifice di-
ameter and shape are critical factors in determining powder
flow rate.
(3) The method of measuring powder flow rate. Flow rate
can be measured continuously using an electronic balance
and with some sort of recording device (strip chart recorder,
computer). It can also be measured in discrete samples (for
example, the time it takes for 100 grams of powder to pass
through the orifice to the nearest tenth of a second or the
amount of powder passing through the orifice in 10 seconds
to the nearest tenth of a gram).
3.2 Variations in Methods for Flow through an Orifice
Either mass flow rate or volume flow rate can be deter-
mined. Mass flow rate is the easier of the methods, but it
biases the results in favor of high-density materials. Since die
fill is volumetric, determining volume flow rate may be
preferable.
3.3 General Scale of Flowability for Flow through an
Orifice
No general scale is available because flow rate is critically
dependent on the method used to measure it.
3.4 Experimental Considerations for Flow through an
Orifice
Flow rate through an orifice is not an intrinsic property of
the powder. It is very much dependent upon the methodology
used. The existing literature points out several important con-
siderations affecting these methods:
•The diameter and shape of the orifice
•The type of container material (metal, glass, plastic)
•The diameter and height of the powder bed.
3.5 Recommended Procedure for Flow through an
Orifice
Flow rate through an orifice can be used only for materials
that have some capacity to flow. It is not useful for cohesive
materials. Provided that the height of the powder bed (the
'head' of powder) is much greater than the diameter of the
orifice, the flow rate is virtually independent of the powder
head. Use a cylinder as the container because the cylinder
material should have little effect on flow. Powder flow rate
often increases when the height of the powder column is less
than two times the diameter of the column. The orifice should
be circular and the cylinder should be free of vibration.
General guidelines for dimensions of the cylinder are as fol-
lows:
•Diameter of opening >6 times the diameter of the particles
• Diameter of the cylinder > 2 times the diameter of the open-
ing
Use of a hopper as the container may be appropriate and
representative of flow in a production situation. It is not ad-
visable to use a funnel, particularly one with a stem, because
flow rate will be determined by the size and length of the stem
as well as the friction between the stem and the powder. A
truncated cone may be appropriate, but flow will be in-
fluenced by the powder — wall friction coefficient, thus, selec-
tion of an appropriate construction material is important.
For the opening in the cylinder, use a flat-faced bottom
plate with the option to vary orifice diameter to provide maxi-
mum flexibility and better ensure a powder-over-powder flow
pattern. Rate measurement can be either discrete or continu-
ous. Continuous measurement using an electronic balance
can more effectively detect momentary flow rate variations.
4. Shear Cell Methods
A variety of powder shear testers and methods that permit
more thorough and precisely defined assessment of powder
flow properties have been developed. Shear cell methodology
has been used extensively in the study of pharmaceutical
materials. From these methods, a wide variety of parameters
can be obtained, including the yield loci representing the
shear stress-shear strain relationship, the angle of internal
friction, the unconfined yield strength, the tensile strength,
and a variety of derived parameters such as the flow factor
and other flowability indices. Because of the ability to more
precisely control experimental parameters, flow properties
can also be determined as a function of consolidation load,
time, and other environmental conditions.
4.1 Basic Methods for Shear Cell
One type of shear cell is the cylindrical shear cell which is
split horizontally, forming a shear plane between the lower
stationary base and the upper movable portion of the shear
cell ring. After powder bed consolidation in the shear cell (us-
ing a well-defined procedure), the force necessary to shear the
powder bed by moving the upper ring is determined. Annular
shear cell designs offer some advantages over the cylindrical
shear cell design, including the need for less material. A dis-
advantage, however, is that because of its design, the powder
bed is not sheared as uniformly because material on the out-
side of the annulus is sheared more than material in the inner
region. A third type of shear cell (plate-type) consists of a
thin sandwich of powder between a lower stationary rough
surface and an upper rough surface that is moveable.
All of the shear cell methods have their advantages and dis-
advantages. A significant advantage of shear cell methodolo-
gy in general is a greater degree of experimental control. The
methodology generally is rather time-consuming and requires
significant amounts of material and a well-trained operator.
4.2 Recommendations for Shear Cell
1732 Preservatives-Effectiveness / General Information
JP XV
Because of the diversity of available equipment and ex-
perimental procedures, no specific recommendations regard-
ing methodology are presented in this chapter. It is recom-
mended that the results of powder flow characterization using
shear cell methodology include a complete description of eq-
uipment and methodology used.
References
1) Carr R.L.: Evaluating flow properties of solids. Chem.
Eng., 72: 163-168(1965).
19. Preservatives-Effectiveness
Tests
The purpose of the Preservatives-Effectiveness Tests is to
assess microbiologically the preservative efficacy, either due
to the action of product components themselves or any added
preservative(s), for multi-dose containers. The efficacy of the
preservatives is assessed by direct inoculation and mixing of
the test strains in the product, and titration of survival of the
test strains with time.
Preservatives must not be used solely to comply with GMP
for drugs or to reduce viable aerobic counts. In addition,
preservatives themselves are toxic substances. Therefore,
preservatives must not be added to products in amounts
which might jeopardize the safety of human beings, and con-
sideration must be given to minimizing the amounts of
preservative used. These tests are commonly used to verify
that products maintain their preservative effectiveness at the
design phase of formulation or in the case of periodic
monitoring. Although these tests are not performed for lot
release testing, the efficacy of the preservative present in the
product packaged in the final containers should be verified
throughout the entire dating period.
1. Products and their Categories
The products have been divided into two categories for
these tests. Category I products are those made with aqueous
bases or vehicles, and Category II products are those made
with nonaqueous bases or vehicles. Oil-in-water emulsions
are considered Category I products, and water-in-oil emul-
sions Category II products. Category I is further divided into
three subtypes depending on the dosage forms.
Category IA: Injections and other sterile parenterals
Category IB: Nonsterile parenterals
Category IC: Oral products made with aqueous bases (in-
cluding syrup products to be dissolved or suspended before
use)
Category II: All the dosage forms listed under Category I
made with nonaqueous bases or vehicles.
2. Test Microorganisms and Culture Media
The following strains or those considered to be equivalent
are used as the test microorganisms.
Escherichia coli ATCC 8739, NBRC 3972
Pseudomonas aeruginosa ATCC 9027, NBRC 13275
Staphylococcus aureus ATCC 6538, NBRC 13276
Candida albicans ATCC 10231, NBRC 1594, JCM 2085
Aspergillus niger ATCC 16404, NBRC 9455
These test microorganisms are representative of those that
might be found in the environment in which the product is
manufactured, used or stored, and they are also recognized
as opportunistic pathogens. In addition to these strains desig-
nated as test microorganisms, it is further recommended to
use strains that might contaminate the product and grow on
or in it, depending on its characteristics. For the test microor-
ganisms received from coordinated collections of microor-
ganisms, one passage is defined as the transfer of microor-
ganisms from an established culture to fresh medium, and
microorganisms subjected to not more than five passages
should be used for the tests. Single-strain challenges rather
than mixed cultures should be used. The test strains can be
harvested by growth on solid agar or liquid media.
Cultures on agar plate media: Inoculate each of the five test
strains on the surface of agar plates or agar slants. For
growth of bacteria, use Soybean-Casein Digest Agar Medi-
um, and for yeasts and moulds, use Sabouraud Agar,
Glucose-Peptone Agar or Potato Dextrose Agar Medium. In-
cubate bacterial cultures at 30°C to 35°C for 18 to 24 hours,
the culture of C. albicans at 20°C to 25 °C for 40 to 48 hours
and the culture of A. niger at 20°C to 25 C C for one week or
until good sporulation is obtained. Harvest these cultured
cells aseptically using a platinum loop, etc. Suspend the col-
lected cells in sterile physiological saline or in 0.1% peptone
water and adjust the viable cell count to about 10 8 microor-
ganisms per mL. In the case of A. niger, suspend the cultured
cells in sterile physiological saline or 0.1% peptone water
containing 0.05 w/v% of polysorbate 80 and adjust the spore
count to about 10 8 per mL. Use these suspensions as the inoc-
ula.
Liquid cultures: After culturing each of the four strains ex-
cept for A. niger in a suitable medium, remove the medium
by centrifugation. Wash the cells in sterile physiological sa-
line or 0.1% peptone water and resuspend them in the same
solution with the viable cell or spore count of the inoculum
adjusted to about 10 8 per mL.
When strains other than the five listed above are cultured,
select a culture medium suitable for growth of the strain con-
cerned. The cell suspension may also be prepared by a
method suitable for that strain. Use the inoculum suspen-
sions within 24 hours after they have been prepared from the
cultivations on agar plate media or in liquid media. Store the
inoculum suspensions in a refrigerator if it is not possible to
inoculate them into the test specimens within 2 hours. Titrate
the viable cell count of the inocula immediately before use,
and then calculate the theoretical viable cell count per mL (g)
of the product present just after inoculation.
3. Test Procedure
3.1 Category I products
Inject each of the cell suspensions aseptically into five con-
tainers containing the product and mix uniformly. When it is
difficult to inject the cell suspension into the container asepti-
cally or the volume of the product in each container is too
small to be tested, transfer aseptically a sufficient volume of
the product into each of alternative sterile containers, and
mix the inoculum. When the product is not sterile, incubate
additional containers containing the uninoculated product as
controls and calculate their viable cell counts (the viable
counts of bacteria and those of yeasts and moulds). A sterile
syringe, spatula or glass rod may be used to mix the cell sus-
pension uniformly in the product. The volume of the suspen-
sion mixed in the product must not exceed 1/100 of the
volume of the product. Generally, the cell suspension is in-
oculated and mixed so that the concentration of viable cells is
JPXV
General Information / Preservatives-Effectiveness 1733
10 5 to 10 6 cells per mL or per gram of the product. Incubate
these inoculated containers at 20°C to 25 °C with protection
from light, and calculate the viable cell count of 1 mL or 1 g
of the product taken at 0, 14 and 28 days subsequent to in-
oculation. Record any marked changes (e.g., changes in color
or the development of a bad odor) when observed in the mix-
ed samples during this time. Such changes should be consi-
dered when assessing the preservative efficacy of the product
concerned. Express sequential changes in the viable counts as
percentages, with the count at the start of the test taken as
100. Titration of the viable cell counts is based, in principle,
on the Pour Plate Methods in "Microbial Limit Tests". In
this case, confirm whether any antimicrobial substance is
present in the test specimen. If a confirmed antimicrobial
substance needs to be eliminated, incorporate an effective in-
activator of the substance in the buffer solution or liquid
medium to be used for dilution of the test specimen, as well
as in the agar plate count medium. However, it is necessary to
confirm that the inactivator has no effect on the growth of the
microorganisms. When the occurrence of the preservative or
the product itself affects titration of the viable cell count and
there is no suitable inactivator available, calculate the viable
cell counts by the Membrane Filtration Method in "Microbi-
al Limit Tests".
3.2 Category II products
The procedures are the same as those described for Catego-
ry I products, but special procedures and considerations are
required for both uniform dispersion of the test microorgan-
ism in the product and titration of viable cell counts in the
samples.
For semisolid ointment bases, heat the sample to 45°C to
50°C until it becomes oily, add the cell suspension and dis-
perse the inoculum uniformly with a sterile glass rod or spat-
ula. Surfactants may also be added to achieve uniform dis-
persion, but it is necessary to confirm that the surfactant
added has no effect on survival or growth of the test microor-
ganisms and that it does not potentiate the preservative effica-
cy of the product. For titration of the viable cell count, a sur-
factant or emulsifier may be added to disperse the product
uniformly in the buffer solution or liquid medium. Sorbitan
monooleate, polysorbate 80 or lecithin may be added to im-
prove miscibility between the liquid medium and semisolid
ointments or oils in which test microorganisms were inoculat-
ed. These agents serve to inactivate or neutralize many of the
most commonly used preservatives.
4. Interpretation
Interpret the preservative efficacy of the product according
to Table 1. When the results described in Table 1 are ob-
tained, the product examined is considered to be effectively
preserved. There is a strong possibility of massive microbial
contamination having occurred when microorganisms other
than the inoculated ones are found in the sterile product to be
examined, and caution is required in the test procedures and/
or the control of the manufacturing process of the product.
When the contamination level in a nonsterile product to be
examined exceeds the microbial enumeration limit specified
in "Microbial Attributes of Nonsterile Pharmaceutical
Products" in General Information, caution is also required
in the test procedures and/or the control of the manufactur-
ing process of the product.
Table 1.
Interpretation
criteria by product category
Product
Microorganisms
Interpretation criteria
category
After 14 days
After 28 days
Bacteria
0.1% of inocu-
Same or less
lum count or
than level after
less
14 days
Category IA
Yeasts /moulds
Same or less
Same or less
than inoculum
than inoculum
count
count
Bacteria
1% of inoculum
Same or less
count or less
than level after
14 days
Category IB
Yeasts /moulds
Same or less
Same or less
than inoculum
than inoculum
count
count
Bacteria
10% of inocu-
Same or less
lum count or
than level after
less
14 days
Category IC
Yeasts /moulds
Same or less
Same or less
than inoculum
than inoculum
count
count
Bacteria
Same or less
Same or less
than inoculum
than inoculum
count
count
Category II
Yeasts /moulds
Same or less
Same or less
than inoculum
than inoculum
count
count
5. Culture Media
Culture media and buffer solution used for Preservatives-
Effectiveness Tests are described below. Other media may be
used if they have similar nutritive ingredients and selective
and growth-promoting properties for the microorganisms to
be tested.
Soybean Casein Digest Agar Medium
Casein peptone 15.0 g
Soybean peptone 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Water 1000 mL
Mix all of the components and sterilize at 121 °C for 15 -
20 minutes in an autoclave. pH after sterilization: 7.1 - 7.3.
Sabouraud Glucose Agar Medium
Peptone (animal tissue and casein) 10.0 g
Glucose 40.0 g
Agar 15.0 g
Water 1000 mL
Mix all of the components and sterilize at 121 °C for 15 -
20 minutes in an autoclave. pH after sterilization: 5.4 - 5.8.
Glucose Peptone (GP) Agar Medium
Glucose
Yeast extract
Magnesium sulfate heptahydrate
20.0 i
2.0 j
0.5 i
1734 Qualification of Animals / General Information
JP XV
Peptone 5.0 g
Monobasic potassium phosphate 1.0 g
Agar 15.0 g
Water 1000 mL
Mix all of the components and sterilize at 121 °C for 15 -
20 minutes in an autoclave. pH after sterilization: 5.6 - 5.8.
Potato Dextrose Agar Medium
Potato extract
Glucose
Agar
Water
4.0 g
20.0 g
15.0 g
1000 mL
Mix all of the components and sterilize at 121 °C for 15 -
20 minutes in an autoclave. pH after sterilization: 5.4-5.8.
0.1% Peptone Water
Peptone 1.0 g
Sodium chloride 8.0 g
Water 1000 mL
Mix all of the components and sterilize at 121 °C for 15 -
20 minutes in an autoclave. pH after sterilization: 7.2 - 7.4.
20. Qualification of Animals as
Origin of Animal-derived Medicinal
Products provided in the General
Notices of Japanese
Pharmacopoeia and
Other Standards
Introduction
The Official Gazette issued on March 29, 2002 announced
that General Notices of the Japanese Pharmacopoeia and
other standards were amended to add a provision that
"When a drug product or a drug substance which is used to
manufacture a drug product, is manufactured from a raw
material of animal origin, the animal in question should be in
principle a healthy subject, if not otherwise provided.".
The Notice Iyaku-hatsu No. 0329001, which was issued on
the same date, provided that "Healthy subject herein pro-
vided is the animal which does not cause any disease or any
infection to human being at an appropriate production proc-
ess and use of the drug product, and as for the oral or exter-
nal drug for example, the animal, as its raw material of
animal origin, should be confirmed at this stage to meet the
Food Standard. It has to be noted that this standard of
healthy subject has to be revised timely taking into account
the up-to-date information with respect to the amphixenosis
infections common between human beings and animals.".
This General Information describes safety assurance
against infection of drugs, which are manufactured from raw
materials of animal origin, to follow up the Notice as men-
tioned above.
1. Basic concept
When drugs derived from raw materials of animal origin
including human are used, it is important to take into ac-
count any possibility that communicable disease agents such
as virus may cause infectious disease or any possible hazards
to patients. In such case, it goes without saying that the pri-
mary subject that has to be considered is the absence of any
infectious agents such as virus in the raw materials of animal
origin including human as the source of the drug. More im-
portant points are whether the drugs derived from such raw
materials are free of such infectious agents and whether there
is any possibility of transmission of infectious agents when
the drugs are administered to patient. The eligibility of
animals including human, as the source of raw materials of
drugs, in other words "the subject which is free from any dis-
ease or transmission of infectious agents that is infectious to
human being at an appropriate production process and use of
the drug product" is that "The drug should be entirely free
from any risk of infections by means of whole procedures
which include evaluation of appropriateness of the animals
including human as the source of their raw materials, estab-
lishment of appropriate production processes and their ap-
propriate control, and strict adherence to the clinical indica-
tions of the final product."
2. Animals including human as the source of raw materials
of drugs
What is the most clear and appropriate preventive meas-
ures against infection to human being due to administration
of drugs which are derived from animals including human is
to assure the absence of any infectious agents such as virus in
its raw materials or an appropriate critical raw material by
each of the followings: (1) the use of raw materials of healthy
animal origin, which are proved to be free from communica-
ble disease agents to human, or (2) the use of appropriate
critical raw materials (e.g., cell substrate, blood plasma,
pooled urine after some treatments) for drug production,
which are proved to be free from communicable disease
agents after certain appropriate processing on raw materials
of animal origin.
As for raw materials of drugs of human origin, cell, tissue,
blood, placenta, urine, etc. are used. Whenever it is sufficient
and possible each donor, as the origin of such raw materials,
should be asked his (her) health condition and undergoes his
(her) medical examination at this stage, so that the ap-
propriateness as a donor can be confirmed from the stan-
dpoint of safety concerning communicable disease agents
such as virus.
For example, "Basic concept on handling and use of a
drug product, etc. which is derived from cell/tissue"
(Attachment 1 of the Notice Iyaku-Hatsu No. 1314 dated De-
cember 26, 2000) and "Guidance for quality and safety assur-
ance of a drug product, etc. which is derived from human cell
/tissue (Attachment 2 of the Notice Iyaku-Hatsu No. 1314
dated December 26, 2000)" issued by the Director-General of
the Medicinal Safety Bureau, Ministry of Health and Wel-
fare, states that since the cell/tissue supplied by a human
donor comes to be applied to patients without processing
through any sufficient inactivation or removal of communica-
ble disease agents, the selection and qualification criteria on
such donor has to be established. These criteria are to be
composed with the respect to the check items on the case
history and the physical conditions as well as the test items on
the various transmission of infectious agents through cell /tis-
sue, and that the appropriateness of these criteria has to be
clarified. Hepatitis Type-B (HBV), Hepatitis Type-C (HCV),
Human Immune Deficiency Viral infections (HIV), Adult
T-Cell Leukemia and Parvovirus B19 Infections should be
denied through the interview to the donor and the tests (sero-
JPXV
General Information / Qualification of Animals 1735
logic test, nucleic-acid amplification test, etc.)- Further, if
necessary, Cytomegalovirus infection and EB Virus infection
should be denied by tests. "Infections caused by bacteria
such as Treponema pallidum, Chlamydia, Gonococci, Tuber-
cule bacillus, etc.", "septicemia and its suspicious case",
"vicious tumor", "serious metabolic or endocrine-related
disorders", "collagenosis and haematological disorder",
"hepatic disease" and "dementia (transmissible spongiform
encephalopathies and its suspicious case)" should be checked
on the case history or by the interview, etc. and the ex-
perience of being transfused or /and transplanted should be
checked to confirm eligibility as a donor. The most appropri-
ate check items and test methods then available are to be
used, which need to be reconsidered at appropriate timing
taking into account the updated knowledge and the progress
of the science and the technologies. At screening of a donor,
reexaminations has to be made at appropriate timing using
the eligible check items and the test methods taking into ac-
count the window period (Initial period after infection, in
which antibody against bacteria, fungi or virus is not detect-
ed.)
In the case of plasma derivatives produced from the donat-
ed blood in Japan, the donor should be checked by means of
self-assessed report about health conditions, and a serologic
check and a nucleic acid amplification test (NAT) on mini
pooled plasma should be performed at the stage of donated
blood. Further, the plasma material (i.e., critical raw materi-
al ) for fractionation should be stored 4 months in minimum
so that the arrangement could be taken based on the informa-
tion available after collection of the blood and the blood in-
fusion to exclude the possibility of using any critical raw
material which might cause infection to patients.
On the other hand, as for the materials such as urine which
are taken from the unspecified number of the donors and
come to be critical raw materials for drug production after
some treatments, it is unrealistic and not practical to conduct
the tests of virus infection, etc. on the individual donor. Con-
sequently, appropriate tests such as virus test has to be per-
formed on such critical raw materials for drug production.
In the case of the animals besides human, the wild ones
should be excluded. Only the animals, which are raised under
well sanitarily controlled conditions taken to prevent bacteri-
al contamination or under the effective bacterial pollution
monitoring systems, have to be used, and it is recommended
that the animals from a colony appropriately controlled un-
der specific pathogen-free (SPF) environment are to be used
as far as possible. Further, for the animals regulated under
the Food Standard, only the animals that met this standard
should be used. It should be confirmed by appropriate tests
that the animals were free from pathogen, if necessary.
The concrete measures to avoid transmittance or spread of
infectivity of prion, which is considered to be the pathogen of
transmissible spongiform encephalopathies (TSEs), as far as
possible are the followings: ® avoidance of use of animals,
which are raised in the areas where high incidence or high risk
of TSEs (Scrapie in sheep and goat, bovine spongiform en-
cephalopathies (BSE) in cattle, chronic wasting disease
(CWD) in deer, new type of Creutzfeldt-Jacob-Disease (C JD)
in human, etc.) is reported, and humans, who have stayed
long time (more than 6 months) in such areas, as raw materi-
als or related substances of drugs; © avoidance of use of any
substances that are derived from the individual infected with
scrapie, BSE, CJD, etc.; © avoidance of using a material
derived from organ, tissue and cell, etc. of high risk of TSEs;
and © taking appropriate measures basing on the informa-
tion collected, which includes incidence of TSEs, the results
of epidemiological investigation and the experimental
research on prion, and incidence of tardive infection on
donors after collecting raw materials, etc.
3. Human or animal cells which are used as critical raw
materials for drug production
Cell substrates derived from humans or animals are used
for drug production. In such case, it is desirable that the
humans or the animals, which are the origins of the cell sub-
strates, are healthy subjects. However, it is considered practi-
cal that viral safety of the drugs derived from the cell sub-
strates are evaluated on the cells, which are so called critical
raw materials for production of such drugs. In such case, the
safety should be confirmed through the test and analysis on
established cell bank thoroughly with respect to virus etc., as
far as possible. The items and the methods of the tests that
have been followed in this case are described in detail in the
Notice of Japanese version on the internationally accepted
ICH Guideline entitled "Viral safety evaluation of
biotechnology products derived from cell lines of human or
animal origin" (Iyakushin No. 329 issued on February 22,
2000 by Director, Evaluation and Licensing Division, Phar-
maceutical and Medical Safety Bureau, Ministry of Health
and Welfare). In the meantime, it is important how to handle
the cell in case that any virus has been detected under the cell
level tests. This Notice describes how to cope with this situa-
tion as follows: "It is recognised that some cell lines used for
the manufacture of product will contain endogenous
retroviruses, other viruses or viral sequences. In such circum-
stances, the action plan recommended for manufacturer is
described in Section V (Rationale and action plan for viral
clearance studies and virus tests on purified bulk) of the No-
tice. The acceptability of cell lines containing viruses other
than endogenous retroviruses will be considered on an in-
dividual basis by the regulatory authorities, by taking into ac-
count a risk/benefit analysis based on the benefit of the
product and its intended clinical use, the nature of the con-
taminating viruses, their potential for infecting humans or
for causing disease in humans, the purification process for
the product (e.g., viral clearance evaluation data), and the ex-
tent of the virus tests conducted on the purified bulk." For
example, it is well known that Type A-, R- and C-endogenous
particles like retrovirus are observed in the cells of the ro-
dents used most often for drug production. It is also known
that they are not infectious to human and is not dangerous,
and CHO cells are generally used for drug production. The
established cell lines (e.g., NAMALWA Cell, BALL-1 Cell,
etc.) derived from cancer patients are sometimes used, but
through the thorough virus tests, etc., their safety are con-
firmed. The established cell lines are assumed to be safer than
the primary cultured cells which are hard to conduct the
thorough virus test.
4. Establishment and control of appropriate production
process and adherence to the clinical indication of final
product for safety assurance
Safety assurance against potential infections at only the
level of animals that are source of raw materials of drugs is
limited. Further, "health of animal" can not be defined
univocally, and the various factors have to be taken into ac-
count. The final goal of this subject is to protect human from
1736 Quality Control of Water / General Information
JP XV
any infectious disease caused by drugs. Achieving this goal,
the establishment and control of appropriate production
processes of each drug and the adherence to the clinical indi-
cations of the final product are important.
As mentioned above, the rodent cells used most often for
the production of the drugs are known to have endogenous
retrovirus sometimes. The reason why such cells can be used
for the production of the drugs is that multiple measures are
applied for safety in the purification stages which include ap-
propriate inactivation or removal processes. There are cases
in which the production procedure involves intentional use of
a virus or a microorganism. In this case, relevant measures
capable of removing or inactivating of such virus or microor-
ganism are appropriately incorporated in the purification
process, so that the risk of infection to human can be fully
denied and its safety can be assured when it is used as a drug.
Further, even in the case that it is difficult to clarify the risk of
contamination of the infectious agents or that the raw materi-
al are contaminated by viruses etc., the raw material in ques-
tion may be used for the production of drugs so long as ap-
propriate inactivation or removal processes are introduced,
their effectiveness can be confirmed and the safety can be as-
sured by appropriate control of the manufacturing processes
under GMP, etc.
5. Conclusion
The qualification of animals including human, as the
source of raw materials of drugs, in other words "the subject
which does not cause any infectious diseases to human being
at an appropriate production process and use of the drug
product" is that "the drug has to be entirely free from any
risk of infections by means of whole procedures which in-
clude evaluation of appropriateness of the animal including
human as the source of their raw materials, establishment of
appropriate production processes and their appropriate con-
trol, and strict adherence to the clinical indication of the final
product."
To cope with this subject, the advanced scientific measures,
which actually reflect the updated knowledge and progress of
the science and the technology about infectious diseases in
human and infection of animal origin, have to be taken into
account timely.
21. Quality Control of Water for
Pharmaceutical Use
Water used for producing pharmaceutical products, clean-
ing pharmaceutical containers and equipment, and the like is
referred to as "Pharmaceutical Water." To consistently as-
sure the quality of pharmaceutical water, it is important to
verify through appropriate validation that water of required
quality is supplied, and to maintain that quality by routine
control.
Types of Pharmaceutical Water
® Water
The specifications for Water are specified in the corre-
sponding monograph of the Japanese Pharmacopoeia. It is
required for Water to meet the quality standards for drinking
water provided by the Japanese Water Supply Law and an
additional requirement for ammonium of "no greater than
0.05 mg/L." In preparing Water at various facilities from
source water such as well water or industrial water, it is need-
ed to assure compliance with specifications set forth for
Water in the Japanese Pharmacopoeia. Furthermore, when
Water is used after storage for a long period of time, microbi-
al proliferation should be prevented.
Water is used as source water for "Purified Water" and
"Water for Injection." It also is used in the production of in-
termediates of active pharmaceutical ingredients (APIs) and
in pre-washing the equipments used for pharmaceutical pur-
poses.
© Purified Water
The specifications of Purified Water are included in "Offi-
cial Monographs" of the Japanese Pharmacopoeia. Purified
Water is prepared from Water by distillation, ion-exchange
treatment, reverse osmosis (RO), ultrafiltration (UF) capable
of removing substances having molecular weights of 6,000
and above, or a combination of these methods. Because Puri-
fied Water contains no components capable of inhibiting
microbial growth, appropriate microbiological control is
needed. Particularly in the case of ion-exchange treatment,
reverse osmosis or ultrafiltration, the appropriate treatment
for microbial growth inhibition or periodical sterilization
should be conducted.
Purified Water which has been sterilized or treated with
chemical agents for the purpose of microbial growth inhibi-
tion or maintaining the endotoxin level within an appropriate
control range should be appropriately controlled in order to
maintain the quality in compliance with standards set forth
for the individual purposes of use. Purified Water that has
been sterilized is referred to as "Sterilized Purified Water."
(3) Water for Injection
The specifications of "Water for Injection" are described
in "Official Monographs" of the Japanese Pharmacopoeia.
Water for Injection is prepared from Water or Purified
Water by distillation, or from Purified Water by reverse os-
mosis (RO), ultrafiltration (UF) capable of removing sub-
stances having molecular weights of 6,000 and above, or a
combination of RO and UF. The quality of processing water
by RO and/or UF should be maintained consistently at the
same level as that prepared by distillation, by the validation
through long-term operation of the process and elaborate
routine control. Because Water for Injection contains no
components that inhibit microbial growth, stringent control
is needed for microorganisms and endotoxins. The standard
for endotoxins requires a level lower than 0.25 EU/mL.
Selection of Pharmaceutical Water
Depending on the purpose of use, pharmaceutical water
can be selected from the above-described group of phar-
maceutical waters that allow us to assure the quality of the fi-
nal product without causing any trouble during the produc-
tion process. Table 1 shows some example criteria for such
selection (in the case of charge-in water for the preparation of
drug products).
For the production of sterile drug products, for which con-
tamination by microorganisms or endotoxins is not permit-
ted, Water for Injection should be used. For ophthalmics and
eye ointments, Water for Injection or Purified Water should
be used. For the production of non-sterile drug products,
water of a quality higher than that of Purified Water should
be used, however for a non-sterile drug product that requires
JPXV
General Information / Quality Control of Water 1737
Table 1. Criteria for Selecting Pharmaceutical Water (Charge-In Water)
Classification
Class of
Pharmaceutical
Water
Application
Remarks
Drug
Product
Water for
Injection
Injections, Ophthalmics, Eye Ointments
Purified Water
Ophthalmics, Eye Ointment
For ophthalmics and eye ointments for
which precautions should be taken against
microbial contamination, purified water
that is subjected to microbiological con-
trol by treatment with sterilization,
ultrafiltration, or the like, should be used.
Aerosols, Liquids and Solutions, Extracts,
Elixirs, Capsules, Granules, Pills, Suspen-
sions and Emulsions, Suppositories,
Powders, Spirits, Tablets, Syrups, Infu-
sions and Decoctions, Plasters, Tinctures,
Troches, Ointments, Cataplasms, Aro-
matic Waters, Liniments, Lemonades,
Fluidextracts, Lotions, and Pharmaceuti-
cal preparations of percutaneous absorp-
tion type
For liquids, solutions, ointments, suspen-
sions, emulsions, aerosols, and the like for
which precautions should be taken against
microbial contamination, select and use
purified water subjected to appropriate
microbiological control.
Active
Pharmaceutical
Ingredient
(API)
Water for
Injection
Sterile APIs, and APIs rendered sterile in
the formulation process
Purified Water
APIs, APIs rendered sterile in the formu-
lation process, and API intermediates
In the production of APIs that are
rendered sterile in the formulation process
and have no subsequent processes capable
of removing endotoxins, Purified Water
that is controlled to maintain endotoxins
at a low level should be used.
Water
API Intermediates
care against microbiological contamination, such as liquids,
ointments, suspensions, emulsions, suppositories, and aer-
osols, water should have a quality appropriately controlled
from microbiological viewpoints, considering the possible
impacts of preservatives formulated in the dosage form.
Water for pre-washing containers or equipment surfaces that
come in direct contact with the drug should be of a quality
higher than that of Water. Water for final rinsing should
have the same quality as that of charge-in water.
For the production of non-sterile drug products, the quali-
ty of water should be higher than that of Purified Water;
however for the production of liquids, ointments, suspen-
sions, emulsions, suppositories, and aerosols for which care
should be taken against microbial contamination, the quality
of water should be controlled appropriately from microbio-
logical viewpoints in consideration of the possible effects of
formulated preservatives. Water for pre-washing containers
or equipment surfaces that come in direct contact with the
product should be of a quality not less than that of Water.
The water for final rinsing should be the same quality as that
of charge-in water.
In selecting pharmaceutical water for Active Pharmaceuti-
cal Ingredient (API), the characteristics and formulation
process of the drug products for which the API is used should
be considered, so that the quality of the final drug products is
maintained at an appropriate level. Water used in the produc-
tion of sterile API or to clean pharmaceutical containers or
equipment surfaces that come in direct contact with the
product should have the quality controlled chemically and
microbiologically at the level of Water or higher, even if the
water is to be used at an earlier stage of a synthetic or extrac-
tion process. In the final purification process, water should
have a quality equal to Purified Water or higher. Water used
for the final rinsing of containers or equipment surfaces that
come in direct contact with the product should have the same
quality as that of the charge-in water. Pharmaceutical water
used in sterile API should be Water for Injection. Similarly,
in the case of APIs for drug products, where endotoxin con-
trol is required and there are no subsequent processes capable
of removing endotoxins, Water for Injetion or Purified
Water, for which endotoxins are controlled to an appropriate
level, should be used.
Quality Control of Pharmaceutical Water
3.1 Outline
Routine control of pharmaceutical water is conducted on
1738 Quality Control of Water / General Information
JP XV
the premise that the production of water of a required quality
has been thoroughly verified by validation studies at an earli-
er stage of the system that produces the pharmaceutical water
(pharmaceutical water system). If these requirements are
met, the following control methods are applicable.
For routine control, the control of electrical conductivity
and Total Organic Carbon (TOC) is markedly useful. Items
to be controlled regularly should be determined according to
the intended use of the pharmaceutical water. In addition to
the above, control should be made for several chemical sub-
stances, viable counts, endotoxins, insoluble foreign parti-
cles, and the like. The measurement frequency should be de-
termined in consideration of the stability of the water quality
in question.
The following shows microbiological control items and
control items for electrical conductivity and Total Organic
Carbon (TOC), for which special attention should be paid.
Similar considerations should be made of the other control
items so that the water quality meets the specifications of
pharmaceutical water.
3.2 Sampling
Monitoring should be conducted at a sufficient enough fre-
quency to ensure that the pharmaceutical water system is in
control and that water of acceptable quality is continuously
produced. Sampling should be made at representative loca-
tions in the production and supply systems of the pharmaceu-
tical water, with particular care that the collected sample
reflects the pharmaceutical water system. Normally, point-of-
use sites are appropriate for the sampling locations. Sampling
frequency is established on the basis of data from validation
studies of the pharmaceutical water system. In making a sam-
pling plan for pharmaceutical water system, it is important to
cover all important points of the system by thoroughly con-
sidering the quality characteristics required for the water to
be sampled. Particularly in microbiological control, attention
should be paid to the difference in conditions among sam-
pling locations.
3.3 Alert and Action Levels
In a pharmaceutical water system, monitoring is done for
microorganisms and other quality attributes that assure water
of acceptable quality is produced during operation within the
designed specifications. Monitoring data thus obtained is
compared against an alert level, action level, other control
levels, and allowable limits for pharmaceutical waters. This
implies the alert level and action level are used for process
control, rather than for judging the acceptance.
Definition of Alert Level
An alert level indicates that, when exceeded, a process may
have drifted from its normal operating condition. Alert levels
constitute a warning and exceeding them does not necessarily
require a corrective action. Alert levels are generally estab-
lished either at a mean + 2cr on the basis of past trend ana-
lyses or at a level of 70% (50% for viable counts) of the ac-
tion criteria, whichever is smaller.
Definition of Action Level
An action level indicates that, when exceeded, a process
has drifted from its normal operating range. Exceeding an ac-
tion level indicates that corrective action must be taken to br-
ing the process back within its normal operating range.
Alert and action levels should be established within process
and product specification tolerances and based on integrated
considerations for technology and product quality. Conse-
quently, exceeding an alert or action level does not necessari-
ly indicate that the product quality has been compromised.
3.4 Microbiological Monitoring
The main purpose of a microbiological monitoring pro-
gram for pharmaceutical water system is to predict any
microbiological quality deterioration of the produced water
and to prevent any adverse effects it may have on product
quality. Consequently, detecting all of the microorganisms
present may not be necessary; however it is required to adopt
a monitoring technique capable of detecting a wide range of
microorganisms, including slow growing microorganisms.
The following indicates culture-based microbiological
monitoring methods for pharmaceutical water systems. In
adopting a rapid detection system for microorganisms, it is
needed to confirm in advance that the obtained microbial
counts are equivalent to or above the data obtained through
incubation.
3.4.1 Media and Incubation Conditions
There are many mesophilic bacteria of heterotrophic type
that are adaptable to poor nutrient water environments. In
many pharmaceutical water systems, heterotrophic bacteria
may form bio-films and cause water quality deterioration. It,
therefore, is useful to monitor the water quality by use of
R2A Agar Medium, which is excellent for growing bacteria
of oligotrophic type. On the other hand, in routine microbial
monitoring, an approach that identifies the trend in
microbiological quality change is widely employed; a stan-
dard agar plate is used for counting the total number of via-
ble microorganisms capable of proliferating at 30 - 35 °C in a
comparatively short period of time.
Table 2 shows examples of measurement methods, mini-
mum sample sizes, media, and incubation periods for es-
timating viable counts.
3.4.2 Media Growth Promotion Test
In the media growth promotion test with R2A Agar Medi-
um, use the strains listed below or other strains considered to
be equivalent to these strains. Prior to the media growth pro-
motion test, inoculate these strains into sterile purified water
and incubate at 20 - 25 C C for 3 days.
Met hylo bacterium extorquens: NBRC 15911
Pseudomonas fluorescens: NBRC 15842, ATCC 17386, or
the like
Dilute the fluid containing the starved strain in purified
water with sterile purified water to prepare a fluid containing
about 50 - 200 cfu per mL. When pipeting 1 ml of the diluted
solution onto the R2A agar medium to be used for incubating
at 20 - 25 °C or 30 - 35 °C for 4 - 7 days, a sufficient number
of recovered colonies of the microorganism must be observed
in comparison with the inoculated cfus.
In the media growth promotion test with standard agar
medium, use the strains listed below or other strains consi-
dered to be equivalent to these strains. When pipeting 1 mL
of the solution containing the microorganism for incubating
at 30 - 35°C for 48 hours, a sufficient number of recovered
colonies of the microorganism must be observed in compari-
son with the inoculated counts.
Staphylococcus aureus: ATCC 6538, NCIMB 9518, CIP
4.83 or NBRC 13276
Pseudomonas aeruginosa: ATCC 9027, NCIMB 8626, CIP
82.118 or NBRC 13275
JPXV
General Information / Quality Control of Water 1739
Table 2. Methods for Assessment of Viable Counts in harmaceutical Water
Method
Pharmaceutical Water
Water
Purified Water in Bulk
Water for Injection in Bulk
Measurement
Method
Pour Plate Method or
Membrane Filtration
Pour Plate Method or Membrane
Filtration
Membrane Filtration
Minimum
Sample Size
1.0 mL
1.0 mL
100 mL
Media
Standard Agar Medium
R2A Agar Medium, Standard
Agar Medium
R2A Agar Medium, Standard
Agar Medium
Incubation
Period
Standard Agar Medium:
48 - 72 hours (or longer)
R2A Agar Medium: 4-7 days
(or longer)
Standard Agar Medium: 48 - 72
hours (or longer)
R2A Agar Medium: 4-7 days
(or longer)
Standard Agar Medium: 48 - 72
hours (or longer)
Incubation
Temperature
Standard Agar Medium:
30-35°C
R2A Agar Medium: 20 - 25 °C or
30-35°C
Standard Agar Medium: 30 - 35°C
R2A Agar Medium: 20 - 25°C or
30-35°C
Standard Agar Medium: 30 - 35°C
Colon bacillus {Escherichia coli):
8545, CIP 53.126 or NBRC 3972
ATCC 8739, NCIMB
3.4.3 Action Levels for Microorganisms in Pharmaceuti-
cal Water System
The following action levels are considered appropriate and
generally applicable to pharmaceutical water systems. Action
levels for Purified Water and Water for Injection should be
those obtained using R2A Agar Medium.
Action Levels for viable counts in various pharmaceutical
waters
Water: 100 cfu/mL (according to the criteria in the Water
Supply Law)
Purified Water: 100 cfu/mL
Water for Injection: lOcfu/lOOmL
(cfu: colony-forming units)
When actual counts exceed such action levels in validation
studies or routine control studies, it is necessary to character-
ize the isolated microorganisms and to sterilize or disinfect
the affected system.
3.5 Physicochemical Monitoring
Physicochemical monitoring of a pharmaceutical water
system is generally performed using electrical conductivity
and Total Organic Carbon (TOC) as indicators. Monitoring
by electrical conductivity can predict probable quantities of
the total inorganic salts present. Monitoring by TOC can esti-
mate the total quantity of the organic compounds present.
Basically, the tests for electrical conductivity and total organ-
ic carbon specified in "General Tests" of the Japanese Phar-
macopoeia are applied as these physicochemical monitoring
methods. However, tests for monitoring are different in pro-
file from those described in "Monographs" of the Japanese
Pharmacopoeia. The following describes the particulars for
completing the part that cannot be worked out with the in-
dividual descriptions in "General Tests" of the Phar-
macopoeia alone. When monitoring is done at a production
facility using electrical conductivity or TOC as an indicator,
appropriate alert and action levels, as well as countermeas-
ures against contingency should be established for each indi-
cator.
3.5.1 Monitoring with an Indicator of Electrical Conduc-
tivity
Measurement of electrical conductivity for monitoring is
usually conducted continuously with an in-line meter having
a flow-through type or pipe-insertion type cell. Alternatively,
batch testing may be done using a dip type cell with samples
taken at point-of-use sites or other appropriate locations of
the pharmaceutical water system. The following guidelines
for the operation control of a pharmaceutical water system
address how to interpret the results of electrical conductivity
tests, and how to approve the continuation of operation, de-
pending on whether measurements are made at the standard
temperature (20°C) or at temperatures outside the standard.
(1) When monitoring is done at the standard temperature
(20°C)
Tests for electrical conductivity of the Japanese Phar-
macopoeia commonly require the tester to measure at the
standard temperature (20°C), however measurement at a
temperature within a range of 20 ± 5°C may be acceptable on
condition that the correct equation is used. When monitoring
electrical conductivity at the standard temperature of Puri-
fied Water and Water for Injection, the recommended allow-
able (action level) conductivity is as follows.
•Action Level 1.0^S/cm (20°C)
The above allowable conductivity is established on the sup-
position of in-line monitoring, so this standard level may be
changed in batch testing.
(2) When monitoring is done at temperatures other than
the standard temperature
When the quality of water is monitored by the measure-
ment of electrical conductivity at a temperature other than
1740 Quality Control of Water / General Information
JP XV
the standard temperature, the following three-stage approach
is applied. The method described below complies with the
description in "General Chapter <645> WATER CONDUC-
TIVITY" of the United States Pharmacopoeia (USP28,
2005), which specifies the establishment of allowable levels of
electrical conductivity individually corresponding to different
measurement temperatures and pH levels of specimens meas-
ured by the three-stage approach (Stage 1 to Stage 3).
Stage 1
1. When a non-temperature-compensated conductivity
instrument is used, measure the temperature of the water
specimen, in addition to the conductivity.
2. Using Table 3, find the conductivity value corre-
sponding to the measured temperature or a temperature listed
in Table 3 just below the measured temperature that can be
used to calculate the allowable electrical conductivity at the
measured temperature.
3. If the measured conductivity is not greater than the al-
lowable conductivity value, the water specimen is judged as
passing the electrical conductivity test. If the conductivity
value exceeds the allowable conductivity value, proceed with
Stage 2.
Table 3 Stage 1 Allowable Electrical Conductivity Meas-
ured at Different Temperatures*
Temperature
(°Q
Allowable
Conductivity
C«S/cm)
Temperature
(°C)
Allowable
Conductivity
(aS/cm)
0.6
5
0.8
55
2.1
10
0.9
60
2.2
15
1.0
65
2.4
20
1.1
70
2.5
25
1.3
75
2.7
30
1.4
80
2.7
35
1.5
85
2.7
40
1.7
90
2.7
45
1.8
95
2.9
50
1.9
100
3.1
*Applied to the conductivity value measured with a non-tem-
perature-compensated conductivity instrument.
Stage 2
1. Transfer a sufficient amount of water specimen to a
suitable container, and stir the specimen. Adjust the tempera-
ture (25 ± 1 C C), and while vigorously agitating the specimen,
measure the conductivity periodically. If the change in con-
ductivity is not greater than 0.1 ^S/cm, due to the uptake of
atmospheric carbon dioxide, interpret the observed value as
the conductivity of the specimen (in equilibrium with the at-
mosphere).
2. If the observed conductivity value at 25 °C (with the
specimen in equilibrium with the atmosphere) is not greater
than 2.1 //S/cm, the specimen is judged as passing the con-
ductivity test. If the observed value is greater than the level,
proceed with Stage 3.
Stage 3
1 . While maintaining the sample at 25 ± 1 °C, perform the
conductivity measurement as explained in Step 1 of Stage 2.
Add 0.3 mL of saturated potassium chloride solution to 100
mL of the water specimen, and determine the pH to the
nearest 0.1 pH.
2. Use Table 4 to find the allowable conductivity value
corresponding to the observed pH. If the observed conductiv-
ity is lower than the allowable level, the water specimen is
judged as passing the electrical conductivity test. If the ob-
served valued is greater than the allowable level, or if the pH
of the specimen is outside of a range of pH 5.0-7.0, the
water specimen is judged as unacceptable.
Table 4 Third Step Allowable Electrical Conductivity at
Different pHs*
pH
Allowable
Conductivity
((/S/cm)
pH
Allowable
Conductivity
(juS/cm)
5.0
4.7
5.1
4.1
6.1
2.4
5.2
3.6
6.2
2.5
5.3
3.3
6.3
2.4
5.4
3.0
6.4
2.3
5.5
2.8
6.5
2.2
5.6
2.6
6.6
2.1
5.7
2.5
6.7
2.6
5.8
2.4
6.8
3.1
5.9
2.4
6.9
3.8
6.0
2.4
7.0
4.6
*Applied only to a sample in equilibrium with the air at
25°C.
3.5.2 Monitoring with an Indicator of Total Organic
Carbon (TOC)
Drinking Water Standards (Prescribed under the Article 4
of the Japanese Water Supply Law) require that TOC should
be "not greater than 5 ppm". However it is preferable for in-
dividual facilities to conduct TOC monitoring on Water with
alert and action levels separately determined for water-quali-
ty control by TOC monitoring.
The Japanese Pharmacopoeia specifies the Test for Total
Organic Carbon, and usually, TOC measurement is conduct-
ed using an apparatus in compliance with the specifications,
however if the apparatus conforms to the apparatus suitabil-
ity test requirements described in "General Chapter <643>
TOTAL ORGANIC CARBON" of the United States Phar-
macopeia (USP28, 2005), otherwise described in "Methods
of Analysis 2.2.44. TOTAL ORGANIC CARBON IN
WATER FOR PHARMACEUTICAL USE" of the Europe-
an Pharmacopoeia (EP 5.0, 2005), the apparatus may be used
for monitoring a pharmaceutical water supplying system,
provided that sufficiently pure water not contaminated with
ionic organic substances or organic substances containing
nitrogen, sulfur or chlorine atoms is used as source water for
the system.
A TOC apparatus, characterized by calculating the amount
of organic carbon from the difference in conductivity before
and after the decomposition of organic substances without
separating carbon dioxide from the sample solution, may be
influenced negatively or positively, when applied to a sample
solution containing ionic organic substances or organic sub-
stances comprised of nitrogen, sulfur, or halogens such as
chlorine and the like; therefore, the apparatus should be
selected appropriately depending on the purity of phar-
JPXV
General Information / Rapid Identification 1741
maceutical water to be measured and on the contamination
risk in the case of apparatus failure.
3.6 Storage of Water for Injection
In storing Water for Injection, the water should be heated
during circulation or other action to inhibit microbial
proliferation. The maximum storage time allowed should
also be appropriately established on the basis of validation
studies, with considerations given to risks of contamination
and quality deterioration.
22. Rapid Identification of
Microorganisms Based on
Molecular Biological Method
This chapter describes the methods for the identification or
estimation of microorganisms (bacteria and fungi), found in
in-process control tests or lot release tests of pharmaceutical
products, at the species or genus level based on their DNA se-
quence homology. The identification of isolates found in the
sterility test or aseptic processing can be helpful for inves-
tigating the causes of contamination. Furthermore, informa-
tion on microorganisms found in raw materials used for
pharmaceutical products, processing areas of pharmaceutical
products, and so on is useful in designing measures to control
the microbiological quality of drugs. For the identification of
microorganisms, phenotypic analysis is widely used, based on
morphological, physiological, and biochemical features and
analysis of components. Commercial kits based on differ-
ences in phenotype patterns have been used for the identifica-
tion of microorganisms, but are not always applicable to
microorganisms found in raw materials used for pharmaceu-
tical products and in processing areas of pharmaceutical
products. In general, the identification of microorganisms
based on phenotypic analysis needs special knowledge and
judgment is often subjective. It is considered that the evolu-
tionary history of microorganisms (bacteria and fungi) is
memorized in their ribosomal RNAs (rRNAs), so that sys-
tematic classification and identification of microorganisms in
recent years have been based on the analysis of these se-
quences. This chapter presents a rapid method to identify or
estimate microorganisms based on partial sequences of diver-
gent regions of the 16S rRNA gene for bacteria and of the in-
ternal transcribed spacer 1 (ITS1) region located between 18S
rRNA and 5.8S rRNA for fungi, followed by comparison of
the sequences with those in the database. Methods described
in this chapter do not take the place of usual other methods
for the identification, and can be modified based on the ex-
aminer's experience, and on the available equipment or
materials. Other gene regions besides those mentioned in this
chapter can be used if appropriate.
Apparatuses
(1) DNA sequencer
Various types of sequencers used a gel board or capillary
can be used.
(2) DNA amplifier
To amplify target DNA and label amplified (PCR)
products with sequencing reagents.
Procedures
The following procedures are described as an example.
1 . Preparation of template DNA
It is important to use a pure cultivated bacterium or fungus
for identification. In the case of colony samples, colonies are
picked up with a sterilized toothpick (in the case of fungi, a
small fragment of colony sample is picked up), and suspend-
ed in 0.3 mL of DNA releasing solution in a 1.5 mL cen-
trifuge tube. In the case of culture fluid, a 0.5 mL portion of
fluid is put in a 1.5 mL centrifuge tube and centrifuged at
10,000 rpm for 10 min. After removal of the supernatant, the
pellet is suspended in 0.3 mL of DNA releasing solution, and
then heated at 100°C for 10 min. In general, PCR can be run
for bacteria and yeasts heated in DNA releasing solution. For
fungi, DNA extraction after treatment with a mixer or ultra-
sonic generator may be necessary before PCR.
2. PCR
Add 2//L of template DNA in PCR reaction solution. Use
10F/800R primers for bacteria and ITS1F/ITS1R primers for
fungi, and then perform 30 amplification cycles at 94°C for
30 sec, 55 °C for 60 sec, and 72°C for 60 sec. DNA fragments
are amplified about 800 bp in the case of bacteria and about
150 - 470 bp depending on the strain in the case of fungi. In-
clude a negative control (water instead of the test solution) in
the PCR.
3. Confirmation of PCR products
Mix 5 /uh of PCR product with 1 jXL of loading buffer
solution, place it in a 1.5 w/v% agarose gel well, and carry
out electrophoresis with TAE buffer solution (1-fold concen-
tration). Carry out the electrophoresis together with
appropriate DNA size markers. After the electrophoresis, ob-
serve PCR products on a trans-illuminator (312 nm) and con-
firm the presence of a single band of the targeted size. If mul-
tiple bands are observed, cut the targeted band out of the gel,
and extract DNA by using appropriate commercial DNA ex-
traction kit.
4. Purification of PCR products
Remove unincorporated PCR primers and deoxynucleo-
side triphosphates (dNTP) from PCR products by using
appropriate purification methods.
5. Quantification of purified DNA
When purified DNA is measured by spectrophotometer,
calculate 1 OD 2 60nm as 50//g/mL.
6. Labeling of PCR products with sequencing reagents
Use an appropriate fluorescence-labeled sequencing
reagent suitable for the available DNA sequencer or its
program and label the PCR products according to the
instructions provided with the reagent.
7. Purification of sequencing reagent-labeled PCR products
Transfer the product in 75 fiL of diluted ethanol (7 in 10)
into a 1 .5 mL centrifuge tube, keep in an ice bath for 20 min,
and centrifuge at 15,000 rpm for 20 min. After removal of
supernatant, add 250 /xh of diluted ethanol (7 in 10) to the
precipitate and centrifuge at 15,000 rpm for 5 min. Remove
the supernatant and dry the precipitate.
8. DNA homology analysis
Place sequencing reagent-labeled PCR products in the
DNA sequencer and read the nucleotide sequences of the
PCR products. Compare the partial nucleotide sequence with
those in the BLAST database.
Judgment
If sequencing data show over 90% identity with a sequence
in the database, in general, judgment may be made as fol-
lows.
1742 SDS-Polyacrylamide Gel / General Information
JP XV
1. In the case of bacteria, compare about 300 nucleotides
between positions 50 to 350 in the product obtained with
the 10F primer, with the BLAST database. Higher
ranked species are judged as identified species or closely
related species.
2. In the case of fungi, compare sequencing data for the
product obtained with the ITS1F primer, with the
BLAST database. Higher ranked species are judged as
identified species or closely related species.
Reagents, Test Solutions
(1) 0.5 mol/L Disodiuin dihydrogen ethylenediamine
tetraacetate TS
Dissolve 18.6 g of disodium dihydrogen ethylenediamine
tetraacetate dihydrate in water to make 100 mL.
(2) 1 mol/L Tris buffer solution, pH 8.0
Dissolve 24.2 g of 2-amino-2-hydroxymethyl-l,3-
propanediol in a suitable amount of water, adjust the pH to
8.0 with 0.2 mol/L hydrochloric acid TS, and add water to
make 200 mL.
(3) TE buffer solution
Mix 1.0 mL of 1 mol/L tris buffer solution, pH 8.0 and 0.2
mL of 0.5 mol/L disodium dihydrogen ethylenediamine
tetraacetate TS, and add water to make 100 mL.
(4) DNA releasing solution
Divide TE buffer solution containing Triton X-100 (1 in
100) into small amounts and store frozen until use.
(5) PCR reaction solution
10-fold buffer solution* 5 /uL
dNTP mixture** 4 fiL
10/imol/L Sense primer 1 fiL
lO^mol/L Anti-sense primer 1 fiL
Heat-resistant DNA polymerase (1 U///L) 1 fiL
Water 36 fiL
* Being composed of lOOmmol/L 2-amino-2-hydrox-
ymethyl-l,3-propanediol hydrochloride, pH 8.4, 500
mmol/L potassium chloride, 20 mmol/L magnesium chlo-
ride and 0.1 g/L gelatin.
** A solution containing 2.5 mmol/L each of dGTP (sodi-
um 2'-deoxyguanosine 5 '-triphosphate), dATP (sodium 2'-
deoxyadenosine 5 '-triphosphate), dCTP (sodium 2'-deox-
ycytidine 5 '-triphosphate) and dTTP (sodium 2'-deox-
ythymidine 5 '-triphosphate). Adequate products containing
these components as described above may be used.
(6) Sequencing reagent
There are many kinds of sequencing methods, such as the
dye-primer method for labeling of primer, the dye-terminator
method for labeling of dNTP terminator and so on. Use an
appropriate sequencing reagent kit for the apparatus and
program to be used.
(7) 50-Fold concentrated TAE buffer solution
Dissolve 242 g of 2-amino-2-hydroxymethyl-l,3-
propanediol in 57.1 mL of acetic acid (100) and 100 mL of
0.5 mol/L disodium dihydrogen ethylenediamine tetraacetate
TS, and add water to make 1000 mL.
(8) 1-Fold concentrated TAE buffer solution
Diluted 50-fold concentrated TAE buffer solution (1 in 50)
prepared before use is referred to as 1-fold concentrated TAE
buffer solution.
(9) Agarose gel
Mix 1.5 g of agarose, 2.0 mL of 50-fold concentrated TAE
buffer solution, 10 fiL of a solution of ethidium bromide (3,8-
diamino-5-ethyl-6-phenylphenanthridinium bromide) (1 in
100) and 100 mL of water. After dissolving the materials by
heating, cool the solution to about 60°C, and prepare gels.
(10) Loading buffer solution (6-fold concentrated)
Dissolve 0.25 g of bromophenol blue, 0.25 g of xylene
cyanol FF and 1.63 g of disodium dihydrogen ethylenedia-
mine tetraacetate dihydrate in 50 mL of water, and add
30 mL of glycerol and water to make 100 mL.
(11) PCR primers
For
Primer
Bacteria 10F 5'-GTTTGATCCTGGCTCA-3'
800R 5'-TACCAGGGTATCTAATCC-3'
Fungi ITS1F 5'-GTAACAAGGT(T/C)TCCGT-3'
ITS1R 5'-CGTTCTTCATCGATG-3'
23. SDS-Polyacrylamide Gel
Electrophoresis
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia.
The SDS-Polyacrylamide Gel Electrophoresis is used for
the characterization of proteins in biotechnological and bio-
logical products and for control of purity and quantitative
determinations.
This technique is a suitable analytical method with which
to identify and to assess the homogeneity of proteins in
biotechnological and biological products. The method is also
routinely used for the estimation of protein subunit molecu-
lar masses and for determining the subunit compositions of
purified proteins.
Ready-to-use gels and reagents are widely available on the
market and can be used instead of those described in this text,
provided that they give equivalent results and that they meet
the validity requirements given below under Validation of the
Test.
1. Characteristics of Polyacrylamide Gels
The sieving properties of polyacrylamide gels are afforded
by the three-dimensional network of fibers and pores which is
formed as the bifunctional bisacrylamide cross-links adjacent
polyacrylamide chains. Polymerization is catalyzed by a free
radical-generating system composed of ammonium
persulfate and A^A^Af'.TV'-tetramethylethylenediamine
(TEMED).
As the acrylamide concentration of a gel increases, its
effective pore size decreases. The effective pore size of a gel is
operationally defined by its sieving properties; that is, by the
resistance it imparts to the migration of macromolecules.
There are limits on the acrylamide concentration that can be
used. At high acrylamide concentrations, gels break much
more easily and are difficult to handle. As the pore size of a
gel decreases, the migration rate of a protein through the gel
decreases. By adjusting the pore size of a gel, through
manipulating the acrylamide concentration, the resolution of
the method can be optimized for a given protein product.
Thus, the physical characteristics of a given gel are deter-
mined by the relative concentrations of acrylamide and bi-
sacrylamide, used in its preparation.
In addition to the composition of the gel, the state of the
JPXV
General Information / SDS-Polyacrylamide Gel 1743
protein is an important determinant of the electrophoretic
mobility. In the case of proteins, the electrophoretic mobility
is dependent on the pK values of the charged groups and the
size of the molecule. It is also influenced by the type, concen-
tration and pH of the buffer, the temperature and the field
strength, as well as by the nature of the support material.
2. Polyacrylamide Gel Electrophoresis under Denaturing
Conditions
The method cited as an example is limited to the analysis of
monomeric polypeptides with a mass range of 14,000 to
100,000 daltons. It is possible to extend this mass range by
various techniques (e.g., by using gradient gels, particular
buffer systems, etc.), but those techniques are not discussed
in this chapter.
Analysis by electrophoresis on sodium dodecyl sulfate
(SDS) polyacrylamide gel (SDS-Polyacrylamide Gel Elec-
trophoresis) under denaturing conditions is the most com-
mon mode of electrophoresis used in assessing the quality of
proteins in biotechnological and biological products, and will
be the focus of the example described here. Typically, analyt-
ical electrophoresis of proteins is carried out in poly-
acrylamide gels under conditions that ensure dissociation of
the proteins into their individual polypeptide subunits and
that minimize aggregation. Most commonly, the strongly an-
ionic detergent SDS is used in combination with heat to dis-
sociate the proteins before they are loaded on the gel. The
denatured polypeptides bind to SDS, become negatively
charged and exhibit a consistent charge-to-mass ratio regard-
less of protein type. Because the amount of SDS bound is
almost always proportional to the molecular mass of the
polypeptide and is independent of its amino acid sequence,
SDS-polypeptide complexes migrate through polyacrylamide
gels with mobilities that are dependent on the size of the poly-
peptides.
The electrophoretic mobilities of the resultant SDS-
polypeptide complexes all assume the same functional
relationship to their molecular masses. Migration of SDS-
complexes occurs toward the anode in a predictable manner,
with low-molecular-mass complexes migrating faster than
larger ones. The molecular mass of a protein can therefore be
estimated from its relative mobility calibrated in SDS-
Polyacrylamide Gel Electrophoresis and the occurrence of a
single band in such a gel is a criterion of purity.
However, modifications to the polypeptide backbone, such
as N- or O-linked glycosylation, have a significant impact on
the apparent molecular mass of a protein, since SDS does not
bind to a carbohydrate moiety in a manner similar to a poly-
peptide. Thus, a consistent charge-to-mass ratio is not main-
tained. The apparent molecular masses of proteins that have
undergone post-translational modifications do not truly
reflect the masses of the polypeptides.
1) Reducing conditions
Polypeptide subunits and three-dimensional structure of
proteins are often fixed, at least in part, by the presence of
disulfide bonds. A goal of SDS-Polyacrylamide Gel Elec-
trophoresis under reducing conditions is to disrupt this struc-
ture by reducing the disulfide bonds. Complete denaturation
and dissociation of proteins by treatment with 2-mercap-
toethanol or dithiothreitol (DTT) will result in unfolding of
the polypeptide backbone and subsequent complexation with
SDS. Under these conditions, the molecular masses of the
polypeptide subunits can be calculated by interpolation in the
presence of suitable molecular-mass standards.
2) Non-reducing conditions
For some analyses, complete dissociation of the protein of
interest into subunit peptides is not desirable. In the absence
of treatment with reducing agents such as 2-mercaptoethanol
or DTT, disulfide covalent bonds remain intact, preserving
the oligomeric form of the protein. Oligomeric SDS-protein
complexes migrate more slowly than their SDS-polypeptide
subunits. In addition, non-reduced proteins may not be com-
pletely saturated with SDS and, hence, may not bind the de-
tergent in the expected mass ratio. This makes molecular-
mass determinations of these molecules by SDS-
Polyacrylamide Gel Electrophoresis less straightforward than
analyses of fully denatured polypeptides, since it is necessary
that both standards and unknown proteins be in similar con-
figurations for valid comparisons. However, the staining of a
single band in such a gel is a criterion of purity.
3. Characteristics of Discontinuous Buffer System Gel Elec-
trophoresis
The most widely used electrophoretic method for the anal-
ysis of complex mixtures of proteins involves the use of a dis-
continuous buffer system consisting of two contiguous, but
distinct gels: a resolving or separating (lower) gel and a stack-
ing (upper) gel. The two gels are cast with different porosities,
pH, and ionic strengths. In addition, different mobile ions are
used in the gel and electrode buffers. The buffer discontinuity
acts to concentrate large-volume samples in the stacking gel,
resulting in improved resolution. When power is applied, a
voltage drop develops across the sample solution which
drives the proteins into the stacking gel. Glycinate ions from
the electrode buffer follow the proteins into the stacking gel.
A moving boundary region is rapidly formed with the highly
mobile chloride ions in the front and the relatively slow
glycinate ions in the rear. A localized high-voltage gradient
forms between the leading and trailing ion fronts, causing the
SDS-protein complex to form into a very thin zone (called the
stack) and to migrate between the chloride and glycinate
phases. Regardless of the height of the applied sample solu-
tion in the wells, all SDS-protein complexes condense within
broad limits and enter the resolving gel as a well-defined, thin
zone of high protein density. The large-pore stacking gel does
not retard the migration of most proteins and serves mainly
as an anticonvective medium. At the interface of the stacking
and resolving gels, the proteins experience a sharp increase in
retardation due to the smaller pore size of the resolving gel.
Once the proteins are in the resolving gel, their mobility con-
tinues to be slowed down by the molecular sieving effect of
the matrix. The glycinate ions overtake the proteins, which
then move in a space of uniform pH formed by the
tris(hydroxymethyl)aminomethane and glycine. Molecular
sieving causes the SDS-polypeptide complexes to separate on
the basis of their molecular masses.
4. Preparing Vertical Discontinuous Buffer SDS-
Polyacrylamide Gels
1) Assembling of the gel moulding cassette
Clean the two glass plates (size: e.g. 10 cm x 8 cm), the
sample comb made of polytetrafluoroethylene, the two
spacers and the silicone rubber tubing (diameter, e.g. 0.6 mm
x 35 cm) with mild detergent and rinse extensively with
water. Dry all the items with a paper towel or tissue. Lubri-
cate the spacers and the silicone rubber tubing with non-sili-
cone grease. Apply the spacers along each of the two short
1744 SDS-Polyacrylamide Gel / General Information
JP XV
sides of the glass plate 2 mm away from the edges and 2 mm
away from the long side corresponding to the bottom of the
gel. Begin to lay the silicone rubber tubing on the glass plate
by using one spacer as a guide. Carefully twist the silicone
rubber tubing at the bottom of the spacer and follow the long
side of the glass plate. While holding the silicone rubber tub-
ing with one finger along the long side again twist the tubing
and lay it on the second short side of the glass plate, using the
spacer as a guide. Place the second glass plate in perfect
alignment and hold the mould together by hand pressure.
Apply two clamps on each of the two short sides of the
mould. Carefully apply four clamps on the longer side of the
gel mould, thus forming the bottom of the gel mould. Verify
that the silicone rubber tubing is running along the edge of
the glass plates and has not been extruded while placing the
clamps.
2) Preparation of the gel
In a discontinuous buffer SDS polyacrylamide gel, it is
recommended to pour the resolving gel, let the gel set, and
then pour the stacking gel, since the compositions of the two
gels in acrylamide-bisacrylamide, buffer and pH are different.
Preparation of the resolving gel: In a conical flask, prepare
the appropriate volume of solution containing the desired
concentration of acrylamide for the resolving gel, using the
values given in Table 1. Mix the components in the order
shown. Where appropriate, before adding the ammonium
persulfate solution and the tetramethylethylenediamine
(TEMED), filter the solution if necessary under vacuum
through a cellulose acetate membrane (pore size: 0.45 //m);
keep the solution under vacuum by swirling the filtration unit
until no more bubbles are formed in the solution. Add ap-
propriate amounts of ammonium persulfate solution and
TEMED as indicated in Table 1, swirl and pour immediately
into the gap between the two glass plates of the mould. Leave
sufficient space for the stacking gel (the length of the teeth of
the sample comb plus 1 cm). Using a pipette, carefully over-
lay the solution with water-saturated isobutanol. Leave the
gel in a vertical position at room temperature to allow poly-
merization to occur.
Preparation of the stacking gel: After polymerization is
complete (about 30 minutes), pour off the isobutanol and
wash the top of the gel several times with water to remove the
isobutanol overlay and any unpolymerized acrylamide. Drain
as much fluid as possible from the top of the gel, and then re-
move any remaining water with the edge of a paper towel.
In a conical flask, prepare the appropriate volume of solu-
tion containing the desired concentration of acrylamide,
using the values given in Table 2. Mix the components in the
order shown. Where appropriate, before adding the ammoni-
um persulfate solution and the TEMED, filter the solution if
necessary under vacuum through a cellulose acetate mem-
brane (pore size: 0.45 fim); keep the solution under vacuum
by swirling the filtration unit until no more bubbles are
formed in the solution. Add appropriate amounts of ammo-
nium persulfate solution and TEMED as indicated in
Table 2, swirl and pour immediately into the gap between the
two glass plates of the mould directly onto the surface of the
polymerized resolving gel. Immediately insert a clean sample
comb into the stacking gel solution, taking care to avoid trap-
ping air bubbles. Add more stacking gel solution to fill com-
pletely the spaces of the sample comb. Leave the gel in a ver-
tical position and allow to polymerize at room temperature.
3) Mounting the gel in the electrophoresis apparatus and
electrophoretic separation
After polymerization is complete (about 30 minutes),
remove the sample comb carefully. Rinse the wells immedi-
ately with water or with the running buffer for SDS-
Polyacrylamide Gel Electrophoresis to remove any un-
polymerized acrylamide. If necessary, straighten the teeth of
the sample comb of the stacking gel with a blunt hypodermic
needle attached to a syringe. Remove the clamps on one short
side, carefully pull out the silicone rubber tubing and replace
the clamps. Proceed similarly on the other short side. Re-
move the silicone rubber tubing from the bottom part of the
gel. Mount the gel in the electrophoresis apparatus. Add the
electrophoresis buffers to the top and bottom reservoirs. Re-
move any bubbles that become trapped at the bottom of the
gel between the glass plates. This is best done with a bent
hypodermic needle attached to a syringe. Never pre-run the
gel before loading solutions, such as samples, since this will
destroy the discontinuity of the buffer systems. Before load-
ing solutions, such as samples, carefully rinse the stacking gel
wells with the running buffer for SDS-Polyacrylamide Gel
Electrophoresis. Prepare the test and reference solutions in
the recommended sample buffer and treat as specified in the
individual monograph. Apply the appropriate volume of
each solution to the stacking gel wells. Start the electrophore-
sis using suitable operating conditions for the electrophoresis
equipment to be used. There are commercially available gels
of different surface area and thickness that are appropriate
for various types of electrophoresis equipment. Electropho-
resis running time and current /voltage may need to be altered
depending on the type of apparatus used, in order to achieve
optimum separation. Check that the dye front is moving into
the resolving gel. When the dye is reaching the bottom of the
gel, stop the electrophoresis. Remove the gel assembly from
the apparatus and separate the glass plates. Remove the
spacers, cut off and discard the stacking gel and immediately
proceed with staining.
5. Detection of Proteins in Gels
Coomassie staining is the most common protein staining
method, with a detection level of the order of 1 ng to 10 /ug of
protein per band. Silver staining is the most sensitive method
for staining proteins in gels and a band containing 10 ng to
100 ng can be detected. All of the steps in gel staining are
done at room temperature with gentle shaking in any con-
venient container. Gloves must be worn when staining gels,
since fingerprints will stain.
1) Coomassie staining
Immerse the gel in a large excess of Coomassie staining TS
and allow to stand for at least 1 hour. Remove the staining
solution.
Destain the gel with a large excess of destaining TS.
Change the destaining solution several times, until the stained
protein bands are clearly distinguishable on a clear back-
ground. The more thoroughly the gel is destained, the smaller
is the amount of protein that can be detected by the method.
Destaining can be speeded up by including 2 to 3 g of anion-
exchange resin or a small sponge in the destaining TS.
NOTE: the acid-alcohol solutions used in this procedure
do not completely fix proteins in the gel. This can lead to loss-
es of some low-molecular-mass proteins during the staining
and destaining of the gel. Permanent fixation is obtainable by
allowing the gel to stand in trichloroacetic acid TS for fixing
for 1 hour before it is immersed in Coomassie staining TS.
JP XV
General Information / SDS-Polyacrylamide Gel 1745
Table 1. Preparation of resolving gel
Solution components
5 mL
Component volumes (mL) per gel mould volume of
10 mL 15 mL 20 mL 25 mL 30 mL 40 mL 50 mL
6% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8)°
100 g/L SDS' 3 *
100 g /LAPS' 4 *
TEMED"'
8% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8)°
100 g/L SDS' 5 '
100 g/L APS' 41
TEMED"*
10% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8)°
100 g/L SDS' 51
100 g/L APS' 41
TEMED"*
12% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8)' 2
100 g/L SDS' 31
100 g/L APS' 41
TEMED"*
14% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8) 12
100 g/L SDS 131
100 g/L APS' 41
TEMED"*
15% Acrylamide
Water
Acrylamide solution'"
1.5 mol/L Tris solution (pH 8.8)°
100 g/L SDS' 31
100 g/L APS' 41
TEMED"*
2.6
5.3
7.9
10.6
13.2
15.9
21.2
26.5
1.0
2.0
3.0
4.0
5.0
6.0
8.0
10.0
1.3
2.5
3.8
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.004
0.008
0.012
0.016
0.02
0.024
0.032
0.04
2.3
4.6
6.9
9.3
11.5
13.9
18.5
23.2
1.3
2.7
4.0
5.3
6.7
8.0
10.7
13.3
1.3
2.5
3.8
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.003
0.006
0.009
0.012
0.015
0.018
0.024
0.03
1.9
4.0
5.9
7.9
9.9
11.9
15.9
19.8
1.7
3.3
5.0
6.7
8.3
10.0
13.3
16.7
1.3
2.5
3.8
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.002
0.004
0.006
0.008
0.01
0.012
0.016
0.02
1.6
3.3
4.9
6.6
8.2
9.9
13.2
16.5
2.0
4.0
6.0
8.0
10.0
12.0
16.0
20.0
1.3
2.5
3.8
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.002
0.004
0.006
0.008
0.01
0.012
0.016
0.02
1.4
2.7
3.9
5.3
6.6
8.0
10.6
13.8
2.3
4.6
7.0
9.3
11.6
13.9
18.6
23.2
1.2
2.5
3.6
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.002
0.004
0.006
0.008
0.01
0.012
0.016
0.02
1.1
2.3
3.4
4.6
5.7
6.9
9.2
11.5
2.5
5.0
7.5
10.0
12.5
15.0
20.0
25.0
1.3
2.5
3.8
5.0
6.3
7.5
10.0
12.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.5
0.002
0.004
0.006
0.008
0.01
0.012
0.016
0.02
(1)
(2)
(3)
(4)
(5)
Acrylamide solution: 30% acrylamide /bisacrylamide (29:1) solution
1.5 mol/L Tris solution (pH 8.8): 1.5 mol/L tris-hydrochloride buffer solution, pH 8.8
100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate
100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive
polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-
tions must be prepared before use.
TEMED : N,N,N' ,iV'-tetramethylethylenediamine
1746 SDS-Polyacrylamide Gel / General Information
JP XV
Table 2. Preparation of stacking gel
Solution components
Component volumes (mL)
per gel
mould volume of
lmL
2mL
3 mL
4mL
5 mL
6 mL
8mL
10 mL
Water
0.68
1.4
2.1
2.7
3.4
4.1
5.5
6.8
Acrylamide solution'"
0.17
0.33
0.5
0.67
0.83
1.0
1.3
1.7
l.Omol/L Tris solution (pH 6.8)°'
0.13
0.25
0.38
0.5
0.63
0.75
1.0
1.25
100 g/L SDS' 3 *
0.01
0.02
0.03
0.04
0.05
0.06
0.08
0.1
100 g /LAPS™
0.01
0.02
0.03
0.04
0.05
0.06
0.08
0.1
TEMED'"
0.001
0.002
0.003
0.004
0.005
0.006
0.008
0.01
(1)
(2)
(3)
(4)
(5)
Acrylamide solution: 30% acrylamide /bisacrylamide (29:1) solution
l.Omol/L Tris solution (pH 6.8): 1 mol/L tris-hydrochloride buffer solution, pH 6.8
100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate
100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive
polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-
tions must be prepared before use.
TEMED: AT,N,iV',Ar'-tetramethylethylenediamine
2) Silver staining
Immerse the gel in a large excess of fixing TS and allow to
stand for 1 hour. Remove the fixing solution, add fresh fixing
solution and incubate either for at least 1 hour or overnight,
if convenient. Discard the fixing solution and wash the gel in
water for 1 hour. Soak the gel for 15 minutes in a 1 vol%
glutaraldehyde solution. Wash the gel twice for 15 minutes in
water. Soak the gel in fresh silver nitrate TS for silver staining
for 15 minutes, in darkness. Wash the gel three times for 5
minutes in water. Immerse the gel for about 1 minute in de-
veloper TS until satisfactory staining has been obtained. Stop
the development by incubation in blocking TS for 15
minutes. Rinse the gel with water.
6. Drying of Stained SDS-Polyacrylamide Gels
Depending on the staining method used, gels are pretreated
in a slightly different way. For Coomassie staining, after the
destaining step, allow the gel to stand in a diluted solution of
concentrated glycerin (1 in 10) for at least 2 hours (overnight
incubation is possible). For silver staining, add to the final
rinsing a step of 5 minutes in a diluted solution of concentrat-
ed glycerin (1 in 50).
Immerse two sheets of porous cellulose film in water and
incubate for 5 to 10 minutes. Place one of the sheets on a
drying frame. Carefully lift the gel and place it on the cellu-
lose film. Remove any trapped air bubbles and pour 2 to 3
mL of water around the edges of the gel. Place the second
sheet on top and remove any trapped air bubbles. Complete
the assembly of the drying frame. Place in an oven or leave at
room temperature until dry.
7. Molecular-Mass Determination
Molecular masses of proteins are determined by compari-
son of their mobilities with those of several marker proteins
of known molecular mass. Mixtures of proteins with pre-
cisely known molecular masses blended for uniform staining
are commercially available for calibrating gels. They are ob-
tainable in various molecular mass ranges. Concentrated
stock solutions of proteins of known molecular mass are
diluted in the appropriate sample buffer and loaded on the
same gel as the protein sample to be studied.
Immediately after the gel has been run, the position of the
bromophenol blue tracking dye is marked to identify the
leading edge of the electrophoretic ion front. This can be
done by cutting notches in the edges of the gel or by inserting
a needle soaked in India ink into the gel at the dye front.
After staining, measure the migration distances of each pro-
tein band (markers and unknowns) from the top of the
resolving gel. Divide the migration distance of each protein
by the distance traveled by the tracking dye. The normalized
migration distances so obtained are called the relative mobili-
ties of the proteins (relative to the dye front) and convention-
ally denoted as Rf. Construct a plot of the logarithm of the
relative molecular masses (M r ) of the protein standards as a
function of the Rf values. Note that the graphs are slightly
sigmoid. Unknown molecular masses can be estimated by
linear regression analysis or interpolation from the curves of
log M r against Rf as long as the values obtained for the
unknown samples are positioned along the linear part of the
graph.
8. Suitability of the Test (Validation)
The test is not valid unless the front end of the molecular
mass marker migrates 80% of the migrating distance of the
dye, and over the required separation range (e.g., the range
covering the product and its dimer or the product and its
related impurities) the separation obtained for the relevant
protein bands shows a linear relationship between the
logarithm of the molecular mass and the Rf as described in 7.
Additional requirements with respect to the solution under
test may be specified in individual monographs.
9. Quantification of Impurities
Where the impurity limit is specified in the individual
monograph, a reference solution corresponding to that level
of impurity should be prepared by diluting the test solution.
For example, where the limit is 5%, a reference solution
would be a 1:20 dilution of the test solution. No impurity
(any band other than the main band) in the electrophoreto-
gram obtained with the test solution may be more intense
than the main band obtained with the reference solution.
Under validated conditions, impurities may be quantified
by normalization to the main band, using an integrating den-
sitometer. In this case, the responses must be validated for
linearity.
JPXV
General Information / Solid and Particle Densities 1747
Test solutions:
Coomassie staining TS Dissolve 125 mg of coomassie
brilliant blue R-250 in 100 mL of a mixture of water,
methanol and acetic acid (100) (5:4:1), and filter.
Developer TS Dissolve 2 g of citric acid monohydrate in
water to make 100 mL. To 2.5 mL of this solution add 0.27
mL of formaldehyde solution and water to make 500 mL.
Fixing TS To 250 mL of methanol add 0.27 mL of for-
maldehyde solution and water to make 500 mL.
Silver nitrate TS for silver staining To 40 mL of sodium
hydroxide TS add 3 mL of ammonia solution (28), then add
dropwise 8 mL of a solution of silver nitrate (1 in 5) while
stirring, and add water to make 200 mL.
Destaining TS A mixture of water, methanol and acetic
acid (100) (5:4:1).
Blocking TS To 10 mL of acetic acid (100) add water to
make 100 mL.
Trichloroacetic acid TS for fixing Dissolve 10 g of
trichloroacetic acid in a mixture of water and methanol (5:4)
to make 100 mL.
24. Solid and Particle Densities
Density of a solid or a powder as a state of aggregation has
different definitions depending on the way of including of the
interparticulate and intraparticulate voids that exist between
the particles or inside the powder. Different figures are ob-
tained in each case, and there are different practical mean-
ings. Generally, there are three levels of definitions of the
solid or powder density.
(1) Crystal density: It is assumed that the system is
homogeneous with no intraparticulate void. Crystal density is
also called true density.
(2) Particle density: The sealed pores or the experimental-
ly non-accessible open pores is also included as a part of the
volumes of the solid or the powder.
(3) Bulk density: The interparticulate void formed in the
powder bed is also included as a part of the volumes of the
solid or the powder. Bulk density is also called apparent
density. Generally, the powder densities at loose packing and
at tapping are defined as the bulk density and the tapped den-
sity, respectively.
Generally, the densities of liquid or gas are affected only by
temperature and pressure, but the solid or powder density is
affected by the state of aggregation of the molecules or the
particles. Therefore, the solid or powder densities naturally
vary depending on crystal structure or crystallinity of the sub-
stance concerned, and also varies depending on the method
of preparation or handling if the sample is amorphous form
or partially amorphous. Consequently, even in a case that
two solids or powders are chemically identical, it may be pos-
sible that the different figures of density are obtained if their
crystal structures are different. As the solid or powder parti-
cle densities are important physical properties for the pow-
dered pharmaceutical drugs or the powdered raw materials of
drugs, the Japanese Pharmacopoeia specifies each density de-
termination as "Powder Particle Density Determination" for
the particle density and as "Determination of Bulk and
Tapped Densities" for the bulk density.
The solid or powder densities are expressed in mass per
unit volume (kg/m 3 ), and generally expressed in g/cm 3 (1
g/cm 3 = 1000kg/m 3 ).
Crystal Density
The crystal density of a substance is the average mass per
unit volume, exclusive of all voids that are not a fundamental
part of the molecular packing arrangement. It is an intrinsic
property concerning the specific crystal structure of the sub-
stance, and is not affected by the method of determination.
The crystal density can be determined either by calculation or
by simple measurement.
A. The calculated crystal density is obtained using:
1) For example, the crystallographic data (volume and
composition of the unit cell) obtained by indexing
the perfect crystal X-ray diffraction data from single
crystal or the powder X-ray diffraction data.
2) Molecular mass of the substance.
B. The measured crystal density is obtained as the mass to
volume ratio after measuring the single crystal mass and
volume.
Particle Density
The particle density takes account both the crystal density
and the intraparticulate porosity (sealed and/or experimen-
tally non-accessible open pores) as a part of the particle
volume. The particle density depends on the value of the
volume determined, and the volume in turn depends on the
method of measurement. Particle density can be determined
either by gas displacement pycnometry or mercury porosi-
metry, but the Japanese Pharmacopoeia specifies the
pycnometry as the "Powder Particle Density Determina-
tion".
A. The pycnometric density is obtained by assuming that
the volume of the gas displaced, which is measured with
the gas displacement pycnometer, is equivalent to that
of a known mass of the powder. In pycnometric density
measurements, any volume with the open pores accessi-
ble to the gas is not included as a part of volume of the
powder, but the sealed pores or pores inaccessible to the
gas is included as a part of the volume of the powder.
Due to the high diffusivity of helium which can penetrate
to most open pores, it is recommendable as the measure-
ment gas of particle density. Therefore, the pycnometric
particle density of a finely milled powder is generally not
very different from the crystal density. Hence, the parti-
cle density by this method is the best estimate of the true
density of an amorphous or partially crystalline sample,
and can be widely used for manufacturing control of the
processed pharmaceutical powder samples.
B. The mercury porosimetric density is also called granular
density. This method also includes the sealed pores as a
part of the volumes of the solid or the powder, but
excludes the volume only from the open pores larger
than some size limit. This pore size limit or minimal
access diameter depends on the maximal mercury
intrusion pressure applied during the measurement and
under normal operating pressure, the mercury does not
penetrate the finenest pores accessible to helium. Since
this method is capable of measuring the density which
corresponds to the pore size limit at each mercury
intrusion pressure, the various granular densities can be
obtained from one sample.
Bulk Density and Tapped Density
The bulk density of a powder includes the contribution of
1748 Sterility Assurance / General Information
JP XV
interparticulate void volume as a part of the volume of the
powder. Therefore, the bulk density depends on both the
powder particle density and the space arrangement of
particles in the power bed. Further, since the slightest
disturbance of the bed may result in variation of the space ar-
rangement, it is often very difficult to determine the bulk den-
sity with good reproducibility. Therefore, it is essential to
specify how the determination was made upon reporting the
bulk density.
The Japanese Pharmacopoeia specifies "Determination of
Bulk and Tapped Densities".
A. The bulk density is determined by measuring the
apparent volume of a known mass of powder sample
that has been passed through a screen in a graduated
cylinder (constant mass method). Separately, the Phar-
macopoeia specifies the method of determining bulk
density by measuring the mass of powder in a vessel hav-
ing a known volume (constant volume method).
B. The tapped density is obtained by mechanically tapping
a measuring cylinder containing a powder sample. After
determining the initial bulk volume, carry out tapping
under a fixed measurement condition (tapping rate and
drop height), and the measurement is carried out repeat-
edly until the bulk volume variation obtained at consecu-
tive two measurements is within an acceptable range
(constant mass method). Separately, the Pharmacopoeia
specifies the method of determining the tapped density
by measuring the mass of a fixed volume of the tapped
powder (constant volume method).
25. Sterility Assurance for
Terminally Sterilized
Pharmaceutical Products
As indicated in the "Terminal Sterilization and Steriliza-
tion Indicators", the pharmaceuticals to which terminal
sterilization can be applied, generally must be sterilized so
that a sterility assurance level of 10~ 6 or less is obtained. The
sterility assurance level of 10 -6 or less can be proven by using
a sterilization process validation based on physical and
microbiological methods, but cannot be proven by sterility
tests of the sterilized products. This chapter deals with the
necessary requirements for the appropriate management of
the important control points of the sterilization process for
the parametric release of products, without performing steril-
ity tests on products which have been subjected to terminal
sterilization (in the case of radiation sterilization, called dosi-
metric release). Parametric release is a method that can be ap-
plied in cases where the sterilization system is clearly defined,
important control points are clearly specified, and the sterili-
zation system process can be validated by microbiological
methods using appropriate biological indicators.
1. Definitions
The definitions of the terminology used in this chapter are
provided below.
1.1 Terminal sterilization
A process whereby a product is sterilized in its final con-
tainer or packaging, and which permits the measurement and
evaluation of quantifiable microbial lethality.
1.2 Validation
A documented procedure for obtaining, recording and in-
terpreting the results needed to show that a process will con-
sistently yield a product complying with predetermined
specifications.
1.3 Periodic re-validation
Validation that is regularly performed to reconfirm that a
process is consistently yielding a product complying with
predetermined specifications. It should confirm that variables
and the acceptable ranges are permissible to yield a product
consistently of the required quality.
1.4 Facility/equipment qualification
This is to provide evidence that the manufacturing facilities
/equipment, measuring equipment, and manufacturing en-
vironment control facilities, etc. have been properly selected,
correctly installed, and are operated in conformity with the
specifications at the time of installation and during opera-
tion.
1.5 Operation qualification
This is to provide evidence to confirm physically, chemical-
ly and microbiologically that equipment, operated in accor-
dance with its operational instructions, operates as specified
and affords a product meeting the specifications.
1.6 Support system for sterilization process
This refers to the facility/equipment that is associated with
the sterilization devices, such as the preconditioning and aer-
ation for ethylene oxide sterilization, the steam supply equip-
ment for moist heat sterilization, and the loading devices for
radiation sterilization.
1.7 Quality system
The procedures, resources and organizational structure of
a manufacturer (responsibilities, authorities and relation-
ships between these) required to implement quality manage-
ment.
1.8 Change control system
A system designed to evaluate all of the changes that may
affect the quality of the pharmaceutical product, in order to
ensure that the process is continuously controlled.
1.9 F value
Assume a value of 10°C for the Z value defined as the num-
ber of degrees of temperature required for a 10-fold change
in the D value. The F value indicates the time (minutes) re-
quired to give the equivalent lethality at T b of the sterilization
heat obtained by integrating the lethality rate (L) over an en-
tire heating cycle.
£ = los
= 10"
T = Temperature inside the chamber or inside the product
to be sterilized
T b = Reference temperature (121 °C)
F„=J Ldt
k
t\ — t — Processing time (minutes)
1.10 Control device
A general term for the devices and measurement equip-
ment, including the equipment for controlling, measuring
and recording the physical parameters that can be measured
(temperature, humidity, pressure, time, radiation dose, etc.).
1.11 Parametric release
A release procedure based on an evaluation of the produc-
JPXV
General Information / Sterility Assurance 1749
tion records and critical parameters of the sterilization
process (temperature, humidity, pressure, time, radiation
dose, etc.) based on the results of validation, in lieu of release
based on testing results of the final product.
2. Sterilization Validation
2.1 Subject of the Implementation
A manufacturer of sterile pharmaceuticals (hereafter,
"manufacturer") must establish a quality system, implement
product sterilization validation for the categories below as a
general rule, and continuously control the sterilization
process based on the results of the sterilization validation.
a. Sterilization process
b. Sterilization process support system
2.2 Documenting Sterilization Validation Procedure
2.2.1 The manufacturer must prepare a "Sterilization
Validation Procedure" defining the items listed below regard-
ing the procedures for managing the sterilization process.
a. Details related to the range of duties of the persons
responsible for the validation, as well as the extent of their
authority
b. Details related to the implementation period for the
sterilization validation
c. Details related to the creation, modification, and ap-
proval of the sterilization validation plan documents
d. Details related to the reporting, evaluation, and ap-
proval of the sterilization validation implementation results
e. Details related to the storage of documentation con-
cerning the sterilization validation
f . Other required matters
2.2.2 The sterilization validation procedure must list the
names of the enactors, the date of enactment, and when there
are revisions, must also list the revisers, date of revisions,
revised sections and reasons for the revisions.
2.2.3 The manufacturer must properly store and main-
tain the sterilization validation procedure after clarifying the
procedures related to alterations and deletions of the contents
of the sterilization validation procedure.
2.3 Persons Responsible for the Validation
The manufacturer must assign persons to be responsible
for the sterilization validation. The responsible parties must
perform each of the duties listed below according to the
sterilization validation procedure.
2.3.1 For products that are to be produced according to
the sterilization validation procedure, a written sterilization
validation implementation plan must be prepared. The im-
plementation plan will specify the following points based on
a consideration of the implementation details of the steriliza-
tion validation.
a. Subject pharmaceutical name (product name)
b. Purpose of the applicable sterilization validation
c. Expected results
d. Verification methods (including inspection results and
evaluation methods)
e. Period of verification implementation
f . Names of persons performing the sterilization valida-
tion (persons-in-charge)
g. Names of the persons who created the plan, creation
date, and in the event of revisions, the names of the revisers,
date of the revisions, revised sections, and reasons for revi-
sion.
h. Technical requirements for the applicable sterilization
validation
i. Other required matters for the implementation of the
applicable sterilization validation
2.3.2 The following sterilization validation is implement-
ed according to the plan defining the items above.
a. When the manufacturing license and additional
(modification) licenses for product production are obtained,
implementation items for the sterilization validation to be ex-
ecuted
1 Product qualification
2 Facility/equipment qualification
1) Installation qualification
2) Operation qualification
3 Performance qualification
1) Physical performance qualification
2) Microbiological performance qualification
b. Sterilization validation to be executed until it is time to
renew the manufacturing license
1 Re-validation when there are changes
2 Periodic re-validation (The items implemented, etc.
must be determined based on a consideration of
relevant factors such as the sterilization method.)
2.3.3 Evaluate the results of the sterilization validation
and verify that sterility is assured.
2.3.4 Make a written report of the results of the steriliza-
tion validation to the manufacturer's authorized person.
2.3.5 Perform the day-to-day management of the sterili-
zation process.
3. Microorganism Control Program
When parametric release is adopted, it is important to con-
trol the bioburden in the raw materials of the product, the
containers and stoppers, and in the product before steriliza-
tion. The bioburden is measured with a previously specified
method and frequency, and when required, surveys of the
characteristics of the isolated microorganisms are made to in-
vestigate their resistance to the applicable sterilization
method. Refer to the "Microbiological Evaluation of Process
Areas for Sterile Pharmaceutical Products" regarding the
method for evaluating the environmental microorganisms in
the processing areas of pharmaceutical products.
4. Sterilization Indicators
Biological indicators (BI), chemical indicators (CI), and
dosimeters are among the means used to monitor a steriliza-
tion process and as indices of sterility (refer to Terminal
Sterilization and Sterilization Indicators). When using sterili-
zation indicators it is important to consider environmental
and human safety, and to take all necessary precautions. The
BI used for sterilization validation and daily process control
must be defined in the specification, and recorded in writing.
When BI are used for daily process control it must be verified
that the loading pattern on the form, product, or simulated
product has a resistance equal to or greater than that used for
the microbiological performance qualification.
5. Establishment of a Change Control System
Changes which have a large effect on the sterile quality,
such as changes in sterilization equipment, loading pattern,
and sterilization conditions, correspond to changes of the
parametric release conditions for the relevant pharmaceutical
product. A change control system must be defined in the
sterilization validation procedure; and when there are
changes in the causes of variation that have been previously
specified, there must be an investigation of the causes of vari-
1750 Sterility Assurance / General Information
JP XV
ation and of acceptable conditions to verify that the phar-
maceutical product is guaranteed always to conform to the
quality standards. Furthermore, before modifications are
made to a sterilization process that has been validated, it is
mandatory to obtain approval for the implementation of the
modifications in question from the appropriate authorized
person.
6. Release Procedure
A release procedure must be created to clarify the condi-
tions required for shipment based on parametric release of
terminally sterilized products. The following points must be
evaluated and recorded when a product is released.
Depending on the sterilization method, some of these items
may be omitted or modified.
a) Batch record
b) Microorganism evaluation data of production en-
vironment
c) Bioburden data for the raw materials and product be-
fore sterilization
d) Data related to the sterilization indicators
e) Data on the maintenance management of the steriliza-
tion process and sterilization process support systems
f ) Data on the management of sterilization parameters
g) Data on the calibrations of measurement equipment
h) Re-validation data
i) Other
7. Critical Control Points
The important control points for each sterilization method
are presented.
7.1 Moist heat sterilization
Moist heat sterilization is a method for killing microorgan-
isms in which saturated water vapor is generated or in-
troduced into a sterilization chamber at the appropriate tem-
perature and pressure, and the chamber is then heated for a
certain period of time. It is roughly classified into saturated
vapor sterilization, in which the target microorganisms are
directly exposed to the saturated vapor, and unsaturated
vapor sterilization, in which the fluid inside a container, such
as an ampule, is subjected to moist heat energy or highfre-
quency energy from the outside.
7.1.1 Important control points
A process control procedure must be created, specifying
the process parameters that affect the sterile quality of the
pharmaceutical product, and the permissible range of varia-
tion for each parameter. The important control points for the
moist heat sterilization are indicated below.
a) Heating history (usually indicated by F Q value)
b) Temperature
c) Pressure
d) Time
e) Product loading format/loading density
f ) Other necessary matters
7.1.2 Utilities
The utilities and control devices required for moist heat
sterilization determine the quality and precision.
a) Quality of the vapor used
b) Quality of the air introduced into the sterilization
chamber to restore pressure, etc.
c) Quality of the water used for cooling
d) Precision of the temperature control devices
e) Precision of the pressure control devices
f ) Precision of the time control devices
g) Other
7.2 Ethylene oxide gas sterilization
Ethylene oxide gas allows sterilization at low temperatures,
so there is typically little injury to the substance being steri-
lized; however, since the gas is toxic it must be handled with
extreme caution. The sterilization process consists of precon-
ditioning, a sterilization cycle, and aeration. The precon-
ditioning is performed before the sterilization cycle to process
the product so that temperature and relative humidity in the
room or container are within the range in the specifications.
The sterilization cycle indicates the stage at which the actual
sterilization is performed, and consists of removal of the air,
conditioning (when used), injection of the sterilization gas,
maintenance of the sterilization conditions, removal of the
sterilization gas, and replacement of the air. The aeration is
the process of eliminating the residual ethylene oxide gas
from the product, either inside the sterilization chamber or in
a separate location.
7.2.1 Important control points
The important control points for the ethylene oxide gas
sterilization are indicated below.
7.2.1.1 Preconditioning (when performed)
a) Time, temperature, humidity
b) Product loading pattern/loading density
c) Sterilization loading temperature and/or humidity
d) Time from the end of preconditioning until the start of
the sterilization
e) Other necessary matters
7.2.1.2 Conditioning
a) If pressure reduction is performed, the pressure
achieved and required time
b) Reduced pressure maintenance period
c) Time, temperature, pressure, humidity
d) Sterilization loading temperature and humidity
e) Other necessary matters
7.2.1.3 Sterilization cycle
a) Pressure increase, injection time, and final pressure
for the injection of the sterilization gas
b) Concentration of the ethylene oxide gas (it is desirable
to analyze directly the gas concentration inside the steriliza-
tion chamber, but the following alternatives are acceptable if
direct analysis is difficult)
i) Mass of gas used
ii) Volume of gas used
iii) Conversion calculation using the initial low pressure
level and the gas injection pressure
c) Temperature within the sterilization chamber
d) Temperature of the loaded products to be sterilized
e) Effect time (exposure time)
f ) Product loading pattern/loading density
g) BI placement points and cultivation results
h) Other necessary matters
7.2.1.4 Aeration
a) Time, temperature
b) Loaded sterilized substance temperature
c) Pressure variation in the sterilization chamber and/or
the aeration room
d) Rate of change of the air or other gases in the aeration
room
e) Other necessary matters
7.2.2 Utilities
The utilities and control devices required for ethylene oxide
sterilization determine the quality and precision.
JPXV
General Information / Tablet Friability Test 1751
a) Quality of the ethylene oxide gas
b) Quality of the injected vapor or water
c) Quality of the replacement air after the completion of
sterilization
d) Quality of the BI
e) Precision of the temperature control devices
f ) Precision of the pressure control devices
g) Precision of the humidity control devices
h) Precision of the time control devices
i) Other
7.3 Irradiation Sterilization
Irradiation sterilization refers to methods of killing
microorganisms through exposure to ionizing radiation. The
types of ionizing radiation used are gamma-rays (y-rays)
emitted from a radioisotope such as 60 Co or 137 Cs, or electron
beams and bremsstrahling (X-ray) generated from an elec-
tron accelerator. In the case of y-rays, the cells are killed by
secondarily generated electrons, while in the case of the elec-
tron beam, the cells are killed by the electrons generated
directly from the electron accelerator. For this reason, the
processing time for electron beam sterilization is generally
shorter than that for y-ray sterilization; but, since the
penetration of the y-rays is better than that of the electron
beam, there must be appropriate consideration of the density
and thickness of the substance being sterilized when choosing
between these methods. For an irradiation sterilization
process, the control procedures primarily make use of
dosimeters and measure the absorbed dose in the substance
being sterilized. This is called dosimetric release.
7.3.1 Important control points
The important control points for the irradiation steriliza-
tion are indicated below.
7.3.1.1 y-ray radiation
a) Irradiation time (timer setting or conveyor speed)
b) Absorbed dose
c) Product loading pattern
d) Other necessary matters
7.3.1.2 Electron beam and X-ray radiation
a) Electron beam characteristics (average electron beam
current, electron energy, scan width)
b) Conveyor speed
c) Absorbed dose
d) Product loading pattern
e) Other necessary matters
7.3.2 Utilities
A traceable calibration, performed according to national
standards, must be performed for the radiation devices and
dose measurement systems. This calibration must be per-
formed as specified in a written plan in order to verify that
the equipment is kept within the required range of accuracy.
7.3.2.1 Required calibration items for gamma-radiation
equipment
a) Cycle time or conveyor speed
b) Weighing device
c) Dose measurement system
d) Other
7.3.2.2 Required calibration items for electron-beam and
X-ray radiation equipment
a) Electron beam characteristics
b) Conveyor speed
c) Weighing device
d) Dose measurement system
e) Other
References
1) Validation Standards, PAB Notification No. 158,
Ministry of Health and Welfare 1995
2) Sterilization Validation Standards, PMSB/IGD Notifi-
cation No.l, Ministry of Health and Welfare 1997
3) Quality Assurance Standards for Medical Devices, PAB
Notification No. 1128, Ministry of Health and Welfare
1994
4) ISO 9000 series, International Standards for Quality
Assurance
5) ISO 11134 Industrial moist heat sterilization
6) ISO 11135 Ethylene oxide sterilization
7) ISO 11137 Radiation sterilization
8) ISO 11138 Biological indicators
9) ISO 11140 Chemical indicators
10) ISO 11737-1 Microbiological Methods Part 1: Estima-
tion of population of microorganisms on products
11) USP <1222> Terminally Sterilized Pharmaceutical
Products - Parametric Release
26. Tablet Friability Test
This test is harmonized with the European Pharmacopoeia
and the U.S. Pharmacopeia.
The Tablet Friability Test is a method to determine the
friability of compressed uncoated tablets. The test procedure
presented in this chapter is generally applicable to most com-
pressed tablets. Measurement of tablets friability supple-
ments other physical strength measurement, such as tablet
crushing strength.
Use a drum, with an internal diameter between 283 and 291
mm and a depth between 36 and 40 mm, of transparent syn-
thetic polymer with polished internal surface, and not subject
to minimum static build-up (see figure for a typical appara-
tus). One side of the drum is removable. The tablets are tum-
bled at each turn of the drum by a curved projection with an
inside radius between 75.5 and 85.5 mm that extends from
the middle of the drum to the outer wall. The outside di-
ameter of the central shaft ring is between 24.5 and 25.5 mm.
The drum is attached to the horizontal axis of a device that
rotates at 25 ± 1 rpm. Thus, at each turn the tablets roll or
slide and fall onto the drum wall or onto each other.
For tablets with a unit mass equal to or less than 650 mg,
take a sample of whole tablets n corresponding as near as
possible to 6.5 g. For tablets with a unit mass of more than
650 mg, take a sample of 10 whole tablets. The tablets should
be carefully dedusted prior to testing. Accurately weigh the
tablet sample, and place the tablets in the drum. Rotate the
drum 100 times, and remove the tablets. Remove any loose
dust from the tablets as before, and accurately weigh.
Generally, the test is run once. If obviously cracked,
cleaved, or broken tablets are present in the tablet sample
after tumbling, the sample fails the test. If the results are
difficult to interpret or if the weight loss is greater than the
targeted value, the test should be repeated twice and the mean
of the three tests determined. A maximum mean weight loss
from the three samples of not more than 1% is considered ac-
ceptable for most products.
If tablet size or shape causes irregular tumbling, adjust the
drum base so that the base forms an angle of abut 10° with
1752 Terminal Sterilization / General Information
JP XV
LiisLclf I'adius
8O.;±5.0 mm
TT77T.
the bench top and the tablets no longer bind together when
lying next to each other, which prevents them from falling
freely.
Effervescent tablets and chewable tablets may have differ-
ent specifications as far as friability is concerned. In the case
of hygroscopic tablets, an appropriate humidity-controlled
environment is required for testing.
Drums with dual scooping projections, or apparatus with
more than one drum, for the running of multiple samples at
one time, are also permitted.
27. Terminal Sterilization and
Sterilization Indicators
Sterilization is a process whereby the killing or removal of
all forms of viable microorganisms in substances is accom-
plished. It is achieved by terminal sterilization or a filtration
method. For substances to which terminal sterilization can be
applied, an appropriate sterilization method should be select-
ed in accordance with the properties of the product, including
the packaging, after full consideration of the advantages and
disadvantages of each sterilization method, from among the
heat method, irradiation method and gas method. After in-
stallation of the sterilizer (including design and development
of the sterilization process), validation is required to confirm
that the sterilization process is properly performing its
designed function, under conditions of loading and unload-
ing of the product, on the basis of sufficient scientific evi-
dence. After the process has been validated and the steriliza-
tion of the product commenced, the process must be con-
trolled correctly, and qualification tests of the equipment and
procedures must be performed regularly. The bioburden per
product, prior to terminal sterilization, must be evaluated
periodically or on the basis of batches. Refer to the ISO stan-
dard (ISO 11737-1) relevant to bioburden estimation. For a
substance to which terminal sterilization can be applied,
generally use sterilization conditions such that a sterility as-
surance level of less than 10~ 6 can be obtained. The propriety
of the sterilization should be judged by employing an ap-
propriate sterilization process control, with the use of a suita-
ble sterilization indicator, and if necessary, based on the
result of the sterility test. The filtration procedure is used for
the sterilization of a liquid product, to which terminal sterili-
zation can not be applied. Concerning the disinfection and/
or sterilization necessary for processing equipment and areas
of pharmaceutical products, and performing microbiological
tests specified in the monographs, see Disinfection and
Sterilization Methods.
1. Definitions
The definitions of the terms used in this text are as follows.
Terminal sterilization: A process whereby a product is
sterilized in its final container or packaging, and which per-
mits the measurement and evaluation of quantifiable
microbial lethality.
Product: A generic term used to describe raw materials, in-
termediate products, and finished products, to be sterilized.
Bioburden: Numbers and types of viable microorganisms
in a product to be sterilized.
Sterility assurance level (SAL): Probability of a viable
microorganism being present in a product unit after exposure
to the proper sterilization process, expressed as 10".
Integrity test: A non-destructive test which is used to
predict the functional performance of a filter instead of the
microorganism challenge test.
D value: The value which shows the exposure time (decimal
reduction time) or absorbed dose (decimal reduction dose) re-
quired to cause a 1 -logarithm or 90% reduction in the popu-
lation of test microorganisms under stated exposure condi-
tions.
Sterilization indicator: Indicators used to monitor the
sterilization process, or as an index of sterility, including bio-
logical indicators (BI), chemical indicators (CI), dosimeters
and the like.
2. Sterilization
2-1. Heat Method
In the heat method, microorganisms are killed by heating.
(i) Moist heat method
Microorganisms are killed in saturated steam under pres-
sure. In this method, factors which may affect the steriliza-
tion include temperature, steam pressure and exposure time.
Therefore, in routine sterilization process control, it is re-
quired to monitor continuously the temperature, steam pres-
sure and exposure time, and they should be included in the
specifications of the sterilizer.
(ii) Dry-heat method
Microorganisms are killed in dry heated air. This method is
usually conducted in a batch-type dry heat sterilizer or a tun-
nel-type dry heat sterilizer. In this method, factors which may
affect the sterilization include temperature and exposure
time. Therefore, in routine sterilization process control, it is
required to monitor continuously the temperature and ex-
posure time, and they should be included in the specifications
of the sterilizer.
2-2. Irradiation method
Microorganisms are directly killed by ionizing radiation, or
by the heat generated by microwave radiation.
(i) Radiation method
Ionizing radiations which may be used are gamma (y) rays
emitted from a radioisotope such as cobalt 60, an electron
beam and bremsstrahlung (X rays) generated from an elec-
tron accelerator. Although any procedure can be applied to
thermally unstable products with no radioactivity residue, it
is necessary to consider the possibility of material degrada-
tion. Although a 25 kGy dose is traditionally used as a sterili-
zation dose, there are some ways to calculate the dose as fol-
lows: the bioburden of the substance to be sterilized is meas-
ured and the sterilization dose is calculated based on the
JPXV
General Information / Terminal Sterilization 1753
mean bioburden and the standard resistance distribution
(Method 1 in ISO 111 37), the dose is calculated from the frac-
tion positive information from a sterility test in which
representative product samples are exposed to a substerilizing
dose (Method 2 in ISO 1 1 1 37), or the dose is calculated based
on the bioburden and D value of the most resistant microor-
ganisms (Log method) (see 5-3). In the case of the radiation
sterilization procedure, factors which may affect the steriliza-
tion include dose (absorbed dose) and exposure time. There-
fore, in y ray sterilization process control, it is required to de-
termine the dose (the absorbed dose) at appropriate intervals
and to monitor continuously the exposure time in terms of
the operating parameters (the conveyer speed, the cycle time).
The dose control mechanism should be included in the
specifications of the sterilizer. In the case of electron beam or
bremsstrahlung irradiation, it is required to monitor the ac-
celeration voltage, the beam current and beam scanning
width besides the above-mentioned items.
(ii) Microwave method
Microorganisms are killed by the heat generated by micro-
wave radiation, usually at the frequency of 2450 ±50 MHz.
This method is applied to liquids or water-rich products in
sealed containers. Since a glass or plastic container may be
destroyed or deformed due to the rise of the inner pressure,
the containers must be certified to be able to withstand the
heat and the inner pressure generated during microwave
sterilization. Leakage of electromagnetic radiation must be at
a sufficiently low level to cause no harm to humans and no in-
terference with radio communications and the like. In this
method, factors which may affect the sterilization include
temperature, processing time and microwave output power.
Therefore, in routine sterilization process control, it is re-
quired to monitor continuously the temperature, time and the
microwave output power, and they should be included in the
specifications of the sterilizer.
2-3. Gas method
Ethylene oxide (EO) is widely used as a sterilization gas.
Since EO gas has an explosive nature, a 10 - 30% mixture
with carbon dioxide is commonly used. Also, as EO gas is a
strong alkylating agent, it can not be applied to the products
which are likely to react with or absorb it. Furthermore, be-
cause EO gas is toxic, the residual concentration of EO gas
and other secondarily generated toxic gases in products steri-
lized with EO gas must be reduced to less than the safe levels
thereof by means of aeration and the like before the product
is shipped. In this method, factors which may affect the
sterilization include temperature, gas concentration (pres-
sure), humidity and exposure time. Therefore, in routine
sterilization process control, it is required to monitor con-
tinuously the temperature, gas concentration (pressure), hu-
midity and exposure time, and they should be included in the
specifications of the sterilizer.
3. Filtration method
Microorganisms are removed by using a sterilizing filter
made of an appropriate material. However, this method is
not intended for microorganisms smaller than bacteria.
Generally, a sterilizing filter challenged with more than 10 7
microorganisms of a strain of Brevundimonas diminuta
(ATCC 19146, NBRC 14213, JCM 2428), cultured under the
appropriate conditions, per square centimeter of effective
filter area should provide a sterile effluent. In this method,
factors which may affect the sterilization include pressure,
flow rate, filter unit characteristics and the like. In routine
filtration process control, it is required to perform integrity
tests of the sterilizing filter after each filtration process (also
prior to the filtration process, if necessary).
4. Sterilization Indicators
4-1. Biological indicator (BI)
A BI is prepared from specific microorganisms resistant to
the specified sterilization process and is used to develop and/
or validate a sterilization process. The dry type BI is classified
into two kinds. In one, bacterial spores are added to a carrier
such as filter paper, glass or plastic and then the carriers are
dried and packaged. In the other, bacterial spores are added
to representative units of the product to be sterilized or to
simulated products. Packaging materials of the BI should
show good heat penetration in dry heat sterilization and good
gas or steam penetration in ethylene oxide and moist heat
sterilizations. It should be confirmed that any carrier does not
affect the D value of the spores. In the case of a liquid
product, the spores may be suspended in the same solution as
the product or in a solution showing an equivalent effect in
the sterilization of biological indicator. However, when the
spores are suspended in liquid, it is necessary to ensure that
the resistance characteristics of the spores are not affected
due to germination.
Typical examples of biological indicator
Sterilization
Representative
microorganisms*
Strain name
Moist heat
method
Geobacillus
stearo-
thermophilus
Dry heat Bacillus
method atrophaeus
Gas method Bacillus
atrophaeus
ATCC 7953, NBRC
13737, JCM 9488
ATCC 12980, NBRC
12550, JCM 2501
ATCC 9372, NBRC 13721
ATCC 9372, NBRC 13721
* In addition to these microorganisms, other microorgan-
isms with the greatest resistance to the sterilization proce-
dure concerned, found in the bioburden, can be used as the
biological indicator.
4-1-1. D value of BI
Methods for determination of the D value include the sur-
vival curve method and the fraction negative method
(Stumbo, Murphy & Cochran procedure, Limited Spearman-
Karber procedure and the like). In using marketed Bis, it is
usually unnecessary to determine the D value before use if the
D value indicated on the label has been determined by a stan-
dardized biological indicator evaluation resistometer (BIER)
under strictly prescribed conditions in accordance with ISO
11138-1. It is acceptable that the D value indicated on the
label shows a scattering of not more than ± 30 seconds.
4-1-2. Setting up procedure of BI
(i) In the case of dry materials
A Dry type BI is placed at predetermined cold spots in the
product to be sterilized or a suitable product showing an
equivalent effect in the sterilization. The Bis are usually pri-
mary packaged in the same way as the product, including a
secondary packaging, if applicable.
(ii) In the case of wet materials
Spores are suspended as the BI in the same solution as the
1754 Test for Trace Amounts / General Information
JP XV
product or in an appropriate similar solution, and should be
placed at cold spots in the sterilizer.
4-1-3. Culture conditions of BI
Soybean casein digest medium is generally used. General
culture conditions are at 55 - 60°C for 7 days in the case of
G. stearothermophilus and at 30 - 35 °C for 7 days in the case
of B. atrophaeus.
4-2. Chemical indicator (CI)
CI is an indicator which shows a color change of a sub-
stance applied to a paper slip, etc. as a result of physical and/
or chemical change due to exposure to heat, gas or radiation.
The CI can be classified into three types. The first is employed
to identify whether or not sterilization has already been im-
plemented, the second is employed to control the sterilization
process (for example, its color changes after sterilization for
a sufficient time), and the third is the Bowie & Dick type used
to evaluate the effectiveness of air removal during the pre-
vacuum phase of the pre-vacuum sterilization cycle.
4-3. Dosimeter
In the radiation (y-ray) method, the sterilization effect de-
pends on the absorbed radiation dose, so the sterilization
process control is mainly performed by measuring the dose.
A dosimeter is installed at a position corresponding to the
minimum dose region of an exposed container or a position
where the dose is in a known relation to that in the above
region. Measurement should be done for each radiation
batch. If there are many containers in the same batch,
dosimeters should be employed so that more than one
dosimeter is always installed at the effective radiation section
of the irradiation chamber. It should be noted that
dosimeters may be affected by environmental conditions
(temperature, humidity, ultraviolet light, time until reading,
etc.) before and during irradiation. Practical dosimeters for
y-ray and bremsstrahlung sterilization include the
dyed polymethylmethacrylate dosimeter, clear poly-
methylmethacrylate dosimeter, ceric-cerous sulfate
dosimeter, alanine-EPR dosimeter and the like. A dosimeter
for gamma radiation can not generally be used for steriliza-
tion process control with an electron beam of less than 3 MeV
energy. Dosimeters for electron beam sterilization include the
cellulose acetate dosimeter, radiochromic film dosimeter and
the like. A practical dosimeter must be calibrated against an
appropriate national or international standard dosimetry sys-
tem.
5. Determination of sterilization conditions using microor-
ganism as an indicator
Taking account of the characteristics upon the sterilization
concerned, bioburden, etc. of a product to be sterilized,
chose a suitable method from the followings and determine
the conditions.
5-1. Half-cycle method
In this method, a sterilization time of twice as long as that
required to inactive all of 10 6 counts of BI placed in the
product is used, regardless of bioburden count in the product
being sterilized or the resistance of the objective microorgan-
isms to the sterilization.
5-2. Overkill method
In this method, a sterilization condition giving a sterility
assurance level of not more than 10~ 6 counts is used, regard-
less of bioburden count in the product being sterilized or the
resistance of the objective microorganisms to the steriliza-
tion. Generally, a sterilization condition providing 12
logarithmic reduction (12D) of a known count of BI of more
than 1.0 D value is used.
5-3. Combination of BI and bioburden
Generally, a count of mean bioburden added three times of
its standard deviation obtained by an extensive bioburden es-
timation is considered as the maximum bioburden count, and
the sterilization time (or radiation dose) is calculated with the
bioburden count based on an objective sterility assurance lev-
el. When this procedure is used, it is required to determine
the resistance of the bioburden to the sterilization as well as
the bioburden count in the product being sterilized. If a more
resistant microorganism than the BI spore is found in the bio-
burden estimation, it should be used as the BI.
N
Sterilization time (or radiation dose) =Dxlog
D: D value of the BI
N: Sterility assurance level
N : Maximum bioburden count in the product
5-4. Absolute bioburden method
The sterilization conditions are determined by employing
the D value of the most resistant microorganism found in the
product or environment by the resistant estimation and being
based on the bioburden count in the product. Generally, a
count of mean bioburden added three times of its standard
deviation obtained by an extensive bioburden estimation is
employed as the bioburden count. When this procedure is
used, it is required to make frequent counting and resistance
determination of microorganisms in daily bioburden estima-
tion.
References
1) ISO 11134 Industrial moist heat sterilization
2) ISO 11135 Ethylene oxide sterilization
3) ISO 11137 Radiation sterilization
4) ISO 11138 Biological indicators
5) ISO 11140 Chemical indicators
6) ISO 11737 Microbiological methods
Part 1: Estimation of population of micro-
organisms on products
28. Test for Trace Amounts of
Aluminum in Trans Parenteral
Nutrition (TPN) Solutions
Trans parenteral nutrition solutions (TPNs) are nutrient
preparations for intravenous injection packed in a large
volume. Since toxic effects to the central nervous system,
bone, etc. due to trace amounts of aluminum have recently
been reported in several countries, testing methods for trace
amounts of aluminum contaminating TPNs are required for
the official standard. The following three analytical methods
are available: (1) High-Performance Liquid Chromatography
using a fluorescence photometric detector (HPLC with
fluorescence detection), (2) Inductivity Coupled Plasma-
Atomic Emission Spectrometry (ICP-AES method), (3) In-
ductivity Coupled Plasma-Mass Spectrometry (ICP-MS
method). Detection sensitivity by HPLC with fluorescence
detection is about 1 //g/L (ppb), while ICP-AES fitted with
special apparatus and ICP-MS have higher sensitivity.
JPXV
General Information / Test for Trace Amounts 1755
Since TPNs are nutrient preparations, they contain many
nutrients such as sugars, amino acids, electrolytes, etc., in
various compositions. Thus, care is needed in the selection of
a suitable analytical method, because these coexisting compo-
nents may affect the measurement of trace amounts of alumi-
num.
In view of the general availability of HPLC apparatus, the
present general information describes procedures for the de-
termination of trace levels of aluminum in TPNs by means of
HPLC with a fluorescence photometric detector, using two
kinds of fluorescent chelating agents, i.e., (1) Quinolinol
complexing method, (2) Lumogallion complexing method.
(1) Quinolinol complexing method
After forming a complex of aluminum ion in the sample
solution with quinolinol, the assay for aluminum by HPLC
fitted with a fluorescence photometer is performed.
Preparation of sample solution
Pipet 1 mL of the sample (TPNs) exactly, and after adding
10 /xL of water for aluminum test, make up the sample solu-
tion to 10 mL exactly by adding the mobile phase.
Preparation of a series of standard solutions for calibra-
tion curve
Pipet 1 mL of water for aluminum test exactly, and after
adding 10 /xL each of standard solutions of aluminum (1)??
(5), make up the standard solutions for calibration curve to
10 mL (Aluminum concentration: 0, 1.25, 2.5, 5.0, and 10.0
ppb).
Standard testing method
Pipet 0.1 mL each of the sample solution and standard so-
lutions, and perform the test by HPLC under the following
conditions. Calculate the aluminum cotent in the sample so-
lution using a calibration curve method.
Operating conditions —
Detector: Fluorescence photometer(excitation wavelength:
380 nm, emission wavelength: 520 nm)
Column: A stainless steel column 4.6 mm in inside di-
ameter and 15 cm in length, packed with phenylsilanized sili-
ca gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: A mixture of 8-quinolinol in acetonitrile (3
in 100) and diluted 0.5 mol/L ammonium acetate TS (2 in 5)
(1:1).
Flow rate: Adjust the flow rate so that the retention time of
aluminum/8-quinolinol complex is about 9 minutes.
System suitability —
The correlation coefficient of the calibration curve, which
is prepared using a series of standard solutions, is not less
than 0.99.
Furthermore there is an alternative method, in which the
chelating agent 8-quinolinol is not included in the mobile
phase. In this method also, aluminum is detected as a com-
plex with 8-quinolinol in the sample solution by using HPLC
fitted with fluorescence photometer. But it is necessary to
form a more stable aluminum/8-quinolinol complex in the
sample solution, because the chelating agent is not included
in the mobile phase. Further, since the analytical wavelength
for the fluorescence detection is different from that in the
standard method, excitation WL: 370 nm, emission WL: 504
nm, the detection sensitivity is different. Thus, it is appropri-
ate to obtain the calibration curve between 0-25 ppb of alu-
minum. Other than the above- mentioned differences, the size
of column, column temperature, and the mobile phase are
also different from those used in the standard method, so
suitable analytical conditions should be established for per-
forming precise and reproducible examinations of trace
amounts of aluminum in the sample specimen.
(2) Lumogallion complexing method
After forming a complex of aluminum ion in the sample
specimen with the fluorescent reagent of lumogallion, the so-
lution is examined by HPLC fitted with a fluorescence pho-
tometer.
Preparation of sample solution
Pipet 70 /uL of the sample specimen (TPN) exactly, add
0.15 mL of lumogallion hydrochloric acid TS and 0.6 mL of
buffer solution for aluminum test, pH 7.2 exactly, then mix
the solution. After this solution has been allowed to stand for
4 hours at 40°C, it can be used for the measurement as a sam-
ple solution.
Preparation of a series of standard solutions for calibration
curve
Pipet 1 mL each of standard aluminum solutions (1) - (5)
exactly, and add diluted nitric acid for aluminum test (1 in
100) to make exactly 100 mL. Pipet 70 /xL each of these solu-
tions exactly, and add exactly 0.15 mL of lumogallion
hydrochloric acid TS and exactly 0.6 mL of buffer solution
for aluminum test, pH 7.2 then allow to stand for 4 hours at
40°C to make a series of standard solutions for obtaining the
calibration curve (Aluminum: 0, 1.07, 2.13, 4.27, and 8.54
ppb).
Standard examination method
Take 0.1 mL each of the sample solution and standard alu-
minum solutions for the calibration curve, and perform
HPLC analysis under the following conditions. Calculate the
aluminum content in the sample solution by using a calibra-
tion curve method.
Operating conditions —
Detector: Fluorescence photometer(excitation wavelength
505 nm, emission wavelength 574 nm)
Column: A stainless steel column 6.0 mm in inside di-
ameter and 10 cm in length, packed with octylsilanized silica
gel for liquid chromatography (5 /xm in particle diameter).
Column temperature: A constant temperature of about
40°C.
Mobile phase: Take 100 mL of 2-propanol, and add a
diluted 1 mol/L acetic acid-sodium acetate buffer solution of
pH 5.0 (1 in 10) to make 1000 mL.
Flow rate: Adjust the flow rate so that the retention time of
aluminum/lumogallion complex is about 5 minutes.
System suitability —
The correlation coefficient of the calibration curve, which
is prepared using a series of standard solutions, is not less
than 0.99.
The following points should be noted in connection with
measuring trace amounts of aluminum in TPN preparations.
1) Regarding water, solvents, reagents, vessels and other
tools used for the examination, select those not contami-
nated with aluminum. Further, keep the testing environ-
ment clean and free from dust in the testing room.
2) Before the measurement, it is necessary to confirm that the
characteristic properties of the sample do not affect the for-
mation of the complex.
1756 Total Protein Assay / General Information
JP XV
3) Reference substances of river water for analysis of trace
elements, distributed by the Japan Society for Analytical
Chemistry, contain certified amounts of aluminum: JSAC
0301-1 and JSAC 0302 (a known amount of aluminum is
artificially added to JSAC 0301-1).
Standard Solutions, Reagents and Test Solutions
Other than the standard solutions, reagents and test solu-
tions specified in the Japanese Pharmacopoeia, those de-
scribed below can be used in this test.
iV,./V-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acid C 6
H 15 N0 5 S White crystals or powder.
Hydrochloric acid for aluminum test Same as the reagent
Hydrochloric acid. Further, it contains not more than 1 ppb
of aluminum.
Lumogallion [5-Chloro-2-hydroxy-3(2,4-dihydrox-
yphenylazo)benzenesulfonic acid] Ci2H 9 ClN 2 6 S Red-brown
to dark brown powder. Further, it contains not more than 1
ppm of aluminum.
Lumogallion hydrochloric acid TS Dissolve 0.86 g of lu-
mogallion in 300 mL of 2-propanol, and add 350 mL of dilut-
ed Hydrochloric acid for aluminum test (9 in 50) and Water
for aluminum test to make 1000 mL exactly.
Nitric acid for aluminum test Same as the reagent Nitric
acid. Further, it contains not more than 1 ppb of aluminum.
pH buffer solution for aluminum test, pH 7.2 Dissolve
106.6 g of A f ,A r -bis(2-hydroxyethyl)-2-aminoethane sulfonic
acid in 800 mL of Water for aluminum test, adjust the pH 7.2
by using Tetramethylammonium hydroxide aqueous solu-
tion, and add Water for aluminum test to make 1000 mL.
Standard aluminum solution Pipet a constant volume
each of Water for aluminum test or the Standard aluminum
stock solution, dilute and adjust the aluminum concentration
to 0, 1.25, 2.5, 5.0, and 10 ppm by using diluted Nitric acid
for aluminum test (1 in 100), to make Standard aluminum so-
lutions (1) - (5).
Tetramethylammonium hydroxide TS (CH 3 ) 4 NOH
It is a 25% aqueous solution, prepared for aluminum test.
Further, it contains not more than 1 ppb of aluminum.
Water for aluminum test Same as the monograph Puri-
fied Water. Further, it contains not more than 1 ppb of alu-
minum.
29. Total Protein Assay
This test is harminized with the European Pharmacopoeia
and the U.S. Pharmacopeia. The parts of the text that are not
harmonized are marked with symbols (* ♦).
The following procedures are provided as illustrations of
the determination of total protein content in pharmacopoeial
preparations. Other techniques, such as HPLC, are also ac-
ceptable if total protein recovery is demonstrated. Many of
the total protein assay methods described below can be per-
formed successfully using kits from commercial sources.
Note: Where water is required, use distilled water.
Method 1
Protein in solution absorbs UV light at a wavelength of 280
nm, due to the presence of aromatic amino acids, mainly
tyrosine and tryptophan. This property is the basis of this
method. Protein determination at 280 nm is mainly a func-
tion of the tyrosine and tryptophan content of the protein. If
the buffer used to dissolve the protein has a high absorbance
relative to that of water, there is an interfering substance in
the buffer. This interference can be compensated for when
the spectrophotometer is adjusted to zero buffer absorbance.
If the interference results in a large absorbance that
challenges the limit of sensitivity of the spectrophotometer,
the results may be compromised. Furthermore, at low con-
centrations protein can be absorbed onto the cuvette, thereby
reducing the content in solution. This can be prevented by
preparing samples at higher concentrations or by using a non-
ionic detergent in the preparation.
Note: Keep the Test Solution, the Standard Solution, and
the buffer at the same temperature during testing.
Standard Solution Unless otherwise specified in the
individual monograph, prepare a solution of the reference
standard or reference material for the protein under test in
the same buffer and at the same concentration as the Test
Solution.
Test Solution Dissolve a suitable quantity of the protein un-
der test in the appropriate buffer to obtain a solution having a
concentration of 0.2 to 2 mg per mL.
Procedure Concomitantly determine the absorbances of the
Standard Solution and the Test Solution in quartz cells at a
wavelength of 280 nm, with a suitable spectrophotometer,
using the buffer as the blank. To obtain accurate results, the
response should be linear in the range of protein concentra-
tions to be assayed.
Light-Scattering The accuracy of the UV spectroscopic
determination of protein can be decreased by the scattering
of light by the test specimen. If the proteins in solution exist
as particles comparable in size to the wavelength of the meas-
uring light (250 to 300 nm), scattering of the light beam
results in an apparent increase in absorbance of the test speci-
men. To calculate the absorbance at 280 nm due to light-scat-
tering, determine the absorbances of the Test Solution at
wavelengths of 320, 325, 330, 335, 340, 345, and 350 nm. Us-
ing the linear regression method, plot the log of the observed
absorbance versus the log of the wavelength, and determine
the standard curve best fitting the plotted points. From the
graph so obtained, extrapolate the absorbance value due to
light-scattering at 280 nm. Subtract the absorbance from
light-scattering from the total absorbance at 280 nm to ob-
tain the absorbance value of the protein in solution. Filtra-
tion with a filter having a 0.2-//m porosity or clarification by
centrifugation may be performed to reduce the effect of light-
scattering, especially if the solution is noticeably turbid.
Calculations Calculate the concentration, C u , of protein in
the test specimen by the formula:
C V = C S (A U /As),
in which C s is the concentration of the Standard Solution;
and A v and A s are the corrected absorbances of the Test
Solution and the Standard Solution, respectively.
Method 2
This method, commonly referred to as the Lowry assay, is
based on the reduction by protein of the phosphomolybdic-
JP XV
General Information / Total Protein Assay 1757
tungstic mixed acid chromogen in the Folin-Ciocalteu's
phenol reagent, resulting in an absorbance maximum at
750 nm. The Folin-Ciocalteu's phenol reagent (Folin's TS)
reacts primarily with tyrosine residues in the protein, which
can lead to variation in the response of the assay to different
proteins. Because the method is sensitive to interfering
substances, a procedure for precipitation of the protein from
the test specimen may be used. Where separation of interfer-
ing substances from the protein in the test specimen is
necessary, proceed as directed below for Interfering
Substances prior to preparation of the Test Solution. The
effect of interfering substances can be minimized by dilution
provided the concentration of the protein under test remains
sufficient for accurate measurement. Variations of the Lowry
test that are indicated in national regulatory documents 1 ' can
be substituted for the method described below.
Standard Solutions Unless otherwise specified in the
individual monograph, dissolve the reference standard or
reference material for the protein under test in the buffer used
to prepare the Test Solution. Dilute portions of this solution
with the same buffer to obtain not fewer than five Standard
Solutions having concentrations between 5 and 100 //g of
protein per mL, the concentrations being evenly spaced.
Test Solution Dissolve a suitable quantity of the protein un-
der test in the appropriate buffer to obtain a solution having a
concentration within the range of the concentrations of the
Standard Solutions. An appropriate buffer will produce a pH
in the range of 10 to 10.5.
Blank Use the buffer used for the Test Solution and the
Standard Solutions.
Reagents and Solutions —
Copper Sulfate Reagent Dissolve 100 mg of copper (II)
sulfate pentahydrate and 200 mg of sodium tartrate dihy-
drate in water, dilute with water to 50 mL, and mix. Dissolve
10 g of anhydrous sodium carbonate in water to a final
volume of 50 mL, and mix. Slowly pour the sodium car-
bonate solution into the copper sulfate solution with mixing.
Prepare this solution fresh daily.
5% SDS TS Dissolve 5 g of sodium dodecyl sulfate in
water, and dilute with water to 100 mL.
Alkaline Copper Reagent Prepare a mixture of 5% SDS
TS, Copper Sulfate Reagent, and Sodium Hydroxide
Solution (4 in 125) (2:1:1). This reagent may be stored at
room temperature for up to 2 weeks.
Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mL
of water. Store in an amber bottle, at room temperature.
Procedure To 1 mL of each Standard Solution, the Test So-
lution, and the Blank, add 1 mL of Alkaline Copper Reagent,
and mix. Allow to stand at room temperature for 10 minutes.
Add 0.5 mL of the Diluted Folin's TS to each solution, and
mix each tube immediately after the addition, and allow to
stand at room temperature for 30 minutes. Determine the ab-
sorbances of the solutions from the Standard Solutions and
the Test Solution at the wavelength of maximum absorbance
at 750 nm, with a suitable spectrophotometer, using the solu-
tion from the Blank to set the instrument to zero.
Calculations [Note: The relationship of absorbance to
protein concentration is nonlinear; however, if the standard
curve concentration range is sufficiently small, it will
approach linearity.] Using the linear regression method, plot
* J) Example: the Minimum Requirements for Biological Products and in-
dividual monograph of JP.«
the absorbances of the solutions from the Standard Solutions
versus the protein concentrations, and determine the stan-
dard curve best fitting the plotted points. From the standard
curve so obtained and the absorbance of the Test Solution,
determine the concentration of protein in the Test Solution.
Interfering Substances In the following procedure, deoxy-
cholate-trichloroacetic acid is added to a test specimen to
remove interfering substances by precipitation of proteins be-
fore testing. This technique also can be used to concentrate
proteins from a dilute solution.
Sodium Deoxycholate Reagent Prepare a solution of
sodium deoxycholate in water having a concentration of
150 mg in 100 mL.
Trichloroacetic Acid Reagent Prepare a solution of
trichloroacetic acid in water having a concentration of 72 g in
100 mL.
Procedure Add 0.1 mL of Sodium Deoxycholate Reagent
to 1 mL of a solution of the protein under test. Mix on a
vortex mixer, and allow to stand at room temperature for 10
minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, and
mix on a vortex mixer. Centrifuge at 3000 x g for 30 minutes,
decant the liquid, and remove any residual liquid with a
pipet. Redissolve the protein pellet in 1 mL of Alkaline Cop-
per Reagent. Proceed as directed for Test Solution. [Note:
Color development reaches a maximum in 20 to 30 minutes
during incubation at room temperature, after which there is a
gradual loss of color. Most interfering substances cause a
lower color yield; however, some detergents cause a slight in-
crease in color. A high salt concentration may cause a
precipitate to form. Because different protein species may
give different color response intensities, the standard protein
and test protein should be the same.]
Method 3
This method, commonly referred to as the Bradford assay,
is based on the absorption shift from 470 nm to 595 nm ob-
served when the brilliant blue G-250 dye binds to protein.
The Coomassie Brilliant Blue G-250 dye binds most readily to
arginyl and lysyl residues in the protein, which can lead to
variation in the response of the assay to different proteins.
Standard Solutions Unless otherwise specified in the
individual monograph, dissolve the reference standard or the
reference material for the protein under test in the buffer used
to prepare the Test Solution. Dilute portions of this solution
with the same buffer to obtain not fewer than five Standard
Solutions having concentrations between 100 n% and 1 mg of
protein per mL, the concentrations being evenly spaced.
Test Solution Dissolve a suitable quantity of the protein un-
der test in the appropriate buffer to obtain a solution having a
concentration within the range of the concentrations of the
Standard Solutions.
Blank Use the buffer used to prepare the Test Solution and
the Standard Solutions.
Coomassie Reagent Dissolve 100 mg of brilliant blue G-250
2 > in 50 mL of ethanol (95). [Note: Not all dyes have the same
brilliant blue G content, and different products may give
different results.] Add 100 mL of phosphoric acid, dilute with
water to 1000 mL, and mix. Filter the solution through filter
paper (Whatman No.l or equivalent), and store the filtered
reagent in an amber bottle at room temperature. [Note: Slow
precipitation of the dye will occur during storage of the rea-
2> Dye purity is important in the reagent preparation.
1758 Total Protein Assay / General Information
JP XV
gent. Filter the reagent before use.]
Procedure Add 5 mL of the Coomassie Reagent to 100 /xL
of each Standard Solution, the Test Solution, and the Blank,
and mix by inversion. Avoid foaming, which will lead to poor
reproducibility. Determine the absorbances of the solutions
from the Standard Solutions and the Test Solution at 595
nm, with a suitable spectrophotometer, using the Blank to set
the instrument to zero.
[Note: Do not use quartz (silica) spectrophotometer cells:
the dye binds to this material. Because different protein
species may give different color response intensities, the
standard protein and test protein should be the same.] There
are relatively few interfering substances, but detergents and
ampholytes in the test specimen should be avoided. Highly
alkaline specimens may interfere with the acidic reagent.
Calculations [Note: The relationship of absorbance to
protein concentration is nonlinear; however, if the standard
curve concentration range is sufficiently small, it will
approach linearity.] Using the linear regression method, plot
the absorbances of the solutions from the Standard Solutions
versus the protein concentrations, and determine the stan-
dard curve best fitting the plotted points. From the standard
curve so obtained and the absorbance of the Test Solution,
determine the concentration of protein in the Test Solution.
Method 4
This method, commonly referred to as the bicinchoninic
acid or BCA assay, is based on reduction of the cupric (Cu 2+ )
ion to cuprous (Cu + ) ion by protein. The bicinchoninic acid
reagent is used to detect the cuprous ion. The method has few
interfering substances. When interfering substances are
present, their effect may be minimized by dilution, provided
that the concentration of the protein under test remains
sufficient for accurate measurement.
Standard Solutions Unless otherwise specified in the
individual monograph, dissolve the reference standard or the
reference material for the protein under test in the buffer used
to prepare the Test Solution. Dilute portions of this solution
with the same buffer to obtain not fewer than five Standard
Solutions having concentrations between 10 and 1200 fig of
protein per mL, the concentrations being evenly spaced.
Test Solution Dissolve a suitable quantity of the protein un-
der test in the appropriate buffer to obtain a solution having a
concentration within the range of the concentrations of the
Standard Solutions.
Blank Use the buffer used to prepare the Test Solution and
the Standard Solutions.
Reagents and Solutions —
BCA Reagent Dissolve about 10 g of bicinchoninic acid,
20 g of sodium carbonate monohydrate, 1.6 g of sodium
tartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g of
sodium hydrogen carbonate in water. Adjust, if necessary,
with sodium hydroxide or sodium hydrogen carbonate to a
pH of 11.25. Dilute with water to 1000 mL, and mix.
Copper Sulfate Reagent Dissolve about 2 g of copper (II)
sulfate pentahydrate in water to a final volume of 50 mL.
Copper-BCA Reagent Mix 1 mL of Copper Sulfate
Reagent and 50 mL of BCA Reagent.
Procedure Mix 0.1 mL of each Standard Solution, the Test
Solution, and the Blank with 2 mL of the Copper-BCA
Reagent. Incubate the solutions at 37°C for 30 minutes, note
the time, and allow to come to room temperature. Within 60
minutes following the incubation time, determine the absor-
bances of the solutions from the Standard Solutions and the
Test Solution in quartz cells at 562 nm, with a suitable
spectrophotometer, using the Blank to set the instrument to
zero. After the solutions are cooled to room temperature, the
color intensity continues to increase gradually. If substances
that will cause interference in the test are present, proceed as
directed for Interfering Substances under Method 2. Because
different protein species may give different color response in-
tensities, the standard protein and test protein should be the
same.
Calculations [Note: The relationship of absorbance to pro-
tein concentration is nonlinear; however, if the standard
curve concentration range is sufficiently small, it will
approach linearity.] Using the linear regression method, plot
the absorbances of the solutions from the Standard Solutions
versus the protein concentrations, and determine the stan-
dard curve best fitting the plotted points. From the standard
curve so obtained and the absorbance of the Test Solution,
determine the concentration of protein in the Test Solution.
Method 5
This method, commonly referred to as the Biuret assay, is
based on the interaction of cupric (Cu 2 + ) ion with protein in
an alkaline solution and the resultant development of absor-
bance at 545 nm.
Standard Solutions Unless otherwise specified in the in-
dividual monograph, prepare a solution of Albumin Human
for which the protein content has been previously determined
by nitrogen analysis (using the nitrogen-to-protein conver-
sion factor of 6.25) or of the reference standard or reference
material for the protein under test in sodium chloride solu-
tion (9 in 1000). Dilute portions of this solution with sodium
chloride solution (9 in 1000) to obtain not fewer than three
Standard Solutions having concentrations between 0.5 and 10
mg per mL, the concentrations being evenly spaced. [Note:
Low responses may be observed if the sample under test has
significantly different level of proline than that of Albumin
Human. A different standard protein may be employed in
such cases.]
Test Solution Prepare a solution of the test protein in
sodium chloride solution (9 in 1000) having a concentration
within the range of the concentrations of the Standard
Solutions.
Blank Use sodium chloride solution (9 in 1000).
Biuret Reagent Dissolve about 3.46 g of copper (II) sulfate
pentahydrate in 10 mL of water, with heating if necessary,
and allow to cool (Solution A). Dissolve about 34.6 g of
sodium citrate dihydrate and 20.0 g of anhydrous sodium
carbonate in 80 mL of water, with heating if necessary, and
allow to cool (Solution B). Mix Solutions A and B, and dilute
with water to 200 mL. This Biuret Reagent is stable at room
temperature for 6 months. Do not use the reagent if it de-
velops turbidity or contains any precipitate.
Procedure To one volume of the Standard Solutions and a
solution of the Test Solution add an equal volume of sodium
hydroxide solution (6 in 100), and mix. Immediately add a
volume of Biuret Reagent equivalent to 0.4 volume of the
Test Solution, and mix. Allow to stand at a temperature
between 15 C C and 25°C for not less than 15 minutes. Within
90 minutes after the addition of the Biuret Reagent, deter-
mine the absorbances of the Standard Solutions and the
solution from the Test Solution at the wavelength of maxi-
mum absorbance at 545 nm, with a suitable spectrophotome-
JPXV
General Information / Total Protein Assay 1759
ter, using the Blank to set the instrument to zero. [Note: Any
solution that develops turbidity or a precipitate is not accept-
able for calculation of protein concentration.]
Calculations Using the least-squares linear regression
method, plot the absorbances of the Standard Solutions
versus the protein concentrations, and determine the stan-
dard curve best fitting the plotted points, and calculate the
correlation coefficient for the line. [Note: Within the given
range of the standards, the relationship of absorbance to pro-
tein concentration is approximately linear.] A suitable system
is one that yields a line having a correlation coefficient of not
less than 0.99. From the standard curve and the absorbance
of the Test Solution, determine the concentration of protein
in the test specimen, making any necessary correction.
Interfering Substances To minimize the effect of interfering
substances, the protein can be precipitated from the initial
test specimen as follows. Add 0.1 volume of 50% trichloroa-
cetic acid to 1 volume of a solution of the test specimen,
withdraw the supernatant layer, and dissolve the precipitate
in a small volume of 0.5 mol/L sodium hydroxide TS. Use
the solution so obtained to prepare the Test Solution.
Comments This test shows minimal difference between
equivalent IgG and albumin samples. Addition of the sodium
hydroxide and the Biuret Reagent as a combined reagent, in-
sufficient mixing after the addition of the sodium hydroxide,
or an extended time between the addition of the sodium
hydroxide solution and the addition of the Biuret Reagent
will give IgG samples a higher response than albumin sam-
ples. The trichloroacetic acid method used to minimize the
effects of interfering substances also can be used to determine
the protein content in test specimens at concentrations below
500 /ug per mL.
Method 6
This fluorometric method is based on the derivatization of
the protein with o-phthalaldehyde (OP A), which reacts with
the primary amines of the protein (i.e., NH 2 -terminal amino
acid and the e-amino group of the lysine residues). The
sensitivity of the test can be increased by hydrolyzing the pro-
tein before testing. Hydrolysis makes the a-amino group of
the constituent amino acids of the protein available for reac-
tion with the OPA reagent. The method requires very small
quantities of the protein.
Primary amines, such as tris(hydroxymethyl)amino-
methane and amino acid buffers, react with OPA and must be
avoided or removed. Ammonia at high concentrations will
react with OPA as well. The fluorescence obtained when a-
mine reacts with OPA can be unstable. The use of automated
procedures to standardize this procedure may improve the ac-
curacy and precision of the test.
Standard Solutions Unless otherwise specified in the
individual monograph, dissolve the reference standard or the
reference material for the protein under test in the buffer used
to prepare the Test Solution. Dilute portions of this solution
with the same buffer to obtain not fewer than five Standard
Solutions having concentrations between 10 and 200 /ug of
protein per mL, the concentrations being evenly spaced.
Test Solution Dissolve a suitable quantity of the protein un-
der test in the appropriate buffer to obtain a solution having a
concentration within the range of the concentrations of the
Standard Solutions.
Blank Use the buffer used to prepare the Test Solution and
the Standard Solutions.
Reagents and Solutions —
Borate Buffer Dissolve about 61.83 g of boric acid in
water, and adjust with potassium hydroxide to a pH of 10.4.
Dilute with water to 1000 mL, and mix.
Stock OPA Reagent Dissolve about 120 mg of o-
phthalaldehyde in 1.5 mL of methanol, add 100 mL of
Borate Buffer, and mix. Add 0.6 mL of polyoxyethylene (23)
lauryl ether, and mix. This solution is stable at room temper-
ature for at least 3 weeks.
OPA Reagent To 5 mL of Stock OPA Reagent add 15 /uL
of 2-mercaptoethanol. Prepare at least 30 minutes prior to
use. This reagent is stable for one day.
Procedure Adjust each of the Standard Solutions and the
Test Solution to a pH between 8.0 and 10.5. Mix 10 /uL of the
Test Solution and each of the Standard Solutions with 1 00 juL
of OPA Reagent, and allow to stand at room temperature for
15 minutes. Add 3 mL of 0.5 mol/L sodium hydroxide TS,
and mix. Using a suitable fluorometer, determine the fluores-
cent intensities of solutions from the Standard Solutions and
the Test Solution at an excitation wavelength of 340 nm and
an emission wavelength between 440 and 455 nm. [Note: The
fluorescence of an individual specimen is read only once be-
cause irradiation decreases the fluorescent intensity.]
Calculations The relationship of fluorescence to protein
concentration is linear. Using the linear regression method,
plot the fluorescent intensities of the solutions from the Stan-
dard Solutions versus the protein concentrations, and deter-
mine the standard curve best fitting the plotted points. From
the standard curve so obtained and the fluorescent intensity
of the Test Solution, determine the concentration of protein
in the test specimen.
Method 7
This method is based on nitrogen analysis as a means of
protein determination. Interference caused by the presence of
other nitrogen-containing substances in the test protein can
affect the determination of protein by this method. Nitrogen
analysis techniques destroy the protein under test but are not
limited to protein presentation in an aqueous environment.
Procedure A Determine the nitrogen content of the protein
under test as directed elsewhere in the Pharmacopoeia. Com-
mercial instrumentation is available for the Kjeldahl nitrogen
assay.
Procedure B Commercial instrumentation is available for
nitrogen analysis. Most nitrogen analysis instruments use
pyrolysis (i.e., combustion of the sample in oxygen at
temperatures approaching 1000°C), which produces nitric
oxide (NO) and other oxides of nitrogen (NO*) from the
nitrogen present in the test protein. Some instruments
convert the nitric oxides to nitrogen gas, which is quantified
with a thermal conductivity detector. Other instruments mix
nitric oxide (NO) with ozone (0 3 ) to produce excited nitrogen
dioxide (N0 2 ), which emits light when it decays and can be
quantified with a chemiluminescence detector. A protein
reference standard or reference material that is relatively pure
and is similar in composition to the test proteins is used to op-
timize the injection and pyrolysis parameters and to evaluate
consistency in the analysis.
Calculations The protein concentration is calculated by
dividing the nitrogen content of the sample by the known
nitrogen content of the protein. The known nitrogen content
of the protein can be determined from the chemical composi-
tion of the protein or by comparison with the nitrogen
1760 Validation of Analytical / General Information
JP XV
content of the appropriate reference standard or reference
material.
30. Validation of Analytical
Procedures
The validation of an analytical procedure is the process of
confirming that the analytical procedure employed for a test
of pharmaceutics is suitable for its intended use. In other
word, the validation of an analytical procedure requires us to
demonstrate scientifically that risks in decision by testing
caused by errors from analytical steps are acceptably small.
The performance of an analytical procedure is established by
various kinds of validation characteristics. The validity of a
proposed analytical procedure can be shown by demonstrat-
ing experimentally that the validation characteristics of the
analytical procedure satisfy the standards set up according to
the acceptable limits of testing.
When an analytical procedure is to be newly carried in the
Japanese Pharmacopoeia, when a test carried in the Japanese
Pharmacopoeia is to be revised, and when the test carried in
the Japanese Pharmacopoeia is to be replaced with a new test
according to regulations in general notices, analytical proce-
dures employed for these tests should be validated according
to this document.
Required data for analytical procedures to be carried in the
Japanese Pharmacopoeia
(1) Outline
This section should provide a brief explanation of the prin-
ciple of a proposed analytical procedure, identify the necessi-
ty of the analytical procedure and its advantage compared
with other procedures, and summarize the validation. When
an analytical procedure is revised, the limitation of the cur-
rent analytical procedure and the advantage offered by the
new analytical procedure should be described.
(2) Analytical procedure
This section should contain a complete description of the
analytical procedure to enable skilled persons to evaluate cor-
rectly the analytical procedure and replicate it if necessary.
Analytical procedures include all important operating proce-
dures for performing analyses, the preparation of standard
samples, reagents and test solutions, precautions, procedures
to verify system suitability (e.g. the verification of the
separating performance of a chromatographic system), for-
mulas to obtain results, the number of replications and so
forth. Any instruments and apparatus that are not stated in
the Japanese Pharmacopoeia should be described in detail.
The physical, chemical or biological characteristics of any
new reference standards should be clarified and their testing
methods should be established.
(3) Data showing the validity of analytical procedures
This section should provide complete data showing the
validity of the analytical procedures. This includes the ex-
perimental designs to determine the validation characteris-
tics, experimental data, calculation results and results of
hypothesis tests.
Validation characteristics
The definition of typical validation characteristics to be as-
sessed in validation of analytical procedures and examples of
assessing procedures are given below.
The terminology and definitions of the validation charac-
teristics may possibly vary depending upon the fields to which
analytical procedures are applied. The terminology and defi-
nitions shown in this document are established for the pur-
pose of the Japanese Pharmacopoeia. Typical methods for
assessing the validation characteristics are shown in the item
of assessment. Various kinds of methods to determine the
validation characteristics have been proposed and any
methods that are widely accepted will be accepted for the
present purpose. However, since values of the validation
characteristics may possibly depend upon methods of deter-
mination, it is required to present the methods of determining
the validation characteristics, the data and calculation
methods in sufficient detail.
Although robustness is not listed as a validation charac-
teristic, it should be considered during the development of
analytical procedures. Studying the robustness may help to
improve analytical procedures and to establish appropriate
analytical conditions including precautions.
(1) Accuracy/Trueness
Definition: The accuracy is a measure of the bias of ob-
served values obtained by an analytical procedure. The ac-
curacy is expressed as the difference between the average
value obtained from a large series of observed values and the
true value.
Assessment: The estimate of accuracy of an analytical
method is expressed as the difference between the total mean
of observed values obtained during investigation of the
reproducibility and the true value. The theoretical value is
used as the true value (e.g., in the case of titration methods,
etc.). When there is no theoretical value or it is difficult to ob-
tain a theoretical value even though it exists, a certified value
or a consensus value may be used as the true value. When an
analytical procedure for a drug product is considered, the ob-
served value of the standard solution of the drug substance
may be used as the consensus value.
It may be inferred from specificity data that an analytical
procedure is unbiased.
The estimate of accuracy and a 95% confidence interval of
the accuracy should be calculated using the standard error
based on the reproducibility (intermediate precision). It
should be confirmed that the confidence interval includes zero
or that the upper or lower confidence limits are within the
range of the accuracy required of the analytical procedure.
(2) Precision
Definition: The precision is a measure of the closeness of
agreement between observed values obtained independently
from multiple samplings of a homogenous sample and is ex-
pressed as the variance, standard deviation or relative stan-
dard deviation (coefficient of variation) of observed values.
The precision should be considered at three levels with
different repetition conditions; repeatability, intermediate
precision and reproducibility.
(i) Repeatability/Intra-assay precision
The repeatability expresses the precision of observed values
obtained from multiple samplings of a homogenous sample
over a short time interval within a laboratory, by the same
analyst, using the same apparatus and instruments, lots of
reagents and so forth (repeatability conditions).
(ii) Intermediate precision
The intermediate precision expresses the precision of ob-
served values obtained from multiple samplings of a
homogenous sample by changing a part of or all of the oper-
JPXV
General Information / Validation of Analytical 1761
ating conditions including analysts, experimental dates, ap-
paratus and instruments and lots of reagents within a labora-
tory (intermediate precision condition).
(iii) Reproducibility
The reproducibility expresses the precision of observed
values obtained from multiple samplings of a homogenous
sample in different laboratories (reproducibility condition).
Assessment: A sufficient volume of a homogenous sample
should be prepared before studying the precision. The solu-
tion is assumed to be homogenous. When it is difficult to ob-
tain a homogenous sample, the following samples may be
used as homogenous samples; e.g., a large amount of drug
products or mixture of drug substance and vehicles that are
crushed and mixed well until they can be assumed to be
homogenous.
Suitable experimental designs such as one-way layout may
be employed when more than one level of precision is to be
investigated simultaneously. A sufficient number of repeti-
tions, levels of operating conditions and laboratories should
be employed. Sources of variations affecting analytical results
should be evaluated as thoroughly as possible through the
validation.
It is required to show the variance, standard deviation and
relative standard deviation (coefficient of variation) of each
level of precision. The 90% confidence interval of the vari-
ance and corresponding intervals of the standard deviation
and relative standard deviation should also be established.
The validity of the proposed analytical procedure for its in-
tended use may be confirmed by comparing obtained values
with the required values of the analytical procedure. Whether
the proposed analytical procedure is acceptable may normal-
ly be decided based on the reproducibility.
(3) Specificity
Definition: The specificity is the ability of an analytical
procedure to measure accurately an analyte in the presence of
components that may be expected to be present in the sample
matrix. The specificity is a measure of discriminating ability.
Lack of specificity of an analytical procedure may be com-
pensated by other supporting analytical procedures.
Assessment: It should be confirmed that the proposed ana-
lytical procedure can identify an analyte or that it can ac-
curately measure the amount or concentration of an analyte
in a sample. The method to confirm the specificity depends
very much upon the purpose of the analytical procedure. For
example, the specificity may be assessed by comparing analyt-
ical results obtained from a sample containing the analyte
only with results obtained from samples containing ex-
cipients, related substances or degradation products, and in-
cluding or excluding the analyte. If reference standards of
impurities are unavailable, samples that are expected to con-
tain impurities or degradation products may be used (e.g.
samples after accelerated or stress tests).
(4) Detection limit
Definition: The detection limit is the lowest amount or con-
centration of the analyte in a sample that is detectable, but
not necessarily quantifiable.
Assessment: The detection limit should be normally deter-
mined so that producer's and consumer's risks are less than
5%. The detection limit may be calculated using the standard
deviation of responses of blank samples or samples contain-
ing an analyte close to the detection limit and the slope of the
calibration curve close to the detection limit. The following
equation is an example to determine the detection limit using
the standard deviation of responses of blank samples and the
slope of the calibration curve.
DL = 3.3a/slope
DL: detection limit
a: the standard deviation of responses of blank
samples
slope: slope of the calibration curve
The noise level may be used as the standard deviation of
responses of blank samples in chromatographic methods. It
should be ensured that the detection limit of the analytical
procedure is lower than the specified limit for testing.
(5) Quantitation limit
Definition: The quantitation limit is the lowest amount or
concentration of the analyte in a sample that can be deter-
mined. The precision expressed as the relative standard devia-
tion of samples containing an analyte at the quantitation
limit is usually 10%.
Assessment: The quantitation limit may be calculated us-
ing the standard deviation of responses of blank samples or
samples containing an analyte close to the quantitation limit
and the slope of the calibration curve close to the quantita-
tion limit. The following equation is an example to determine
the quantitation limit using the standard deviation of
responses of blank samples and the slope of the calibration
curve.
QL = lOa/slope
QL: quantitation limit
a: the standard deviation of responses of blank
samples
slope: slope of the calibration curve
The noise level may be used as the standard deviation of
responses of blank samples in chromatographic methods. It
should be ensured that the quantitation limit of the analytical
procedure is lower than the specified limit for testing.
(6) Linearity
Definition: The linearity is the ability of an analytical
procedure to elicit responses linearly related to the amount or
concentration of an analyte in samples. A well-defined
mathematical transformation may sometimes be necessary to
obtain a linear relationship.
Assessment: Responses are obtained after analyzing sam-
ples with various amounts or concentrations of an analyte ac-
cording to described operating procedures. The linearity may
be evaluated in terms of the correlation coefficient, and the
slope and y-intercept of the regression line. It may be also
helpful for evaluating the linearity to plot residual errors
from the regression line against the amount or concentration
and to confirm that there is no particular tendency in the
graph. Samples with five different amounts or concentrations
of an analyte should be usually investigated.
(7) Range
Definition: The range for the validation of analytical
procedures is the interval between the lower and upper limits
of the amount or concentration of an analyte providing
sufficient accuracy and precision. The range for the valida-
tion of analytical procedures for an analytical procedure with
linearity is the interval between the lower and upper limits
providing sufficient accuracy, precision and linearity.
Assessment: When the range for the validation of analyti-
cal procedures is investigated, 80 to 120% of specified limits
1762 Validation of Analytical / General Information
JP XV
of testing should be usually considered. The accuracy, preci-
sion and linearity should be evaluated using samples contain-
ing the lower and upper limits and in the middle of the range.
Categories of tests employing analytical procedures
Tests covered with this document are roughly classified
into three categories shown below according to their pur-
poses. The table lists the normally required validation charac-
teristics to be evaluated in the validation of analytical proce-
dures used in these tests. This list should be considered to
represent typical validation characteristics. A different ap-
proach to validating analytical procedures should be consi-
dered depending upon the characteristics of analytical proce-
dures and their intended use.
Type I Identification. Tests for identifying major compo-
nents in pharmaceuticals according to their characteristics.
Type II Impurity tests. Tests for determination of impur-
ities in pharmaceuticals.
Type III Tests for assaying drug substances, active in-
gredients, and major components in pharmaceuticals.
(Additives such as stabilizing agents and preservatives are in-
cluded in major components.) Tests for determining perfor-
mance of pharmaceuticals, such as dissolution testing.
Table Lists of validation characteristics required to be
evaluated in tests of each type
^^ Type of test
Validation ^^.
Type I
Type
II
Type III
Quantitation
Limit
characteristics ^^
test
test
Accuracy/Trueness
-
+
-
+
Precision
Repeatability
-
+
-
+
Intermediate precision
-
_ *
-
_ *
Reproducibility
-
+ *
-
+ *
Specificity**
+
+
+
+
Detection limit
-
-
+
-
Quantitation limit
-
+
-
-
Linearity
-
+
-
+
Range
—
+
—
+
- Usually need not to be evaluated.
+ Usually need to be evaluated.
* Either intermediate precision or reproducibility should be
evaluated depending upon circumstances in which analytical
procedures or tests are performed. The latter should be normally
evaluated in the validation of analytical procedures proposed to
be included in the Japanese Pharmacopoeia.
** The lack of the specificity of an analytical procedure may be
compensated by other relevant analytical procedures
Terminology used in the validation of analytical procedures
Analytical procedure: This document covers analytical
procedures applied to identification, and ones that provides
responses depending upon the amount or concentration of
analytes in samples.
Laboratory: The laboratory means an experimental room
or facility where tests are performed. In this document differ-
ent laboratories are expected to perform an analytical proce-
dure using different analysts, different experimental appara-
tus and instruments, different lots of reagents and so forth.
Number of replications: The number of replications is one
that is described in analytical procedures. An observed value
is often obtained by more than one measurement in order to
achieve good precision of analytical procedures. Analytical
procedures including the number of replications should be
validated. This is different from repetition in the validation of
analytical procedures to obtain accuracy or precision.
Observed value: The value of a characteristic obtained as
the result of performing an analytical procedure.
Consumer's risk: This is the probability that products out
of the specification of tests are decided to be accepted after
testing. It is usually expressed as /?, and is called the probabil-
ity of type II error or the probability of false negative in im-
purity tests.
Producer's risk: This is the probability that products satis-
fying the specification of tests are decided to be rejected after
testing. It is usually expressed as a, and is called the probabil-
ity of type I error or the probability of false positive in im-
purity tests.
Robustness: The robustness is a measure of the capacity to
remain unaffected by small but deliberate variations in ana-
lytical conditions. The stability of observed values may be
studied by changing various analytical conditions within suit-
able ranges including pH values of solutions, reaction tem-
perature, reaction time or amount of reagents added. When
observed values are unstable, the analytical procedure should
be improved. Results of studying robustness may be reflected
in the developed analytical procedure as precautions or sig-
nificant digits describing analytical conditions.
Test: Tests mean various tests described in general tests
and official monographs in the Japanese Pharmacopoeia such
as impurity tests and assay. They includes sampling methods,
specification limits and analytical procedures.
Atomic Weight Table (2004)
In 1961 it was decided that the atomic weights of the
elements would be based on values relative to the mass
number of 12 (no fractions) for carbon ( 12 C). Ever
since, there has been a marked improvement in the
quality and quantity of data on the nuclide masses and
isotope ratios of the elements using physical methods
such as mass spectrometry. The Commission on
Atomic Weights and Isotope Abundances (CAWIA) of
the International Union of Pure and Applied Chemis-
try (IUPAC) collected and examined newly measured
data and then published an atomic weight table. Based
on this table, in April of each year the Atomic Weight
Subcommittee of the Chemical Society of Japan also
publishes an atomic weight table. A simple explanation
is provided below so that the table can be used correctly
and effectively. For a more detailed explanation, the
user is referred to a report" and a review 2 ' published by
the CAWIA.
The atomic weight values of each of the elements
shown in the atomic weight tables are, as stated in the
preface to the table, for elements that originate on
Earth and are present in substances that exist naturally.
Atomic weights are, with the exception of single
nuclide elements (elements consisting of one stable
nuclide), not natural constants like the speed of light,
but rather change depending on a variety of factors,
such as the method of treatment or the origin of the
substance containing that element. This is because the
atomic weight is dependent on the relative frequency
(isotope ratio) of the stable nuclides comprising each of
the respective elements. Due to advancements in
measurement techniques, the isotopic frequencies of
each of the elements are not necessarily constant, and
fluctuate due to a variety of processes that occur on the
Earth. We have come to learn that this is reflected in
the atomic weights. The result of this is that differences
have arisen in the accuracy of the atomic weights
between elements. The figures in parentheses that
follow the atomic weight values in the atomic weight
tables represent the uncertainty with respect to the last
digit in the atomic weight. For example, in the case of
hydrogen, 1.00794(7) means 1.00794 + 0.00007.
The atomic weight of a single nuclide element is the
most accurate and the precision is also high. This is
because it is not necessary to consider the isotope ratio
since single nuclide elements do not possess a multiple
number of stable isotopes. The atomic weights of such
elements are determined based on the mass 3 ' of each
nuclide determined by physical techniques, taking into
consideration the uncertainty with constant criteria.
Among the elements, the majority of samples
gathered on Earth exhibit a constant isotope composi-
tion, however, some specific samples have isotope
compositions that are different to these. These kinds of
elements are indicated by a "g", which means the value
in the atomic weight table cannot be used as is, depend-
ing on the sample, as the atomic weight of these
elements. In relation to this, oxygen for example exists
in a number of forms on Earth, such as in air, salt
water, fresh water, and in rocks, and because the
isotope compositions fluctuate among these sub-
stances, oxygen is not an element for which only one
value can be used. Thus, an "r" is attached to an
element for which a precise atomic weight cannot be
given, no matter how much progress is made in
techniques for measuring the isotope composition. On
the other hand, it is also possible, depending on the
element, to use an isotope that has undergone artificial
fractionation as a reagent. Typical elements that are
representative include hydrogen, lithium, boron, and
uranium. This type of element is identified by an "m",
and particularly in cases where the atomic weight is a
problem, it is necessary to be careful by referring to the
label of the reagent.
1) IUPAC Inorganic Chemistry Division, CAWIA: Atomic
Weights of the Elements 2001. Pure App I. Chem., 75, 1107
(2003).
2) J. R. De Laeter et al.: Atomic Weights of the Elements:
Review 2000. Pure App I. Chem., 75, 683 (2003).
3) G. Audi and A. H. Wapstra: The 1993 Atomic Mass
Evaluation (I). Atomic Mass Table. Nucl. Phys., A565. 1
(1993).
1763
1764 Appendix
JP XV
Standard Atomic Weights 2004
(scaled to Ar{ n C) = \2, where n C is a neutral atom in its nuclear and electronic ground state)
The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The standard values of
Ar(E) and the uncertainties (in parentheses, following the last significant figure to which they are attributed) apply to elements of natural
terrestrial origin. The footnotes to this Table elaborate the types of variation which may occur for individual elements and which may be
larger than the listed uncertainties of values of Ar(E). Names of elements with atomic number 112 to 116 are provisional.
Atomic
Atomic
Atomic
Atomic
Name
Symbol
Number
Weight
Footnotes
Name
Symbol
Number
Weight
Footnt
Hydrogen
11
1
1.00794(7)
gmr
Praseodymium
Pr
59
140.90765(2}
Helium
He
2
4.002602(2)
g f
Neodymium
Nd
60
144,24(3)
9
Lithium
Li
3
[6.941(2]] :
gmr
Promethium*
Pm
61
Bsrylium
Be
4
9,012182(3)
Samarium
Sm
62
150.36(3}
9
Boron
B
5
10.811(7)
gmr
Europium
Ru
63
151.964(1}
g
Carbon
C
6
12.0107(8)
9 '
Gadolinium
Gd
64
157.25(3)
9
Nitrogen
N
7
14.0067(2)
g r
Terbium
Tb
65
158.92534(2)
Oxygen
8
15.9994(3)
g r
Dysprosium
Dy
66
162.500(1)
9
Fluorine
F
9
18.9984032(5)
Holmium
Ho
67
164.93032(2)
Neon
Ne
10
20.1797(6)
gm
Erbium
Lr
68
167.259(3)
9
Sodium
\a
11
22.989770(2)
Thulium
Tm
69
168.93421(2)
Magnesium
Mg
12
24.3050(6)
Ytterbium
Yh
70
173.04(3)
g
Aluminium
Al
13
26.981538(2)
Lutetium
Lu
71
174.967(1)
9
Silicon
Si
14
28.0855(3)
r
Hafnium
Hf
72
178.49(2)
Phosphorus
P
15
30.973761(2)
Tantalum
Ta
73
180.9479(1)
Sulfur
s
16
32.065(5)
9 '
Tungsten
W
74
183.84(1)
Chlorine
CI
17
35.453(2)
gm
(Wolfram)
Argon
Ar
18
39.948(1)
g '
Rhenium
Re
75
186.207(1)
Potassium
K
19
39.0983(1)
Osmium
Os
76
190.23(3)
g
Calcium
Ca
20
40.078(4)
g
Iridium
lr
77
192.217(3)
Scandium
Sc
21
44.955910(8)
Platinum
1't
78
195.078(2)
Titanium
Ti
22
47,867(1)
Gold
Au
79
196.96655(2)
Vanadium
V
23
50.9415(1)
Mercury
Hg
80
200.59(2)
Chromium
Cr
24
51.9961(6)
Thallium
Tl
81
204.3833(2)
Manganese
Mn
25
54.938049(9)
Lead
Pb
82
207.2(1)
9 i
Iron
Fc
26
55.845(2)
Bismuth
Bi
83
208.98038(2)
Cobalt
Co
27
58,933200(9)
Polonium"
Po
34
Nickel
Ni
28
58,6934(2}
Astatine*
At
85
Copper
Cu
29
63.546(3)
r
Radon*
Rn
86
Zinc
Zn
30
65.409(4}
Francium*
Kr
87
Gallium
Ga
31
69,723(1)
Radium*
Ra
88
Germanium
Ge
32
72.64(1}
Actinium*
Ac
89
Arsenic
As
33
74.92160(2)
Thorium*
Th
90
232.0381(1)
9
Selenium
Se
34
78.96(3)
r
Protactinium*
Pa
91
231.03588(2)
Bromine
Br
35
79.904(1)
Uranium*
(J
92
238.02891 (3)
gm
Krypton
Kr
36
83.798(2)
gm
Neptunium"
Np
93
Rubidium
Rb
37
85.4678(3)
g
Plutonium*
l'u
94
Strontium
Sr
38
87.62(1)
g '
Americium*
Am
95
Yttrium
Y
39
88.90585(2)
Curium*
Cm
96
Zirconium
Zr
40
91 .224(2)
9
Berkelium*
Bk
97
Niobium
Kb
41
92.90638(2)
Californium*
Cf
98
Molybdenum
Mo
42
95.94(2)
3
Einsteinium'
Es
99
Technetium*
Tc
43
Fermium*
Fm
100
Ruthenium
Ru
44
101.07(2)
g
Mendetevium*
Md
101
Rhodium
Rh
45
102.90550(2)
Nobelium'
No
102
Palladium
Pd
46
106.42(1)
g
Lawrencium'
Lr
103
Silver
As
47
107.8682(2)
g
Rutherfordium"
Rf
104
Cadmium
Cd
48
112.411(8)
g
Dubnium*
Db
105
Indium
In
49
114.818(3)
Seaborgium*
Sa
106
Tin
Sn
50
118.710(7)
g
Bohrium*
Bh
107
Antimony
Sb
51
121.760(1)
g
Hassium*
Us
108
Tellurium
Te
52
127.60(3)
g
Meitnerium*
Mt
109
Iodine
I
53
126.90447(3)
Darmstadtium*
Ds
110
Xenon
Xe
54
131.293(6)
gm
Roentgenium*
Rs
111
Caesium
Cs
55
132.90545(2)
Ununbium*
(Jnb
112
Barium
Ba
56
137.327(7)
Ununquadium*
Uua
114
Lanthanum
La
57
138.9055(2)
g
Ununhexium*
Uuh
116
Cerium
Ce
58
140.116(1)
9
Element has no stable isotopes.
Commercially available Li materials have atomic weights that range between 6.939 and 6.996; if a more accurate value is required, it must be determined for
the specific material.
geological specimens are known in which the element has an isotopic composition outside the limits for normal material. The difference between the atomic
weight of the element in such specimens and that given in the Table may exceed the stated uncertainty.
modified isotopic compositions may be found in commercially available material because it has been subjected to an undisclosed or inadvertent isotopic fraction-
ation. Substantial deviations in atomic weight of the element from that given in the Table can occur.
range in isotopic composition of normal terrestrial material prevents a more precise Ar(E) being given: the tabulated Ar(E) value should be applicable to any
normal material.
©Atomic Weights Subcommittee of the Chemical Society of Japan
INDEX
Absorptive Ointment, 265
Acacia, 1251
Acebutolol Hydrochloride, 265
Aceglutamide Aluminum, 266
Acetaminophen, 267
Acetazolamide, 268
Acetic Acid, 268
Acetohexamide, 269
Acetylcholine Chloride for Injection,
271
Acetylsalicylic Acid, 318
Tablets, 319
Achyranthes Root, 1252
Aclarubicin Hydrochloride, 272
Acrinol
and Zinc Oxide Oil, 273
and Zinc Oxide Ointment, 274
Hydrate, 274
Actinomycin D, 275
Adrenaline, 276
Injection, 276
Solution, 277
Adsorbed
Diphtheria Toxoid for Adult Use,
596
Diphtheria-Purified Pertussis-Teta-
nus Combined Vaccine, 596
Diphtheria-Tetanus Combined
Toxoid, 596
Habu-venom Toxoid, 716
Hepatitis B Vaccine, 716
Purified Pertussis Vaccine, 980
Tetanus Toxoid, 1156
Agar, 1252
Ajmaline, 278
Tablets, 279
Akebia Stem, 1253
Alacepril, 279
Tablets, 280
Albumin Tannate, 282
Alcohol, 638
for Disinfection, 640
Aldioxa, 282
Alimemazine Tartrate, 283
Alisma Rhizome, 1253
Allopurinol, 284
Aloe, 1254
Alpinia Officinarum Rhizome, 1256
Alprazolam, 284
Alprenolol Hydrochloride, 285
Alprostadil, 286
Alfadex, 287
Alum, 292
Solution, 293
Aluminum
Acetylsalicylate, 319
Monostearate, 291
Potassium Sulfate Hydrate, 292
Sucrose Sulfate Ester, 1118
Amantadine Hydrochloride, 293
Ambenonium Chloride, 294
Amidotrizoic Acid, 295
Amikacin Sulfate, 296
Aminoacetic Acid, 704
Aminobenzylpenicillin, 307
Sodium, 308
Aminophylline
Hydrate, 297
Injection, 297
Amitriptyline Hydrochloride, 298
Tablets, 299
Ammonia Water, 299
Amobarbital, 300
Sodium for Injection, 300
Amomum Seed, 1256
Amorphous Insulin Zinc Injection
(Aqueous Suspension), 764
Amoxapine, 301
Amoxicillin Hydrate, 302
Amphotericin B, 303
for Injection, 304
Syrup, 305
Tablets, 305
Ampicillin
Ethoxycarbonyloxyethyl Hydro-
chloride, 329
Hydrate, 307
Sodium, 308
Ampicillinphthalidyl Hydrochloride,
1138
Amyl Nitrite, 309
Anemarrhena Rhizome, 1256
Anesthamine, 644
Anesthetic Ether, 641
Angelica Dahurica Root, 1257
Anhydrous
Aminobenzylpenicillin, 306
Ampicillin, 306
Caffeine, 386
Citric Acid, 514
Dibasic Calcium Phosphate, 396
Ethanol, 639
Lactose, 802
Antipyrine, 310
Apricot
Kernel, 1257
Kernel Water, 1257
Arbekacin
Sulfate, 311
Sulfate Injection, 312
Areca, 1258
Arginine Hydrochloride, 313
Injection, 314
L-Arginine, 313
Hydrochloride, 313
Hydrochloride Injection, 314
Aromatic Castor Oil, 415
Arotinolol Hydrochloride, 314
Arsenical Paste, 315
Arsenic Trioxide, 316
Arsenous Acid, 316
Artemisia Capillaris Flower, 1258
Ascorbic Acid, 316
Injection, 317
Powder, 317
Asiasarum Root, 1259
Asparagus Tuber, 1259
L-Aspartic Acid, 318
Aspirin, 318
Aluminum, 319
Tablets, 319
Aspoxicillin Hydrate, 320
Astragalus Root, 1260
Astromicin Sulfate, 322
Atenolol, 323
Atractylodes
Lancea Rhizome, 1261
Rhizome, 1260
Atropine
Sulfate Hydrate, 324
Sulfate Injection, 324
Azathioprine, 325
Tablets, 326
Azithromycin Hydrate, 327
Aztreonam, 328
Afloqualone, 277
B
Bacampicillin Hydrochloride, 329
Bacitracin, 330
Baclofen, 331
Tablets, 332
Bamethan Sulfate, 333
Barbital, 333
Barium Sulfate, 334
Bearberry Leaf, 1262
Bear Bile, 1262
Beclometasone Dipropionate, 335
Beef Tallow, 336
Bekanamycin Sulfate, 337
Belladonna
Extract, 1263
Root, 1263
Benidipine Hydrochloride, 338
1765
1766
Index
JP XV
Tablets, 339
Benincasa Seed, 1264
Benoxinate Hydrochloride, 951
Benserazide Hydrochloride, 340
Bentonite, 341
Benzalkonium Chloride, 342
Concentrated Solution 50, 343
Solution, 342
Benzbromarone, 343
Benzethonium Chloride, 344
Solution, 345
Benzocaine, 644
Benzoic Acid, 345
Benzoin, 1265
Benzyl
Alcohol, 346
Benzoate, 347
Benzylpenicillin
Benzathine Hydrate, 348
Potassium, 349
Berberine
Chloride Hydrate, 351
Tannate, 352
Betahistine Mesilate, 353
Tablets, 354
Betamethasone, 355
Dipropionate, 358
Sodium Phosphate, 359
Tablets, 356
Valerate, 360
Valerate and Gentamicin Sulfate
Cream, 361
Valerate and Gentamicin Sulfate
Ointment, 362
Bethanechol Chloride, 363
Bezafibrate, 364
Sustained Release Tablets, 365
Bifonazole, 366
Biperiden Hydrochloride, 366
Bisacodyl, 367
Suppositories, 368
Bismuth
Subgallate, 368
Subnitrate, 369
Bitter
Cardamon, 1265
Orange Peel, 1265
Tincture, 1266
Bleomycin
Hydrochloride, 370
Sulfate, 372
Boric Acid, 374
Bromazepam, 374
Bromhexine Hydrochloride, 375
Bromocriptine Mesilate, 376
Bromovalerylurea, 377
Bucillamine, 377
Bucumolol Hydrochloride, 378
Bufetolol Hydrochloride, 379
Bufexamac, 380
Cream, 380
Ointment, 381
Bumetanide, 382
Bunazosin Hydrochloride, 383
Bupleurum Root, 1266
Bupranolol Hydrochloride, 383
Burdock Fruit, 1267
Burnt Alum, 292
Busulfan, 384
Butropium Bromide, 385
Butyl Parahydroxybenzoate, 386
Cacao Butter, 386
Calciferol, 624
Calcium
Chloride Hydrate, 390
Chloride Injection, 390
Folinate, 391
Gluconate Hydrate, 391
Hydroxide, 392
Lactate Hydrate, 393
Leucovorin, 391
Oxide, 393
Pantothenate, 394
Paraaminosalicylate Granules, 394
Paraaminosalicylate Hydrate, 395
Polystyrene Sulfonate, 398
Stearate, 400
Calumba, 1267
Camellia Oil, 400
Camostat Mesilate, 400
tf-Camphor, 401
(//-Camphor, 402
Capsicum, 1268
and Salicylic Acid Spirit, 1270
Tincture, 1269
Capsules, 403
Captopril, 403
Carbamazepine, 404
Carbazochrome Sodium Sulfonate
Hydrate, 405
Carbetapentane Citrate, 974
Carbetapentene Citrate, 974
Carbidopa Hydrate, 406
Carbolic Acid, 985
for Disinfection, 986
L-Carbocisteine, 407
Carbon Dioxide, 407
Carboxymethylcellulose, 408
Calcium, 409
Sodium, 410
Cardamon, 1271
Carmellose, 408
Calcium, 409
Sodium, 410
Carmofur, 411
Carnauba Wax, 411
Carteolol Hydrochloride, 412
Carumonam Sodium, 412
Cassia Seed, 1271
Castor Oil, 414
Catalpa Fruit, 1271
Caffeine
and Sodium Benzoate, 388
Hydrate, 387
Cefaclor, 415
Capsules, 416
Compound Granules, 419
Fine Granules, 417
Cefadroxil, 421
Cefalexin, 422
Cefalotin Sodium, 423
Cefapirin Sodium, 424
Cefatrizine Propylene Glycolate, 425
Cefazolin Sodium, 426
Hydrate, 428
Cefbuperazone Sodium, 429
Cefcapene Pivoxil Hydrochloride
Fine Granules, 432
Hydrate, 430
Tablets, 433
Cefdinir, 434
Capsules, 435
Fine Granules, 436
Cefditoren Pivoxil, 437
Fine Granules, 438
Tablets, 438
Cefepime Dihydrochloride
for Injection, 441
Hydrate, 439
Cefmenoxime Hydrochloride, 443
Cefmetazole Sodium, 445
Cefminox Sodium Hydrate, 446
Cefodizime Sodium, 447
Cefoperazone Sodium, 448
Cefotaxime Sodium, 449
Cefotetan, 451
Cefotiam
Hexetil Hydrochloride, 453
Hydrochloride, 455
Hydrochloride for Injection, 456
Cefozopran Hydrochloride, 457
for Injection, 458
Cefpiramide Sodium, 458
Cefpirome Sulfate, 460
Cefpodoxime Proxetil, 461
Cefroxadine Hydrate, 462
Cefsulodin Sodium, 464
Ceftazidime Hydrate, 466
Cefteram Pivoxil, 468
Fine Granules, 469
Ceftibuten Hydrate, 470
Ceftizoxime Sodium, 471
Ceftriaxone Sodium Hydrate, 472
Cefuroxime
Axetil, 474
Sodium, 476
Cellacefate, 480
Celmoleukin (Genetical Recombina-
tion), 481
Cetanol, 484
Cetraxate Hydrochloride, 485
Cefixime, 442
Chenodeoxycholic Acid, 486
Chloral Hydrate, 487
Chloramphenicol, 487
Palmitate, 488
JPXV
Index
1767
Sodium Succinate, 489
Chlordiazepoxide, 490
Powder, 491
Tablets, 492
Chlorhexidine
Gluconate Solution, 493
Hydrochloride, 493
Chlorinated Lime, 494
Chlormadinone Acetate, 494
Chlorobutanol, 495
Chlorphenesin Carbamate, 496
Chlorpheniramine
and Calcium Powder, 497
Maleate, 498
Maleate Injection, 499
Maleate Powder, 499
Maleate Tablets, 500
(/-Chlorpheniramine Maleate, 501
Chlorpromazine Hydrochloride, 502
Injection, 503
Tablets, 503
Chlorpropamide, 504
Tablets, 505
Cholecalciferol, 506
Cholera Vaccine, 506
Cholesterol, 507
Chorionic Gonadotrophin, 707
for Injection, 708
Chrysanthemum Flower, 1271
Ciclacillin, 507
Ciclosporin, 509
A, 509
Cilastatin Sodium, 510
Cilostazol, 511
Tablets, 512
Cimetidine, 513
Cimicifuga Rhizome, 1272
Cinchocaine Hydrochloride, 569
Cinnamon
Bark, 1272
Oil, 1273
Cisplatin, 513
Citric Acid Hydrate, 515
Citrus Unshiu Peel, 1273
Clarithromycin, 516
Tablets, 517
Clemastine Fumarate, 519
Clematis Root, 1274
Clindamycin Hydrochloride, 519
Capsules, 520
Clindamycin Phosphate, 521
Clinofibrate, 522
Clocapramine Hydrochloride Hydrate,
523
Clofedanol Hydrochloride, 524
Clomifene Citrate, 526
Tablets, 527
Clomipramine Hydrochloride, 528
Clonazepam, 528
Clonidine Hydrochloride, 529
Cloperastine Hydrochloride, 530
Clotiazepam, 531
Clotrimazole, 532
Clove, 1274
Oil, 1275
Cloxacillin Sodium Hydrate, 533
Cloxazolam, 534
Clofibrate, 525
Capsules, 526
CMC, 408
Calcium, 409
Sodium, 410
Cnidium
Monnieri Fruit, 1275
Rhizome, 1275
Cocaine Hydrochloride, 535
Coconut Oil, 535
Codeine Phosphate
Hydrate, 536
Tablets, 537
\% Codeine Phosphate Powder, 536
10% Codeine Phosphate Powder, 537
Cod Liver Oil, 538
Coix Seed, 1276
Colchicine, 538
Colistin
Sodium Methanesulfonate, 540
Sulfate, 541
Colophonium, 1347
Compound
Acrinol and Zinc Oxide Oil, 273
Diastase and Sodium Bicarbonate
Powder, 567
Hycodenone Injection, 952
Iodine Glycerin, 770
Methyl Salicylate Spirit, 879
Oxycodone and Atropine Injection,
953
Oxycodone Injection, 952
Phellodendron Powder for
Cataplasm, 1332
Rhubarb and Senna Powder, 1346
Salicylic Acid Spirit, 1080
Scopolia Extract and Diastase Pow-
der, 1355
Thianthol and Salicylic Acid Solu-
tion, 1165
Vitamin B Powder, 1230
Concentrated
Glycerin, 703
Glycerol, 703
Condurango, 1276
Fluidextract, 1277
Coptis Rhizome, 1277
Corn
Oil, 542
Starch, 542
Cornus Fruit, 1279
Cortisone Acetate, 543
Corydalis Tuber, 1279
Creosote, 544
Cresol, 544
Solution, 545
Croconazole Hydrochloride, 546
Croscarmellose Sodium, 546
Crude Glycyrrhiza Extract, 1297
Crystalline Insulin Zinc Injection
(Aqueous Suspension), 765
Crystal Violet, 881
Cyanamide, 548
Cyanocobalamin, 548
Injection, 549
Cyclopentolate Hydrochloride, 549
Cyclophosphamide Hydrate, 550
Cycloserine, 551
Cyperus Rhizome, 1280
Cyproheptadine Hydrochloride Hy-
drate, 551
Cytarabine, 552
D
Daiokanzoto Extract, 1280
Dantrolene Sodium Hydrate, 553
Daunorubicin Hydrochloride, 553
Deferoxamine Mesilate, 554
Dehydrated Alcohol, 639
Dehydrocholate Sodium Injection,
557
Dehydrocholic Acid, 556
Injection, 557
Demethylchlortetracycline Hydro-
chloride, 558
Dental
Antiformin, 310
Iodine Glycerin, 771
Paraformaldehyde Paste, 969
Phenol with Camphor, 987
Sodium Hypochlorite Solution, 310
Triozinc Paste, 1210
Dermatol, 368
Deslanoside, 559
Injection, 560
Dexamethasone, 560
Dextran
40, 561
40 Injection, 562
70, 562
Sulfate Sodium Sulfur 18, 565
Sulfate Sodium Sulfur 5, 564
Dextrin, 565
Dextromethorphan Hydrobromide
Hydrate, 566
Diagnostic Sodium Citrate Solution,
1100
Diastase, 567
and Sodium Bicarbonate Powder,
567
Diazepam, 567
Dibasic
Calcium Phosphate Hydrate, 396
Sodium Phosphate Hydrate, 568
Dibekacin Sulfate, 569
Dibucaine Hydrochloride, 569
Dichlorphenamide, 571
Tablets, 572
Diclofenac Sodium, 570
Diclofenamide, 571
Tablets, 572
1768
Index
JP XV
Dicloxacillin Sodium Hydrate, 573
Diethylcarbamazine Citrate, 574
Tablets, 574
Diethylstilbestrol Diphosphate, 683
Tablets, 684
Difenidol Hydrochloride, 575
Digenea, 1280
Digitoxin, 576
Tablets, 576
Digoxin, 577
Injection, 578
Tablets, 580
Dihydrocodeine Phosphate, 581
1% Dihydrocodeine Phosphate Pow-
der, 582
10% Dihydrocodeine Phosphate Pow-
der, 582
Dihydroergotamine Mesilate, 583
Dihydroergotoxine Mesilate, 584
Dilazep Hydrochloride Hydrate, 586
Diltiazem Hydrochloride, 586
Dilute
Hydrochloric Acid, 726
Iodine Tincture, 769
Diluted Opium Powder, 940
Dimemorfan Phosphate, 588
Dimenhydrinate, 588
Tablets, 589
Dimercaprol, 590
Injection, 590
Dimorpholamine, 591
Injection, 591
Dinoprost, 592
Dionin, 648
Dioscorea Rhizome, 1282
Diphenhydramine, 593
and Bromovalerylurea Powder, 593
Hydrochloride, 594
Tannate, 595
Phenol and Zinc Oxide Liniment,
595
Diphenylhydantoin, 991
Powder, 992
Sodium for Injection, 992
Tablets, 992
Diphtheria
Toxoid, 596
Tetanus Combined Toxoid, 596
Dipyridamole, 597
Disodium Edetate Hydrate, 598
Disopyramide, 598
Distigmine Bromide, 599
Tablets, 600
Disulfiram, 600
Dobutamine Hydrochloride, 601
Dolichos Seed, 1282
Dopamine Hydrochloride, 602
Injection, 602
Dover's Powder, 1324
Doxapram Hydrochloride Hydrate,
603
Doxifluridine, 604
Capsules, 604
Doxorubicin Hydrochloride, 605
Doxycycline Hydrochloride Hydrate,
606
Dried
Aluminum Hydroxide Gel, 288
Aluminum Hydroxide Gel Fine
Granules, 289
Aluminum Potassium Sulfate, 292
Sodium Carbonate, 1096
Sodium Sulfite, 1109
Thyroid, 1171
Yeast, 1241
Droperidol, 608
Dydrogesterone, 609
Tablets, 610
E
Ecarazine Hydrochloride, 1185
Ecothiopate Iodide, 610
Edrophonium Chloride, 611
Injection, 612
EDTA Sodium Hydrate, 598
Elcatonin, 612
Eleutherococcus Senticosus Rhizome,
1283
Enoxacin Hydrate, 616
Enviomycin Sulfate, 616
Enflurane, 615
Eperisone Hydrochloride, 618
Ephedra Herb, 1283
Ephedrine Hydrochloride, 619
Injection, 619
Powder, 620
Tablets, 621
10% Ephedrine Hydrochloride Powder,
620
Epimedium Herb, 1284
Epinephrine, 276
Injection, 276
Solution, 277
Epirizole, 621
Epirubicin Hydrochloride, 622
Ergocalciferol, 624
Ergometrine Maleate, 625
Injection, 625
Tablets, 626
Ergotamine Tartrate, 627
Erythromycin, 627
Ethylsuccinate, 629
Lactobionate, 629
Stearate, 630
Estazolam, 631
Estradiol Benzoate, 631
Injection, 632
Injection (Aqueous Suspension),
633
Estriol, 633
Injection (Aqueous Suspension),
634
Tablets, 635
Etacrynic Acid, 636
Tablets, 636
Ethacridine Lactate, 274
Ethambutol Hydrochloride, 637
Ethanol, 638
for Disinfection, 640
Ethenzamide, 640
Ether, 641
Ethinylestradiol, 642
Tablets, 642
Ethionamide, 643
Ethosuximide, 644
Ethoxybenzamide, 640
Ethyl
Aminobenzoate, 644
Cysteine Hydrochloride, 645
L-Cysteine Hydrochloride, 645
Icosapentate, 646
Parahydroxybenzoate, 647
Ethylenediamine, 648
Ethylmorphine Hydrochloride Hy-
drate, 648
Etidronate Disodium, 649
Tablets, 650
Etilefrine Hydrochloride, 650
Tablets, 651
Etizolam, 652
Etodolac, 653
Etoposide, 653
Eucalyptus Oil, 654
Eucommia Bark, 1284
Evodia Fruit, 1285
Exsiccated Gypsum, 1298
Famotidine, 655
for Injection, 656
Powder, 657
Tablets, 657
Faropenem Sodium
for Syrup, 659
Hydrate, 658
Tablets, 660
Fenbufen, 660
Fennel, 1285
Oil, 1286
Fentanyl Citrate, 661
Ferrous Sulfate Hydrate, 661
Flavin Adenine Dinucleotide Sodium,
662
Flavoxate Hydrochloride, 664
Flomoxef Sodium, 664
for Injection, 666
Flopropione, 667
Capsules, 667
Flucytosine, 668
Fludiazepam, 669
Flunitrazepam, 670
Fluocinolone Acetonide, 670
Fluocinonide, 672
Fluorescein Sodium, 673
Fluorometholone, 673
Fluorouracil, 674
Fluoxymesterone, 675
JPXV
Index
1769
Fluphenazine Enanthate, 676
Flurazepam, 677
Capsules, 678
Hydrochloride, 678
Flurbiprofen, 679
Foeniculated Ammonia Spirit, 1286
Folic Acid, 680
Injection, 681
Tablets, 681
Formalin, 681
Water, 682
Formoterol Fumarate Hydrate, 682
Forsythia Fruit, 1286
Fosfestrol, 683
Tablets, 684
Fosfomycin
Calcium Hydrate, 685
Sodium, 686
Sodium for Injection, 687
Fradiomycin Sulfate, 688
Freeze-dried
BCG Vaccine (for Percutaneous
Use), 335
Botulism Antitoxin, Equine, 374
Diphtheria Antitoxin, Equine, 596
Habu Antivenom, Equine, 716
Inactivated Tissue Culture Rabies
Vaccine, 1054
Japanese Encephalitis Vaccine, 786
Live Attenuated Measles Vaccine,
843
Live Attenuated Mumps Vaccine,
902
Live Attenuated Rubella Vaccine,
1074
Mamushi Antivenom, Equine, 841
Smallpox Vaccine, 1091
Smallpox Vaccine Prepared in Cell
Culture, 1091
Tetanus Antitoxin, Equine, 1156
Fritillaria Bulb, 1287
Fructose, 689
Injection, 689
Furosemide, 690
Tablets, 691
Fursultiamine Hydrochloride, 692
Gabexate Mesilate, 693
/?-Galactosidase
(Aspergillus), 694
(Penicillium), 695
Gallium ( 67 Ga) Citrate Injection, 696
Gambir, 1287
Gardenia Fruit, 1288
Gas Gangrene Antitoxin, Equine, 696
Gastrodia Tuber, 1289
Gelatin, 696
Gentamicin Sulfate, 698
Gentian, 1290
and Sodium Bicarbonate Powder,
1290
Geranium Herb, 1291
Ginger, 1291
Ginseng, 1292
Glacial Acetic Acid, 269
Glehnia Root, 1294
Glibenclamide, 699
Glucose, 700
Injection, 701
Glutathione, 701
Glycerin, 702
and Potash Solution, 704
Glycerol, 702
Glyceryl Monostearate, 704
Glycine, 704
Glycyrrhiza, 1295
Extract, 1296
Gonadorelin Acetate, 705
Gramicidin, 712
Griseofulvin, 713
Guaiacol Glyceryl Ether, 714
Guaifenesin, 714
Guanabenz Acetate, 715
Guanethidine Sulfate, 716
Gypsum, 1297
H
Haloperidol, 717
Tablets, 718
Halothane, 719
Haloxazolam, 719
Hemp Fruit, 1298
Heparin Sodium, 721
Injection, 722
Hochuekkito Extract, 1298
Homatropine Hydrobromide, 722
Homochlorcyclizine Hydrochloride,
723
Honey, 1301
Houttuynia Herb, 1302
Human
Chorionic Gonadotrophin, 707
Chorionic Gonadotrophin for
Injection, 708
Menopausal Gonadotrophin, 708
Normal Immunoglobulin, 724
Hycoato Injection, 953
Hydralazine Hydrochloride, 724
for Injection, 724
Powder, 725
Tablets, 725
Hydrochloric Acid, 725
Lemonade, 727
Hydrochlorothiazide, 727
Hydrocortisone, 728
Acetate, 729
and Diphenhydramine Ointment,
730
Butyrate, 730
Sodium Phosphate, 731
Sodium Succinate, 732
Succinate, 733
Hydrocotarnine Hydrochloride
Hydrate, 734
Hydrogenated Oil, 735
Hydrophilic
Ointment, 735
Petrolatum, 981
Hydrous Lanolin, 807
Hydroxocobalamin Acetate, 736
Hydroxypropylcellulose, 736
Hydroxypropylmethylcellulose, 741
Hydroxyzine
Hydrochloride, 739
Pamoate, 739
Hymecromone, 740
Hypromellose, 741
Phthalate, 743
I
Ibuprofen, 744
Ichthammol, 744
Idarubicin Hydrochloride, 745
for Injection, 746
Idoxuridine, 747
Ophthalmic Solution, 747
Ifenprodil Tartrate, 748
Imipenem
and Cilastatin for Injection, 750
Hydrate, 749
Hydrochloride, 751
Imipramine Hydrochloride, 751
Tablets, 752
Immature Orange, 1302
Imperata Rhizome, 1302
Indenolol Hydrochloride, 753
Indigocarmine, 754
Injection, 755
Indium ( m In) Chloride Injection, 755
Indometacin, 755
Capsules, 756
Suppositories, 757
Insulin, 758
Human (Genetical Recombination),
760
Injection, 762
Zinc Injection (Aqueous Suspen-
sion), 763
Zinc Protamine Injection (Aqueous
Suspension), 766
Influenza HA Vaccine, 758
Iodamide, 767
Iodinated ( 131 I) Human Serum Albumin
Injection, 768
Iodine, 768
Salicylic Acid and Phenol Spirit,
772
Tincture, 769
Iodoform, 773
Iopamidol, 773
Iotalamic Acid, 774
Iotroxic Acid, 775
Ipecac, 1303
Syrup, 1304
Ipratropium Bromide Hydrate, 776
1770
Index
JP XV
Iproveratril Hydrochloride, 1225
Isepamicin Sulfate, 777
L-Isoleucine, 780
Isoniazid, 780
Injection, 781
Tablets, 781
Isophane Insulin Injection (Aqueous
Suspension), 762
/Tsoprenaline Hydrochloride, 782
Isopropanol, 783
Isopropyl Alcohol, 783
Isopropylantipyrine, 783
Isosorbide, 784
Dinitrate, 785
Dinitrate Tablets, 786
Isotonic
Salt Solution, 1098
Sodium Chloride Injection, 1098
Sodium Chloride Solution, 1098
Isoflurane, 778
Japanese
Angelica Root, 1305
Encephalitis Vaccine, 786
Gentian, 1306
Valerian, 1306
Josamycin, 786
Propionate, 788
Jujube, 1307
Seed, 1307
K
Kainic Acid
and Santonin Powder, 790
Hydrate, 789
Kakkonto Extract, 1308
Kallidinogenase, 790
Kamishoyosan Extract, 1309
Kanamycin
Monosulfate, 793
Sulfate, 794
Kaolin, 795
Ketamine Hydrochloride, 795
Ketoprofen, 796
Ketotifen Fumarate, 797
Kitasamycin, 798
Acetate, 799
Tartrate, 800
Lactic Acid, 801
Lactose, 803
Hydrate, 803
Lactulose, 804
Lanatoside C, 805
Tablets, 806
Lard, 808
Latamoxef Sodium, 809
Lauromacrogol, 810
Lenampicillin Hydrochloride, 810
L-Leucine, 812
Leucomycin, 798
Acetate, 799
Tartrate, 800
Levallorphan Tartrate, 813
Injection, 814
Levodopa, 814
Levomepromazine Maleate, 815
Levothyroxine Sodium
Hydrate, 816
Tablets, 817
Lidocaine, 818
Hydrochloride Injection, 818
Injection, 818
Light
Anhydrous Silicic Acid, 1087
Liquid Paraffin, 967
Limaprost Alfadex, 819
Lincomycin Hydrochloride Hydrate,
820
Lindera Root, 1313
Liothyronine Sodium, 821
Tablets, 822
Liquefied
Carbolic Acid, 986
Phenol, 986
Liquid Paraffin, 966
Lisinopril
Hydrate, 823
Tablets, 824
Lithium Carbonate, 826
Lithospermum Root, 1313
Live Oral Poliomyelitis Vaccine, 1005
Longgu, 1313
Lonicera Leaf and Stem, 1314
Loquat Leaf, 1314
Lorazepam, 827
Low Substituted Hydroxypropylcellu-
lose, 738
Loxoprofen Sodium Hydrate, 828
Lycium
Bark, 1315
Fruit, 1315
Lysine Hydrochloride, 829
L-Lysine Hydrochloride, 829
Lysozyme Hydrochloride, 830
M
Macrogol
400, 830
1500, 831
4000, 832
6000, 832
20000, 833
Ointment, 833
Magnesium
Carbonate, 834
Oxide, 835
Silicate, 836
Stearate, 837
Sulfate Hydrate,
838
Sulfate Injection, 839
Sulfate Mixture, 839
Magnolia
Bark, 1315
Flower, 1317
Mallotus Bark, 1317
Maltose Hydrate, 840
D-Mannite Injection, 842
D-Mannitol, 841
Injection, 842
Maprotiline Hydrochloride, 842
Meclofenoxate Hydrochloride, 843
Mecobalamin, 844
Medazepam, 845
Medicinal
Carbon, 845
Soap, 846
Mefenamic Acid, 847
Mefruside, 849
Tablets, 849
Meglumine, 850
Amidotrizoate Injection, 851
Iotalamate Injection, 852
Sodium Amidotrizoate Injection,
853
Sodium Iodamide Injection, 854
Melphalan, 855
Menatetrenone, 855
Mentha
Herb, 1317
Oil, 1318
Water, 1318
(//-Menthol, 856
/-Menthol, 857
Mepenzolate Bromide, 857
Mepirizole, 621
Mepitiostane, 858
Mepivacaine Hydrochloride, 859
Injection, 860
Mequitazine, 861
Merbromin, 862
Solution, 863
Mercaptopurine Hydrate, 861
Mercurochrome, 862
Solution, 863
Meropenem Hydrate, 863
Mestranol, 864
Metenolone
Acetate, 865
Enanthate, 866
Enanthate Injection, 866
Metformin Hydrochloride, 867
Tablets, 867
Methamphetamine Hydrochloride,
868
L-Methionine, 869
Methotrexate, 869
Methoxsalen, 870
Methylbenactyzium Bromide, 871
Methylcellulose, 871
Methyldopa
Hydrate, 873
Tablets, 874
JPXV
Index
1771
tf/-Methylephedrine Hydrochloride,
875
Powder, 876
10% c?/-Methylephedrine Hydrochlo-
ride Powder, 876
Methylergometrine Maleate, 876
Tablets, 877
Methyl
Parahydroxybenzoate, 878
Salicylate, 881
Methylprednisolone, 879
Succinate, 880
Methylrosanilinium Chloride, 881
Methyltestosterone, 882
Tablets, 883
Meticrane, 884
Metildigoxin, 885
Metoclopramide, 886
Tablets, 886
Metoprolol Tartrate, 887
Tablets, 888
Metronidazole, 889
Tablets, 889
Metyrapone, 890
Mexiletine Hydrochloride, 891
Mefloquine Hydrochloride, 848
Miconazole, 892
Nitrate, 893
Microcrystalline Cellulose, 477
Micronomicin Sulfate, 893
Midecamycin, 894
Acetate, 895
Migrenin, 896
Minocycline Hydrochloride, 897
Mitomycin C, 898
Monobasic Calcium Phosphate
Hydrate, 397
Monosodium Trichloroethyl Phos-
phate, 1202
Syrup, 1203
Morphine
and Atropine Injection, 899
Hydrochloride Hydrate, 900
Hydrochloride Injection, 901
Hydrochloride Tablets, 902
Moutan Bark, 1318
Mulberry Bark, 1320
Mupirocin Calcium Hydrate, 902
N
Nadolol, 904
Nalidixic Acid, 905
Naloxone Hydrochloride, 906
Naphazoline
and Chlorpheniramine Solution,
906
Hydrochloride, 907
Nitrate, 908
Naproxen, 908
Narcotine, 936
Hydrochloride, 937
Natamycin, 994
Natural Aluminum Silicate, 289
Nelumbo Seed, 1320
Neomycin Sulfate, 688
Neostigmine Methylsulfate, 909
Injection, 910
Netilmicin Sulfate, 910
Nicardipine Hydrochloride, 911
Injection, 912
Nicergoline, 913
Powder, 914
Tablets, 915
Niceritrol, 916
Nicomol, 917
Tablets, 918
Nicorandil, 919
Nicotinamide, 919
Nicotinic Acid, 920
Injection, 921
Nifedipine, 922
Nilvadipine, 923
Tablets, 924
Nitrazepam, 925
Nitrendipine, 926
Tablets, 927
Nitrogen, 928
Nitroglycerin Tablets, 928
Nitrous Oxide, 929
Noradrenaline, 930
Hydrochloride Injection, 931
Noradrenaline Injection, 931
Norepinephrine, 930
Hydrochloride Injection, 931
Injection, 931
Norethisterone, 932
Norgestrel, 933
and Ethinylestradiol Tablets, 934
Nortriptyline Hydrochloride, 935
Norfloxacin, 932
Noscapine, 936
Hydrochloride Hydrate, 937
Notopterygium Rhizome, 1321
Nuphar Rhizome, 1321
Nux Vomica, 1321
Extract, 1322
Extract Powder, 1323
Tincture, 1323
Nystatin, 937
o
Olive Oil, 939
Operidine, 980
Injection, 981
Ophiopogon Tuber, 1324
Opium
Ipecac Powder, 1324
Tincture, 940
Opium Alkaloids
and Atropine Injection, 942
and Scopolamine Injection, 943
Hydrochlorides, 941
Hydrochlorides Injection, 942
Orange
Oil, 946
Peel Syrup, 1325
Peel Tincture, 1325
Orciprenaline Sulfate, 946
Oriental Bezoar, 1325
Oxapium Iodide, 947
Oxaprozin, 948
Oxazolam, 948
Oxetacaine, 949
Oxethazaine, 949
Oxprenolol Hydrochloride, 950
Oxybuprocaine Hydrochloride, 951
Oxycodone Hydrochloride Hydrate,
951
Oxydol, 954
Oxygen, 955
Oxymetholone, 956
Oxytetracycline Hydrochloride, 956
Oxytocin, 958
Injection, 960
Oyster Shell, 1326
Ofloxacin, 938
Panax Japonicus Rhizome, 1326
Pancreatin, 961
Pancuronium Bromide, 962
Panipenem, 962
Pantethine, 964
Papaverine Hydrochloride, 965
Injection, 965
Paracetamol, 267
Paraformaldehyde, 968
Paraffin, 966
Parnaparin Sodium, 969
Pas-calcium
Granules, 394
Hydrate, 395
Peach Kernel, 1327
Peanut Oil, 971
Penbutolol Sulfate, 972
Penicillin G Potassium, 349
Pentazocine, 972
Pentobarbital Calcium, 973
Pentoxyverine Citrate, 974
Peony Root, 1328
Peplomycin Sulfate, 975
Perilla Herb, 1329
Perphenazine, 977
Maleate, 978
Maleate Tablets, 979
Tablets, 977
Pethidine Hydrochloride, 980
Injection, 981
Petroleum Benzin, 982
Pharbitis Seed, 1330
Phellodendron,
Albumin Tannate and Bismuth Sub-
nitrate Powder, 1332
Bark, 1330
Phenazone, 310
Phenethicillin Potassium, 983
1772 Index
JP XV
Phenobarbital, 984
Powder, 985
10% Phenobarbital Powder, 985
Phenol, 985
and Zinc Oxide Liniment, 987
for Disinfection, 987
Phenolated Water, 987
for Disinfection, 986
Phenolsulfonphthalein, 988
Injection, 988
L-Phenylalanine, 989
Phenylbutazone, 990
Phenylephrine Hydrochloride, 990
Phenytoin, 991
Powder, 992
Sodium for Injection, 992
Tablets, 992
Phytomenadione, 993
Phytonadione, 993
Picrasma Wood, 1333
Pilocarpine Hydrochloride, 994
Pimaricin, 994
Pindolol, 995
Pinellia Tuber, 1333
Pipemidic Acid Hydrate, 996
Piperacillin Sodium, 997
for Injection, 998
Piperazine
Adipate, 999
Phosphate Hydrate, 999
Phosphate Tablets, 1000
Pirarubicin, 1000
Pirenoxine, 1001
Pirenzepine Hydrochloride Hydrate,
1002
Piroxicam, 1003
Pivmecillinam Hydrochloride, 1004
Plantago
Herb, 1334
Seed, 1334
Platycodon
Fluidextract, 1334
Root, 1334
Polyethylene Glycol
400, 830
1500, 831
4000, 832
6000, 832
20000, 833
Ointment, 833
Polygala Root, 1335
Polygonatum Rhizome, 1336
Polygonum Root, 1336
Polymixin B Sulfate, 1006
Polyoxyethylene Lauryl Alcohol Ether,
810
Polyoxyl 40 Stearate, 1007
Polyporus Sclerotium, 1336
Polysorbate 80, 1007
Polyvidone, 1015
Polyvinylpyrrolidone, 1015
Poria Sclerotium, 1337
Potash Soap, 1007
Potassium
Bromide, 1008
Canrenoate, 1008
Carbonate, 1009
Chloride, 1009
Clavulanate, 1010
Guaiacolsulf onate , 1011
Hydroxide, 1012
Iodide, 1013
Permanganate, 1013
Sulfate, 1014
Potato Starch, 1014
Povidone, 1015
Iodine, 1017
Powdered
Acacia, 1251
Agar, 1253
Alisma Rhizome, 1253
Aloe, 1255
Amomum Seed, 1256
Atractylodes Lancea Rhizome,
1261
Atractylodes Rhizome, 1260
Calumba, 1268
Capsicum, 1269
Cellulose, 480
Cinnamon Bark, 1273
Clove, 1274
Cnidium Rhizome, 1276
Coix Seed, 1276
Coptis Rhizome, 1278
Cyperus Rhizome, 1280
Dioscorea Rhizome, 1282
Fennel, 1285
Gambir, 1287
Gardenia Fruit, 1288
Gentian, 1290
Geranium Herb, 1291
Ginger, 1292
Ginseng, 1293
Glycyrrhiza, 1296
Ipecac, 1303
Japanese Angelica Root, 1305
Japanese Gentian, 1306
Japanese Valerian, 1307
Magnolia Bark, 1316
Moutan Bark, 1319
Opium, 939
Oyster Shell, 1326
Panax Japonicus Rhizome, 1327
Peach Kernel, 1327
Peony Root, 1329
Phellodendron Bark, 1331
Picrasma Wood, 1333
Platycodon Root, 1335
Polygala Root, 1335
Polyporus Sclerotium, 1337
Poria Sclerotium, 1337
Processed Aconite Root, 1339
Rhubarb, 1345
Rose Fruit, 1346
Scutellaria Root, 1359
Senega, 1361
Senna Leaf, 1362
Smilax Rhizome, 1364
Sophora Root, 1364
Sweet Hydrangea Leaf, 1365
Swertia Herb, 1366
Tragacanth, 1369
Zanthoxylum Fruit, 1371
Pranoprofen, 1018
Pravastatin Sodium, 1018
Prazepam, 1020
Tablets, 1021
Precipitated Calcium Carbonate, 389
Prednisolone, 1021
Acetate, 1023
Sodium Succinate for Injection,
1025
Succinate, 1024
Tablets, 1022
Primidone, 1026
Probenecid, 1027
Tablets, 1027
Procainamide Hydrochloride, 1028
Injection, 1029
Tablets, 1029
Procaine Hydrochloride, 1030
Injection, 1030
Procarbazine Hydrochloride, 1031
Procaterol Hydrochloride Hydrate,
1032
Prochlorperazine Maleate, 1033
Tablets, 1033
Processed
Aconite Root, 1338
Ginger, 1341
Progesterone, 1034
Injection, 1034
Proglumide, 1035
Promethazine Hydrochloride, 1036
Propantheline Bromide, 1036
Propranolol Hydrochloride, 1037
Tablets, 1038
Propylene Glycol, 1039
Propyl Parahydroxybenzoate, 1039
Propylthiouracil, 1040
Tablets, 1040
Propyphenazone, 783
Prostaglandin
El 286
Ei a-Cyclodextrin Clathrate Com-
pound, 287
F 2a , 592
Protamine Sulfate, 1041
Injection, 1042
Prothionamide, 1042
Protirelin, 1043
Tartrate Hydrate, 1044
Prunella Spike, 1341
Pueraria Root, 1341
Pullulan, 1045
Purified
Dehydrocholic Acid, 556
Gelatin, 697
Lanolin, 808
JPXV
Index
1773
Shellac, 1085
Water, 1236
Pyrantel Pamoate, 1045
Pyrazinamide, 1046
Pyridostigmine Bromide, 1047
Pyridoxine Hydrochloride, 1047
Injection, 1048
Pyroxylin, 1049
Pyrrolnitrin, 1049
Q
Quick Lime, 393
Quinidine Sulfate Hydrate, 1050
Quinine
Ethyl Carbonate, 1051
Hydrochloride Hydrate, 1052
Sulfate Hydrate, 1053
R
Ranitidine Hydrochloride, 1054
Rape Seed Oil, 1055
Red Ginseng, 1342
Rehmannia Root, 1343
Reserpine, 1055
Injection, 1056
Powder, 1057
Tablets, 1057
0.1% Reserpine Powder, 1057
Retinol
Acetate, 1058
Palmitate, 1059
Rhubarb, 1344
Ribostamycin Sulfate, 1063
Riboflavin, 1059
Butyrate, 1060
Phosphate, 1061
Phosphate Injection, 1062
Powder, 1060
Sodium Phosphate, 1061
Sodium Phosphate Injection, 1062
Rice Starch, 1346
Rifampicin, 1064
Capsules, 1065
Ringer's Solution, 1066
Ritodrine Hydrochloride, 1067
Tablets, 1068
Rokitamycin, 1069
Rose Fruit, 1346
Rosin, 1347
Roxatidine Acetate Hydrochloride,
1070
Extended-release Capsules, 1071
Roxithromycin, 1073
Ryokeijutsukanto Extract, 1347
Saccharated Pepsin, 1074
Saccharin, 1075
Sodium, 1076
Sodium Hydrate, 1076
Saireito Extract, 1349
Salazosulfapyridine, 1077
Salbutamol Sulfate, 1078
Salicylated Alum Powder, 1078
Salicylic Acid, 1079
Adhesive Plaster, 1080
Spirit, 1080
Santonin, 1081
Saponated Cresol Solution, 545
Saposhnikovia Root, 1352
Sappan Wood, 1352
Saussurea Root, 1352
Saffron, 1349
Safflower, 1348
Schisandra Fruit, 1352
Schizonepeta Spike, 1353
Scopolamine
Butylbromide, 1082
Hydrobromide Hydrate, 1083
Scopolia Extract, 1353
Papaverine and Ethyl Aminobenzo-
ate Powder, 1356
and Carbon Powder, 1355
and Ethyl Aminobenzoate Powder,
1355
and Tannic Acid Suppositories,
1357
Powder, 1353
Rhizome, 1357
Scutellaria Root, 1358
Senega, 1360
Syrup, 1361
Senna Leaf, 1361
Serrapeptase, 1083
Serum Gonadotrophin, 710
for Injection, 711
Sesame Oil, 1084
Siccanin, 1086
Silver
Nitrate, 1088
Nitrate Ophthalmic Solution, 1088
Protein, 1088
Protein Solution, 1089
Simple
Ointment, 1089
Syrup, 1089
Sinomenium Stem, 1363
Sisomicin Sulfate, 1090
Slaked Lime, 392
Smilax Rhizome, 1364
Sodium
Acetate Hydrate, 1091
Aurothiomalate, 1092
Benzoate, 1093
Bicarbonate, 1093
Bicarbonate and Bitter Tincture Mix-
ture, 1364
Bicarbonate Injection, 1094
Bisulfite, 1094
Borate, 1095
Bromide, 1095
Carbonate Hydrate, 1096
Chloride, 1097
Chromate ( 51 Cr) Injection, 1099
Citrate Hydrate, 1099
Citrate Injection for Transfusion,
1099
Cromoglicate, 1100
Fusidate, 1101
Hydrogen Sulfite, 1094
Hydroxide, 1101
Iodide, 1102
Iodide ( 123 I) Capsules, 1103
Iodide ( 131 I) Capsules, 1103
Iodide ( 131 I) Solution, 1103
Iodohippurate ( 131 I) Injection, 1103
Iotalamate Injection, 1103
Lauryl Sulfate, 1104
Metabisulfite, 1108
Pertechnetate ( 99m Tc) Injection,
1105
Picosulfate Hydrate, 1105
Polystyrene Sulfonate, 1106
Prasterone Sulfate Hydrate, 1107
Pyrosulfite, 1108
Salicylate, 1108
Thiosulfate Hydrate, 1109
Thiosulfate Injection, 1110
Valproate, 1110
0.9% Sodium Chloride Injection,
1098
10% Sodium Chloride Injection, 1098
Sophora Root, 1364
Sorbitan Sesquioleate, 1111
D-Sorbitol, 1112
Solution, 1113
Soybean Oil, 1113
Spectinomycin Hydrochloride Hydrate,
1114
Spiramycin Acetate, 1114
Spironolactone, 1116
Stearic Acid, 1116
Stearyl Alcohol, 1117
Sterile Purified Water, 1236
Streptomycin Sulfate, 1117
Sucralfate Hydrate, 1118
Sucrose, 1120
Sulbactam Sodium, 1122
Sulbenicillin Sodium, 1123
Sulfadiazine Silver, 1124
Sulfafurazole, 1128
Sulfamethizole, 1125
Sulfamethoxazole, 1126
Sulfamonomethoxine Hydrate, 1126
Sulfasalazine, 1077
Sulfobromophthalein Sodium, 1129
Injection, 1130
Sulfur, 1130
Salicylic Acid and Thianthol Oint-
ment, 1131
and Camphor Lotion, 1130
Sulpiride, 1131
Capsules, 1132
Tablets, 1132
Sulpyrine
Hydrate, 1133
1774 Index
JP XV
Injection, 1134
Sultamicillin Tosilate Hydrate, 1134
Sultiame, 1136
Sulfinpyrazone , 1 1 27
Tablets, 1128
Sulfisomezole, 1126
Sulfisoxazole, 1128
Suxamethonium Chloride
for Injection, 1137
Hydrate, 1137
Injection, 1138
Sweet Hydrangea Leaf, 1365
Swertia
and Sodium Bicarbonate Powder,
1367
Herb, 1365
Synthetic
Aluminum Silicate, 290
Camphor, 402
Talampicillin Hydrochloride, 1138
Talc, 1139
Tamsulosin Hydrochloride, 1140
Tannic Acid, 1141
Tartaric Acid, 1141
Taurine, 1142
Teceleukin
(Genetical Recombination), 1143
for Injection (Genetical Recombina-
tion), 1148
Tegafur, 1149
Teicoplanin, 1150
Terbutaline Sulfate, 1153
Termeric, 1367
Testosterone
Enanthate, 1154
Enanthate Injection, 1154
Propionate, 1155
Propionate Injection, 1155
Tetracaine Hydrochloride, 1156
Tetracycline Hydrochloride, 1157
Thallium ( 201 T1) Chloride Injection,
1158
Theophylline, 1158
Thiamazole, 1159
Tablets, 1160
Thiamine
Chloride Hydrochloride, 1160
Chloride Hydrochloride Injection,
1161
Chloride Hydrochloride Powder,
1162
Nitrate, 1162
Thiamylal Sodium, 1163
for Injection, 1164
Thianthol, 1165
Thiopental Sodium, 1166
for Injection, 1167
Thioridazine Hydrochloride, 1168
Thiotepa, 1168
L-Threonine, 1169
Thrombin, 1170
Thymol, 1170
Tiaramide Hydrochloride, 1171
Tablets, 1172
Ticlopidine Hydrochloride, 1173
Timepidium Bromide Hydrate, 1174
Timolol Maleate, 1174
Tinidazole, 1175
Tipepidine Hibenzate, 1176
Tablets, 1177
Titanium Oxide, 1178
Tizanidine Hydrochloride, 1179
Toad Venom, 1368
Tobramycin, 1180
Tocopherol, 1181
Acetate, 1182
Calcium Succinate, 1183
Nicotinate, 1184
(//-a-Tocopherol, 1181
Acetate, 1182
Nicotinate, 1184
Todralazine Hydrochloride Hydrate,
1185
Tofisopam, 1186
Tolazamide, 1187
Tolbutamide, 1188
Tablets, 1188
Tolnaftate, 1189
Solution, 1189
Tolperisone Hydrochloride, 1190
Tragacanth, 1369
Tranexamic Acid, 1191
Capsules, 1192
Injection, 1193
Tablets, 1193
Trapidil, 1194
Trepibutone, 1195
Tretoquinol Hydrochloride, 1209
Triamcinolone, 1195
Acetonide, 1196
Triamterene, 1197
Tribulus Fruit, 1369
Trichlormethiazide, 1198
Tablets, 1199
Trichomycin, 1201
Trichosanthes Root, 1369
Triclofos Sodium, 1202
Syrup, 1203
Trihexyphenidyl Hydrochloride, 1204
Tablets, 1204
Trimebutine Maleate, 1206
Trimetazidine Hydrochloride, 1207
Tablets, 1207
Trimethadione, 1208
Tablets, 1209
Trimetoquinol Hydrochloride Hydrate,
1209
Tropicamide, 1211
L-Tryptophan, 1211
Tubocurarine Chloride Hydrochloride
Hydrate, 1212
Injection, 1213
Tubocurarine Hydrochloride, 1212
Injection, 1213
Tulobuterol Hydrochloride, 1213
Turpentine Oil, 1214
u
Ubidecarenone, 1214
Ulinastatin, 1215
Uncaria Hook, 1370
Urapidil, 1217
Urea, 1218
Urokinase, 1219
Ursodeoxycholic Acid, 1220
Uva Ursi Fluidextract, 1371
L-Valine, 1221
Vancomycin Hydrochloride, 1221
for Injection, 1223
Vasopressin Injection, 1223
Verapamil Hydrochloride, 1225
Tablets, 1226
Vinblastine Sulfate, 1226
for Injection, 1227
Vincristine Sulfate, 1228
Vitamin A
Acetate, 1058
Capsules, 1230
Oil, 1229
Oil Capsules, 1230
Palmitate, 1059
Vitamin Bj
Hydrochloride, 1160
Hydrochloride Injection, 1161
Hydrochloride Powder, 1162
Nitrate, 1162
Vitamin B 2 , 1059
Phosphate Ester, 1061
Phosphate Ester Injection, 1062
Powder, 1060
Vitamin B 6 , 1047
Injection, 1048
Vitamin B 12 , 548
Injection, 549
Vitamin C, 316
Injection, 317
Powder, 317
Vitamin D 2 , 624
Vitamin D 3 , 506
Vitamin E, 1181
Acetate, 1182
Calcium Succinate, 1183
Nicotinate, 1184
Vitamin K,, 993
Voglibose, 1231
Tablets, 1232
w
Warfarin Potassium, 1233
Tablets, 1234
Water, 1235
JP XV
Index
1775
for Injection, 1235
Weak Opium Alkaloids and Scopola-
mine Injection, 945
Weil's Disease and Akiyami Combined
Vaccine, 1237
Wheat Starch, 1237
White
Beeswax, 336
Ointment, 1238
Petrolatum, 981
Shellac, 1085
Soft Sugar, 1121
Whole Human Blood, 1238
Wine, 1238
Xylitol, 1240
Injection, 1241
Yellow
Beeswax, 336
Petrolatum, 982
Zaltoprofen, 1242
Tablets, 1243
Zanthoxylum Fruit, 1371
Zedoary, 1372
Zinc
Chloride, 1244
Oxide, 1245
Oxide Oil, 1245
Oxide Ointment, 1246
Oxide Starch Powder, 1246
Sulfate Hydrate, 1246
Sulfate Ophthalmic Solution, 1247
Zinostatin Stimalamer, 1247
INDEX IN LATIN NAME
E
Achyranthis Radix, 1252
Adeps
Lanae Rurificatus, 808
Suillus, 808
Agar, 1252
Pulveratum, 1253
Akebiae Caulis, 1253
Alismatis
Rhizoma, 1253
Rhizoma Pulveratum, 1253
Aloe, 1254
Pulverata, 1255
Alpiniae
Fructus, 1265
officinari Rhizoma, 1256
Semen, 1256
Semen Pulveratum, 1256
Maydis, 542
Oryzae, 1346
5o/o«/, 1014
7Wf/cz', 1237
Anemarrhenae Rhizoma, 1256
flatf/x, 1305
Radix Pulverata, 1305
Arctii Fructus, 1267
y4recae Semen, 1258
Armeniacae Semen, 1257
Artemisiae Capillaris Flos, 1258
Asiasari Radix, 1259
Asparagi Tuber, 1259
Astragali Radix, 1260
yl tractylodis
Lanceae Rhizoma, 1261
Lanceae Rhizoma Pulveratum,
1261
Rhizoma, 1260
Rhizoma Pulveratum, 1260
y4«ra«?z7
Bobilis Pericarpium, 1273
Fructus Immaturus, 1302
Pericarpium, 1265
B
Belladonnae Radix, 1263
Benincasae Semen, 1264
Benzoinum, 1265
Bezoar Bovis, 1325
Bufonis Venenum, 1368
Bupleuri Radix, 1266
Calumbae
Radix, 1267
Radix Pulverata, 1268
Cannabis Fructus, 1298
Capsici
Fructus, 1268
Fructus Pulveratus, 1269
Cardam om i Fructus, 1 27 1
Carthami Flos, 1348
Caryophylli
Flos Pulveratus, 1274
F/os, 1274
Cassiae Semen, 1271
Catalpae Fructus, 127 '1
Cellulose Acetate Phthalate, 480
Cera
y4/6a, 336
Carnauba, 411
Flava, 336
Chrysanthemi Flos, 1271
Cimicifugae Rhizoma, 1272
Cinnamomi
Cortex, 1272
Cortex Pulveratus, 1273
Clematidis Radix, 1214
Cnidii
Monnieris Fructus, 1275
Rhizoma, 1275
Rhizoma Pulveratum, 1276
Coz'cis
Semen, 1276
Semen Pulveratum, 1276
Condurango Cortex, 1276
Copt id is
Rhizoma, 1277
Rhizoma Pulveratum, 1278
Corni Fructus, 1279
Corydalis Tuber, 1279
Crocus, 1349
Curcumae Rhizoma, 1367
Cyperi
Rhizoma, 1280
Rhizoma Pulveratum, 1280
D
Digenea, 1280
Z>/o«:oreae
Rhizoma, 1282
Rhizoma Pulveratum, 1282
Dolichi Semen, 1282
Eleutherococci senticosi Rhizoma,
1283
£p A drae Herba , 1283
Epimedii Herba, 1284
Eriobotryae Folium , 1314
Eucommiae Cortex, 1284
Evodiae Fructus, 1285
Extractum Belladonnae, 1263
Fe/ £//-.»', 1262
Foeniculi
Fructus, 1285
Fructus Pulveratus, 1285
Forsythiae Fructus, 1286
Fossilia Ossis Mastodi, 1313
Fritillariae Bulbus, 1287
Gambir, 1287
Pulveratum, 1287
Gardeniae
Fructus, 1288
Fructus Pulveratus, 1288
Gastrodiae Tuber, 1289
Ge«i7a«ae
/?arf«, 1290
Radix Pulverata, 1290
Scabrae Radix, 1306
Scabrae Radix Pulverata, 1306
Gera«n
7/erda, 1291
Herba Pulverata, 1291
G/wseMg
i?arf«, 1292
Radix Pulverata, 1293
Radix Rubra, 1342
Glehniae Radix cum Rhizoma, 1294
Glycyrrhizae
Radix, 1295
Radix Pulverata, 1296
GwffJOTZ
Arabicum, 1251
Arabicum Pulveratum, 1251
Gypsum
Exsiccatum, 1298
Fibrosum, 1297
H
Houttuyniae Herba, 1302
1777
1778 Index in Latin name
JP XV
Hydrangeae
Dulcis Folium, 1365
Dulcis Folium Pulveratum,
I
1302
Imperatae Rhizoma
Ipecacuanhae
Radix, 1303
Radix Pulverata, 1303
Linderae Radix, 1313
Lithospermi Radix, 1313
Lonicerae Folium Cum Caulis,
Lycii
Cortex, 1315
Fructus, 1315
M
Magnoliae
Cortex Pulveratus, 1316
Flos, 1317
Malloti Cortex, 1317
Mel, 1301
Menthae Herba, 1317
Mori Cortex, 1320
M out an
Cortex, 1318
Cortex Pulveratus, 1319
N
Nelumbis Semen, 1320
Notopterygii Rhizoma, 1321
Nupharis Rhizoma, 1321
o
Arachidis, 971
Aurantii, 946
Cacao, 386
Camelliae, 400
Caryophylli, 1275
Cinnamomi, 1273
Cocois, 535
Eucalypti, 654
Foeniculi, 1286
Maydis, 542
Menthae Japonicae, 1318
Ortvae, 939
Popae, 1055
Ricini, 414
Sesami, 1084
So/ae, 1113
Terebinthinae, 1214
Ophiopogonis Tuber, 1324
Opium Pulveratum, 939
Ostreae
Testa, 1326
1365 Testa Pulverata, 1326
PaeoMzae
Parfix:, 1328
Radix Pulverata, 1329
Panacis Japonici
Rhizoma, 1326
Rhizoma Pulveratum, 1327
Perillae Herba, 1329
Perezcae
Semen, 1327
1314 Semen Pulveratum, 1327
Pharbitidis Semen, 1330
Phellodendri
Cortex, 1330
Cortex Pulveratus, 1331
Picrasmae
Lignum, 1333
Lignum Pulveratum, 1333
Pinelliae Tuber, 1333
Plantaginis
Herba, 1334
Semen, 1334
Platycodi
Radix, 1334
Radix Pulverata, 1335
Po/yga/ae
Patfz'x, 1335
Radix Pulverata, 1335
Polygonati Rhizoma, 1336
Polygoni Multiflori Radix, 1336
Polyporus, 1336
Pulveratus, 1337
Pow, 1337
Pulveratum, 1337
Process/
Aconiti Radix Pulverata, 1339
y4 coniti Radix, 1338
Prunellae Spica, 1341
Puerariae Radix, 1341
R
Rehmanniae Radix, 1343
Resina Pini, 1347
Rhei
Rhizoma, 1344
Rhizoma Pulveratum, 1345
Posae
Fructus, 1346
Fructus Pulveratus, 1346
Saposhnikoviae Radix, 1352
Sappan Lignum, 1352
Saussureae Radix, 1352
Schisandrae Fructus, 1352
Schizonepetae Spica, 1353
Scopoliae Rhizoma, 1357
Scutellariae
Radix, 1358
Radix Pulverata, 1359
SeMegae
Radix, 1360
Radix Pulverata, 1361
Sewnae
Folium, 1361
Folium Pulveratum, 1362
Sevum Bovinum, 336
Sinomeni Caulis et Rhizoma, 1363
Smilacis
Rhizoma, 1364
Rhizoma Pulveratum, 1364
Sophorae
Paofo, 1364
Radix Pulverata, 1364
Strychni Semen, 1321
Swez-fz'ae
Z/erda, 1365
Herba Pulverata, 1366
Syrupus
Ipecacuanha, 1304
Senegae, 1361
Tinctura Amara, YLbb
Tragacantha, 1369
Pulverata, 1369
Tribuli Fructus, 1369
Trichosanthis Radix, 1369
u
Uncariae Uncis Cum Ramulus, 1370
Uvae Ursi Folium, 1262
Valerianae
Radix, 1306
Radix Pulverata, 1307
Zanthoxyli
Fructus, 1371
Fructus Pulveratus, 1371
Zedoariae Rhizoma, 1372
Zingiberis
Processum Rhizoma, 1341
Rhizoma, 1291
Rhizoma Pulveratum, 1292
Zizyphi
Fructus, 1307
Semen, 1307
INDEX IN JAPANESE
7
i7- v:
'V 1246
1246
SFt-ft^St 929
y-fttU^/V 1317
7?f/?f>yD 275
T^^iVKi/ySli 272
7y u/-;y7yy#i 273
T y D / -;!/■ ffil&^ftV 274
y?y/-;wMPti 274
77*777 'Jy 325
Tf^t^JVi 326
Mfi|g|7 5 ;l/ 309
7^Xn-7^f-/y tK| Pfe 327
T-yfyii^^yy 999
7-y7'jy 278
7^'jyi 279
77.n)Hfy| 316
77n;l/£y^ffic 317
7X3;m*yiaifI 317
T7,Yu-?4>yvlHmM 322
L-7X/^^y| 318
7Xt"'Jy 318
7y.f'Jy7^5-'7A 319
7Xt!ijyi 319
T X 7° 7 7 7 U V *f m 320
7XH/ttA 328
7-tr^;l/?3F7;l/;-')A 266
7-tr?V*7SF 268
7-feb75/7iV 267
7-trh'\W3F 269
7-fe7>n-;l/^^^ 265
7tyt^ 1287
T-fey-t^yfc 1287
7r/n-)l' 323
7 F D t7ygft^yjtftl% 324
7 h n fcTyffltgfciaaifiK 324
7Fl/t'Jy 276
7Fl/t'JyI 277
7Fl/t'JVffi*fS 276
Itl/U? 315
Tynyrny 277
r^sy-hnyffc 1324
7^y7;W n/fF-7bn 7y£#f
« 942
7 / Ny7^n/f F-^3!K5 5yft
#t« 943
7^\y7Wn^FM 941
7^y7Wn^ Fmk&MW. 942
T^yffc 940
7^y7y7 940
7 / \y* 939
7^7^ 1365
7^7^* 1365
7?y?yySlI 293
75*yyifl 296
75F'J 77 U VW®& 298
7 5 F U 77 U y^^mge 299
7 5 F F U V'm 295
7 5 F F D 7S?7 F U 7 A / 7;1/ 5 V
£#)* 853
7 5 F F u y'i/^y 5 yffiWit
851
75/$lSilfy 644
75/77J y*fP^f 297
75/77J yftJH* 297
7 A7-7- U 7 y B y P - y 7 305
7A77UyyB§E 305
7A77'jyyB 303
7-t777y 301
7-t7yy uy7MPti 302
7-t/^i/7y-y 300
77-fe7'jy 279
7y-tr7'J7i/§E 280
7 7 £7 '=f A 1251
7 7t737J 1251
7U/^yyS5^it 283
M*K**7FU7A 1094
L-7;l/7-y 313
L-7;l/7-yit^it 313
L-7;l/7-ytt^ita#tS 314
7;u7777 282
7^7777 A 284
7;l/^y/n-;HiS 285
7y7nyyyy 286
7Jl/7*nX?77)77T7X
287
7 y^7yy Sft®?^ 311
7;K*yy«^SftM 312
7ni 1254
7nx| 1255
7Dfyn-)l/S|I 314
7D7*'J/-y 284
•RMMW. 345
K,§.#^7FU7A 1093
SS»^7 F U 7 7 7 7 x 4 V 388
Slfi^y^y 347
7yV-y37 1265
7yf7jy 3io
7y7yjy7FU77 308
7yt'7jy*» 307
7y^7-^AStt 294
7y-t-7-7^7a7» 1286
7yt"7* 299
4t7 1130
^77-7yy;i/n-y g v 1130
4 77 -7 U 7H77y F -;Hfc
1131
^7y7A
^t?7A
1103
^7^77
^7Foy
^77? F
y.w-
i 774
E7F U7Aft#Hg
M7;l/5y^#tS 852
I 775
;l/ 773
■^yy-t-y 744
7^yHl7H7;T)l' 646
^/W^yyftii 777
^yy;i/7F 784
7;-7yF 780
77-7yFt 781
^y-77F£JH* 781
-Y y 7 x y -f y 7 u y*tt!isa*j*
762
y;7J7y 778
l- 4 y 7 y 7 y y^g^ 782
yyyp/V-y 783
7;7nn"y7yft*'Jy 783
L-/fVU/fyy 780
0>t'7yIlS 745
^F'yyyyyy 747
4 f 7 7. 7 y y y j£8B$ 747
-f7iy7a71/fi51I 748
77o7iy 744
07hn \±VJ*%\\M-%M%0 776
■^S77 5yiil 751
-f 5 77 3y^^§g 752
^5^7A7KfP% 749
-fW-try 1274
^yyn^)7y 754
^fyyn^y^yMS 755
7/X'jy 758
^ y 7 U yffi#&*ttJS$ffiAfi£ 763
^yyyy&tf« 762
^y7yn7 1258
7yr7n-;l/iil 753
^"yF/yyy 755
7yF/?-yyA7*y 756
7yF7 7yy« 757
7y7;nyfHAy77y 758
^ya7#y 1284
7^7 a 7 1285
7^7a7S 1285
V^avi 1286
7ny 1367
777 1313
77 77;!/ 1217
7U7xy7y 1215
7;i/y77773— ;y^ 1220
7077—7 1219
77 7;i/y 1262
1779
1780 Index in Japanese
JP XV
777;W/$fLX^7 1371
X
X — y-)]/ 641
X.JV"y 1346
xyv-y* 1346
It7i/-;l/ 986
xn^y-A— F 3 7-ft4t! 610
XXyy'^A 631
631
xy h5^-;v^IfSx^r;i'*
ttMS^JH* 633
xy h^^-^lfSx^r^a
#t« 632
XXh'Jt-;!/ 633
X^h'Jt-;!^ 635
iXh'Jt- ;l/7JCt4i*M^#t?« 634
x y y u vBfc 636
X y y U V^ie 636
xyy-;y 638
x?yyh-;l/Sti 637
X^^-^5 F 643
XfV5A 652
XfFD VM^-J- F U 7 A 649
xfFDyirth'J r )Ai 650
If^JUXl-^?^* 642
lf-;l/XXh7y^-)l/i 642
L-ifil/VXfyySIS 645
Z-?-lV^)}/\L*-mB&%M%0 648
Xfl/7'JVliI 650
z-i-vywmkmx 65i
xfl/y^T?y 648
xy-V-tf 5 F 640
Ifhlt F U 7 AyMflt) 598
X F X y -> 5 F 644
X F F y y 653
XF^VF 653
lFD*r.7AStt 611
iFQ*x7AMttaifl 612
xy^i/vyjcftit/ 616
Xt°ijy*— ;l/ 621
XHVUfi/Vffi^^ 622
l7iF'JyffilS 619
!7iF'JySSSi 621
xy x f u vmm.m£%\wt 619
X7xF'JyIlI10% 620
i^'JV'yiii 618
I'J^D'7/f-yv 627
xU xn-7-fyyifjl/3/\7li7,
ryl/ 629
x u y d? -f y yy 77 U ySS
630
xuyn-7-f yv^y F t'tySS
629
xjWhxy 612
i;i/3^y>7iD-* 624
xy^^yygEiS 627
x;i/n y f U y-7 y y v^ffi 625
xji/n y f u y? y y v^MS 626
xji/3* y f u w y y v®fc&ttl?H&
625
MkMIB 1244
MikWm 1245
Mt^ y77A(lllIn)£#fS 755
Mt*'J7A 1009
Mk/7;yyyA7MPti 390
Mt#;Fy7A£#F« 390
Mty 'J7A(201Tl)£#Jit 1158
Mty-FuyA 1097
10% mt-T F U 7AMt 1098
xyrf'+fy 1279
^ 725
Wk*——* 1052
i&^F"/Uv£#t$ 602
i^-^HX^ffiHift 901
^u-ty--^ 727
iyfc't^'fyylSS 616
XV7;l/5V 615
;t7=^ 1260
^-y=f'y 1358
t7n*y| 1359
Hfey-feiJV 982
jfy-ty 1336
^-y/^y 1330
^-y/sy • y yty^'y- ify.-77.ifc
1332
^7/^7* 1331
t7Fy 1277
jfyyyyfc 1278
tW/^A 948
^-^r-tlX'yAHy'fbtl 947
t^yyoyy 948
t*ynF V&Bfc&*W& 95 1
t+yrF7+)-'fy'jytiS 956
sT^i/FW 958
t^yF-yyftltl 960
■t^i/Y—)^ 954
t+y77D*^yMS 951
t^yy t-ny 956
^-dr-t-y-tf y y 949
^-yyy°yyn-;i/it^^ 950
tyn^yy 938
t'J7l 939
yyyyyty ylts 946
sM/yvift 946
tyy 1335
tyy* 1335
A-fxyl^yhxyft 790
y^xy^RfRti 789
yyy-uv 795
##;rtl 386
SnSixvyrfi 415
73 y 7 1341
^7j7 1336
jnyjt^y uv 8 07
7)-yny 1341
MUMZ-^T* 1308
ii^y *y7Alf FU7A (99mTc)
&^m 1105
Hit 689
mg&MM 689
yy-y^yyy— sf L ii^ 793
y;y^yyv«i^it 794
*ynV7 1306
*yny7* 1307
* 7 x y y yfcfPti 387
y;y-fe;F 403
y7y°Fy°u;y 403
i|vy;#yM#y7A 1013
SnBKIigffcx^y 1309
«x?7Fyy;HS 400
#y-vvy:f-H? 790
*'J5^-y 1007
#;l/x5j-n—;i/&Bfc& 412
y7;F-yy/^ny 411
ts ;w \ y" y n a y ;i/7 v^-y f y 7 a
TXWt; 405
#;WW-1f £V 404
y;;l/ty F^yKfPti 406
L-*y*'yyr'fy 407
y;;i/yp-7 408
#;l/yn — y^;yy7A 409
A;MCi-7tF 'J7A 410
^MtAtF'J7A 412
ft;^?— ;i/ 411
#nnv 1369
#V^a7 1341
fg«ti^y^tt#* 716
^M*ti-Ly^JrC«* 841
tiy7f'J77^ttll 596
fat tF y U 7 y 7 ^ ttwSl 374
fpaS*«L®?:F F U7A 1109
fgiS¥tt)S 1171
£*il£# 1241
igiSfflssit^s^-yyy^v 1091
902
$ai^«^|siLA7 7f-y 1074
ijQillS£jftl^7y^y 843
^MyJc^b7;y5x7A^-yy 288
igS*®rfk 77F 3 x 7AW|fi
289
&mmmim^ft{t&JKm r 7 y ^v
1054
f&mptWi-fh U7A 1096
iglSS^-5 7y^V 1091
l&JI 0*1^7 75^7 786
fg»ffif«a7^irc?
1156
fatSfESiyy 3x7 AT; U 7 A
^iBCG77fy 335
#V7*7 1295
*y77x+7 1296
y7vy"7fix^y 1297
#77*7* 1296
*7tV 1252
AVfy^ 1253
di-usy )]/ 402
d-UW)]/ 401
ffRft 538
y7V7y^7;U7A 1008
•%¥ 3.V 1372
292
JPXV
Index in Japanese 1781
U7siL%;V?$mM 696
P-U=>9 hi/?— fe*(TX^;I/^r;l/7.)
694
$-11=7 9 \- y ?—•& (^->'J9A)
695
^•JB^^i/V 1074
#^S? 726
4^3 7 1334
3^ 3 7* 1335
Wa-JllW 1334
^7# 1271
^r-9-y-y 1271
^v"J b— ;l/ 1240
^'Jb-iVffilfi 1241
3^77 1302
dpyy^yyy 798
W^'f-yv SSKia soo
^?+)-7'fyyiil^r^ 799
^--^x^;^^ 1051
^x-^^tM^ 1053
^-^yylil*fi 1050
EStR^* 265
^ 3 7#y 1321
^3>>-y 1257
^3>)-yic 1257
#3 — F^V^ 769
&?*-# U V n |£y h U 7 A 1092
^flg 336
7iyW 'J 7 A (67Ga) ftttS 69<
^xylth'J^A 1099
^iySttSf 515
7ny 1315
?v?y 1364
^yyS 1364
^VfSMif. 1364
^Sfet^V^ 1266
7 7^7y®*!J^A 1010
y7 yxn-7/fyv 516
?7'Jxn-7/fyvi 517
yjyy^yy-h 522
y y y?"-7 y yy y yiixyy
521
y y y ?■* 4 y y *£®^ 519
y y y?y^yySit*7'-try
520
yy^-y— b 544
yyy*— ;y 544
^yy-M 545
yyy*-;y6y-y® 545
^y7Xfy77ii/ti 519
y * 7° 5 5 y &Bfcfi*#i$r 523
y p^yy y v-r v y 7 atM^
533
yp^yy*7A 534
^nnty-iViiS 546
ypy*;i//n-xt h y 7 a 546
ypyy-tf'yA 531
y^y^fyii
y-?V4vmMk
y^yyyilffil
ynhy-^y*— ;i/ 532
ypy-tfAA 528
yn-yyi|| 529
yay^y'y— b 525
7P7w7*y— b#"y*-tr;i/ 526
y n 7 x y* y -)i<&i& 524
^n^ 7 xfySiS 530
7nS7iy7iyfi 526
7Q57iy^iylig 527
yp57°7 5yitsfe^: 528
y d A®?y h y 7 a (5icr) mM
1099
yn-tyy y^y h y yA 1100
ynyA7x — a— iu 487
?D7A7i-n-yn/\yliyf
yyh'jyA 489
707A7i-n-iW^Sfy|l
yyy 488
y nyyr-tf' ^y F 490
ypyyy-tf^y Fifc 491
ypyyy-tftf^y Fig 492
y n;i/7x — y 5 y-Tyyy 7 AS
497
yn;i/7x — y
498
yn;y7x-y
499
y n;l/7x — y
500
y n;l/7x — y
S 499
^□;i/7i*yy*)W^y|x7r
y 496
rf-y n;y7 x -y yy?y-f yit
501
y n;l/7°nA5 F 504
yn;l/7°nA5 Fig 505
7D;l/7n7-yytii 502
^njl/y'n^yySSM 503
7D;l/y'n'7yyiiift|H 503
7 n;i/Atyyy^;l/nyiM
493
7Dji/A+yyySiS 493
^D^y/ysiixfy 494
ynn7yy— y 495
y*yy7xyyy 714
yyt^yylii 715
^r^fyylil 716
yyy p-yyy^yK* y 7 a
1011
y*y?yyy 712
y*y yy 704
yyyyyytx'y 713
y* y -t y y 702
yy-tyyyjyit 704
y'y^yyy? F 699
yypy^*yy7A7jcfpti 391
7~)V?3-tV 701
yyy/y 1353
gPi*''Jt7 7fy 1005
yy^yyyyA 836
gfi*7jcyyK 1087
gR^ES)/^y7wy 967
yyyy 1272
>TA\L% 1273
yyyy& 1273
yy^yit^tt 795
|g H B B -lryn-y 477
!£ ditty yy y vffi»*tt«»ftifis
765
jfc-SttttBlf'ja^y-ty 710
yyyyy 1271
^hf7iy7'7;Hi 797
yh7°P7xy 796
y/yy^yn-yi 486
try 31/ 1330
«SttS* 1236
y y y -7 y y vUW& 698
yyfyt 1290
yyfTf'lfft 1290
yyyyfS 1290
yyyya7n 1291
yyyy g 7ns 1291
p77? 1348
WfcM 735
nyyy 1342
n77*y 1280
n77*y* 1280
n7*'7 1315
37tyj 1316
p 77 y y^^ig 535
PA^y'yyy 1237
p yy'y 7°y 1346
p y xfyy y yyy*ylt h y 7
A 540
n'JXfylil 541
nyfyyftiiyyy 543
nytfy 538
nl/*yy7in-;V 506
3V7rn-y 507
n777yyy 506
nnyt; 1267
3Dy**i 1268
nyXyyn* 1276
^yX'yynli^y 1277
^•)Syy^yy-5-7A 290
y^"7 1325
yyy 1252
yyii 1285
705
y**7y 1267
y-7?S 1084
yyy 1352
v-
-y-yyp-tyy 551
-y-yp 1266
-y-yyy 1259
*?y§Xdr7 1349
1782 Index in Japanese
JP XV
mm 268
SStb'J^A*^ 1091
+Ky#lJV 1075
^yA'JVtl U 7 AtMP% 1076
t7 5V 1349
+>"57$& 494
■tj-^-y^'yo 1 ) 336
77 7777 r n° y -^y 1077
•y-'J^-;l/-5 3^/^VtSc 1078
ifU^-;Ht 1079
^'J^Htl-'JW 1108
777.71177.7 88i
W3-)VW% 1080
•9" U ^l/gfc^HIW 1080
^^I'^^—ft/WBM. 1078
^#777 A 393
SMb^V 1178
81^7^77 A 835
1tV^5^ 1364
ifV+5^* 1364
7®rfb7it 316
+7777 1288
+7777* 1288
D-y^il 1279
+J-V7a7 1371
1+77 a 7* 1371
BM 955
•tfyy^-v 1307
WY—V 1081
+)-yt? 1282
7V77* 1282
fJl/h^n7iy 1242
+fil/b7'n7iyi 1243
7777 F 548
■y7/n^75y 548
77y3/^5yftM 549
Mfl775 L *;i/5y 310
iM-ffl F U 77y?/U? 1210
M-ffl^ydvi/A/^y? 969
Wffl7i/-;V'*y7;i' 987
tffla-Fy'Jt'Jy 771
y 7 7 y u y 507
yyny^uv 509
■y^n^yhy-HiS 549
770:7777-5 F*W% 550
yay 1313
73*7) 1283
7777fy 513
yy^77lft^ 1090
yyyy'v 552
■y«;*-y 1086
yyjy 1369
y 7° n /\ y 7 y y&Sfcfekf pfe 551
'y/^-yy 513
y-^yy-y 1328
^^9^9% 1329
y-r-tfyy 1334
y-r-y'vyy 1334
A-fb^yyA 1008
Wbyh'jyA 1095
981
lfh'J7AS 1100
567
t*$r 567
■yvyxv?
•>i7y-Y 1256
y^yy-y* 1256
m^6M 1141
y a 77 a 7 1291
7 a 77 a 7* 1292
S^^yy;i/tfF 785
i§^yy;i/tf f§8 786
«« 1088
«*£« 1088
7 a 777 1271
itix^7-;l/ 640
iliffl7i/-;V 986
!Sffi7x/-M 987
yg 1 )^ 1272
-y777fyf b'J7A 510
ynyyy— y 511
ynyyy*-;i/§£ 512
7n y 7|7 7 D^AtF 'J7A
659
yv7 1317
JS*«C* 735
IMM7XVS
7777- fcf
•7777- tf-lr
yy-tf7-\A 567
574
7ify»iW^7y^iy|II
574
yyy 1343
777 77V 576
777 77V§e 576
77nt77 U yf F U 7 ATRfPf
573
yyoyxyyyFuyy 570
7^D7it3 F 571
7^D7it3Fi 572
73.yf 1315
yyy-yv 577
yy+yvlg sso
7nyyyffiltS 578
W§St:"7-7 7 369
77f75yMt 599
-7Xf^5Vl«i 600
yy;yyyyy 600
yyn°y?F 598
yFoyyynv 609
yFoyyynvig 610
•7/7?fy yyvy-7— 1247
y/7°n7F 592
7H F nx;l/3 7 7 V/i/fr&M
583
y hfdi ;i/ y f y- y v / y 7^
584
ytFnnryyjyifi 581
y y f n a y 7 v u vK^Sc 10%
582
y y f n n y 7 y y v ®9Sft 1 %
582
yyyy-t— v 597
77t-F-;l4tS 575
77iytF53y 593
77 i7t F7 7 -y-r\V V )]/W3
593
77 i7h F'757-7i/- 7-
¥ u — y y f 595
7 7 x y y F y 7 y &Bfc& 594
y77U7hyy7F 596
77 7 U 7W.%MM% F7V-f F
596
yy'n^v^BM. 569
7^*77ilii 569
*'Sf?it'7-77 368
77^7 7 yj VMM 588
yy7#7*n— 7 590
y/777°n — ll&Mffl. 590
y/vyFuy-F 588
y/vyFuy-hie 589
7*^*7 3 7 591
y7777 5vffiW?t 591
ii7^vyy*n7 F-7n^y =
£#f$ 945
7^7 a 77 1275
y^yyy 1302
??7R 1235
7g7"7y>y 786
7 a y-7 7 7 V7°D ty7i!7
788
7y-y'7°^^^7MPfe 586
y;777-yy^^^ 586
A±ltm 1238
7KK-fb*U7A 1012
TKK-fb^yyyy 392
TK^-fbyFuyy 1101
7W/ h^7Alft**W#l
777/ F-7Al«ffiM
1137
1138
-FtMW
WbTKitf
777777-
73*7 5 7
1083
7r7U777n-Jl/
777 yy
777 yy
777 77
777 yy
7F77F
yfy^yyyi
yy°nyyy f v
1118
1117
1116
A777A 400
7°U77y7 40 1007
-77*7-777 837
yyyllS 1117
1777 1114
1116
1114
7.77 7 7 U 7 F y;F^it7jcfP%
1134
7777A 1136
77/\7 7A7F'J7A 1122
777 U F 1131
777U F77°77 1132
77H°U Fie 1132
7;l/y°U77joiflt/ 1133
777uy&tjig H34
777 t77 7711 1124
777 7/ : fy*-7 1125
777 r y f ^7-7—7 1126
777y7/7 F d r7V7MPtl 1126
JPXV
Index in Japanese 1783
771/7 W VH°5W 1127
771/7 ^ yK7 v /vi 1128
771/7^ V^-9-y— ;l/ 1128
yM^yyythij^A 1123
yji/^7*nt7?y^yth U7A
1129
#t« 1130
iAI»^7f U 7 F ^ V ^ F
596
mm^v? loss
fitUHf^V 697
muf^-Y^^-^m 556
mUy/W 808
Sit* 1236
ttKfiii 1120
^S^itS 1098
5i^yyy 982
•tyy— 71/ 484
•fc-yn7 1297
•feby^- bi&Bm 485
•fe^y/ 1360
■t^*'yn-y7' 1361
*.^13% 1361
-tr7T707 415
-t7r^oW7*-fe;V 416
■fe7 7^n;Hg 417
■fe7 7 7D;l/MIg 419
•b77V"Jytb'J7A 426
•fe 7 r V" U yth'J7A 7MP4tl 428
•fc7r h U ?y7"nn - yy^ij n— ;1/
425
•fe7r Fn+yJl' 421
■fe77f l JyfF l J7A 424
■t7 7y+yy 422
■t7 7 0fyf |-'J7A 423
•fe7^7A 442
•fe7iH°A^^7Mn% 439
•fe7*V'^A-7-b U7A 447
*7 * 7'7'7y£iS 457
•fe7*^^yA7-h'J7A 449
•fe7^-7 t 7A^\^r-fe^;l/itK^ 453
•fe7*^7AttKit 455
•t7*7^V 451
-t7f^7V'yth l J7A 448
432
•fe77J^V £tf^77l/:^M? 433
•fe7 77^V H°^dri/;l/ttKit7MPtl
430
•fe77hyy £4^7 ;i/ 437
-tr77F77 t.°#+77l/fflf*A 438
•fc7 7F77 H°tf*^77l/§g 438
•fe7 7— ;l/ 434
•fe77— ;l/77 7°-fe^ 435
•fe77— 7l/jffl$ 436
■fe7Xn-yytb U7A 464
•iT7^v'7A7MPtl 466
•fe 7 5^7*^7.^:7- h U 7 A 471
•fe7f-7777MP% 470
•t!7f7At'*'+y)l' 468
-t?7 77A £/t*^77l/Hf£ 469
*7 h U 7+Vyt b U 7A7jOfPtl
472
-t?7t:°7 3 F7F U7A 458
-tr7t°nABt^:!& 460
•fe 7 7*^ 7 7*7 "7" h U 7 A 429
•fe7*F= ! f-yA7'n :! f-fef^ 461
•fe75/7Xf h'J7 A7KfP% 446
•fe7/?y-;yth'J7A 445
-t7/y d r7AitKit 443
•fe 7 O d r^777j 1 CfP% 462
•t7n + yA7 :! f-fefiV 474
-t70 d r7A7h U7A 476
■t7-fe7i-h 480
-tr^^y^r — -if 1083
^7l/^n7+7Gtl77f"-|iJI7) 481
■fey+a7 1275
ty+a-7i 1276
•feya'7 1321
•tryy 1368
•tyf 1361
•tytS 1362
-t?77'J 1365
-tyyy-awfs B67
-t77"US 1366
•tf'77-7 696
V
V 7^7 a 7 1261
y77i'7j 1261
V77\yt: 1320
y#y 1352
y 3 7 1329
yii/tyyty+ty^yixyriv
mi
D-y;i/t'h-iV 1112
D-yji/t.'h-;H iii3
y-fyy 1307
yyjv 1142
yyy^ 1253
?yyt* 1253
?A7nyyllS 1140
y7VH°7U7tt^it 1138
y;uy 1139
#»y7'J7A 1009
#«7F U7A7MPtl 1096
M?yy^y7A 834
M'JfyA 826
M^**^-FU7A 1093
mfcifM-y \- u 7 a£#« 1094
#yn- y y 1089
i^fylF 1089
?V — VW. 1141
77-7^771/7*3 7 282
?y-y!77iytF7 5y 595
y y^yf^M u 7 352
yy^-y 1344
y-Y^-ys 1345
1280
ytJ?-H7pL?ix^7
yVXri 1113
y 7 y ;i/ 1* 7 7 1£^ is 553
y* v hnyyth'J 7 A*frtf
553
5^-7^7— 7F 1159
9*-7^7*— 71/^ 1160
f757-^th'J7A 1163
9*-7 3 VW(MUW& H60
7^7 3 i/MkQimESl&WL 1162
9*-7 3 V itft^it^ i£ffi#tS 1161
9*-73V#fty#/ 1162
f7 7 5FIiI 1171
f-7 7 3 Fit^itie 1172
5^77 F— 71/ 1165
f-^-77-x 1168
ft^y?-il/tF U7A 1166
f-^'jy'77^^it 1168
T^St^T" h U 7 ATKfRtl 1109
f-^Sf L K7 h U 7AMS mo
fy-ty-y-yy 1326
fyty-y-yyj 1327
fynfyyfiis ii73
^•9 ! -77itKtt 1179
^St 928
9^-r.y*7— 71/ 1175
5^£77t^7X^a 1176
f^f-yyt^yXili 1177
T^y^yyA^-fbtiTKfRti 1174
5^ 1256
5^—71/ 1170
5*-^ea-7l/T 7 ^fvSt& 1174
MI7-fef;i/3'JyMkti 271
£ "WH 7A*T'j-yyB 304
ft#t>17- : e7 7l/Ky-77 h U 7A
300
ffiWH ^ 7' 7 £ 7 7 %$& 746
[/f 3^7.A-777yf-v7F
U7A 750
!7Wyhr.7Al« 1137
ft#tll-fe7xt:Aitftit 441
ft#tll-fe7*y"77Vtt^^ 458
g#f/i"t?7*^7A'£^'£ 456
Mff75 7-ytF'J7A 1164
Mffif^y?-ii/tF U7A
1167
ft#tH7-fen^^7 cae^iasi 7 )
1148
ffiM^yn^AyyllS 1223
708
ffi#tn n f 7 7 7 vmmm. 724
£#f H t°7 9 7 U 7 7 F U 7 A 998
^117 7^77 656
Mffl7i-F-fytF'J7A 992
Mffl7nWt77F'J7A 666
£#1/177 F-ynyt F U 7An7N
y|xyf;v 1025
ffiIi*7*?^yytF U7A
687
£tfffljft#l4tti!M»7^7 711
1784 Index in Japanese
JP XV
■m^myK 1235
r77y->F 559
F
ffi*Mfft^ H* V 7 y 7 7 V
1227
7^77/7 F£#t$ 560
F
7 a 77 1274
TtFD3-jl/l 556
F
7 a 77* 1274
rtFnn-jl/iftM 557
F
7 a 77$) 1275
r*7inW5y/-7iFS£ 554
F
7 3 7F7p7 1370
r/fi^n^rF^^OUyii
L-
7a W 1336
ig 558
F
7a W* 1337
F
777* 1245
h
F
ttHNi-Sh^V^ F 716
W
»i^7? U TH&MWm-t b 7 V A F
F77/7 1264
F
596
F77/77 1268
F
»»®m b*^77'j rm%WM
F7#77-+»-y7;l^fS 1270
F
£-7 7 77 596
F7^7'7fy^ 1269
F
ttHMS*WH-ft#7^^V
980
F>7 7?7-yS 1269
F
W$J^7J;17/7A 389
F77 1305
F
»Iffth+V'f F 115(
F77S 1305
F
ik&Bmjft&yy^-v 7i6
b^ — y 1327
F
fyf 1273
F7-y* 1327
7
yA7?fi
400
vfcvy
J vWtffoWkM.-fcfa
1212
7 77 7
j ymit^mmm&ki
1213
-yn7f
a-)l,m.W& 1213
T
f^37'7-y 1150
ffiBSIffik: FD^-y^nfiF-t^o-
7 738
rt7-f 'Jy 1158
7#7 — )\/ 1149
r7 F7rnyiry FSxXril/
1154
r7 F7fnyityF|x7fW
WS 1154
r7F7fny7Dftyix7rJF
1155
rXF7rny7'nftylx/^r;F
ffiW* 1155
77P^7yGtf77liJI7) 1143
rF5*'fySiS 1156
7F777 7'JV:^^& 1157
777>l)76i«i£ 1153
rl/UV'i 1214
^Ir^lTiF^X^A 289
TV? 1289
rytyF7 1259
7^77 7 7*7 560
777 b 77^x7777 F 'J7A
•Y77 5 564
f>X F y Vffl&J- F'J7A-ft7
18 565
f>7F7y40 561
y=^y F77 40£#f« 562
r+7F7y70 562
"f^y b U y 565
r+7 F P / b 77 r V%fc&MW&
7jCfP% 566
F 7 \L 1265
F7tyn,y7 1325
F'JllrV^ 1325
Fvtnnyfyyv 542
F^-tnn-yi 542
Fp~7iP — )]/ 1181
F 37in-)l/n/\^lxyfWiF
■77A 1183
F 37in-;l/xafy|x7fiF
1184
Fn7in-)l/Six7f)F 1182
Fay 1303
Fnyyn. y 7 1304
Fny| 1303
Ffa7 1284
FF77-7 VWkW&fafy H85
F7-fV^A 1186
F 7*7^ 77 1180
F7^/yF 1369
F7*'yFJ 1369
F7f5F 1187
F7^7A^ 1191
F 77777^7777 1192
F77777^§e 1193
F77777^£#f« 1193
F7 7°77 1194
F'JTA-y/ny 1195
F U7Ai//PV7-feF-F 1196
F'J 77777 1197
FiJ^D^XtF'J^A 1202
F 'J?n*xtF 'J^A-yn *, 7
1203
F'J7D)F/f7> i F 1198
F'J^njMfyyF'i 1199
Fija^^yy 1201
L-F l J7*F7rV 1211
F'J'stv'yi-^Hifcl 1204
F'jA+y7ixy ;WtKitig i 204
F'j/?yty 1208
F'j/?-yryt 1209
F u/ ?yyylit 1207
bVJ?i?V vWkWfe 1 207
F U / F 7 / -;l/^»^*ffi% 1209
F U /- 7*7 y? 7 ^ yli 1206
777? — F 1189
;yt7?-FS ii89
777? 5 F 1188
;l/7*7 3F§£ 1188
M'jyySSI 1190
F7;t-V 1169
yix'y'hv 1195
P7'7 3F 1211
PVlx'V 1170
m 808
^-y-yyAit^tt7jcfpti 603
ty-y-O'J VitKit 7jC^P % 606
^y7)F'jyy 604
7 y 7 y y y v 77 y -t 7 604
^y;i/b'-yyISi 605
/^yylli 602
7?5VliS 601
P^U F— 7 608
t-
7^7777 937
7y?7$) 1055
7 HP — 7 904
77 r 7 U V- 7 P^7 x — y 3 vft
906
t7 7V*'jylis 907
77r7'Jy5i^it 908
t7'n+-ty 908
t777 7l 905
tn + yyMI 906
-7771x°y^^^ 911
xi7 777y^:^£#Ji$ 912
—■ft* 1333
xiT/7* 1333
xnfyf 920
xnfyiTSF 919
xnfyiftM 921
xin-t— 7 917
r.n't— ;1/§e 918
X3 577F 919
-BMfc^H 407
x-feij FP— ;l/ 916
xHr77'Jy 913
xnr;y=njyfg: 914
xi-te;l/7yy|£ 915
xi F 77'77 925
xFyyyt'y 926
xiF7y77viE 927
xi F P 7 U 7 U 7§g 928
xi7x77y 922
0*JK^.777V 786
f\M 801
IL^7777 77MPt; 393
%mJkffl%0 803
MM 1218
xi;l//-\77y 923
xi;l//-\77v|g 924
xyyy 1292
xy77* 1293
JPXV
Index in Japanese 1785
Fy 1314
7°Pt:°y 1039
/3-)V 878
910
yyy^y yyft
/i-)VWBM. 909
910
y
343
937
931
g^ U -fe U V 703
g^V-tf ;l/n - ^ hJ&AYMfc 50
/X#H°V 936
/^TFUt'JV 930
7)H«rny 932
y*nhv* 933
^--;l/^ 934
y ;y f u yy u y*&®^ 935
/;i/7DWyy 932
/\
6fe-fe7-y7 1085
Sfey-irUv 981
fifcttW 1238
a« 1121
/\f5'V 1301
Ay^J 1317
?\yU& 1318
/^y#?ft 1318
/\-?yiy7 7 1294
/\n^+ry*yA 719
/NPyV 719
/\pyy F — ;l/ 717
/\n^u F— ;l/§| 718
/\yy-" 1333
A^y 1287
/^yti/'jylSi
/^tyF 1 ) 1324
/^D7iy 331
A?D7iy| 332
AyFyyy 330
yyy°yyy£#fS
yyyy«E^it 333
L-M'JV 1221
/■yi/ti*y — ;l/ 333
A^nifb'J^A 1110
A'i//fy 3 fy7*y 1014
/v/n^/fi/ySKI 1221
^v7°mW]-*V^7lk 1332
Ar^^A 962
/^^ijylSi 965
/V^gylKtaM 965
A775 y-yyTyi/KyyyyyTRfp
% 395
A775 yyyyyKyyyyAKfi
394
/^*ty$Ifiif;v 647
Ayyyy^llS^yyy 386
329
1223
/^yy~yy^li :
/^yy~yy^li ;
^7 7-fy 966
/'?7*M7;l'fllF 968
/^^'Jythy^A 969
Ay^U7fy 96i
Ayy"D-7Altt 962
Ayffy 964
/•?yFryl*;vy7A 394
n F y y 7 u y ( jtisy-l§.i& £ ) 760
t FTS#ttffll$iJgc*;l/- : &y 708
HFM^ttttloffl^tf-yy^v 707
A^fi7n7"'Jy 724
HF75^yIifi 724
hF77 yySlitt 725
tF77-7 v&afc^g 725
HF'ntyyy/^il 739
HF'ntyyySSl 739
\L Fn^V7°DH°;l/-fe;yn— 7 736
nFnty^/^rySlI 736
tFP7nnf77F 727
n f o 3 y ;y- vWkWtf&tfo 734
tFnn;l/fyy 728
n F D3;i/f)/y'^7 x vn F y 3
yfyff 730 l-:
tFn3)l/fy'y3/\^8i7f)l' 7.
733 7.
tFo3)l/f7yn/\yiiXr^t 7.
F U y A 732
H F on;l/fy*y u ylxyf;l/t F
|J7A 731
H F □n;Vfy*yiSi/;T^ 729
\L F on)l/fy'yS|i7r;l/ 730
nynyn— y 741
t7'PFn-77?yix7fll/
743
t^^t 414
yyypyy 740
*» 269
K'+J-nyy 367
yypyy^Sl 368
tyyyAi 1229
yy 5vAft77 7°-fe;y 1230
£^ U rylSi 366
d*^y-y*— ;i/ 366
t>77 1257
t>^7a7 1260
tf^yy^y* 1260
H'7 3 7 1314
£yy 'jxfylil
yyyyyy-ySftKit
yypyy 1258
H° a 7 ;l/7 r - F 7" F U y AtMW
1105
n°yyyuyAttKii
t^ry 'Jyf F
ny^yyjyi
1228
1226
1004
996
U y A 997
1000
y°yyyy f 1046
t°y;i/H*yy 1000
tyyry/^it 1045
n°u F^yyllS 1047
y°u Fyyyt£S?Mx7Wi« 1048
n° u f y y y 3 y A-f t % 1 047
tyy+yy 1001
yVyyyyt^itTRfpti 1002
H°n— ;l/r.FUy 1049
^nIMth'J7A 1108
y°pyyyyit®?it 994
fn^yTJA 1003
fn+yjy 1049
t'ypn-y 995
1
7 7W7y 655
7r : &7 : -yyt5: 657
7 7*^778 657
7rn^Wfh'J7Ai 660
7 7 n^Ut F 'J7A*|0ft 658
7^ Fyyy-y 993
7x— MV 991
7x-F^Vffc 992
7 x - f y y|g 992
7x-;yyyy"y 990
L-7iX;l/75-7 989
7i-l'7 l JyISi 990
7i*fyjy* l J7A 983
y— ;l/ 985
7i7-;V-II5* , J->yF 987
7x7-yy;y*y7yyyy 988
7 x y -)i/7ii/*y 7 y y y v&MW.
7 x y— ;l/7fC 987
yy/^my-y 984
7xy/yyy*y-y& 10% 985
'iV^-MlVil 661
yy77iy 660
CTjTy uy-y-yyy?fi 273
itj y y y p F y • t f p y yffiWS
953
173 y- y y p F y a Atf& 952
iTY-y-uy-y^yy-yft 879
iTjy U i-)VWM 1080
fiWf& 567
yK 1346
t'y y y y u y^^*fq%
ty^yy 994
999
STjyyyy— t?
S73y*yy-y--ty
S73f77F-;V-
1165
STjyy yy b
K73 a - f ■ y u y u
IfiTjp-Fxyy-y
1355
yyyy^y f >J7A
yyyyr^yyySf L K^ 688
7 y y*y yyy y y 7 7 y y y f y f
yyy 662
yy^'yy-Fit^^ 664
yyyyyyyypy 675
yyyyy-F 672
yyyyypyyy F-F 670
yyy-yy-i'yy f uyy 673
1230
770
y y —tfi
1101
1786 Index in Japanese
JP XV
7 )]/ifUy ?%/)]/ 674
777P7 Fpv 673
777 FW 668
7)\>V7 J d^k> 669
7iVX;l/f73yIiI 692
77x F 77777 670
7Ji/7it^yitvHi7TJi/
676
7777777 677
7;l/5^f/^A*y-fe;l/ 678
7)\zy J t£^lJ£BM. 678
7;Wl/t"7*D7iy 679
7P-75 F 690
70*5 F§e 691
7o7nt'*V 667
7P7°P7°7777 7°77 667
7nW-t7th 'J7A 664
f^-77P-7 480
7;7 ; eu-;vSiS 378
7*7 U a 7 1337
7*7 U g 7* 1337
77 1338
77S 1339
777 5 V 377
7777 rV 384
7fJl/73* , 7SyMft 1082
7*hDH°7A^b% 385
7*F7fi 1238
7*F7« 700
7*F7$t&U* 701
7*t7*-yylMS 383
77x77^7 380
77iW-7^7'J-A 380
? 7x77^7i
,'W
381
i 379
i 383
' 7 x F P —)VWb
? 7 7 7 p— ;l/^S
'V7— F 382
'"l/*'7'f7ylil 370
? y^-{yylil 372
? pv7'77 374
'nA'x+yySIS 375
'"nt7 U 77v 7 7;l/^ 376
r P77 7y;I/RJ| 377
'°7 77PVSf L Kx77V7F U7A
*fpife 1107
'°77'77 1020
'°7-tf/-\A^ 1021
'°7 7 7°P7xV 1018
'7^7?rytb'J7A 1018
'°U 5 FV 1026
01/7 V 1045
'i/F- y'pv 1021
'1/ F-7nyn;\7ii7T^
1024
'yp-7nyi 1022
'l/F-ynyiii^7-;V 1023
'°p77 V75 FffiSfciS 1028
" P 7 7 V 7 5 F itK^ie 1 029
" P 77 4 V7 3 F it®ffi«« 1 029
'u-fiJvMBM. 1030
'°py?7 ylKiffilffi 1030
'n^fn-)HS*|ft 1032
7P
7°P
7°P
7°P
7°P
7°P
7P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
7°P
ynMy
1033
7nM7
1033
7*7-5 F
777PV
777P7
y 5 V 7 V
ttS 766
y 5 VfflESt
y 5 v«les
777 5 F
77 U V
fi/'jyl
7 7VIB
7 7 vmm
/"yr'Jy
7° 77 7 7
7°7777
H°777*U
77 7 P —
77 7 P —
^7F
^77 Fie
yywm
yy?y/fyii
777y7y|li
1035
1034
£#f« 1034
y u vffiftdcttJBWffi
J£ 1041
i££#j« 1042
1042
1043
5K^7)Cfttl 1044
1088
1089
%it 1036
7 77 1040
7 77IS 1040
P— ;l/ 1039
;l/&^ 1037
7it^it|g 1038
1027
1027
IS 1036
722
365
353
354
p p7j;1//77vJ
1031
^/7JV7FU77 721
A/"'jytF ^AftM
AVX 1282
^y?7^7 77ifLSHt 337
^7 P y y y*77°P 7°7V||X777
335
^•^7^7*7— F 364
^+f7^7*7-F#;#t§e
^7^n-iHtt 363
^y^77vy 771
^7t7fy^ 77^1*
^yyyy'v 355
^777 7"v77°p 7°7V^X777
358
^y y y 7v y 7^x7777 f u 7
7 359
^7/?7y?7?
^7/?77f|?
/Kyy yyv±]=r
-7 7 yyll
^yyyy'vii 356
■^-i?ifyii| 338
^-VVl^WkWm 339
^7 F 77x77 1263
^7F'77p7 1263
^7/^5 7S^it 1225
^77 5 7Sg?iM8 1226
^7-77PX77M!y$J 342
^yf)^-7AMkfS 342
^77777p— )]/ 346
7yy;Kr.yij V77 >J 77 349
^y7M-y y v^Vlf 7v*f0
348
^777D"7Dy 343
|l7ry 360
^X777-7vy
gyj-A 36i
gx777-7vy
&*» 362
^y-fe7 7FSiS 340
^y-fe7-7AStt 344
^V-7 F -7AMfS 345
^ V F 7 7 F 341
^7^ U vmttyl&fiity) 351
^fVytii 980
^77vit^)7#Jit 981
^7'n-7-f yylfl 975
777 x77 V 977
71/7it7y7 7 7yi£ 978
7l/7x77y-77/fyiJ|i 979
^77x77v§? 977
^<-y?yi/-y 972
^v F +yy u vy x vllffi 974
^y F/~"7i7'y
7v7Fp-
77 7777 973
mM 972
7731 374
77# 1095
W^ny-Jl/ 487
777x7 FD-;V 683
777x7 FP-VIS 684
777-7 7 7v777777jcfP%
685
777-77 7V7 F U 77 686
M^^M?©X77 1298
7-7 F P 7° vMAtiKWSiM 722
75 7 1321
7 5 7x77 1322
7 5 7X77& 1323
7 5 7777 1323
77 7 p 77 y u yySil 723
7 U 7- F 77777 391
77^ UV 681
77-7 U V* 682
7 77 7 P - 7 7 ^ 7e?S7jCf Pt!
682
77 7 1363
77 PV 1302
777 7 1352
77 U 7-7 1231
777 7-718 1232
77Vt° 1318
7yv7°S 1319
77 7 1326
77 7* 1326
7I7TV 1015
7KFV3— F 1017
7 U 7777777 V^77777
398
7U 777V777V^7 F U 77
1106
7u y;i/y- f 80 1007
7 U 5 7 7 V B Sft^iS 1006
-7— 7aPyP7 862
-7— 7a Py PAS 863
-7 7 F-777VC 898
-777 1283
JPXV
Index in Japanese 1787
■77'J 1280
•?^n^-;l/tf 833
?7nJ- ;M500 831
T^Drf— ;l/ 20000 833
■7^3- 7 400 830
■7^3- 7 4000 832
*?9U-d— ;l/6000 832
-?7 — V 1298
ftS^^X-x^ 641
■^^DfijySlI 842
^;i/l"X7jcfP4t; 840
D-r/"h-;i/ 841
D-7y- F — 7£#fS 842
3 7 n / t A y y Sft^ffi 893
5 7*yxy 896
Snty-;V 892
S^ty-iWIlS 893
5 7 n 7 336
5r*-7^>y 894
^fs^^yyiii^fii' 895
5 7-y-O'J VMBM. 897
5 a ^/Sy* 293
A
imtt^ y x u ysl&7jcttM^ft#«
764
te*7yt°yjy 306
&7KX7/ — }]/ 639
1**71^7 386
l7R7xyj!i 514
i7jC#LS 802
ixK u vWKM #;yy 7 a 396
A n° d y y 7 y y 7 a Tjcf p % 902
y
^->vfySIS 89i
7 777y 861
/^n7i/W-HiS 843
7 771/ 3 y 850
ynAy^y 844
7 '»H£^y+t- F 693
/Xhyy-jV 864
^?y7i?5Viai 868
7 7-77 ~\ A 845
L-^t-y 869
^^77 884
/t^5^y 890
/f)H;l/n7 F ijy?l//fy9i
876
y-^yx;yyy f uy-7 7^fyiSg
877
tf/-7 f;H7 x F U y^ffi 875
tf/-7 f;H7 x F u ylilt 10%
876
/f^7n*+yy 885
737l/-fe;l/D — 7s 871
/f;l/f7h7fay 882
^Jl/fX h7fnyi 883
7 971/F/^R 874
7yyF7y,RfRti 873
/f-^yF- v* □ y 879
7 571/77 F-ynynA^ixxf
;y 880
7771^777-7 7 A^{kti 87i
7-37i/n+fxyy:^ft% 88i
/r/nyitv Hx7ril/ 866
/r/n y x-y y F ixyfil/ffilfS
866
/r/nyiiiXfil/ 865
7 hW7y 870
-/±#t
1^ 887
F 886
F§e 886
F 869
'm 867
iWMIfe 867
-y 889
859
y h^n77
y 77077
7 F F 777
y i-7nn- y
y h7nn- y
/ hW'yl
y Fnx^y-
y FP-yy-y^
7 7-r-Fy/y 855
/t'ftX^y 858
t&^AvWkW&M®. 860
/7itA| 847
/yyyF 849
/yyyFIS 849
/7oyyiiS 848
7 y y y" 9 - F %\\M 857
7777 77 uy7jcfp4fj 86i
yi/777'y 855
7ny^A tK|P% 863
<#-7yF— ;l/ 856
mvf— ;i/ 857
•t y y y 1253
■7-y7J7 1352
-t/7r7 l Jyi7)l/5-7A 291
■t/7r7 l J7l 7 U 7 U y 704
■777 7-T hnyyMS 899
77ti7it®:i&§E 902
777 7it®;i&7MPtl 900
1*^7 1298
yyy 1265
lffl57y 846
HfflK 845
yy$i 535
X — 7U)tt 654
x7 7x 1262
1099
xtfr^y/y 1214
3—^*5 F 767
3-7*3 F7F 'J7A/7)l/;
S 854
a - F -it U 77l/®7 7 x 7 -;HB
772
3 — F^y^ 769
3 — F4vi/A 773
3 7ft*'J7A 1013
3 7 -ftyAjo.it 7)1/7? y (1311) mm.
768
37-^77777 1102
3 y-fty f u 7 a (1231) 7777
1103
3 711x7 F U 7 A (1311)$ 1103
3 y-fty f u y a (1311) 777/7
1103
3 7-fttyyy^yF uyA(i3ii)ffi#t
« 1103
mm 680
MM§Z 681
HKf7#f« 681
371 768
3 77-y 1276
3 7 7-yS 1276
"7
7 7 77*^7 7 U7A
77n"77n7— y 810
@§KU7'y7t;'y 1060
777P-7 804
7777777 7 U7A
7-y^-fe-fi 971
7 7 F 7 F C 805
77F7FCiE 806
5X5 L 7yIIS 1054
1104
809
jy-y
J 77
'J 7 7
l- U 7
■j yy
J F F
'J F F
'J F 7?
'J F #
'J77
'J77
■j y*y
■j 7*y
u^y
A
JtK7
A
'j y^y
j -77
Ja7
P-y7FU7A 821
P-y7FU7A§E 822
7U7§e 824
7 U 77]Cf P% 823
yitK^ 829
-AitFtit 830
U y^K^ 1067
U yit^itie 1068
7 y 818
7y£#fS 818
yfyy 1064
y7°yy7777 1065
7^7 7ySfL^it 1063
7 7'y 1059
7 7'y U y|i7r;l/t F U 7
1061
7 7'y U y|i7r;l/t F U 7
£#J1$ 1062
7 7'yt'c 1060
oyF 7)1/77777 819
ay 1313
1788 Index in Japanese
JP XV
MM7 ;l/5-^A»'J ? AtWp %
292
Sft^U^A 1014
«/^J?A 334
SEiK^^A* 839
IftSI^ 7* * V ? AtKI Pfe 838
Sft^^y^AftlM 839
mMM^y^m 1246
SKKMIB^IS® 1247
mkwuuwyd 66i
'JaWV 1306
'Ja^^yJ 1306
ffiM^?7 4V 966
'Ja^a^ 1256
^STttH'SxdrX 1347
U yy;Vf 1066
■JV37^> V i£g^*f R% 820
VVWt^rJVlfclOX 537
'jyfcf^yliX 536
'jyfnf'fyi 537
'Jyi^r-f V 7M P% 536
ij VW£%3])W2 AtMP% 396
ij V^tJC*^- h U ? AtMP% 568
U Vg£— tRU* ;l/ V 7 A*ft]# 397
l/-fe;l/£v 1055
t/-fe;l/£vtfe0.1% 1057
i/-fe;i/£v§e 1057
i/-fe;yn'vfttj-S 1056
i/fy-^/^y5fylxxr;y
1059
i/ty^yjylKi 8io
l/Aa;i/7 7 yi5ig 813
l/An;l/7 7 yIEitftltt 814
U^fD^yth'J^At 817
816
L/3^F/-\ 814
Uf/T-n^^yvlz/fylS 815
Uy^a 1 ) 1286
UV—7 1320
O— hX^X 1353
D-bX^^-7^X^5VfSC 1355
D-hX3o*.-#--^Vffe 1355
D-ntx-?y-y*| 1357
D-HW-/W^yy'7^?5
Vffc 1356
□ -H«t 1353
D-hny 1357
L-Q/fyy 812
p^tf^yiixxf;HiI
1070
□ ^^yygi^xXT^^ilt*
y?7°-t;y 1071
n+yxn-T/f-yy 1073
□*yy'a7iyth u7Ayjcfti%
828
o+?-7^-yy io69
n-yy 1347
O^-tf/^A 827
7 4 frffifo^&m&yjrf-V
7;y7 7 u yyju^A 1233
7;y7 7 uy^y^Ai 1234
1237
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